Edited by Janos Fischer,
C. Robin Ganellin and
David P. Rotella
Analogue-based Drug Discovery III
Related Titles
Fischer, J., Ganellin, C. R. (Eds.)
Analogue-based Drug Discovery II
2010
ISBN: 978-3-527-32549-8
IUPAC, Fischer, J., Ganellin, C. R. (Eds.)
Analogue-based Drug Discovery
2006
ISBN: 978-3-527-31257-3
Abraham, D. J., Rotella, D. P.
Burger's Medicinal Chemistry, Drug Discovery and Development
8 Volume Set
2010
ISBN: 978-0-470-27815-4
Li, J. J., Johnson, D. S. (eds.)
Modern Drug Synthesis
2010
ISBN: 978-0-470-52583-8
Lednicer, D.
Strategies for Organic Drug Synthesis and Design
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Chorghade, M. S. (ed.)
Drug Discovery and Development
2 Volume Set
2007
ISBN: 978-0-471-39846-2
Edited by Janos Fischer, C. Robin Ganellin
and David P. Rotella
Analogue-based Drug Discovery III
The Editors
Prof. Dr. Janos Fischer
Gedeon Richter Plc.
Gyömröi ut 30
1103 Budapest
Hungary
Prof. Dr. C. Robin Ganellin
University College London
Department of Chemistry
20 Gordon Street
London WC1H 0AJ
United Kingdom
Prof. Dr. David P. Rotella
Montclair State University
Department of Chemistry & Biochemistry
Montclair, NJ 07043
USA
Supported by the international Union of
Pure and Applied Chemistry (IUPAC)
Chemistry and Human Health Division
PO Box 13757
Research Triangle Park, NC 2770-3757
USA
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Contents
Preface XIII
List of Contributors XV
Part I General Aspects 1
1 Pioneer and Analogue Drugs 3
Janos Fischer, C. Robin Ganellin, and David P. Rotella
1.1 Monotarget Drugs 5
1.1.1 H2 Receptor Histamine Antagonists 5
1.1.2 ACE Inhibitors 6
1.1.3 DPP IV Inhibitors 7
1.1.4 Univalent Direct Thrombin Inhibitors 8
1.2 Dual-Acting Drugs 10
1.2.1 Monotarget Drugs from Dual-Acting Drugs 10
1.2.1.1 Optimization of Beta-Adrenergic Receptor Blockers 10
1.2.2 Dual-Acting Drugs from Monotarget Drugs 11
1.2.2.1 Dual-Acting Opioid Drugs 11
1.3 Multitarget Drugs 12
1.3.1 Multitarget Drug Analogue to Eliminate a Side Effect 12
1.3.1.1 Clozapine and Olanzapine 12
1.3.2 Selective Drug Analogue from a Pioneer Multitarget Drug 13
1.3.2.1 Selective Serotonin Reuptake Inhibitors 13
1.4 Summary 16
Acknowledgments 16
References 16
2 Competition in the Pharmaceutical Drug Development 21
Christian Tyrchan and Fabrizio Giordanetto
2.1 Introduction 21
2.2 Analogue-Based Drugs: Just Copies? 22
jV
2.3 How Often Does Analogue-Based Activity Occur? Insights from the
GPCR Patent Space 25
References 32
3 Metabolic Stability and Analogue-Based Drug Discovery 37
Amit S. Kalgutkar and Antonia F. Stepan
List of Abbreviations 37
3.1 Introduction 37
3.2 Metabolism-Guided Drug Design 39
3.3 Indirect Modulation of Metabolism by Fluorine Substitution 42
3.4 Modulation of Low Clearance/Long Half-Life via Metabolism-Guided
Design 45
3.5 Tactics to Resolve Metabolism Liabilities Due to Non-CYP Enzymes 46
3.5.1 Aldehyde Oxidase 46
3.5.2 Monoamine Oxidases 48
3.5.3 Phase II Conjugating Enzymes (UGTand Sulfotransferases) 49
3.6 Eliminating RM Liabilities in Drug Design 51
3.7 Eliminating Metabolism-Dependent Mutagenicity 51
3.8 Eliminating Mechanism-Based Inactivation of CYP Enzymes 54
3.9 Identification (and Elimination) of Electrophilic Lead Chemical
Matter 60
3.10 Mitigating Risks of Idiosyncratic Toxicity via Elimination of RM
Formation 61
3.11 Case Studies on Elimination of RM Liability in Drug Discovery 62
3.12 Concluding Remarks 67
References 68
4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin,
a Potential Ghrelin Agonist for Gastrointestinal Motility Disorders 77
Mark L. Peterson, Hamid Hoveyda, Graeme Fraser, Eric Marsault,
and Rene Gagnon
4.1 Introduction 77
4.1.1 Ghrelin as a Novel Pharmacological Target for GI Motility
Disorders 77
4.1.2 Macrocycles in Drug Discovery 79
4.1.3 Tranzyme Technology 80
4.2 High-Throughput Screening Results and
Hit Selection 82
4.3 Macrocycle Structure–Activity Relationships 83
4.3.1 Preliminary SAR 83
4.3.2 Ring Size and Tether 83
4.3.3 Amino Acid Components 87
4.3.4 Further Tether Optimization 89
4.4 PK–ADME Considerations 92
4.5 Structural Studies 95
4.6 Preclinical Evaluation 96
VIjContents
4.6.1 Additional Compound Profiling 97
4.6.2 Additional Pharmacokinetic Data 98
4.6.3 Animal Models for Preclinical Efficacy 100
4.7 Clinical Results and Current Status 100
4.8 Summary 103
References 104
Part II Drug Classes 111
5 The Discovery of Anticancer Drugs Targeting Epigenetic Enzymes 113
A. Ganesan
List of Abbreviations 113
5.1 Epigenetics 114
5.2 DNA Methyltransferases 116
5.3 5-Azacytidine (Azacitidine, Vidaza) and 5-Aza-20
-deoxycytidine
(Decitabine, Dacogen) 118
5.4 Other Nucleoside DNMT Inhibitors 122
5.5 Preclinical DNMT Inhibitors 123
5.6 Zinc-Dependent Histone Deacetylases 124
5.7 Suberoylanilide Hydroxamic Acid (SAHA, Vorinostat, Zolinza) 125
5.8 FK228 (Depsipeptide, Romidepsin, Istodax) 127
5.9 Carboxylic Acid and Benzamide HDAC Inhibitors 131
5.10 Prospects for HDAC Inhibitors 132
5.11 Epigenetic Drugs – A Slow Start but a Bright Future 133
Acknowledgments 133
References 134
6 Thienopyridyl and Direct-Acting P2Y12 Receptor Antagonist Antiplatelet
Drugs 141
Joseph A. Jakubowski and Atsuhiro Sugidachi
List of Abbreviations 141
6.1 Introduction 142
6.1.1 Platelet Involvement in Atherothrombosis 142
6.2 Thienopyridines 143
6.2.1 Ticlopidine: 5-[(2-Chlorophenyl)methyl)-4,5,6,7-tetrahydrothieno[3,2-c]
pyridine 144
6.2.2 Clopidogrel: (þ)-(S)-a-(2-Chlorophenyl)-6,7-dihydrothieno[3,2-c]
pyridine-5(4H) acetate 145
6.2.3 Prasugrel: 5-[(1RS)-2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4,5,6,7tetrahydrothieno[3,2-c]pyridin-2-yl
acetate 147
6.3 Direct-Acting P2Y12 Antagonists 152
6.3.1 Nucleoside-Containing Antagonists 152
6.3.1.1 Cangrelor: [Dichloro-[[[(2R,3S,4R,5R)-3,4-dihydroxy-5-[6-(2-
methylsulfanylethylamino)-2-(3,3,3-trifluoropropylsulfanyl)purin-9-yl]
oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]methyl]
phosphonic acid 153
Contents jVII
6.3.1.2 Ticagrelor: (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-Difluorophenyl)
cyclopropylamino]-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-
3-yl]-5-(2-hydroxyethoxy)cyclopentane-1,2-diol 154
6.3.2 Non-Nucleoside P2Y12 Antagonists 157
6.3.2.1 Elinogrel: N-[(5-Chlorothiophen-2-yl)sulfonyl]-N0
-{4-[6-fluoro-
7-(methylamino)-2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl]phenyl}
urea 157
6.4 Summary 158
References 158
7 Selective Estrogen Receptor Modulators 165
Amarjit Luniwal, Rachael Jetson, and Paul Erhardt
List of Abbreviations 165
7.1 Introduction 166
7.1.1 Working Definition 166
7.1.2 Early ABDD Leading to a Pioneer SERM 167
7.1.3 Discovery and Development of Clomiphene 169
7.1.4 SERM-Directed ABDD: General Considerations 170
7.2 Tamoxifen 171
7.2.1 Early Development 171
7.2.2 Clinical Indications and Molecular Action 172
7.2.3 Pharmacokinetics and Major Metabolic Pathways 174
7.2.4 Clinical Toxicity and New Tamoxifen Analogues 175
7.3 Raloxifene 175
7.3.1 Need for New Antiestrogens 176
7.3.2 Design and Initial Biological Data on Raloxifene 176
7.3.3 RUTH Study 177
7.3.4 STAR Study 177
7.3.5 Binding to the Estrogen Receptor 178
7.3.6 ADME 179
7.3.7 Further Research 179
7.4 Summary 179
References 180
8 Discovery of Nonpeptide Vasopressin V2 Receptor Antagonists 187
Kazumi Kondo and Hidenori Ogawa
List of Abbreviations 187
8.1 Introduction 187
8.2 Peptide AVP Agonists and Antagonists 188
8.3 Lead Generation Strategies 189
8.4 Lead Generation Strategy-2, V2 Receptor Affinity 192
8.5 Lead Optimization 197
8.6 Reported Nonpeptide Vasopressin V2 Receptor Antagonist
Compounds 199
8.6.1 Sanofi 199
VIIIjContents
8.6.2 Astellas (Yamanouchi) 199
8.6.3 Wyeth 201
8.6.4 Johnson & Johnson 201
8.6.5 Wakamoto Pharmaceutical Co. Ltd 202
8.6.6 Japan Tobacco Inc. 202
8.7 Conclusions 203
References 203
9 The Development of Cysteinyl Leukotriene Receptor Antagonists 211
Peter R. Bernstein
List of Abbreviations 211
9.1 Introduction 212
9.2 Scope of the Drug Discovery Effort on Leukotriene Modulators 214
9.3 Synthetic Leukotriene Production and Benefits Derived from this
Effort 215
9.4 Bioassays and General Drug Discovery Testing Cascade 216
9.5 Development of Antagonists – General Approaches 218
9.6 Discovery of Zafirlukast 218
9.7 Discovery of Montelukast 224
9.8 Discovery of Pranlukast 227
9.9 Comparative Analysis and Crossover Impact 229
9.10 Postmarketing Issues 231
9.11 Conclusions 232
Acknowledgment 232
Disclaimer 232
References 233
Part III Case Studies 241
10 The Discovery of Dabigatran Etexilate 243
Norbert Hauel, Andreas Clemens, Herbert Nar, Henning Priepke,
Joanne van Ryn, and Wolfgang Wienen
List of Abbreviations 243
10.1 Introduction 243
10.2 Dabigatran Design Story 246
10.3 Preclinical Pharmacology Molecular Mechanism of Action of
Dabigatran 254
10.3.1 In Vitro Antihemostatic Effects of Dabigatran 255
10.3.2 Ex Vivo Antihemostatic Effects of Dabigatran/Dabigatran Etexilate 256
10.3.3 Venous and Arterial Antithrombotic Effects of Dabigatran/Dabigatran
Etexilate 256
10.3.4 Mechanical Heart Valves 257
10.3.5 Cancer 257
10.3.6 Fibrosis 257
10.3.7 Atherosclerosis 258
Contents jIX
10.4 Clinical Studies and Indications 258
10.4.1 Prevention of Deep Venous Thrombosis 259
10.4.2 Therapy of Venous Thromboembolism 259
10.4.3 Stroke Prevention in Patients with Atrial Fibrillation 260
10.4.4 Prevention of Recurrent Myocardial Infarction in Patients with Acute
Coronary Syndrome 260
10.5 Summary 260
References 261
11 The Discovery of Citalopram and Its Refinement to Escitalopram 269
Klaus P. Bøgesø and Connie Sanchez
List of Abbreviations 269
11.1 Introduction 270
11.2 Discovery of Talopram 271
11.3 Discovery of Citalopram 272
11.4 Synthesis and Production of Citalopram 275
11.5 The Pharmacological Profile of Citalopram 276
11.6 Clinical Efficacy of Citalopram 277
11.7 Synthesis and Production of Escitalopram 278
11.8 The Pharmacological Profile of the Citalopram Enantiomers 279
11.9 R-Citalopram’s Surprising Inhibition of Escitalopram 279
11.10 Binding Site(s) for Escitalopram on the Serotonin Transporter 283
11.11 Future Perspectives on the Molecular Basis for Escitalopram’s
Interaction with the SERT 286
11.12 Clinical Efficacy of Escitalopram 287
11.13 Conclusions 288
References 288
12 Tapentadol – From Morphine and Tramadol to the Discovery
of Tapentadol 295
Helmut Buschmann
List of Abbreviations 295
12.1 Introduction 296
12.1.1 Pain and Current Pain Treatment Options 297
12.1.2 Pain Research Today 300
12.1.3 The Complex Mode of Action of Tramadol 301
12.2 The Discovery of Tapentadol 302
12.2.1 From the Tramadol Structure to Tapentadol 303
12.2.2 Synthetic Pathways to Tapentadol 306
12.3 The Preclinical and Clinical Profile of Tapentadol 310
12.3.1 Preclinical Pharmacology of Tapentadol 311
12.3.2 Clinical Trials 312
12.3.3 Pharmacokinetics and Drug–Drug Interactions of Tapentadol 314
12.4 Summary 315
References 315
XjContents
13 Novel Taxanes: Cabazitaxel Case Study 319
Herve Bouchard, Dorothee Semiond, Marie-Laure Risse, and Patricia Vrignaud
List of Abbreviations 319
13.1 Introduction 320
13.1.1 Isolation and Chemical Synthesis of Taxanes 321
13.1.2 Drug Resistance and Novel Taxanes 322
13.2 Cabazitaxel Structure–Activity Relationships and Chemical
Synthesis 323
13.2.1 Chemical and Physical Properties 323
13.2.2 Structure–Activity Relationships of Cabazitaxel 324
13.2.3 Chemical Synthesis of Cabazitaxel 325
13.3 Cabazitaxel Preclinical and Clinical Development 328
13.3.1 Preclinical Development 328
13.3.2 Clinical Studies 330
13.3.2.1 Phase I and II Studies 332
13.3.2.2 Clinical Pharmacokinetics 333
13.3.2.3 Phase III Trial 334
13.3.3 Other Ongoing Trials 335
13.4 Summary 336
Acknowledgments 337
References 337
14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role
of Structure-Based Drug Design 343
Srikanth Venkatraman, Andrew Prongay, and George F. Njoroge
List of Abbreviations 343
References 359
15 A New-Generation Uric Acid Production Inhibitor: Febuxostat 365
Ken Okamoto, Shiro Kondo, and Takeshi Nishino
List of Abbreviations 365
15.1 Introduction 365
15.2 Xanthine Oxidoreductase – Target Protein for Gout Treatment 367
15.3 Mechanism of XOR Inhibition by Allopurinol 368
15.4 Development of Nonpurine Analogue Inhibitor of XOR:
Febuxostat 369
15.5 Mechanism of XOR Inhibition by Febuxostat 370
15.6 Excretion of XOR Inhibitors 372
15.7 Results of Clinical Trials of Febuxostat in Patients with
Hyperuricemia and Gout 372
15.8 Summary 373
15.9 Added in proof 373
References 373
Index 377
Contents jXI
Preface
The editors of the third volume of the book series “Analogue-Based Drug Discovery”
thank the International Union of Pure and Applied Chemistry (IUPAC) for
supporting this book project. We also thank the coworkers at Wiley-VCH, Dr. Frank
Weinreich and Waltraud W€ust, for their excellent help, and last but not least we are
grateful to all the contributors of this book. Special thanks are due to the following
reviewers who helped both the authors and the editors: Klaus Peter Bøgesø,
Helmut Buschmann, Paul W. Erhardt, Staffan Erickson, Susan B. Horwitz,
Manfred Jung, Amit Kalgutkar, Danijel Kikelj, Andrew MacMillan, Eckhard Ottow,
Jens-Uwe Peters, Henning Priepke, John Proudfoot, Stephen C. Smith, Bernard
Testa, and Han van de Waterbeemd.
Analogue-based drug discovery is a basic principle of drug research. In this book
series, we focused on analogues of existing drugs. In the first volume (2006), we
discussed structural and pharmacological analogues, whereas the second volume
(2010) also included analogues with pharmacological similarities.
In this volume, we continued the same concept, recognizing that in several cases
there is only a narrow gap between a pioneer and an analogue drug because of the
strong competitive environment in the industry. A new promising molecular biological
target inspires parallel research efforts at several companies. It can happen that
the first discovery does not lead to a marketed drug; instead, a molecule discovered
later proves to be the first to be launched. As a result of the strong competition, it can
also happen that two pioneer drugs are introduced nearly simultaneously in the market,
and these drugs often have chemical and pharmacological similarities.
The third volume of Analogue-Based Drug Discovery consists of three parts.
Part I (General Aspects)
The introductory chapter discusses the relationship between the pioneer and analogue
drugs, where their overlapping character can be observed. A chapter by
Christian Tyrchan and Fabrizio Giordanetto (AstraZeneca) analyzes competition in
pharmaceutical drug development. Amit S. Kalgutkar and Antonia F. Stepan
(Pfizer) study the important role of metabolic stability in drug research. Mark L.
Peterson, Hamit Hoveyda, Graeme Fraser, Eric Marsault, and Rene Gagnon
jXIII
(Tranzyme Pharma Inc.) write on the use of peptide-based macrocycles in drug
design exemplified with the discovery of ulimorelin.
Part II (Drug Classes)
A. Ganesan (University of East Anglia) gives an overview of discovery research
into anticancer epigenetic drugs. Joseph A. Jakubowski (Lilly) and Atsushiro
Sugidachi (Daiichi Sankyo) evaluate the structurally diverse drug class of the antithrombotic
P2Y12 receptor antagonists. Paul Erhardt, Amarjit Luniwal, and Rachael
Jetson (University of Toledo, USA) summarize the medicinal chemistry of selective
estrogen receptor modulators. Kazumi Kondo and Hidenori Ogawa (Otsuka Phramaceutical
Co., Japan) describe the discovery of aquaretics that are vasopressin V2 receptor
antagonists. Peter R. Bernstein (PhaRmaB LLC) evokes the discovery of cysteinyl
leukotriene receptor antagonists that are important in the treatment of asthma.
Part III (Case Histories)
Norbert Hauel, Andreas Clemens, Herbert Nar, Henning Priepke, Joanne van Ryn,
and Wolfgang Wienen (Boehringer Ingelheim, Biberach, Germany) report on the
discovery of dabigatran etexilate, an oral direct thrombin inhibitor approved for use
in the treatment of acute thrombosis. Klaus P. Bøgesø and Connie Sanchez
(Lundbeck) describe the discovery of escitalopram, which is one of the most successful
selective serotonin reuptake inhibitors in the treatment of depressive disorders.
Helmut Buschmann (Pharma-Consulting, Aachen, Germany) analyzes the
discovery of tapentadol, a novel centrally acting synthetic analgesic with a dual
mechanism of action. Herve Bouchard, Drothee Semiond, Marie-Laure Risse, and
Patricia Vrignaud (Sanofi) describe the discovery of cabazitaxel, a novel semisynthetic
taxane, a new anticancer drug. Srikanth Venkatraman, Andrew Prongay,
and George F. Njoroge (Merck) summarize the discovery of boceprevir and narlaprevir,
hepatitis C protease inhibitors. Ken Okamoto, Shiro Kondo, and Takeshi
Nishino (Nippon Medical School, Teijin Ltd, and University of Tokyo) describe the
discovery of febuxostat, a new uric acid production inhibitor.
The above 15 chapters of the book with 40 authors from 9 countries bring important
and successful drug discoveries closer to the medicinal chemists and to all who
are interested in the complicated history of drug discoveries.
The major parts of the chapters are written by key inventors.
We hope that also the third volume of this book series will be well received by
people interested in medicinal chemistry.
May 2012
Budapest, Hungary Janos Fischer
London, UK C. Robin Ganellin
Montclair, NJ, USA David P. Rotella
XIVjPreface
List of Contributors
Peter R. Bernstein
PhaRmaB LLC
14 Forest View Road
Rose Valley, PA 1908-6721
USA
Klaus P. Bøgesø
H. Lundbeck A/S
Lundbeck Research Denmark
9 Ottiliavej
2500 Valby
Denmark
Herve Bouchard
Sanofi
LGCR/Natural Product & Protein
Chemistry (NPPC)
13 quai Jules Guesde
94400 Vitry-sur-Seine
France
Helmut Buschmann
Sperberweg 15
52076 Aachen
Germany
Andreas Clemens
Boehringer Ingelheim Pharma
GmbH & Co. KG
Department of Medicinal Chemistry
Birkendorfer Straße 65
88400 Biberach an der Riß
Germany
Paul Erhardt
The University of Toledo
College of Pharmacy
2801 West Bancroft Street
Toledo, OH 43606-3390
USA
Janos Fischer
Gedeon Richter Plc.
Department of Medicinal Chemistry
Gy€omr€oi ut 19/21
1103 Budapest
Hungary
Graeme Fraser
Tranzyme Pharma, Inc.
3001, 12th Avenue Nord
Sherbrooke, Quebec J1H 5N4
Canada
Rene Gagnon
Tranzyme Pharma, Inc.
3001, 12th Avenue Nord
Sherbrooke, Quebec J1H 5N4
Canada
C. Robin Ganellin
University College London
Department of Chemistry
20 Gordon Street
London WC1H 0AJ
UK
jXV
A. Ganesan
School of Pharmacy
University of East Anglia
Norwich NR4 7TJ
United Kingdom
Fabrizio Giordanetto
AstraZeneca CV/GI
Lead Generation Department
Pepparedsleden 1
43150 Mölndal
Sweden
Norbert Hauel
Boehringer Ingelheim Pharma
GmbH & Co. KG
Department of Medicinal Chemistry
Birkendorfer Straße 65
88400 Biberach an der Riß
Germany
Hamid Hoveyda
Tranzyme Pharma, Inc.
3001, 12th Avenue Nord
Sherbrooke, Quebec J1H 5N4
Canada
Joseph A. Jakubowski
Eli Lilly and Company
Lilly Research Laboratories
Indianapolis, IN 46285
USA
Rachael Jetson
The University of Toledo
College of Pharmacy
2801 West Bancroft Street
Toledo, OH 43606-3390
USA
Amit S. Kalgutkar
Pfizer Worldwide Research and
Development
Department of Pharmacokinetics,
Dynamics, and Metabolism
MS 8220-3529
Groton CT 06340
USA
Kazumi Kondo
Otsuka Pharmaceutical Co. Ltd.
Qs’ Research Institute
463-10 Kagasuno, Kawauchicho
Tokushima 771-0192
Japan
Shiro Kondo
Teijin Ltd.
Kondo Laboratory
4-3-2 Asahigaoka, Hino
Tokyo 191–8512
Japan
Amarjit Luniwal
The University of Toledo
College of Pharmacy
2801 West Bancroft Street
Toledo, OH 43606-3390
USA
Eric Marsault
Tranzyme Pharma, Inc.
3001, 12th Avenue Nord
Sherbrooke, Quebec J1H 5N4
Canada
Herbert Nar
Boehringer Ingelheim Pharma
GmbH & Co. KG
Department of Medicinal Chemistry
Birkendorfer Straße 65
88400 Biberach an der Riß
Germany
Takeshi Nishino
The University of Tokyo
Graduate School of Agricultural and
Life Sciences
Department of Applied Biological
Chemistry
1-1-1 Yayoi, Bunkyo-Ku
Tokyo 113–8657
Japan
XVIjList of Contributors
George F. Njoroge
Eli Lilly and Company
Lilly Research Laboratories
Indianapolis, IN 46285
USA
Hidenori Ogawa
Otsuka Pharmaceutical Co. Ltd.
Qs’ Research Institute
463-10 Kagasuno, Kawauchicho
Tokushima 771-0192
Japan
Ken Okamoto
Nippon Medical School
Department of Biochemistry
1-1-5 Sendagi, Bunkyo-ku
Tokyo 113-8602
Japan
Mark L. Peterson
Tranzyme Pharma, Inc.
3001, 12th Avenue Nord
Sherbrooke, Quebec J1H 5N4
Canada
Henning Priepke
Boehringer Ingelheim Pharma
GmbH & Co. KG
Department of Medicinal Chemistry
Birkendorfer Straße 65
88400 Biberach an der Riß
Germany
Andrew Prongay
Merck Research Laboratories
Chemical Research
2015 Galloping Hill Road
Kenilworth, NJ 07033
USA
Marie-Laure Risse
Sanofi Oncology
Centre de Recherche de
Vitry-Alfortville
13 quai Jules Guesde
94400 Vitry-sur-Seine
France
David P. Rotella
Montclair State University
Department of Chemistry &
Biochemistry
1 Normal Ave
Montclair, NJ 07043
USA
Connie Sanchez
Lundbeck Research USA
Department of Neuroscience
215 College Road
Paramus, NJ 07652
USA
Dorothee Semiond
Sanofi
Centre de Recherche de
Vitry-Alfortville
Department of Disposition, Safety
and Animal Research (DSAR)
13 quai Jules Guesde
94400 Vitry-sur-Seine
France
Antonia F. Stepan
Pfizer Worldwide Research and
Development
Neuroscience Medicinal Chemistry
700 Main Street
Cambridge, MA 02139
USA
List of Contributors jXVII
Atsuhiro Sugidachi
Daiichi Sankyo Co., Ltd.
Biological Research Laboratories
1-2-58 Hiromachi, Shinagawa-ku
Tokyo 140-8710
Japan
Christian Tyrchan
AstraZeneca CV/GI
Lead Generation Department
Pepparedsleden 1
43150 Mölndal
Sweden
Joanne van Ryn
Boehringer Ingelheim Pharma
GmbH & Co. KG
Department of Medicinal Chemistry
Birkendorfer Straße 65
88400 Biberach an der Riß
Germany
Srikanth Venkatraman
Schering-Plough Research Institute
Department of Medicinal Chemistry
2015 Galloping Hill Road
Kenilworth, NJ 07033-1335
USA
Patricia Vrignaud
Sanofi Oncology
Centre de Recherche de
Vitry-Alfortville
13 quai Jules Guesde
94400 Vitry-sur-Seine
France
Wolfgang Wienen
Boehringer Ingelheim Pharma
GmbH & Co. KG
Department of Medicinal Chemistry
Birkendorfer Straße 65
88400 Biberach an der Riß
Germany
XVIIIjList of Contributors
Part I
General Aspects
j1
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
1
Pioneer and Analogue Drugs
Janos Fischer, C. Robin Ganellin, and David P. Rotella
A pioneer drug (“first in class”) represents a breakthrough invention that affords a
marketed drug where no structurally and/or pharmacologically similar drug was
known before its introduction. The majority of drugs, however, are analogue drugs,
which have structural and/or pharmacological similarities to a pioneer drug or, as
in some cases, to other analogue drugs.
The aim of this chapter is to discuss these two drug types [1].
The term “pioneer drug” is not used very often, because only a small fraction of
drugs belongs to this type and in many cases the pioneer drugs lose their importance
when similar but better drugs are discovered. A pioneer drug and its analogues
form a drug class in which subsequent optimization may be observed.
Analogue drugs typically offer benefits such as improved efficacy and/or side
effect profiles or dose frequency than a pioneer drug to be successful on the
market.
The discovery of both pioneer and analogue drugs needs some serendipity. A pioneer
drug must clinically validate the safety and efficacy of a new molecular target
and mechanism of action based on a novel chemical structure. In the case of an
analogue drug, it is helpful that a pioneer or an analogue exists; nevertheless, some
serendipity is needed to discover a new and better drug analogue, because there are
no general guidelines on how such molecules can be identified preclinically. The
analogue approach is very fruitful in new drug research, because there is a higher
probability of finding a better drug than to discover a pioneer one. A significant risk
with this approach is based on the potential for one of the many competitors in the
drug discovery area to succeed prior to others.
The similarity between two drugs cannot be simply defined. Even a minor modification
of a drug structure can completely modify the properties of a molecule.
Levodopa (1) and methyldopa (2) are applied in different therapeutic fields; however,
their structures differ only in a methyl group. Both molecules have the same
stereochemistry as derivatives of L-tyrosine. Levodopa [2] is used for the treatment
of Parkinson’s disease as a dopamine precursor, whereas methyldopa [3] was an
important antihypertensive agent before safer and more efficacious molecules
(e.g., ACE inhibitors) appeared on the market.
j3
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
Methyldopa (first synthesized at Merck Sharp & Dohme) has a dual mechanism
of action: it is a competitive inhibitor of the enzyme DOPA decarboxylase and its
metabolite acts as an a-adrenergic agonist.
HO
HO
O
OH
NH2
levodopa
1
HO
HO
O
OH
NH2
methyldopa
2
Levodopa and methyldopa are not analogues from the viewpoint of medicinal
chemistry. Both are pioneer drugs in their respective therapeutic fields and can be
considered as stand-alone drugs, because they have no successful analogues.
There are several examples, and it is a usual case that a minor modification of a
drug molecule affords a much more active drug in the same therapeutic field. The
pioneer drug chlorothiazide (3) and its analogue hydrochlorothiazide (4) from
Merck Sharp & Dohme differ only by two hydrogen atoms; however, the diuretic
effect of hydrochlorothiazide [4] is 10 times higher than that of the original drug.
The pioneer drug chlorothiazide is rarely used, but its analogue, hydrochlorothiazide,
is an important first-line component in current antihypertensive therapy as a
single agent and in combination with other compounds.
N
NH
S
Cl
NH2-SO2
OO
chlorothiazide
3
N
H
NH
S
Cl
NH2-SO2
OO
hydrochlorothiazide
4
Chlorothiazide and hydrochlorothiazide are direct analogues, which term emphasizes
their close relationship.
The terms “pioneer drugs” and “analogue drugs” will be discussed in the following
sections.
4j1 Pioneer and Analogue Drugs
1.1
Monotarget Drugs
1.1.1
H2 Receptor Histamine Antagonists
Before the launch of cimetidine (1976), only short-acting neutralization of gastric
acid was possible by administration of various antacids (e.g., sodium bicarbonate,
magnesium hydroxide, aluminum hydroxide, etc.) that did not affect gastric acid
secretion. Cimetidine [5], the first successful H2 receptor histamine antagonist, a
pioneer drug for the treatment of gastric hyperacidity and peptic ulcer disease, was
discovered by researchers at Smith, Kline & French. The inhibition of histaminestimulated
gastric acid secretion was first studied in rats. Burimamide (5) was the
first lead compound, a prototype drug, that also served as a proof of concept for
inhibition of acid secretion in human subjects when administered intravenously,
but its oral activity was insufficient. Its analogue, metiamide (6), was orally active,
but its clinical studies had to be discontinued because of a low incidence of granulocytopenia.
Replacing the thiourea moiety in metiamide with a cyanoguanidino
moiety afforded cimetidine (7). Its use provided clinical proof for inhibition of gastric
acid secretion and ulcer healing and was a great commercial and clinical success
in the treatment of peptic ulcer disease.
HN N
N
H NH-CH3
S
burimamide
5
HN N
S
N
H NH-CH3
S
metiamide
6
HN N
S
N
H NH-CH3
N CN
cimetidine
7
1.1 Monotarget Drugs j5
Although cimetidine was very effective for the treatment of peptic ulcer disease
and related problems of acid hypersecretion, there were some side effects associated
with its use, albeit at a very low level. A low incidence of gynecomastia in men
can occur at high doses of cimetidine due to its antiandrogen effect. Cimetidine
also inhibits cytochrome P450, an important drug metabolizing enzyme. It is therefore
advisable to avoid coadministration of cimetidine with certain drugs such as
propranolol, warfarin, diazepam, and theophylline.
Cimetidine led to the initiation of analogue-based drug research affording more
potent analogue drugs such as ranitidine (8) and famotidine (9) that lack the above
side effects of cimetidine.
O
NCH3
CH3
S
N
H
N
H
CH3
NO2
ranitidine
8
S
N
S N
NH2
SO2NH2
N
NH2
NH2
famotidine
9
Ranitidine [6] also has a pioneer character, because ranitidine is the first H2
receptor histamine antagonist that has no antiandrogen adverse effect and does not
inhibit the cytochrome CYP450 enzymes. Famotidine is the most potent member
of this drug class, which has been discussed in Volume I of this series [7].
Summary:
Pioneer H2 receptor histamine antagonist: cimetidine.
First H2 receptor histamine antagonist with no antiandrogen adverse effects and
without inhibition of P450 enzymes: ranitidine.
1.1.2
ACE Inhibitors
A natural product, the nonapeptide teprotide (10), was the pioneer drug for angiotensin-converting
enzyme (ACE) inhibitors. Teprotide [8] was used as an active antihypertensive
drug in patients with essential hypertension. It could only be
administered parenterally, which is a great drawback for chronic use of a drug. A
breakthrough occurred with the approval of the first orally active ACE inhibitor captopril
(11) in 1980 by Squibb. Captopril [9] has a short onset time (0.5–1 h), and its
duration of action is also relatively short (6–12 h); as a result, two to three daily
doses are necessary. Captopril can be regarded as a pharmacological analogue of
teprotide, but it is also the pioneer orally active ACE inhibitor. Captopril’s discovery
6j1 Pioneer and Analogue Drugs
initiated intensive research by several other drug companies to discover longer acting
ACE inhibitors. Enalapril (12) was introduced by Merck in 1984. Enalapril [10]
can be regarded as the first long-acting oral ACE inhibitor. The long-acting ACE
inhibitors are once-daily antihypertensive drugs. There are several long-acting ACE
inhibitors, whose differences have been discussed in the first volume of this book
series [11].
pyro-Glu - Trp - Pro - Arg - Pro - Gln - Ile - Pro - Pro -OH
teprotide
10
N
COOHO
HS
captopril
11
N
COOHO
N
H
EtOOC CH3
enalapril
12
Summary:
Pioneer ACE inhibitor drug: teprotide.
First orally active ACE inhibitor drug: captopril.
First orally long-acting ACE inhibitor drug: enalapril.
1.1.3
DPP IV Inhibitors
Sitagliptin (13) [12], a pioneer dipeptidyl peptidase IV (DPP IV) inhibitor, was
launched in 2006 by Merck for the treatment of type 2 diabetes. The medicinal
chemistry team began its research in 1999 when some DPP IV inhibitor molecules
were known as substrate-based analogues. The lead molecule derived from this
research was vildagliptin (14) [13]; discovered at Novartis in 1998, it was the second
compound to be introduced to the market.
NN
N
N
O
NH2 F
F
F
CF3
sitagliptin
13
1.1 Monotarget Drugs j7
N
CNO
N
H
OH
vildagliptin
14
The pioneer drug sitagliptin is a commercial success with 2010 sales greater than
USD 3 billion. Vildagliptin was the first successful discovery in this drug class, but
its development time was longer and it was introduced in 2007, after sitagliptin.
Vildagliptin is only moderately selective over DPP-8 and DPP-9 compared to sitagliptin
that is a highly selective DPP IV inhibitor. Based on long-term safety studies,
these selectivity differences do not influence the toxicity of vildagliptin.
Sitagliptin and vildagliptin show similar clinical efficacies. Vildagliptin has a short
half-life (3 h) and its dosing regimen is twice a day, whereas sitagliptin has a long
half-life (12 h) and once-daily dosing is used. DPP IV inhibitors are typical earlyphase
analogues that result from a highly competitive industry, and not the first
candidate (vildagliptin) but a follow-on drug (sitagliptin) became the pioneer drug
on the market (“first-in-class drug”). Further DPP IV inhibitors are available (alogliptin,
saxagliptin, and linagliptin) and the individual compounds differ significantly
in their mode of metabolism and excretion and these differences help the
treatment of patients with type 2 diabetes in an individual way [14] (see Chapter 5
of Volume II of this book series).
Summary:
Pioneer DPP IV inhibitor drug: sitagliptin (long-acting inhibitor).
First DPP IV inhibitor analogue drug: vildagliptin (short-acting inhibitor).
1.1.4
Univalent Direct Thrombin Inhibitors
Thrombin is a serine protease enzyme whose inhibition plays an important role in
the mechanism of several anticoagulants. Univalent direct thrombin inhibitors bind
only to the active site of the enzyme, whereas bivalent direct thrombin inhibitors
(e.g., hirudin and bivalirudin) block thrombin at both the active site and exosite 1.
The pioneer univalent direct thrombin inhibitor is argatroban monohydrate
(15) [15] that was launched by Daiichi Pharmaceutical and Mitsubishi Pharma in
1990. Argatroban was approved by the FDA for prophylactic anticoagulation in the
treatment of thrombosis in patients with heparin-induced thrombocytopenia. Argatroban
is a rather selective reversible inhibitor for human thrombin. Despite its
low molecular weight, argatroban is administered parenterally due to the presence
of the highly basic guanidine moiety that prevents absorption from the
8j1 Pioneer and Analogue Drugs
gastrointestinal tract. This characteristic limits the clinical use of the compound.
The first oral direct thrombin inhibitor was ximelagatran (17) [16], which was introduced
in 2004 by AstraZeneca. Ximelagatran is a double prodrug derivative of melagatran
(16) with a bioavailability of about 20%, a measurable improvement
compared to melagatran with oral bioavailability of 5.8%. Ximelagatran was withdrawn
from the market in 2006 because of unacceptable hepatic side effects (alanine
aminotransferase increased threefold and bilirubin level increased twofold
above the normal upper limit) [17]. In this drug class, dabigatran etexilate (18) [18]
was discovered by Boehringer Ingelheim as a new direct thrombin inhibitor without
adverse liver effects [19] (see Chapter 10)
NH
SO2
N
H
N
O
N
H
NH2
NH
O OH
H2O
argatroban monohydrate
15
N
NH2
NH
N
H
O O
N
H
O
OH
melagatran
16
N
N
H
NH
N
H
O
HO
O
N
H
O
O
ximelagatran
17
N
N
N
H
NH2
N
OO
N
O
N
O
O
dabigatran etexilate mesylate
CH3-SO2OH
18
1.1 Monotarget Drugs j9
Summary:
Pioneer univalent direct thrombin inhibitor drug: argatroban.
First orally active univalent thrombin inhibitor: ximelagatran.
First orally active univalent thrombin inhibitor without adverse liver affects: dabigatran
etexilate.
1.2
Dual-Acting Drugs
1.2.1
Monotarget Drugs from Dual-Acting Drugs
1.2.1.1 Optimization of Beta-Adrenergic Receptor Blockers
James W. Black and coworkers at ICI invented propranolol as a product of analoguebased
drug discovery (ABDD) using their prototype drug, pronethalol (19), as a lead
compound. Pronethalol [20] was an active drug for the treatment of angina pectoris
in humans, but its development was discontinued because it proved to be carcinogenic
in mice in long-term toxicology studies. Continuation of the analogue-based
drug discovery afforded propranolol (20), where an oxymethylene link was inserted
between the 1-napthyl group and the secondary alcohol moiety of pronethalol. Propranolol
[21] was more potent than pronethalol. Propranolol became the pioneer
nonselective b-adrenergic receptor antagonist, a true antagonist without partial agonist
properties (intrinsic sympathomimetic activity). It was a breakthrough discovery
for the treatment of arrhythmias, angina pectoris, and hypertension.
OH
N
H
pronethalol
19
O N
H
OH
propranolol
20
O
O
NH2
OH
N
H
atenolol
21
10j1 Pioneer and Analogue Drugs
The pioneer drug propranolol has equal antagonist affinity for b1 and b2 adrenergic
receptors; however, b1 receptors are located only in the heart and the nonselective
propranolol also blocks b2 receptors in bronchial smooth muscle.
Therefore, propranolol is not used in patients with bronchial asthma. Several analogues
have been tested in a battery of in vivo pharmacological tests resulting in the
discovery of atenolol (21) [22]. A guinea pig bronchospasm test served for investigation
and demonstration of b1 selectivity. Atenolol had no intrinsic sympathomimetic
effect (partial agonism), similar to propranolol (see Chapter 8 of Volume I
(Part II) of this book series).
Summary:
Pioneer dual-acting (b1 and b2) beta-adrenergic receptor antagonist: propranolol.
First b1 selective antagonist drug without intrinsic sympathomimetic activity:
atenolol.
1.2.2
Dual-Acting Drugs from Monotarget Drugs
1.2.2.1 Dual-Acting Opioid Drugs
Most ligands designed from the morphine template are mu (m) opioid receptor
(MOP) agonists. A simplified version of the morphine skeleton afforded tramadol
that is marketed in its racemic form. The (þ)-isomer is a weak MOP agonist,
whereas the (À)-isomer inhibits neurotransmitter reuptake. Tramadol (22) [23] was
discovered by Gr€unenthal and was introduced in 1977 for the treatment of moderate
to severe pain.
N
OH
O
tramadol
22
N
OH
H
CH3
tapentadol
23
1.2 Dual-Acting Drugs j11
Gr€unenthal continued analogue-based drug research using tramadol as a
starting compound, and out of several analogues, tapentadol (23) [24] was
selected and developed. It was introduced in 2009 to the market as a new opioid
analgesic drug with dual activity: a MOP agonist and an inhibitor of norepinephrine
reuptake. Tramadol is thousands of times less potent than morphine
on the mu opioid receptor, whereas tapentadol’s analgesic activity is
comparable to that of oxycodone with reduced constipation and respiratory
depression (see Chapter 12).
Summary:
Pioneer dual-acting (MOP agonist and norepinephrine reuptake inhibitor) opioid
drug racemate: tramadol.
First dual-acting (MOP agonist and norepinephrine reuptake inhibitor) opioid
drug in a single molecule: tapentadol.
1.3
Multitarget Drugs
1.3.1
Multitarget Drug Analogue to Eliminate a Side Effect
1.3.1.1 Clozapine and Olanzapine
Clozapine (24) [25] is the pioneer drug in the class of atypical antipsychotic agents.
It was a serendipitous discovery by researchers at Wander in Switzerland in 1960
from the structural analogue antidepressant amoxapine (25). Its discovery was
unexpected from the structurally very close analogue and therapeutically it had a
great advantage over the typical antipsychotic drugs such as chlorpromazine and
haloperidol because clozapine produced no extrapyramidal side effects (EPS). Clozapine
causes agranulocytosis in about 1% of the patients, and this side effect limited
its application. Analogue-based drug design afforded quetiapine (26) [26], a
clozapine analogue without this side effect. It was discovered at ICI in 1986 and it
became one of the main products of AstraZeneca. Instead of the dibenzodiazepine
nucleus of clozapine, the analogue quetiapine has a dibenzothiazepine scaffold.
Both clozapine and quetiapine have affinity for a number of receptors. The antipsychotic
activity is believed to be associated primarily by virtue of affinity for D2 and
5-HT2A receptors. Chapter 3 discusses metabolic aspects that may contribute to the
distinct adverse event profiles of these two drugs (see the chapter of Volume I on
clozapine analogues).
Summary:
Pioneer atypical antipsychotic drug: clozapine.
First atypical clozapine-like antipsychotic drug without the side effect of agranulocytosis:
quetiapine.
12j1 Pioneer and Analogue Drugs
N
N
H
N
N
Cl
CH3
clozapine
24
N
O
N
N
H
Cl
amoxapine
25
N
S
N
N
O
OH
quetiapine
26
1.3.2
Selective Drug Analogue from a Pioneer Multitarget Drug
1.3.2.1 Selective Serotonin Reuptake Inhibitors
From a retrospective viewpoint, imipramine (27) was the pioneer antidepressant
drug with a multitarget receptor profile, where serotonin and norepinephrine
reuptake inhibition played an important role, but no in vitro activities were known
at the time of its serendipitous discovery. Researchers at Geigy first synthesized the
molecule in 1948. It was an analogue of the antipsychotic chlorpromazine (28), but
the Swiss psychiatrist Roland Kuhn [27] found imipramine to be an effective antidepressant
drug. It was launched by Geigy in 1959.
1.3 Multitarget Drugs j13
N
N
imipramine
antidepressant
27
S
N
N
Cl
chlorpromazine
antipsychotic
28
Imipramine and the analogue tricyclic antidepressants inhibit the reuptake of
serotonin and norepinephrine but they also exhibit a variety of side effects. The
anticholinergic side effects include dry mouth, blurred vision, and sinus tachycardia.
The histamine H1 receptor antagonist activity likely contributes to the sedative
effects associated with the compound.
N
N
Br
zimelidine
29
Arvid Carlsson initiated research on selective serotonin reuptake inhibitors
(SSRIs) in order to get new antidepressants with less side effects. The first SSRI
was zimelidine (29) [28]. It was launched by Astra in 1982, but it had to be withdrawn
shortly afterward because of serious peripheral nerve side effects. Further
research was continued at several pharmaceutical companies. The first successful
SSRIs were fluoxetine (30) [29] (Lilly, 1988) and citalopram (31) [30] (Lundbeck,
1989). The antihistamine diphenhydramine (32) served as a lead compound for
fluoxetine, whereas talopram (33) was the lead structure for citalopram. The
14j1 Pioneer and Analogue Drugs
discovery of citalopram and its refinement to escitalopram is discussed in
Chapter 11.
O
N
H
CH3
F3C
fluoxetine
30
O
NC
F
N
CH3
CH3
citalopram
31
O N CH3
CH3
diphenhydramine
(lead for fluoxetine)
32
O
CH3
CH3
NHCH3
talopram
(lead for citalopram)
33
Summary:
Pioneer multitarget nonselective serotonin/norepinephrine reuptake inhibitor
antidepressant drug: imipramine.
First (but unsuccessful) selective serotonin reuptake inhibitor: zimelidine.
First selective serotonin reuptake inhibitors: fluoxetine and citalopram.
1.3 Multitarget Drugs j15
1.4
Summary
Pioneer drugs open up new therapeutic treatments. They are also called “first-inclass”
drugs.
Analogue-based drug discovery is a very important part of medicinal chemistry,
because the analogues frequently are intended to optimize drug therapy. There is a
continuous development in a drug class and in several cases the pioneer drugs disappear
from the market and analogue drugs achieve a dominant role. It is the main reason
why so many successful drugs are among the analogue drugs. The above examples
focused on some cases where these drugs have a unique character in a drug class.
For early-phase analogues, it is possible that a pioneer drug derives from the analogue-based
drug discovery for different reasons; for example, a more convenient
lead compound or a more successful optimization can strongly influence which
drug will be introduced to the market as a pioneer drug.
There are several examples where a new prototype drug candidate was discontinued
at the late phase of drug research and then an analogue was introduced to the
market as a successful pioneer drug.
The properties of pioneer and analogue drugs overlap (Figure 1.1). The drug analogues
preserve some properties of the pioneer drug and they have to achieve some
new and better properties in order to be successful on the market.
Acknowledgments
We thank Klaus Peter Bøgesø, Helmut Buschmann, Jens-Uwe Peters, and Henning
Priepke for carefully reading and reviewing the manuscript.
References
1 Fischer, J. (2011) Pioneer and analogue
drugs. 43rd IUPAC World Chemistry
Congress, August 3, 2011, San Juan,
Puerto Rico, Abstract No. 848.
2 Carlson, A. (1971) Recent Advances in
Parkinson’s Disease (eds F.H. McDowett
and C.H. Markham), Davis, Philadelphia,
PA, pp. 1–10.
Figure 1.1 Pioneer drugs and drug analogues have overlapping properties.
16j1 Pioneer and Analogue Drugs
3 Oates, J.A., Gillespie, S., Udenfriend, S.,
and Sjoerdsma, A. (1960) Decarboxylase
inhibition and blood pressure reduction of
alpha methyl-3,4-dihydroxy-DLphenylalanine.
Science, 131, 1890–1891.
4 DeStevens, G., Werner, L.H.,
Holamandris, A., and Ricca, S., Jr.
(1958) Dihydrobenzothiazine dioxides
with potent diuretic effect. Experientia,
14, 463.
5 Ganellin, C.R. (1982) Cimetidine, in
Chronicles of Drug Discovery, vol. 1 (eds J.S.
Nindra and D. Lednicer), John Wiley and
Sons, Inc., New York, pp. 1–38.
6 Pearce, P. and Funder, J.W. (1980)
Histamine H2-receptor antagonist:
radioreceptor assay for antiandrogenic
side effects. Clin. Exp. Pharmacol. Physiol.,
7, 442.
7 Ganellin, C.R. (2006) Development of
antiulcer H2-receptor histamine
antagonists, in Analogue-Based Drug
Discovery (eds J. Fischer and C.R.
Ganellin), Wiley-VCH Verlag GmbH,
Weinheim, pp. 71–80.
8 Bakhle, Y.S. (1972) Inhibition of
converting enzyme by venom peptides, in
Hypertension (eds J. Genest and E. Koiw),
Springer, Berlin, pp. 541–547.
9 Ondetti, M.A., Rubin, B., and Cushman,
D.W. (1977) Design of specific inhibitors
of angiotensin-converting enzyme: new
class of orally active antihypertensive
agents. Science, 196, 441–444.
10 Patchett, A.A., Harris, E., Tristram, E.W.,
Wyvratt, M.J., Wu, M.T., Taub, D.,
Peterson, E.R., Ikeler, T.J., Ten Broeke, J.,
Payne, G., Ondeyka, D.L., Thorsett, E.D.,
Greenlee, W.J., Lohr, N.S., Hoffsommer,
R.D., Joshua, H., Ruyle, J., Rothrock, W.,
Aster, S.D., Maycock, A.L., Robinson,
F.M., Hirschmann, R., Sweet, C.S., Ulm,
E.H., Gross, D.M., Vassil, C., and Stone,
C.A. (1980) A new class of angiotensinconverting
enzyme inhibitors. Nature,
288, 280–283.
11 Alf€oldi, S. and Fischer, J. (2006)
Optimizing antihypertensive therapy by
angiotensin converting enzyme
inhibitors, in Analogue-Based Drug
Discovery (eds J. Fischer and C.R.
Ganellin), Wiley-VCH Verlag GmbH,
Weinheim, pp. 169–179.
12 Biftu, T., Scapin, G., Singh, S., Feng, D.,
Becker, J.W., Eiermann, G., He, H., Lyons,
K., Patel, S., Petrvov, A., Sinha-Roy, R.,
Zhang, B., Wu, J., Zhang, X., Doss, G.A.,
Thornberry, N.A., and Weber, A.E. (2007)
Rational design of a novel, potent and
orally bioavailable cyclohexylamine DPP-4
inhibitor by application of molecular
modeling and X-ray crystallography of
sitagliptin. Bioorg. Med. Chem. Lett., 17,
3384–3387.
13 Villhauer, E.B., Brinkmann, J.A., Naderi,
G.B., Burkey, B.F., Dunning, B.E., Prasad,
K., Mangold, B.L., Russell, M.E., and
Hughes, T.E. (2003) 1-[[(3-Hydroxy-1-
adamantyl)amino]acetyl]-2-cyano-(S)pyrrolidine:
a potent, selective, and orally
bioavailable dipeptidyl peptidase IV
inhibitor with antihyperglycemic
properties. J. Med. Chem., 46, 2774–2789.
14 Peters, J.-U. and Mattei, P. (2010)
Dipeptidyl peptidase IV inhibitors for the
treatment of type 2 diabetes, in AnalogueBased
Drug Discovery II (eds J. Fischer and
C.R. Ganellin), Wiley-VCH Verlag GmbH,
Weinheim, pp. 109–134.
15 Jeske, W., Walenga, J.M., Lewis, B.E., and
Fareed, J. (1999) Pharmacology of
argatroban. Expert Opin. Invest. Drugs, 8
(5), 625–654.
16 Gustafsson, D. and Elg, M. (2003) The
pharmacodynamics and pharmacokinetics
of the oral direct thrombin inhibitor
ximelagatran and its active metabolite
melagatran: a mini-review. Thromb. Res.,
109 (Suppl. 1), S9–S15.
17 Lee, W.M., Larrey, D., Olsson, R., Lewis, J.
H., Keisu, M., Auclert, L., and Shet, S.
(2005) Hepatic findings in long-term
clinical trials of ximelagatran. Drug Saf.,
28 (4), 351–370.
18 Hauel, N.H., Nar, H., Priepke, H., Ries,
U., Stassen, J.M., and Wienen, W. (2002)
Structure-based design of novel potent
nonpeptide thrombin inhibitors. J. Med.
Chem., 45, 1757–1766.
19 Eikelboom, J.W. and Weitz, J.I. (2010)
Update on antithrombotic therapy. New
anticoagulants. Circulation, 121, 1523–1532.
20 Black, J.W. and Stephenson, J.S. (1962)
Pharmacology of a new adrenergic betareceptor-blocking
compound. Lancet, 2
(7251), 311–314.
References j17
21 Black, J.W., Crowther, A.F., Shanks, R.G.,
Smith, L.H., and Dornhorst, A.C. (1964) A
new adrenergic beta-receptor antagonist.
Lancet, 1 (7342), 1080–1081.
22 Barrett, A.M., Carter, J., Fitzgerald, J.D.,
Hull, R., and Le Count, D. (1973) A new
type of cardioselective adrenoceptive
blocking drug. Br. J. Pharmacol., 48, 3408.
23 Flick, K., Frankus, E., and Friderichs, E.
(1978) Studies of chemical structure and
analgetic activity of phenyl substituted
aminomethylcyclohexanoles. Arzneim.Forsch./Drug
Res., 28 (I), 107–113.
24 Buschmann, H., Strassburger, W., and
Friderichs, F. (2001) 1-Phenyl-3dimethylaminopropane
compounds with
a pharmacological effect. EP 693 475.
25 Schmutz, J. and Eichenberger, E. (1982)
Clozapine, in Chronicles of Drug
Discoveries, vol. 1 (eds J.S. Bindra and D.
Lednicer), John Wiley & Sons, Inc.,
New York, pp. 39–60.
26 Szegedi, A., Wiesner, J., Hillert, A.,
Hammes, E., Wetzel, H., and Benkert, O.
(1993) ICI 204,636, a putative “atypical”
antipsychotic, in the treatment of
schizophrenia with positive
symptomology: an open clinical trial.
Pharmacopsychiatry, 26, 197.
27 Kuhn, R. (1957) €Uber die Behandlung
depressiver Zust€ande mit einem
Iminodibenzyl Derivat (G 22355).
Schweiz. Med. Wochensch., 87, 1135.
28 Montgomery, S.A., McAuley, R., Rani, S.J.,
Roy, D., and Montgomery, D.B. (1981) A
double blind comparison of zimelidine
and amitriptyline in endogenous
depression. Acta Psychiatr. Scand. Suppl.,
290, 314–327.
29 Wong, D.T., Horng, J.S., Bymaster, F.P.,
Hauser, K.L., and Malloy, B.B. (1974) A
selective inhibitor of serotonin uptake:
Lilly 110140, 3-(p-trifluoromethylphenoxy)N-methyl-3-phenylpropylamine.
Life Sci.,
15, 471–479.
30 Bigler, A.J., Bùgesù, K.P., Toft, A., and
Hansen, V. (1977) Quantitative structure–
activity relationships in a series of
selective 5-HTuptake inhibitors. Eur. J.
Med. Chem., 12, 289–295.
18j1 Pioneer and Analogue Drugs
Janos Fischer is Senior Research Scientist at Richter Plc.
(Budapest, Hungary). He received his education in Hungary
with M.Sc. and Ph.D. degrees in Organic Chemistry from
Eotvos University of Budapest with Professor A. Kucsman.
Between 1976 and 1978, he was a Humboldt Fellow at the
University of Bonn with Professor W. Steglich. He has
worked at Richter Plc. since 1981, where he participated in
the research and development of leading cardiovascular
drugs in Hungary. His main current interest is analoguebased
drug discovery. He is author of some 100 patents and
scientific publications. In 2012, he was reelected Titular Member of the Chemistry
and Human Health Division of IUPAC. He received an honorary professorship at
the Technical University of Budapest.
C. Robin Ganellin studied chemistry at London University,
receiving a Ph.D. in 1958 under Professor Michael Dewar,
and was a Research Associate at MIT with Arthur Cope in
1960. He then joined Smith, Kline & French Laboratories in
the United Kingdom and was one of the co-inventors of the
revolutionary drug cimetidine (also known as Tagamet1
),
eventually becoming Vice President for Research. In 1986,
he was made a Fellow of the Royal Society and appointed to
the SK&F Chair of Medicinal Chemistry at University College
London, where he is now Professor Emeritus of Medicinal Chemistry. Professor
Ganellin is co-inventor on over 160 patents and has authored over 250 scientific
publications. He was President of the Medicinal Chemistry Section of IUPAC and
is currently Chairman of the IUPAC Subcommittee on Medicinal Chemistry and
Drug Development.
David P. Rotella is the Margaret and Herman Sokol Professor
of Medicinal Chemistry at Montclair State University.
He earned a B.S. Pharm. degree at the University of
Pittsburgh (1981) and a Ph.D. (1985) at The Ohio State
University with Donald. T. Witiak. After postdoctoral studies
in organic chemistry at Penn State University with
Ken S. Feldman, he worked as an Assistant Professor at
the University of Mississippi. David worked at Cephalon,
Bristol-Myers, Lexicon, and Wyeth, where he was involved
in neurodegeneration, schizophrenia, cardiovascular, and metabolic disease
drug discovery projects.
References j19
2
Competition in the Pharmaceutical Drug Development
Christian Tyrchan and Fabrizio Giordanetto
2.1
Introduction
Granted patents give the right to exclude others from making, using, selling, offering
for sale, or importing the claimed invention for a given time period that can
vary between countries. A filed patent in the United States, in principle, expires
20 years after the filing date. In the European Union (EU), applicants are required
to disclose their inventions in a sufficiently clear and complete way so that the innovative
step can be carried out by a person skilled in the art. In the United States,
inventors are additionally required to include the “best mode” of making or practicing
the invention. Failure to include such “best mode,” which is the best available at
the time of filing of the application, has been a potential ground for invalidation of
the patent.
In such encouraged competitive environment, innovation is more likely to stem
from small, incremental cycles, following an evolutionary rather than revolutionary
path. Indeed, the patent system stimulates innovation by rewarding improvements
to be published, and by this recognizing the reality of incremental innovation [1].
Consequently, in the pharmaceutical field, patents that claim similar structural
spaces normally occur. This is because of different interpretations of commonly
available knowledge, or because of convergence from different starting points as
key scientific publications.
In this respect, it is important to recognize that what is normally referred to as
pioneer, first-in-class, or breakthrough drug is the first compound reaching the
market following the approval of the regulatory offices and not the first filed in a
patent specification (the term “patent specification” is used throughout the chapter
as a general reference to a patent text be it application or granted patent). These
compounds then constitute a new class of drugs in that they introduce a novel
mode of action (MoA) or provide a significant improvement over the standard therapy,
if not directly enabling a therapy, in terms of efficacy and safety. An analoguebased
drug (also termed follow-on drug) is normally a chemical entity that has the
same pharmacological MoA as the pioneer drug but it is registered afterward [2],
regardless of its patent publication date.
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
j21
The ever-changing and demanding health economic environment and the
increasing costs and attrition of drug development have pressed pharmaceutical
companies to constantly look for opportunities to de-risk their programs [2–7].
Here, the aim toward developing “lower risk” analogues rather than “high-risk”
pioneer drugs has been fueled by the steady increase in external and internal
knowledge around a specific MoA, as from publications such as peer-reviewed journals,
patents, and clinical trial results’ analyses. However, because the information
is publicly available, the ability to differentiate the final products against the
expected competitors becomes key to success. Accordingly, any analogous drug is
sought to become a best-in-class drug.
2.2
Analogue-Based Drugs: Just Copies?
The approach to analogue-based drug design is controversially discussed in the literature,
most notably in terms of different value propositions such as innovation,
risk of failure, marketing costs, and price competition, among others [2, 8, 9].
In two seminal publications, DiMasi et al., from the Tufts Center for the Study
of Drug Development, discussed the economics of first-in-class and follow-on
drugs and the kind of competitiveness in pharmaceutical R&D from a marketed
drug space perspective [2, 10]. Two important questions are raised and
answered:
 Are pioneer drugs best in class, in terms of treatment value?
 Can analogue-based drugs deliver therapeutic benefit?
First, they report that the period of marketing exclusivity until an analogous drug
reaches the market has fallen in the United States (from time of launch) for the
pioneer drug from a median of 10.2 years in the 1970s to 1.2 years in the late
1990s. Furthermore, they find that approximately one-third of the overall analoguebased
drugs have received a priority rating from the FDA. This priority rating is
granted by the FDA for compounds that are considered to add significant value
compared to the standard therapy. These findings are reported for 57% of the
defined drug classes and clearly point toward the continuous evolution in a drug
class. Consequently, analogous drugs can provide true therapeutic benefit,
although this will only be apparent after substantial investigation in a clinical
setting [2, 10].
In some cases, an analogue drug appears later on the US market than the first-inclass
compound of the same drug class. Its reasons were discussed by Agarwal and
Gort [11]. The authors conclude that the main factors are increased mobility of
skilled labor, rapid diffusion of scientific and technical information (information
age), increased competition, and expansion of emerging markets [2, 11]. Taken
together, these data reflect a highly dynamic race among competitors rather than a
simple imitation exercise. The following examples, supported by the original patent
publication dates, further reinforce this notion.
22j2 Competition in the Pharmaceutical Drug Development
An interesting molecular pair is provided by the prostaglandin (PG) analogues
latanoprost and unoprostone. Latanoprost (XalatanTM
) was approved as a first-inclass
treatment for elevated intraocular pressure (IOP) in 1996, while unoprostone
(ResculaTM
) received approval in 1994 as a follow-on PG analogue. Nevertheless,
unoprostone was actually disclosed in the patent literature prior to latanoprost:
1988 and 1990, respectively [12, 13]. While the reasons for the faster developmentto-launch
course of latanoprost are not known (cf. 6 and 12 years, Figure 2.1), its
structural similarity to unoprostone is striking (Figure 2.2). Both latanoprost and
unoprostone are prostaglandin F2a (PGF2a) derivatives, where the omega chain of
PGF2a has been the subject of chemical modifications, as shown in Figure 2.2.
Interestingly, researchers at R-Tech Ueno Ltd modified PGF2a to mimic existing
PGF2a metabolites where the hydroxyl group at position 15 is oxidized to the
Figure 2.1 The time difference in years from patent filing to US approval for 20 pioneer drugs
(diamond) with their second entrant (square). The triangles denote the time difference in years
between the patent filing date of the first-in-class and second-entry drug.
Prostaglandin
analogue
(ophthalmic)
HO
HO
OH
O
OH
alpha
omega
15
Prostaglandin F2α
HO
HO
O
O
OH
Latanoprost (Xalatan™)
Launch: 1996-06-05
Published Patent: 1990-03-22
HO
HO
O
O
O
Unoprostone (Rescula™)
Launch: 2000-08-03
Published Patent: 1988-11-02
Figure 2.2 First-in-class and follow-on prostaglandin analogue drug pair and the natural agonist
PGF2a.
2.2 Analogue-Based Drugs: Just Copies? j23
corresponding ketone [12]. Scientists at Pharmacia AB resolved to introduce an aromatic
ring as a way to circumvent the irritative effects of the naturally occurring
PGF2a. Although both groups started from the same chemical matter and opted
for the same prodrug approach, the medicinal chemistry strategies to modify the
omega chain of PGF2a were different and resulted in two safe and well-tolerated
drugs. Despite the largely similar chemical structures and safety profiles, several
differences in terms of metabolic fate and pharmacokinetics [14, 15], and more
importantly therapeutic effects, discriminate unoprostone from latanoprost, the latter
being more efficacious in a clinical setting [16].
Even though anastrozole (ArimidexTM
) was patented later than letrozole, it won
the race to be the first-in-class third-generation aromatase inhibitor (AI) to be
launched in the United States by merely 1 year, as depicted in Figure 2.3. Anastrozole
was launched in 1996 for the treatment of advanced breast cancer in postmenopausal
women. Previous first- and second-generation AIs suffered from
limited enzyme selectivity and clinical efficacy. Letrozole was then launched in
1997 despite being patented earlier than anastrozole: 1987 and 1988, respectively
[17, 18]. Scientists at Ciba-Geigy discovered letrozole through the iterative
optimization of fadrozole [19], a second-generation AI originated in the same laboratories
[20, 21]. Here, the original innovation step revolved around the mechanistic
hypothesis that imidazole-containing compounds could be selective P450
inhibitors [20], analogously to what was observed in a parallel discovery program
against thromboxane A2 synthetase [22]. In an effort to further improve upon
fadrozole selectivity, simplification of fadrozole’s structure through ring opening
and chiral center removal, followed by the subsequent azaheterocycle optimization,
resulted in the discovery of letrozole [19], a totally selective aromatase inhibitor.
Simultaneous research activities by medicinal chemists at ICI resulted in anastrozole,
another safe and effective 1,2,4-triazole-based AI with similar structure to
letrozole [23]. Interestingly though, the chemical starting point that led to the
Aromatase
inhibitor
N
N
N
Fadrozole (Ciba-Geigy’s starting point)
Published Patent: 1985-06-17
OH
N
N
ICI starting point [24]
NN
N
N
N
Anastrozole (Arimidex™)
Launch: 1995-12-27
Published Patent: 1988-12-28
N
N
N
N
N
Letrozole (Femara™)
Launch: 1997-07-25
Published Patent: 1987-09-16
Figure 2.3 First-in-class and follow-on third-generation AI pair. Fadrozole, a second-generation
AI and the initial starting point for AI discovery at ICI [24] are shown as a reference.
24j2 Competition in the Pharmaceutical Drug Development
discovery of anastrozole was already identified in late 1970s through random
screening of the ICI collection [24].
Moreover, design principles aimed at increasing selectivity over other P450
enzymes led to the final selection of anastrozole for subsequent development in a
very “intense competitive situation,” in the author’s own words [24]. While anastrozole
and letrozole are classified as first-in-class and follow-on third-generation AIs,
their discoveries originated and followed fairly different tracks [19, 20, 24]. The timings
of initial discoveries, program sanctions, patent publications, and market
launch further reflect a highly competitive race rather than imitation [10]. In these
settings, their structural similarity is even more remarkable and could only serve as
inspiration to medicinal chemists in their next design round. Nevertheless, those
limited structural differences are still responsible for the different pharmacokinetic
profiles of the two AIs, as well as the superior preclinical pharmacodynamic properties
of letrozole [25]. The currently ongoing, randomized head-to-head trial in early
breast cancer (id: NCT00248170) will hopefully set the dispute over which one is
the best AI.
In a similar fashion, “best in class” represents the ultimate goal. As market exclusivity
is no longer an advantage due to increased competition, and different groups
have faster access to increasing amounts of information, even the smallest chemical
variations over existing chemotypes need to demonstrate tangible experimental
advantages. The fact that 70% of the first-in-class drug–analogous drug pairs analyzed
by Giordanetto et al. [26] differed minimally from a structural perspective, yet
diverged in their experimental properties, further highlights such opportunities.
2.3
How Often Does Analogue-Based Activity Occur? Insights from the GPCR Patent
Space
Considering the highly competitive environment, follow-on of external information
remains a vital activity for any drug researcher. As outlined above, this is simply the
result of the incremental nature of research, where knowledge and insight evolve
with time. Indeed, the chemical information landscape is changing rapidly with a
yearly increase of over 1 million new compounds and more than 700 000 publications
related to chemistry [27].
Accordingly, the chemical space claimed by patent specifications is becoming
more crowded and competitive, especially for target classes such as protein kinases
and G-protein-coupled receptors (GPCRs). Exploring the chemical space covered by
relevant journals and patent specifications is an important activity in medicinal
chemistry projects due to the constant need to convert information into knowledge
to support business decisions. This is also greatly aided by the unprecedented
access to large quantities of data. A variety of commercial institutions now supply
databases with high-quality, manually extracted structures of patent examples [28].
Following the advances in text mining technologies and improvements of the
chemical named entity recognition (NER) process, several databases containing
2.3 How Often Does Analogue-Based Activity Occur? Insights from the GPCR Patent Space j25
automatically extracted chemical entities from patent specifications are also available
[29–32]. Consequently, computational methods developed in the fields of chemoinformatics
or patent informatics [33] can be applied for data analysis and
visualization to support medicinal chemistry teams in evaluating and exploiting
the available information.
To investigate the occurrence of drug discovery follow-on activities as captured by
the pharmaceutical patent space (in contrast to the marketed drug space), we
abstracted 11 827 patent specifications that are linked to a GPCR target with a
defined Entrez Gene ID from the GVKBIO databases (as of November 2010) [28].
The GVKBIO Medicinal Chemistry and Target Class databases capture explicit relationships
between published documents, compounds, assay results, and targets
extracted by manual curation. In a next step, we focused on small molecules by
removing all peptides, based on substructural and physicochemical properties’
analysis. By focusing on the top 100 companies, in terms of number of published
patent specifications, this resulted in a total of 10 253 patents. The set was further
consolidated by associating patent specifications based on known mergers and
acquisitions until 2008. Subsequently, all possible patent specification combinations
were created and a competitive activity was recorded if two patent specifications
shared the same gene name and were published by different companies
within a 6-year interval.
Similarity descriptors such as the Shannon entropy (SE, based on the number of
atoms of the joined substructures, using 26 bins) and corresponding scores could
be generated for 1 570 381 out of a total of 1 608 368 patent specification pairs
(97.6%) [34]. All calculations were performed with Pipeline Pilot 7.5.2 and Python
scripts [35].
Using a conservative cutoff (SE  4) to identify patent specifications sharing
at least one compound with high structural similarity, we found 6516 (64%)
patents linked to analogue-based activities (from 49 638 patent pairs over 28
years and 433 different gene ids). These patent specifications were further
classified as follows:
 Originator: a patent specification that is linked to a later patent specification by
structural similarity.
 Follower: a patent specification that is linked by structural similarity to an earlier
patent specification and is published between 2 and 6 years of the earlier patent
specification.
 Competitor: a patent specification that is linked by structural similarity and is
published in the same year.
The results shown in Figures 2.4 and 2.5 demonstrate that pharmaceutical
research is indeed highly competitive and dynamic. Sixty-four percent of the published
patent specifications targeting a specific GPCR are linked by significant
structural similarity to other patent specifications for the same GPCR. Most of the
follow-on patent specifications (74%) are published within the first 2 years of the
originator. This stresses the importance of speed (and commitment) when reacting
to an external patent specification. Strategically, this would translate in a maximum
26j2 Competition in the Pharmaceutical Drug Development
period of 6 months from the publication date of the originator patent specification
to the follow-on’s patent specification submission.
It is interesting to note that, overall, out of the final set of 6561 patent specifications
involved in follow-on activities, only 1705 patent specifications (26%) are classified
as originator or competitor or combination thereof, whereas 4811 (74%)
Figure 2.4 Number of patent specifications published in the GPCR field: overall (diamond), top
100 companies (square), and linked to follow-on activities (triangle).
Figure 2.5 Frequency distribution of patent specification pair linked to analogue-based activities
based on time between patent specification publications.
2.3 How Often Does Analogue-Based Activity Occur? Insights from the GPCR Patent Space j27
patent specifications belong to the follower class. It is of course tempting to ask the
question whether there are certain organizations that are patenting more from an
“originator” position than others. While Merck and Takeda display the highest
number of originator patent specifications, due to the limitations of the current
data set, only small, statistically nonsignificant differences between the companies
can be observed (Figure 2.6).
Some originator patent specifications are more followed on than others. This
might reflect the level of competitiveness in the specific area, if not the available
confidence in the MoA or chemical equity. The most followed-on patent specification
in the present GPCR data set is WO200046209, published in 2000 from
researchers at Sanofi-Aventis (now Sanofi). In the 6 years after its original publication,
it has inspired 74 follow-on patent specifications from 9 of the top 100 companies
analyzed here, as shown in Table 2.1.
WO200046209 disclosed cannabinoid receptor 1 (CB1) antagonists closely
related to rimonabant, as shown in Figure 2.8. Although the original patent specification
covering rimonabant was published in 1993 (EP0576357A1), no follow-on
patent specifications could be identified within the 6 years window considered
here. As a result, WO200046209 figures as the “originator.”
Analysis of the structural relationships between patent specifications from a given
company is very useful. It captures the evolution of the chemical asset in terms of
medicinal chemistry practices as well as patent strategy. In the Sanofi-Synthelabo
Figure 2.6 Top 20 companies and their ratio of follow-on (diamond), originator (square), and
competitor (triangle) patents.
28j2 Competition in the Pharmaceutical Drug Development
case, after the original WO200046209 patent, two interesting applications followed
in 2001 and 2003, respectively, as shown in Figure 2.7. The first one describes an
interesting ring closure strategy linking the pyrazole to one of its phenyl substituents
to afford new tricyclic derivatives as CB1 receptor antagonists. It is noteworthy that
Table 2.1 Number of published patent specifications (WIPO) from the top 20 companies
following on Sanofi-Synthelabo’s WO200046209.
Company 2000 2001 2003 2004 2005 2006 Total
Sanofi-Synthelabo (Sanofi-Aventis) 1 1 1 2 5 11
Merck 5 2 2 9
Solvay (Abbott) 4 1 7 12
AstraZeneca 1 5 4 1 11
Roche 3 2 1 6
Pfizer 13 9 2 24
BMS 4 1 5
Janssen (J&J) 1 3 4
Bayer (Bayer-Schering) 1 1
Schering (Bayer-Schering) 1 1
Neurogen 1 1
1993 2000 2001 2003 2005
N
N
O NH
N
Cl
Cl
Cl
N
O NH
N
ClBr
Cl
Cl
N
N
O NH
N
Cl
Cl
Cl
O
N
H
N
Cl
Cl
S
O NH
N
ClCl
Cl
Rimonabant
EP0576357A1 WO2005035488WO2003084943WO200132663WO200046209
2006
N
Cl Cl
O NH
N
Cl
O
NH
S
Cl
ClCl
S
O
O
NH
S
Cl
ClCl
S
Cl
NH
O
Cl
Cl
N
Cl
Cl
NH
O
Cl
WO2006087476WO2006084975WO2006077320WO2006070106WO2006024777
N
Figure 2.7 Number of published patent specifications (WIPO) targeting CB1 receptor antagonists
from Sanofi-Synthelabo during the 2000–2006 period. Rimonabant is shown as a reference.
2.3 How Often Does Analogue-Based Activity Occur? Insights from the GPCR Patent Space j29
such tricyclic systems could also contain different heteroatoms in the central ring, as
exemplified by compounds bearing sulfur atoms in various oxidation states. The second
application, dated 2003, discloses a more conservative “scaffold hop” from the
central pyrazole scaffold of rimonabant to a trisubstituted phenyl core structure, as
shown in Figure 2.7.
The years 2005 and 2006 witnessed a total of six additional WIPO applications
from Sanofi-Aventis covering CB1 receptor antagonists. The structural resemblance
of the exemplified compounds to the original rimonabant structure is
immediate, as displayed in Figure 2.7. Here, the original pyrazole framework is
again swapped for similar five-membered heterocycles, notably substituted thiophenes
and pyrroles. The first interpretations of amide bond bioisosteres from
Sanofi-Aventis appeared as well. Sulfonamides (WO2006077320) and elongated
inverse amides (WO2006070106, WO2006084975, and WO2006087476) replaced
the original carboxamide derivative from EP 0576357A1. These probably represent
defensive applications following the surge in CB1 receptor antagonist disclosures
during 2004, as listed in Figure 2.7.
Merck was the first of the top 100 companies analyzed here to patent close analogues
of the original CB1 receptor antagonist chemotype from Sanofi-Synthelabo.
Their first patent specification is dated 2003 and discloses imidazole-containing
CB1 receptor antagonists showing an elegant scaffold change from pyrazole to
imidazole, as shown in Figure 2.8. Following the same strategy, Merck chemists
also patented pyridine analogues of rimonabant (WO2003082191). Here, their initial
imidazole core structure was replaced by a trisubstituted pyridine. In addition
to the typical heterocycle “hop,” three applications covered a very interesting ringopening
paradigm. Here, the central pyrazole ring was cut open to a 1,2disubstituted
propyl chain, and the original carboxamide was inverted to bear
1993 2003
N
N
O NH
N
ClCl
Cl
N N
O NH
N
Cl Cl
Cl
Cl Cl
N
H
O
Cl Cl
N
H
O
O
N
Cl
O
N
H
N
Cl
Cl
Cl Cl
N
H
O
N NH
Rimonabant
EP0576357A1
WO2003087037WO2003082191WO2003082190WO2003077847WO2003007887
2004 2005
NH2
N
O
O N
Cl
ClCl
N
N
Cl
N
H
O
Cl
Cl
NH
SO
O
Cl
ClCl
N
N
O
Cl
ClCl
NH2
WO2005047285WO2005027837WO2004029204WO2004012671
Figure 2.8 Number of published patent applications (WIPO) targeting CB1 receptor antagonists
from Merck during the 2000–2006 period. Rimonabant is shown as a reference.
30j2 Competition in the Pharmaceutical Drug Development
different types of substituents: aliphatic, spirocyclic, and aromatic
(WO2003077847, WO2003082190, and WO2003087037, respectively), as displayed
in Figure 2.8. In 2004, Merck followed up their core modification with an additional
hop from pyridine to pyrimidine (cf. WO2003082191 and WO2004029204, Figure
2.8). They also disclosed novel derivatives bearing a central furo[2,3-b]pyridine
scaffold (WO2004012671) as a fused heterocyclic system alternative to rimonabant’s
pyrazole. During 2005, the 1,2-disubstituted propyl-based chemotype was
revisited by Merck, this time being decorated with a sulfonamide functionality in
lieu of the previous amide (cf. WO2003087037 and WO2005027837, Figure 2.8).
Analogously, the initial furo[2,3-b]pyridine core published in 2004 was expanded to
a 1,8-naphthyridin-2-one (cf. WO2005047285 and WO2004012671).
The CB1 receptor antagonist case culminated in the discovery and launch of
rimonabant, as an effective anti-obesity agent. As shown above, several other companies
were inspired by the early work from Sanofi-Aventis. Unfortunately, the
withdrawal of rimonabant from the market, due to the severe CNS-mediated
side effects observed, prompted most of the follow-on companies to either terminate
their internal programs or evaluate different compound profiles, thus reinforcing
how dynamic and competitive the follow-on race actually is. A number of
companies have thus actively engaged in research aiming at developing peripherally
restricted CB1 receptor antagonist, as a way to mitigate the safety hazard
related to CNS exposure. Aptly, researchers at 7TM Pharma disclosed compounds
useful for modulating peripheral CB1 receptors that may have a reduced propensity
to induce psychiatric and nervous system side effects (WO2009074782). Among
these “second-generation” CB1 receptor antagonists, the amide derivative
TM38328 (Figure 2.9) demonstrated weight reduction effects comparable to that of
rimonabant in diet-induced obese (DIO) mice [36]. This pharmacological efficacy
was achieved despite much reduced brain exposure (plasma/brain ratios: 0.42 and
10.2 for rimonabant and TM38328, respectively) [36]. Interestingly, as displayed in
Figure 2.9, TM38328 is a rimonabant analogue, where the original, lipophilic 4methylpyrazole
substituent was further derivatized to a more polar acetamide as a
way to reduce passive permeability. This, coupled with increased molecular weight
1993 2009 2010
N
N
O NH
N
ClCl
Cl N
N
O N
Cl
Cl
NH2
O
OH
F
N
N
O NH
Cl
Cl
N
N
S
O O
Rimonabant
EP0576357A1
TM38328
WO2009074782
AM6545
WO2010104488
Figure 2.9 Selected “second-generation” CB1 receptor antagonists with reduced CNS
distribution.
2.3 How Often Does Analogue-Based Activity Occur? Insights from the GPCR Patent Space j31
on position 3 of the pyrazole ring, provided additional gain in plasma/brain separation
[36]. In 2010, scientists at the University of Connecticut published novel CB1
neutral antagonists acting preferentially on CB1 receptors located in the peripheral
nervous system (WO2010104488). One of these disclosed compounds, AM6545
(Figure 2.9), was extensively profiled to assess its pharmacological, metabolic, and
behavioral potential [37]. AM6545 displayed limited brain penetrance after acute
administration to mice (brain/plasma ratio: 0.03 versus 0.8 for rimonabant) and
was effective at reducing weight in DIO mice, but, contrarily to rimonabant, did
not affect behavioral responses in mice [36]. AM6545 is also a rimonabant analogue
with higher hydrophilicity (clogP: 3.3 versus 6.4 for rimonabant). As shown in Figure
2.9, replacement of a methylene by a sulfone and of a chlorine atom by a cyanobut-1-ynyl
side chain are responsible for the reduced CNS exposure. It is
noteworthy that these molecular changes do not alter passive diffusion but, rather,
trigger active extrusion by the ABC transporter P-glycoprotein, as elegantly demonstrated
by both in vitro and in vivo experiments [37].
The analysis of originator and follow-on patents can be valuable to highlight
interesting chemical permutations, as summarized in Figures 2.7–2.9. Indeed, a
large arsenal of medicinal chemistry tactics is on display: from ring opening to ring
closure, from ring homologation and contraction to substituent walking and fine
tuning, every modification could have a profound effect on key optimization
parameters, as exemplified by the “second-generation” peripheral ligands. The
CB1 antagonist example provides several different interpretations of the original
pyrazole-3-carboxamide derivatives. While the general applicability of these variations
is routinely evaluated in medicinal chemistry programs, these follow-on
examples represent an important source of inspiration and medicinal chemistry
experience that is now electronically available.
As patent informatics develops based on the availability of more comprehensive
databases, more exhaustive enumeration techniques, and more effective structural
comparison methods, direct evaluation of medicinal chemistry precedents and follow-on
practices will be easier to implement. Following the PG analogues, and AI
and CB1 receptor antagonist examples presented here, experimental evidence,
time, and significant investments will single out the best-in-class compound from
the follow-on crowd in an innovation race that does not seem to become any easier
with time.
References
1 Miller, C.P. and Evans, M.J. (2010) The
Chemist’s Companion Guide to Patent
Law, John Wiley & Sons, Inc.,
Hoboken, NJ.
2 DiMasi, J.A. and Paquette, C. (2004) The
economics of follow-on drug research and
development: trends in entry rates and the
timing of development.
Pharmacoeconomics, 22 (2 Suppl. 2),
1–14.
3 Schachter, A.D. and Ramoni, M.F. (2007)
From the analyst’s couch: clinical
forecasting in drug development. Nat. Rev.
Drug Discov., 6, 107–108.
4 DiMasi, J.A., Hansen, R.W., and
Grabowski, H.G. (2003) The price of
32j2 Competition in the Pharmaceutical Drug Development
innovation: new estimates of drug
development costs. J. Health Econ., 22,
151–185.
5 DiMasi, J.A., Feldman, L., Seckler, A., and
Wilson, A. (2010) Trends in risks
associated with new drug development:
success rates for investigational drugs.
Clin. Pharmacol. Ther., 87 (3), 272–277.
6 Kola, I. and Landis, J. (2004) Can the
pharmaceutical industry reduce attrition
rates? Nat. Rev. Drug Discov., 3 (8),
711–715.
7 Sams-Dodd, F. (2007) Research & market
strategy: how choice of drug discovery
approach can affect market position. Drug
Discov. Today, 12 (7–8), 314–318.
8 Hollis, A. (2005) Comment on “The
economics of follow-on drug research and
development: trends in entry rates and the
timing of development”.
Pharmacoeconomics, 23 (12), 1187–1192,
discussion 1193–1202.
9 Cohen, J., Cabanilla, L., and Sosnov, J.
(2006) Role of follow-on drugs and
indications on the WHO Essential Drug
List. J. Clin. Pharm. Ther., 31 (6), 585–592.
10 Dimasi, J.A. and Faden, L.B. (2011)
Competitiveness in follow-on drug R&D: a
race or imitation? Nat. Rev. Drug Discov.,
10 (1), 23–27.
11 Agarwal, R. and Gort, M. (2001) First-mover
advantage and the speed of competitive
entry. J. Law Econ., 44, 161–177.
12 Ueno, R., Ueno, R., and Oda, T. (1998)
Preparation of prostaglandin F derivatives
as vasopressors. European Patent
Application EP 289349A1.
13 Stjernschantz, J. and Resul, B. (1990)
Prostaglandin derivatives for the
treatment of glaucoma or ocular
hypertension. PCT International
Application WO9002553A1.
14 Kashiwagi, K., Iizuka, Y., and Tsukuhara,
S. (1999) Metabolites of isopropyl
unoprostone as potential ophthalmic
solutions to reduce intraocular pressure in
pigmented rabbits. Jpn. J. Pharmacol., 81,
56–62.
15 Sjoquist, B., Basu, S., Byding, P., Bergh,
K., and Stjernschantz, J. (1998) The
pharmacokinetics of a new antiglaucoma
drug, latanoprost, in the rabbit. Drug
Metab. Dispos., 26, 745–754.
16 Jampel, H.D., Bacharach, J., Sheu, W.,
Wohl, L.G., Solish, A.M., and Christie, W.
(The Latanoprost/Unoprostone Study
Group) (2002) Randomized clinical trial of
latanoprost and unoprostone in patients
with elevated intraocular pressure. Am. J.
Ophthalmol., 134, 863–871.
17 Bowman, R.M., Steele, R.E., and Browne,
L.J. (1987) Preparation and testing of
alpha-heterocyclyltolunitriles as aromatase
inhibitors. European Patent Application
EP 0236940A2.
18 Edwards, P.N. and Large, M.S. (1988)
Preparation and formulation of
(substituted aralkyl) heterocyclic
compounds as aromatase inhibitors.
European Patent Application EP 296749A1.
19 Lang, M., Batzl, C., Furet, P., Bowman, R.,
Hausler, A., and Bhatnagar, A.S. (1993)
Structure–activity relationships and binding
model of novel aromatase inhibitors.
J. Steroid Biochem. Mol. Biol., 44, 421–428.
20 Browne, L.J., Gude, C., Rodriguez, H.,
and Steele, R.E. (1991) Fadrozole
hydrochloride: a potent, selective,
nonsteroidal inhibitor of aromatase for
the treatment of estrogen-dependent
disease. J. Med. Chem., 34 (2), 725–736.
21 Browne, L.J. (1985) Substituted fused
imidazole compounds as aromatase
inhibitors. European Patent Application
EP 165904A2.
22 Ford, N.F., Browne, L.J., Campbell, T.,
Gemenden, C., Goldstein, R., Gude, C.,
and Wasley, J.W.F. (1985) Imidazo[1,5-a]
pyridines: a new class of thromboxane A2
synthetase inhibitors. J. Med. Chem., 28,
164.
23 Plourde, P.V., Dyroff, M., and Dukes, M.
(1994) Arimidex: a potent and selective
fourth-generation aromatase inhibitor.
Breast Cancer Res. Treat., 30, 103–111.
24 Edwards, P.N. (2006) A partial view of the
quest for Arimidex, a potent, selective
inhibitor of aromatase, in Smith and
Williams’ Introduction to the Principles of
Drug Design and Action, 4th edn, CRC
Press, Boca Raton, FL, pp. 233–256.
25 Bhatnagar, A.S. (2007) The discovery and
mechanism of action of letrozole. Breast
Cancer Res. Treat., 105, 7–17.
26 Giordanetto, F., Bostr€om, J., and Tyrchan,
C. (2011) Follow-on drugs: how far should
References j33
chemists look? Drug Discov. Today, 16,
722–732.
27 Engel, T. (2006) Basic overview of
chemoinformatics. J. Chem. Inform.
Model, 46, 2267–2277.
28 GVKBIO (2011) http://www.gvkbio.com/
informatics.html (accessed December 8,
2011).
29 Surechem (2011) http://www.surechem.
org (accessed December 8, 2011).
30 IBM (2011) http://www.ibm.com/patent
(accessed December 8, 2011).
31 Spangler, S., Ying, C., Kreulen, J., Boyer,
S., Griffin, T., Alba, A., Kato, L., Lelescu,
A., and Yan, S. (2011) Exploratory analytics
on patent data sets using the SIMPLE
platform. World Patent Inform., 33 (4),
328–339.
32 Banville, D.L. (ed.) (2009) Chemical
Information Mining: Facilitating LiteratureBased
Discovery, CRC Press, Boca Raton,
FL.
33 Trippe, A.J. (2003) Patinformatics: tasks to
tools. World Patent Inform., 25 (3),
211–221.
34 Godden, J.W. and Bajorath, J. (2001)
Differential Shannon entropy as a
sensitive measure of differences in
database variability of molecular
descriptors. J. Chem. Inform. Comput. Sci.,
4, 1060–1066.
35 Accelrys (2011) http://accelrys.com/
products/scitegic (accessed December 8,
2011).
36 Receveur, J.-M., Murray, A., Linget, J.-M.,
Norregard, P.K., Cooper, M., Bjurling, E.,
Aadal Nielsen, P., and H€ogberg, T. (2010)
Conversion of 4-cyanomethyl-pyrazole-3carboxamides
into CB1 antagonists with
lowered propensity to pass the blood–
brain-barrier. Bioorg. Med. Chem. Lett., 20,
453–457.
37 Tam, J., Vemuri, K., Liu, J., Batkai, S.,
Mukhopadhyay, B., Godlewski, G., OseiHyiaman,
D., Ohnuma, S., Ambudkar,
S.V., Pickel, J., Makriyannis, A., and
Kunos, G. (2010) Peripheral CB1
cannabinoid receptor blockade improves
cardiometabolic risk in mouse models of
obesity. J. Clin. Invest., 120, 2953–2966.
34j2 Competition in the Pharmaceutical Drug Development
Fabrizio Giordanetto studied medicinal chemistry at the University
of Genoa and obtained his Ph.D. in Computational
Chemistry in 2003 from the University of London. He joined
the Computational Sciences section of Pharmacia (Pfizer
Group, Nerviano, Italy) in 2002, and then moved to the
Medicinal Chemistry Department of AstraZeneca (M€olndal,
Sweden) in 2004, where he is currently working as Principal
Scientist in addition to his role as Project Leader in Drug Discovery.
From hit finding to clinical candidate selection, he has
a keen interest in everything that has to do with drug design, from rational chemical
modifications to serendipitous realizations.
Christian Tyrchan studied chemistry with focus on pharmacology
and bioinformatics, and received his Ph.D. in 2002
from the Department of Biochemistry at the University of
Cologne, Germany. After holding a postdoctoral position at
the Research Group for Molecular Informatics at the Cologne
University BioInformatics Center (CUBIC), he joined in
2004 as a computational chemist at Euroscreen S.A. in Gosselies,
Belgium, first in a postdoctoral position and then as a
research scientist. In 2007, he moved to a postdoctoral position
at the Chemoinformatics Group in the Discovery Enabling Capabilities and
Sciences unit (DECS) of AstraZeneca in M€olndal, Sweden. Since 2008, he has been
a member of the Computational Chemistry Group in the Medicinal Chemistry
Department of AstraZeneca CV/GI in M€olndal, Sweden.
References j35
3
Metabolic Stability and Analogue-Based Drug Discovery
Amit S. Kalgutkar and Antonia F. Stepan
List of Abbreviations
ADME absorption, distribution, metabolism, and excretion
AO aldehyde oxidase
CLint,app apparent intrinsic clearance
CYP cytochrome P450
DDI drug–drug interaction
DME drug metabolizing enzyme
GSH reduced glutathione
HIV human immunodeficiency virus
HLM human liver microsomes
IADR idiosyncratic adverse drug reaction
5-LO 5-lipoxygenase
MAO monoamine oxidases
NADPH nicotinamide adenine dinucleotide phosphate, reduced form
NAPQI N-acetyl-p-benzoquinone imine
PPAR-c peroxisome proliferator-activated receptor-c
QD once a day (from Latin quaque die)
RM reactive metabolite
SAR structure–activity relationship
SULT sulfotransferases
UGT uridine glucuronosyl transferases
3.1
Introduction
A major focus in preclinical drug discovery involves the identification of safe and
efficacious drug candidates that can be administered orally at low daily doses. During
the hit-to-lead stage of drug discovery, most new chemical entities demonstrate
high oxidative metabolic instability in human hepatic tissue (e.g., human liver
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
j37
microsomes (HLMs) and human hepatocytes), principally mediated by cytochrome
P450 (CYP) enzymes. The in vivo manifestation of high oxidative instability is suboptimal
pharmacokinetics (high clearance, short elimination half-life, and poor oral
bioavailability) due to extensive intestinal and first-pass metabolism by CYP
enzymes [1]. Consequently, designing drug candidates with the optimal balance
between efficacy and oral pharmacokinetics usually involves the introduction of
metabolic resistance within the unstable lead chemical matter. For human pharmacokinetic
predictions in drug discovery, much attention has been focused on the
role of CYPs, since the particular enzyme system has been implicated in the metabolism
of about 90% of marketed drugs. For this purpose, a high-throughput screening
to facilitate structure–activity relationship (SAR) studies around these oxidative
drug metabolizing enzymes (DMEs) is usually a practical starting point in most
drug discovery programs. While such in vitro metabolic stability assays provide
convenient means for “rank ordering” large numbers of compounds, information
pertaining to the cause of oxidative instability cannot be discerned from such
studies, and require customized mechanistic studies including the identification
of metabolic “soft spots.” Furthermore, it is not uncommon to find that as metabolic
liability due to CYP enzymes is decreased, the molecules render themselves
as substrates for other DMEs that may not be represented in the in vitro metabolism
vector (e.g., HLM). Under such circumstances, when examined in vivo, an
apparent in vitro–in vivo disconnect is revealed (i.e., greater in vivo metabolism is
observed than would be predicted from the in vitro studies). The role of non-CYP
enzymes in oxidative metabolism has been the subject of several recent
reviews [2, 3]. Though not an exhaustive list, non-CYP DMEs that consistently
reveal themselves as possible contributors to metabolism of pharmaceutical
agents include (a) monoamine oxidases (MAO), (b) aldehyde oxidase (AO), and
(c) uridine glucuronosyl transferases (UGTs). Unfortunately, in vitro tools for
these enzymes that allow for high-throughput screening and prediction of human
pharmacokinetics are typically not as readily available nor do they possess the
fidelity for predicting human pharmacokinetics in a similar way that liver microsomes
do for CYP enzymes.
In addition to manipulation of physicochemical properties (e.g., log P, log D,
molecular weight, etc.), rational design of drug candidates with improved metabolic
resistance also relies heavily on identification of metabolically labile sites for both
phase I and phase II DMEs. Within this chapter, we will present several examples
wherein SAR information from in vitro metabolism studies has enabled the conversion
of metabolically labile lead chemical matter into “drug-like” molecules with
improved absorption, distribution, metabolism, and excretion (ADME) characteristics.
The value of metabolism-directed studies in the design of short-acting drug
candidates (i.e., compounds with a short half-life) will also be discussed. Finally,
the chapter will dwell into an analysis of rational discovery strategies/approaches
for eliminating metabolism-dependent safety liabilities (e.g., reactive metabolite
(RM) formation leading to genotoxicity, mechanism-based CYP inactivation, and
idiosyncratic toxicity) using knowledge gathered from mechanistic drug metabolism
studies.
38j3 Metabolic Stability and Analogue-Based Drug Discovery
3.2
Metabolism-Guided Drug Design
Reducing metabolic liability in the course of hit-to-lead optimization studies
requires a proactive approach between medicinal chemists and drug metabolism
scientists. The drug metabolism scientists identify sites of metabolism using
appropriate in vitro metabolism vectors, information that is used by chemists to
make rational structural modifications including the elimination of metabolic “soft
spots” (e.g., replacement of a metabolically labile methyl group with the metabolically
inert trifluoromethyl substituent), deactivating electron-rich aromatic rings
prone to oxidation (e.g., substitution of phenyl with fluorophenyl or fluoropyridyl
rings), and/or generally manipulating the physicochemical properties (e.g., reducing
molecular weight and lipophilicity) to attenuate metabolic instability. In practice,
however, this exercise is not trivial; medicinal chemistry tactics to optimize
clearance could confer a detrimental effect on primary pharmacology (e.g., changes
in agonist/antagonist behavior and/or subtype selectivity for target receptor or
enzyme) and/or pharmacokinetic attributes. Thus, chemical intervention strategies
to optimize ADME properties are often an iterative process. Of course, it will be
recognized that changes in one parameter are generally not independent of others
and it is likely that chemists will ultimately achieve an acceptable balance of properties
rather than optimization of each aspect of the molecule. For example, excessive
reduction in lipophilicity can reduce metabolism and decrease passive permeability
simultaneously, which can ultimately translate into poor oral absorption via
inability of the compound to permeate cellular membranes.
In the case of the major constitutively expressed human CYP isoform, that is,
CYP3A4, the predominant interaction with substrates is via hydrophobic forces
and overall lowering of lipophilicity can reduce metabolic lability to the enzyme.
The trend for lower metabolic lability with lower lipophilicity holds regardless of
structure and metabolic route [4]. An illustration of a medicinal chemistry strategy
to attenuate CYP3A4 oxidative instability is evident in structure–metabolism studies
on a series of N-arylsulfonamide-based c-secretase inhibitors, wherein the lead
compound exemplified by the lipophilic analogue 1 (Figure 3.1) suffered from
extensive oxidations on the cyclohexyl motif by CYP3A4, which translated into a
very high apparent intrinsic clearance (CLint,app) in HLM [5]. Modulation of overall
lipophilicity through reduction in size and introduction of polarity on the cycloalkyl
region generated the corresponding tetrahydropyrans (compounds 4–6) and tetrahydrofurans
(compounds 7 and 8), wherein the 3- and 4-substituted variants exhibited
greater stability in HLM relative to their 2-substituted counterparts in both
cases (Figure 3.1). To understand the greater metabolic resistance of 3-substituted
cyclic ethers relative to the 2-substituted analogues, metabolite identification studies
were performed in HLM with tetrahydropyrans 4 and 5 and tetrahydrofurans 7
and 8. As seen in Figure 3.2, oxidative metabolism of these compounds still
occurred primarily on the cycloether ring. For the tetrahydropyran and tetrahydrofuran
rings, the characterization of stable hydroxycarboxylic acid metabolites was
consistent with an initial CYP3A4-catalyzed oxidation(s) on the carbon a to the
3.2 Metabolism-Guided Drug Design j39
oxygen atom, which would lead to unstable hemiacetal intermediates followed by
hydrolysis to the corresponding aldehyde derivatives and further oxidation to the
corresponding hydroxy acid metabolites. Based on these observations, it was speculated
that the greater stability of the 3-substituted cyclic ethers (relative to the
2-substituted variants) resulted from a slower overall rate of metabolism caused by
a diminished affinity for CYP binding due to a reduction in log D and/or
unfavorable active site interactions with the 3-oxo substituent. Guided by these
results, the lipophilicity of the ring system was reduced by further reducing the
ring size and introducing the oxetane ring system. It was found that, relative to the
other cyclic ethers, the oxetane-containing sulfonamides 2 and 3 possessed the
highest degree of HLM stability and retained c-secretase inhibitory potency in the
desired nanomolar range. The relationship between oxetane ring substitution and
oxidative metabolic stability followed similar trends as seen with the tetrahydropyran
and tetrahydrofuran derivatives. Thus, 3-substituted oxetane 3 (CLint,app
¼ 28.6 ml/min/kg) was relatively more stable in HLM than the two 2-substituted
oxetane 2 (CLint,app
¼ 62.1 ml/min/kg).
An example of metabolic blocking approach to minimize oxidative metabolism
by CYP3A4 is evident from SAR work on human immunodeficiency virus (HIV)
protease inhibitors for the treatment of HIV-1 infections. As a structural class, protease
inhibitors, exemplified by indinavir, generally suffer from extensive first-pass
metabolism in the small intestine and liver, which is mediated by CYP3A4.
Attempts to develop agents with improved pharmacokinetics focused on blocking
the sites of CYP3A4-mediated metabolism. Cheng et al. [6] deconstructed the metabolic
liabilities of indinavir and designed in attributes that significantly enhanced
O
O
O O
O
O
O
R =
IC50
(Aβ42, nM)
Cl
S OO
N
O NH2
CF3R
12.3 27.9 45.2 8.58 44.5 11.2 7.99 16.9
CLint,app
(ml/min/kg)
176 >300 136 114 210 69.8 62.1 28.6
Compound 41 65 87 32
tetrahydropyrans tetrahydrofurans oxetanes
Figure 3.1 In vitro c-secretase inhibition and HLM apparent intrinsic clearance (CLint,app) data for
a representative set of N-arylsulfonamides.
O
41
O
5
O
7
O
8
O
2
O
3
100% 100%
95%
5%
100%
76%
24% 65%
35%
100%
Cl
S OO
N
O NH2
CF3R
tetrahydropyrans tetrahydrofurans oxetanes
Figure 3.2 Sites of oxidative metabolism in HLM for representative N-arylsulfonamides.
40j3 Metabolic Stability and Analogue-Based Drug Discovery
their disposition attributes. Indinavir undergoes metabolism at two regions of the
molecule designated as P0
2 and P3 (Figure 3.3). Specifically, oxidation takes place at
the benzylic position of the aminoindanol moiety ðP0
2Þ, in addition to the pyridine
nitrogen and the methylene linker (P3). Incorporation of the gem-dimethyl and pyridylfuran
functionalities in P3 compound 9), along with replacement of the aminoindanol
with an aminochromanol moiety (compound 10), led to analogues shown
in Figure 3.3. In two markers of pharmacological activity ((i) inhibition of cleavage
of a substrate by the wild-type HIV-1 protease enzyme and (ii) inhibition of the
spread of viral infection in MT4 human T-lymphoid cells infected by the NL4-3
virus), both compounds showed enhanced potency compared to indinavir. When
normalized for dose, compound 9 also showed twofold improvement in oral systemic
exposure in dogs, while compound 10 showed a marginal improvement.
The calcium channel blocker diltiazem (Figure 3.4) is extensively metabolized
via distinct pathways that include N-demethylation, ester hydrolysis, and
O-demethylation. The enzyme responsible for the major route (N-demethylation)
of clearance has been shown to be CYP3A4. Although widely used in therapy, diltiazem
has a relatively short duration of action. In the search for metabolically resistant
analogues, Floyd et al. [7] substituted the trifluoromethyl benzazepinone ring
structure for the benzothiazepinone of diltiazem. Metabolism studies on this class
of compound showed that the principal elimination routes were similar to that for
diltiazem with N-demethylation, ester hydrolysis, and O-demethylation all occurring.
Interestingly, the N-desmethyl metabolite was pharmacologically equipotent
to the parent, and was much more resistant to metabolism. Consequently, N-1-pyrrolidinyl
derivatives (exemplified by compound 11) were designed to achieve metabolic
stability both by a decrease in stability of the secondary amine radical (relative
to tertiary amines) and by steric hindrance afforded by b-substitution (Figure 3.4).
N
N
N
H
N
O NH
OH
O
OH
P3
P2'
N
N NHR
O NH
OH
O
O
N
OH
O
HO
R =
9 10
Indinavir
Figure 3.3 Improving oxidative metabolic liability in the HIV-1 protease inhibitor indinavir
through blocking of metabolic sites.
3.2 Metabolism-Guided Drug Design j41
The success of this strategy indicates how vulnerable alkyl-substituted nitrogen
functionalities can be stabilized to oxidative N-dealkylation.
3.3
Indirect Modulation of Metabolism by Fluorine Substitution
The effects of fluorine on ADME properties, including metabolism, have been
known and exploited by medicinal chemists for some time [8]. Substituting hydrogen
with fluorine at metabolically labile positions, for example, is a common
approach to attenuate metabolism, although deuterium has also been considered.
In the case of deuterium, the rate of metabolism via a specific pathway is attenuated,
but the rate of overall substrate consumption or overall clearance is not significantly
altered, due to a compensatory increase in the rate of formation of an
alternate metabolite [9]. Moorjani et al. [10] reported on the use of fluorine substitution
to enhance metabolic stability in a series of non-xanthine-selective A2a antagonists
for the potential treatment of Parkinson’s disease. Lead compound 12
(Figure 3.5) demonstrated good antagonist activity and selectivity for the A2a receptor,
but was unstable in HLM. Introduction of a fluorine atom in the aromatic ring
in 12 furnished 13, which was resistant to oxidative metabolism in HLM while
exhibiting single-digit nanomolar potency for the A2A receptor. In this scenario, the
electron-withdrawing effects of the fluorine atom most likely rendered the phenyl
group resistant to CYP oxidation. A second example relates to the metabolismdirected
lead optimization of N-(3,3-diphenylpropyl)nicotinamide (14), a potent
inhibitor of soluble epoxide hydrolase (Figure 3.5) [11]. ADME profiling of 14
revealed a short metabolic half-life in HLM and rat liver microsomes, which was
consistent with rapid clearance in rats. Metabolite identification studies indicated
extensive hydroxylation on the pendant phenyl groups of the benzhydryl motif,
which led to the design of the corresponding bis-(4-fluorophenyl) analogue 15 in
order to block the metabolically vulnerable sites with fluorine atoms. Examination
of the in vitro metabolic profile of 15, however, revealed an apparent metabolic
S
N
O
O
N
CH3
H3C
OCH3
*
*
*
*
N
O
O
OCH3
CF3
HN
Diltiazem 11
O
CH3
O
CH3
* = sites of metabolism
Figure 3.4 Structures of diltiazem and its benzazepinone analogue 11, which is inert to
metabolism.
42j3 Metabolic Stability and Analogue-Based Drug Discovery
switch in the major route of metabolism to pyridine ring hydroxylation, a feature
that was consistent with lack of change in microsomal half-lives. Hypothesizing
that increase in overall polarity could decrease metabolism, analogues with polar
pyridine ring substituents were synthesized. From this exercise emerged 16, which
retained the soluble epoxide hydrolase inhibitory potency of 14, and was practically
inert to oxidative metabolism in microsomes. However, 16 revealed poor in vivo
oral absorption in rats due to its poor aqueous solubility. Guided by the analysis of
the costructure of 14 with soluble epoxide hydrolase, one fluorine atom in 16 was
substituted for an additional polar substituent on the right-hand side benzhydryl
motif with simultaneous modifications on the pyridine ring to balance physicochemical
properties. These efforts culminated in the identification of potent and in
vitro metabolically stable epoxide hydrolase inhibitors with excellent oral absorption
as exemplified with compound 17.
Burgey et al. also reported on the metabolism-directed optimization of a series of
3-(2-phenethylamino)-6-methylpyrazinone acetamide thrombin inhibitors (exemplified
by compound 18, Figure 3.5), which involved the use of fluorine atoms to
stabilize oxidative metabolism resulting in orally bioavailable compounds [12].
Metabolic profiling of 18 revealed three principal sites of metabolism – oxidation of
NN
H
N CH3
O
N
N CH3
H3C
O
R
N
H3C
12: R = H
13: R = F
N
N
H
O
14
Human soluble epoxide hydrolase IC50 = 7 nM
HLM t1/2 = 13 min; RLM t1/2 = 3.0 min
N
N
H
O
15
Human soluble epoxide hydrolase IC50 = 8 nM
HLM t1/2 = 12 min; RLM t1/2 = 12 min
F
F
N
N
H
O
16
Human soluble epoxide hydrolase IC50 = 5 nM
HLM t1/2 > 300 min; RLM t1/2 > 300 min
F
FOF3C
N
N
H
O
17
Human soluble epoxide hydrolase IC50 = 7 nM
HLM t1/2 > 300 min; RLM t1/2 = 161 min
F
SS
H3C
O O
O
O
CH3
N
H
N
N
O
N
H
O
NH3C NH2
CH3
18
*
*
*
* = Sites of metabolism
P1
P3
P2
IC50 (thrombin IIa) = 0.8 nM
IC50 (trypsin) = 1800 nM
N N
H
N
N
O
N
H
O
NH3C NH2
CH3
19
IC50 (thrombin IIa) = 0.042 nM
IC50 (trypsin) = 800 nM
F F
N N
H
N
N
O
N
H
O
N
CH3
20
IC50 (thrombin IIa) = 4.2 nM
IC50 (trypsin) = 252 nM
F F
N N
H
N
N
O
N
H
O
N
CN
21
IC50 (thrombin IIa) = 2.3 nM
IC50 (trypsin) = 213 nM
F F
F
F
Figure 3.5 Modulation of oxidative metabolism by fluorine substitution.
3.3 Indirect Modulation of Metabolism by Fluorine Substitution j43
the P3 and P2 benzylic positions and phase II conjugation of the P1 basic amino
group (Figure 3.5). A study to examine the effect of P3 benzylic substitution on
thrombin inhibitory potency was initiated with the goal of sterically blocking oxidative
metabolism. Incorporation of a 2-pyridyl and gem-difluoro modifications
afforded 19, with improved thrombin inhibitory potency and lack of obvious metabolic
soft spots. Attempts to address phase II metabolism in 18 via removal of the
amino group led to dramatic loss of thrombin inhibitory potency, which was
regained to some degree via installation of the optimized 2,2-difluoro-2-pyridylethylamino
P3 modification. Incorporation of a meta-substituted fluorine atom on the
P1 aryl group led to 20 with inhibitory potency in the targeted range for efficacy.
Compound 20 also demonstrated moderate to good oral bioavailability in rats,
dogs, and monkeys. Metabolism of the P2 benzylic group was finally addressed via
replacement of the problematic methyl group with a cyano functionality leading to
compound 21 with excellent pharmacology and animal pharmacokinetics.
The final example involves optimization of the cholesterol absorption inhibitor
(À)-SCH 48461. Metabolism studies indicated that there were four primary sites
of metabolism for (À)-SCH 48461 (Figure 3.6), with metabolites resulting from
O-demethylation, hydroxylation, and/or oxidation, and combinations thereof. The
metabolism data along with the SAR studies were used as a guide in designing a
metabolically more stable analogue of (À)-SCH 48461 without affecting the potency
of the compound [13]. Initial attempts to prevent metabolism via substitution of the
C0
-3 carbon in (À)-SCH 48461 with an oxygen atom (compound 22) were
unsuccessful since this step produced an electron-rich phenoxy motif in comparison
to the original phenyl group and possibly made this function more amenable to
aromatic hydroxylation. Blocking aromatic oxidation with a fluorine atom introduced
in the para position was required to produce the eventual more stable substitution
((À)-SCH 53079). Additional SAR led to the design of even more stable and
N
O
OCH3
OCH3
(-)-SCH 48461
*
*
*
*
* = sites of oxidative metabolism
O
N
O
OH
(-)-SCH 57939
F
O
N
O
OCH3
OCH3
*
O
N
O
OCH3
(-)-SCH 53079
F
OH
F
N
O
OH
Ezetimibe
F
F
OH
22
Figure 3.6 Use of fluorine to stabilize oxidative metabolism in cholesterol absorption inhibitors.
44j3 Metabolic Stability and Analogue-Based Drug Discovery
pharmacologically potent analogues exemplified by (À)-SCH 57939, and ultimately
led to the discovery of ezetimibe [14, 15].
3.4
Modulation of Low Clearance/Long Half-Life via Metabolism-Guided Design
While optimization of ADME attributes usually focuses on reducing clearance and
increasing half-lives, in some scenarios (e.g., mechanism-based safety concerns),
engagement of the pharmacological target requires short-acting agents and/or molecules
with specific clearance and half-life values. Metabolism-guided drug design
has also proven very valuable in such efforts. For instance, the known lability of
benzylic positions on electron-rich heteroatoms toward CYP metabolism has been
exploited to decrease the unacceptably low clearance and resultant long half-life of
selective cyclooxygenase-2 inhibitor lead compounds. Specifically, the anti-inflammatory
agent celecoxib (Figure 3.7) exhibits a half-life of 3.5 h in rats, whereas early
structural leads, represented by compounds in which celecoxib’s benzylic methyl
was replaced with a halogen substituent (compound 23, Figure 3.7), possessed
half-life values >220 h in rats [16]. In another illustration, short-acting calciumsensing
receptor antagonists were designed by incorporating the metabolically
labile thiomethyl functionality into the metabolically stable, zwitterionic amino
alcohol leads (e.g., compound 24, Figure 3.7) to increase oxidative clearance by
CYP3A4, while delivering a pharmacologically inactive sulfoxide metabolite. The
efforts led to the discovery of clinical candidates 25 and 26 for the potential treatment
of osteoporosis [17].
S
N
N
CF3
F
H3C
O O
S
N
N
CF3
H3C
H3C
O O
23 Celecoxib
COOH
CH3
O
CH3
OH
N
H
CH3
F
COOH
CH3
O
CH3
OH
N
H
S
R
CH3
S
R
CH3
O
Metabolically inactive
sulfoxide
24
R = H (25); R = F (26)
O
N
H3C CH3
R1
R2
27: R1 = NHSO2CH3; R2 = CF3
28: R1 = F; R2 = SCH3
29: R1 = SO2NH2; R2 = SCH3
30: R1 = SO2NH2; R2 = S(O)CH3
Figure 3.7 Metabolism-guided strategies to enable the design of short-acting clinical agents.
3.4 Modulation of Low Clearance/Long Half-Life via Metabolism-Guided Design j45
In a slightly different approach, Middleton et al. report a strategy on purposely
redirecting metabolism away from an undesired site on a series of serotonin
reuptake inhibitors [18]. Lead compound 27 (Figure 3.7), although a promising
lead, was found to undergo CYP2D6-mediated N-demethylation to the secondary
amine metabolite, which was pharmacologically active and possessed a relatively
long half-life, a phenomenon that was inconsistent with the laboratory objectives of
this program. A simple thiomethyl analogue 28 was designed to explore the concept
that CYP-mediated thioalkyl S-oxidation can be a rapid metabolic process and
could potentially compete with N-demethylation. Further expansion of the SAR led
to the sulfonamide 29, which was potent and selective for serotonin over dopamine
and norepinephrine reuptake inhibition. In vitro, the sulfoxide 30 was the predominant
metabolite (>90%) and showed only weak pharmacological activity (IC50 > 1
mM). In vivo, in both rat and dog pharmacokinetic studies, the parent compound 29
retained the desired ADME properties of 27, and indeed as predicted by the in vitro
studies, the sulfoxide 30 was the predominant metabolite. Compound 29 possessed
the desired short half-life and, furthermore, was shown to be inactive against a
broad panel of other receptors, enzymes, and ion channels. Based on the cumulative
knowledge, sulfonamide 29 was progressed into clinical development.
3.5
Tactics to Resolve Metabolism Liabilities Due to Non-CYP Enzymes
3.5.1
Aldehyde Oxidase
In addition to CYP isoforms, oxidative metabolic liabilities can also be encountered
with AO and MAO enzymes. AO is a member of a family of enzymes referred to as
molybdenum cofactor-containing enzymes, which also includes xanthine oxidase.
Liver has by far the highest AO expression in all species including humans. Typical
substrates of AO are compounds containing an aldehyde function, nitro/nitroso
derivatives, and nitrogen-containing heterocycles. AO catalyzes the a- or c-carbon
oxidation of the imine motif (R1R2C ¼ NR3) in nitrogen-containing heterocycles
(e.g., pyridine, pyrimidine, quinoline, quinoxaline, etc.), resulting in the formation
of the corresponding lactam (a-carbon oxidation) or aminoenone (c-carbon oxidation)
metabolites, respectively [2]. Even though AO utilizes molecular oxygen as the
ultimate electron acceptor, the oxygen atom that is incorporated into the product
during AO-mediated hydroxylation is derived from water and not molecular oxygen.
Consequently, a- and c-carbon atoms adjacent to electron-withdrawing nitrogen
atoms in nitrogen heterocycles preferentially are oxidized by AO. While the
order of AO activity among animal species may vary depending on the substrate, it
generally seems to be high in monkeys and humans and low in rats, whereas dogs
are to a large extent deficient in activity [2].
Efforts to confer metabolic resistance into aromatic rings by virtue of introducing
polar heteroatoms such as nitrogen often render the compounds susceptible to AO
46j3 Metabolic Stability and Analogue-Based Drug Discovery
metabolism. In a recent account, Magee et al. disclosed a series of heterocyclesubstituted
azetidinyl ketolide derivatives as potent antibacterial agents with minimal
turnover in CYPs and little to no inhibitory effects against the human CYP3A4
isoform [19]. An extensive investigation into heterocycle analogues identified a
number of systems that appeared to combine favorable efficacy and metabolism
properties. Compound 31 (Figure 3.8) was predicted to have low human clearance,
which combined with its favorable in vitro profile led to pharmacokinetic studies in
clinical trials. However, upon oral dosing at the predicted efficacious dose range of
300–1000 mg, the measured plasma exposures were $20% of the predicted area
under the plasma concentration–time curve values, leading to the termination of
further clinical investigation with the agent. Human liver cytosolic assessment of
turnover with and without raloxifene (a selective inhibitor of AO) revealed that 31
was a substrate of human AO. The 1,8-naphthyridine ring system in 31 was subsequently
shown to be vulnerable to AO, although the regiochemistry of hydroxylation
was not established. The AO liability was not circumvented by incorporation
of polar functionalities such as hydroxyl (e.g., compound 32), but was avoided
through modification of the heteroatom arrangement. For instance, the 1,5-naphthyridine
system (compound 33) was stable in human liver cytosol and the
O
O
N(CH3)2
OH
O
O
O
O
N
O
O
N
31
N
CH3
N
O
O
N(CH3)2
OH
O
O
O
O
N
O
O
N
32
N
N
HO
O
O
N(CH3)2
OH
O
O
O
O
N
O
O
N
33
N
N
HO
N
H
O
N N
N
O
N
CN
34
N
H
O
N N
O
N
CN
35
N
H
O
N N
N
O
N
CN
36
OCH3
*
* = site of AO metabolism
OCH3
OCH3
OCH3
Figure 3.8 SAR for AO-mediated oxidation of azetidinyl ketolide derivatives.
3.5 Tactics to Resolve Metabolism Liabilities Due to Non-CYP Enzymes j47
compound was selected for clinical development [19]. In another account, AOmediated
metabolism on the imidazo[1,2-a]pyrimidine moiety in the androgen
receptor antagonist and anticancer agent 34 was prevented by blocking the site of
metabolism as well as alteration in the heterocycle as highlighted with analogues 35
and 36, respectively (Figure 3.8) [20].
3.5.2
Monoamine Oxidases
MAO isoforms MAO-A and MAO-B are mitochondrial flavoenzymes that catalyze
the oxidative deamination of structurally diverse alkyl- and arylalkyl amines [3].
Elaborate SAR studies with arylalkylamine substrates and inhibitors suggest that
MAO-A and MAO-B are efficient catalysts for amines, which possess aryl group 1–
3 carbons away from the amine nitrogen. Although primary and secondary amines
are deaminated indiscriminately by both isozymes, tertiary amines generally display
selectivity toward one form of the enzyme. From a drug metabolism perspective,
MAO plays a dominant role in the clearance of the triptan class of
antimigraine drugs. This class of compounds constitutes novel and highly selective
serotonin receptor agonists that are used in the acute, oral treatment of migraine
and cluster headaches. Members of this group of drugs include acyclic tertiary
amines such as sumatriptan, almotriptan, zolmitriptan, and rizatriptan, cyclic tertiary
amines such as eletriptan and naratriptan, and a secondary amine (frovatriptan).
MAO-A is the principal enzyme responsible for the clearance of sumatriptan
and almotriptan leading to the formation of the corresponding indoleacetic acid
metabolites (Figure 3.9) [21, 22]. In the case of sumatriptan, in vitro studies have
clearly demonstrated the involvement of MAO by using the MAO-A-specific inactivator
clorgyline, which prevents sumatriptan metabolism. Rizatriptan also undergoes
MAO-catalyzed oxidation leading to the indoleacetic acid derivative as the
major metabolite in humans (Figure 3.9) [23]. The structurally related zolmitriptan
is cleared in humans primarily via N-demethylation and N-oxidation that is mediated
by CYP1A2 [24]. The N-demethylated metabolite undergoes selective MAO-Acatalyzed
oxidation to yield the corresponding indoleacetic acid derivative (Figure
3.9) [24]. Despite their structural similarity to sumatriptan, zolmitriptan, and
rizatriptan, the cyclic tertiary amines eletriptan and naratriptan and the secondary
amine frovatriptan (Figure 3.9) are not substrates of MAO-A and/or MAO-B. Eletriptan
is metabolized via N-demethylation by CYP2D6 and CYP3A4, whereas frovatriptan
is metabolized by CYP1A2 [25, 26]. Naratriptan is mostly eliminated in
the urine in the unchanged form (naratriptan product information). The half-lives
of naratriptan, eletriptan, and, in particular, frovatriptan range from 26 to 30 h, and
are longer than that of sumatriptan ($2 h) [27]. As such, most basic amine drugs do
not have MAO-catalyzed metabolism as a primary clearance pathway with the
exception of the triptans. The findings are probably a reflection of the low lipophilicity
of the triptans and their stability to oxidative enzymes such as CYP. Overall,
the findings on the MAO-A substrate properties of sumatriptan, almotriptan,
rizatriptan, and the N-dealkylated metabolite of zolmitriptan are not surprising
48j3 Metabolic Stability and Analogue-Based Drug Discovery
considering the structural similarity with the endogenous neurotransmitter serotonin,
which is a preferred MAO-A substrate [3].
3.5.3
Phase II Conjugating Enzymes (UGT and Sulfotransferases)
Conjugation with glucuronic acid or sulfate requires a nucleophilic substituent
(e.g., hydroxyl, amine, etc.) present in the molecule. UGT substrates include phenol,
primary, secondary, or even tertiary alcohol, or carboxylic acid derivatives,
whereas substrates for sulfation catalyzed by sulfotransferases (SULTs) are usually
phenolic compounds [28, 29]. In some cases, primary alcohols can also form sulfate
conjugates. The most “reactive” functionality is the phenol and a simple rule of the
thumb is to eliminate such a functionality unless absolutely essential for pharmacological
activity. For instance, the peroxisome proliferator-activated receptor-c
(PPAR-c) agonist troglitazone (Figure 3.10) is principally metabolized in humans
via sulfation of its phenol group [30]. Related PPAR agonists pioglitazone and rosiglitazone
(Figure 3.10) do not undergo this metabolic fate since they do not contain
the phenol substituent. It is also noteworthy to point out that pioglitazone and rosiglitazone
are significantly more potent than the PPAR-c agonist troglitazone indicating
that the phenol motif is not essential for primary pharmacology.
S
O O
N
H
N
H3C
CH3
HN
S
O O
N
H
N
CH3
NaratriptanEletriptan
R
N
H
N
H3C
CH3
MAO-A
or
CYP (N-Dealkylation)
then MAO-A
R
N
H
Indoleacetic acid metabolite
H3 SC
H
N
O O
O
Sumatriptan
N S
O
O
Almotriptan
N
N
N
Rizatriptan
O
H
NO
Zolmitriptan
R
COOH
R
N
H
N
H3C
R
R
N
H
O
N
H
H2N
O
NH
H3C
Frovatriptan
Not subject to MAO metabolism
Figure 3.9 Oxidative deamination of the triptan class of antimigraine drugs by MAO-A.
3.5 Tactics to Resolve Metabolism Liabilities Due to Non-CYP Enzymes j49
An example of minimizing phase II conjugation while maintaining pharmacological
potency is evident with 4-(3-hydroxylphenylamino)pyrrolotriazine-based
inhibitors of vascular endothelial growth factor-2 receptor [31]. The lead compounds
exemplified by 37 (Figure 3.11) suffered from extensive phenol glucuronidation
resulting in poor oral activity. Hence, SAR efforts were directed toward
finding functionalities that would suppress glucuronidation. The solvent-exposed
R2 region of the pyrrolotriazine was considered to be the most suitable for manipulations.
In the course of the SAR studies, it was observed that compounds with
neutral R2 groups (e.g., compound 37) with basic pKa < 6 exhibited high rates of
glucuronidation. In contrast, introduction of a basic amino group with a calculated
basic pKa between 7 and 10 on an appendage attached to the R2 position via different
functionalities (e.g., compound 38 in Figure 3.11) led to a substantial decrease
in glucuronidation rates. A second example is evident from SAR studies on a second
generation of N-hydroxyurea-based 5-lipoxygenase (5-LO) inhibitors, wherein
duration of 5-LO inhibition after oral administration in animals was optimized by
S
NH
O
O
O
R
Troglitazone: R =
CH3
H3C
HO
CH3
O
Rosiglitazone: R =
N
N
CH3
Pioglitazone: R =
N
CH3
Figure 3.10 PPAR-c agonists troglitazone, rosiglitazone, and pioglitazone. Sulfation of the
phenol group in troglitazone is the primary pathway of elimination in humans.
S
N
CH3
HO
NH2
O
Zileuton
Monkey t1/2 < 1.0 h
N
CH3
HO
NH2
O
O
39
Monkey t1/2 = 3.1 h
N
CH3
HO
NH2
O
(R)
OO
F
40
Monkey t1/2 = 7.4 h
N
N
N
R1
R2
HN
CH3
OH
Glucuronidation rates in HLM
0.90 nmol/min/mg
0.08 nmol/min/mg
37: R1 = R2 =
N
N
N
O
O
HN
38: R1 = R2 = N
O
O
HN
Figure 3.11 Strategies for reducing excessive glucuronidation.
50j3 Metabolic Stability and Analogue-Based Drug Discovery
reducing glucuronidation of the N-hydroxyurea motif [32]. The primary route of
metabolism of zileuton (Figure 3.11), the first N-hydroxyurea-based 5-LO inhibitor,
in humans involves O-glucuronidation of the N-hydroxyurea group, resulting in a
relatively short plasma half-life of $3 h after oral dosing [33]. Clinical trials with
zileuton in asthmatics demonstrated efficacy with oral administration of 600 mg
four times daily. Validation of the therapeutic application of 5-LO inhibitors in
asthma patients has provided impetus to identify an optimized compound with
greater potency and longer duration of action that would in turn provide a lower
and less frequent daily dose. Duration of 5-LO inhibition after oral administration
in monkeys (as a model for human pharmacokinetics) was optimized by identification
of structural features in the proximity of the N-hydroxyurea (e.g., compounds
39 and 40), which correlated to low in vitro glucuronidation rates. For instance,
compound 40 (phenoxyphenyl-substituted template in combination with the
butynyl link) proved to be a compatible match that enhanced inhibitory potency
and provided resistance to glucuronidation.
3.6
Eliminating RM Liabilities in Drug Design
Metabolic activation (also referred to as bioactivation) of innate functional groups
into electrophilic RMs is considered to be an undesirable trait of drug candidates
on the grounds of evidence linking this liability with genotoxicity [34], drug–drug
interactions (DDIs) [35], end organ toxicity, and possibly immune-mediated idiosyncratic
adverse drug reactions (IADRs) [36–39]. Most pharmaceutical companies
have implemented procedures to evaluate a drug candidate’s potential to form RMs
with the goal of eliminating or minimizing this liability by rational structural modifications.
Qualitative assessment of CYP-mediated RM formation in vitro usually
involves “trapping” studies conducted with NADPH-supplemented HLM in the
presence of reduced glutathione (GSH), amines (e.g., semicarbazide and methoxylamine),
and/or cyanide ion. Microsomes can be replaced by alternate metabolism
systems (e.g., liver cytosol, liver S9 fractions, hepatocytes, neutrophils, etc.), to evaluate
the participation of non-CYP enzymes in RM formation. In case of RM positives,
characterization of the nucleophile–RM structure provides insight into the
structure of the reactive species and the mechanism leading to its formation. The
information is then used, as appropriate, to modify the structure of the RM positives
in order to eliminate the liability.
3.7
Eliminating Metabolism-Dependent Mutagenicity
The role of metabolism in generating RMs capable of covalently adducting with
nucleic acids and leading to mutagenic lesions is well established for many endogenous
and exogenous xenobiotics (including drugs). Virtually, any molecule that
3.7 Eliminating Metabolism-Dependent Mutagenicity j51
forms RMs possesses the propensity to modify DNA and elicit a genotoxic/carcinogenic
response. Genetic toxicology testing is usually conducted in the early stages
of drug development with the intent of identifying hazards associated with both the
parent molecule and its metabolites. Identification of genotoxic metabolites in
in vitro test systems is accomplished by employing metabolic activation systems
(e.g., Aroclor 1254-induced rat liver S9). The Salmonella reverse mutation assay has
become an integral part of the safety evaluation of drug candidates and is required
by regulatory agencies for drug approvals worldwide. The mutagenic potential of
small-molecule drug candidates is generally evaluated in genetically different
strains of the Salmonella typhimurium, such as TA98, TA100, TA1535, and TA1537.
These test strains all carry some type of defective (mutant) gene that prevents them
from synthesizing the amino acid histidine in a minimal bacterial culture medium.
In the presence of mutagenic chemicals, the defective gene may be mutated back to
the functional state that allows the bacterium to grow in the medium. Because positive
findings in the Salmonella reverse mutation assay have a good correlation with
the outcome of rodent carcinogenicity testing, a positive result leads to the discontinuation
of development, particularly for drugs intended for non-life-threatening
indications.
RM trapping studies have proven useful in elucidating mutagenic mechanisms
of drug candidates, and the mechanistic insights gathered have been used in the
rational design of follow-on compounds that are devoid of genotoxic response. An
example is evident with the anti-obesity agent and 5-hydroxytryptamine 2C agonist
41 (Figure 3.12), which was mutagenic in the bacterial Salmonella Ames assay in an
Aroclor 1254-induced rat liver S9/NADPH-dependent fashion [40, 41]. In the Salmonella
assay, 41 produced a significant increase of mutations only in strains
TA100 and TA1537 that are known to be sensitive to mainly base-pair and frameshift
mutagens, consistent with covalent adduction to DNA (rather than a simple
intercalation). Studies with ½14
CŠ-41 revealed the irreversible and concentrationdependent
incorporation of radioactivity in calf thymus DNA in an S9/NADPHdependent
fashion confirming that 41 was metabolized to a DNA-reactive metabolite.
RM trapping studies in S9/NADPH incubations led to the detection of GSH
and amine conjugates. Structural elucidation of these conjugates allowed an insight
into the mechanism leading to the formation of DNA-reactive metabolites (Figure
3.12). The mass spectrum of the methoxylamine conjugate of 41 was consistent
with condensation of amine with an electrophilic, aldehyde metabolite derived
from piperazine ring scission in 41, whereas the mass spectrum of the GSH conjugate
suggested a bioactivation pathway involving initial aromatic ring hydroxylation
on the 3-chlorobenzyl motif in 41 followed by b-elimination to an electrophilic quinone
methide species that reacted with GSH. The observation that methoxylamine
and GSH reduced mutagenicity suggested that the trapping agents competed with
DNA toward reaction with the RMs. Overall, the exercise provided indirect information
on the structure of DNA-reactive intermediates leading to mutagenic
response with 41 and hence a rationale on which to base subsequent chemical
intervention strategy for designing nonmutagenic 5-hydroxytryptamine 2C agonists
such as compound 42 (Figure 3.12) [41]. In contrast with 41, 42 cannot form the
52j3 Metabolic Stability and Analogue-Based Drug Discovery
3.7 Eliminating Metabolism-Dependent Mutagenicity j53
S9/NADPH
Cl
CH2
O GSH ClHO
Squinone methide
CH3ONH2
S9/NADPH
aldehyde
N
N N
NH
O
F
F 42
N
N
N O
HN
Cl
41
N
N
N O
HN
OH
N
N
N O
O
H2N
N
N
N O
N
H2N Cl
H3CO
N
N
N O
Cl
OH
HN
N
H
N
N O
HN
+
NH
O
H
N COOH
O
COOH
NH2
Figure 3.12 Insights into the S9/NADPH-dependent mutagenic response with the 5-hydroxytryptamine 2C agonist 41 that led to the design of nonmutagenic
analogue and clinical candidate 42.
reactive quinone methide species. Likewise, introduction of the methyl group is
thought to shunt metabolism from piperazine ring opening to hydroxylation of the
methyl substituent.
Another account of medicinal chemistry tactics to resolve metabolism-dependent
mutagenicity is evident with the arylindenopyrimidine series of selective dual
A2A/A1 adenosine receptor antagonists exemplified by 2-amino-4-phenyl-8-pyrrolidin-1-ylmethyl-indeno[1,2-d]pyrimidin-5-one
(43) (Figure 3.13) [42]. Compound 43
was shown to be genotoxic in both the Ames and mouse lymphoma L5178Y assays
in a rat liver S9/NADPH-dependent fashion, and also demonstrated irreversible
covalent binding to calf thymus DNA in the presence of Aroclor 1254-induced rat
liver S9 and NADPH. Profiling for RM formation in rat liver S9 indicated that 43
was bioactivated on its pyrrolidine ring to several electrophilic species that included
endocyclic iminium ion, amino aldehyde, epoxide, and a,b-unsaturated carbonyl
intermediates as judged from the detection of corresponding cyano, oxime, and
GSH conjugates (Figure 3.13). The endocyclic iminium ion and amino aldehyde
species appear to be the likely candidates responsible for genotoxicity based on,
first, the protection afforded by both cyanide ion and methoxylamine, which
reduced the potential to form covalent adducts with DNA, and, second, analogues
of 43 (e.g., compounds 44 and 45) designed with low probability to form these RMs
were not genotoxic. It was concluded that 43 also had the potential to be mutagenic
in humans based on the formation of the endocyclic iminium ion following incubation
with a human liver S9 preparation and the commensurate detection of DNA
adducts.
3.8
Eliminating Mechanism-Based Inactivation of CYP Enzymes
Modulation of CYP activity via induction or inhibition by xenobiotics including
drugs can lead to clinical DDIs with consequences ranging from loss of efficacy to
the introduction of adverse effects, respectively, for coadministered “victim” drugs.
DDIs arising from CYP inhibition are more frequent and, in some cases, have
resulted in the withdrawal of the marketed “perpetrator” drug (e.g., the antihypertensive
agent mibefradil), especially those drugs associated with potent inhibition
of the major constitutively expressed human CYP enzyme, CYP3A4. CYP inhibitors
can be categorized as reversible (competitive or noncompetitive), quasiirreversible,
and irreversible in nature [35]. Reversible inhibition usually involves
two or more agents competing for metabolism at the active site of a CYP isozyme,
with one drug inhibiting the metabolism of the other agent. In contrast, drugs converted
to electrophilic RMs by CYP may interact with the CYP quasi-irreversibly or
irreversibly leading to enzyme destruction. Quasi-irreversible inactivation occurs
when the reactive species complexes with the ferrous form of heme (product of
reduction of resting state ferric enzyme by NADPH-CYP reductase) in a tight, noncovalent
interaction. CYP inactivation can also arise through covalent adduction of
the RM to the heme prosthetic group and/or to an active site amino acid residue in
54j3 Metabolic Stability and Analogue-Based Drug Discovery
3.8 Eliminating Mechanism-Based Inactivation of CYP Enzymes j55
N
NH2N
O
O
N43
N
NH2N
O
N
N
NH2N
NH
O
iminium
amino aldehyde
Covalent Modification of DNA
CH3ONH2
N
NH2N
O
NH
N-OCH3
CN
N
NH2N
O
N
NC
N
NH2N
O
Nenamine
N
OH
N
OH
O
N
O
GSH
N
O
SG
GSH
N
OH
GS
OH
N
NH2N
O
N44
N
NH2N
O
45
N
Ames Negative
Ames Positive
DNA
Figure 3.13 Circumventing metabolism-dependent genotoxicity of a pyrrolidine-substituted arylindenopyrimidine derivative 43.
the apoprotein. As such, CYP isozymes inactivated via these mechanisms are rendered
catalytically incompetent, and must be replenished by newly synthesized protein.
The phenomenon is oftentimes referred to as time-dependent inactivation,
mechanism-based inactivation, and/or suicide inactivation. Compared to reversible
CYP inhibition, drug-induced time-dependent inactivation of CYPs presents a
greater safety concern because of the increased propensity for pharmacokinetic
interactions upon multiple dosing and the sustained duration of these interactions
after discontinuation of the mechanism-based inactivator. Furthermore, depending
upon the fraction of the mechanism-based inactivator that is metabolized by the
inactivated CYP, an additional clinical consequence could involve supraproportional
increases in systemic exposure of the inactivator itself after multiple doses.
Finally, covalent modification of CYP enzymes can also lead to hapten formation
and can, in some cases, trigger an autoimmune response in susceptible patients
leading to idiosyncratic toxicity.
Identification of CYP inactivators with corresponding insights into the
inactivation mechanism(s) is required for the rational design of compounds, devoid
of the liability. Recently, several publications have demonstrated tactics to minimize
and/or eliminate time-dependent inactivation of CYP enzymes without affecting
primary pharmacology and/or disposition characteristics. Solutions range from
modulation of lipophilicity resulting in diminished rates of metabolism (and bioactivation
to RMs) to virtually eliminating the potential for RM formation via functional
group manipulations. Paroxetine (Figure 3.14) is a selective serotonin
reuptake inhibitor that inhibits CYP2D6 in a time- and concentration-dependent
fashion consistent with mechanism-based inactivation [43]. Mechanism-based
inactivation of CYP2D6 by paroxetine occurs during metabolism of the 1,3-benzdioxole
ring. Thus, hydrogen atom abstraction from the methylene carbon or elimination
of water from a hydroxymethylene intermediate generates a carbene
intermediate, which forms a MI complex with the ferrous form of the heme iron
atom in CYP2D6 resulting in a catalytically inactive enzyme (Figure 3.14) [44]. As
such, 1,3-benzdioxole ring scission by CYP2D6 also represents the major metabolic
pathway of paroxetine in humans, leading to the formation of a catechol intermediate,
which is converted to the corresponding guaiacol isomers via the action of catechol-O-methyltransferase
[45]. GSH conjugates resulting from Michael addition of
the thiol nucleophile to electrophilic ortho-quinone species have also been characterized
in HLM incubations of paroxetine, and may represent an additional pathway
of enzyme inactivation [46]. Because CYP2D6 is responsible for paroxetine
metabolism, mechanism-based inactivation of CYP2D6 activity by paroxetine is
associated with nonstationary PK in CYP2D6 extensive metabolizers [47]. Likewise,
DDIs with coadministered drugs whose clearance is mediated by CYP2D6 have
been demonstrated [48–50]. Against this backdrop, in vitro metabolism studies
with the doubly deuterated paroxetine analogue CTP-347 (Figure 3.14) demonstrated
little to no time-dependent inactivation of CYP2D6, apparently due to a dramatic
reduction in the formation of the reactive carbene intermediate [51]. CTP-347
is currently in clinical trials for the treatment of hot flashes in menopausal women.
CTP-347 has been studied in a 96-patient single- and multiple-ascending dose
56j3 Metabolic Stability and Analogue-Based Drug Discovery
3.8 Eliminating Mechanism-Based Inactivation of CYP Enzymes j57
paroxetine
+
O
HO
H3CO O
H3CO
HO
O
HO
HO
SG
O
HO
HO
SG
SG
O
N
S
SG
O
N
H
O
HOOC
COMT
human metabolites of paroxetine
CYP2D6
MI complex with CYP2D6
(enzyme inactivation)
O
O O
HO
H2O
CYP2D6
O
O O
carbene
OHO
OO
OHO
HO
paroxetine catechol
OO
O
paroxetine ortho-quinone
GSH
GSH
GSH
H
N
O
F
O
O
D
D
CTP-347
H
N
O
F
O
O
H
H
N N
NN
Fe+2
O
C
O
MI complex
Figure 3.14 Attenuating CYP mechanism-based inactivation liability through deuterium incorporation at the metabolically labile site as illustrated with the
antidepressant paroxetine.
clinical trial. The multiple-dose subjects initially received a single dose of dextromethorphan
(a probe CYP2D6 substrate), followed by 14 days of treatment with CTP-
347, and then another single dose of dextromethorphan. Subjects receiving CTP-
347 at 20–40 mg once a day (QD) retained substantially greater ability to metabolize
dextromethorphan compared to treatment with paroxetine administered at 20 mg
QD ($15–20% inhibition of CYP2D6-catalyzed dextromethorphan metabolism
to dextrorphan versus $55–60% inhibition of CYP2D6 activity with paroxetine).
As such, this DDI represents the first clinical demonstration that deuteration can
be utilized to ameliorate DDI liabilities due to mechanism-based inactivation of
CYP enzymes.
PF-562271 (Figure 3.15) is a dual inhibitor of the focal adhesion kinase and proline-rich
tyrosine kinase 2 with potential utility in the treatment of ovarian cancer
and osteoporosis [52]. In vitro metabolism studies in HLM revealed that PF-562271
is a CYP3A4 substrate and a time-dependent inactivator suggesting that the agent
could inhibit its own clearance mechanism in vivo. The mechanism of CYP3A4
inactivation was shown to occur via a two-electron oxidation of the dianiline motif
to a reactive diimine species, which was trapped with GSH (Figure 3.15) [53]. Furthermore,
PF-562271 demonstrated supraproportional increases in oral systemic
exposure upon repeated dosing in clinical trials, a phenomenon that was consistent
with self-inactivation of its clearance mechanism. Likewise, PF-562271 also caused
greater than threefold increase in the systemic exposure of the probe CYP3A4 substrate
midazolam in a clinical DDI study. Insights into the mechanism of CYP3A4
inactivation allowed a rational medicinal chemistry strategy to reduce/eliminate
enzyme inactivation liability [53]. Thus, simply exchanging the 5-aminooxindole
nitrogen and carbonyl groups in the parent compound afforded analogues (e.g., 46
N
NHN
NH
O
CF3
NH
N
N
S
CH3
O O
N
NHN
CF3
NH N
S
CH3
O O
S
CH3
N
NHN
CF3
NH
N
N
N
S
CH3
O O
HN
O
PF-0562271
CYP3A4
N
NN
N
O
CF3
NH
N
N
S
CH3
O O
diimine
GSH
N
NHN
NH
O
CF3
NH
N
N
S
CH3
O O
SG
CYP3A4 inactivator
46 47
devoid of CYP3A4 inactivation
devoid of RM formation
O
O
Figure 3.15 Strategies to eliminate CYP3A4 time-dependent inactivation by virtue of eliminating
reactive diimine formation with oxindole-based focal adhesion kinase inhibitors.
58j3 Metabolic Stability and Analogue-Based Drug Discovery
and 47) that were virtually devoid of CYP3A4 inactivation, while maintaining
kinase potency. In general, disruption of the electron-rich dianiline structure present
in PF-562271 also mitigated the formation of reactive diimine metabolites in
the course of SAR studies [53].
In the course of SAR studies on a series of 1-amino-1,2,3,4-tetrahydronaphthalene-2-carboxylic
acid derivatives as selective agonists for the human melanocortin-
4 receptor and potential anti-obesity agents, lead compound 48 (Figure 3.16) demonstrated
potent time- and concentration-dependent inactivation of CYP3A4 in a
NADPH-dependent fashion, which was consistent with mechanism-based
inactivation [54, 55]. Upon concomitant administration with CYP3A4 substrates in
the clinic, increases in the area under the plasma concentration versus time curve
for the victim drugs were expected to range from 4- to 10-fold as a result of the
inactivation. The projected degree of clinical DDI was considered to be
unacceptable for an anti-obesity indication and, as a result, 48 was eliminated from
consideration as a development candidate. Studies were undertaken to understand
the mechanism underlying 48-mediated CYP3A4 inactivation to guide medicinal
chemistry efforts. UV spectral analysis of incubations of 48 with recombinant
CYP3A4 provided evidence for the formation of an MI complex between heme and
NH2
H
N
O
Cl
ON
NHO
NH2
H
N
O
Cl
ON
NHO
48 51
N
N
O
R
R =
H
N
O
Cl
ON
NHO
R
NH2
48
CYP3A4
R
HN
OH
CYP3A4
R
N
O
R
N
OH
nitroso intermediate
oxime
[O]
R
N
O
nitroso carbocation
CN
50
49
CYP3A4 inactivator devoid of CYP3A4 inactivation
Figure 3.16 A strategy to eliminate CYP3A4 MBI liability in the selective melanocortin-4 receptor
agonist 48.
3.8 Eliminating Mechanism-Based Inactivation of CYP Enzymes j59
a RM of 48. The RM was proposed to be the nitroso intermediate 49, which is
obtained from a two-electron oxidation of the initially formed hydroxylamine
metabolite (Figure 3.16). Characterization of a geminal cyanonitroso adduct 50 in
incubations of 48 in NADPH- and potassium cyanide-supplemented HLM was consistent
with the proposed bioactivation pathway, wherein the nitroso intermediate
would exist in a tautomeric equilibrium with the corresponding oxime derivative,
which could be oxidized to an electrophilic nitroso carbocation and trapped by a
cyanide ion [55]. Based on the insights gained from metabolism studies, it was
envisioned that introducing an alkyl group a to the basic amine motif would shield
this functionality from oxidation through steric hindrance, thereby reducing the
probability of MI complex formation and CYP3A4 inactivation. Indeed analogue 51
(Figure 3.16) was synthesized and shown to be devoid of CYP3A4 inactivation
while retaining the potent and selective melanocortin-4 receptor agonism observed
with 48 [55].
3.9
Identification (and Elimination) of Electrophilic Lead Chemical Matter
Innately electrophilic compounds represent a significant liability in drug discovery
because they often possess chemical reactivity similar to affinity labels
(e.g., alkyl halides, Michael acceptors, and N-substituted maleimides); such
compounds can alkylate proteins, DNA, and/or GSH, leading to toxicity.
Because of the potential safety concerns, electrophilic functional groups are
generally avoided in drug design. Examination of the medicinal chemistry literature,
however, reveals several examples of seemingly “chemically inert” compounds,
which are prone to nucleophilic displacement by GSH under
nonenzymatic (pH 7.4, phosphate buffer, 37 
C) and/or enzymatic conditions.
In the case of enzyme-assisted reactions, GSH conjugation to electrophilic centers
is mediated by microsomal, cytosolic, and/or mitochondrial glutathione Stransferases.
RM trapping studies have been effectively used to identify intrinsically
electrophilic compounds in discovery programs. An example was
recently disclosed with the oncology clinical candidate AMG458 (Figure 3.17),
which covalently adducted to liver microsomal proteins in the absence of
NADPH [56]. Subsequent incubations of AMG458 with GSH and N-acetylcysteine
led to the identification of thiol conjugates derived from the nucleophilic
displacement of an unanticipated electrophilic site on the molecule. Likewise,
in the glucokinase activator program for treatment of type 2 diabetes, a unique
metabolic liability of the 4-sulfonyl-2-pyridone ring system (e.g., compound 52,
Figure 3.17) was identified, wherein the heterocycle readily underwent conjugation
with GSH under nonenzymatic as well as enzyme-catalyzed conditions
[57, 58]. Identification of the electrophilic nature of 4-sulfonyl-2-pyridone
motif prevented further investment in this series, and instead led to efforts
centered on the optimization of a series of benzofuran derivatives, from which
emerged the clinical candidate 53 (see Figure 3.17) [59].
60j3 Metabolic Stability and Analogue-Based Drug Discovery
3.10
Mitigating Risks of Idiosyncratic Toxicity via Elimination of RM Formation
While the potential for DDI and genotoxicity can be examined directly from in vitro
assays, the same does not often hold true for immune-mediated IADRs (e.g., hepatotoxicity,
skin rashes, agranulocytosis, and aplastic anemia) in drug-treated
patients. IADRs are unrelated to known drug pharmacology, and are generally dose
independent (a notable exception is the dose-dependent hepatotoxicity caused by
acetaminophen). Because the incidence of IADRs is very low (1 in 10 000 to 1 in
100 000), these reactions are often not detected, until the drug has gained broad
exposure in a large patient population. The concept of human drug-induced hepatotoxicity
via RM formation was provided from studies on the anti-inflammatory
agent and hepatotoxin acetaminophen [60]. Mechanistic studies that established
NH3CO
O
N
H
N
O
N
N
CH3
O
OH
AMG458
NH3CO
SR
N
OS
H3C
O O
H
N
O
N
N
CH3
52
N
OSG
H
N
O
N
N
CH3
GSH N
O
S
SG
H3C
O O
H3C S
OH
O
Meisenheimer complex
+
HO
N
H
N
O
N
N
CH3
O
OHRSH
R (glutathione) =
R (N-acetylcysteine) =
H
N
NH
O
HOOC
O
COOH
NH2
H
N
O
OH
O
CH3
H
N
O
N
N
CH3
O
H3C
O
N
N
N
O
H3C
CH3
53
Clinical candidate
Figure 3.17 Identification of innately electrophilic lead chemical matter in drug discovery.
3.10 Mitigating Risks of Idiosyncratic Toxicity via Elimination of RM Formation j61
the CYP-mediated oxidation of acetaminophen to a reactive quinone imine intermediate
NAPQI [61], capable of depleting GSH levels and/or binding covalently to
liver macromolecules, have served as a paradigm for drug toxicity assessment over
the decades.
An understanding of the biochemical basis for drug toxicity has aided to replace
the vague perception of a chemical class effect with a sharper picture of individual
molecular peculiarity. There are several instances of prototype drugs associated
with IADRs that also form RM(s) and elimination of RM liability in follow-on successor
agent(s) markedly improves the safety profile. Although the evidence is most
often anecdotal in nature, a compelling case for chemotype-based toxicity can be
inferred from such structure–toxicity analyses [62]. For instance, while clozapine
use is limited by a high incidence of agranulocytosis, hepatotoxicity, and myocarditis,
quetiapine and loxapine are not associated with these adverse events. Clozapine
exhibits covalent binding to human neutrophil proteins in vitro, via myeloperoxidase-mediated
oxidation of the dibenzodiazepine ring to a reactive iminium ion,
which covalently binds to the target tissues and also reacts with GSH (Figure
3.18) [63]. Proteins covalently modified with clozapine have been observed in
neutrophils of patients being treated with clozapine, which reaffirms the relevance
of the in vitro studies [64]. In contrast, quetiapine and loxapine cannot form a
reactive iminium [65], since the bridging nitrogen atom is replaced with a sulfur or
oxygen atom, respectively (Figure 3.18). In the case of the nonsteroidal anti-inflammatory
drug sudoxicam, hepatotoxicity observed in the clinic that led to its suspension
in clinical trials has been attributed to thiazole ring scission yielding a reactive
acylthiourea metabolite capable of oxidizing GSH and proteins (Figure 3.18) [66].
The structurally related anti-inflammatory drug meloxicam does not possess the
hepatotoxic liability associated with sudoxicam. Although introduction of a methyl
group at the C-5 position on the thiazole ring in meloxicam is the only structural
difference, the change dramatically alters the metabolic profile such that oxidation
of the C-5 methyl group to the alcohol (and carboxylic acid) metabolite(s) constitutes
the principal metabolic fate of meloxicam in humans (Figure 3.18).
Because of the inability to predict and quantitate the risk of IADRs and because
RMs as opposed to the parent molecules from which they are derived are thought
to be responsible for the pathogenesis of many IADRs, detection of RM in the
course of metabolism is considered to be an undesirable trait in preclinical drug
discovery.
3.11
Case Studies on Elimination of RM Liability in Drug Discovery
Examples of medicinal chemistry tactics to eliminate RM formation are abundant
in the primary literature. A recent example, which focuses on eliminating RM liability
within a lead chemical series, is evident with pyrazinone-based corticotropinreleasing
factor-1 receptor antagonists and potential antidepressant/anti-anxiolytic
drugs [67]. A major portion of the clearance mechanism of the lead pyrazinone 54
62j3 Metabolic Stability and Analogue-Based Drug Discovery
3.11 Case Studies on Elimination of RM Liability in Drug Discovery j63
Loxapine:
X
N
N
N
R1
R2
X = O; R1 = CH3; R2 = Cl
Quetiapine: X = S; R1 = (CH2)2O(CH2)2OH; R2 = H
N
H
N
N
N
CH3
Cl
CYP or peroxidase
N
N
N
N
CH3
Cl GSH
N
H
N
N
N
CH3
Cl
SG
S
N
O O
OH
CH3
O
N
H
S
N
Clozapine iminium
Sudoxicam
CYP
S
N
O O
OH
CH3
O
N
H
S
N O
S
N
O O
OH
CH3
O
N
H
S
N
OH
OH
O
O
S
N
O O
OH
CH3
O
N
H
NH2
S
acylthiourea
epoxide
S
N
O O
OH
CH3
O
N
H
S
N
Meloxicam
CH3
S
N
O O
OH
CH3
O
N
H
S
N
OH
S
N
O O
OH
CH3
O
N
H
S
N
OH
O
CYP
Figure 3.18 Structure–toxicity relationships for idiosyncratic drug toxicity.
in rats comprised of conjugation with GSH, consistent with RM formation in
vivo [68]. Two distinct metabolic activation pathways were deciphered (Figure 3.19):
(a) oxidative metabolism on the chloropyrazinone ring to yield an electrophilic
epoxide and (b) O-dealkylation of the difluoromethylphenoxy moiety to yield a phenol
metabolite 55, two electrons of which generated a reactive quinone imine species.
To eliminate RM formation on the 2,6-dichloroaniline motif, the pyridyl group
was incorporated as a bioisosteric replacement. To prevent epoxidation on the 5chloropyrazinone
ring, the chlorine atom was replaced with more strongly electron-withdrawing
groups (e.g., cyano). Out of this iterative medicinal chemistry
exercise emerged 56 (Figure 3.19) with sufficiently diminished RM formation in
liver microsomes from rats and humans. Consistent with the in vitro finding, <2–
4% of GSH conjugates were recovered in rat bile following in vivo administration
of 56. Compound 56 also retained all of the primary pharmacology and pharmacokinetic
properties of 54.
A second example is depicted with nonpeptidyl thrombopoietin receptor agonists
exemplified by 57 [69]. In the course of routine metabolite identification studies, it
was found that 4-(4-fluoro-3-(trifluoromethyl)phenyl)thiazol-2-amine (58), the product
of carboxylesterase-mediated hydrolysis of 57, formed RMs in HLM. GSH trapping
studies led to the formation of two conjugates derived from the addition of the
thiol nucleophile to 58 and a thiazole-S-oxide metabolite of 58 (i.e., compound 59)
(Figure 3.20). Mass spectral fragmentation and 1
H NMR analysis indicated that the
site of attachment of the GSH moiety in both conjugates was the C-5 position in the
thiazole ring. Based on the structures of the GSH conjugates, two metabolic activation
pathways were proposed, one involving b-elimination of an initially formed
hydroxylamine metabolite and the other involving direct two-electron oxidation of
the electron-rich 2-aminothiazole system to RM. The mechanistic insights gained
from the metabolism studies influenced subsequent medicinal chemistry strategies,
which involved blocking the C-5 position with a fluorine atom (compound 60)
N
N O
NHNC
N
H3C
CH3
O
F
F
O
56
N
N O
NHCl
Cl
O
F
F
54
Cl
CYP
N
N O
N
H
Cl
O
epoxide
GSH
N
N O
N
H
Cl
SG
N
N O
NHCl
Cl
OH
55
Cl
N
Cl
O
Cl
quinone imine
NH
Cl
OH
Cl
SG
Bioactivation
Minimized
GSH
Figure 3.19 Eliminating RM liability with a series of pyrazinone-based corticotropin-releasing
factor-1 receptor antagonists.
64j3 Metabolic Stability and Analogue-Based Drug Discovery
or replacing the thiazole ring with a 1,2,4-thiadiazole group (compound 61) (Figure
3.20). These structural changes not only abrogated RM liability associated
with 58 but also resulted in compounds that retained the attractive pharmacological
and pharmacokinetic attributes of the prototype agent. Compound 60 was considered
for further preclinical profiling as a clinical candidate [70].
Strategies toward eliminating metabolic activation of the electron-rich 4-hydroxyaniline
motif have also been described by Park and coworkers with the antimalarial
agent amodiaquine. The clinical use of amodiaquine is somewhat restricted by
several cases of hepatotoxicity and agranulocytosis; the detection of IgG antibodies
in patients exposed to the drug is consistent with an immune-mediated hypersensitivity
reaction [71]. The immune-mediated toxicity is believed to arise from
the metabolism of amodiaquine to a reactive quinone imine species that can covalently
bind to cellular proteins or GSH (Figure 3.21) [72, 73]. Exchanging the
C0
-4-phenolic OH group with a fluorine atom results in compound 62 that prevents
RM formation (Figure 3.21) [74]. An alternate approach to prevent RM formation
involved the isomerization of the 30
- and 40
-substituents in amodiaquine; from this
exercise emerged analogues 63 and 64, which were devoid of RM formation
(judged from the lack of formation of GSH conjugates) and were advanced to further
profiling as the next generation of antimalarials based on their favorable pharmacokinetic
and pharmacodynamic attributes (Figure 3.21) [75, 76].
N
HOOC
N
Cl
N
H
O
N
S
F
CF3
57
Esterases
H2N N
S
F
CF3
N
HOOC
N
Cl
O
OH
58
H2N N
S
F
CF3O
GSH GSH
H2N N
S
F
CF3
SG
H2N N
S
F
CF3
SG
O
CYP
59
N
HOOC
N
Cl
N
H
O
N
S
F
CF3
60
F
N
HOOC
N
Cl
N
H
O
N
NS
F
CF3
61
Devoid of RM formation
Figure 3.20 Eliminating RM liability with acetamido-thiazolyl nonpeptidyl thrombopoietin
receptor agonists.
3.11 Case Studies on Elimination of RM Liability in Drug Discovery j65
In the course of discovery efforts leading to the discovery of taranabant (Figure
3.22), a selective and potent inhibitor of the cannabinoid-1 (CB-1) receptor, and
phase III clinical candidate for the treatment of obesity, lead compound 65 (Figure
3.22) revealed a high level of covalent binding to HLM in a NADPH-dependent
fashion, consistent with RM formation. Elucidation of the structure of the GSH
conjugate suggested that the RM was an arene oxide intermediate derived from
epoxidation of the electron-rich phenoxy ring [77]. Replacement of the phenoxy
ring with the trifluoromethylpyridyl ring afforded taranabant, which was devoid of
RM formation, while retaining potency against the CB-1 receptor.
The final illustration concerns the antiepileptic drug felbamate, which is associated
with several cases of aplastic anemia and hepatotoxicity [78]. Evidence has
been presented that links felbamate-induced toxicity to its metabolism to the highly
reactive a,b-unsaturated carbonyl compound atropaldehyde (2-phenylpropenal, 68)
(Figure 3.23) [79, 80]. The rate-limiting step for the formation of 68 involves amidase-mediated
hydrolysis of one of the carbamate groups in felbamate to yield the
corresponding primary alcohol 66. Alcohol dehydrogenase-catalyzed oxidation
of 66 yields the aldehyde intermediate 67, which undergoes b-elimination to
yield 68, carbon dioxide, and ammonia. Compound 68, being a a,b-unsaturated
carbonyl derivative, readily reacts with GSH to afford conjugate 69. Downstream
NCl
HN
OH
(H3CH2C)2N
CYP
NCl
N
O
(H3CH2C)2N
quinone imineAmodiaquine
NCl
HN
R1
62: R1 = F; R2 = CH2N(CH2CH3)2
63: R1 = CH2NHC(CH3)3; R2 = OH
64: R1 = F; R2 = CH2NHC(CH3)3
R2
Devoid of RM formation
Figure 3.21 Metabolism-directed efforts in the discovery of amodiaquine backup clinical
candidates.
66j3 Metabolic Stability and Analogue-Based Drug Discovery
mercapturic acid conjugates (breakdown products of GSH such as cysteine and
N-acetylcysteine) of 68 have been detected in urine from patients treated with felbamate
that supports the mechanistic pathway [81]. The mechanistic information on
felbamate bioactivation to the electrophilic species 68 has been utilized to design
fluorofelbamate, which cannot undergo the b-elimination chemistry to the reactive
a,b-unsaturated carbonyl intermediate because of the replacement of the acidic
benzylic hydrogen with a fluorine atom (see Figure 3.23) [82].
3.12
Concluding Remarks
It is clear from our own experience and from the general literature that the application
of metabolism studies and concepts has been readily embraced by the pharmaceutical
industry as an approach to mitigating attrition due to poor
pharmacokinetics [83]. The real value of metabolism studies in drug discovery lies
N
H
CH3
Cl
O
O
CH3
CH3
65
RM positive (arene oxide)
Covalent binding to HLM
N
H
CH3
Cl
O
O
N
CH3
CH3
Taranabant
CB-1 IC50 = 0.3 nMCB-1 IC50 = 2.0 nM
RM negative
No covalent binding to HLM
CF3
CN
Figure 3.22 Elimination of RM liability in the discovery of taranabant, a selective and potent CB-1
receptor antagonist and anti-obesity agent.
O
NH2O
O
O
NH2
Felbamate
Amidases
H2O
HO
O
O
NH2
Alcohol
Dehydrogenase
O
O
O
NH2
H
O
O
O
NH2
Keto-enol tautomerism
O
H
H
GSH
66
68
O
NH2O
O
O
NH2
Fluorofelbamate
F
H
67
O H
69
SG
CO2, NH3
Figure 3.23 Designing a felbamate analogue that is devoid of RM formation.
3.12 Concluding Remarks j67
in the identification of relevant clearance mechanisms for lead chemical matter,
which then dictates the choice of in vitro models for making projections of human
pharmacokinetics prior to first-in-human testing. Similarly, the value in identifying
metabolic hot spots is not the structural characterization itself, but in enabling the
understanding of SAR. Recently, much effort has gone into understanding RM,
and although the link between metabolism and safety is elusive, it remains prudent
to continue to explore these relationships as early as possible in drug development,
if for no other reason than to give chemists the option of pursuing alternate chemical
series devoid of such concerns if possible.
References
1 Riley, R.J. (2001) The potential
pharmacological and toxicological impact
of P450 screening. Curr. Opin. Drug
Discov. Dev., 4 (1), 45–54.
2 Pryde, D.C., Dalvie, D., Hu, Q., Jones, P.,
Obach, R.S., and Tran, T.D. (2010)
Aldehyde oxidase: an enzyme of emerging
importance in drug discovery. J. Med.
Chem., 53 (24), 8441–8460.
3 Kalgutkar, A.S., Dalvie, D.K., Castagnoli,
N., Jr., and Taylor, T.J. (2001) Interactions
of nitrogen-containing xenobiotics with
monoamine oxidase (MAO) isozymes A
and B: SAR studies on MAO substrates
and inhibitors. Chem. Res. Toxicol., 14 (9),
1139–1162.
4 Bu, H.Z. (2006) A literature review of
enzyme kinetic parameters for CYP3A4mediated
metabolic reactions of 113
drugs in human liver microsomes:
structure–kinetics relationship
assessment. Curr. Drug Metab., 7 (3),
231–249.
5 Stepan, A.F., Karki, K., McDonald, W.S.,
Dorff, P.H., Dutra, J.K., Dirico, K.J., Won,
A., Subramanyam, C., Efremov, I.V.,
O’Donnell, C.J., Nolan, C.E., Becker, S.L.,
Pustilnik, L.R., Sneed, B., Sun, H., Lu, Y.,
Robshaw, A.E., Riddell, D., O’Sullivan,
T.J., Sibley, E., Capetta, S., Atchison, K.,
Hallgren, A.J., Miller, E., Wood, A., and
Obach, R.S. (2011) Metabolism-directed
design of oxetane-containing
arylsulfonamide derivatives as c-secretase
inhibitors. J. Med. Chem., 54 (22),
7772–7783.
6 Cheng, Y., Zhang, F., Rano, T.A., Lu, Z.,
Scheif, W.A., Gabryelski, L., Olsen, D.B.,
Stahlhut, M., Rutkowski, C.A., Lin, J.H.,
Jin, L., Emini, E.A., Chapman, K.T., and
Tata, J.R. (2002) Indinavir analogues with
blocked metabolism sites as HIV protease
inhibitors with improved pharmacological
profiles and high potency against PIresistant
viral strains. Bioorg. Med. Chem.
Lett., 12 (17), 2419–2422.
7 Floyd, D.M., Kimball, S.D., Krapcho, J.,
Das, J., Turk, C.F., Moquin, R.V., Lago, M.
W., Duff, K.J., Lee, V.G., White, R.E.,
Ridgewell, R.E., Moreland, S., Brittain,
R.J., Normandin, D.E., Hedberg, S.A., and
Cucinotta, G.C. (1992) Benzazepinone
calcium channel blockers. 2. Structure–
activity and drug metabolism studies
leading to potent antihypertensive agents.
Comparison with benzothiazepinones. J.
Med. Chem., 35 (4), 756–772.
8 Park, B.K., Kitteringham, N.R., and
O’Neill, P.M. (2001) Metabolism of
fluorine-containing drugs. Annu. Rev.
Pharmacol. Toxicol., 41, 443–470.
9 Fisher, M.B., Henne, K.R., and Boer, J.
(2006) The complexities inherent in
attempts to decrease drug clearance by
blocking sites of CYP-mediated
metabolism. Curr. Opin. Drug Discov. Dev.,
9 (1), 101–109.
10 Moorjani, M., Luo, Z., Lin, E., Vong, B.G.,
Chen, Y., Zhang, X., Rueter, J.K., Gross,
R.S., Lanier, M.C., Tellew, J.E., Williams,
J.P., Lechner, S.M., Malany, S., Santos, M.,
Crespo, M.I., Dõaz, J.-L., Saunders, J., and
Slee, D.H. (2008) 2,6-Diaryl-4acylaminopyrimidines
as potent and
selective adenosine A2A antagonists with
improved solubility and metabolic
68j3 Metabolic Stability and Analogue-Based Drug Discovery
stability. Bioorg. Med. Chem. Lett., 18 (20),
5402–5405.
11 Eldrup, A.B., Soleymanzadeh, F., Taylor,
S.J., Muegge, I., Farrow, N.A., Joseph, D.,
McKellop, K., Man, C.C., Kukulka, A., and
De Lombaert, S. (2009) Structure-based
optimization of arylamides as inhibitors of
soluble epoxide hydrolase. J. Med. Chem.,
52 (19), 5880–5895.
12 Burgey, C.S., Robinson, K.A., Lyle, T.A.,
Sanderson, P.E.J., Lewis, S.D., Lucas, B.J.,
Krueger, J.A., Singh, R., Miller-Stein, C.,
White, R.A., Wong, B., Lyle, E.A.,
Williams, P.D., Coburn, C.A., Dorsey, B.
D., Barrow, J.C., Stranieri, M.T., Holahan,
M.A., Sitko, G.R., Cook, J.J., McMasters,
D.R., McDonough, C.M., Sanders, W.M.,
Wallace, A.A., Clayton, F.C., Bohn, D.,
Leonard, Y.M., Detwiler, T.J., Jr., Lynch,
J.J., Jr., Yan, Y., Chen, Z., Kuo, L., Gardell,
S.J., Shafer, J.A., and Vacca, J.P. (2003)
Metabolism-directed optimization of 3aminopyrazinone
acetamide thrombin
inhibitors. Development of an orally
bioavailable series containing P1 and P3
pyridines. J. Med. Chem., 46 (4), 461–473.
13 Dugar, S., Yumibe, N., Clader, J.W.,
Vizziano, M., Huie, K., Van Heek, M.,
Compton, D.S., and Davis, H.R., Jr. (1996)
Metabolism and structure activity data
based drug design: discovery of
(À)-SCH53079, an analog of the potent
cholesterol absorption inhibitor
(À)-SCH48461. Bioorg. Med. Chem. Lett., 6
(11), 1271–1274.
14 Kirkup, M.P., Rizvi, R., Shanker, B.B.,
Dugar, S., Clader, J.W., McCombie, S.W.,
Lin, S.-I., Yumibe, N., Huie, K., Van Heek,
M., Compton, D.S., Davis, H.R., Jr., and
McPhail, A.T. (1996) (À)-SCH 57939:
synthesis and pharmacological properties
of a potent, metabolically stable
cholesterol absorption inhibitor. Bioorg.
Med. Chem. Lett., 6 (17), 2069–2072.
15 Rosenblum, S.B., Huynh, T., Afonso, A.,
Davis, H.R., Jr., Yumibe, N., Clader, J.W.,
and Burnett, D.A. (1998) Discovery of 1-(4-
fluorophenyl)-(3R)-[3-(4-fluorophenyl)-
(3S)-hydroxypropyl]-(4S)-(4hydroxyphenyl)-2-azetidinone
(SCH
58235): a designed, potent, orally active
inhibitor of cholesterol absorption. J. Med.
Chem., 41 (6), 973–980.
16 Penning, T.D., Talley, J.J., Bertenshaw,
S.R., Carter, J.S., Collins, P.W., Docter, S.,
Graneto, M.J., Lee, L.F., Malecha, J.W.,
Rogers, R.S., Rogier, D.J., Yu, S.S.,
Anderson, G.D., Burton, E.G., Cogburn,
J.N., Gregory, S.A., Koboldt, C.M.,
Perkins, W.E., Seibert, K., Veenhuizen, A.
W., Zhang, Y.Y., and Isakson, P.C. (1997)
Synthesis and biological evaluation of the
1,5-diarylpyrazole class of cyclooxygenase-
2 inhibitors: identification of 4-[5-(4-
methylphenyl)-3-(trifluoromethyl)-1Hpyrazol-1-yl]benzenesulfonamide
(SC-
58635, celecoxib). J. Med. Chem., 40 (9),
1347–1365.
17 Southers, J.A., Bauman, J.N., Price, D.A.,
Humphries, P.S., Balan, G., Sagal, J.F.,
Maurer, T.S., Zhang, Y., Oliver, R., Herr,
M., Healy, D.R., Li, M., Kapinos, B., Fate,
G.D., Riccardi, K.A., Paralkar, V.M.,
Brown, T.A., and Kalgutkar, A.S. (2010)
Metabolism-guided design of short-acting
calcium-sensing receptor antagonists.
ACS Med. Chem. Lett., 1, 219–223.
18 Middleton, D.S., Andrews, M., Glossop,
P., Gymer, G., Hepworth, D., Jessiman,
A., Johnson, P.S., MacKenny, M., Stobie,
A., Tang, K., Morgan, P., and Jones, B.
(2008) Designing rapid onset selective
serotonin re-uptake inhibitors. Part 3.
Site-directed metabolism as a strategy to
avoid active circulating metabolites:
structure–activity relationships of
(thioalkyl)phenoxy benzylamines. Bioorg.
Med. Chem. Lett., 18 (19), 5303–5306.
19 Magee, T.V., Ripp, S.L., Li, B., Buzon,
R.A., Chupak, L., Dougherty, T.J., Finegan,
S.M., Girard, D., Hagen, A.E., Falcone,
M.J., Farley, K.A., Granskog, K., Hardink,
J.R., Huband, M.D., Kamicker, B.J.,
Kaneko, T., Knickerbocker, M.J., Liras,
J.L., Marra, A., Medina, I., Nguyen, T.T.,
Noe, M.C., Obach, R.S., O’Donnell, J.P.,
Penzien, J.B., Reilly, U.D., Schafer, J.R.,
Shen, Y., Stone, G.G., Strelevitz, T.J., Sun,
J., Tait-Kamradt, A., Vaz, A.D., Whipple,
D.A., Widlicka, D.W., Wishka, D.G.,
Wolkowski, J.P., and Flanagan, M.E.
(2009) Discovery of azetidinyl ketolides for
the treatment of susceptible and
multidrug resistant community-acquired
respiratory tract infections. J. Med. Chem.,
52 (23), 7446–7457.
References j69
20 Linton, A., Kang, P., Ornelas, M., Kephart,
S., Hu, Q., Pairish, M., Jiang, Y., and Guo,
C. (2011) Systematic structure
modifications of imidazo[1,2-a]pyrimidine
to reduce metabolism mediated by
aldehyde oxidase (AO). J. Med. Chem., 54
(21), 7705–7712.
21 Dixon, C.M., Park, G.R., and Tarbit, M.H.
(1994) Characterization of the enzyme
responsible for the metabolism of
sumatriptan in human liver. Biochem.
Pharmacol., 47 (7), 1253–1257.
22 McEnroe, J.D., and Fleishaker, J.C. (2005)
Clinical pharmacokinetics of almotriptan,
a serotonin 5-HT(1B/1D) receptor agonist
for the treatment of migraine. Clin.
Pharmacokinet., 44 (3), 237–246.
23 Vyas, K.P., Halpin, R.A., Geer, L.A., Ellis,
J.D., Liu, L., Cheng, H., Chavez-Eng, C.,
Matuszewski, B.K., Varga, S.L., Guiblin,
A.R., and Rogers, J.D. (2000) Disposition
and pharmacokinetics of the antimigraine
drug, rizatriptan, in humans. Drug Metab.
Dispos., 28 (1), 89–95.
24 Wild, M.J., McKillop, D., and Butters, C.J.
(1999) Determination of the human
cytochrome P450 isoforms involved in the
metabolism of zolmitriptan. Xenobiotica,
29 (8), 847–857.
25 Evans, D.C., O’Conner, D., Lake, B.G.,
Evers, R., Allen, C., and Hargreaves, R.
(2003) Eletriptan metabolism by human
hepatic CYP450 enzymes and transport by
human P-glycoprotein. Drug Metab.
Dispos., 31 (7), 861–869.
26 Buchan, P., Keywood, C., Wade, A., and
Ward, C. (2002) Clinical pharmacokinetics
of frovatriptan. Headache, 42 (Suppl. 2),
S54–S62.
27 Tfelt-Hansen, P., De Vries, P., and Saxena,
P.R. (2000) Triptans in migraine: a
comparative review of pharmacology,
pharmacokinetics and efficacy. Drugs, 60
(6), 1259–1287.
28 Fisher, M.B., Paine, M.F., Strelevitz, T.J.,
and Wrighton, S.A. (2001) The role of
hepatic and extrahepatic UDPglucuronosyltransferases
in human drug
metabolism. Drug Metab. Rev., 33 (3–4),
273–297.
29 Lindsay, J., Wang, L.L., Li, Y., and Zhou,
S.F. (2008) Structure, function and
polymorphism of human cytosolic
sulfotransferases. Curr. Drug Metab., 9 (2),
99–105.
30 Honma, W., Shimada, M., Sasano, H.,
Ozawa, S., Miyata, M., Nagata, K., Ikeda,
T., and Yamazoe, Y. (2002) Phenol
sulfotransferase, ST1A3, as the main
enzyme catalyzing sulfation of
troglitazone in human liver. Drug Metab.
Dispos., 30 (8), 944–949.
31 Borzilleri, R.M., Cai, Z.W., Ellis, C.,
Fargnoli, J., Fura, A., Gerhardt, T., Goyal,
B., Hunt, J.T., Mortillo, S., Qian, L.,
Tokarski, J., Vyas, V., Wautlet, B., Zheng,
X., and Bhide, R.S. (2005) Synthesis and
SAR of 4-(3-hydroxyphenylamino)pyrrolo
[2,1-f][1,2,4]triazine based VEGFR-2
kinase inhibitors. Bioorg. Med. Chem. Lett.,
15 (5), 1429–1433.
32 Stewart, A.O., Bhatia, P.A., Martin, J.G.,
Summers, J.B., Rodriques, K.E., Martin,
M.B., Holms, J.H., Moore, J.L., Craig, R.
A., Kolasa, T., Ratajczyk, J.D., Mazdiyasni,
H., Kerdesky, F.A., DeNinno, S.L., Maki,
R.G., Bouska, J.B., Young, P.R., Lanni, C.,
Bell, R.L., Carter, G.W., and Brooks, C.D.
(1997) Structure–activity relationships of
N-hydroxyurea 5-lipoxygenase inhibitors.
J. Med. Chem., 40 (13), 1955–1968.
33 Awni, W.M., Braeckman, R.A.,
Granneman, G.R., Witt, G., and Dube,
L.M. (1995) Pharmacokinetics and
pharmacodynamics of zileuton after oral
administration of single and multiple
dose regimens of zileuton 600 mg in
healthy volunteers. Clin. Pharmacokinet.,
29 (Suppl. 2), 22–33.
34 Dobo, K.L., Obach, R.S., Luffer-Atlas, D.,
and Bercu, J.P. (2009) A strategy for the
risk assessment of human genotoxic
metabolites. Chem. Res. Toxicol., 22 (2),
348–356.
35 Kalgutkar, A.S., Obach, R.S., and Maurer,
T.S. (2007) Mechanism-based inactivation
of cytochrome P450 enzymes: chemical
mechanisms, structure–activity
relationships and relationship to clinical
drug–drug interactions and idiosyncratic
adverse drug reactions. Curr. Drug Metab.,
8 (5), 407–447.
36 Stepan, A.F., Walker, D.P., Bauman, J.,
Price, D.A., Baillie, T.A., Kalgutkar, A.S.,
and Aleo, M.D. (2011) Structural
alert/reactive metabolite concept as
70j3 Metabolic Stability and Analogue-Based Drug Discovery
applied in medicinal chemistry to mitigate
the risk of idiosyncratic drug toxicity: a
perspective based on the critical
examination of trends in the top 200 drugs
marketed in the United States. Chem. Res.
Toxicol., 24 (9), 1345–1410.
37 Park, B.K., Boobis, A., Clarke, S.,
Goldring, C.E., Jones, D., Kenna, J.G.,
Lambert, C., Laverty, H.G., Naisbitt, D.J.,
Nelson, S., Nicoll-Griffith, D.A., Obach, R.
S., Routledge, P., Smith, D.A., Tweedie, D.
J., Vermeulen, N., Williams, D.P., Wilson,
I.D., and Baillie, T.A. (2011) Managing the
challenge of chemical reactive metabolites
in drug development. Nat. Rev. Drug
Discov., 10 (4), 292–306.
38 Kumar, S., Kassahun, K., Tschirret-Guth,
R.A., Mitra, K., and Baillie, T.A. (2008)
Minimizing metabolic activation during
pharmaceutical lead optimization:
progress, knowledge gaps and future
directions. Curr. Opin. Drug Discov. Dev.,
11 (1), 43–52.
39 Guengerich, F.P. and MacDonald, J.S.
(2007) Applying mechanisms of chemical
toxicity to predict drug safety. Chem. Res.
Toxicol., 20 (3), 344–369.
40 Kalgutkar, A.S., Dalvie, D.K., Aubrecht, J.,
Smith, E.B., Coffing, S.L., Cheung, J.R.,
Vage, C., Lame, M.E., Chiang, P.,
McClure, K.F., Maurer, T.S., Coelho, R.V.,
Jr., Soliman, V.F., and Schildknegt, K.
(2007) Genotoxicity of 2-(3-
chlorobenzyloxy)-6-(piperazinyl)pyrazine,
a novel 5-hydroxytryptamine 2C receptor
agonist for the treatment of obesity: role of
metabolic activation. Drug Metab. Dispos.,
35 (6), 848–858.
41 Kalgutkar, A.S., Bauman, J.N., McClure,
K.F., Aubrecht, J., Cortina, S.R., and
Paralkar, J. (2009) Biochemical basis for
differences in metabolism-dependent
genotoxicity by two diazinylpiperazinebased
5-HT2C receptor agonists. Bioorg.
Med. Chem. Lett., 19 (6), 1559–1563.
42 Lim, H.-K., Chen, J., Sensenhauser, C.,
Cook, K., Preston, R., Thomas, T., Shook,
B., Jackson, P.F., Rassnick, S., Rhodes, K.,
Gopaul, V., Salter, R., Silva, J., and Evans,
D.C. (2011) Overcoming the genotoxicity
of a pyrrolidine substituted
arylindenopyrimidine as a potent dual
adenosine A2A/A1 antagonist by
minimizing bioactivation to an iminium
ion reactive intermediate. Chem. Res.
Toxicol., 24 (7), 1012–1030.
43 Bertelsen, K.M., Venkatakrishnan, K., Von
Moltke, L.L., Obach, R.S., and Greenblatt,
D.J. (2003) Apparent mechanism-based
inhibition of human CYP2D6 in vitro by
paroxetine: comparison with fluoxetine
and quinidine. Drug Metab. Dispos., 31 (3),
289–293.
44 Murray, M. (2000) Mechanisms of
inhibitory and regulatory effects of
methylenedioxyphenyl compounds on
cytochrome P450-dependent drug
oxidation. Curr. Drug Metab., 1 (1), 67–84.
45 Haddock, R.E., Johnson, A.M., Langley, P.
F., Nelson, D.R., Pope, J.A., Thomas, D.
R., and Woods, F.R. (1989) Metabolic
pathways of paroxetine in animals and
man and the comparative pharmacological
properties of its metabolites. Acta
Psychiatr. Scand. Suppl., 350, 24–26.
46 Zhao, S.X., Dalvie, D.K., Kelly, J.M.,
Soglia, J.R., Frederick, K.S., Smith, E.B.,
Obach, R.S., and Kalgutkar, A.S. (2007)
NADPH-dependent covalent binding of
[3H]paroxetine to human liver
microsomes and S-9 fractions:
identification of an electrophilic quinone
metabolite of paroxetine. Chem. Res.
Toxicol., 20 (11), 1649–1657.
47 Kaye, C.M., Haddock, R.E., Langley, P.F.,
Mellows, G., Tasker, T.C., Zussman, B.D.,
and Greb, W.H. (1989) A review of the
metabolism and pharmacokinetics of
paroxetine in man. Acta Psychiatr. Scand.
Suppl., 350, 60–75.
48 Hemeryck, A., Lefebyre, R.A., De Vriendt,
C., and Belpaire, F.M. (2000) Paroxetine
affects metoprolol pharmacokinetics and
pharmacodynamics in healthy volunteers.
Clin. Pharmacol. Ther., 67 (3), 283–291.
49 Spina, E., Avenoso, A., Facciola, G.,
Scordo, M.G., Ancione, M., and Madia, A.
(2001) Plasma concentrations of
risperidone and 9-hydroxyrisperidone
during combined treatment with
paroxetine. Ther. Drug Monit., 23 (3),
223–227.
50 Belle, D.J., Ernest, C.S., Sauer, J.M.,
Smith, B.P., Thomasson, H.R., and
Witcher, J.W. (2002) Effect of potent
CYP2D6 inhibition by paroxetine on
References j71
atomoxetine pharmacokinetics. J. Clin.
Pharmacol., 42 (11), 1219–1227.
51 Yarnell, A.T. (2009) Heavy-hydrogen drugs
turn heads, again. Chem. Eng. News, 87
(25), 36–39.
52 Wendt, M.K., and Schiemann, W.P. (2009)
Therapeutic targeting of the focal
adhesion complex prevents oncogenic
TGF-beta signaling and metastasis. Breast
Cancer Res., 11 (5), R68.
53 Walker, D.P., Bi, F.C., Kalgutkar, A.S.,
Bauman, J.N., Zhao, S.X., Soglia, J.R.,
Aspnes, G.E., Kung, D.W., KlugMcLeod,
J., Zawistoski, M.P., McGlynn,
M.A., Oliver, R., Dunn, M., Li, J.C.,
Richter, D.T., Cooper, B.A., Kath, J.C.,
Hulford, C.A., Autry, C.L., Luzzio, M.J.,
Ung, E.J., Roberts, W.G., Bonnette,
P.C., Buckbinder, L., Mistry, A., Griffor,
M.C., Han, S., and Guzman-Perez, A.
(2008) Trifluoromethylpyrimidine-based
inhibitors of proline-rich tyrosine
kinase 2 (PYK2): structure–activity
relationships and strategies for the
elimination of reactive metabolite
formation. Bioorg. Med. Chem. Lett., 18
(23), 6071–6077.
54 Bakshi, R.K., Hong, Q., Olson, J.T., Ye, Z.,
Sebhat, I.K., Weinberg, D.H., MacNeil, T.,
Kalyani, R.N., Tang, R., Martin, W.J.,
Strack, A., McGowan, E., Tamvakopoulos,
C., Miller, R.R., Stearns, R.A., Tang, W.,
MacIntyre, D.E., van der Ploeg, L.H.T.,
Patchett, A.A., and Nargund, R.P. (2005)
1-Amino-1,2,3,4-tetrahydronaphthalene-2carboxylic
acid as a Tic mimetic:
application in the synthesis of potent
human melanocortin-4 receptor selective
agonists. Bioorg. Med. Chem. Lett., 15 (14),
3430–3433.
55 Tang, W., Stearns, R.A., Wang, R.W.,
Miller, R.R., Chen, Q., Ngui, J., Bakshi, R.
K., Nargund, R.P., Dean, D.C., and Baillie,
T.A. (2008) Assessing and minimizing
time-dependent inhibition of cytochrome
P450 3A in drug discovery: a case study
with melanocortin-4 receptor agonists.
Xenobiotica, 38 (11), 1437–1451.
56 Teffera, Y., Colletti, A.E., Harmange, J.C.,
Hollis, L.S., Albrecht, B.K., Boezio, A.A.,
Liu, J., and Zhao, Z. (2008) Chemical
reactivity of methoxy 4-o-aryl quinolines:
identification of glutathione displacement
products in vitro and in vivo. Chem. Res.
Toxicol., 21 (11), 2216–2222.
57 Pfefferkorn, J.A., Lou, J., Minich, M.L.,
Filipski, K.J., He, M., Zhou, R., Ahmed,
S., Benbow, J., Perez, A.G., Tu, M.,
Litchfield, J., Sharma, R., Metzler, K.,
Bourbonais, F., Huang, C., Beebe, D.A.,
and Oates, P.J. (2009) Pyridones as
glucokinase activators: identification of a
unique metabolic liability of the
4-sulfonyl-2-pyridone heterocycle. Bioorg.
Med. Chem. Lett., 19 (12), 3247–3252.
58 Litchfield, J., Sharma, R., Atkinson, K.,
Filipski, K.J., Wright, S.W., Pfefferkorn, J.
A., Tan, B., Kosa, R.E., Stevens, B., Tu, M.,
and Kalgutkar, A.S. (2010) Intrinsic
electrophilicity of the 4-methylsulfonyl-2pyridone
scaffold in glucokinase
activators: role of glutathione-Stransferases
and in vivo quantitation of a
glutathione conjugate in rats. Bioorg. Med.
Chem. Lett., 20 (21), 6262–6267.
59 Pfefferkorn, J.A., Guzman-Perez, A.,
Oates, P.J., Litchfield, J., Aspnes, G.,
Basak, A., Benbow, J., Berliner, M.A.,
Bian, J., Choi, C., Freeman-Cook, K.,
Corbett, J.W., Didiuk, M., Dunetz, J.R.,
Filipski, K.J., Hungerford, W.M., Jones,
C.S., Karki, K., Ling, A., Li, J.-C., Patel, L.,
Perreault, C., Risley, H., Saenz, J., Song,
W., Tu, M., Aiello, R., Atkinson, K.,
Barucci, N., Beebe, D., Bourassa, P.,
Bourbounais, F., Brodeur, A.M., Burbey,
R., Chen, J., D’Aquila, T., Derksen, D.R.,
Haddish-Berhane, N., Huang, C., Landro,
J., Lapworth, A.L., MacDougall, M.,
Perregaux, D., Pettersen, J., Robertson, A.,
Tan, B., Treadway, J.L., Liu, S., Qiu, X.,
Knafels, J., Ammirati, M., Song, X.,
DaSilva-Jardine, P., Liras, S., Sweet, L.,
and Rolph, T.P. (2011) Designing
glucokinase activators with reduced
hypoglycemia risk: discovery of N,N-
dimethyl-5-(2-methyl-6-((5-methylpyrazin-
2-yl)-carbamoyl)benzofuran-4-yloxy)
pyrimidine-2-carboxamide as a clinical
candidate for the treatment of type 2
diabetes mellitus. Med. Chem. Commun.,
2, 828–839.
60 Liu, Z.X., and Kaplowitz, N. (2006) Role of
innate immunity in acetaminopheninduced
hepatotoxicity. Expert Opin. Drug
Metab. Toxicol., 2 (4), 493–503.
72j3 Metabolic Stability and Analogue-Based Drug Discovery
61 Dahlin, D.C., Miwa, G.T., Lu, A.Y., and
Nelson, S.D. (1984) N-Acetyl-pbenzoquinone
imine: a cytochrome P-450mediated
oxidation product of
acetaminophen. Proc. Natl. Acad. Sci.
USA, 81 (5), 1327–1331.
62 Kalgutkar, A.S., and Didiuk, M.T. (2009)
Structural alerts, reactive metabolites, and
protein covalent binding: how reliable are
these attributes as predictors of drug
toxicity. Chem. Biodivers., 6 (11),
2115–2137.
63 Uetrecht, J.P. (1992) Metabolism of
clozapine by neutrophils. Possible
implications for clozapine-induced
agranulocytosis. Drug Saf., 7 (Suppl. 1),
51–56.
64 Gardner, I., Leeder, J.S., Chin, T., Zahid,
N., and Uetrecht, J.P. (1998) A
comparison of the covalent binding of
clozapine and olanzapine to human
neutrophils in vitro and in vivo. Mol.
Pharmacol., 53 (6), 999–1008.
65 Uetrecht, J., Zahid, N., Tehim, A., Fu, J.M.,
and Rakhit, S. (1997) Structural features
associated with reactive metabolite
formation with clozapine analogues. Chem.
Biol. Interact., 104 (2–3), 117–129.
66 Obach, R.S., Kalgutkar, A.S., Ryder, T.F.,
and Walker, G.S. (2008) In vitro
metabolism and covalent binding of enolcarboxamide
derivatives and antiinflammatory
agents sudoxicam and
meloxicam: insights into the
hepatotoxicity of sudoxicam. Chem. Res.
Toxicol., 21 (9), 1890–1899.
67 Hartz, R.A., Ahuja, V.T., Zhuo, X.,
Mattson, R.J., Denhart, D.J., Deskus, J.A.,
Vrudhula, V.M., Pan, S., Ditta, J.L., Shu, Y.
Z., Grace, J.E., Lentz, K.A., Lelas, S., Li, Y.
W., Molski, T.F., Krishnananthan, S.,
Wong, H., Qian-Cutrone, J., Schartman,
R., Denton, R., Lodge, N.J., Zaczek, R.,
Macor, J.E., and Bronson, J.J. (2009) A
strategy to minimize reactive metabolite
formation: discovery of (S)-4-(1-
cyclopropyl-2-methoxyethyl)-6-[6-
(difluoromethoxy)-2,5-dimethylpyridin-3-
ylamino]-5-oxo-4,5-dihydropyrazine-2carbonitrile
as a potent, orally bioavailable
corticotropin-releasing factor-1 receptor
antagonist. J. Med. Chem., 52 (23),
7653–7668.
68 Zhuo, X., Hartz, R.A., Bronson, J.J.,
Wong, H., Ahuja, V.T., Vrudhula, V.M.,
Leet, J.E., Huang, S., Macor, J.E., and Shu,
Y.Z. (2010) Comparative
biotransformation of pyrazinonecontaining
corticotropin-releasing factor
receptor-1 antagonists: minimizing the
reactive metabolite formation. Drug
Metab. Dispos., 38 (1), 5–15.
69 Kalgutkar, A.S., Driscoll, J., Zhao, S.X.,
Walker, G.S., Shepard, R.M., Soglia, J.R.,
Atherton, J., Yu, Li., Mutlib, A.E.,
Munchhof, M.J., Reiter, L.A., Jones, C.S.,
Doty, J.L., Trevena, K.A., Shaffer, C.L., and
Ripp, S.L. (2007) A rational chemical
intervention strategy to circumvent
bioactivation liabilities associated with a
nonpeptidyl thrombopoietin receptor
agonist containing a 2-amino-4arylthiazole
motif. Chem. Res. Toxicol., 20
(12), 1954–1965.
70 Antipas, A.S., Blumberg, L.C., Brissette,
W.H., Brown, M.F., Casavant, J.M., Doty,
J.L., Driscoll, J., Harris, T.M., Jones, C.S.,
McCurdy, S.P., McElroy, E., Mitton-Fry,
M., Munchhof, M.J., Reim, D.A., Reiter, L.
A., Ripp, S.L., Shavnya, A., Smeets, M.I.,
and Trevena, K.A. (2010) Structure–
activity relationships and hepatic safety
risks of thiazole agonists of the
thrombopoietin receptor. Bioorg. Med.
Chem. Lett., 20 (14), 4069–4072.
71 Tingle, M.D., Jewell, H., Maggs, J.L.,
O’Neill, P.M., and Park, B.K. (1995) The
bioactivation of amodiaquine by human
polymorphonuclear leucocytes in vitro:
chemical mechanisms and the effects of
fluorine substitution. Biochem.
Pharmacol., 50 (7), 1113–1119.
72 Maggs, J.L., Tingle, M.D., Kitteringham,
N.R., and Park, B.K. (1988) Drug–protein
conjugates. 14. Mechanisms of formation
of protein-arylating intermediates from
amodiaquine, a myelotoxin and
hepatotoxin in man. Biochem. Pharmacol.,
37 (2), 303–311.
73 Tingle, M.D., Jewell, H., Maggs, J.L.,
O’Neill, P.M., and Park, B.K. (1995) The
bioactivation of amodiaquine by human
polymorphonuclear leucocytes in vitro:
chemical mechanisms and the effects of
fluorine substitution. Biochem.
Pharmacol., 50 (7), 1113–1119.
References j73
74 O’Neill, P.M., Harrison, A.C., Storr, R.C.,
Hawley, S.R., Ward, S.A., and Park, B.K.
(1994) The effect of fluorine substitution
on the metabolism and antimalarial
activity of amodiaquine. J. Med. Chem., 37
(9), 1362–1370.
75 O’Neill, P.M., Shone, A.E., Stanford, D.,
Nixon, G., Asadollahy, E., Park, B.K.,
Maggs, J.L., Roberts, P., Stocks, P.A.,
Biagini, G., Bray, P.G., Davies, J., Berry,
N., Hall, C., Rimmer, K., Winstanley, P.A.,
Hindley, S., Bambal, R.B., Davis, C.B.,
Bates, M., Gresham, S.L., Brigandi, R.A.,
Gomez-de-Las-Heras, F.M., Gargallo, D.
V., Parapini, S., Vivas, L., Lander, H.,
Taramelli, D., and Ward, S.A. (2009)
Synthesis, antimalarial activity, and
preclinical pharmacology of a novel series
of 40
-fluoro and 40
-chloro analogues of
amodiaquine. Identification of a suitable
“back-up” compound for N-tert-butyl
isoquine. J. Med. Chem., 52 (7), 1828–
1844.
76 O’Neill, P.M., Park, B.K., Shone, A.E.,
Maggs, J.L., Roberts, P., Stocks, P.A.,
Biagini, G.A., Bray, P.G., Gibbons, P.,
Berry, N., Winstanley, P.A., Mukhtar, A.,
Bonar-Law, R., Hindley, S., Bambal, R.B.,
Davis, C.B., Bates, M., Hart, T.K.,
Gresham, S.L., Lawrence, R.M., Brigandi,
R.A., Gomez-delas-Heras, F.M., Gargallo,
D.V., and Ward, S.A. (2009) Candidate
selection and preclinical evaluation of
N-tert-butyl isoquine (GSK369796), an
affordable and effective 4-aminoquinoline
antimalarial for the 21st century. J. Med.
Chem., 52 (5), 1408–1415.
77 Hagmann, W.K. (2008) The discovery of
taranabant, a selective cannabinoid-1
receptor inverse agonist for the treatment
of obesity. Arch. Pharm., 341, 405–411.
78 Nightingale, S.L. (1994) Recommendation
to immediately withdraw patients from
treatment with felbamate. J. Am. Med.
Assoc., 272 (13), 995.
79 Thompson, C.D., Kinter, M.T., and
Macdonald, T.L. (1996) Synthesis and in
vitro reactivity of 3-carbamoyl-2phenylpropionaldeyde
and 2phenylpropenal:
putative reactive
metabolites of felbamate. Chem. Res.
Toxicol., 9 (8), 1225–1229.
80 Diekhaus, C.M., Thompson, C.D., Roller,
S.G., and MacDonald, T.L. (2002)
Mechanisms of idiosyncratic drug
reactions: the case of felbamate. Chem.
Biol. Interact., 142 (1–2), 99–117.
81 Thompson, C.D., Barthen, M.T., Hopper,
D.W., Miller, T.A., Quigg, M., Hudspeth,
C., Montouris, G., Marsh, L., Perhach, J.
L., Sofia, R.D., and Macdonald, T.L. (1999)
Quantitation in patient urine samples of
felbamate and three metabolites: acid
carbamate and two mercapturic acids.
Epilepsia, 40 (6), 769–776.
82 Roecklein, B.A., Sacks, H.J., Mortko, H.,
and Stables, J. (2007) Fluorofelbamate.
Neurotherapeutics, 4 (1), 97–101.
83 Smith, D.A., Allerton, C., Kalgutkar, A.,
van de Waterbeemd, H., and Walker, D.K.
(2012) Pharmacokinetics and Metabolism in
Drug Design, 3rd edn, Wiley-VCH Verlag
GmbH, Weinheim.
74j3 Metabolic Stability and Analogue-Based Drug Discovery
Amit S. Kalgutkar is a Research Fellow in the Drug Metabolism
Group at Pfizer. He earned his Ph.D. (1993) in Organic
Chemistry from Virginia Tech, and carried out postdoctoral
research at the Department of Biochemistry, Vanderbilt University
School of Medicine before joining Pfizer. His research
interest primarily focuses on medicinal chemistry and mechanistic
drug metabolism centered on biotransformation. He
has been a member of the editorial board of Chemical
Research in Toxicology (American Chemical Society) and
is currently on the editorial board of Drug Metabolism and
Disposition and Xenobiotica. Besides his current position at Pfizer, he is also an
Adjunct Professor at the Department of Biomedical and Pharmaceutical Sciences,
School of Pharmacy, University of Rhode Island. He has coauthored over 110 peerreviewed
papers, reviews, and book chapters and holds several patents in the area
of selective COX-2 inhibition.
Antonia F. Stepan is a Principal Scientist in the Neuroscience
Medicinal Chemistry Department at Pfizer. She earned
her Ph.D. in Organic Chemistry from the University of
Cambridge (UK) in 2006 and then carried out postdoctoral
research at The Scripps Research Institute in La Jolla, California.
In 2008, she joined the Antibacterials Medicinal Chemistry
Department at Pfizer. Her research interest focuses on the discovery
of novel medicines for the treatment of neurodegenerative
diseases. She has coauthored 15 peer-reviewed
papers, reviews, and patents.
References j75
4
Use of Macrocycles in Drug Design Exemplified with Ulimorelin,
a Potential Ghrelin Agonist for Gastrointestinal Motility
Disorders
Mark L. Peterson, Hamid Hoveyda, Graeme Fraser, Eric Marsault, and Rene Gagnon
4.1
Introduction
4.1.1
Ghrelin as a Novel Pharmacological Target for GI Motility Disorders
Ghrelin is a 28-amino acid peptide hormone isolated originally from the stomach
of rats with the orthologue subsequently identified in humans, distinguished by an
unusual n-octanoyl group modification on Ser3
(Figure 4.1) [1]. The existence of
this hormone in a wide range of species suggests a conserved and important role
in normal physiological function. Indeed, since the isolation and characterization
of the peptide in 1999, numerous studies have identified a range of endocrine and
nonendocrine processes in which the hormone is involved [2].
The ghrelin peptide has been demonstrated to be the endogenous ligand for a
previously orphan G protein-coupled receptor (GPCR), the type 1 growth hormone
secretagogue receptor (GHS-R1a) [3], which, not surprisingly, was reclassified as
the ghrelin receptor (GRLN) in 2005 [4]. Although ghrelin is produced primarily in
the stomach [5], GRLN is found predominantly in the brain, with the highest level
in the pituitary, but also is expressed throughout the gut, and a number of other
tissues and organs [6]. The GHS-R1a receptor had been the focus of a significant
amount of pharma industry interest in the latter part of the twentieth century due
primarily, as its name suggests, to its role in stimulating growth hormone (GH)
release. The goal was to develop small molecules, generically termed growth hormone
secretagogues (GHS), which could activate the receptor and thereby act as
oral mimics of GH, a “holy grail”-type objective that motivated drug discovery
efforts by essentially all major pharmaceutical firms [7]. Despite significant success,
initially with peptidomimetics, and then with traditional small molecules, in finding
a number of potent, orally available GHS, the inability to generate sustained
effects from these molecules eventually resulted in a waning of interest in the pharmaceutical
potential of the receptor by the time ghrelin itself was identified [8, 9].
Since then, however, the myriad actions of ghrelin have led to a renaissance in
exploring modulation of this receptor for therapeutic purposes [10]. Indeed,
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
j77
renewed attention to many of the GHS (i.e., ghrelin agonists) developed from
these previous efforts has occurred, albeit often by smaller companies that have
in-licensed these compounds from the innovator firms. Interestingly, only those
therapeutic indications related to the original focus on promoting GH release were
being actively pursued prior to the work reported herein. For example, ghrelin and
ghrelin agonists have been demonstrated to have positive effects in wasting syndromes,
such as cachexia [11, 12], and as agents to counteract some effects of
aging [13]. In addition, antagonism of the ghrelin receptor has been identified as a
strategy for the treatment of metabolic diseases due to the intimate involvement of
ghrelin in the control of energy balance and appetite [14].
In contrast, we were drawn to the effects ghrelin displayed in the gastrointestinal
(GI) system [15], in large part due to the dearth of new products being developed in
that therapeutic space despite GI diseases and disorders being among the most
prevalent illnesses in the general population. In the United States alone, it has
been estimated that there were over 104 million visits to primary care physicians
with a diagnosis of a GI disorder in 2004 imposing an annual cost burden greater
than $141 billion including both direct and indirect expenditures [16]. Motility disorders,
which negatively impact the normal contractions and relaxation of the GI
tract to ensure orderly transit of food and liquids, are among the most common of
these disorders, which include gastroparesis, functional dyspepsia, constipation,
and irritable bowel syndrome (IBS).
Ghrelin is intimately connected to the GI tract. It is produced mainly by oxyntic
cells of the stomach [17] and has a highly potent and direct role in the stimulation
of GI motility [18]. This GI prokinetic activity appears to be independent of the GH
secretory action and is likely mediated by the vagal cholinergic muscarinic pathway
[19]. Ghrelin acts locally in the stomach to activate and coordinate the firing of
vagal afferent neurons and thereby induce gut motility. The peptide has been
shown to accelerate gastric emptying in animal models [20] and successfully treat
postoperative ileus (POI) in both rats and dogs [21]. These results suggested that
ghrelin agonists would be able to provide a novel mechanism of action for therapeutic
purposes in GI disorders characterized by dysmotility through duplicating
the effects of ghrelin and accelerating the normalization of gut function [22]. This
was later confirmed through studies with earlier developed GHS/ghrelin agonist
compounds [23]. Also subsequent to the initiation of our work, clinical studies conclusively
demonstrated the GI promotility effects of ghrelin, acting by promoting
phase III of the migrating motor complex (MMC), in humans [24, 25].
Most previous efforts to develop pharmacotherapies for GI motility disorders
have focused on modulation of serotonin [26] and dopamine receptors [27].
Serotonin and dopamine are major mediators of various physiological functions,
both inside and outside the CNS, and exert their actions, including the stimulation
Gly-Ser-Ser(Oct)-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg
ghrelin (human)
Figure 4.1 Primary sequence of human ghrelin.
78j4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin
of GI motility, through multiple different receptor subtypes. Unfortunately, a number
of these agents have been removed from the market, or had their distribution
highly restricted, due to deleterious side effects, often caused by nonselective activity
at these subtypes or other receptors. For example, cisapride, a serotonin receptor
modulator, originally used for nocturnal GERD, was removed from the market in
2000 due to the potential of serious cardiac arrhythmias [28]. Another serotonin
receptor modulator, tegaserod, developed as a treatment for IBS with constipation,
was withdrawn in 2007 due to an increased risk of ischemic events, including heart
attacks and strokes [29]. Even one of the few remaining available drugs, metoclopramide,
a mixed dopamine and serotonin receptor modulator with modest efficacy,
had a black box warning added to its label in 2009 because of the risk of tardive
dyskinesia with extended use [30]. Given this checkered history with established
targets, coupled with the unmet need in the area, we felt that exploration of the
potential of activation of the ghrelin receptor as a new pharmacological approach
for the generation of novel GI prokinetic agents was well warranted.
4.1.2
Macrocycles in Drug Discovery
Despite the promise of combinatorial chemistry in conjunction with the many
advances in genomics/proteomics, drug discovery productivity has not experienced
the significant improvement originally anticipated. One of the reasons for this has
been suggested to be a lack of sufficient diversity in the number of chemotypes
typically investigated [31]. Hence, attention has been directed toward mining new
or underexplored chemical scaffolds for interesting pharmacological activity.
One of the emerging compound classes in the underexplored category is macrocycles.
Due to their presence in a wide variety of natural products that exhibit an
equally broad range of bioactivity, such structures have long been recognized as a
potentially fruitful pharmaceutical compound class. However, the generation of
libraries of macrocycles, screening of which would be the typical starting point for
the majority of current medicinal chemistry efforts, has been an impediment due
to the lack of appropriate synthetic methods for their construction in a highly parallel
manner. Further, concerns about the ability of these structures to possess appropriate
“drug-like” properties, since typically one or more parameters fall outside the
acceptable “rule of five” ranges [32], have deterred wider investigation.
Nonetheless, macrocyclic compounds possess highly attractive characteristics,
including
 diverse, often complex, functionality;
 defined topological display of functionality;
 ability to interact at otherwise intractable targets.
These features often lead to stronger affinity and greater selectivity than traditional
structures. Further, macrocycles usually possess enhanced metabolic stability
versus the comparable linear compounds. Indeed, increasingly over the past decade,
macrocyclic compounds have been reported that display biological activity
4.1 Introduction j79
against a wide range of pharmacological targets and recent reviews have summarized
these efforts [33]. In many cases, such structures were accessed as a means of
improving activity of linear analogues through conformational restriction, although
more examples have begun to appear where macrocycles are being utilized as the
core scaffold as well. With the successful discovery and development of technologies
that permit larger screening libraries of macrocycles to be prepared, including
that described herein, the use of macrocycles as a de novo source of novel chemical
entities is expected to continue to expand.
4.1.3
Tranzyme Technology
Tranzyme has discovered and developed a proprietary technology for the generation
of peptidomimetic macrocycles of the general structure shown in Figure 4.2 in
a highly parallel fashion, thereby enabling the creation of the first library of macrocyclic
structures available for screening for novel bioactivity [34].
Several features of these structures merit further comment as they have proven
very advantageous for their use in drug discovery. The molecules are comprised of
four building block units, three from amino acids (or their equivalents) and one a
unique element, acting as a bridge between the ends of the peptidic segment, that
we termed a “tether.” This tether more importantly functions to constrain and
define the conformation(s) of the resulting structure. In this manner, topological
diversity is introduced into these compounds and can be investigated simultaneously
with chemical diversity. Although initially designed in order to control and
modulate the conformational population, subsequent studies have demonstrated
that the tether functions as a recognition site for binding to the target as well. Further,
the tether can significantly influence the physicochemical properties of the
macrocycle as the current work will also illustrate.
In addition, the use of three amino acids maintains the total molecular weight
within the small molecule range (400–600), yet with the capability of interacting
over significant regions of three-dimensional space. Although additional amino
acids can be incorporated, the screening library used for the ghrelin receptor only
utilized macrocycles of the general structure of Figure 4.2. Substantial chemical
diversity is provided by the standard amino acid side chains that display, as a group,
a variety of polarities, hydrophobicities/hydrophilicities, sizes, and charges.
HN
H
N
NH
HN
Tether
O
O
O
R1
R2
R3
Figure 4.2 Generic structure of Tranzyme macrocyclic library (stereochemistry for illustration
purposes only).
80j4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin
Importantly, their side chains have unquestioned biological relevance. This diversity
is further enhanced through using both the natural and unnatural stereoisomers
in the library construction. Our modeling efforts have shown that the amino
acid configurations can have significant effects on the conformations of the resulting
macrocycles, enabling the sampling of different regions of space simply by
using a variety of configurations. Finally, the secondary nitrogen atom provides a
handle from which further modifications can be incorporated during optimization
or for salt formation to enhance solubility.
The synthetic approach utilized for these structures as shown in Scheme 4.1 has
several salient features: (i) performed on solid phase to permit high numbers of
compounds to be made simultaneously; (ii) utilizes a Fukuyama–Mitsunobu
reaction to attach the tether; (iii) employs standard amino acid coupling chemistry
for high-yielding building block assembly; (iv) incorporates a linker that permits
concomitant ring closure and cleavage from the resin (i.e., cyclative release);
(v) uses radiofrequency tagging methodology for compound tracking [35]; and (vi)
conducts the final steps as an array in microtiter format to prepare individual, discrete
compounds in each well for ease of transfer to screening. The use of cyclative
release also biases the compounds toward better crude purity as only cyclized
compounds are released from the resin. Difficult macrocyclizations are thus
reflected in low yields rather than multiple impurities. Indeed, the primary
impurity observed in most cases, as with most such cyclizations, is the macrocyclic
dimer. The synthesis success rate is typically quite high, over 95%, even for a
variety of amino acids and configurations.
As a final point on the general drug discovery strategy using these macrocycles,
modified synthetic methods have been developed that can be used to incorporate
N
H
O
STrt
Reagents and conditions:
(a) 10% TFA, TES, DCM; (b) Ddz-AA3-OH, PyBOP, DIPEA, NMP;
(c) 1-2% TFA, TES, DCM; (d) Ddz-AA2-OH, HBTU, DIPEA, NMP;
(e) 1-2% TFA, TES, DCM; (f) Bts-AA1-OH, HBTU, DIPEA, NMP;
(g) A, PPh3, DIAD, THF; (h) 1-2% TFA, TES, DCM; (i) DIPEA,
THF, MP-carbonate, Ag(OCOCF3) (optional); (j) PS-thiophenol,
KOTMS, THF:EtOH; (k) 50% TFA, TES, DCM.
PS
S
O
H
N
O
N
H
O
R3
R2
N
R1
Tether
Bts
S
O
H
N
R3
Ddz S
O
H
N
O
N
H
O
R3
R2
HN
R1
Tether
Bts
TFA • H2N
NH
O
HN
NH
O
O
HN
R1
R2
R3
Tether
a,b
DdzNH OH
c-f
g,h
i-k
STrt
Cyclization-
release
S
O
O S
N
Bts
O
O
O
O
Ddz
A
Scheme 4.1 Macrocyclic library synthetic method.
4.1 Introduction j81
peptide bond isosteres into these structures. Since such an approach did not produce
desirable effects in the ghrelin agonist program, they will not be discussed
further, but does illustrate the additional, more significant, changes that can be
made to these structures during hit-to-lead optimization.
4.2
High-Throughput Screening Results and Hit Selection
As with all of our discovery efforts, the search for agonists of the ghrelin receptor
(GRLN) began with high-throughput screening (HTS) of the company’s HitCreateTM
Library of proprietary macrocyclic small molecules (Figure 4.2). In surveying the
potential platforms available for this effort, we decided on the aequorin assay, a
robust system that has proven quite useful for GPCR targets [36]. In particular, the
wide dynamic range and high signal-to-noise ratio, as well as low cost per assay,
were attractive benefits for screening of this new compound class. Importantly,
screening for agonist and antagonist activity is performed in series with the same
samples, allowing a complete characterization of the functional activity to be
obtained from the primary screening. As a secondary, confirmatory screen, a standard
radioligand binding assay was employed.
Screening of a set of approximately 10 000 compounds led to two different series
of validated hits, represented in Figure 4.3 with their most active members, 1
and 2. The high affinity and functional activity obtained for compounds directly
from the HTS efforts were quite remarkable, although a similar level was obtained
in our motilin antagonist program as well [37]. The requirement for the cyclic structure
was confirmed in that linear analogues of 1 and 2 were devoid of any activity.
These compounds exhibited selectivity (>100-fold) versus the motilin receptor
(MLNR), the most closely homologous GPCR, plus did not display cytotoxicity in
HepG2 cells at concentrations up to 100 mM.
After preliminary follow-up libraries on both series, series A, despite being initially
slightly weaker in bioactivity, proved to be the most fruitful for further optimization
and will be the focus of the remainder of the discussion [38].
O
NH
O
N
O
NH
NH
O
Ki 86 nM
EC50 134 nM
1
(series A)
O
NH
O
NH
O
NH
N
H
O
Ki 41 nM
EC50 90 nM
2
(series B)
Figure 4.3 Active series obtained from HTS.
82j4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin
4.3
Macrocycle Structure–Activity Relationships
4.3.1
Preliminary SAR
With four individual components available for optimization and a wealth of potential
diversity elements to choose from, the focus of the first phase of SAR development
was to determine (1) the preferred configurations at each of the amino acid
positions, (2) the steric requirements at the amino acid sites, (3) the optimal ring
size, and (4) the best tether component.
In order to gain initial insight into the SAR, a library of approximately 100 compounds
was prepared with each member designed to interrogate one of these questions.
The same solid-phase methodology as for the synthesis of the library
(Scheme 4.1) was applied to access these analogues. All compounds were purified
using automated preparative HPLC to >95% purity. Selections from this library
and their binding results are provided in Table 4.1.
Investigation of L- and D-stereoisomers at each of the three amino acid positions
revealed strikingly different results depending on the site, although the configurations
as in 1 were confirmed as preferred in all instances. For AA1 and AA2, a clear
stereochemical preference was found, while for AA3 the opposite stereocenter to
that in 1 was much less disfavored, suggesting that this was the most promiscuous
site on the structure.
From these results, the following salient characteristics of the SAR regarding the
general requirements at the four diversity sites of the structure became evident
(compounds that demonstrate the point shown in parentheses):
 The double bond in the tether was not necessary (3).
 For AA1, the L-stereochemistry was definitely preferred (4) with an apparent preference
for larger aliphatic side chains (5 versus 6).
 The D-configuration was the choice at AA2 (10 versus 11 and 13 versus 14) with a
limitation on the size of the side chain evident (15 and 16).
 The N-methyl group was required for AA2 (9).
 AA3 exhibited a preference for the D-stereochemistry (18) and it appeared that
aromatic moieties were somewhat advantageous (22 and 23).
In particular, the benefits of Ile at AA1 and D-NMeAla at AA2 were considered to
be significant enough to often utilize such elements as the standard reference at
these positions as the optimization efforts progressed.
4.3.2
Ring Size and Tether
Turning attention next to the tether component, which allowed the exploration of
different ring sizes as well, a number of alterative tether moieties to that of 1 were
examined to determine if a change in the macrocyclic conformation would be
4.3 Macrocycle Structure–Activity Relationships j83
beneficial. This also provided an opportunity to investigate heteroaromatic rings
and modified polarities. The results of these efforts are shown in Table 4.2.
As can be seen, the majority of these alternative tether structures had dramatic
detrimental effects on binding affinity to GRLN. Smaller rings also were definitely
disadvantageous. Hence, the results suggest that the conformation of the macrocycle
of the original hit 1 must already be at or near the optimal range. This nicely
demonstrates one of the strengths of the underlying macrocyclic technology in
being able to scan topological space simultaneously with chemical space to accelerate
hit-to-lead-to-clinic optimization.
Therefore, more subtle modifications around the tether were tested, including
shifting position of the phenyl ring, necessity of the oxygen atom, and increasing
Table 4.1 Preliminary SAR selections.
Compound AA1 AA2 AA3 Ki (nM)
1a
Nva Sar D-Phe 86
3 Nva Sar D-Phe 68
4 D-Nva Sar D-Phe 1200
5 Ala Sar D-Phe 4300
6 Nle Sar D-Phe 67
7 Phe Sar D-Phe 670
8 Ser Sar D-Phe >10 000
9 Nva Gly D-Phe 820
10 Nva Ala D-Phe 5000
11 Nva D-Ala D-Phe 500
12 Ile D-Ala D-Phe 177
13 Nva NMeAla D-Phe 580
14 Nva D-NMeAla D-Phe 14
15 Nva D-Leu D-Phe >10 000
16 Nva D-Phe D-Phe 8300
17 Nva Aib D-Phe 640
18 Nva Sar Phe 380
19 Nva Sar D-Ala >10 000
20 Nva Sar Gly >10 000
21 Nva Sar D-Nle 400
22 Nva Sar D-Tyr 120
23 Nva Sar D-Trp 160
a) Double bond in tether (see Figure 4.2).
84j4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin
Table 4.2 Tether SAR part 1: alternative structures.
Compound Tether Ring size Ki (nM)
24 15 >10 000
25 16 >10 000
26 16 $ 10 000
27 15 9200
28 16 >10 000
29 17 650
30 15 >10 000
31 15 >10 000
32a
18 >10 000
a) AA1 ¼ Ile.
4.3 Macrocycle Structure–Activity Relationships j85
or decreasing ring size by one atom, although the more substantial removal of the
phenyl ring was also assessed (Table 4.3).
The outcome of this series was more revealing, with additional features of the
SAR becoming evident:
 Removal of the phenyl was definitely unfavorable (41 and 42).
 Reintroduction of a conformational constraint in the form of a double bond (43)
did restore binding, although at a level still an order of magnitude less than with
the aromatic ring, confirming the importance of that moiety.
 The oxygen atom was beneficial, but not critical, as the methylene substitution
only had a slightly negative effect on binding (1 versus 38 and 33 versus 34),
which was recovered if an additional double bond was introduced (39). However,
the oxygen-containing tether is much superior in terms of GRLN-to-MLNR selectivity
(>1500 : 1) in comparison with the olefinic tether ( 30 : 1).
 Smaller rings were somewhat detrimental (33 and 34), while larger ones (40) did
not offer any improvement over the original 18-membered macrocycle.
 Interestingly, shifting the position of the phenyl ring with respect to the rest of
the structure proved to be mixed, with positive results closer to the amine nitrogen
(36) and negative in the opposite direction toward the amide (35).
Table 4.3 Tether SAR part 2: phenyl ring presence and position.
Compound X Y Z Ring size Ki (nM)
14a
(CH2)2O (CH2)3 18 14
33 (CH2)3 (CH2)2 17 400
34 (CH2)2O (CH2)2 17 210
35 (CH2)4 (CH2)2 18 230
36 CH2 (CH2)5 18 18
38 (CH2)3 (CH2)3 18 81
39 (CH2)3 CH ¼ CHCH2 18 10
40 (CH2)3 (CH2)4 19 78
41 (CH2)7 17 >10 000
42 (CH2)8 18 1500
43 (CH2)4CHÀÀÀÀCH(CH2)2 18 160
a) AA1 ¼ Nva for reference.
86j4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin
Overall, these results led us to retain the tether structure of 3 and 14 as the basis
for further work. Hence, it appeared that substitution on the tether, either on the
phenyl ring or on the aliphatic connecting chains (“arms”), would need to be evaluated
as the next stage in the optimization of this moiety.
4.3.3
Amino Acid Components
With the preferred stereochemical configuration at each amino acid position determined,
although this was subjected to spot checks as analogue development continued,
which confirmed the original biases, the next phase of the investigation was to
conduct a thorough survey of the functionality and polarity accessible via the side
chains of the proteinogenic amino acids, as well as the substantial collection of
unnatural amino acids that are accessible either commercially or through synthesis.
At this stage and beyond, the solid-phase procedure was often supplanted by an
alternative solution-phase approach (Scheme 4.2) for the synthesis of analogues
where one of the building blocks needed to be specially prepared or was available
in only limited quantities.
Key results from these extensive analogue development efforts are summarized
graphically in Figure 4.4. Building on the results of the preliminary SAR described
NH
a, b
O
O
+
H2N
R1
R1
OH
O
Cbz
N
O
OH
H2N
O
OtBu
R3
H
N
O
OtBu
R3O
N
Hc, d
+
+
R2
e, f g, (h)
a. KI, Na2CO3, DMF, 105ºC; b. LiOH, THF:H2O, :MeOH; c. HATU or EDCI/6Cl-HOBt, DIPEA, NMP;
d. H2, Pd/C, AcOEt; e. HATU, HOAt, DIPEA, NMP; f. 50% TFA/5%TES/DCM; g. DEPBT, DIPEA,
THF; h. Deprotect side chains (if necessary)
LG-Tether-NHBoc
Boc-NH-Tether
R2
R4
R4
HN
R1
O
HN
O R3
O
N
R2
R4
Tether
HN
H
N
R1
O
HN
O R3
O
N
R2
R4
Tether
BocNH
HO
(LG = leaving group)
B
C
B C
Scheme 4.2 Solution-phase route to macrocycles.
4.3 Macrocycle Structure–Activity Relationships j87
earlier, this study indicated greater restrictions to allowable groups at AA1 and AA2,
with the latter very highly limited, while the other position, AA3, was indeed more
promiscuous.
For AA1, a preference for aliphatic side chains was confirmed with the presence
of basic (Lys), acidic (Glu), polar (Ser), and aromatic (Phe) groups detrimental.
Slightly larger groups than methyl proved more favorable with branched alkyl moieties
(Ile, Tle, and Leu) being advantageous. Of particular interest was the positive
results obtained with the small cyclic amino acid cyclopropylglycine (Cpg), which
had additional benefits as will be discussed later in Section 4.4.
A very tight SAR was determined for the AA2 constituent. Charged (D-Glu and
D-Lys) amino acids were essentially inactive, as were aromatic (D-Phe) side chains.
Only smaller aliphatic groups, such as methyl or ethyl, were tolerated for the side
chain, while the N-methyl group of the original hit 1 was clearly beneficial, likely
due to its effect on overall molecular conformation. Since N-ethyl and N-isopropyl
analogues did not result in any activity improvement and were more difficult to
access synthetically, further attention at this site was deemed not to be warranted.
It should be noted that N-methyl substitutions at the other amide nitrogens of the
macrocycle proved to be unfavorable. The potential of D-Pro in this position as an
alternative constrained amino acid was tested, but not found to be a fruitful avenue
(Ki ¼ 330 nM).
O
NH
O
N
O
NH
NH
O
(D)-Phe: 14 nM
(L)-Phe: 280 nM
(D)-Hfe: 43 nM
(D)-Phe(4-Cl): 1 nM
(D)-Phe(4-F): 2 nM
(D)-Phe(4-OMe): 3 nM
(D)-Tyr: 119 nM
(D)-Phe(3-Cl): 13 nM
(D)-Phe(2-Cl): 2 nM
(D)-2-Pal: 24 nM
(D)-1-Nal: 2 nM
(D)-Dip: 32 nM
(D)-Bip: 67 nM
(D)-Trp: 163 nM
(D)-His: 257 nM
(D)-Thz: 119 nM
(D)-2-Thi: 10 nM
(D)-NMeAla
14 nM
(D)-NEtAla
11 nM
N
O
N
O
N
O
N
O
N
O
NEtGly
137 nM
(D)-NMeAbu
35 nM
(D)-NMeVal
384 nM
NMeVal
> 1 µM
N
O
(D) or (L)-
NMeNva
> 1 µM
Cpg
7 nM
Tle
4 nM
Ile
6 nM
Leu
25 nM
Nle
67 nM
Val
107 nM
HO C
Glu
>10 µM
H N
Lys
1 µM
Phe
667 nM
Acc
>10 µM
HO
Ser
>10 µM
AA3
AA2
AA1
3
Ki = 68 nM
Figure 4.4 SAR amino acid components.
88j4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin
Contrastingly, a variety of different groups are permitted at the AA3 position; however,
some limitations were evident. Acidic (D-Glu), basic (D-Orn), and polar (D-Ser)
derivatives, for example, failed to show GRLN binding. Small aliphatic moieties (Gly
and D-Ala) were similarly ineffective; however, larger alkyl side chains (D-Nle) did
exhibit binding affinity, albeit a little weaker than the worst aromatic moiety (D-His).
Therefore, of all the derivatives tested at this stage, the phenyl group as originally in
1 still appeared to offer the best scaffold for additional optimization, although we
were cognizant of the detrimental effects of the moiety on clogP, making this
parameter higher than is typically desirable. However, as alluded to in the introduction,
these macrocyclic molecules can be considered rule breakers, which do not
lend themselves to standard measures of suitability for drug development.
Fortunately, many substituted phenylalanine building blocks were readily available
for investigation as were alternative isosteres, such as pyridylalanine. As seen in
Figure 4.4, the presence of halogen or methoxy substitution in the 4-position of the
phenyl ring was particularly effective in increasing potency by an order of magnitude,
with 2-halo substituents showing similar results. This provided several
options to be investigated with respect to optimization of the pharmacokinetic (PK)
profile (Section 4.4).
4.3.4
Further Tether Optimization
With the optimization of the three amino acid diversity elements achieved, at least
with regard to the pharmacodynamic (PD) profile of the macrocycle, the tether
component became the focus area. Based on the previous results (Section 4.3.2),
the core tether structure of compound 3 appeared to still be the appropriate starting
point for further investigation. Hence, the strategy that was taken was twofold: (1)
introducing additional conformational rigidity into the arm through methyl substitutions
and (2) modulation of the electronic properties of the phenyl ring through
appropriate substituents. A selection of such tether variations together with their
impact on the PD profile of the resulting macrocycles is presented in Table 4.4.
In considering where best to start this investigation, the all aliphatic arm connecting
to the amide nitrogen did not seem to be the more fruitful avenue since
removing the olefinic conformational restriction from that portion of the molecule
had a minimal, actually positive, effect on activity. Hence, attention was placed on
the arm joining the ether oxygen to the amine. Systematic substitution along that
region led to very different results. A methyl group adjacent to the amine nitrogen
(45) retained the binding level of the unsubstituted analogue (44), but hurt the
functional activity1)
. In contrast, stereospecific methyl substitutions at the adjacent
carbon atom resulted in both good binding affinity and functional efficacy (EC50),
with the (R)-derivative about 3.5-fold more potent than its epimer (47). Introducing
1) For SAR development, the focus was placed on receptor binding affinity (Ki), rather than on
functional activity, since the latter is subject to potential bias due to the choice of the signal
transduction pathway, as well as constitutive GRLN signaling and allosteric activation (also
see Ref. [38]).
4.3 Macrocycle Structure–Activity Relationships j89
Table4.4TetherSAR:tetherbackbonesubstitutions.
CompoundTetherKi(nM)EC50(nM)CompoundTetherKi(nM)EC50(nM)
447.3ND490.9085
459.07605019ND
461629512.627
90j4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin
4756265258ND
4812Partialagonista
5361ND
ND:notdetermined.
a)$40%agonistresponseat3mMtestconcentration.
4.3 Macrocycle Structure–Activity Relationships j91
an additional methyl group on that atom to give the gem-disubstituted analogue
(48), although displaying good binding, yielded a partial agonist, whereas all other
compounds tested were full agonists. Additional restrictions on conformation by
connecting the methyl to the phenyl moiety to create a dihydrobenzopyran ring
provided chiral derivatives 49 and 50. Consistent with the trends observed with the
methyl derivatives, the (R)-analogue proved to be more potent than the (S)-isomer,
although with a much greater effect here, exhibiting over a 20Â difference in activity
versus 3.5Â for 46 versus 47.
The second aspect of this investigation, substitution on the ring of the tether,
displayed a clear SAR. Introduction of a fluorine substituent at the para-benzylic
tether position (51) resulted in more than a twofold improvement in binding as
compared with the unsubstituted compound (44). Shifting the fluorine substitution
to the para-phenoxy position reduced the activity by over 20Â (52 versus 51). Surprisingly,
an analogue that combined both the tether (R)-methyl and phenyl fluorine
beneficial substitutions (53) displayed worse potency than either of its singly
substituted progenitors. The unexpected impact of these tether structural variants
on the PK is described in the next section.
4.4
PK–ADME Considerations
Although not addressed in the discussion to this point, an assessment of the PK–
ADME properties of the molecules actually was initiated at an early stage of the
discovery process. Since these small molecule macrocycles represent an essentially
unexplored chemical class, this work represents one of the first opportunities to
extensively explore their properties. The target therapeutic indications for these
ghrelin agonists were those characterized by acute GI dysmotility, such as those
occurring after surgery (termed postoperative ileus), serious gastroparesis, and gastric
stasis in critical care patients. For such applications, intravenous (i.v.) administration
would actually be preferred since patients suffering from these conditions
cannot easily take or tolerate oral medications. Hence, the primary focus was ensuring
that the PK–ADME properties were optimal for i.v. administration. However,
further development of a second generation of this compound class for chronic GI
disorders was anticipated and these studies were expected to assist in establishing
the groundwork for that effort as well. Despite the failure to meet certain traditional
measurements required for oral pharmaceutical agents, such as Lipinski’s rule of
five referred to earlier, these macrocycles did prove amenable to optimization not
only toward acceptable plasma half-life (T1/2) and low systemic clearance rate (CL),
but also toward good oral bioavailability (%F) to support their eventual development
for chronic clinical applications2)
.
Shown in Table 4.5 are the PK data obtained in rats for a selection of analogues
described previously. Two tether modifications in particular proved to
2) This second-generation ghrelin agonist, TZP-102, is currently being evaluated in a
multinational phase 2b clinical trial as an oral treatment for diabetic gastroparesis.
92j4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin
Table4.5PKprofile(i.v.administration)inrats.a
CompoundAA1XTetherKi(nM)EC50(nM)
i.v.,2mg/kg
T1/2(min)Vdss(1/kg)CL(ml/min/kg)
44CpgFA7.3ND141.472
46CpgFB1629501.724
47CpgFC5626222.164
49CpgFD0.7085591.113
51CpgFE2.627441.828
53CpgFF61ND660.99
54NvaFB34ND291.741
55ValFB104ND342.040
56IleFB32ND442.540
57CpgClB1518411.424
58CpgHB34ND321.325
59ChgFB0.3849744.743
60IleClA0.3520333.267
ND:notdetermined.
a)MaleSprague-Dawleyratswereadministeredcompoundsasanintravenous(i.v.)bolusat2mg/kginphysiologicalsalinewith9%2-hydroxypropyl-b-cyclodextrin.
4.4 PK–ADME Considerations j93
have substantial impact on the PK profile: (i) stereospecific (R)-methyl substitution
at the tether position adjacent to the oxygen atom (46) and (ii) substitution
of the phenyl at the tether para-benzylic position (51). Indeed, a
combination of these features (53) led to additional improvement of CL and
half-life, unfortunately at the expense of potency ($4-fold reduction compared
with 46). Furthermore, an interesting difference in PK was observed for different
configurations of the methyl substituent on the tether in the ether-containing
arm. Analogue 46 with an (R)-methyl had a relatively low clearance
and acceptable half-life, whereas the corresponding (S)-stereoisomer (47) was
cleared at much higher rates with a much shorter T1/2. Consistent with this
trend, the benzopyran with an (R)-configuration at the same center (49) also
possessed an improved PK profile with an even further reduced clearance
rate, longer half-life, and a volume of distribution nearly twice that of total
body water. Therefore, the additional conformational restriction introduced
with the benzopyran benefited both binding affinity (18Â improvement) and
improved clearance. Nonetheless, analogue 46, in comparison with 49, possessed
a threefold advantage in functional potency and, further, the synthesis
of 49 was more complex than that for 46. In contrast, compound 51 seemed
to have similar functional activity and PK properties to 46. However, after
the additional in vivo evaluations described in Section 4.6, compound 46
(ulimorelin) proved to be a superior candidate for advancement into clinical
development.
Although the tether structure had a significant impact on the PK profile of
these macrocyclic compounds, there was an additional critical factor that contributed
to the desirable PK profile for 46 of reduced CL and longer T1/2.
Indeed, this improved profile resulted from the (R)-methyl-substituted tether
together with the cyclopropyl side chain on AA1. Comparison of the PK results
obtained with analogues 54–56, which differ solely in the nature of the AA1
side chain (n-propyl (Nva), isopropyl (Val), and isobutyl (Ile), respectively) highlights
this apparent cooperative effect. Thus, 46 displayed $2-fold improved
clearance rate versus 54–56 for which significantly higher CL values were
obtained. Similarly, analogue 44, which contains only AA1 ¼ Cpg, but not the
(R)-methyl substitution on the tether, displayed essentially flow-limited clearance
along with a short T1/2. Further, these results suggest that it is not simply
metabolic blockade of that particular tether position that leads to the improved
PK profile of 46. A potential rationale for this vital cooperativity effect observed
with this analogue is provided in Section 4.5.
In contrast to the crucial role of the AA1 side chain in modulating PK, variations
in the AA3 side chain were much less impactful. For example, analogue 57 with a
4-chlorophenyl side chain at AA3 exhibited a similar CL to 46, containing a
4-fluorophenyl in that position. In fact, even the unsubstituted AA3 phenyl ring
(58) showed similar clearance rates to 46 and 57. These results indicated that
metabolism of the AA3 phenyl ring was likely not a major elimination pathway in
these macrocycles as was later confirmed from more detailed studies conducted on
ulimorelin (46).
94j4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin
4.5
Structural Studies
In order to potentially gain insight into the PD and PK profiles observed with these
unique macrocycles, as well as obtain confirmation of the underlying conformational
control principles on which the technology was originally based, the solidstate
structure of compound 46 (as its hydrochloride salt) was determined using
single-crystal X-ray crystallography. In addition, solution conformation analysis of
46ÁHCl was performed by a combination of NMR spectroscopy and molecular
modeling3)
. Upon analysis of bond distance and torsion angle data from the X-ray
structure, the following information regarding the conformation(s) of the macrocycle
could be gleaned: (i) the Ca(i)–Ca(i þ 3) distances are less than 7 A

, indicative
of a b-turn [39], and (ii) the torsion angles in the tripeptide segment revealed a
(nonideal) closed type I0
b-turn [40]. These same features were also identified in the
NMR-derived consensus solution structure where only a single conformational
family was consistent with all the experimental constraints. Indeed, a very good
correlation between the solid-state and solution-state structures was obtained,
suggesting that the conformation was well optimized and rigidified in the final
macrocycle (Figure 4.5).
Further comparison of the X-ray and NMR structures revealed another major
similarity, the presence of an intramolecular hydrogen bond between the tether
oxygen and the protonated secondary amine at the AA1 position [41]. This intramolecular
H-bond imparts additional conformational rigidity on the macrocyclic
structure that, perhaps not surprisingly, does appear to have an impact on the activity.
Recall that fluorine substitution at the para-phenoxy position in 52 reduced the
activity by over 20Â relative to the analogue with a para-benzylic fluoro substituent.
This result is consistent with substitution of an electron-withdrawing group para to
the oxygen lowering the basicity of that atom with concomitant weakening of this
intramolecular H-bond. The possible influence that the presence of this H-bond
has on the PK profile of the molecules is discussed below. Finally, in the NMR
structure, the ammonium hydrogen appears associated with the carbonyl oxygen
of AA1, adding to the conformational constraints in 46.
Additional insights into the potential role of the intramolecular H-bond between
the tether oxygen and the AA1 amine in 46 can be obtained from consideration of
the somewhat unique nature of the cyclopropyl side chain. The cyclopropyl ring is
almost as strong an electron-donating moiety as the cyclohexyl group4)
. The effect
of such electron donation would be to enhance the basicity of the secondary amine
at AA1, concomitantly strengthening the intramolecular H-bond5)
. In addition, the
relatively low lipophilicity of the cyclopropyl group could positively impact hepatic
metabolism rates as well as the solubility. Indeed, 46 has aqueous solubility
values in the 1–8 mg/ml range (at about pH 5), while related structures with
3) For a more detailed description of these studies, see Ref. [38].
4) Based upon a comparison of the Hammett sp and Hansch p values.
5) This is confirmed by the measured pKa values for analogues with Cpg at AA1, where the
amine basicity is enhanced by !0.3 log units versus derivatives with an n-propyl side chain.
4.5 Structural Studies j95
AA1 ¼ Nva (54) or Ile (56) gave significantly lower aqueous solubility (0.1–
0.5 mg/ml range) with the solubility of the Chg analogue (59) extremely poor
( 0.01 mg/ml) under similar conditions.
Thus, the strong electron-donating ability of the cyclopropyl moiety at the AA1
position appears to enhance the conformational rigidity in 46 by strengthening the
intramolecular H-bond seen in both the X-ray and NMR structures with minimal
impact on the overall lipophilicity. Further, the topological positioning of the (R)-Me
tether substitution and the AA1 cyclopropyl side chain on opposite faces of the core
macrocyclic framework provides hydrophobic shielding of this intramolecular
H-bond from both sides of the molecule, possibly protecting it from disruption by
encroaching water molecules. Hence, the electronic and steric shielding effects provided
by these two moieties work together to stabilize the intramolecular H-bond
and further rigidify the structure, providing a potential explanation for the observed
PK cooperative effect observed in 46, which was lacking in its close analogues.
4.6
Preclinical Evaluation
A number of in vitro assays have become fixtures in drug discovery research in
order to improve the evaluation of early stage compounds and direct attention
Figure 4.5 Comparison of NMR (orange) and X-ray structures for 46 (ulimorelin).
96j4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin
toward compounds with favorable properties for further development [42]. In addition,
the lack of an existing knowledge base regarding this particular compound
class and our desire to eventually develop an oral gastroprokinetic agent prompted
us to investigate applicable physicochemical parameters and oral PK of these molecules
as well. To access sufficient material for more complete profiling and for in
vivo studies, a synthetic route amenable to larger amounts of material first needed
to be developed. Based upon the solution-phase procedure presented earlier,
Scheme 4.3 illustrates the resulting process, which has proved viable for even clinical
quantities of 46.
4.6.1
Additional Compound Profiling
One common screen for membrane permeability, used as a predictor for oral
bioavailability, is the Caco-2 assay [43]. The results in Table 4.6 demonstrate
that very good membrane permeability could be obtained with the small variety
of substituents tested and that the variations seen in the PK for these molecules
were not duplicated in this test. Further, the near-equivalent flux rates
in both directions for 46 and 60, both of which displayed moderate permeability,
indicate that neither appears to be a P-glycoprotein (Pgp) substrate. However,
the results with 51 suggest that at least moderate Pgp-mediated active
transport is likely occurring.
For metabolic stability, however, differences between 46 and 60 are again evident.
Analogue 46 displayed greater in vitro stability in rat (RLM) and human hepatic
liver microsomes (HLMs) as compared to 60. This indicates that the improved CL
and oral bioavailability obtained for 46 could be due to improved first-pass hepatic
O
H
N COOH
NHCbz
HN
H
N
OBn
O
O
F
63
64
HATU, DIPEA
THF
DCM, rt, 18 h
O
H
N
NHCbz
N
H
N
OBn
O
O
O
F
65
H2, Pd/C
THF
O
H
N
NH2
N
H
N
OH
O
O
O
F
66
O
HNNH
N HN
Me
O
O
O
F
46
DEPBT, DIPEA
THF
rt, 16–18 h
46•HCl
O
NHCbz
OH 1. TsCl, DMAP, TEA
0°C > rt, O/N
2. KI, DIPEA
PrCN-DMF
100°C, 48 h
3. LiOH, THF
rt, 24 h
1. HCl, EtOH
rt -> 0 °C
2. recrystallization
H2N COOMe
61
62
Scheme 4.3 Synthesis of ulimorelin (46).
4.6 Preclinical Evaluation j97
and/or intestinal metabolism, perhaps mediated by P450 enzymes. In addition,
improved RLM and HLM stability was observed for 51, which contains a fluorophenyl
tether, in comparison with 46 and 60. Overall, these results imply that the
improved rat PK profile obtained with para-fluorobenzylic tether moieties (51 and
53) may be due to both improved first-pass clearance and enhanced absorption
assisted by Pgp active transport.
Following a 30 min i.v. infusion at a target dose level of 6 mg/kg in SpragueDawley
rats, 14
C-labeled 46 was efficiently excreted in the feces unchanged with a
recovery of 95% of the administered dose6)
. These results are consistent
with hepatobiliary elimination [44] and suggests that 46 would be subjected to liver
first-pass metabolism after PO dosing. It is also noteworthy that 46 displayed a
reasonably high unbound fraction in rat plasma (fu ¼ 13%) compared to human
plasma (fu ¼ 1.6%)7)
. Subsequent studies have determined that 46 is actually even
more strongly protein bound than this in man (!99%), primarily to
a1-acid glycoprotein [45].
4.6.2
Additional Pharmacokinetic Data
Shown in Tables 4.7 and 4.8 are PK data in rats and monkeys, respectively, for various
analogues that displayed good overall PD and PK profiles with the most
advanced compounds only being examined in the latter species.
Whereas 46 displayed identical oral bioavailability in monkeys and rats, the bioavailability
of compound 51 in monkeys was nearly fivefold lower than that
observed in the rodent species. Parallel trends for clearance followed for these two
Table 4.6 Physiochemical profile, permeability, and metabolic stability of advanced analogues.
Caco-2 flux (nm/s)b
RLMc
HLMd
Compound
MW
(g/mol) clogPa
H-bond
donors
H-bond
acceptors s-to-me
m-to-se
%cmpdf
T1/2 (min)
46 538.6 5.3 3 5 140 150 68 19
51 542.6 5.1 3 5 125 198 54 41
60 557.1 6.4 3 5 150 140 86 5
a) Calculated using ChemDrawUltra v. 12.0.
b) For reference, propranolol, a high-permeability compound, displays Caco-2 flux values of
$350 nm/s.
c) RLM: rat liver microsomes.
d) As a reference, human liver microsome (HLM) T1/2 value for propranolol was 59 min.
e) s-to-m and m-to-s represent serosal-to-mucosal and mucosal-to-serosal, respectively.
f) %cmpd refers to % of compound metabolized after 15 min.
6) Compound was dosed in 5% dextrose in water containing sodium acetate buffer.
7) Equilibrium protein binding results were obtained using an ultracentrifugation method.
98j4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin
compounds; thus, in monkeys, 46 was cleared 2.3 times more slowly than 51, representing
a greater disparity versus the same trends observed in rats in which both
analogues cleared at similar rates.
The majority of drug–drug interactions are metabolism based and, moreover,
these interactions typically involve inhibition of cytochrome P450s. Six CYP450
enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) appear
to be commonly responsible for the metabolism of most drugs and binding to these
and other isoforms are often evaluated to determine potential risk from drug–drug
interactions [46]. The overall cytochrome P450 profile for 46 appeared somewhat
better than that of 51 (Table 4.9) even though both analogues displayed suboptimal
CYP3A4 profiles that are nonetheless acceptable for the acute treatment regimen
envisioned.
The data from these profiling efforts taken together with the in vivo efficacy
results (Section 4.6.3) affirmed 46 as the clinical candidate of choice.
Table 4.8 PK profiles of advanced analogues in monkey.a
Compound
PO, 8 mg/kg IV, 2 mg/kg
%Fb
Cmax
(mM)
AUC0À1
(mM min)
T1/2
(min)
Vdss
(l/kg)
CL
(ml/min/kg)
46 1.4 75.4 23 0.23 8.8 24
51 0.09 10.3 29 0.66 20 7
a) Adult male cynomolgus monkeys were administered compounds either as an i.v. bolus at 0.5 mg/kg
in physiological saline with 9% 2-hydroxypropyl-b-cyclodextrin or PO (gavage) at 1.5 mg/kg in physiological
saline.
b) %F: absolute oral bioavailability.
Table 4.7 PK profiles of representative analogues in rat.a
Compound
PO, 8 mg/kg i.v., 2 mg/kg
%Fb
Cmax
(mM)
AUC0À1
(mM min)
T1/2
(min) Vdss (l/kg)
CL
(ml/min/kg)
46 0.39 82.0 50 1.7 24 24
51 0.90 99.9 44 1.8 28 36
53 0.75 125 66 0.9 9 15
57 0.67 73.8 41 1.4 24 22
60 0.06 3.51 33 3.2 67 3
a) Male Sprague-Dawley rats were administered compounds either as an i.v. bolus at 2 mg/kg in physiological
saline with 9% 2-hydroxypropyl-b-cyclodextrin or PO (gavage) at 8 mg/kg in physiological
saline.
b) %F: absolute oral bioavailability.
4.6 Preclinical Evaluation j99
4.6.3
Animal Models for Preclinical Efficacy
Based upon the output from the medicinal chemistry efforts and the additional
profiling just described, the most promising macrocycles were advanced into
proof-of-concept testing in an animal model of gastric emptying (Table 4.10). Gratifyingly,
these compounds displayed potent effects on the stimulation of GI motility
as was expected from these ghrelin agonists.
As further evidence of in vivo prokinetic activity of ulimorelin (46), its effectiveness
as a treatment in a rat model of POI has been reported [47]. In contrast to
other classes of ghrelin agonists, ulimorelin did not appear to stimulate concurrent
GH secretion in these animal models [48].
4.7
Clinical Results and Current Status
Based upon the promising preclinical data, ulimorelin (compound 46) was
advanced into the clinic in early 2006 as an intravenous treatment for restoration of
GI motility following surgery. Table 4.11 summarizes the six clinical trials conducted,
completed, and reported to date for ulimorelin.
Table 4.10 Gastric emptying data (i.v. administration) in rats for advanced analogues.
Compound
Gastric emptyinga
(i.v.)b
,
EC50 (mg/kg)c
46 0.91
51 1.20
53 1.38
57 0.84
60 0.39
a) Conducted with overnight-fasted male Wistar rats given a 2% methylcellulose meal containing
10% phenol red administered orally at 2 ml/animal and sacrificed 15 min later.
Stomach contents were measured at sacrifice by colorimetric analysis (560 nm) for determination
of phenol red remaining.
b) Administered coincident with the meal in 9% 2-hydroxypropyl-b-cyclodextrin.
c) For reference, ghrelin peptide in this assay has EC50 ¼ 0.04 mg/kg.
Table 4.9 Human cytochrome P450 profile of advanced analogues.
Compound
CYP P450 IC50 (mM)
3A4 2D6 2C9 2C19 1A2
46 2.4 84 66 >100 >100
51 1.2 31 >100 >100 >100
100j4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin
Table4.11Clinicalevaluationofulimorelin.
Clinical
phaseDesignandsizeUlimorelindoselevelsResults
Phase
1a[49]
Prospective,randomized,double-blind,placebo-
controlled,doseescalationtrialtoassesssafety,
tolerability,pharmacokinetics,and
pharmacodynamicsin48healthyvolunteers
Single30mini.v.infusion,sixdose
rangesfrom20to600mg/kgorplacebo
Alldoseswerewelltolerated,PKanalysisrevealed
lessthandose-proportionalbehaviorofdrugwith
lowclearance(approximately7ml/h/kg),small
volumeofdistribution(approximately114ml/kg),
andhalf-lifevaluesofapproximately13h,which
wereindependentofdose.Pharmacodynamic
analysessuggestedulimorelin,atdosesaslowas
40mg/kg,expressedactivityatthereceptor.
Phase
1b[50]
Dose-escalatingdouble-blindstudyin12healthy
subjects,withsix(6)additionalatthehighestdose,
forevaluationofsafety,tolerability,
pharmacokinetics,andpharmacodynamics
30mini.v.infusionofoneofthreedose
levels(80,320,and600mg/kg)or
placeboforfiveconsecutivedays
Ulimorelinwaswelltoleratedandsafeafter
infusionsacrossseveraldoselevelswithnodose-
dependentdrug-relatedeventsobserved.PKwas
linear,butnotdoseproportional,possiblyasaresult
ofconcentration-dependentproteinbinding.
Confirmedlowclearanceandlowvolumeof
distributionwithplasmahalf-lifeof20h.
Phase1
(QTc)
Double-blind,randomized,parallelstudyto
comparetheECGeffectsofulimorelintoplacebo
andmoxifloxacin(apositivecontrolknownto
increasetheQTinterval)in160healthymenand
women
Twodosesdailyforfiveconsecutive
days,therapeutic(160mg/kg)and
supratherapeuticdose(600mg/kg)
Noeffectoncardiacparametersormorphology.
Measuredamultitudeofelectrophysiological
parameterswithnoapparentdeleteriouseffecton
anyofthem.
Phase
2a[51]
Double-blind,crossovertrialin10subjectswith
moderate-to-severediabeticgastroparesis
30mini.v.infusionofoneoffourdoses
(80,160,320,or600mg/kg)orplacebo
Ulimorelinshowedastatisticallysignificant
accelerationofgastricemptying(p¼0.043)and
reductionoflatencytimes(p¼0.037)ofasolid
radiolabeledmealrelativetoplacebo.
(continued)
4.7 Clinical Results and Current Status j101
Table4.11(Continued)
Clinical
phaseDesignandsizeUlimorelindoselevelsResults
Phase
2b[52]
Double-blind,randomized,placebo-controlledtrial
in76diabeticpatientswithmoderate-to-severe
symptomsduetodiabeticgastroparesis
Single30mini.v.infusionofoneofsix
dosesrangingfrom20to600mg/kgor
placebooncedailyforfourconsecutive
days
Symptomswereevaluateddailywiththe
GastroparesisCardinalSymptomIndex(GCSI,
consistingofninepatient-ratedsymptom
assessmentsforquantifyingtheseverityof
gastroparesis.The80mg/kgdosewasidentifiedas
themosteffectivedose;statisticallysignificant
improvementcomparedwithplacebointheseverity
ofGCSIlossofappetiteandvomitingscoresforthat
dosegroup(p¼0.034andp¼0.006).Inaddition,
theproportionofpatientswithatleast50%
improvementinvomitingscorewassignificantly
higherwithulimorelin(p¼0.019)comparedwith
placebo.
Phase
2b[53]
Multinational,multicenter,double-blind,dose-
ranging,placebo-controlledtrialemployingan
adaptiverandomizationdesignin236patients
undergoingopenpartialcolectomy
Oneofsevendoselevels,20–600mg/kg,
orplacebo,singledailydoseby30min
i.v.infusionforuptosevenconsecutive
days
Ulimorelinacceleratedthetimetofirstbowel
movementinallgroups,withp¼0.056forthelow
efficaciousdose(80mg/kg)andp¼0.03forthemost
efficaciousdose(480mg/kg).Themediantimeto
firstbowelmovementwasreducedinalltreatment
groupsby10–22hversusplacebo.Atthe480mg/kg
dose,themediantimetoreadinessforhospital
dischargewassignificantlyacceleratedby20.4h
comparedwithplacebo(p¼0.03).Agreaternumber
ofpatientswhoreceivedulimorelinachieved
recoveryby72hpostsurgerycomparedwithplacebo
(p0.001).
102j4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin
In summary, these studies have shown that ulimorelin
 has potent GI motility effects in humans;
 is efficacious in the treatment of two disorders characterized by GI dysmotility,
POI and diabetic gastroparesis;
 possesses better PK parameters in humans than had been anticipated based
upon the effects in preclinical animal models;
 gives PK profiles in both healthy subjects and patients that were less than dose
proportional, most likely because of concentration-dependent protein binding;
 exhibits very good safety in humans, with no indication of the side effects that
have plagued previous GI promotility agents.
Ulimorelin has just completed two multinational phase 3 studies for the treatment
of restoration of GI function following partial bowel resection surgery in the
United States and Europe. Each trial targeted 360 patients and was conducted at
approximately 50 sites in North America and Europe. Patients in these doubleblind,
placebo-controlled trials were evenly randomized to one of three treatment
groups: placebo, 160 mg/kg, or 480 mg/kg, doses which were selected based on data
from the phase 2 trial. Topline results from these trials have been reported, which
disappointingly failed to meet its primary and secondary efficacy end points [54].
While analysis of the data from these trials continues, all other NDA development
activities for ulimorelin have been placed on hold.
4.8
Summary
The discovery and development of ulimorelin admirably demonstrates the tremendous
utility of mining underexplored chemotypes in the search for new pharmacological
activity. Drug discovery using these macrocycles proceeds with both
differences and similarities to efforts with more traditional scaffolds. Although
these compounds do not typically conform to certain standard measurements of
druglikeness, such as the Lipinski rules, they do match better with the revised considerations
advocated by Veber that emphasize polar surface area (PSA) and rotatable
bond count [55].
Further, in many traditional efforts, moving from hit to clinical candidate has
been seen to add molecular weight and increase lipophilicity [56]. With this macrocyclic
scaffold, however, significant property modification can be achieved without
such increases. Indeed, the MW of ulimorelin (46) was merely 9% higher than analogue
1 and clogP increased only 12%.
In conclusion, starting from a library of unique, conformationally defined macrocyclic
molecules, a promising hit series was progressed through a series of approximately
500 analogues, based upon a systematic investigation of each of the four
diversity components of the structures, to ultimately produce a viable clinical candidate
with a novel mechanism of action, ghrelin receptor agonism, for gastrointestinal
disorders. In addition, the capacity for the nature of one of these
4.8 Summary j103
components, the tether, to have profound effects on the PK–ADME parameters
suggests that there will be much new knowledge to be gained as additional investigations
are conducted on this structural class.
References
1 Kojima, M., Hosoda, H., Date, Y.,
Nakazato, M., Matsuo, H., and Kangawa,
K. (1999) Ghrelin is a growth-hormonereleasing
acylated peptide from stomach.
Nature, 402, 656–660.
2 (a) Broglio, F., Gottero, C., Arvat, E., and
Ghigo, E. (2003) Endocrine and nonendocrine
actions of ghrelin. Horm. Res.,
59, 109–117. (b) Hosoda, H., Kojima, M.,
and Kangawa, K. (2006) Biological,
physiological, and pharmacological
aspects of ghrelin. J. Pharmacol. Sci., 100,
398–410.
3 Howard, A.D., Feighner, S.D., Cully, D.F.,
Arena, J.P., Liberator, P.A., Rosenblum,
C.I., Hamelin, M., Hreniuk, D.L., Palyha,
O.C., Andrerson, J., Paress, P.S., Diaz, C.,
Chou, M., Liu, K.K., McKee, K.K., Pong,
S.-S., Chaung, L.-Y., Elbrecht, A.,
Dashkevicz, M., Heavens, R., Rigby, M.,
Sirinathsighji, D.J.S., Dean, D.C., Melillo,
D.G., Patchett, A.A., Nargund, R., Griffin,
P.R., DeMartino, J.A., Gupta, S.K.,
Schaeffer, J.M., Smith, R.G., and Van der
Ploeg, L.H.T. (1996) A receptor in pituitary
and hypothalamus that functions in
growth hormone release. Science, 273,
974–977.
4 Davenport, A.P., Bonner, T.I., Foord, S.M.,
Harmar, A.J., Neubig, R.R., Pin, J.P.,
Spedding, M., Kojima, M., and Kangawa,
K. (2005) International Union of
Pharmacology. LVI. Ghrelin receptor
nomenclature, distribution, and function.
Pharmacol. Rev., 57, 541–546.
5 Ghelardoni, S., Carnicelli, V., Frascarelli,
S., Ronca-Testoni, S., and Zucchi, R. (2006)
Ghrelin tissue distribution: comparison
between gene and protein expression.
J. Endocrinol. Invest., 29, 115–121.
6 Gnanapavan, S., Kola, B., Bustin, S.A.,
Morris, D.G., McGee, P., Fairclough, P.,
Bhattacharya, S., Carpenter, R.,
Grossman, A.B., and Korbonits, M. (2002)
The tissue distribution of the mRNA of
ghrelin and subtypes of its receptor,
GHS-R, in humans. J. Clin. Endocrinol.
Metab., 87, 2988–2991.
7 (a) Svensson, J. (2000) Growth hormone
secretagogues. Exp. Opin. Ther. Pat., 10,
1071–1080. (b) Nargund, R.P., Patchett,
A.A., Bach, M.A., Murphy, M.G., and
Smith, R.G. (1998) Peptidomimetic
growth hormone secretagogues. Design
considerations and therapeutic potential.
J. Med. Chem., 41, 3103–3127. (c) Ghigo,
E., Arvat, E., and Camanni, F. (1998)
Orally active growth hormone
secretagogues: state of the art and clinical
perspective. Ann. Med., 30, 159–168.
(d) Smith, R.G., Van der Ploeg, L.H.T.,
Howard, A.D., Feighner, S.D., Cheng, K.,
Hickey, G.J., Wyvratt, M.J., Jr., Fisher, M.
H., Nargund, R.P., and Patchett, A.A.
(1997) Peptidomimetic regulation of
growth hormone secretion. Endocr. Rev.,
18, 621–645.
8 (a) Smith, R.G., Sun, Y.X., Beatancourt, L.,
and Asnicar, M. (2004) Growth hormone
secretagogues: prospects and pitfalls. Best
Pract. Res. Clin. Endocrinol. Metab., 18,
333–347. (b) Fehrentz, J.-A., Martinez, J.,
Boeglin, D., Guerlavais, V., and
Deghenghi, R. (2002) Growth hormone
secretagogues: past, present and future.
IDrugs, 5, 804–814.
9 A peptidomimetic GHS, NN703, was also
shown to cause irreversible inhibition of
cytochrome P450 enzymes: Zdravkovic,
M., Olse, A.K., Christiansen, T., Schulz,
R., Taub, M.E., Thomsen, M.S.,
Rasmussen, M.H., and Ilondo, M.M.
(2003) A clinical study investigating the
pharmacokinetic interaction between
NN703 (tabimorelin), a potential inhibitor
of CYP3A4 activity, and midazolam, a
CYP3A4 substrate. Eur. J. Clin.
Pharmacol., 58, 683–688.
10 (a) Kojima, M. and Kangawa, K. (2006)
Drug insight: the functions of ghrelin and
104j4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin
its potential as a multitherapeutic
hormone. Nat. Clin. Pract. Endocrinol.
Metab., 2, 80–88. (b) Akamizu, T. and
Kangawa, K. (2006) Translational research
on the clinical applications of ghrelin.
Endocr. J., 53, 585–591. (c) Leite-Moreira,
A.F. and Soares, J.-B. (2007) Physiological,
pathological and potential therapeutic
roles of ghrelin. Drug Discov. Today, 12,
276–288. (d) Katergari, S.A., Milousis, A.,
Pagonopoulou, O., Asimakopoulos, B.,
and Nikolettos, N.K. (2008) Ghrelin in
pathological conditions. Endocr. J., 55,
439–453.
11 (a) Kamiji, M.M. and Inui, A. (2008) The
role of ghrelin and ghrelin analogues in
wasting disease. Curr. Opin. Clin. Nutr.
Metab. Care, 11, 443–451. (b) DeBoer, M.
D. (2008) Emergence of ghrelin as a
treatment for cachexia syndromes.
Nutrition, 24, 806–814.
12 (a) Garcia, J.M. and Polvino, W.J. (2007)
Effect on body weight and safety of RC-
1291, a novel, orally available ghrelin
mimetic and growth hormone
secretagogue: results of a phase I,
randomized, placebo-controlled, multipledose
study in healthy volunteers.
Oncologist, 12, 594–600. (b) Strasser, F.,
Lutz, T.A., and Maeder, M.T. (2008) Safety,
tolerability and pharmacokinetics of
intravenous ghrelin for cancer-related
anorexia/cachexia: a randomised, placebocontrolled,
double-blind, double-crossover
study. Br. J. Cancer, 98, 300–308.
(c) Garcia, JM. and Polvino, W.J. (2009)
Pharmacodynamic hormonal effects of
anamorelin, a novel oral ghrelin mimetic
and growth hormone secretagogue in
healthy volunteers. Growth Horm. IGF
Res., 19, 267–273.
13 Smith, R.G., Sun, Y., Jiang, H., AlbarranZeckler,
R., and Timchenko, N. (2007)
Ghrelin receptor (GHS-R1A) agonists
show potential as interventive agents
during aging. Ann. N. Y. Acad. Sci., 1119,
147–164.
14 (a) Horvath, T.L., Casta~neda, T., TangChristensen,
M., Pagotto, U., and Tsch€op,
M.H. (2003) Ghrelin as a potential antiobesity
target. Curr. Pharm. Des., 9, 1383–
1395. (b) Cummings, D.E. (2006) Ghrelin
and the short- and long-term regulation of
appetite and body weight. Physiol. Behav.,
89, 71–84.
15 Murray, C.D.R., Kamm, M.A., Bloom, S.
R., and Emmanuel, A.V. (2003) Ghrelin
for the gastroenterologist: history and
potential. Gastroenterology, 125, 1492–
1502.
16 Everhart, J.E. (ed.) (2008) The Burden of
Digestive Diseases in the United States, NIH
Publication 09-6433, National Institute of
Diabetes and Digestive and Kidney
Diseases, U.S. Department of Health and
Human Services, Bethesda, MD.
17 (a) Kojima, M. Hsoda, H., and Kangawa,
K. (2001) Purification and distribution of
ghrelin: the natural endogenous ligand for
the growth hormone secretagogue
receptor. Horm. Res., 56 (Suppl. 1), 93–97.
(b) Ariyasu, H., Takaya, K., Tagami, T.,
Ogawa, Y., Hosoda, K., Akamizu, T., Suda,
M., Koh, T., Natsui, K., Toyooka, S.,
Shirakami, G., Usui, T., Shimatsu, A.,
Doi, K., Hosoda, H., Kojima, M.,
Kangawa, K., and Nakao, K. (2001)
Stomach is a major source of circulating
ghrelin, and feeding state determines
plasma ghrelin-like immunoreactivity
levels in humans. J. Clin. Endocrinol.
Metab., 86, 4753–4758.
18 Inui, A., Asakawa, A., Bowers, C.Y.,
Mantovani, G., Laviano, A., Meguid, M.
M., and Fujimiya, M. (2004) Ghrelin,
appetite, and gastric motility: the
emerging role of the stomach as an
endocrine organ. FASEB J., 18, 439–456.
19 (a) Peeters, T.L. (2003) Central and
peripheral mechanisms by which ghrelin
regulates gut motility. J. Physiol.
Pharmacol., 54 (Suppl. 4), 95–103.
(b) Edholm, T., Levin, F., Hellstr€om, P.M.,
and Schmidt, P.T. (2004) Ghrelin
stimulates motility in the small intestine
of rats through intrinsic cholinergic
neurons. Regul. Pept., 121, 25–30.
20 (a) Masuda, Y., Tanaka, T., Inomata, N.,
Ohnuma, N., Tanaka, S., Itoh, Z., Hosoda,
H., Kojima, M., and Kangawa, K. (2000)
Ghrelin stimulates gastric acid secretion
and motility in rats. Biochem. Biophys. Res.
Commun., 276, 905–908. (b) Fujino, K.,
Inui, A., Asakawa, A., Kihara, N.,
Fujimura, M., and Fujimiya, M. (2003)
Ghrelin induces fasting motor activity of
References j105
the gastrointestinal tract in conscious fed
rats. J. Physiol., 550, 227–240.
21 (a) Trudel, L., Tomasetto, C., Rio, M.C.,
Bouin, M., Plourde, V., Eberling, P., and
Poitras, P. (2002) Ghrelin/motilin-related
peptide is a potent prokinetic to reverse
gastric postoperative ileus in rats. Am. J.
Physiol., 282, G948–G952. (b) Trudel, L.,
Bouin, M., Tomasetto, C., Eberling, P.,
St-Pierre, S., Bannon, P., L’Heureux, M.
C., and Poitras, P. (2003) Two new
peptides to improve post-operative gastric
ileus in dog. Peptides, 24, 531–534.
22 (a) This is now a well-established
paradigm: Peeters, T.L. (2006) Potential of
ghrelin as a therapeutic approach for
gastrointestinal motility disorders. Curr.
Opin. Pharmacol., 6, 553–558. (b) Sanger,
G.J. (2008) Motilin, ghrelin and related
neuropeptides as targets for the treatment
of GI diseases. Drug Discov. Today, 13,
234–239. (c) Venkova, K. and GreenwoodVan
Meerveld, B. (2008) Application of
ghrelin to gastrointestinal diseases. Curr.
Opin. Invest. Drugs, 9, 1103–1107.
(d) DeSmet, B., Mitselos, A., and
Depoortere, I. (2009) Motilin and ghrelin
as prokinetic drug targets. Pharmacol.
Ther., 123, 207–223. (e) Greenwood-Van
Meerveld, B., Kriegsman, M., and Nelson,
R. (2011) Ghrelin as a target for
gastrointestinal motility disorders.
Peptides, 32, 2352–2356.
23 (a) Kitazawa, T., De Smet, B., Verbeke, K.,
Depoortere, I., and Peeters, T.L. (2005)
Gastric motor effects of peptide and nonpeptide
ghrelin agonists in mice in vivo
and in vitro. Gut, 54, 1078–1084.
(b) Poitras, P., Polvino, W.J., and
Rocheleau, B. (2005) Gastrokinetic effect
of ghrelin analog RC-1139 in the rat.
Effect on post-operative and on morphine
induced ileus. Peptides, 26, 1598–1601.
24 (a) Murray, C.D.R., Martin, N.M.,
Patterson, M., Taylor, S., Ghatei, M.A.,
Kamm, M.A., Johnston, C., Bloom, S.R.,
and Emmanuel, A.V. (2005) Ghrelin
enhances gastric emptying in diabetic
gastroparesis: a double blind, placebo
controlled, crossover study. Gut, 54,
1693–1698. (b) Tack, J., Depoortere, I.,
Bisschops, R., Verbeke, K., Janssens, J.,
and Peeters, T. (2005) Influence of ghrelin
on gastric emptying and meal-related
symptoms in idiopathic gastroparesis.
Aliment. Pharmacol. Ther., 22, 847–853.
(c) Binn, M., Albert, C., Gougeon, A.,
Maerki, H., Coulie, B., Lemoyne, M.,
Rabasa Lhoret, R., Tomasetto, C., and
Poitras, P. (2006) Ghrelin gastrokinetic
action in patients with neurogenic
gastroparesis. Peptides, 27, 1603–1606.
25 Tack, J., Depoortere, I., Bisschops, R.,
Delporte, C., Coulie, B., Meulemans, A.,
Janssens, J., and Peeters, T. (2006)
Influence of ghrelin on interdigestive
gastrointestinal motility in humans. Gut,
55, 327–333.
26 (a) For representative reviews, see
Gershon, M.D. and Tack, J. (2007) The
serotonin signaling system: from basic
understanding to drug development for
functional GI disorders. Gastroenterology,
132, 397–414. (b) Spiller, R. (2008)
Serotonin and GI clinical disorders.
Neuropharmacology, 55, 1072–1080.
27 Fernandez, AG. and Massingham, R.
(1985) Peripheral receptor populations
involved in the regulation of
gastrointestinal motility and the
pharmacological actions of
metoclopramide-like drugs. Life Sci.,
36, 1–14.
28 Quigley, E.M. (2011) Cisapride: what can
we learn from the rise and fall of a
prokinetic? J. Dig. Dis., 12, 147–156.
29 Al-Judaibi, B., Chande, N., and Gregor, J.
(2010) Safety and efficacy of tegaserod
therapy in patients with irritable bowel
syndrome or chronic constipation. Can. J.
Clin. Pharmacol., 17, e194–200.
30 Pasricha, P.J., Pehlivanov, N., Sugumar,
A., and Jankovic, J. (2006) Drug Insight:
from disturbed motility to disordered
movement – a review of the clinical
benefits and medicolegal risks of
metoclopramide. Nat. Clin. Pract.
Gastroenterol. Hepatol., 3, 138–148.
31 Drewry, D.H. and Macarron, R. (2010)
Enhancements of screening collections to
address areas of unmet medical need: an
industry perspective. Curr. Opin. Chem.
Biol., 14, 289–298.
32 Lipinski, C.A., Lombardo, F., Dominy, B.
W., and Feeney, P.J. (1997) Experimental
and computational approaches to estimate
106j4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin
solubility and permeability in drug
discovery and development settings. Adv.
Drug Deliv. Rev., 23, 3–25.
33 (a) Driggers, E.M., Hale, S.P., Lee, J., and
Terrett, N.K. (2008) The exploration of
macrocycles for drug discovery – an
underexploited structural class. Nat. Rev.
Drug Discov., 7, 608–624. (b) Terrett, N.K.
(2010) Methods for the synthesis of
macrocycle libraries for drug discovery.
Drug Discov. Today, 7 (2), e97–e104.
(c) Marsault, E.M. and Peterson, M.L.
(2011) Macrocycles are great cycles:
applications, opportunities, and
challenges of synthetic macrocycles in
drug discovery. J. Med. Chem., 54, 1961–
2004.
34 Marsault, E., Hoveyda, H.R., Gagnon, R.,
Peterson, M.L., Vezina, M., Saint-Louis,
C., Landry, A., Pinault, J.-F., Ouellet, L.,
Beauchemin, S., Beaubien, S., Mathieu,
A., Benakli, K., Wang, Z., Brassard, M.,
Lonergan, D., Bilodeau, F., Ramaseshan,
M., Fortin, N., Lan, R., Li, S., Galaud, F.,
Plourde, V., Champagne, M., Doucet, A.,
Bherer, P., Gauthier, M., Olsen, G.,
Villeneuve, G., Bhat, S., Foucher, L.,
Fortin, D., Peng, X., Bernard, S., Drouin,
A., Deziel, R., Berthiaume, G., Dory, Y.L.,
Fraser, G.L., and Deslongchamps, P.
(2008) Efficient parallel synthesis of
macrocyclic peptidomimetics. Bioorg.
Med. Chem. Lett., 18, 4731–4735.
35 Xiao, X.Y., Li, R., Zhuang, H., Ewing, B.,
Karunaratne, K., Lillig, J., Brown, R., and
Nicolaou, K.C. (2000) Solid-phase
combinatorial synthesis using MicroKan
reactors, Rf tagging, and directed sorting.
Biotechnol. Bioeng., 71, 44–50.
36 Le Poul, E., Hisada, S., Mizuguchi, Y.,
Dupriez, V.J., Burgeon, E., and Detheux,
M. (2002) Adaptation of aequorin
functional assay to high throughput
screening. J. Biomol. Screen., 7, 57–65.
37 Marsault, E., Hoveyda, H.R., Peterson, M.
L., Saint-Louis, C., Landry, A., Vezina, M.,
Ouellet, L., Wang, Z., Ramaseshan, M.,
Beaubien, S., Benakli, K., Beauchemin, S.,
Deziel, R., Peeters, T., and Fraser, G.L.
(2006) Discovery of new class of
macrocyclic antagonists to the human
motilin receptor. J. Med. Chem., 49,
7190–7197.
38 For additional discussions on the
development of ulimorelin, see Hoveyda,
H.R., Marsault, E., Gagnon, R., Mathieu,
A.P., Vezina, M., Landry, A., Wang, Z.,
Benakli, K., Beaubien, S., Saint-Louis, C.,
Brassard, M., Pinault, J.F., Ouellet, L.,
Bhat, S., Ramaseshan, M., Peng, X.,
Foucher, L., Beauchemin, S., Bherer, P.,
Veber, D.F., Peterson, M.L., and Fraser, G.
L. (2011) Optimization of the potency and
pharmacokinetic properties of a
macrocyclic ghrelin receptor agonist. Part
I. Development of ulimorelin (TZP-101)
from hit to clinic. J. Med. Chem., 54,
8305–8320.
39 Rose, G.D., Gierasch, L.M., and Smith,
J.A. (1985) Turns in peptides and proteins.
Adv. Protein Chem., 37, 1–109.
40 Tyndall, J.D.A., Pfeiffer, B., Abbenante,
G., and Fairlie, D.P. (2005) Over one
hundred peptide-activated G protein
coupled receptors recognize ligands with
turn structure. Chem. Rev., 105, 793–826.
41 Since distances are significantly shorter
than the sum of the van der Waals radii:
Bondi, A. (1964) van der Waals volumes
and radii. J. Phys. Chem., 68, 441–451.
42 (a) Di, L. and Kerns, E.H. (2003) Profiling
drug-like properties in discovery research.
Curr. Opin. Chem. Biol., 7, 402–408. (b) Li,
A.P. (2005) Preclinical in vitro screening
assays for drug-like properties. Drug
Discov. Today Technol., 2, 179–185.
43 van Breemen, R.B. and Li, Y. (2005) Caco-
2 cell permeability assays to measure drug
absorption. Expert Opin. Drug Metab.
Toxicol., 1, 175–185.
44 (a) Biliary excretion is commonly observed
in cyclic peptides or peptidomimetics; see,
for example, Veber, D.F., Saperstein, R.,
Nutt, R.F., Freidinger, R.M., Brady, S.F.,
Curley, P., Perlow, D.S., Paleveda, W.J.,
Colton, C.D., Zacchei, A.G., Tocco, D.J.,
Hoff, D.A., Vandlen, R.L., Gerich, J.E.,
Hall, L., Mandarino, L., Cordes, E.H.,
Anderson, P.S., and Hirschmann, R.
(1984) A super active cyclic hexapeptide
analog of somatostatin. Life Sci., 34, 1371–
1378. (b) Llinas-Burnet, M., Bailey, M.D.,
Bolger, G., Brochu, C., Faucher, A.-M.,
Ferland, J.M., Garneau, M., Ghiro, E.,
Gorys, V., Grand-Ma^õtre, C., Halmos, T.,
Lapeyre-Paquette, N., Liard, F., Poirier,
References j107
M., Rheaume, M., Tsantrizos, Y.S., and
Lamarre, D. (2004) Structure–activity
study on a novel series of macrocyclic
inhibitors of the hepatitis C virus NS3
protease leading to the discovery of BILN
2061. J. Med. Chem., 47, 1605–1608.
45 Wargin, W., Thomas, H., Clohs, L., StLouis,
C., Ejskjaer, N., Gutierrez, M.,
Shaughnessy, L., and Kosutic, G. (2009)
Contribution of protein binding to the
pharmacokinetics of the ghrelin receptor
agonist TZP-101 in healthy volunteers and
adults with symptomatic gastroparesis:
two randomized, double-blind studies and
a binding profile study. Clin. Drug Invest.,
29, 409–418.
46 White, R.E. (2000) High-throughput
screening in drug metabolism and
pharmacokinetic support of drug
discovery. Annu. Rev. Pharmacol. Toxicol.,
40, 133–157.
47 (a) Venkova, K., Fraser, G., Hoveyda, H.R.,
and Greenwood-Van Meerveld, B. (2007)
Prokinetic effects of a new ghrelin
receptor agonist TZP-101 in a rat model of
postoperative ileus. Dig. Dis. Sci., 52,
2241–2248. (b) Fraser, G.L., Venkova, K.,
Hoveyda, H.R., Thomas, H., and
Greenwood-Van Meerveld, B. (2009) Effect
of the ghrelin receptor agonist TZP-101
on colonic transit in a rat model of
postoperative ileus. Eur. J. Pharmacol.,
604, 132–137.
48 Fraser, G.L., Hoveyda, H.R., and
Tannenbaum, G.S. (2008) Pharmacological
demarcation of the growth hormone, gut
motility and feeding effects of ghrelin
using a novel ghrelin receptor agonist.
Endocrinology, 149, 6280–6288.
49 Lasseter, K.C., Shaughnessy, L.,
Cummings, D., Pezzullo, J.C., Wargin, W.,
Gagnon, R., Oliva, J., and Kosutic, G.
(2008) Ghrelin agonist (TZP-101): safety,
pharmacokinetics and pharmacodynamic
evaluation in healthy volunteers: a phase I,
first-in-human study. J. Clin. Pharmacol.,
48, 193–202.
50 Gutierrez, M., Shaughnessy, L.,
Cummings, D., Pezzullo, J., Reeve, R.,
Reilly, R., and Kosutic, G. (2007) Ghrelin
agonist (TZP-101): pharmacokinetics and
pharmacodynamics: a multi-dose
evaluation in healthy volunteers.
Neurogastrol. Motil., 19, 428–429.
51 Ejskjaer, N., Vestergaard, E.T., Hellstr€om,
P.M., Gormsen, L.C., Madsbad, S.,
Madsen, J.L., Jensen, T.A., Pezzullo, J.C.,
Christiansen, J.S., Shaughnessy, L., and
Kosutic, G. (2009) Ghrelin receptor
agonist (TZP-101) accelerates gastric
emptying in adults with diabetes and
symptomatic gastroparesis. Aliment.
Pharmacol. Ther., 29, 1179–1187.
52 (a) Ejskjaer, N., Dimcevski, G., Wo, J.,
Hellstr€om, P.M., Gormsen, L.C., Sarosiek,
I., Sùfteland, E., Nowak, T., Pezzullo, J.C.,
Shaughnessy;, L., Kosutic, G., and
McCallum, R. (2010) Safety and efficacy of
ghrelin agonist TZP-101 in relieving
symptoms in patients with diabetic
gastroparesis: a randomized, placebocontrolled
study. Neurogastroenterol. Motil.,
22, 1069–1078. (b) Wo, J.M., Ejskjaer, N.,
Hellstr€om, P.M., Malik, R.A., Pezzullo, J.
C., Shaughnessy, L., Charlton, P., Kosutic,
G., and McCallum, R.W. (2011)
Randomised clinical trial: ghrelin
agonist TZP-101 relieves gastroparesis
associated with severe nausea and
vomiting – randomised clinical study
subset data. Aliment. Pharmacol. Ther., 33,
679–688.
53 Popescu, I., Fleshner, P.R., Pezzullo,
J.C., Charlton, P.A., Kosutic, G., and
Senagore, A.J. (2010) The ghrelin agonist
TZP-101 for management of postoperative
ileus after partial colectomy: a
randomized, dose-ranging, placebocontrolled
clinical trial. Dis. Colon Rectum,
53, 126–134.
54 Tranzyme (2012) Corporate Press Release.
Available at http://ir.tranzyme.com/
releasedetail.cfm?ReleaseID¼656438
(last accessed March 12, 2012).
55 Veber, D.F., Johnson, S.R., Cheng, H.Y.,
Smith, B.R., Ward, K.W., and Kopple, K.D.
(2002) Molecular properties that influence
the oral bioavailability of drug candidates.
J. Med. Chem., 45, 2615–2623.
56 Keser€u, G.M. and Makara, G.M. (2009)
The influence of lead discovery strategies
on the properties of drug candidates. Nat.
Rev. Drug Discov., 8, 203–212.
108j4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin
Graeme Fraser has more than 17 years of industry experience
in both management and technical roles. Prior to his current
position as Director, Drug Discovery at Euroscreen SA, Dr.
Fraser was Vice President, Drug Discovery at Tranzyme
Pharma, where he led the discovery and preclinical development
activities for a pipeline of GPCR small-molecule programs.
Earlier, he managed preclinical development
activities at Viron Therapeutics (London, Ontario) and led
specific GPCR target validation activities at Astra Pain Control
AB (S€odert€alje, Sweden and Montreal, Quebec). In total, he has directed
research activities for three products currently in clinical development. Dr. Fraser
received a Ph.D. from McGill University and is an author of over 50 publications
and research abstracts, including nine patents and two IND filings.
Rene Gagnon earned his Ph.D. in Organic Chemistry from
the University of Laval. He conducted postdoctoral studies
with Stanley Roberts at the University of Exeter (UK). After
research positions with the Armand-Frappier Institute and
the University of Sherbrooke (UdeS), he joined NeoKimia
(now Tranzyme Pharma), where he eventually became Manager,
Analytical Chemistry & QC. He has expertise in the use
of enzymes in organic synthesis, total synthesis, small-molecule
library characterization, drug substance and drug product
analysis, pharmacokinetic and stability studies, and purification and
formulation of new chemical entities. After leaving Tranzyme, he worked as an
Associate Professor at the Biomedical Genetics Department (UdeS) and is presently
Manager of the Mass Spectrometry Facility at the UdeS Chemistry
Department.
Hamid Hoveyda is currently the Director of Medicinal
Chemistry & Development at Euroscreen SA, Belgium. He
received his Ph.D. from the University of British Columbia
(UBC) and conducted postdoctoral research at Harvard. His
first contribution to clinical candidate discovery was an insulin
mimetic agent discovered during his doctoral studies at
UBC. He began his industrial career in 1997 at Affymax
Research Institute, where he worked on applications of diversity-oriented
synthesis in drug discovery. Later, at Tranzyme
Pharma, he contributed to improvements in the proprietary macrocyclic chemistry
platform technology and then led several medicinal chemistry projects that culminated
in the discovery of ulimorelin and the follow-up oral candidate TZP-102. Dr.
Hoveyda is an author/inventor on 25 peer-reviewed papers and over 50 patent
applications.
References j109
Eric Marsault is an Associate Professor at the Institut de
Pharmacologie de Sherbrooke (IPS). He earned his Ph.D. in
Organic Chemistry from McGill University. After working
for Sanofi (Milan, Italy) as a Visiting Scientist and performing
postdoctoral studies at the University of Sherbrooke
(UdeS), he became a Research Scientist at NeoKimia (now
Tranzyme Pharma), where he rose through the ranks, culminating
as Director of Medicinal Chemistry. In 2009, he joined
the faculty at UdeS in the Pharmacology Department. His
research focuses on validating emerging pharmacological targets and emerging
approaches to existing targets, with the goal to provide new avenues for drug discovery.
Particular research interests include macrocycles, peptidomimetics, GPCR
signaling in metabolic disorders, infectious diseases, and pain, as well as emerging
antibacterial targets.
Mark L. Peterson is currently Vice President, IP and Operations
at Tranzyme Pharma. He was previously with Monsanto
and Advanced ChemTech, where he worked in a
variety of research areas including structure-based design,
solid-phase organic chemistry, combinatorial libraries, synthetic
automation, heterocycles, unnatural amino acids, peptides,
and peptidomimetics. With Tranzyme Pharma since
1999, Dr. Peterson led the chemistry R&D efforts during the
technology development stage of the company and the initiation
of its drug discovery programs. He received his Ph.D. in Organic Chemistry
from Washington State University and conducted postdoctoral research at the University
of Minnesota. He is author or coauthor of over 80 publications and
abstracted presentations plus two book chapters, as well as co-inventor on over 20
patents.
110j4 Use of Macrocycles in Drug Design Exemplified with Ulimorelin
Part II
Drug Classes
j111
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
5
The Discovery of Anticancer Drugs Targeting Epigenetic
Enzymes
A. Ganesan
List of Abbreviations
5-azaC 5-azacytidine
5-aza-dC 5-aza-20
-deoxycytidine
AML acute myelogenous leukemia
CpG cytosine phosphate guanine dinucleotide
CTCL cutaneous T-cell lymphoma
DMSO dimethyl sulfoxide
DNMT DNA methyltransferase
EGFR epidermal growth factor receptor
FDA Food and Drug Administration
HATU (2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
HDAC histone deacetylase
HER2 human epidermal growth factor receptor
IL interleukin
IND investigational new drug
MBD methyl binding domain
MDS myelodysplastic syndromes
NAD nicotinamide adenine dinucleotide
NCI National Cancer Institute
QT Q and T waves
SAHA suberoylanilide hydroxamic acid
SAM S-adenosylmethionine
ST S and T waves
STAT signal transducer and activator of transcription
j113
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
5.1
Epigenetics
Sixty years ago, Conrad Waddington first defined epigenetics as “the branch of biology
which studies the causal interactions between genes and their products” in his article
titled “The epigenotype” [1]. Subsequently, our understanding of gene structure and
function has led to the currently accepted definition of epigenetics as the study of
heritable changes in an organism’s phenotype without underlying changes in genotype.
For example, there are dramatic differences between humans although we share
99% identity at the genome level. Within a single individual, there are dramatic differences
between tissues and organs in the body although the genome is 100% identical.
These phenotypic variations are due to gene regulation as not all genes are equally
expressed in different cells or different individuals. The physical basis for gene regulation
in eukaryotes is the science of epigenetics and at the molecular level it takes place
by a complex pattern of chemical alterations of DNA and their associated histone proteins
at specific regions of a chromosome. Unlike the genome of DNA sequences, the
epigenome of DNA/histone modifications is dynamically changing in response to signals
and the environment and it can be partially transmitted to future generations.
From the drug discovery perspective, there is one major difference between genetics
and epigenetics: once a gene mutation has occurred, it is permanent and cannot be
reversed. Epigenetic modifications, on the other hand, are catalyzed by enzymes. In
principle, they are reversible by a small molecule enzyme inhibitor that can then
reverse a disease state back to the normal phenotype.
In eukaryotic cell nuclei, the genetic material is tightly packaged with histone
proteins. The basic repeating unit is the nucleosome composed of 146 base pairs of
DNA wound around four pairs of histone proteins H2A, H2B, H3, and H4 stapled
with the linker histone H1A. Together with linker DNA, the nucleosomes form
“beads on a string” that leads to the higher order structures of chromatin and chromosomes.
Chromatin exists in two forms: heterochromatin is formed when
nucleosomes are tightly condensed and genes are transcriptionally silent, whereas
euchromatin is a relaxed state that allows access to the transcription machinery and
gene expression. Recently, it has become evident that the transition between different
states of chromatin is due to extensive and reversible structural modifications
of the DNA and histone proteins. In DNA, the major alteration is the conversion of
cytosine to 5-methylcytosine that has a gene silencing effect (Figure 5.1).
N
N
NH2
O
DNA
methylation N
N
NH2
O
DNA
CH3
Figure 5.1 The methylation of cytosine residues in DNA to 5-methylcytosine.
114j5 The Discovery of Anticancer Drugs Targeting Epigenetic Enzymes
In histone proteins, the basic N-terminal regions are subject to a plethora of posttranslational
modifications including methylation, acetylation, malonylation, crotonylation,
biotinylation, polyADP-ribosylation, ubiquitinylation, and sumoylation of
lysine; methylation and polyADP-ribosylation of arginine; phosphorylation of serine
and threonine; and cis–trans isomerization of proline (Figure 5.2).
Together, this complex pattern of DNA and histone modification regulates gene
expression in two ways. First, the modifications can alter the intrinsic affinity
between DNA and histones leading to unwinding of the nucleic acid off the nucleosome
into a transcriptionally active state. Second, the modifications serve as chemical
signals that are recognized by specific binding partners that act as activators or
repressors of transcription. The pattern of DNA/histone modification is often
referred to as the “epigenetic code” [2]. The code, if it exists, is highly complex as
there are multiple sites for DNA and histone modification and their consequences
for gene regulation can be context dependent. Nevertheless, it is already clear that
the epigenome is at least partially heritable in addition to the genetic sequence and
that aberrant changes in the epigenome contribute to many human diseases.
histone
H3N
histone
H2N
CH3
histone
NH
O
acetylation
methylation
histone
OH
histone
O
PO
O
O
phosphorylation
histone
NH
HN
NH3
histone
NH
HN
NH
O
HO
HO
O
P
PO
O
O
O
O
O
OH
HO
N
N
N
N
H2N
ADP ribosylation
Figure 5.2 Examples of histone post-translational modification.
5.1 Epigenetics j115
There are three major sets of proteins involved in epigenetics:
1) Writers: enzymes that chemically modify DNA or histone proteins, for example,
DNA methyltransferases and histone acetyltransferases.
2) Erasers: enzymes that remove the modifications introduced by writers, for
example, histone deacetylases and lysine demethylases.
3) Readers: protein domains that recognize and bind to modified DNA or histones,
for example, methyl-binding proteins that recognize 5-methylcytosine
and bromodomains that recognize acetyl-lysine residues.
All three classes of epigenetic modulators are of great interest as drug discovery
targets. Attention has been primarily focused on epigenetic modulation for anticancer
therapy as there is genetic and clinical evidence that readers, writers, and
erasers are overexpressed in a variety of human cancers. In vitro proof-of-concept
studies with small molecules have confirmed the anticancer effects of epigenetic
modulators and in some cases this has progressed to in vivo models and proof of
efficacy in humans. At the present time, two inhibitors of DNA methyltransferase
and two inhibitors of Zn-dependent histone deacetylases have received FDA
approval as anticancer drugs [3].
5.2
DNA Methyltransferases
The DNMTs catalyze the C-5 methylation of cytosine by the cofactor S-adenosylmethionine
(SAM). The reaction primarily occurs within CpG dinucleotides present
in highly repeated transposable elements in DNA. The methylated cytosine
residues protrude into the DNA major groove and interfere with binding of the
transcription machinery and gene transcription. Furthermore, methylated cytosine
is specifically recognized by methyl CpG binding domains (MBDs) that function as
adaptors between DNA and other chromatin modifying enzymes. Together, these
effects lead to gene silencing. Programmed changes in DNA methylation are essential
during normal embryological development and play a crucial role in chromatin
remodeling, X chromosome inactivation, and genomic imprinting. Cancer cells
are globally hypomethylated compared to normal cells, but hypermethylated
upstream of CpG islands of silenced genes [4]. These silenced genes often encode
for tumor suppressors and a DNMT inhibitor would activate their transcription
leading to re-establishment of pathways of cell repair, apoptosis, and suppression
of proliferation.
In mammals, three DNA methyltransferases have been identified: DNMT1 and
DNMT3A/B. Among these enzymes, DNMT1 is ubiquitous and the most abundant
and primarily methylates hemimethylated DNA. Thus, it is referred to as a maintenance
methyltransferase responsible for maintaining the pattern of methylation
during DNA replication. DNMT3A/B methylate both nonmethylated and hemimethylated
DNA and are hence de novo methyltransferases that establish methylation
patterns during embryogenesis. In addition, there are nonfunctional DNMTs:
116j5 The Discovery of Anticancer Drugs Targeting Epigenetic Enzymes
DNMT2 is a tRNA methyltransferase while DNMT3L has a mutated active site that
is catalytically inactive and is involved in recruiting other binding partners to
chromatin.
In double-stranded DNA, Watson–Crick base pairing and stacking with adjacent
base pairs place the purine and pyrimidine heterocycles in the interior and
unexposed toward enzymatic reaction. X-ray crystallographic studies with DNMTs
show that the enzyme flips the cytosine substrate out of the double helix in order
for catalysis to take place. The DNMTmechanism (Figure 5.3) is composed of three
steps: (i) Transient protonation of cytosine N-3 by a glutamic acid residue increases
the pyrimidine electrophilicity and promotes Michael addition of an active site cysteine
residue to generate the imino form of the heterocycle. (ii) Nucleophilic attack
of the SAM cofactor by the imine methylates C-5. (iii) Deprotonation of the C-5
Figure 5.3 The catalytic reaction mechanism of DNA methyltransferases.
5.2 DNA Methyltransferases j117
proton by a basic enzyme residue or the DNA backbone causes b-elimination of the
cysteine to release the 5-methylcytosine product and regenerate the enzyme. Overall,
the mechanism of DNMTs is similar to that of RNA methyltransferases and
thymidylate synthase [5].
One strategy for enzyme inhibition would be to compete with SAM for the
cofactor binding pocket. Indeed, the cofactor by-product S-adenosylhomocysteine
is a competitive inhibitor of DNMTs. However, it lacks specificity and would affect
other SAM-dependent enzymes. Another approach is the design of substrate analogues
that are recognized by the enzyme but do not result in catalytic turnover.
Although not rationally designed to work in this manner, the two clinically
approved DNMT inhibitors 5-azacytidine and 5-aza-20
-deoxycytidine (Figure 5.4)
serendipitously follow this principle.
5.3
5-Azacytidine (Azacitidine, Vidaza) and 5-Aza-20
-deoxycytidine (Decitabine,
Dacogen)
The structural elucidation of DNA led to the hypothesis that compounds interfering
with nucleic acid biosynthesis would have a damaging effect on cell division
and inhibit the proliferation of tumor cells. There was a substantial interest by
medicinal chemists in the synthesis of such purine and pyrimidine antimetabolites
and a number of these are widely used anticancer drugs including 5-fluorouracil
and mercaptopurine (Figure 5.5).
With a similar goal in mind of preparing cytostatic antimetabolites, Alois Piskala
and Frantisek Sorm at the Czech Academy of Sciences reported the synthesis of
N N
NH2
ON
O
OHOH
HO
N N
NH2
ON
O
OH
HO
5-azacytidine 5-aza-2'-deoxycytidine
Figure 5.4 Structures of 5-azacytidine and 5-aza-20
-deoxycytidine.
Figure 5.5 Examples of antimetabolite drugs.
118j5 The Discovery of Anticancer Drugs Targeting Epigenetic Enzymes
5-azaC and 5-aza-dC in 1964 [6]. The azapyrimidine heterocycle was constructed
starting from a ribosyl isocyanate (Figure 5.6). Later, 5-azaC was identified as a
natural product from an extract of Streptoverticillium ladakanus [7].
5-AzaC demonstrated the ability to induce chromosomal breakage and had high
activity against lymphatic leukemia in cell lines and mouse in vivo cancer models.
Clinical trials of 5-azaC were initiated in Europe in 1967 and in the United States in
1970. In 1971, the US National Cancer Institute (NCI) filed an FDA application for
IND status for the use of 5-azaC in the treatment of multiple cancers. The clinical
trial data were promising for acute myelogenous leukemia (AML), especially in
patients who did not respond to conventional chemotherapy. However, responses
were low or absent in other cancer types and drug treatment was accompanied by
toxicity with 73% of 745 patients suffering from nausea and vomiting within 3 h of
administration. Consequently, the IND application was rejected.
Interest in 5-azaC was rekindled in 1980 by Peter Jones and Shirley Taylor at the
University of California, Los Angeles (UCLA), who reported that the drug inhibits
DNA methylation [8]. In vivo, 5-azaC acts as a prodrug converted by a series of
phosphorylations to 5-azacytidine triphosphate that can then be incorporated
into RNA. Partially, 20
-reduction of the sugar by ribonucleotide reductase produces
5-aza-20
-deoxycytidine diphosphate that is further phosphorylated to 5-aza-20
-deoxycytidine
triphosphate. The deoxycytidine triphosphate is then incorporated into
DNA and this is the fate of approximately 10% of administered 5-azaC. DNA containing
5-azaC is recognized as a substrate by DNMTs. While the Michael addition
by the enzyme and methyl transfer from SAM can take place, the final elimination
step is impossible and the drug remains as an irreversible covalent adduct leading
to depletion of cellular DNMTand loss of genomic methylation (Figure 5.7).
The growing evidence highlighting the importance of methylation in cancer cells
and the elucidation of DNMT as the molecular target of 5-azaC led to a second IND
application submitted by Pharmion (later acquired by Celgene) for the treatment of
myelodysplastic syndromes (MDS). MDS are a collection of hematopoietic stem
cell disorders characterized by bone marrow dysplasia and peripheral blood cytopenia
with a propensity for transformation into AML. As the standard treatment consists
of supportive measures such as blood transfusions, and administration of
hematopoetic factors and antibiotics to treat opportunistic infections, there is a
pressing need for new therapeutic agents that specifically target MDS. In this predominantly
elderly population, a positive response was observed with low doses of
5-azaC leading to a significant decrease in onset of leukemia and an increase in
Figure 5.6 The Piskala and Sorm synthesis of 5-azacytidine.
5.3 5-Azacytidine (Azacitidine, Vidaza) and 5-Aza-20
-deoxycytidine (Decitabine, Dacogen) j119
quality of life [9]. Based on the Pharmion clinical trial data, in 2004 the FDA
approved 5-azaC (azacitidine, trade name Vidaza) as the first drug for the treatment
of all five subtypes of MDS. Thus, the journey from Piskala and Sorm’s initial synthesis
culminated in clinical approval four decades later. Pharmion has patented a
more efficient route to 5-azaC compared to the original synthesis using Vorbruggen
coupling that can be performed as a one-pot process (Figure 5.8). Clinically, Vidaza
is administered subcutaneously or intravenously at a dose of 100 mg for 7 days per
month and has a half-life of 41 min.
Alongside 5-azaC, Piskala and Sorm had reported 5-aza-20
-deoxycytidine in 1964.
As described above, 5-azaC is a prodrug of 5-aza-dC triphosphate that is the actual
O
O
O
N
N
N
NH2
P
P
O
O O
O O
O
O
O
O
O
N
N
N
NH2
P
P
O
O O
O O
O
O
Me
S Enz
N N
NH2
ON
O
OHOH
HO
5-azacytidine
(Vidaza)
N N
NH2
ON
O
OH
O
5-aza-2'-deoxycytidine
triphosphate
incorporation
into DNA
DNMT
P
O
P
O
P
O
O
O
O
O
O
O
N N
NH2
ON
O
OH
HO
5-aza-2'-deoxycytidine
(Dacogen)
in vivo
phosphorylation and
deoxygenation
in vivo
phosphorylation
Figure 5.7 The mechanism of DNMT inhibition by 5-azacytidine and 5-aza-20
-deoxycytidine.
N N
NH2
ON
O
OHOH
HO
OAc
O
OAcOAc
AcO
N N
NH2
ON
H
TMSCl, HMDS
acetonitrile, reflux 20 h N N
HN
OTMSN
TMSOTf
1)
2) NaOMe, MeOH
TMS
Figure 5.8 The Pharmion route to 5-azacytidine.
120j5 The Discovery of Anticancer Drugs Targeting Epigenetic Enzymes
molecule incorporated into DNA. However, this requires 20
-reduction of the ribose
sugar and the major metabolic pathway is phosphorylation without deoxygenation
to give 5-azaC triphosphate. 5-AzaC triphosphate is incorporated into RNA and
hence the drug has a number of effects independent of DNA methylation including
disruption of ribosome and tRNA biosynthesis. The RNA pathway may be responsible
for some of the side effects as well as some of the clinical benefits of 5-azaC.
Meanwhile, 5-aza-dC offers an opportunity to bypass the formation of 5-azaC triphosphate
as the 20
-deoxysugar can only be incorporated into DNA. Both in vitro
and in vivo evidence indicate that 5-aza-dC shows 10-fold or higher activity compared
to 5-azaC [10]. In 2004, MGI Pharma (later acquired by Eisai) submitted a
new drug application (NDA) to the FDA and in 2006 5-aza-dC (decitabine, trade
name Dacogen) received approval as the only drug for 5-day dosing in previously
treated and untreated, intermediate, and intermediate and high-risk MDS [11].
Dacogen is administered by intravenous infusion for either 3 days in a 6-week cycle
or 5 days in a 4 week cycle.
The success of 5-azaC and 5-aza-dC stems from their novel mechanism of action
as DNMT inhibitors and their clinical value of reactivating silenced tumor suppressors
in the treatment of MDS. These drugs are highly cytotoxic with dose-limiting
myelosuppression and work best when administered at low doses over long periods.
MDS primarily affects elderly patients and there is a dearth of chemotherapy
options available – a situation that favors the prolonged low dosing suitable for the
two drugs. It is likely that DNMTs do not play a special role in the progression of
MDS but rather the high cytotoxicity of azacytosines complicates the more aggressive
dosing needed in other cancers [12].
Besides the high cytotoxicity, another drawback of 5-azaC and 5-aza-dC is their
susceptibility to base hydrolysis and removal of the 5-azapyrimidine. This occurs at
neutral or basic pH and the drug solution has to be freshly prepared for prolonged
infusion [13]. Both the 50
-elaidic acid ester [14] of 5-azaC and the 5-azaCpG
dinucleotide [15] (Figure 5.9) are undergoing evaluation as more stable and bioavailable
derivatives of 5azaC.
Figure 5.9 5-AzaC derivatives with improved bioavailability.
5.3 5-Azacytidine (Azacitidine, Vidaza) and 5-Aza-20
-deoxycytidine (Decitabine, Dacogen) j121
5.4
Other Nucleoside DNMT Inhibitors
The poor stability, lack of oral bioavailability, and high cytotoxicity are shortcomings
of 5-azaC and 5-aza-dC. Several other nucleoside DNMT inhibitors that work by a
similar mechanism were investigated and have reached clinical trials (Figure 5.10).
In 1979, Beisler et al. reported the synthesis of fazarabine, the 5-azaC analogue with
arabinose instead of ribose as the sugar moiety [16]. In parallel, Beisler et al. had
synthesized dihydro-5-azacytidine whereby the 5,6-imino bond of the pyrimidine is
reduced [17]. This leads to improved aqueous stability over 5-azaC and the opportunity
to dose the drug by slow infusion to avoid acute toxicity. Like 5-azaC, fazarabine
and dihydro-5-azacytidine are prodrugs that are partially metabolized in vivo to
the 20
-deoxysugar triphosphate that is incorporated into DNA and inhibits DNMT.
Both compounds are active against cancer cell lines and tumor xenograft animal
models. However, a lack of activity was observed in phase II trials and further clinical
development of these agents has been discontinued [18].
Zebularine is a cytidine analogue lacking the exocyclic C-4 amine and was first
synthesized in 1961 by Funakoshi et al. [19]. Initial investigations focused on its
bacteriostatic properties and later on inhibition of cytidine deaminase [20] before
its potential as a DNMT inhibitor was realized [21]. Zebularine is stable and orally
bioavailable unlike 5-azaC and 5-aza-dC. Following conversion to 20
-deoxyzebularine
triphosphate, it is incorporated into DNA. The absence of the pyrimidine
amino group results in loss of one hydrogen bond in Watson–Crick base pairing to
the adjacent DNA strand. Consequently, there is less of an energy penalty for the
base to be flipped out and bind to DNMTs. Compared to cytosine, the absence of
the C-4 amine increases the reactivity toward Michael addition of the DNMT active
site cysteine while inhibiting the b-elimination release of the methylated DNA.
20
-Deoxyzebularine itself is not phosphorylated in vivo and is inactive. To bypass
this metabolic block and the inefficient 20
-reduction needed to reach zebularine,
the 30
- and 30
,50
-phosphoramidite analogues of 20
-deoxyzebularine have been
prepared [22].
Based on the antitumor properties of 5-fluorouracil, the analogous 5-fluoro-20
deoxycytidine
was expected to have similar activity and its synthesis was reported
in 1961 [23]. 5-Fluoro-20
-deoxycytidine is incorporated into DNA, whereupon it acts
N
ON
O
OHOH
HO
N
ON
O
OH
HO
F
NH2
zebularine
O
OH
HO
N
N
N
NH2
O
HO
HN N
NH2
ON
O
OHOH
HO
fazarabine dihydro-5-azacytidine 5-fluoro-2'-deoxycytidine
Figure 5.10 Nucleoside DNMT inhibitors that have reached clinical trials.
122j5 The Discovery of Anticancer Drugs Targeting Epigenetic Enzymes
as a DNMT inhibitor [24]. The Michael addition of cysteine occurs, but the absence
of the C-5 proton makes the final elimination step impossible and the drug forms a
covalent adduct. The mechanism is identical to the inhibition of thymidylate synthase
by 5-fluorouracil. As the drug is already a 20
-deoxysugar, its metabolism is not
complicated by incorporation into RNA. Clinical trials with zebularine and 5-fluoro-
20
-deoxycytidine are ongoing. Like the other nucleoside prodrugs, the active
triphosphate is only formed after a series of metabolic conversions.
5.5
Preclinical DNMT Inhibitors
A diverse range of natural products and some known drugs have been reported as
DNMT inhibitors (Figure 5.11) [25]. Although these leads are at the early stages of
drug discovery, it is possible that further optimization may result in a clinical candidate.
Unlike the nucleoside DNMT inhibitors, they bind directly to DNMT without
being incorporated into nucleic acids. The known antihypertensive drug hydralazine
and the anesthetic procainamide are DNMT inhibitors. Molecular modeling
suggests that hydralazine binds to the active site by a set of hydrogen bonds similarly
to the cytosine substrate [26]. Among the natural products reported to be
DNMT inhibitors are epigallocatechin gallate, a component of green tea, and
nanaomycin A that is selective for inhibition of DNMT3b [27]. Molecular modeling
and virtual screening of compound libraries have led to DNMT inhibitors such as
the tryptophan derivative RG108 designed to bind to the active site of DNMT1 [28].
The quinoline SGI-1027 is a micromolar inhibitor of DNMTs and competes with
the SAM cofactor [29].
O
O
OH
OH
OH
O
OH
HO
OH
OH
OH
N
N
HN
NH2
hydralazine
H2N
N
H
O
N
procainamide epigallocatechin gallate
OOH
O
O
CO2H
nanaomycin A
N
H
N
CO2H
O
O
RG108
N
HN
N
H
O
H
N
N N
NH2
SGI-1027
Figure 5.11 Examples of preclinical non-nucleoside DNMT inhibitors.
5.5 Preclinical DNMT Inhibitors j123
5.6
Zinc-Dependent Histone Deacetylases
Acetylation of lysine residues is an important epigenetic mark. It converts the positively
charged lysine to a neutral acetamide, thus reducing its affinity for the negatively
charged DNA backbone. In addition, protein bromodomains specifically
recognize acetyllysine and help recruit binding partners involved in gene transcription.
The chemical reversal of acetyllysine to lysine is catalyzed by histone deacetylases.
There are 18 HDACs in the human genome and these are subdivided
according to their catalytic mechanism, sequence homology, and localization [30].
HDACs 1–11 are metallohydrolases that employ zinc as the active site catalyst
while the sirtuins SIRTs 1–7 use the cofactor NADþ
for catalysis. Both activators
and inhibitors of sirtuins are of interest as therapeutic agents. As these are at the
preclinical stage, they will not be discussed further [31]. The zinc-dependent
HDACs are further classified as class I (HDACs 1, 2, 3, and 8), class II (HDACs 4,
5, 6, 7, 9, and 10), and class IV (HDAC 11). Class I HDACs are localized in the cell
nucleus whereas the larger class II HDACs shuttle between the nucleus and cytoplasm.
HDAC 11, the sole member of class IV, has similarities to both class I and
class II HDACs and is primarily nuclear. The primary effect of a HDAC inhibitor
on cancer cells is to reactivate repair pathways that are suppressed leading to cell
differentiation and apoptosis [32]. In this respect, the phenotypic outcome is similar
to that of DNMT inhibitors. It is important to appreciate that the HDACs do not
exclusively deacetylate histones but have a large number of other substrates in the
nucleus and cytoplasm. While the nuclear class I HDACs that act on histones
appear to be the most important in cancer, some of the therapeutic benefits and
side effects of HDAC inhibitors may be due to effects on nonhistone proteins [33].
The X-ray structures of several zinc-dependent HDACs have been solved and
indicate a catalytic mechanism similar to other metallohydrolases such as matrix
metalloproteinases and angiotensin-converting enzyme [34]. The acetyllysine substrate
is accommodated in a narrow tunnel approximately 12 A

in length with the
active site zinc atom at the end coordinated to two aspartic acid residues and one
histidine. The carbonyl group of the acetyllysine then binds to the zinc and a conserved
tyrosine or histidine residue and this serves to activate the amide bond
toward hydrolysis (Figure 5.12). The attacking water molecule is made more nucleophilic
by interactions with the zinc and charge relay with the active site residues
and addition to the carbonyl gives a tetrahedral intermediate that collapses to the
hydrolysis products.
The inhibition of metallohydrolases in drug discovery is usually accomplished by
designing molecules that reversibly bind to the active site metal cation [35]. Achieving
selectivity between metalloenzymes needs to take advantage of differences in
active site geometry. In the case of zinc-dependent HDACs, inhibitors can be
described by a simple dumbbell-shaped pharmacophore consisting of a warhead, a
spacer, and a cap. At one end of the molecule is a reversible zinc binding warhead
and at the other is a “cap”. In between is a linear spacer that fits into the narrow
hydrophobic substrate binding channel. The critical interaction between a HDAC
124j5 The Discovery of Anticancer Drugs Targeting Epigenetic Enzymes
inhibitor and its enzyme target is binding to the active site zinc. Discrimination
between HDACs and other metalloenzymes is promoted by the long linear spacer
that is typically not accommodated by unrelated enzymes. Discrimination within
the HDAC family is through the “cap” that interacts with a region of the enzyme
referred to as the “rim” at the top of the narrow channel. The channel and active
site are relatively conserved among HDACs, whereas the isoforms diverge from
one another in the rim. Differential binding to the rim can thus give rise to a selective
HDAC inhibitor. Although the understanding of enzyme structure has aided
the structure-based design of inhibitors, the early compounds were developed without
the hindsight of this information. Indeed, the two currently approved HDAC
inhibitors were discovered on the basis of phenotypic activity before the human
HDACs were even identified.
5.7
Suberoylanilide Hydroxamic Acid (SAHA, Vorinostat, Zolinza)
Remarkably, the lead for the first approved HDAC inhibitor SAHA was the simple
molecule DMSO. Charlotte Friend had observed that DMSO caused differentiation
of murine leukemia cells and this sparked collaboration between Paul Marks and
Ronald Breslow at Columbia University to understand the mechanism. It was discovered
that other polar small molecules could induce differentiation although
none were particularly potent. N-Methylacetamide had an optimal effect at 30 mM
and Breslow then synthesized a series of bisacetamides with a linker of 2–8 methylene
units. The compound with a spacer of six methylenes, hexamethylene bisacetamide
(Figure 5.13), induced differentiation of 95% of the cells at 5 mM [36]. The
bisamides with seven and eight methylenes were slightly more potent but more
cytotoxic. On the basis of promising in vitro and in vivo results, an IND was filed
for hexamethylene bisacetamide and the compound reached phase II clinical
trials [37]. While some remissions were observed in MDS and AML, these were
reversed upon cessation of treatment and higher doses were not tolerated by
patients. Additional bis-, tri-, and tetraamides were investigated although further
Zn2+
protein
NHO
HDAC
protein
NHO
O
Tyr
H
His
Zn2+
protein
NH2
Tyr
His
O
O
Figure 5.12 The catalytic reaction mechanism of zinc-dependent HDACs.
5.7 Suberoylanilide Hydroxamic Acid (SAHA, Vorinostat, Zolinza) j125
gains of potency were not achieved. It was then postulated that the amides might
act by hydrogen bonding or metal binding at the receptor. This led to studies with a
hydroxamic acid series as an amide isostere with a linker of 2–10 methylene units.
The compound with six methylene units, suberic bishydroxamic acid, was significantly
more potent than the bisacetamide with an optimum concentration of 30 mM
in cell differentiation [38]. Further optimization involved the synthesis of more
than 600 compounds. One design consideration was that the distance between the
two hydroxamic acids was too large for simultaneous binding to a metal ion and
one should be sufficient. Compounds with a hydroxamic acid at one end and a
hydrophobic amide at the other were prepared and SAHA emerged as the candidate,
inducing differentiation at 2 mM [39].
Up to this point, the molecular targets of the bisamides and hydroxamic acids
were unknown. Meanwhile, Minoru Yoshida in Japan had characterized the natural
product trichostatin A as a HDAC inhibitor [40]. The structural resemblance
between trichostatin A and SAHA (Figure 5.14) suggested that the latter too was a
HDAC inhibitor and this was quickly verified by in vitro studies. Interestingly, the
lead precursor hexamethylene bisacetamide is not a HDAC inhibitor and its target
remains unidentified. An X-ray structure of SAHA bound to a HDAC-like bacterial
protein confirmed that the hydroxamic acid is chelated to the active site zinc while
the phenyl ring extends into the enzyme rim [41]. Thus, SAHA fits the HDAC pharmacophore
of warhead, spacer, and cap and its potency is driven primarily by zinc
binding and occupancy of the enzyme channel. The small phenyl cap does not
Figure 5.13 The evolution of SAHA from DMSO with potencies in cell differentiation indicated.
H
N
N
H
OH
O
O
SAHA
N
H
OH
OO
N
Me
Me
trichostatin A
Figure 5.14 Comparison between SAHA and trichostatin A.
126j5 The Discovery of Anticancer Drugs Targeting Epigenetic Enzymes
discriminate between isoforms and SAHA is a pan-HDAC inhibitor with IC50
values 50 nM against individual HDACs.
SAHA analogues with higher potency were discovered in the course of Breslow’s
optimization. However, they also had higher toxicity and SAHA was deliberately
chosen as the clinical candidate with medium potency and efficacy without an
unacceptable level of side effects. SAHA progressed to phase I clinical trials in
2000 and the compound was licensed to the spinout company Aton Pharmaceuticals
(acquired by Merck in 2004). SAHA (vorinostat, trade name Zolinza) received
FDA approval for the treatment of cutaneous T-cell lymphoma (CTCL) in 2006 [42].
While the initial trials used intravenous infusion, Zolinza is now administered
orally with a maximum dose of 400 mg once daily with food. The drug is 71%
bound to plasma proteins and has a half-life of 2 h with two major metabolic pathways:
O-glucuronidation and hydrolysis of the hydroxamic acid followed by b-oxidation.
Synthetically, Zolinza can be prepared in one step from the bis-acid chloride
of suberic acid by reaction with aniline and hydroxylamine (Figure 5.15) [43].
The identification of trichostatin A and Zolinza as HDAC inhibitors and the
availability of X-ray crystallographic data spurred many medicinal chemistry programs
directed at this target. More than 10 hydroxamic acid HDAC inhibitors have
reached clinical trials (Figure 5.16) [44]. They range from pyroxamide, a closely
related Zolinza analogue with a pyridine replacing the aniline, to more complex
structures such as CHR-3996. Besides the aliphatic hydroxamic acid motif present
in Zolinza, these compounds feature more rigid variants such as cinnamoyl, benzoyl,
and hetaryl hydroxamic acids while the presence of a larger cap provides gains
in potency and Class I isoform stability for JNJ-26481585 and CHR-3996. The
second-generation hydroxamic acids usually have increased oral bioavailability
compared to Zolinza. The Curis compound CUDC-101 is unique in being rationally
designed as a dual-acting HDAC and tyrosine kinase inhibitor combining
erlotinib and vorinostat with nanomolar IC50 values against HDACs, HER2 and
EGFR. Numerous other hydroxamic acid HDAC inhibitors at the preclinical stage
have been reported with varying degrees of isoform selectivity.
5.8
FK228 (Depsipeptide, Romidepsin, Istodax)
A high-throughput screening campaign at Fujisawa Pharmaceutical Co., Ltd. investigated
natural product extracts for the ability to revert a ras-transformed phenotype
to normal cells. They reported the isolation of the cyclic depsipeptide FR901228
Figure 5.15 One-pot synthesis of Zolinza.
5.8 FK228 (Depsipeptide, Romidepsin, Istodax) j127
(later named FK228) based on this assay from a culture of Chromobacterium violaceum
No. 968 in 1994 [45]. The compound displayed potent antitumor activity in
vitro and in murine xenograft models while its structure (Figure 5.17) was
unrelated to known natural products. Mechanistic studies by Yoshida et al. later
confirmed that FK228, like trichostatin A, was a HDAC inhibitor [46]. At first sight,
the structure does not fit the HDAC pharmacophore of warhead, spacer, and cap.
The natural product is in fact a prodrug that undergoes intracellular reductive
N
H
OH
O
O
H
N
O
O
N
abexinostat (Pharmacyclics)
N
H
OH
O
N
H
O
O
N
givinostat (ItalFarmaco)
N
H
OH
O
N
NN
H
NN
JNJ-26481585 (Johnson & Johnson)
N
H
OH
O
N
NN
H
H
N
H
N
F CHR-3996 (Chroma)
N
H
OH
O
N
NN
N
S
O O R306465 (Johnson & Johnson)
pyroxamide (NCI)
H
N
N
H
OH
O
O
N O N
H
OH
O
N
N
NH
OMe
CUDC-101 (Curis)
N
H
OH
O
H
N
HN panobinostat (Novartis)
N
H
OH
O
N
HN
OH
dacinostat (Novartis)
N
H
OH
O
S
N
H O
O
belinostat (TopoTarget)
N
H
OH
O
N
N
N
pracinostat (S*Bio)
N
H
OH
O
NS
O O
N
resminostat (4SC)
N
H
OH
O
N
H
O
AR-42 (Arno)
Figure 5.16 Hydroxamic acid HDAC inhibitors that have reached clinical trials.
128j5 The Discovery of Anticancer Drugs Targeting Epigenetic Enzymes
cleavage of the disulfide bridge. This releases a free thiol-bearing side chain and it
is the thiol that binds to the active site zinc. Recently, support for this mechanism
has been obtained from the X-ray structure of HDAC8 bound to largazole, a depsipeptide
natural product with the same thiol zinc binding warhead as FK228 [47].
Compared to the synthetic HDAC inhibitor SAHA, FK228 has a number of
unique features. The zinc binding warhead is a thiol rather than the higher affinity
bidentate chelating hydroxamic acid. Nevertheless, FK228 is a more potent inhibitor
of HDACs and cancer cells in vitro with an IC50  1nM. This is because FK228 has
a large macrocyclic scaffold as the cap and this extends out into the HDAC rim to
provide additional binding interactions with the enzyme. Furthermore, as the rim is
divergent between HDACs, FK228 is isoform selective and is particularly potent in
the inhibition of class I HDACs – fortuitously the ones that are linked to cancer.
FK228 was licensed by Fujisawa to Gloucester Pharmaceuticals (later acquired by
Celgene) and phase I clinical trials for CTCL were initiated in 2004. As asymptomatic
T-wave flattening and ST segment depression were observed in the phase I
trials, cardiac evaluation was incorporated into the phase II study. While there was
no evidence of acute or cumulative cardiac toxicity, one sudden death occurred and
the protocol excluded patients with heart disease. FK228 (romidepsin, trade name
Istodax) received FDA approval for the treatment of CTCL in 2009, becoming the
second HDAC inhibitor to reach the market [48]. A dose of 10 mg is administered
intravenously over 4 h on days 1, 8, and 15 of a 28-day cycle. Due to the potential
risk of cardiac side effects, potassium and magnesium electrolyte levels should be
within the normal range before administration and cardiac monitoring is recommended
for patients at risk of QT prolongation. Some cardiac effects have been
observed in clinical trials of other HDAC inhibitors of unrelated structure, suggesting
that this is a common on-target side effect of the mechanism of action. Romidepsin
is 92–94% bound to plasma proteins and has a half-life of 3 h. In vivo
metabolism is complex with over a dozen metabolites identified [49]. Romidepsin
is a substrate of P-glycoprotein and transporter-mediated efflux appears to be the
major mechanism of drug resistance [50].
Industrially, romidepsin is produced by bacterial fermentation. Several total syntheses
have been reported that involve Mitsunobu esterification to assemble the
cyclic depsipeptide. However, the reaction needs careful optimization and an alternative
route (Figure 5.18) uses macrolactamization instead [51]. The synthesis
involves building up a D-Val-D-Cys-Dha tripeptide that is combined with a fragment
O
O
O
N
H
O
N
HN
H
O
HN
O
S
S
in vivo
reduction
O
O
O
N
H
O
N
HN
H
O
HN
O
SH
SH
Figure 5.17 Structure of FK228 and the active drug formed by metabolism.
5.8 FK228 (Depsipeptide, Romidepsin, Istodax) j129
containing the zinc binding warhead in protected form. The linear seco-amino acid
undergoes HATU mediated intramolecular coupling to give the macrocycle. In the
final step, oxidation forms the disulfide bridge.
Although total synthesis of the depsipeptides is unlikely to compete with the fermentation
process in efficiency, it provides access to unnatural analogues. These
have shed valuable light on the structure–activity relationship (SAR) of the natural
product (Figure 5.19) [52]. The macrocyclic ester is essential as linear uncyclized
compounds are inactive. The stereochemistry of the ester is essential as the epimer
is virtually inactive. The amino acids present in the peptide macrocycle can be
substituted by others without loss in activity. The dehydroalanine Michael acceptor
is not needed and it is speculated that its function is to tie up the thiol from the Cys
residue upon disulfide ring opening. The zinc-binding thiol warhead needs to be
protected to ensure efficient cell uptake, whether as a disulfide as in FK228 or as a
thioester in synthetic analogues and the related natural product largazole.
NHN
H
STr
O
O
NH2
NH
O
STr
O
O
OH O
HATU, i-Pr2NEt
54%
N
H
H
N CO2H
NH
Fmoc
O
STr
O
Me3Si
O STr
O O O
H2N
+
PyBop, i-Pr2NEt
75%
NHN
H
STr
O
O
NH
NH
O
STr
O
O
O OMe3Si
Fmoc
1. piperidine
2. Bu4NF
I2, MeOH
50%
O
O
O
N
H
O
N
HN
H
O
HN
O
S
S
O
O
O
N
H
O
N
HN
H
O
HN
O
STr
STr
Figure 5.18 A macrolactamization-based total synthesis of FK228.
Figure 5.19 Key structural features of FK228.
130j5 The Discovery of Anticancer Drugs Targeting Epigenetic Enzymes
Subsequent to the discovery of FK228, other natural product HDAC inhibitors
containing the same zinc binding warhead have been isolated. Two noteworthy
examples are largazole from the marine cyanobacterium Symploca sp. and burkholdac
B from Burkholderia thailandensis (Figure 5.20). Largazole features extensive
modification of the peptide backbone to form a thiazole and thiazoline ring. Unlike
FK228, the zinc binding warhead is protected as a thioester prodrug rather than a
disulfide. Largazole is exceptionally potent in HDAC enzyme assays with a picomolar
IC50 and many academic groups have worked on its total synthesis as well
as analogue preparation [53]. Despite its potent HDAC inhibition, largazole suffers
from two shortcomings. First, there is no gain in potency of antiproliferative effects
compared to FK228 and other depsipeptides indicating a loss of activity in
cells relative to HDAC inhibition. Second, the thioester prodrug is highly labile in
liver microsomes and undergoes hydrolysis in less than 2 min [54]. The disulfide in
FK228 appears to be a more stable prodrug and a better approach for protection of
the zinc binding thiol. Burkholdac B (¼ thailandepsin A) is the most recent member
of the depsipeptide HDAC inhibitors to be isolated. In cell proliferation assays,
it outperforms FK228 with a picomolar IC50 and is the most potent compound
within this class [55]. The initial report did not assign the stereochemistry of the
chiral centers and this was later unambiguously determined by the total synthesis
of multiple diastereomers [56].
5.9
Carboxylic Acid and Benzamide HDAC Inhibitors
Besides the hydroxamic acid present in Zolinza and the thiol in FK228, a variety of
other zinc binding warheads have been employed in HDAC inhibitors. In an example
of drug repositioning, three short-chain carboxylic acids – butyric, valproic, and
phenylbutyric acid – as well as a butyrate prodrug have progressed to clinical trials
(Figure 5.21). These compounds are already used for other indications and their
pharmacokinetics is well understood. Nevertheless, the short-chain carboxylic acids
are modest micromolar HDAC inhibitors at best and have short half-lives. This is
to be expected from a consideration of the HDAC pharmacophore as they contain a
O
O
O
N
H
NN
O
HN
S S
S
O
largazole
O
O
O
N
H
N
HN
H
O
S
S
HO
O
SMe
burkholdac B
Figure 5.20 Structure of largazole and burkholdac B.
5.9 Carboxylic Acid and Benzamide HDAC Inhibitors j131
relatively weak monodentate zinc binding warhead and lack a cap. The only carboxylic
acid HDAC inhibitor with a submicromolar IC50 is the marine natural product
azumamide E [57]. In this case, the macrocyclic cap provides additional binding
to the enzyme rim. Synthetic replacement of the carboxylic acid by the superior
hydroxamic acid warhead provided an azumamide E derivative with enhanced
HDAC inhibition [58].
Benzamides are the most recent family of HDAC inhibitors to reach clinical trials.
The two nitrogens coordinate to the active site zinc in a bidentate manner similar
to hydroxamic acids. In vitro and in vivo, benzamides are low micromolar to
nanomolar in efficacy. Development of the early example tacedinaline is now discontinued
due to toxicity while second-generation compounds (Figure 5.22) are
currently in clinical trials.
5.10
Prospects for HDAC Inhibitors
Like DNMT inhibitors, the clinical trial data for HDAC inhibitors have been disappointing
for solid and hematological tumors. Zolinza and Istodax are approved only
O
OH
O
OH
butyric acid
valproic acid
O
OH
phenylbutyric acid
O
O
AN-9
O
O
NH
N
H
H
N
HN
O
O
O
O
OHO
azumamide E
Figure 5.21 Examples of carboxylic acid HDAC inhibitors.
NH2H
N
O
H
N
O
tacedinaline (Pfizer)
NH2H
N
O
N
H
O
O
N
entinostat (Syndax)
NH2H
N
O
N
H
N
N
N
mocetinostat (MethylGene)
NH2H
N
O
N
H
O
N
Fchidamide (Chipscreen)
Figure 5.22 Examples of benzamide HDAC inhibitors.
132j5 The Discovery of Anticancer Drugs Targeting Epigenetic Enzymes
for the treatment of CTCL and even in this indication only a minority of patients
show complete response. The underlying biology that enables HDAC inhibitors to
succeed against this particular cancer is unclear. In vitro studies show that HDAC
inhibitors strongly downregulate cytokine signaling in CTCL, particularly IL-10,
IL-2, IL-4, and STAT3 and this may be the reason for their antiproliferative
effect [59]. For the future, two major developments can be foreseen. First, the true
potential of HDAC inhibitors may lie in combination therapy rather than use as
single agents. Multiple clinical trials are currently underway to test this hypothesis
and early results are promising [60]. Second, there is growing evidence from in vitro
and in vivo models for the use of HDAC inhibitors in other therapeutic indications
besides cancer [61]. Such applications will benefit from the ongoing development
of isoform-selective HDAC inhibitors.
5.11
Epigenetic Drugs – A Slow Start but a Bright Future
The importance of epigenetics and the identification of epigenetic targets for drug
discovery is a recent phenomenon. In spite of the short timeline, four epigenetic
drugs are already approved. These DNMT and HDAC inhibitors were earmarked
for development based solely on their promising phenotypic effects in inhibition of
cell proliferation without knowledge of the mechanism of action. Three of the
drugs originated from academic laboratories and all reached the clinic championed
by small biotech firms rather than big pharma. Interestingly, all these biotech firms
were later acquired by larger organizations. The clinical development owed a lot to
the NCI running pivotal clinical trials and working out appropriate dosing
schedules.
Today, the situation has changed dramatically. Many of the molecular players in
epigenetics have been identified and it is now possible to run high-throughput
screening campaigns against such targets. This approach is being pursued by a
number of biotech firms that focus exclusively on epigenetics as well as big
pharma. At the time of writing, GlaxoSmithKline’s bromodomain inhibitor [62] is
about to enter clinical trials while there are promising preclinical compounds targeting
lysine methyltransferase [63] and lysine demethylase [64], among others.
Epigenetics has become an accepted strategy in drug discovery and is now as competitive
a field as any other. One can expect many more epigenetic drugs to reach
the clinic and to receive approval for multiple therapeutic indications. In addition to
targeted drugs, nutraceuticals can play a role as many nutrients and food components
have been shown to inhibit specific epigenetic enzymes [65].
Acknowledgments
I am grateful to Professors Manfred Jung (University of Freiburg, Germany) and
Andrew MacMillan (University of Alberta, Canada) for helpful comments on the
5.11 Epigenetic Drugs – A Slow Start but a Bright Future j133
chapter and to the COST Action TD0905 “Epigenetics: From Bench to Bedside” for
financial assistance.
References
1 Waddington, C.H. (1942) The
epigenotype. Endeavour, 1, 18–20.
2 (a) Szyf, M. (2009) Epigenetics, DNA
methylation, and chromatin modifying
drugs. Annu. Rev. Pharmacol. Toxicol., 49,
243–263. (b) Chi, P., Allis, C.D., and
Wang, G.G. (2010) Covalent histone
modifications – miswritten,
misinterpreted and mis-erased in human
cancers. Nat. Rev. Cancer, 10, 457–469.
(c)Portela, A. and Esteller, M. (2010)
Epigenetic modifications and human
disease. Nat. Biotechnol., 28, 1057–1068.
(d) Henikoff, S. and Shilatifard, A. (2011)
Histone modification: cause or cog? Trends
Genet., 10, 389–396.
3 (a) Mai, A. and Altucci, L. (2009) Epi-drugs
to fight cancer: from chemistry to cancer
treatment, the road ahead. Int. J. Biochem.
Cell Biol., 41, 199–213. (b) Ganesan, A.,
Nolan, L., Crabb, S.J., and Packham, G.
(2009) Epigenetic therapy: histone
acetylation, DNA methylation and anticancer
drug discovery. Curr. Cancer Drug
Targets, 2, 963–981. (c) J€ungel, A., Ospelt,
C., and Gay, S. (2010) What can we learn
from epigenetics in the year 2009? Curr.
Opin. Rheumatol., 22, 284–292.
4 Esteller, M. (2008) Cancer epigenomics:
DNA methylomes and histonemodification
maps. Nat. Rev. Genet.,
8, 286–298.
5 (a) Christman, J.K. (2002) 5-Azacytidine
and 5-aza-20
-deoxycytidine as inhibitors of
DNA methylation: mechanistic studies
and their implications for cancer therapy.
Oncogene, 21, 5483–5495. (b) Gowher, H.
and Jeltsch, A. (2004) Mechanism of
inhibition of DNA methyltransferases by
cytidine analogs in cancer therapy. Cancer
Biol. Ther., 3, 1062–1068.
6 (a) Piskala, A. and Sorm, F. (1964)
Nucleic acids components and their
analogues. LI. Synthesis of 1-glycosyl
derivatives of 5-azauracil and
5-azacytosine. Collect. Czech. Chem.
Commun., 29, 2060–2076. (b) Pliml, J.
and Sorm, F. (1964) Synthesis of 20
-
deoxy-D-ribofuranosyl-5-azacytosine.
Collect. Czech. Chem. Commun., 29,
2576–2577. (c) Sorm, F., Piskala, A.,
Cihak, A., and Vesely, J. (1964) 5Azacytidine,
a new, highly effective
cancerostatic. Experientia, 20, 202–203.
7 Hanka, L.J., Evans, J.S., Mason, D.J., and
Dietz, A. (1966) Microbiological
production of 5-azacytidine. I. Production
and biological activity. Antimicrob. Agents
Chemother., 6, 619–624.
8 Jones, P.A. and Taylor, S.M. (1980)
Cellular differentiation, cytidine analogs
and DNA methylation. Cell, 20, 85–93.
9 (a) Oki, Y. and Issa, J.P.J. (2007) Treatment
options in advanced myelodysplastic
syndrome, with emphasis on epigenetic
therapy. Int. J. Hematol., 86, 306–314.
(b) Howell, P.M., Jr., Liu, Z., and Khong,
H.T. (2010) Demethylating agents in the
treatment of cancer. Pharmaceuticals, 3,
2022–2044.
10 Lyko, F. and Brown, R. (2005) DNA
methyltransferase inhibitors and the
development of epigenetic cancer
therapies. J. Natl. Cancer Inst., 97,
1498–1506.
11 Gore, S.D., Jones, C., and Kirkpatrick, P.
(2006) Decitabine. Nat. Rev. Drug Discov.,
5, 891–892.
12 Issa, J.-P.J., Kantarjian, H.M., and
Kirkpatrick, P. (2005) Azacitidine. Nat.
Rev. Drug Discov., 4, 275–276.
13 Stresemann, C. and Lyko, F. (2008) Modes
of action of the DNA methyltransferase
inhibitors azacytidine and decitabine. Int.
J. Cancer, 123, 8–13.
14 Brueckner, B., Rius, M., Markelova, M.R.,
Fichtner, I., Hals, P.-A., Sandvold, M.L.,
and Lyko, F. (2010) Delivery of
5-azacytidine to human cancer cells by
elaidic acid esterification increases
therapeutic drug efficacy. Mol. Cancer
Ther., 9, 1256–1264.
134j5 The Discovery of Anticancer Drugs Targeting Epigenetic Enzymes
15 Yoo, C.B., Jeong, S., Egger, G., Liang, G.,
Phiasivongsva, P., Tang, C., Redkar, S.,
and Jones, P.A. (2007) Delivery of
5-aza-20
-deoxycytidine to cells using
oligodeoxynucleotides. Cancer Res., 67,
6400–6408.
16 Beisler, J.A., Abbasi, M.M., and Driscoll, J.
S. (1979) Synthesis and antitumor activity
of 5-azacytosine arabinoside. J. Med.
Chem., 22, 1230–1234.
17 Beisler, J.A., Abbasi, M.M., Kelley, J.A.,
and Driscoll, J.S. (1977) Synthesis and
antitumor activity of dihydro-5azacytidine,
a hydrolytically stable
analogue of 5-azacytidine. J. Med. Chem.,
20, 806–812.
18 Goffin, J. and Eisenhauer, E. (2002) DNA
methyltransferase inhibitors – state of the
art. Ann. Oncol., 13, 1699–1716.
19 Funakoshi, R., Irie, M., and Ukita, T.
(1961) Synthesis of unnatural pyrimidine
nucleosides. Chem. Pharm. Bull., 9,
406–409.
20 Kim, C.H., Marquez, V.E., Mao, D.T.,
Haines, D.R., and McCormack, J.J. (1986)
Synthesis of pyrimidin-2-one nucleosides
as acid-stable inhibitors of cytidine
deaminase. J. Med. Chem., 29, 1374–1380.
21 (a) Hurd, P.J., Whitmarsh, A.J., Baldwin,
G.S., Kelly, S.M., Waltho, J.P., Price, N.C.,
Connolly, B.A., and Hornby, D.P.
(1999) Mechanism-based inhibition of
C5-cytosine DNA methyltransferases by 2H
pyrimidinone. J. Mol. Biol., 286, 389–
401. (b) Zhou, L., Cheng, X., Connolly, B.
A., Dickman, M.J., Hurd, P.J., and Hornby,
D.P. (2002) Zebularine: a novel DNA
methylation inhibitor that forms a covalent
complex with DNA methyltransferases. J.
Mol. Biol., 321, 591–599.
22 Yoo, C.B., Valente, R., Congiatu, C.,
Gavazza, F., Angel, A., Siddiqui, M.A.,
Jones, P.A., McGuigan, C., and Marquez,
V.E. (2008) Activation of p16 gene
silenced by DNA methylation in cancer
cells by phosphoramidate derivatives of
20
-deoxyzebularine. J. Med. Chem., 51,
7593–7601.
23 Wempen, I., Duschinsky, R., Kaplan, L.,
and Fox, J.J. (1961) Thiation of
nucleosides. IV. The synthesis of 5-fluoro-
20
-deoxycytidine and related compounds.
J. Am. Chem. Soc., 83, 4755–4766.
24 Osterman, D.G., DePillis, G.D., Wu, J.C.,
Matsuda, A., and Santi, D.V. (1988)
5-Fluorocytosine in DNA is a mechanismbased
inhibitor of HhaI methylase.
Biochemistry, 27, 5204–5210.
25 Martinet, N., Michel, B.Y., Bertrand, P.,
and Benhida, R. (2012) Small molecules
DNA methyltransferases inhibitors. Med.
Chem. Commun., 3, 263–273.
26 Singh, N., Duenas-Gonzalez, A., Lyko, F.,
and Medina-Franc, J.L. (2009) Molecular
modeling and molecular dynamics studies
of hydralazine with human DNA
methyltransferase 1. ChemMedChem, 4,
792–799.
27 Kuck, D., Caulfield, T., Lyko, F., and
Medina-Franco, J.L. (2010) Nanaomycin A
selectively inhibits DNMT3B and
reactivates silenced tumor suppressor
genes in human cancer cells. Mol. Cancer
Ther., 9, 3015–3023.
28 Brueckner, B., Garcia, B.R., Siedlecki, P.,
Musch, T., Kliem, H.C., Zielenkiewicz, P.,
Suhai, S., Wiessler, M., and Lyko, F. (2005)
Epigenetic reactivation of tumor
suppressor genes by a novel smallmolecule
inhibitor of human DNA
methyltransferases. Cancer Res., 65,
6305–6311.
29 Datta, J., Ghoshal, K., Denny, W.A.,
Gamage, S.A., Brooke, D.G.,
Phiasivongsa, P., Redkar, S., and Jacob, S.
(2009) A new class of quinoline-based
DNA hypomethylating agents reactivates
tumor suppressor genes by blocking DNA
methyltransferase 1 activity and inducing
its degradation. Cancer Res., 69,
4277–4285.
30 (a) Yang, X.J. and Seto, E. (2008) The
Rpd3/Hda1 family of lysine
deacetylases: from bacteria and yeast to
mice and men. Nat. Rev. Mol. Cell Biol.,
9, 206–218. (b) Imai, S.I. and Guarente,
L. (2010) Ten years of NAD-dependent
SIR2 family deacetylases: implications
for metabolic diseases. Trends Pharmacol.
Sci., 31, 212–220.
31 Carafa, V., Nebbioso, A., and Altucci, L.
(2012) Sirtuins and disease: the
road ahead. Front. Pharmacol.,
3, 4.
32 Witt, O., Deubzer, H.E., Milde, T., and
Oehme, I. (2009) HDAC family: what are
References j135
the cancer relevant targets? Cancer Lett.,
277, 8–21.
33 (a) Choudary, C., Kumar, C., Gnad, F.,
Nielsen, M.L., Rehman, M., Walther, T.C.,
Olsen, J.V., and Mann., M. (2009) Lysine
acetylation targets protein complexes and
co-regulates major cellular functions.
Science, 325, 834–840. (b) Singh, B.N.,
Zhang, G.H., Hwa, Y.L., Li, J.P., Dowdy, S.
C., and Jiang, S.W. (2010) Nonhistone
protein acetylation as cancer therapy
targets. Expert Rev. Anticancer Ther., 10,
935–954.
34 Paris, M., Porcelloni, M., Binaschi, M.,
and Fattori, D. (2008) Histone deacetylase
inhibitors: from bench to clinic. J. Med.
Chem., 51, 1505–1529.
35 (a) Jacobsen, F.E., Lewis, J.A., and Cohen,
S.M. (2007) The design of inhibitors for
medicinally relevant metalloproteins.
ChemMedChem, 2, 152–171. (b) Rouffet,
M. and Cohen, S.M. (2011) Emerging
trends in metalloprotein inhibition.
Dalton Trans., 40, 3445–3454.
36 Reuben, R.C., Wife, R.L., Breslow, R.,
Rifkind, R.A., and Marks, P.A. (1976) A
new group of potent inducers of
differentiation in murine erythroleukemia
cells. Proc. Natl. Acad. Sci. USA, 73,
862–866.
37 Andreeff, M., Stone, R., Michaeli, J.,
Young, C.W., Tong, W.P., Sogoloff, H.,
Ervin, T., Kufe, D., Rifkind, R.A., and
Marks, P.A. (1992) Hexamethylene
bisacetamide in myelodysplastic
syndrome and acute myelogenous
leukemia: a phase II clinical trial with a
differentiation-inducing agent. Blood,
80, 2604–2609.
38 Breslow, R., Jursic, B., Yan, Z.F.,
Friedman, E., Leng, L., Ngo, L., Rifkind,
R.A., and Marks, P.A. (1991) Potent
cytodifferentiating agents related to
hexamethylenebisacetamide. Proc. Natl.
Acad. Sci. USA, 88, 5542–5546.
39 Richon, V.M., Webb, Y., Merger, R.,
Sheppard, T., Jursic, B., Ngo, L., Civoli, F.,
Breslow, R., Rifkind, R.A., and Marks,
P.A. (1996) Second generation hybrid
polar compounds are potent inducers of
transformed cell differentiation.
Proc. Natl. Acad. Sci. USA, 93,
5705–5708.
40 Yoshida, M., Kijima, M., Akita, M., and
Beppu, T. (1990) Potent and specific
inhibition of mammalian histone
deacetylase both in vivo and in vitro by
trichostatin A. J. Biol. Chem., 265, 17174–
17179.
41 Finnin, M.S., Donigian, J.R., Cohen, A.,
Richon, V.M., Rifkind, R.A., Marks, P.A.,
Breslow, R., and Pavletich, N.P. (1999)
Structures of a histone deacetylase
homologue bound to the TSA and SAHA
inhibitors. Nature, 401, 188–193.
42 (a) Grant, S., Easley, C., and Kirkpatrick, P.
(2007) Vorinostat. Nat. Rev. Drug Discov.,
6, 21–22. (b) Marks, P.A. and Breslow, R.
(2007) Dimethyl sulfoxide to vorinostat:
development of this histone deacetylase
inhibitor as an anticancer drug. Nat.
Biotechnol., 25, 84–90.
43 Breslow, R., Marks, P.A., Rifkind, R.A.,
and Jursic, B. (1991) Potent inducers of
terminal differentiation and methods of
use thereof. US Patent 5,369,108.
44 Jones, P. (2012) Development of second
generation epigenetic agents. Med. Chem.
Commun., 3, 135–161.
45 (a) Ueda, H., Nakajima, H., Hori, Y.,
Fujita, T., Nishimura, M., Goto, T., and
Okuhara, M. (1994) FR901228, a novel
antitumor bicyclic depsipeptide produced
by Chromobacterium violaceum No. 968. I.
Taxonomy, fermentation, isolation,
physico-chemical and biological
properties, and antitumor activity. J.
Antibiot., 47, 301–310. (b) Shigematsu, N.,
Ueda, H., Takase, S., Tanaka, H.,
Yamamoto, K., and Tada, T. (1994)
FR901228, a novel antitumor bicyclic
depsipeptide produced by
Chromobacterium violaceum No. 968. II.
Structure determination. J. Antibiot., 47,
311–314. (c) Ueda, H., Manda, T.,
Matsumoto, S., Mukumoto, S., Nishigaki,
F., Kawamura, I., and Shimomura, K.
(1994) FR901228, a novel antitumor
bicyclic depsipeptide produced by
Chromobacterium violaceum No. 968. III.
Antitumor activities on experimental
tumors in mice. J. Antibiot., 47, 315–323.
46 (a) Nakajima, H., Kim, Y.B., Terano, H.,
Yoshida, M., and Horinouchi, S. (1998)
FR901228, a potent antitumor antibiotic,
is a novel histone deacetylase inhibitor.
136j5 The Discovery of Anticancer Drugs Targeting Epigenetic Enzymes
Exp. Cell Res., 241, 126–133. (b) Furumai,
R., Matsuyama, A., Kobashi, N., Lee,
K.-H., Nishiyama, N., Nakajima, H.,
Tanaka, A., Komatsu, Y., Nishino, N.,
Yoshida, M., and Horinouchi, S. (2002)
FK228 (depsipeptide) as a natural prodrug
that inhibits class I histone deacetylases.
Cancer Res., 62, 4916–4921.
47 Cole, K.E., Dowling, D.P., Boone, M.A.,
Phillips, A.J., and Christianson, D.W.
(2011) Structural basis of the
antiproliferative activity of largazole, a
depsipeptide inhibitor of the histone
deacetylases. J. Am. Chem. Soc., 133,
12474–12477.
48 VanderMolen, K.M., McCulloch, W.,
Pearce, C.J., and Oberlies, N.H. (2011)
Romidepsin (Istodax, NSC 630176,
FR901228, FK228, depsipeptide): a natural
product recently approved for cutaneous
T-cell lymphoma. J. Antibiot., 64,
525–531.
49 (a) Xiao, J.J., Byrd, J., Marcucci, G.,
Grever, M., and Chan, K.K. (2003)
Identification of thiols and glutathione
conjugates of depsipeptide FK228
(FR901228), a novel histone protein
deacetylase inhibitor, in the blood. Rapid
Commun. Mass Spectrom., 17, 757–766.
(b) Tozuka, Z., Strupat, W.M., Shiraga, T.,
Ishimura, R., Hashimoto, T., Kawamura,
A., and Kagayama, A. (2005) LTQ FT MS
accurate mass and SRM data dependent
exclusion MSn measurements for
structure determination of FK228 and its
metabolites. J. Mass Spectrom. Jpn., 53,
89–99.
50 Robey, R.W., Chakraborty, A.R., Basseville,
A., Luchenko, V., Bahr, J., Zhan, Z., and
Bates, S.E. (2011) Histone deacetylase
inhibitors: emerging mechanisms of
resistance. Mol. Pharm., 8, 2021–2031.
51 Wen, S., Packham, G., and Ganesan, A.
(2008) Macrolactamization versus
macrolactonization: total synthesis of
FK228, the depsipeptide histone
deacetylase inhibitor. J. Org. Chem., 73,
9353–9361.
52 Yurek-George, A., Cecil, A., Mo, A.H.K.,
Wen, S., Rogers, H., Habens, F., Maeda,
S., Yoshida, M., Packham, G., and
Ganesan, A. (2007) The first biologically
active synthetic analogues of FK228, the
depsipeptide histone deacetylase inhibitor.
J. Med. Chem., 50, 5720–5726.
53 Hong, J. and Luesch, H. (2012) Largazole:
from discovery to broad-spectrum therapy.
Nat. Prod. Rep., 29, 449–456.
54 Benelkebir, H., Marie, S., Hayden, A.L.,
Lyle, J., Loadman, P.M., Crabb, S.J.,
Packham, G., and Ganesan, A. (2011)
Total synthesis of largazole and analogues:
HDAC inhibition, antiproliferative activity
and metabolic stability. Bioorg. Med.
Chem., 19, 3650–3658.
55 (a) Biggins, J.B., Gleber, C.D., and Brady,
S.F. (2011) Acyldepsipeptide HDAC
inhibitor production induced in
Burkholderia thailandensis. Org. Lett., 13,
1536–1539. (b) Wang, C., Henkes, L.M.,
Doughty, L.B., He, M., Wang, D., MeyerAlmes,
F.J., and Cheng, Y.Q. (2011)
Thailandepsins: bacterial products with
potent histone deacetylase inhibitory
activities and broad-spectrum
antiproliferative activities. J. Nat. Prod.,
74, 2031–2038.
56 Benelkebir, H., Donlevy, A.M., Packham,
G., and Ganesan, A. (2011) Total synthesis
and stereochemical assignment of
burkholdac B, a depsipeptide HDAC
inhibitor. Org. Lett., 13, 6334–6337.
57 Nakao, Y., Yoshida, S., Matsunaga, S.,
Shindoh, N., Terada, Y., Nagai, K.,
Yamashita, J.K., Ganesan, A., van Soest,
R.W.M., and Fusetani, N. (2006)
Azumamides A-E: histone deacetylase
inhibitory cyclic tetrapeptides from the
marine sponge Mycale izuensis. Angew.
Chem., Int. Ed., 45, 7553–7557.
58 Wen, S., Carey, K.L., Nakao, Y., Fusetani,
N., Packham, G., and Ganesan, A. (2007)
Total synthesis of azumamide A and
azumamide E, evaluation as histone
deacetylase inhibitors, and design of a
more potent analogue. Org. Lett., 9,
1105–1108.
59 Tiffon, C.E., Adams, J.E., van der Fits, L.,
Wen, S., Townsend, P.A., Ganesan, A.,
Hodges, E., Vermeer, M.H., and
Packham, G. (2011) The histone
deacetylase inhibitors vorinostat and
romidepsin downmodulate IL-10
expression in cutaneous T-cell
lymphoma cells. Br. J. Pharmacol., 162,
1590–1602.
References j137
60 (a) Ellis, L. and Pili, R. (2010) Histone
deacetylase inhibitors: advancing
therapeutic strategies in hematological
and solid malignancies. Pharmaceuticals,
3, 2441–2469. (b) Kim, H.J. and Bae, S.C.
(2011) Histone deacetylase inhibitors:
molecular mechanisms of action and
clinical trials as anti-cancer drugs. Am.
J. Transl. Res., 3, 166–179.
61 (a) Dinarello, C.A., Fossati, G., and
Mascagni, P. (2011) Histone deacetylase
inhibitors for treating a spectrum of
diseases not related to cancer. Mol. Med.,
17, 333–352. (b) Ververis, K. and
Karagiannis, T.C. (2011) Potential nononcological
applications of histone
deacetylase inhibitors. Am. J. Transl. Res.,
3, 454–467.
62 Prinjha, R.K., Witherington, J., and Lee,
K. (2012) Place your BETs: the therapeutic
potential of bromodomains. Trends
Pharmacol. Sci., 33, 146–153.
63 Yost, J.M., Korboukh, I., Liu, F., Gao, C.,
and Jin, J. (2011) Targets in epigenetics:
inhibiting the methyl writers of the
histone code. Curr. Chem. Genomics,
5, 72–84.
64 Suzuki, T. and Miyata, N. (2011) Lysine
demethylases inhibitors. J. Med. Chem.,
54, 8236–8250.
65 Huang, J., Plass, C., and Gerhauser,
C. (2011) Cancer chemoprevention by
targeting the epigenome. Curr. Drug
Targets, 12, 1925–1956.
138j5 The Discovery of Anticancer Drugs Targeting Epigenetic Enzymes
A. Ganesan received his B.Sc. degree from the National
University of Singapore (1986), followed by a Ph.D. from the
University of California-Berkeley (1992), and postdoctoral
studies at Harvard University. Between 1993 and 1999,
he worked at the Institute of Molecular and Cell Biology
in Singapore, heading the Medicinal and Combinatorial
Chemistry Group. In 1999, he joined the University of Southampton’s
Combinatorial Centre of Excellence as a Reader.
Since 2011, he is Professor of Chemical Biology in the School
of Pharmacy at the University of East Anglia in Norwich (UK). He is a co-founder
of Karus Therapeutics, a drug discovery spinout company.
References j139
6
Thienopyridyl and Direct-Acting P2Y12 Receptor Antagonist
Antiplatelet Drugs
Joseph A. Jakubowski and Atsuhiro Sugidachi
List of Abbreviations
AA arachidonic acid
ACS acute coronary syndrome
ADP adenosine 50
-diphosphate
AMP adenosine 50
-monophosphate
ASA acetyl salicylic acid (aspirin)
AUC area under time–concentration curve
BRIDGE maintenance of platelet inhibition with cangrelor after discontinuation
of thienopyridines in patients undergoing surgery
CABG coronary artery bypass graft
CAD coronary artery disease
CAPRIE clopidogrel versus aspirin in patients at risk of ischemic events
CD cluster of differentiation
CHAMPION cangrelor versus standard therapy to achieve optimal management
of platelet inhibition
Cmax maximum observed concentration
CURE clopidogrel in unstable angina to prevent recurrent events
CYP cytochrome P450
ECLIPSE elinogrel to clarify the optimal inhibition of platelets in secondary
prevention
ED50 effective dose that reduces response by 50%
FDA Food and Drug Administration
hCE human carboxyl esterase
INNOVATE-PCI randomized, double-blind, active-controlled trial to evaluate
intravenous and oral PRT060128, a selective and reversible
P2Y12 inhibitor, versus clopidogrel, as a novel antiplatelet therapy
in patients undergoing non-urgent PCI
MI myocardial infarction
PCI percutaneous coronary intervention
PD pharmacodynamic
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
j141
PK pharmacokinetic
PLATO the study of platelet inhibition and patient outcomes
SH sulfhydryl (reactive thiol)
TIMI thrombolysis in myocardial infarction
Tmax observed time of Cmax
TRITON trial to assess improvement in therapeutic outcomes by optimizing
platelet inhibition with prasugrel
TXA2 thromboxane A2
wt wild type
6.1
Introduction
6.1.1
Platelet Involvement in Atherothrombosis
Atherothrombosis is a term coined to describe the formation of a partially or fully
occlusive arterial thrombus at sites of atherosclerotic plaque. Multiple lines of evidence
support the involvement of platelets both in the formation and progression
of atherosclerotic plaques and in thrombus formation subsequent to plaque disruption.
Platelets initially adhere to exposed subendothelial collagen, von Willebrand
factor, and other adhesive proteins at sites of vascular injury or atherosclerotic plaque
erosion/rupture (Figure 6.1) [1–3]. Platelet activation following these initial
adhesive interactions results in the secretion from the activated platelets of adenosine
50
-diphosphate (ADP), thromboxane A2 (TXA2), and other mediators. Released
ADP promotes platelet activation via the G-protein-coupled P2Y1 and P2Y12 purinergic
receptors leading to further platelet activation, aggregation, and other platelet
functions such as platelet shape change and granular secretion [4–6].
Thromboxane A2, generated de novo upon platelet activation, similarly amplifies
platelet activation, aggregation, and other platelet functions, albeit via a separate set
of receptors and signaling pathways. These pathways just described are largely
responsible for recruiting additional platelets to sites of coronary plaque rupture
forming aggregates that may lead to platelet-rich thrombi, vascular occlusion, tissue
ischemia, and myocardial necrosis in what is collectively known as acute coronary
syndrome (ACS). Accordingly, agents that inhibit platelet activation and
aggregation (antiplatelet agents) are widely used in patients with atherothrombotic
disease [7–9]. Understanding the above roles of ADP and TXA2 in platelet recruitment
and aggregation, it is not surprising that the most commonly used antiplatelet
agents target the ADP and TXA2 pathways, namely, P2Y12 antagonists and
aspirin, respectively. P2Y12 antagonists will be discussed in detail below. Aspirin
interrupts the TXA2 pathway by irreversible inhibition of platelet cyclooxygenase,
the enzyme responsible for the initial step of TXA2 formation. Aspirin and P2Y12
antagonists individually have shown efficacy in the treatment of ischemic cardiovascular
disease; however, their distinct modes of action also allow for combination
142j6 Thienopyridyl and Direct-Acting P2Y12 Receptor Antagonist Antiplatelet Drugs
therapy (dual antiplatelet therapy), typically resulting in efficacy that is greater than
that provided by either agent alone. Given the platelets’ physiological role in the
promotion of hemostasis, antiplatelet therapy is associated with a bleeding liability.
6.2
Thienopyridines
The thienopyridines are the best-known family of P2Y12 targeted oral antiplatelet
agents. They are all prodrugs and require in vivo metabolism to an active
metabolite. Each active metabolite contains a reactive thiol (SH) group that binds
specifically to the platelet P2Y12 ADP receptor. Platelets also contain a second
G-protein-coupled purinergic (ADP) receptor, P2Y1, but this is unaffected by
the thienopyridines. Much of our more detailed mechanistic information about the
mechanism of action of the thienopyridines has been obtained from studies of
clopidogrel and prasugrel. It is of interest to note that less specific information is
available for ticlopidine since at the time of its development, clinical evaluation,
and regulatory approval neither the structure of the active receptor antagonist nor
the target receptor itself had been described.
The thienopyridine prodrugs, by definition, are all inactive in vitro as platelet
aggregation inhibitors. While several publications describe in vitro activities of the
parent molecules, these observations are mostly at very high concentrations that
would not be sustained following administration at approved doses. Thus, it is generally
accepted that single active metabolite for each thienopyridine provides
the established pharmacological effects of the thienopyridines. The labile reactive
thiol-containing active metabolites bind irreversibly to P2Y12 via cysteines 97 and
175 that are close to the ligand binding site in the transmembrane region of
Figure 6.1 Pathways of platelet activation and aggregation. (Modified from Ref. [10].)
6.2 Thienopyridines j143
P2Y12 [11, 12]. Thus, when administered at doses that singly result in submaximal
inhibition, the magnitude of inhibition increases on a daily basis up to the target
level of inhibition that ultimately reflects the daily dose given [13]. Steady-state
platelet inhibition is typically reached in 3–7 days. This “cumulative inhibition”
reflects the irreversible nature of active metabolite binding to the platelet and the
circulating platelet’s inability to generate and express new P2Y12. Recovery of platelet
function after cessation of thienopyridine administration also reflects the
irreversibility of AM binding to P2Y12 and the requisite entry of new platelets into
the circulation. Accordingly, recovery of platelet function may take up to 7 days following
the last administered dose of thienopyridine. Full restoration of platelet
function, which is not necessarily required to provide hemostasis, reflects the level
of inhibition present at the time of discontinuation [14].
6.2.1
Ticlopidine: 5-[(2-Chlorophenyl)methyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine
The discovery of ticlopidine, which has been addressed elsewhere by Savi and Herbert
[15], resulted from an in vivo/ex vivo screening process in which rodents were
orally administered the test agents and ex vivo platelet aggregation response was
used as the bioassay. Screened agents with antiplatelet activity would have shown
an attenuated aggregation response compared to control values. Interestingly, while
the bioassay was inhibition of platelet aggregation, this was used as a surrogate to
identify molecules with potential anti-inflammatory utility. A detailed structure–
activity relationship has not been reported. However, ticlopidine was ultimately
developed and approved as an antiplatelet agent. Following evaluation of ticlopidine
in animal models of ex vivo platelet inhibition and in vivo thrombus formation, it
moved into the clinical arena and first reports of its phase 1 studies confirmed its
antiplatelet activity in man [16, 17]. Following a series of phase 2 and phase 3 efficacy
trials, ticlopidine was approved for clinical use with indications for use in the
primary and secondary prevention of stroke. As would be expected with administration
of a platelet inhibitory agent, ticlopidine use was associated with increased
bleeding. However, more troublesome side effects included neutropenia, which
occurred at an overall rate of 2.4% of which 0.8% were considered severe, and
thrombotic thrombocytopenic purpura1)
. While not approved for such cases, ticlopidine
was widely used as adjunctive therapy in percutaneous coronary artery intervention
(PCI) and stent placement. This use emerged from several studies
indicating that ticlopidine was more effective at reducing recurrent ischemia
and with fewer hemorrhagic events compared with standard therapy at the
time [18–21].
The approved daily dose of ticlopidine is 500 mg given as 250 mg twice per day.
After several daily doses, steady-state platelet inhibition is achieved and maintained
thereafter at approximately 40% inhibition of ADP-induced platelet
1) Ticlid (US package insert). Available at http://www.accessdata.fda.gov/drugsatfda_docs/nda/
2001/19-979S018_Ticlid_prntlbl.pdf (accessed May 11, 2012).
144j6 Thienopyridyl and Direct-Acting P2Y12 Receptor Antagonist Antiplatelet Drugs
aggregation [22]. Relatively little information is available on the pharmacokinetic
profile of ticlopidine and its active metabolite. This in part reflects the lack of
knowledge of ticlopidine’s active metabolite at the time of its initial development
and clinical evaluation. While initial FDA approval for ticlopidine was obtained in
October of 1991, it was not until 2000 that its active metabolite structure was
deduced following identification of clopidogrel’s and prasugrel’s active metabolite
structures [23, 24] and later more definitively disclosed [25]. The pathway from
ticlopidine to its active metabolite is shown in Figure 6.2. The initial step from
ticlopidine to its 2-oxo intermediate is mediated by cytochrome P450 (CYP) 2C19
and CYP2B6 [26, 27]; this metabolite is not active at P2Y12 and presumably requires
further oxidation by members of the CYP enzyme family to the SH-containing
active metabolite.
Despite the limitations of ticlopidine (lack of understanding of its mode of action
and target receptor, bid dosing, and serious hematological side effects), ticlopidine
played an early, pivotal role in enabling the widespread adoption of coronary artery
stenting. However, a “next-generation” molecule that would provide the clinical
benefit of ticlopidine’s antiplatelet effects but with a more acceptable safety and
“convenience” profile resulted in the development of clopidogrel. It is considered
one of the “blockbuster” cardiovascular drugs of the twenty-first century with
annual sales in 2011 of approximately 8 billion dollars.
6.2.2
Clopidogrel: (þ)-(S)-a-(2-Chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)
acetate
As may be seen from the chemical structures (Figure 6.3), clopidogrel has a different
substitution pattern at the benzylic carbon, namely, a methoxycarbonyl (methyl
ester) group. This molecule was originally described (as PCR 4099) together with
its increased potency over ticlopidine by Maffrand et al. [28] and subsequently
Defreyn et al. [29] and has been extensively reviewed (e.g., see Ref. [15]). Like ticlopidine,
clopidogrel itself is inactive in vitro and was identified based on its platelet
inhibition profile following administration to rodents and assessment of ex vivo
platelet aggregation. Introduction of the methyl ester at the benzylic center introduces
a chiral center into the molecule that was not present in its predecessor ticlopidine.
However, preclinical studies indicated that the R-enantiomer lacked in vivo
activity; accordingly, the S-enantiomer advanced for clinical evaluation. Preclinical
studies in rodents indicated that following oral dosing clopidogrel was substantially
more potent than ticlopidine in terms of ex vivo inhibition of platelet aggregation
Figure 6.2 Structure and activation of ticlopidine.
6.2 Thienopyridines j145
and inhibition of thrombus formation [23]. Early clinical pharmacology trials in
humans confirmed the increased activity of clopidogrel compared to ticlopidine
and, at steady-state platelet inhibition, the levels of inhibition associated with ticlopidine’s
approved dosing regimen of 500 mg/day were achieved with clopidogrel at
daily doses of just 50–75 mg [22]. Based on further clinical evaluation, 75 mg of
clopidogrel was chosen as the daily dose to explore in pivotal phase 3 trials. This
dose was in part chosen on the basis of it providing similar levels of inhibition as
ticlopidine at its approved doses with the anticipation that similar clinical efficacy
would be achieved but in the absence of the blood dyscrasias associated with ticlopidine.
In addition, once a day dosing would be preferable to the bid dosing
required for ticlopidine. Ultimately several phase 3 studies of clopidogrel demonstrated
that clopidogrel did have a superior side effect profile than ticlopidine [30].
While 75 mg is the approved daily (maintenance) dose of clopidogrel, providing
approximately 40% inhibition of ADP-induced platelet aggregation, achieving this
steady-state level of inhibition takes several days of dosing. Accordingly in those
situations where higher levels of inhibition are required more rapidly, loading
doses of 300–600 mg are used. Clopidogrel has a wide range of indications in
patients with ischemic cardiovascular disease either as monotherapy or, in combination
with aspirin, as dual antiplatelet therapy. Based on the results of the CAPRIE
clinical trial, clopidogrel gained its first regulatory approval as monotherapy for the
secondary prevention of atherothrombotic disease [31]. Its indications were broadened
as a result of the CURE trial [32] and associated CURE-PCI [33], the collective
results of which documented the superiority of clopidogrel plus aspirin over aspirin
alone. Further details of the clinical trials supporting these and other indications
can be found elsewhere [15, 34]. Despite the similarity in structure of clopidogrel
to ticlopidine, clopidogrel therapy is associated with much lower rates of neutropenia
than those found with ticlopidine (see above). Given that there is no generally
accepted mechanistic basis for the neutropenia associated with ticlopidine, it is not
possible to explain the different rates found among the thienopyridine family of
compounds.
Figure 6.3 Structure and metabolism of clopidogrel.
146j6 Thienopyridyl and Direct-Acting P2Y12 Receptor Antagonist Antiplatelet Drugs
Like ticlopidine, clopidogrel is converted to its active metabolite by metabolism of
the thiophene ring at the position adjacent to the sulfur (Figure 6.3). Initial oxidation
to the 2-oxo derivative is mediated by CYP enzymes with CYP2C19, CYP1A2,
and CYP2B6 playing major roles [35]. As with ticlopidine’s 2-oxo intermediate,
2-oxo-clopidogrel is devoid of platelet inhibitory activity and requires further oxidation
to open the thiophene ring generating the SH (“reactive thiol”)-containing
active metabolite. The CYP enzymes involved in this second step include CYP3A,
CYP2B6, CYP2C9, and CYP2C19 [35]. The structure of the active metabolite as
shown in Figure 6.3 was first definitively described in 2000 [24] with follow-on studies
on its stereochemistry appearing subsequently [36].
While introduction of the methyl ester group increased the in vivo antiplatelet
activity, this substitution also renders clopidogrel susceptible to hydrolysis by human
carboxyl esterases (hCEs). Carboxyl esterases are ubiquitous in humans and other
species and are acknowledged to act on a wide range of ester- and amide-containing
xenobiotics [37]. Thus, approximately 80% of the administered dose of clopidogrel is
hydrolyzed to an inactive carboxylic acid metabolite2)
[38] (Figure 6.3). Recent evidence
indicates that 2-oxo-clopidogrel, the intermediate en route to the active metabolite,
also appears susceptible to hydrolysis [38]. Overall, the consequence of hCE
activities would appear to be a reduction in the efficiency of conversion of the
administered dose clopidogrel to its active metabolite with perhaps as little as 10%
of the administered dose ending up as the active metabolite. It was only recently
that reliable assays for the active metabolite of clopidogrel were developed [39].
As with many other pharmaceutical agents, genetic polymorphisms resulting in
reduced function alleles encoding for the CYP enzyme lead to altered pharmacokinetics
of clopidogrel. Notably, Brandt et al. initially demonstrated that polymorphisms
in CYP2C19, which is involved in both oxidative steps of clopidogrel’s
transformation to its active metabolite, and to a lesser extent CYP2C9, resulted in
reduced exposure to clopidogrel’s active metabolite and reduced platelet inhibition
[40]. Consistent with these observations on impaired activation of clopidogrel
and reduced platelet inhibition was the finding that patients treated with clopidogrel
who had at least one loss of function allele in CYP2C19 had an approximately
1.55-fold increase in the rate of death, heart attack, and stroke compared to noncarriers
[41, 42]. The above association of loss of function alleles for CYP2C19 and
reduced clinical effectiveness are currently reflected in the prescribing information
for clopidogrel2)
.
6.2.3
Prasugrel: 5-[(1RS)-2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl
acetate
Prasugrel (originally known as CS-747 and LY640315), which is inactive in vitro,
was identified by an in vivo/ex vivo screening approach and first described by
2) Plavix (US package insert). Available at http://www.accessdata.fda.gov/drugsatfda_docs/label/
2011/020839s055lbl.pdf (accessed May 11, 2012).
6.2 Thienopyridines j147
Sugidachi et al. [23]. Prasugrel’s structure is shown in Figure 6.4; in contrast to
clopidogrel, the benzylic carbon is substituted with a cyclopropyl carbonyl group.
In addition, the individual R and S enantiomers at this position in prasugrel
undergo spontaneous racemization in solution. Accordingly, prasugrel was developed
as the racemate rather than as one of the individual enantiomers. Further
differences in prasugrel compared to clopidogrel are found in the substitution
pattern in the thiophene ring in which prasugrel contains an acetoxy group at position
2 and in the halide moiety in the phenyl ring (fluoro- versus chloro-).
Early rodent studies demonstrated that following oral dosing, prasugrel produced
dose-related inhibition of ADP-induced platelet aggregation that was approximately
10- and 100-fold more potent than clopidogrel and ticlopidine, with ED50 doses of
1.2, 16, and >300 mg/kg, respectively [23]. The antithrombotic effects of prasugrel
were also investigated and compared to clopidogrel and ticlopidine in a rat arteriovenous
shunt thrombosis model. Consistent with the relative platelet inhibitory
profile, prasugrel, on a mg/kg basis, had approximately 10 and 100 times more
antithrombotic activity compared with clopidogrel and ticlopidine, exhibiting ED50
values of 0.68, 6.2, and >300 mg/kg, respectively (Figure 6.5). Further preclinical
evaluation of prasugrel indicated that combined administration of prasugrel and
Figure 6.4 Structure and activation of prasugrel.
Figure 6.5 Inhibition of thrombus formation in the rat arteriovenous shunt model following oral
dosing with thienopyridines. (Derived from Ref. [23].)
148j6 Thienopyridyl and Direct-Acting P2Y12 Receptor Antagonist Antiplatelet Drugs
aspirin produced greater inhibition of both platelet aggregation and thrombus formation
compared to either drug alone in rats [43]. These data are consistent with
the alternate pathways that these agents inhibit.
A series of phase 1 and phase 2 clinical studies that assessed the safety, pharmacokinetics,
and pharmacodynamics of both loading and maintenance doses of prasugrel,
the majority of which included clopidogrel as the comparator, have been
completed [10]. In summary, these studies indicated that prasugrel was well tolerated
and exhibited an acceptable side effect profile. The most common observed
side effect being the impaired hemostasis that would be expected with effective
platelet inhibition. Consistent with the rodent studies, following oral administration
in humans prasugrel was found to be approximately 10- and 100-fold more
potent than clopidogrel [10, 13] and ticlopidine, respectively, at inhibiting ADPinduced
platelet aggregation. Figure 6.6 illustrates data collected from two studies
that collectively illustrate the relative platelet inhibitory activities of the three thienopyridines.
As described earlier, the approved maintenance dose of clopidogrel
(75 mg) provides approximately the same level of ADP-induced platelet aggregation
as the approved maintenance dose of ticlopidine (500 mg), while prasugrel provides
similar levels of inhibition at a dose of 5 mg/day.
Based on the results of phase 1 and 2 studies, a loading dose of 60 mg and maintenance
doses of 10 mg/day were chosen to take forward into the pivotal phase 3
study of prasugrel. This study, Trial to Assess Improvement in Therapeutic Outcomes
by Optimizing Platelet Inhibition with Prasugrel Thrombolysis in
Figure 6.6 Levels of inhibition of human platelet aggregation achieved with clinically relevant
doses of thienopyridines. (Derived from (1) Ref. [22] and (2) Ref. [13].)
6.2 Thienopyridines j149
Myocardial Infarction (TRITON-TIMI 38), was a randomized, double-blind, parallel
group, multinational clinical trial that randomized a total of 13 608 ACS patients
undergoing PCI to prasugrel or standard dose clopidogrel. The primary objective
was to test the hypothesis that prasugrel is superior to clopidogrel on a background
of aspirin in the treatment of patients with ACS who are to undergo PCI measured
by the composite end point of cardiovascular death, nonfatal MI, or nonfatal stroke.
The results showed that the primary efficacy end point occurred in 12.1% of
patients receiving clopidogrel and 9.9% of patients receiving prasugrel, a statistically
significantly reduction of 19% [44]. However, prasugrel-treated patients experienced
significantly more major bleeding than the clopidogrel-treated patients
(2.8% versus 1.4%, respectively).
Detailed metabolic, pharmacokinetic, pharmacodynamic, and pharmacogenomic
studies have provided great insight into the relative disposition of prasugrel and
clopidogrel. These studies were in part enabled by the development of reliable
assays for the determination of plasma concentrations of both clopidogrel’s and
prasugrel’s active metabolites [39, 45]. Of note, these assays were not available during
the clinical development of ticlopidine and clopidogrel. Figure 6.7 shows the
notable difference in exposure to the aforementioned active metabolites following
loading dose administration in humans. It can be seen that despite being administered
at one-fifth the dose of clopidogrel (i.e., 60 mg versus 300 mg), prasugrel
resulted in substantially greater active metabolite exposure than did clopidogrel.
This was reflected in the Cmax and AUC for prasugrel’s active metabolite that were
approximately 14- and 12-fold greater than those for clopidogrel’s active metabolite,
and greater and more consistent platelet inhibition [46, 47]. On a dose-adjusted
basis, this represents an approximately 60-fold greater active metabolite exposure
following prasugrel.
Numerous factors contribute to the greater exposure to prasugrel’s active
metabolite [48, 49]. In contrast to the methyl ester found at the benzylic carbon
of clopidogrel that is susceptible to esterase activity, the cyclopropyl carbonyl
group at the corresponding position in prasugrel cannot be hydrolyzed by hCE
activity. Conversely, the acetoxy group at position 2 in prasugrel is susceptible to
hydrolysis, albeit by different carboxyl esterases (hCE-2, present in the intestinal
wall) resulting in rapid generation of prasugrel’s intermediate metabolite (prasugrel
thiolactone). Thus, following absorption/hydrolysis, only a single CYPmediated
oxidative step is required to generate prasugrel’s active metabolite.
This oxidative step is mediated by CYP3A4 and CYP2B6, and to a lesser extent
by CYP2C9 and CYP2C19 [50]. In contrast to CYP2C19, genetic variation in
CYP3A4 does not result in a meaningful loss of function phenotype. The lack
of impact of CYP2C19 loss of function alleles on prasugrel’s pharmacokinetics/
pharmacodynamics is illustrated in Figure 6.8. It can be seen that for clopidogrel,
compared to fully functional CYP2C19 (wt/wt), the presence of one loss of
function allele (wt/Ã
2) results in decreased active metabolite exposure (a) and
resulting loss of platelet inhibition (c), and with the presence of two loss of
function alleles (Ã
2/Ã
2) the effect is further increased. In contrast, neither
hetero- nor homozygous loss of function results in altered active metabolite
150j6 Thienopyridyl and Direct-Acting P2Y12 Receptor Antagonist Antiplatelet Drugs
exposure or platelet inhibition in prasugrel-treated subjects (Figure 6.8b and c).
Taken together, the above factors explain in large part the more efficient conversion
of prasugrel to its active metabolite compared to the fate of clopidogrel
following administration to humans. It should be noted that loss of function
alleles are found in approximately 30% of the Caucasian population and at a
higher frequency in certain Asian populations. As described above, the negative
impact of CYP2C19 loss of function on exposure to clopidogrel’s active metabolite
and inhibition of platelet aggregation were consistent with poor outcome in
patients treated with clopidogrel [41]. Similar analysis for prasugrel likewise
demonstrated that the PK/PD data were consistent with the clinical course in
that CYP2C19 loss of function did not lead to a poorer outcome in ACS patients
undergoing intervention and stent placement [42].
Figure 6.7 Exposure to clopidogrel’s and prasugrel’s active metabolites following oral dosing:
Cmax, maximum concentration; AUC, area under the concentration–time curve; Tmax, time of Cmax.
(Modified from Ref. [10].)
6.2 Thienopyridines j151
More detailed reviews of prasugrel’s early and later stage development program
can be found elsewhere [10, 51].
6.3
Direct-Acting P2Y12 Antagonists
6.3.1
Nucleoside-Containing Antagonists
The early observation that ATP was an antagonist of ADP-induced platelet aggregation
[52] led to the original quest for more rationally designed antagonists of the
platelet ADP receptor. While ATP is an antagonist, it would be in its own right
unsuitable as a clinically useful antagonist given its interaction with many different
purinergic receptors and its rapid degradation by the ecto-ADPase CD39 to metabolites
such as ADP, AMP, and adenosine that themselves possess diverse pharmacological
activities.
Figure 6.8 Influence of loss of function allele
(Ã
2) in CYP2C19 on the pharmacokinetics of
clopidogrel’s (a) and prasugrel’s (b) active
metabolites, and on inhibition of ADP-induced
platelet aggregation (c) (clopidogrel, white bars;
prasugrel, black bars). Integrated data from
crossover studies in which all subjects received
loading doses of both thienopyridines. (Derived
from Ref. [40].)
152j6 Thienopyridyl and Direct-Acting P2Y12 Receptor Antagonist Antiplatelet Drugs
6.3.1.1 Cangrelor: [Dichloro-[[[(2R,3S,4R,5R)-3,4-dihydroxy-5-[6-(2-methylsulfanylethy-
lamino)-2-(3,3,3-trifluoropropylsulfanyl)purin-9-yl]oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]methyl]phosphonic
acid
Cangrelor (formerly AR-C69931MX) is a direct-acting and competitive antagonist
of ADP-induced platelet aggregation. Its similarity to the naturally occurring purinergic
ligand ATP is readily apparent in Figure 6.9. Modifications to ATP were
aimed to improve potency, specificity, and stability and resulted in a molecule with
nM affinity for the P2Y12 (originally known as P2T) ADP receptor [53]. In contrast to
the thienopyridines that are inactive in vitro, several in vitro studies have shown that
cangrelor is a direct-acting inhibitor of ADP-induced platelet aggregation that does
not require activation in vivo [54]. The rapid recovery of platelet function after termination
of cangrelor administration made it suitable as an acute intravenous agent.
Pharmacologically, cangrelor infusions in ACS patients resulted in time- and dosedependent
inhibition of ADP-induced platelet aggregation with doses of
2 mg/kg/min and above resulting in near maximal inhibition of platelet aggregation
[55]. Following termination of infusion, recovery of reactivity to ADP was both
dose and time dependent with full recovery being approached at 1 h at doses up to
2 mg/kg/min; however, at doses of 4 mg/kg/min 40% platelet inhibition was still
present 1 h after termination of infusion [55]. Plasma concentrations dropped rapidly
after ending infusions and appeared to somewhat outpace recovery of platelet
inhibition. Further studies with cangrelor in patients with acute myocardial infarction
suggested utility of cangrelor for this indication [56]. Two phase 3 efficacy trials
of cangrelor in ACS patients undergoing PCI were recently completed. The trials
were designed to assess whether cangrelor, when administered acutely at the time
of PCI, improved short-term outcomes.
Figure 6.9 Structures of adenosine, the adenine nucleotides ADP and ATP, and the P2Y12
antagonist analogues thereof.
6.3 Direct-Acting P2Y12 Antagonists j153
The clinical efficacy of cangrelor was assessed in the CHAMPION trials. CHAMPION
PCI randomized patients either to a bolus and short-term infusion of cangrelor
or to a 600 mg loading dose of clopidogrel. At the end of the cangrelor infusion,
a 600 mg loading dose of clopidogrel was administered to the cangrelor patients.
The primary composite end point of major adverse cardiovascular events in the
cangrelor group was not significantly different from the clopidogrel arm [57]. A
similar lack of differentiation between clopidogrel loading dose and cangrelor
infusion was found in a parallel phase 3 PCI study in ACS [58]. As will be discussed
later, it would appear that dyspnea is a common side effect noted with the administration
of direct-acting P2Y12 antagonists, and this was also observed in the CHAMPION
studies.
It has been proposed that a possible reason for the failure of cangrelor to show
efficacy over clopidogrel in the CHAMPION trials resulted from the presence of
residual cangrelor on the platelet at the time of subsequent clopidogrel loading. As
has previously been demonstrated, cangrelor prevents the binding of clopidogrel’s
active metabolite to platelet P2Y12 [59]. This hindrance, combined with a relatively
short exposure to clopidogrel’s active metabolite, raises the possibility that by the
time cangrelor is fully cleared from the platelet there is insufficient active metabolite
of clopidogrel present to effectively inhibit the platelet P2Y12 receptor. Support
for this competitive interaction has indeed been provided in ex vivo studies of clopidogrel
administration at the time of cangrelor termination [60]. Such an interaction
does not occur when switching among the thienopyridines [61].
Cangrelor’s relatively short half-life is consistent with its utility in weaning
patients off of oral P2Y12 antagonists in preparation for major surgery where dual
antiplatelet therapy may pose a serious bleeding risk. However, extended periods of
time while “washing out” the oral agent, resulting in less effective platelet inhibition
in patients with recent ACS events, may result in the recurrence of ischemic
events, thus the concept of maintaining patients on a cangrelor infusion during the
washout phase and termination of the cangrelor infusion just proximal to surgery.
The feasibility of such a study was recently tested in the BRIDGE study [62].
6.3.1.2 Ticagrelor: (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-Difluorophenyl)cyclopropyla-
mino]-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)cyclo-
pentane-1,2-diol
Ticagrelor (formerly AZD6140) is a direct-acting nucleoside analogue-containing
P2Y12 antagonist. As described previously, the identification of cangrelor resulted
from modifications to the natural ligand ATP. In turn, the discovery of ticagrelor
reflects further chemical modifications to cangrelor aimed at identifying an orally
available agent while maintaining potency and selectivity. In brief, the route from
ATP to ticagrelor has been described by Springthorpe et al. [63] as follows:
(a) introduction of affinity-enhancing 5,7-hydrophobic substituents;
(b) replacement of the labile triphosphate group; (c) changing the core purine to a
triazolopyrimidine, increasing affinity >100-fold; (d) finding the first non-acidic
reversible antagonists; (e) introduction of a trans-2-phenylcyclopropylamino substituent,
increasing affinity >10-fold; and (f) identifying metabolically stable neutral
154j6 Thienopyridyl and Direct-Acting P2Y12 Receptor Antagonist Antiplatelet Drugs
compounds by modifying the hydrophobic phenylcyclopropyl group and the
hydroxylic side chain substituent.
As can be seen in Figure 6.9, ticagrelor retains elements of the naturally occurring
adenine nucleotides and the intravenous P2Y12 antagonist cangrelor and is
similarly classified as a reversible antagonist. However, in contrast to cangrelor,
ticagrelor is available by the oral route; it also differs from cangrelor in that its interaction
with P2Y12 is noncompetitive whereas cangrelor under certain assay conditions
would appear to be competitive [64]. While ticagrelor has inherent P2Y12
receptor antagonistic activity, following oral administration a major active metabolite
is formed by O-dealkylation mediated by CYP3A4/5 isoforms [65]. This metabolite
(AR-C124910XX) has P2Y12 antagonist activity approximately equal to the
parent and approximates 40% of the exposure to ticagrelor (Figure 6.10). The halflives
of ticagrelor and AR-C124910XX are 8.4 and 11.5 h, respectively.
Detailed pharmacokinetic and pharmacodynamic studies of ticagrelor have been
reported [66, 67] and indicate that reduction in ADP-induced platelet aggregation
occurs within 2 h after oral dosing consistent with peak plasma concentrations
being achieved at 2 h after dosing. More consistent inhibition of platelet aggregation
was achieved with twice daily dosing (bid) and at doses >50 mg bid inhibition
was greater than that achieved by standard clopidogrel dosing. Additional studies in
aspirin-treated CAD patients indicated that doses of !90 mg bid dosing provided
platelet inhibition that was consistently greater than that achieved by
clopidogrel [68].
Phase 3 evaluation of ticagrelor in aspirin-treated patients with ACS managed
both invasively and noninvasively was performed in the PLATO study. The results
indicated that ticagrelor (180 mg loading dose and 90 mg bid maintenance dose)
Figure 6.10 Pharmacokinetics of ticagrelor and its major circulating active metabolite (ARC124910XX),
following ticagrelor administration to healthy subjects. (Modified from Ref. [65].)
6.3 Direct-Acting P2Y12 Antagonists j155
plus aspirin resulted in a 16% relative risk reduction in the composite of vascular
death, MI, or stroke compared to clopidogrel plus aspirin, including a statistically
significant reduction in all-cause mortality. Ticagrelor was associated with an
increase in major bleeding and compared to clopidogrel TIMI non-CABG major
bleeding was increased by 25% [69]. As with prasugrel, genetic polymorphisms
associated with CYP loss of function did not compromise the benefit of ticagrelor
over clopidogrel [70]. In detailed parallel pharmacodynamic studies in CAD
patients at doses of ticagrelor and clopidogrel utilized in PLATO, Gurbel et al. [71]
have demonstrated rapid induction of platelet inhibition following the 180 mg loading
dose with high levels being maintained with 90 mg bid (Figure 6.11). Interestingly,
following cessation of ticagrelor, recovery of platelet function was delayed
requiring approximately 5 days to be fully restored to baseline levels. This is in
apparent contrast to the pharmacokinetic profile of ticagrelor and its active metabolite
that are undetectable at 48 h postdosing (Figure 6.10) and suggests that other
factors are at play at the platelet and/or P2Y12 level.
There has been much speculation over the possibility that ticagrelor may be
metabolized to adenosine in vivo and that adenosine mediates off-target side
effects [72]. Chemically, this is unlikely; however, the discussion has been driven by
the observation of increased rates of dyspnea, ventricular pauses, and
Figure 6.11 Time course of induction of and recovery from platelet inhibition following ticagrelor
administration. (Used with permission from Ref. [71].)
156j6 Thienopyridyl and Direct-Acting P2Y12 Receptor Antagonist Antiplatelet Drugs
hyperuricemia in clinical trials of ticagrelor [69, 73, 74]. While these may indeed be
side effects of adenosine, there is, to date, no evidence that increased plasma adenosine
levels explain the manifestation of these side effects in ticagrelor-treated
patients (see also discussion of elinogrel below). An alternative mechanism for
adenosine-like side effects is the possibility that local concentrations of adenosine
may increase as a result of ticagrelor inhibiting adenosine reuptake by the erythrocyte
[75]. However, the potency of this effect may be too low and has recently been
questioned and alternative mechanisms proposed [76].
6.3.2
Non-Nucleoside P2Y12 Antagonists
6.3.2.1 Elinogrel: N-[(5-Chlorothiophen-2-yl)sulfonyl]-N0
-{4-[6-fluoro-7-(methylamino)-
2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl]phenyl}urea
Elinogrel (formerly PRT060128 or PRT128), as may be appreciated from the structure
below (Figure 6.12), is neither a thienopyridine nor a nucleoside-containing
antagonist. Elinogrel thus constitutes a third chemical class, and is a substituted
quinazolinedione phenyl thiophenyl sulfonylurea P2Y12 antagonist [77].
Elinogrel differs from the other direct-acting agents in that it is a competitive
inhibitor of P2Y12 and may be administered either via the intravenous or via oral
route, with the latter route requiring twice daily dosing. The requirement for twice
daily dosing is consistent with the pharmacokinetic profile of orally administered
elinogrel that, in response to a 60 mg dose, shows peak plasma concentrations
being achieved approximately 4 h after dosing and returning to negligible levels by
24 h [78]. In contrast to other direct-acting P2Y12 antagonists, there would appear to
be a consistent relationship between exposure and pharmacodynamic effect with
inhibition of platelet reactivity to ADP reaching a peak at 4 h and fully recovering
by 24 h. Intravenous bolus doses of 10–60 mg were well tolerated in ACS patients
undergoing primary PCI [79].
Phase 2 studies of elinogrel (INNOVATE-PCI) indicated that high levels of platelet
inhibition could be achieved in 30–120 min following combined administration
of an intravenous bolus and concomitant oral formulation of elinogrel and that
inhibition could be maintained with oral twice daily dosing of 100–150 mg/day [80].
Figure 6.12 Structure of elinogrel.
6.3 Direct-Acting P2Y12 Antagonists j157
Consistent with the reversible nature of the antagonist, partial recovery of platelet
function to those levels associated with clopidogrel administration was evident
between doses. Interestingly, in this phase 2 study elinogrel was associated with an
increased rate of dyspnea, which while being reported as mild and transient was
three times more common in patients treated with elinogrel compared to those
treated with clopidogrel. Since elinogrel is a non-nucleoside P2Y12 antagonist,
induction of dyspnea would appear to not be the result of adenosine, as has been
speculated for ticagrelor, but rather an off (platelet) target effect(s) of the direct-acting,
non-thienopyridine, P2Y12 antagonists. An additional concern raised by the
INNOVATE-PCI trial was an approximately four- to fivefold increase in liver
enzyme levels [80].
At present, the future development program for elinogrel is unclear following the
recent company announcement that it would not proceed with its planned phase 3
study of elinogrel in patients with prior history of MI, the ECLIPSE study.
6.4
Summary
Following the serendipity of the initial development of ticlopidine, newer generations
of P2Y12 antagonists with improved pharmacodynamic profiles have been
developed. This progress has in part been achieved with the development and use
of a variety of technologies applied in an integrated fashion [81]. As a result, today’s
cardiologist has a broader armamentarium of agents that can be used in patients
with coronary artery diseases.
References
1 Roth, G.J. and Majerus, P.W. (1975) The
mechanism of the effect of aspirin on
human platelets. I. Acetylation of a
particulate fraction protein. J. Clin. Invest.,
56, 624–632.
2 de Gaetano, G. (2001) Historical overview
of the role of platelets in hemostasis and
thrombosis. Haematologica, 86, 349–356.
3 McNicol, A. and Israels, S.J. (2003)
Platelets and antiplatelet therapy. J.
Pharmacol. Sci., 93, 381–396.
4 Hollopeter, G., Jantzen, H.M., Vincent,
D., Li, G., England, L., Ramakrishnan, V.,
Yang, R.B., Nurden, P., Nurden, A., Julius,
D. et al. (2001) Identification of the platelet
ADP receptor targeted by antithrombotic
drugs. Nature, 409, 202–207.
5 Dorsam, R.T. and Kunapuli, S.P. (2004)
Central role of the P2Y12 receptor in
platelet activation. J. Clin. Invest., 113,
340–345.
6 Gachet, C. and Hechler, B. (2005) The
platelet P2 receptors in thrombosis.
Semin. Thromb. Hemost., 31, 162–167.
7 Braunwald, E., Antman, E.M., Beasley,
J.W., Califf, R.M., Cheitlin, M.D.,
Hochman, J.S., Jones, R.H., Kereiakes,
D., Kupersmith, J., Levin, T.N. et al.
(2002) ACC/AHA Guidelines for the
Management of Patients with Unstable
Angina Non-ST Segment Elevation
Myocardial Infarction. Available at
http://www.acc.org/qualityandscience/
clinical/guidelines/unstable/
incorporated/UA
:
incorporated.pdf
(accessed May 2, 2007).
8 Massberg, S., Schulz, C., and Gawaz, M.
(2003) Role of platelets in the
158j6 Thienopyridyl and Direct-Acting P2Y12 Receptor Antagonist Antiplatelet Drugs
pathophysiology of acute coronary
syndrome. Semin. Vasc. Med., 3, 147–162.
9 Antman, E.M., Anbe, D.T., Armstrong, P.
W., Bates, E.R., Green, L.A., Hand, M.,
Hochman, J.S., Krumholz, H.M.,
Kushner, F.G., Lamas, G.A. et al. (2004)
ACC/AHA Guidelines for the
Management of Patients with ST-Elevation
Myocardial Infarction. Available at http://
www.acc.org/qualityandscience/clinical/
guidelines/stemi/Guideline1/index.pdf
(accessed May 1, 2007).
10 Jakubowski, J.A., Winters, K.J.,
Naganuma, H., and Walletin, L. (2007)
Prasugrel: a novel thienopyridine
antiplatelet agent. A review of
preclinical and clinical studies and the
mechanistic basis for its distinct
antiplatelet profile. Cardiovasc. Drug
Rev., 25, 358–374.
11 Algaier, I., Jakubowski, J.A., Asai, F., and
von K€ugelgen, I. (2008) Interaction of the
active metabolite of prasugrel, R-138727,
with cysteine 97 and cysteine 175 of the
human P2Y12 receptor. J. Thromb.
Haemost., 11, 1908–1914.
12 Ding, Z., Bynagari, Y.S., Mada, S.R.,
Jakubowski, J.A., and Kunapuli, S.P.
(2009) Studies on the role of the
extracellular cysteines and oligomeric
structures of the P2Y12 receptor when
interacting with antagonists. J. Thromb.
Haemost., 7, 232–234.
13 Jakubowski, J.A., Matsushima, N., Asai,
F., Brandt, J.T., Hirota, T., Freestone, S.,
and Winters, K.J. (2007) A multiple dose
study of prasugrel (CS-747), a novel
thienopyridine P2Y12 inhibitor, compared
with clopidogrel in healthy humans. Br. J.
Clin. Pharmacol., 63, 421–430.
14 Price, M.J. and Teirstein, P.S. (2008)
Dynamics of platelet functional recovery
following a clopidogrel loading dose in
healthy volunteers. Am. J. Cardiol., 102,
790–795.
15 Savi, P. and Herbert, J.-M. (2005)
Clopidogrel and ticlopidine. Semin.
Thromb. Haemost., 31, 174–183.
16 Thebault, J.J., Blatrix, C.E., Blanchard,
J.F., and Panak, E.A. (1975) Effects of
ticlopidine, a new platelet aggregation
inhibitor in man. Clin. Pharmacol. Ther.,
18, 485–490.
17 Thebault, J.J., Blatrix, C.E., Blanchard,
J.F., and Panak, E.A. (1977) The
interactions of ticlopidine and aspirin
in normal subjects. J. Int. Med. Res., 5,
405–411.
18 Sch€omig, A. et al. (1996) A randomized
comparison of antiplatelet and
anticoagulant therapy after the placement
of coronary-artery stents. N. Engl. J. Med.,
334, 1084–1089.
19 Leon, M.B. et al. (1998) A clinical trial
comparing three antithrombotic-drug
regimens after coronary-artery stenting.
Stent Anticoagulation Restenosis Study
Investigators. N. Engl. J. Med., 339,
1665–1671.
20 Bertrand, M.E. et al. (1998)
Anticoagulation versus aspirin and
ticlopidine (FANTASTIC) study.
Antiplatelet therapy in unplanned and
elective coronary stenting. Circulation, 98,
1597–1603.
21 Urban, P. et al. (1998) Randomized
evaluation of anticoagulation versus
antiplatelet therapy after coronary stent
implantation in high-risk patients: the
multicenter aspirin and ticlopidine trial
after intracoronary stenting (MATTIS).
Circulation, 98, 2126–2132.
22 Thebault, J.J., Kieffer, G., Lowe, W.S., and
Cariou, R. (1999) Repeated dose
pharmacodynamics of clopidogrel in
healthy subjects. Semin. Thromb. Hemost.,
25 (Suppl. 2), 9–14.
23 Sugidachi, A., Asai, F., Ogawa, T., Inoue,
T., and Koike, H. (2000) The in vivo
pharmacological profile of CS-747, a novel
antiplatelet agent with platelet ADP
receptor antagonist properties. Br. J.
Pharmacol., 129, 1439–1446.
24 Savi, P., Pereillo, J.M., Uzabiaga, M.F.
et al. (2000) Identification and biological
activity of the active metabolite of
clopidogrel. Thromb. Haemost., 84,
891–896.
25 Yoneda, K., Iwamura, R., Kishi, H. et al.
(2004) Identification of the active
metabolite of ticlopidine from rat in vitro
metabolites. Br. J. Pharmacol., 142,
551–557.
26 Ha-Duong, N.T., Dijols, S., Macherey, A.C.
et al. (2001) Ticlopidine as a selective
mechanism-based inhibitor of human
References j159
cytochrome P450 2C19. Biochemistry, 40,
12112–12122.
27 Richter, T., M€urdter, T.E., Heinkele, G.
et al. (2004) Potent mechanism based
inhibition of human CYP2B6 by
clopidogrel and ticlopidine. J. Pharmacol.
Exp. Ther., 308, 189–197.
28 Maffrand, J.P., Vallee, E., Bernat, A. et al.
(1985) Animal pharmacology of PCR
4099. A new thienopyridine compound.
Thromb. Haemost., 54, 133.
29 Defreyn, G., Bernat, A., Delebassee, D.,
and Maffrand, J.-P. (1989) Pharmacology
of ticlopidine: a review. Semin. Thromb.
Hemost., 15, 159–166.
30 Bhatt, D.L. et al. (2002) Meta-analysis of
randomized and registry comparisons of
ticlopidine with clopidogrel after stenting.
J. Am. Coll. Cardiol., 39, 9–14.
31 CAPRIE Steering Committee (1996) A
randomised, blinded, trial of clopidogrel
versus aspirin in patients at risk of
ischaemic events (CAPRIE). Lancet, 348,
1329–1339.
32 Yusuf, S. et al. (2001) Effects of clopidogrel
in addition to aspirin in patients with
acute coronary syndromes without STsegment
elevation. N. Engl. J. Med., 345,
494–502.
33 Mehta, S.R. et al. (2001) Effects of
pretreatment with clopidogrel and aspirin
followed by long-term therapy in patients
undergoing percutaneous coronary
intervention: the PCI-CURE study. Lancet,
358, 527–533.
34 Yousuf, O. and Bhatt, D.L. (2001) The
evolution of antiplatelet therapy in
cardiovascular disease. Nat. Rev. Cardiol.,
8, 547–559.
35 Kazui, M. et al. (2010) Identification of the
human cytochrome P450 enzymes
involved in the two oxidative steps in the
bioactivation of clopidogrel to its
pharmacologically active metabolite. Drug
Metab. Dispos., 38, 92–99.
36 Pereillo, J.M., Maftouh, M., Andrieu, A.
et al. (2002) Structure and stereochemistry
of the active metabolite of clopidogrel.
Drug Metab. Dispos., 30, 1288–1295.
37 Satoh, T. and Hosokawa, M. (1998) The
mammalian carboxylesterases, from
molecules to functions. Annu. Rev.
Pharmacol. Toxicol., 38, 257–288.
38 Hagihara, K., Kazui, M., Yoshiike, M. et al.
(2008) A possible mechanism of poorer
and more variable response to clopidogrel
than prasugrel. Circulation, 118 (Suppl. 2),
S820.
39 Takahashi, M., Pang, H., Kawabata, K.
et al. (2008) Quantitative determination of
clopidogrel active metabolite in human
plasma by LC–MS/MS. J. Pharm. Biomed.
Anal., 48, 1219–1224.
40 Brandt, J.T., Close, S.L., Iturria, S.J. et al.
(2007) Common polymorphisms of
CYP2C19 and CYP2C9 affect the
pharmacokinetic and pharmacodynamic
response to clopidogrel but not prasugrel.
J. Thromb. Haemost., 5, 2429–2436.
41 Mega, J.L. et al. (2009) Cytochrome P-450
polymorphisms and response to
clopidogrel. N. Engl. J. Med., 360, 354–362.
42 Mega, J.L., Close, S.L., Wiviott, S.D. et al.
(2009) Cytochrome P450 genetic
polymorphisms and the response to
prasugrel: relationship to
pharmacokinetic, pharmacodynamic, and
clinical outcomes. Circulation, 119,
2553–2560.
43 Niitsu, Y., Jakubowski, J.A., Sugidachi, A.,
and Asai, A. (2005) Pharmacology of CS-
747 (prasugrel, LY640315), a novel, potent
antiplatelet agent with in vivo P2Y12
receptor antagonist activity. Semin.
Thromb. Hemost., 31, 184–194.
44 Wiviott, S.D. et al. (2007) Prasugrel versus
clopidogrel in patients with acute coronary
syndromes. N. Engl. J. Med., 357,
2001–2015.
45 Farid, N.A., McIntosh, M., Garofolo, F.
et al. (2007) Determination of the active
and inactive metabolites of prasugrel in
human plasma by liquid
chromatography/tandem mass
spectrometry. Rapid Commun. Mass
Spectrom., 21, 169–179.
46 Payne, C.D., Brandt, J.T., Weerakkody,
G.J., Farid, N.A., Small, D.S., Ernest, C.S.,
Jansen, M., Jakubowski, J.A., Naganuma,
H., Wiviott, S.D. et al. (2005) Superior
inhibition of platelet aggregation
following a loading dose CS-747
(prasugrel, LY640315) versus clopidogrel:
correlation with pharmacokinetics of
active metabolite generation. J. Thromb.
Haemost., 3 (Suppl. 1), P0952.
160j6 Thienopyridyl and Direct-Acting P2Y12 Receptor Antagonist Antiplatelet Drugs
47 Brandt, J.T., Payne, C.D., Wiviott, S.D.,
Weerakkody, G., Farid, N.A., Small, D.S.,
Jakubowski, J.A., Naganuma, H., and
Winters, K.J. (2007) A comparison of
prasugrel and clopidogrel loading doses
on platelet function: magnitude of platelet
inhibition is related to active metabolite
formation. Am. Heart J., 153,
66.e9–66.e16.
48 Farid, N.A., Kurihara, A., and Wrighton,
S.A. (2010) Metabolism and disposition
of the thienopyridine antiplatelet drugs
ticlopidine, clopidogrel, and prasugrel in
humans. J. Clin. Pharmacol., 50, 126–142.
49 Small, D.S., Farid, N.A., Payne, C.D.,
Konkoy, C.S., Jakubowski, J.A., Winters,
K.J., and Salazar, D.E. (2010) Effect of
intrinsic and extrinsic factors on the
clinical pharmacokinetics and
pharmacodynamics of prasugrel. Clin.
Pharmacokinet., 49, 777–798.
50 Rehmel, J.L., Eckstein, J.A., Farid, N.A.
et al. (2006) Interactions of two major
metabolites of prasugrel, a thienopyridine
antiplatelet agent, with the cytochromes
P450. Drug Metab. Dispos., 34, 600–607.
51 Jakubowski, J.A., Riesmeyer, J.R., Close,
S.L., Leishman, A.G., and Erlinge, D.
(2011) TRITON and beyond: new insights
into the profile of prasugrel. Cardiovasc.
Ther., 30 (4), e174–e182.
52 McFarlane, D. and Mills, D. (1975) The
effect of ATP on platelets: evidence against
the central role of released ADP in
primary aggregation. Blood, 46, 309–320.
53 Ingall, A.H., Dixon, J., Bailey, A., Coombs,
M.E., Cox, D., McInally, J.I., Hunt, S.F.,
Kindon, N.D., Teobald, B.J., Willis, P.A.,
Humphries, R.G., Leff, P., Clegg, J.A.,
Smith, J.A., and Tomlinson, W. (1999)
Antagonists of the platelet P2Treceptor: a
novel approach to antithrombotic therapy.
J. Med. Chem., 42, 213–220.
54 Storey, R.F., Wilcox, R.G., and Heptinstall,
S. (2002) Comparison of the
pharmacodynamic effects of the platelet
ADP receptor antagonists clopidogrel and
AR-C69931MX in patients with ischaemic
heart disease. Platelets, 13, 407–412.
55 Storey, R.F., Oldroyd, K.G., and Wilcox,
R.G. (2001) Open multicentre study of the
P2Treceptor antagonist AR-C69931MX
assessing safety, tolerability and activity in
patients with acute coronary syndromes.
Thromb. Haemost., 85, 401–407.
56 Greenbaum, A.B., Grines, C.L., Bittl, J.A.
et al. (2006) Initial experience with an
intravenous P2Y12 platelet receptor
antagonist in patients undergoing
percutaneous coronary intervention:
results from a 2-part, phase II,
multicenter, randomized, placebo and
active-controlled trial. Am. Heart J., 151,
689.e1–689.e10.
57 Harrington, R.A., Stone, G.W., McNulty,
S. et al.(CHAMPION PCI Investigators)
(2009) Platelet inhibition with cangrelor in
patients undergoing PCI. N. Engl. J. Med.,
361, 2318–2329.
58 Bhatt, D.L., Lincoff, A.M., Gibson, C.M.
et al.(CHAMPION PLATFORM
Investigators) (2009) Intravenous platelet
blockade with cangrelor during PCI. N.
Engl. J. Med., 361, 2330–2334.
59 Dovlatova, N., Jakubowski, J., Sugidachi,
A. et al. (2008) The reversible P2Y12
antagonist cangrelor influences the ability
of the active metabolites of clopidogrel
and prasugrel to produce irreversible
inhibition of platelet function. J. Thromb.
Haemost., 6, 1153–1159.
60 Steinhubl, S.R., Oh, J.J., Oestreich, J.H.
et al. (2008) Transitioning patients from
cangrelor to clopidogrel: pharmacodynamic
evidence of a competitive effect. Thromb.
Res., 121, 527–534.
61 Jakubowski, J.A., Payne, C.D., Li, Y.G.,
Farid, N.A., Brandt, J.T., Small, D.S.,
Salazar, D.E., and Winters, K.J. (2008) The
use of the VerifyNow P2Y12 point-of-care
device to monitor platelet function across
a range of P2Y12 inhibition levels
following prasugrel and clopidogrel
administration. Thromb. Haemost., 99,
409–415.
62 Angiolillo, D.J., Firstenberg, M.S., Price,
M.J., Tummala, P.E., Hutyra, M., Welsby,
I.J., Voeltz, M.D., Chandna, H., Ramaiah,
C., Brtko, M., Cannon, L., Dyke, C., Liu,
T., Montalescot, G., Manoukian, S.V.,
Prats, J., and Topol, E.J. (BRIDGE
Investigators) (2012) Bridging antiplatelet
therapy with cangrelor in patients
undergoing cardiac surgery: a randomized
controlled trial. J. Am. Med. Assoc., 18, 307,
265–274.
References j161
63 Springthorpe, B., Bailey, A., Barton, P.,
Birkinshaw, T.N., Bonnert, R.V., Brown,
R.C., Chapman, D., Dixon, J., Guile, S.D.,
Humphries, R.G., Hunt, S.F., Ince, F.,
Ingall, A.H., Kirk, I.P., Leeson, P.D., Leff,
P., Lewis, R.J., Martin, B.P., McGinnity, D.
F., Mortimore, M.P., Paine, S.W.,
Pairaudeau, G., Patel, A., Rigby, A.J., Riley,
R.J., Teobald, B.J., Tomlinson, W.,
Webborn, P.J., and Willis, P.A. (2007)
From ATP to AZD6140: the discovery of
an orally active reversible P2Y12 receptor
antagonist for the prevention of
thrombosis. Bioorg. Med. Chem. Lett., 17,
6013–6018.
64 van Giezen, J.J.J. and Humphries, R.G.
(2005) Preclinical and clinical studies with
selective reversible direct P2Y12
antagonists. Semin. Thromb. Haemost., 31,
195–204.
65 Teng, R., Oliver, S., Hayes, M.A., and
Butler, K. (2010) Absorption, distribution,
metabolism, and excretion of ticagrelor in
healthy subjects. Drug Metab. Dispos., 38,
1514–1521.
66 Husted, S., Emanuelsson, H., Heptinstall,
S., Sandset, P.M., Wickens, M., and
Peters, G. (2006) Pharmacodynamics,
pharmacokinetics, and safety of the oral
reversible P2Y12 antagonist AZD6140
with aspirin in patients with
atherosclerosis: a double-blind
comparison to clopidogrel with aspirin.
Eur. Heart J., 27, 1038–1047.
67 Butler, K. and Teng, R. (2010)
Pharmacokinetics, pharmacodynamics,
safety and tolerability of multiple ascending
doses of ticagrelor in healthy volunteers.
Br. J. Clin. Pharmacol., 70, 65–77.
68 Storey, R.F., Husted, S., Harrington, R.A.,
Heptinstall, S., Wilcox, R.G., Peters, G.,
Wickens, M., Emanuelsson, H., Gurbel,
P., Grande, P., and Cannon, C.P. (2007)
Inhibition of platelet aggregation by
AZD6140, a reversible oral P2Y12
receptor antagonist, compared with
clopidogrel in patients with acute coronary
syndromes. J. Am. Coll. Cardiol., 50,
1852–1856.
69 Wallentin, L., Becker, R.C., Budaj, A. et al.
(2009) Ticagrelor versus clopidogrel in
patients with acute coronary syndromes.
N. Engl. J. Med., 361, 1045–1057.
70 Wallentin, L., James, S., Storey, R.F. et al.
(2010) Effect of CYP2C19 and ABCB1
single nucleotide polymorphisms on
outcomes of treatment with ticagrelor
versus clopidogrel for acute coronary
syndromes: a genetic substudy of the
PLATO trial. Lancet, 376, 1320–1328.
71 Gurbel, P.A., Bliden, K.P., Butler, K. et al.
(2009) Randomized double-blind
assessment of the onset and offset of the
antiplatelet effects of ticagrelor versus
clopidogrel in patients with stable
coronary artery disease: the
ONSET/OFFSETstudy. Circulation, 120,
2577–2585.
72 Serebruany, V. (2011) Adenosine release: a
potential explanation for the benefits of
ticagrelor in the PLATelet Inhibition and
Clinical Outcomes trial? Am. Heart J., 161,
1–4.
73 Cannon, C.P., Husted, S., Harrington,
R.A. et al. (2007) Safety, tolerability, and
initial efficacy of AZD6140, the first
reversible oral adenosine diphosphate
receptor antagonist, compared with
clopidogrel, in patients with non-STsegment
elevation acute coronary
syndrome: primary results of the
DISPERSE-2 trial. J. Am. Coll. Cardiol., 50,
1844–1851.
74 Storey, R.F., Bliden, K., Patil, S.B. et al.
(2010) Incidence of dyspnea and
assessment of cardiac and pulmonary
function in patients with stable coronary
artery disease receiving ticagrelor,
clopidogrel or placebo in the ONSET/
OFFSETstudy. J. Am. Coll. Cardiol., 56,
185–193.
75 van Giezen, J.J.J., Sidaway, J., Glaves, P.,
Kirk, I., and Bjorkman, J.A. (2012)
Ticagrelor inhibits adenosine uptake in
vitro and enhances adenosine-mediated
hyperemia responses in a canine model.
J. Cardiovasc. Pharmacol. Ther., 17 (2),
164–172.
76 Ohman, J., Kudira, R., Albinsson, S.,
Olde, B., and Erlinge, D. (2012) Ticagrelor
induces adenosine triphosphate release
from human red blood cells. Biochem.
Biophys. Res. Commun., 418, 754–758.
77 Ueno, M., Rao, S.V., and Angiolillo, D.J.
(2010) Elinogrel: pharmacological
principles, preclinical and early phase
162j6 Thienopyridyl and Direct-Acting P2Y12 Receptor Antagonist Antiplatelet Drugs
clinical testing. Future Cardiol., 6,
445–453.
78 Gurbel, P., Bliden, K., Antonino, M. et al.
(2009) The effect of elinogrel on high
platelet reactivity during dual antiplatelet
therapy and the relation to CYP 2C19Ã
2
genotype: first experience in patients. J.
Thromb. Haemost., 8, 43–53.
79 Berger, J.S., Roe, M.T., Gibson, C.M.,
Kilaru, R., Green, C.L., Melton, L.,
Blankenship, J.D., Metzger, D.C.,
Granger, C.B., Gretler, D.D., Grines, C.L.,
Huber, K., Zeymer, U., Buszman, P.,
Harrington, R.A., and Armstrong, P.W.
(2009) Safety and feasibility of adjunctive
antiplatelet therapy with intravenous
elinogrel, a direct-acting and reversible
P2Y12 ADP-receptor antagonist, before
primary percutaneous intervention in
patients with ST-elevation myocardial
infarction: the Early Rapid ReversAl of
platelet thromboSis with intravenous
Elinogrel before PCI to optimize
reperfusion in acute Myocardial Infarction
(ERASE MI) pilot trial. Am. Heart J., 158,
998.e1–1004.e1.
80 Rao, S. (2010) A randomized, doubleblind,
active controlled trial to evaluate
intravenous and oral PRT060128
(elinogrel), a selective and reversible
P2Y12 receptor inhibitor, vs. clopidogrel,
as a novel antiplatelet therapy in patients
undergoing non-urgent percutaneous
coronary interventions (INNOVATE-PCI).
Presented at the 2010 ESC Congress,
August 28–September 1, 2010,
Stockholm, Sweden.
81 Jakubowski, J.A. (2011) Platelet functional
testing: value in the aggregate. Biomarkers
Med., 5, 5–8.
References j163
Joseph A. Jakubowski received an M.Sc. degree in Nutrition
from the University of London (UK) and Ph.D. in Clinical
Science from the Australian National University, Canberra,
Australia, studying human platelet reactivity. He continued
his platelet studies at Boston University School of Medicine
where he was Assistant Professor of Medicine. Dr. Jakubowski
joined Lilly Research Laboratories in 1988 and established
a Platelet Research Group that was responsible for the identification
and clinical evaluation of several antiplatelet agents.
He has published extensively on the pharmacological control of platelet reactivity
by antiplatelet agents and holds several issued patents. He is an Adjunct Assistant
Professor at Indiana University School of Medicine and is an elected member of
the European Society of Cardiology (ESC) Working Group on Thrombosis.
Atsuhiro Sugidachi studied biochemistry at the Faculty of
Pharmaceutical Sciences, Tohoku University, Japan. In 1989,
he joined the Thrombosis Research Group, Biological
Research Laboratories, Sankyo Co., Ltd., participating in the
P2Y12 antagonist project. In 1996, he received his Ph.D. in
Pharmaceutical Sciences from Tohoku University, Japan.
Between 2000 and 2002, he was a Visiting Scientist in the
laboratory of Dr. Lawrence F. Brass at The University of
Pennsylvania School of Medicine. In 2005, he became Group
Director of The Pharmacology and Molecular Biology Research Laboratories of
Sankyo Co., Ltd., overseeing projects on cardiovascular and metabolic diseases.
Since 2007, he has been a Senior Director of The Biological Research Laboratories
of Daiichi Sankyo Co., Ltd., where his group has been working on several drug
targets for cardiovascular diseases.
164j6 Thienopyridyl and Direct-Acting P2Y12 Receptor Antagonist Antiplatelet Drugs
7
Selective Estrogen Receptor Modulators
Amarjit Luniwal, Rachael Jetson, and Paul Erhardt
List of Abbreviations
ABDD analogue-based drug discovery
CoAc coactivators
CoRe corepressors
CYP cytochrome P450
DES diethylstilbestrol
ER estrogen receptor
ERE estrogen responsive elements
FDA Food and Drug Administration
FMO flavin-containing monooxygenase
GnRH gonadotropin releasing hormone
HERS Heart Estrogen/Progesterone Replacement Study
HRT hormone replacement therapy
HSP heat shock proteins
ICI Imperial Chemical Industries
MORE Multiple Outcomes of Raloxifene Evaluation
NDA New Drug Application
PCOS polycystic ovarian syndrome
RUTH Raloxifene’s Use for The Heart
SAR structure–activity relationship
SERD selective estrogen receptor downregulator
SERM selective estrogen receptor modulator
STAR Study of Tamoxifen and Raloxifene
WHI The Women’s Health Initiative
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
j165
7.1
Introduction
7.1.1
Working Definition
Coined by workers at Eli Lilly during the 1990s [1], the acronym “SERM” (selective
estrogen receptor modulator) has come to serve as the common classification for
molecules that can display a multiplicity of effects due to differential modulation of
the estrogen receptor (ER) pathways at different sites [2]. Scheme 7.1 conveys how
the interaction of various SERM ligands with ERs can lead to different responses
at numerous points along the cascade normally triggered by estrogens like 17bestradiol
(1 in Figure 7.1) so as to eventually lead to DNA transcription. Unlike
estrogens that are uniformly agonists, and antiestrogens that are uniformly antagonists,
the SERMs exert selective agonist or antagonist effects on various estrogen
target tissues [3]. Thus, a drug like fulvestrant, 2, which is sometimes referred to as
a “selective antagonist” or a “selective estrogen receptor downregulator” (SERD), is
not a true SERM because it demonstrates only a pure antiestrogenic profile on all
genes and in all tissues [2]. Following this definition in its strictest sense,
L + ER
CoAc/
CoRe
ERE et al DNA
Cell Effects
ER Express.
ER Degrad.
(L-ER)2
HSP
Scheme 7.1 Simplified cartoon of how SERM
ligands (L) can interact with estrogen receptors
(ER) to cause a multiplicity of effects. This
depiction represents a composite of
information conveyed in several reviews and
additional references cited therein [1–6]. Points
that can lead to variable effects include the
following: (1) there are two types of ER (namely
ERa and ERb) differentially populated in time
across normal tissues, as well as in various
pathologies. (2) Relative to the estrogens, L can
begin to bind with ER as agonists, partial
agonists, or antagonists of varying competitive
or noncompetitive potency. (3) Binding may
sometimes accommodate more than one L and
in all cases continues to proceed through
distinct mutual molding processes wherein
there are unique conformational subtleties for
each SERM that represent a continuum of
graded possibilities ranging between the initial
agonist and antagonist modes of interaction.
(4) Binding releases heat shock proteins (HSP)
from their association with ER and causes the
ER to dimerize ((L-ER)2) in either a homo- or
hetero-fashion. The type of dimer varies
according to the expression levels and
degradation rates of the two ER. (5) Depending
upon the nature of the still maturing complex,
different coactivators (CoAc) or corepressors
(CoRe) can be additionally recruited. Types and
concentrations of CoAc and CoRe vary in time
across normal cells, as well as in various
pathologies. Differences in the latter are
especially important to recruitment and the
eventual responses that become generated. (6)
Interaction of the final complex with DNA
primarily engages estrogen responsive
elements (ERE) but can also involve other
responsive elements. (7) DNA signaling can be
either enhanced or attenuated relative to
various transcriptional pathways. (8) Likewise,
expression of ER can be either induced or
inhibited, as can expression of factors
associated with the pathway that degrades ER.
166j7 Selective Estrogen Receptor Modulators
compounds that are purely estrogenic or purely antiestrogenic will only be mentioned
in passing as the SERMs become further elaborated within this chapter.
7.1.2
Early ABDD Leading to a Pioneer SERM
Analogue-based drug discovery (ABDD) leading to SERM molecules can be traced
back to a period well before the 1990s. For example, the work of Dodds et al. during
the late 1930s [7, 8] established fundamental structure–activity relationships (SAR)
that demonstrated that estrogenic activity could be produced by compounds lacking
the steroid ring characteristic of the estrogens (e.g., 1), those efforts culminating in
diethylstilbestrol (DES, 3) that eventually became marketed as a nonsteroidal estrogen.
Chlorotrianisene (TACE, 4) was discovered shortly thereafter and it represents
the first triphenylethylene-containing synthetic estrogen to eventually reach the
market in the early 1950s [4]. Alternatively, work by Lerner et al. during this
period [9] demonstrated that the same type of scaffold could be substituted so as
to produce an antiestrogenic compound, namely, ethamoxytriphetol (MER-25, 5).
Although MER-25 never became a commercially available pharmaceutical product,
it underwent preliminary clinical investigation during the late 1950s and early
1960s as an estrogen antagonist [4]. Essentially devoid of estrogenic activity, an
exciting property of MER-25 was its antifertility actions in laboratory animals, an
observation that prompted a search for more potent agents by numerous pharmaceutical
companies [1]. Subsequent research in the field of nonsteroidal antiestrogens
tended to retain a focus upon the triphenylethylene nucleus, its cyclized
counterparts, and their reduced versions [10]. Clomiphene (6 in Scheme 7.2),
HO
OH
OH
HO
Cl
OMe
MeO
MeO
17β-Estradiol (1): R = H
Fulvestrant (2):
R = (CH2)9SO(CH2)3CF2CF3
Diethylstilbestrol (DES) (3)
Chlorotrianisene (TACE) (4)
O
MeO
Ethamoxytriphetol (MER-25) (5)
HO
N
R
Figure 7.1 One of the endogenous estrogens (1), a synthetic steroid analogue (2), and three of
the early nonsteroidal analogues (3–5). See the text for details.
7.1 Introduction j167
O O
O
N
Ormeloxifene (11)
HO
O
N
Lasofoxifene (13)
OH
SHO
O
O
N
OH
N
HO
O
N
Raloxifene (9)
Bazedoxifene (12)
O
O
N
Nafoxidine (7)
Cl
O
N
Clomiphene (6)
O
N
Tamoxifen (8)
O
N
Cl
Toremifene (10)
O
Cl
HO
Ophena (14)
Phase III
O
N
Afimoxifene (15)
HO
Scheme 7.2 Evolution of marketed SERMs suggested by ABDD analyses (see the text for details).
Compounds 14 and 15 are progressing favorably through phase III clinical studies such that they
may become marketed in the near future.
168j7 Selective Estrogen Receptor Modulators
nafoxidine (7), and tamoxifen (8) were all the result of that search, but clinical application
as postcoital contraceptives was found to be inappropriate [1]. Instead,
because they induced ovulation in subfertile women, 6 and initially 8 were eventually
approved as profertility drugs for the induction of ovulation [1]. While several
compounds from the composite of drugs arising during this period might be
regarded as the “pioneer SERM,” our attempt to trace the lineage of these agents
by tracking their initial syntheses, patents, and introductions to the marketplace
moved clomiphene to the forefront. Thus, a brief description of its specific discovery
and pharmacological properties follows.
7.1.3
Discovery and Development of Clomiphene
Palopoli et al. first synthesized 6 in the mid-1950s as its citrate salt [4], the latter still
being deployed effectively in today’s formulations. Coupled with preliminary biological
data, a patent pertaining to this immediate family of analogues was issued
in 1959 [11]. Based upon clinical studies associated with TACE and MER-25, criteria
to select a lead compound from among various development stage analogues
became (i) less estrogenic than TACE; (ii) more pituitary gonadotropin inhibiting
activity than MER-25; (iii) some estrogen antagonizing action; and (iv) a dosage
approaching that of TACE and hopefully about one-tenth of that necessary with
MER-25 that was still undergoing clinical trials at that time [4]. Clomiphene was
selected because it displayed the best overall profile across these criteria. Among its
potential clinical indications that included oral contraception, use in certain types
of estrogen-dependent tumors, and induction of ovulation, the last one was pursued
at the highest priority [4]. Initial clinical results were reported in 1961 [12]:
although the mechanism of action was not clear, 6 held exceptional promise of
inducing ovulatory type menses with considerable regularity in anovulatory
women. A New Drug Application (NDA) was filed in 1965 and within 2 years 6 was
approved for marketing by prescription in the United States [4].
Clomiphene is marketed as a mixture of its E and Z stereoisomers in about a 6 : 4
ratio. Initially given the wrong designations as isomers “A” and “B” [12], this situation
was corrected in 1976 by Ernst et al. [13] and the names “enclomiphene” and
“zuclomiphene” were instead adopted [14, 15]. Enclomiphene is essentially antiestrogenic
while zuclomiphene is mildly estrogenic but also capable of serving as an
antiestrogen in certain settings. Importantly, zuclomiphene is about five times
more potent in inducing ovulation. Metabolism is subject to considerable individual
variation based, in part, on body weight [16]. As a result, dosage is sometimes
required to be adjusted for body weight. In order to optimize penetration into the
brain so as to occupy central and pituitary receptor sites, the total daily dose of 6
must be taken at one time. The drug is excreted primarily through the intestines,
51% being accounted for within 5 days after oral administration. However, excretion
then continues for at least another 6 weeks [17]. Thus, repeated administration
at 28-day intervals is cumulative with base levels increasing by nearly 50% per
month [17] and 6 becomes more effective during the second and later cycles of
7.1 Introduction j169
treatment [4]. The beneficial mechanisms are thought to involve competitive antagonism
of estradiol (1) at the level of the cytoplasmic nuclear receptor in the hypothalamic
arcuate nucleus that, in turn, leads to an increase in gonadotropin
releasing hormone (GnRH) [18]. In a complimentary fashion, 6 also increases the
pituitary sensitivity to GnRH in a manner similar to 1 [19] such that follicle stimulating
hormone and luteinizing hormone secretions are increased [20]. Finally, 6
may have a direct effect on the ovary, rendering granulose cells more sensitive to
pituitary gonadotropin [4].
The introduction of 6 revolutionized the treatment of infertility in general and of
polycystic ovarian syndrome (PCOS) in particular [4]. While early drawbacks
included failed conception despite apparent ovulation and a supposed increase in
natural abortions, additional clinical studies have been able to resolve these issues
by using vaginal ultrasound to select for potentially problematic individuals. Clomiphene
remains an effective drug for women suffering from anovulation and ovulating
women with inadequate follicular or luteal development. Large studies have
subsequently shown that 6 is effective through at least six cycles of treatment, and
that there is no evidence for significant increases in spontaneous abortion, ectopic
pregnancy rates, or decreases in the number of male births [4].
7.1.4
SERM-Directed ABDD: General Considerations
From this backdrop of early activities within the field of estrogen-related medicinal
chemistry and our description of 6 to represent the pioneer SERM among several
allied agents that additionally arose during that period, the remainder of this chapter
can now specifically consider its intended subject matter, namely, SERM-directed
ABDD. Toward this end, Table 7.1 lists several SERMs that either became marketed
or are nearing market launch as a result of pursuing new agents for this class.
Scheme 7.2 provides the structures for these compounds while also suggesting
how they can be related to one another according to ABDD principles. Our ABDD
analyses rely upon a combination of structural similarity and apparent evolution of
the latter through logical advances in medicinal chemistry-derived SAR, coupled
with lineage determinations derived from first-synthesized, first-patented, and
first-marketed dates whenever possible. It must be emphasized, however, that
unexplained gaps in the progression of a given agent at any point in the overall
drug discovery and development process cannot always be identified and accounted
for. Such gaps, in turn, can sometimes lead to misalignment of the associated
structures within these types of ABDD schema. Alternatively and without potential
shortcomings about the perfect evolutionary history of any given agent, Scheme 7.2
does provide a clear view of the overall structural landscape presently occupied by
the marketed SERMs from across the globe. Two additional compounds that
appear to be favorably traversing phase III clinical testing are included as well.
Besides clomiphene (6) and nafoxidine (7), a quick count indicates that there are
eight structures residing in this landscape. Given constraints in chapter length,
just two of these agents have been chosen for a thorough description of their
170j7 Selective Estrogen Receptor Modulators
SERM-directed ABDD, namely, tamoxifen (8) and raloxifene (9). Both of these
agents are taken from the first ABDD tier after clomiphene and both have interesting
medicinal chemistry histories to convey. Readers interested in any of the newer
agents can utilize the associated information listed within Table 7.1 as convenient
starting points for further study. We likewise encourage readers to purview any of
the numerous books and reviews that also cover the earlier period leading to the
discovery of 6, as well as those available for some of clomiphene’s subsequent relatives
[1–6, 21–35]. Although not emphasizing ABDD themes as distinctly intended
herein, many of these reports are extremely comprehensive and provide excellent
discussions about the medicinal chemistry, pharmacology, and clinical data associated
with specific SERMs and the field in general.
7.2
Tamoxifen
7.2.1
Early Development
Tamoxifen (Nolvadex1
; ICI 46474; 8) was discovered nearly 50 years ago by
Imperial Chemical Industries (ICI; now AstraZeneca) who like many other
companies at that time were pursuing the development of nonsteroidal antiestrogens
as oral contraceptives [36]. Studies in laboratory animals with triphenylchloroethylene
and triphenylmethylethylene against breast cancer showed that
the latter had higher estrogenic activity than the chloro analogue. These results,
along with the basic amine found on clomiphene (6; discussed above), led to the
synthesis of 16 (Figure 7.2a) that later was found to possess antifertility effects
in mice at a lower dose than needed for estrogenic activity. This led to the further
design of a series of related structures to be developed as nonsteroidal
Table 7.1 Marketed SERMs.
Generic name Company Launch Country Use
Clomiphene Merrell 1961 USA Anovulation (infertility)
Tamoxifen citrate ICI 1977 USA Breast cancer
Toremifene citrate Orion 1988 EU Breast cancer
Ormeloxifene 3M 1991 India Contraceptive
Raloxifene Eli Lilly 1997 USA Osteoporosis
Fulvestranta
AstraZeneca 2002 USA Breast cancer
Bazedoxifene Wyeth 2009 EU Postmenopausal osteoporosis
Lasofoxifene Pfizer 2009 EU Osteoporosis and vaginal atrophy
Ospemifeneb
Orion Phase III
Afimoxifeneb
ASCEND Phase III
a) Not a SERM by strict definition, but rather a pure “antiestrogen.”
b) These compounds are progressing favorably through phase III clinical studies.
7.2 Tamoxifen j171
contraceptives. Figure 7.2a shows some of the key representatives from this
series [36]. Eventually, it was found that only the “Z” rather than “E” isomers
possessed antiestrogenic activity [37, 38]. For example, after the initial synthesis
of 8 (first reported in 1962 [37]), the structures of its two isomers were resolved
by X-ray crystallography in 1970 so as to make the proper connection with biological
activity [39]. Numerous secondary amines such as 18, “desoxy” analogues
where the ether oxygen is removed, and the saturated alkane derivatives
were prepared as part of the overall series. None of these derivatives offered any
benefit over 8 [36]. During this period, structural modifications to the triphenylethylene
derivative chlorotrianisene (4), a nonsteroidal estrogen, had led to the
discovery of compounds with antiestrogenic activity such as MER-25 (5) [9] and
clomiphene (6), a gonadotropin inhibitor [40]. Moreover, the understanding that
hormone-dependent breast cancer could be intervened by oophorectomy [41],
adrenalectomy [42], or hormonal treatment led to a quest for drugs that antagonize
the actions of estrogen with minimal side effects [43, 44]. This led to testing
of tamoxifen against hormone-dependent breast cancer. Based on the
latter’s success, the US Food and Drug Administration (FDA) approved the use
of tamoxifen for treatment of breast cancer in 1977 [43].
7.2.2
Clinical Indications and Molecular Action
Tamoxifen has been used clinically against breast cancer for almost four decades
and is regarded as a standard therapy. It is currently used (i) to treat
O
N
R
R'
X
ICI 42043 R = R' = Et, X = Me (16)
(a) (b)
ICI 45960 R = R' = Et, X = Et (17)
Tamoxifen R = R' = Me, X = Et (8)
ICI 55548 R= Me, R' = H, X = Et (18)
Figure 7.2 (a) Tamoxifen and its immediate
family of analogues. (b) ERa active site surface
depiction with estradiol (1), 4-hydroxytamoxifen
(15), and raloxifene (9). Nitrogens and oxygens
are shown in red and blue, respectively, the
carbon atoms of protein and 9 are in silver,
while carbon atoms for 1 and 15 are in green
and purple, respectively. Raloxifene bound ERa
structural data [45] were used for receptor
surface generation. ERa bound conformations
of 1 [45], 15 [46], and 9 were used for
superimposition. Several amino acid residues
covering the top of the ligand surface were
removed to provide for better visualization
of the receptor binding pocket. Likewise,
not shown in the case of 1 is the helix 12 that
moves across the northeast quadrant
of the pocket.
172j7 Selective Estrogen Receptor Modulators
metastatic breast cancer; (ii) as an adjuvant for treating node-positive and nodenegative
breast cancer; (iii) to lower the risk of invasive breast cancer; and (iv) as
a preventative agent to decrease breast cancer occurrence in high-risk
women [47]. Tamoxifen is an antiestrogenic prodrug. Scheme 7.3 shows
the key metabolic pathways for 8. The active metabolites, 4-hydroxytamoxifen
(4-OHT; 15) and 4-hydroxy-N-desmethyltamoxifen (endoxifen; 20) bind with
estrogen receptors and block ER-mediated transcriptional activation in breast
tissues. Figure 7.2b depicts comparative binding orientations for 1, 15, and 9
within the ERa binding pocket. 4-OHT has key interactions with Arg394 and
Glu353 through its phenolic hydroxyl group. The basic side chain on 15 extends
out of the active site cavity and establishes weak ionic interactions with
Asp351 [31]. This could distort the receptor conformation in a manner similar
OCH2CH2N(CH3)2OCH2CH2N(CH3)2
Tamoxifen (TAM, 8)4-Hydroxy-TAM
(Afimoxifene, 15)
OCH2CH2NHCH3
N-Desmethyl-TAM (19)
OH
3-Hydroxy-TAM
(Droloxifene, 24)
TAM-catechol (23)
OCH2CH2N(CH3)2
OH
α -Hydroxy-TAM (21)
CYP
CYP
CYP
OCH2CH2N(CH3)2
O
N-Oxide-TAM (22)
FMO
CYP
CYP
CYP
CYP2D6
CYP
4-Hydroxy-N-desmethyl-TAM
(Endoxifen, 20)
OCH2CH2NHCH3
OH
OCH2CH2N(CH3)2OCH2CH2N(CH3)2
OH
OH OH
Scheme 7.3 Key metabolic pathways of tamoxifen.
7.2 Tamoxifen j173
to other partial ER antagonists [45] such that the helix 12 cannot move to a
position that is suitable for coactivator binding with the ER–ligand complex.
Although structural data are unavailable at this point in time, it is believed
that 15 interacts with ERb in a similar fashion. Until recent reports [48, 49], 15
was considered to be the main metabolite that competitively blocked ERs. However,
owing to its higher plasma concentration and equipotency to 15, endoxifen
is now believed to be the key active metabolite. This is further supported by the
observation that the efficacy of 8 shows a dependency upon the CYP2D6
enzyme, which is thought to mediate the biotransformation depicted in
Scheme 7.3. It is likely that endoxifen binds with ERa in a manner that is similar
to what is depicted in Figure 7.2 for 15. Finally, preliminary data suggest
that 8 may enhance the degradation of the ERa receptor, rather than simply
serving as a competitive receptor antagonist in breast cancer cells [50, 51]. This
is based upon Western blot analyses, which indicate that 20’s action in breast
tissues appears to be different from that of 15 because it causes degradation of
the ERa [51].
7.2.3
Pharmacokinetics and Major Metabolic Pathways
Tamoxifen is administered orally as its citrate salt. Steady-state plasma concentrations
for 8 and its major metabolite N-desmethyltamoxifen (19) are achieved in
about 4 and 8 weeks, respectively. Tamoxifen is extensively metabolized by microsomal
enzymes into several active metabolites that are primarily eliminated via the
intestinal route as polar conjugates. The terminal half-lives of 8 and 19 are 5–7 and
9–14 days, respectively [52].
Several studies show that the cytochrome P450 (CYP) system is primarily responsible
for the biotransformation of 8 into various oxidation metabolites
(Scheme 7.3) [53–55]. In addition, hepatic flavin-containing monooxygenase
(FMO) converts 8 into its N-oxide metabolite (22) that can be reduced back to 8.
Thus, 22 could act as a reservoir for the drug in various body tissues [56, 57].
Tamoxifen undergoes extensive biotransformation after oral administration,
mainly into 19 as the major primary metabolite with retention of similar low
potency. 4-Hydroxytamoxifen (15) and a-hydroxytamoxifen (21) are the other
primary metabolites. Alternatively, 20 is the major secondary active metabolite [53].
Although both 20 and 15 show similarly high potency compared to the parent
drug, 20 is more abundant than 15 in the plasma. The significant increase in 8’s
activity after first-pass metabolism is the reason why it is classified as a prodrug. In
this regard, 20 is now considered to be primarily responsible for the clinical efficacy
of 8 [48] even though several reports previously attributed 8’s activity to 15 [1].
Endoxifen (20) is mainly formed via oxidative biotransformation of 19 by CYP2D6
(Scheme 7.3). Consequently, the widespread interpatient variation in the clinical
effectiveness and side effects of 8 can now be attributed to interindividual
variation in CYP2D6 activity due to genetic polymorphism in the CYP2D6
genotype [48, 49, 53, 58].
174j7 Selective Estrogen Receptor Modulators
7.2.4
Clinical Toxicity and New Tamoxifen Analogues
A major side effect of 8 is that it can increase endometrial cancer in women during
adjuvant therapy [59]. Other serious toxicity includes pulmonary embolism and
stroke [52]. Some reports have implicated 8’s propensity toward carcinogenicity to
result from the ability of its metabolites, such as a-hydroxytamoxifen (21) and the
N-desmethyl derivatives, to form DNA adducts within normal cells [31, 60].
Several derivatives of 8 have been evaluated to potentially provide drugs that are
less toxic and more efficacious. Other than toremifene, many have been discontinued
because they were found to be either less efficacious than tamoxifen or still
have serious side effects [31, 47]. For instance, both 3-hydroxytamoxifen ( 24) and
idoxifene (metabolically stable since it has an iodo substitution at the 4 position) [31]
were found to increase the incidence of uterine prolapses [61] and had no significant
improvement in therapeutic efficacy; thus, their development was terminated
[47]. Toremifene (Fareston or chlorotamoxifen, 10) as the citrate salt is
currently used in the clinic against estrogen receptor positive or unknown ER status
metastatic breast cancer. Comparative clinical analyses with 8 showed that both
drugs exhibit similar efficacy and side effect profiles [31, 47]. Alternatively, 10
shows a better lipid profile and lower carcinogenicity in rats than 8. It has been
suggested that the presence of the chloro group sterically hinders a-hydroxylation
and thus forms less DNA adducts in comparison to 8 [62]. Afimoxifene (15) is
being developed as a transdermal gel formulation to treat cyclical mastalgia, and as
a SERM by ASCEND Pharmaceuticals [63]. Finally, ospemifene (OphenaTM
, 14) is
under investigation as a SERM to treat postmenopausal vulvovaginal atrophy [64]. It
is being developed by QuatRx Pharmaceuticals.
7.3
Raloxifene
Raloxifene hydrochloride (Evista1
) is a distinct SERM molecule first developed by
Eli Lilly in the early 1980s [65]. Raloxifene (9, Scheme 7.2) was the first SERM for
the prevention and the treatment of osteoporosis and has since been approved for
the prevention of breast cancer in postmenopausal women [66]. This drug is of
great interest in that it acts as an antagonist at ERa in the breast and uterus and
works as an agonist at ERb in the bone [67]. These effects are accompanied by
fewer side effects and different potencies than tamoxifen [68–70]. Raloxifene is still
being studied extensively for additional uses and it continues to be used as a scaffold
for new SERM development.
This section will first describe the motivation for the design and development
of 9. Information on different raloxifene trials including their relevance and the
key results will be covered next. It will conclude by outlining the strengths and
weaknesses of 9 while assessing its potential as a scaffold for future SERM
development.
7.3 Raloxifene j175
7.3.1
Need for New Antiestrogens
As mentioned in the prior section, the early 1980s saw a heightened search for new
multifunctional antiestrogens. These agents were designed to be nonsteroidal and
have greater antagonism accompanied by lower intrinsic estrogenicity than previous
drug molecules. At that time, 8 had already been developed and information
on its side effects and drug resistance was increasing. It was also known that 8
existed in two different isomers, namely, the E and Z forms as described in the
previous section. The Z metabolite isomer is the active form, yet when administered,
a mixture of E and Z metabolites is formed making dosing difficult. Overall,
it was hoped that new SERM molecules could be developed that would be equally or
more effective against breast cancer, while existing as only one isomeric form and
having fewer side effects [65].
7.3.2
Design and Initial Biological Data on Raloxifene
When designing 9, Eli Lilly wanted an antiestrogen having a structure similar
to 8, but with less potential for isomerization. To do this they made an ABDD
series with added rigidity, which according to previous research could be accomplished
by using a variety of different atoms to form a heterocycle on the scaffold
backbone [65]. In the end, they selected a benzothiophene scaffold, which
had shown to give antiestrogenic effects in past research [71]. Other research
had also shown for 8 that retention of the basic side chain (amine-containing
side chain) contributed to the antiestrogenic effects of the drug, so Eli Lilly’s
overall structural motif retained the basic side chain portion of the molecule,
yet the groups attached to the amine were varied [65]. There was also precedent
to attach the side chain by adding a ketone linker to the phenyl ether as found
in 8 [72].
Synthesis was conducted to form a series of benzothiophene analogues and biological
data were obtained for all of the analogues. These studies looked for estrogenic
and nonestrogenic properties of the compounds using an immature rat
uterotrophic assay and the results were compared to that of a control, estradiol
(1, Figure 7.1), as well as to that of 8. One of the analogues, LY139481, showed
impressive biological properties. It had an uterotrophic effect at a low dose that did
not increase with concentration. It was also able to inhibit the effects of 1 at similar
and higher concentrations [65].
With the exciting results pointing to LY139481 as an antiestrogen lead compound,
the need for an improved synthesis at the multigram level was investigated.
This route needed to be more efficient and provide better yields than what had been
deployed during the front-line screening. This goal was accomplished by Eli Lilly
soon after the original synthesis and was done using a one-pot synthesis that
increased the yield to 75%. The new route allowed for multigram syntheses of the
lead [65].
176j7 Selective Estrogen Receptor Modulators
With a process-scale synthesis available, further tests were conducted on the
hydrochloride form of the analogue of interest to determine its efficacy in uterotrophic
and antitumor assays. The hydrochloride salt was referred to as analogue
LY156758 and later became known as “keoxifene.” Studies were continued on cell
cultures and followed with animal studies using immature rats [73] as well as rats
having N-nitrosomethylurea-induced mammary carcinoma [74]. In these trials, it
was determined that keoxifene was not as effective as 8 in the uterotrophic and
antitumor models and due to these biological results, further studies on the analogue
were almost abandoned [75, 76]. In the late 1980s, it was determined that
antiestrogens have estrogenic effects in the bone and new trials began on keoxifene,
yet this time it was referred to as raloxifene (9) [76]. Raloxifene was studied
for its use in the treatment and prevention of osteoporosis where it was then determined
that it was able to increase spinal bone density in postmenopausal women.
Following these studies, Eli Lilly submitted to the FDA for raloxifene’s approval for
the prevention and treatment of osteoporosis in postmenopausal women. In 1997,
Evista1
was approved by the FDA for the prevention of osteoporosis in postmenopausal
women. In 1999, Evista1
was then approved for the treatment of
osteoporosis in postmenopausal women with osteoporosis [77]. Following the new
excitement of Evista1
’s use for osteoporosis, further studies followed to
determine 9’s potential in combating other diseases, especially for treatment and
prevention of breast cancer and cardiovascular diseases.
7.3.3
RUTH Study
The “Raloxifene’s Use for The Heart” (RUTH) trial began in 1998; this trial was a
double-blind, international study. The trial’s objectives were to determine the
effects of 9 on coronary events as well invasive breast cancer in postmenopausal
women. Over 10 000 women participated and each was given either a 60 mg tablet
of Evista1
or a placebo every day for a period of 1 year. The results of this study
showed that 9 had no effect on coronary heart disease or any other coronary effect,
in that these parameters did not increase or decrease from the amount found in the
placebo. It was found that 9 did significantly reduce the amount of breast cancer
and spinal fractures in comparison to placebo [69]. These data were evaluated initially
by two groups referred to as “Heart Estrogen/Progesterone Replacement
Study” (HERS) and “The Women’s Health Initiative” (WHI). A secondary evaluation
was then performed by “Multiple Outcomes of Raloxifene Evaluation”
(MORE). All studies ended with the same conclusions that 9 showed benefits as a
potential breast cancer treatment [3].
7.3.4
STAR Study
The “Study of Tamoxifen and Raloxifene” (STAR) was a double-blind phase III clinical
trial sponsored by the National Cancer Institute in 1999. The trial was
7.3 Raloxifene j177
conducted to compare the effectiveness of 9 in comparison to 8 in the prevention of
invasive breast cancer in postmenopausal women. This study was one of the largest
cancer prevention clinical trials, enrolling close to 20 000 postmenopausal women
that were at high risk for breast cancer. All participants were given either 60 mg of 9
or 20 mg of 8 daily over a period of 5 years.
Results of this study proved that both 9 and 8 were effective in the prevention
of breast cancer in postmenopausal women with 50% effectiveness for 8
and 35% for 9. Raloxifene had a lower instance of thromboembolic events, cataracts,
and a lower instance of gynecological, bladder, and vasomotor problems
as well as less leg cramping. Tamoxifen had a lower incident of musculoskeletal
problems, dyspareunia, and weight gain. There was no notable change
in physical or mental health for 8 or 9, and both had the same instance of
fractures, other cancers, heart disease, and stroke. At the end of this study, it
was determined that 9 was as effective as 8 in the prevention of invasive breast
cancer in postmenopausal women with fewer major side effects. Following a
variety of additional trials that studied the effectiveness of 9 as a preventative
medication for breast cancer, applications for this new use were submitted
[68, 70]. On September 14, 2007 the FDA announced that they had determined
that the benefits of 9 outweighed the risks and then approved 9 for the
prevention of breast cancer in postmenopausal women that are at high risk for
invasive forms of the disease. This was only the second drug allowed by the
FDA for this use at the time [77].
7.3.5
Binding to the Estrogen Receptor
With expansion of the clinical studies being conducted on raloxifene, data about its
mechanism of action and efficacy grew in importance. The belief is that the mechanism
of action for estrogens was due to binding of 1 at ERa and ERb causing dissociation
from heat shock proteins followed by dimerization and recruitment of
coactivators, resulting in transcription complex [76]. For SERMs, the same overall
mechanism of action would be true, yet they are believed to cause coactivator recruitment
in some tissue and corepressor recruitment in others [78] (Scheme 7.1).
To further justify the mechanism of action while equating binding interactions at
ERs to the necessity of the specific structural features (given in the design portion
of this section), comparisons of binding motifs were done for 9, 1, and 15 (a metabolite
of 8 discussed in Section 7.1) within the ERa pocket. These comparisons are
shown in Figure 7.2b. Binding for 1, 15, and 9 occurs through hydrogen bonding at
amino acids Arg394 and Glu353. Further hydrogen bonding can be formed in the
case of 1 and 9 with His524 residue. The antiestrogenic effects found in 9 and 8 are
attributed to the amine side chain that has an ionic interaction with Asp351 [79].
The interaction with aspartic acid prevents helix 12 from reorienting like it does
when 1 binds, and this, in turn, leads to a conformation that does not allow coactivator
recruitment. In the case of 9, there is also an ionic interaction in ERb between
the amine and Asp303 giving it a similar type of effect as seen in ERa [31, 45, 75].
178j7 Selective Estrogen Receptor Modulators
7.3.6
ADME
The absorption, distribution, metabolism, and excretion of 9 are other important factors
to consider when determining other uses for the drug, as well as when designing
new drug analogues. Raloxifene is administered as a 60mg tablet taken
daily [69]. After oral administration, 9 is absorbed through the gastrointestinal tract
and distributed throughout the body [80]. It is metabolized rapidly through first-pass
phase II metabolism in the intestine, forming primarily glucuronide conjugates [81].
The drug has a very low bioavailability of 2%, yet the half-life is 27.7h after a single
dose and is 32.5 h following multiple doses [80, 82]. Elimination proceeds through
the liver with more than 90% of the metabolites found in the feces [80].
7.3.7
Further Research
Raloxifene continues to serve as one of the SERM classical prototypes. While it was
the first SERM for the treatment of osteoporosis, it was also found to have further
antiestrogenic effects that are useful in breast cancer prevention in postmenopausal
women. Raloxifene has a unique potency that is effective at low concentrations
and its efficacy is not concentration dependent. Raloxifene is also of
importance in that its use is still common today, making it an excellent prototype to
help lead new SERM ABDD.
New developments in the area of 9 itself are also needed in that the drug is not
devoid of its own problems. With the known side effects of the drug, a success rate
lower than that of 8 in the breast, and its very low bioavailability, 9 has specific areas
that need significant improvement. For example, the next tier of ABDD-derived
SERMs, as depicted by the four compounds 10–13 in Scheme 7.2, exhibit differences
in their overall profiles and offer some clinical improvements. Uterine data
for bazedoxifene suggest that it is a more selective SERM than raloxifene such that
it may not stimulate the uterus and breast [83] during postmenopausal, hormone
replacement therapy (HRT) [84]. Nevertheless, raloxifene is a SERM of significant
importance because it remains successful as a therapeutic agent for osteoporosis
and has fewer side effects than most other marketed SERMs.
7.4
Summary
Research and clinical development of nonsteroidal ER ligands over the past 60 years
has gradually evolved from estrogenic compounds intended as hormone replacement
therapies to antiestrogenic compounds initially intended for birth control
and later as treatments for hormone-dependent cancers, and finally to the SERMs
from which their differing selective actions can be redirected toward the above uses
as well as toward a variety of other clinical indications, all with less concomitant
7.4 Summary j179
general ER-related side effects. Exemplifying the latter, clomiphene remains effective
for the induction of ovulation in subfertile women, tamoxifen for the treatment
and prevention of postmenopausal ER-positive breast cancer, and raloxifene for the
treatment and prevention of postmenopausal osteoporosis while affording greater
breast and uterine safety. Several newer agents expand the potential uses even further.
Among the longer standing clinically available SERMs that can be considered
for hormone replacement therapy, two major limitations have arisen, namely, that
they are only weak estrogen agonists and they (can) aggravate hot flashes [3]. The
latter represent a particularly confounding situation in that they are also one of the
frequent symptoms for the prescription of HRT.
Marked by the discovery of ERb as a second estrogenic system, the gradually
increasing knowledge about the complexity of the ER pathways contributed significantly
to the evolution of the SERMs across this period, as well as to our today’s
understanding about how SERMs work. Nevertheless, there still remains no unifying
theory that has explained the target site-specific actions of SERMs [1]. Likewise,
advances in estrogen-related medicinal chemistry and its SAR have served to drive
the evolution of the SERMs to its present structural landscape. However and in
sharp contrast to the variety of biological profiles that have been achieved to date,
structural motifs proceeding to the marketplace have yet to depart significantly
from the original triphenylethylene scaffold arrangement and the latter’s cyclized
possibilities with or without reduction and the use of heteroatoms. While ABDD,
in turn, can certainly be ascribed to contributing toward the present lack of structural
diversity, its “plus” side in this same regard has resulted in the variety of biological
profiles and distinct clinical indications that have been able to be delivered
to the marketplace. Furthermore, the status of SAR and X-ray-derived ER structural
details have now evolved to the point where together they can allow ABDD to also
adopt scaffold hopping explorations in a rational manner. Thus, novel structural
types should soon be on the horizon for the SERMs.
As the clinical performances of the latest agents become elaborated and the
nuances of the ER pathways continue to unfold in parallel with our expanding
knowledge about their associated medicinal chemistry, pursuit of new SERMs by
ABDD geared toward producing novel chemical structures should pave the way
toward the development of “ideal” SERMs for clinical practice [2]. These “next-generation”
SERMs will be distinctly tailored for their specific indications. Ultimately
providing dramatic new paradigms for maintaining the health of women [2], indications
will continue to include induction of ovulation, reduction of menopausal
symptoms, and bone preservation. Toward improving the health of both men and
women, indications will include cholesterol reduction and chemotherapy of hormone-related
cancers.
References
1 Jordan, V.C. (2003) Antiestrogens and
selective estrogen receptor modulators as
multifunctional medicines. 1. Receptor
interactions. J. Med. Chem., 46, 883–908.
180j7 Selective Estrogen Receptor Modulators
2 Osborne, C.K., Zhao, H., and Fuqua, S.A.
(2000) Selective estrogen receptor
modulators: structure, function, and
clinical use. J. Clin. Oncol., 18, 3172–3186.
3 Riggs, B.L. and Hartmann, L.C. (2003)
Selective estrogen-receptor modulators –
mechanisms of action and application to
clinical practice. N. Engl. J. Med., 348,
618–629.
4 Dickey, R.P. and Holtkamp, D.E. (1996)
Development, pharmacology and clinical
experience with clomiphene citrate. Hum.
Reprod. Update, 2, 483–506.
5 Clarke, B.L. and Khosla, S. (2009) New
selective estrogen and androgen receptor
modulators. Curr. Opin. Rheumatol., 21,
374–379.
6 Minutolo, F., Macchia, M.,
Katzenellenbogen, B.S., and
Katzenellenbogen, J.A. (2011) Estrogen
receptor beta ligands: recent advances and
biomedical applications. Med. Res. Rev.,
31, 364–442.
7 Dodds, E.C., Goldberg, L., Lawson, W., and
Robinson, R. (1938) Estrogenic activity of
alkylated stilboestrols. Nature, 142, 34.
8 Dodds, E.C. and Lawson, W. (1938)
Molecular structure in relation to
estrogenic activity: compounds without a
phenanthrene nucleus. Proc. R. Soc. Lond.
Ser. B, 125, 222–232.
9 Lerner, L.J., Holthaus, F.J., Jr., and
Thompson, C.R. (1958) A non-steroidal
estrogen antiagonist 1-(p-2-
diethylaminoethoxyphenyl)-1-phenyl-2-pmethoxyphenyl
ethanol. Endocrinology, 63,
295–318.
10 Jones, C.D., Jevnikar, M.G., Pike, A.J.,
Peters, M.K. et al. (1984) Antiestrogens. 2.
Structure–activity studies in a series of 3aroyl-2-arylbenzo[b]thiophene
derivatives
leading to [6-hydroxy-2-(4-hydroxyphenyl)
benzo[b]thien-3-yl]-[4-[2-(1-piperidinyl)
ethoxy]-phenyl]methanone hydrochloride
(LY156758), a remarkably effective
estrogen antagonist with only minimal
intrinsic estrogenicity. J. Med. Chem., 27,
1057–1066.
11 Allen, R.E., Palopoli, F.P., Schumann,
E.L., and Van Campen, M.G., Jr. (1959)
Amine Derivatives of Triphenylethanol
US Patent 2,914,562 (to the Wm. S.
Merrell Co.).
12 Palopoli, F.P., Feil, V.J., Allen, R.E.,
Holtkamp, D.E., and Richardson, A., Jr.,
(1967) Substituted
aminoalkoxytriarylhaloethylenes. J. Med.
Chem., 10, 84–86.
13 Ernst, S., Hite, G., Cantrell, J.S.,
Richardson, A., Jr., and Benson, H.D.
(1976) Stereochemistry of geometric
isomers of clomiphene: a correction of the
literature and a reexamination of
structure–activity relationships. J. Pharm.
Sci., 65, 148–150.
14 Clark, J.H. and Guthrie, S.C. (1981)
Agonistic and antagonistic effects of
clomiphene citrate and its isomers. Biol.
Reprod., 25, 667–672.
15 Jordan, V.C., Haldemann, B., and Allen,
K.E. (1981) Geometric isomers of
substituted triphenylethylenes and
antiestrogen action. Endocrinology, 108,
1353–1361.
16 Shepard, M.K., Balmaceda, J.P., and Leija,
C.G. (1979) Relationship of weight to
successful induction of ovulation with
clomiphene citrate. Fertil. Steril., 32,
641–645.
17 Mikkelson, T.J., Kroboth, P.D., Cameron,
W.J., Dittert, L.W. et al. (1986) Single-dose
pharmacokinetics of clomiphene citrate in
normal volunteers. Fertil. Steril., 46,
392–396.
18 Clark, J.H. and Markaverich, B.M. (1988)
The Physiology of Reproduction (eds E.
Knobil and J. Neill), Raven Press,
New York, pp. 675–723.
19 Hsueh, A.J., Erickson, G.F., and Yen, S.S.
(1978) Sensitisation of pituitary cells to
luteinising hormone releasing hormone
by clomiphene citrate in vitro. Nature, 273,
57–59.
20 Dickey, R.P., Vorys, N., Stevens, V.C.,
Besch, P.K. et al. (1965) Observations on
the mechanism of action of clomiphene
(MRL-41). Fertil. Steril., 16, 485–494.
21 Bryant, H.U. (2002) Selective estrogen
receptor modulators. Rev. Endocr. Metab.
Disord., 3, 231–241.
22 Fang, H., Tong, W., Shi, L.M., Blair, R.
et al. (2001) Structure–activity
relationships for a large diverse set of
natural, synthetic, and environmental
estrogens. Chem. Res. Toxicol., 14,
280–294.
References j181
23 Jordan, V.C. (1994) Long-Term Tamoxifen
Treatment for Breast Cancer (ed. V.C.
Jordan), University of Wisconsin Press,
Madison, WI, pp. 3–26.
24 Kuiper, G.G., Lemmen, J.G., Carlsson, B.,
Corton, J.C. et al. (1998) Interaction of
estrogenic chemicals and phytoestrogens
with estrogen receptor beta. Endocrinology,
139, 4252–4263.
25 Pettersson, K. and Gustafsson, J.A. (2001)
Role of estrogen receptor beta in estrogen
action. Annu. Rev. Physiol., 63, 165–192.
26 Sutherland, R.L. and Jordan, V.C. (eds)
(1981) Non-Steroidal Antiestrogens:
Molecular Pharmacology and Antitumor
Activity, Academic Press, Sydney,
Australia.
27 Zhao, C., Dahlman-Wright, K., and
Gustafsson, J.A. (2008) Estrogen receptor
beta: an overview and update. Nucl. Recept.
Signal., 6, e003.
28 Agatonovic-Kustrin, S. and Turner, J.V.
(2008) Molecular structural characteristics of
estrogen receptor modulators as
determinants of estrogen receptor selectivity.
Mini-Rev. Med. Chem., 8, 943–951.
29 Blizzard, T.A. (2008) Selective estrogen
receptor modulator medicinal chemistry
at Merck. A review. Curr. Top. Med. Chem.
(Sharjah, United Arab Emirates), 8,
792–812.
30 Henke, B.R. and Heyer, D. (2005) Recent
advances in estrogen receptor modulators.
Curr. Opin. Drug Discov. Dev., 8, 437–448.
31 Jordan, V.C. (2003) Antiestrogens and
selective estrogen receptor modulators as
multifunctional medicines. 2. Clinical
considerations and new agents. J. Med.
Chem., 46, 1081–1111.
32 Meegan, M.J. and Lloyd, D.G. (2003)
Advances in the science of estrogen
receptor modulation. Curr. Med. Chem.,
10, 181–210.
33 Redden, P.R. (2004) Selective estrogen
receptor modulators, pure antiestrogens
and related estrogen receptor ligands.
Expert Opin. Ther. Pat., 14, 337–353.
34 Ullrich, J.W. and Miller, C.P. (2006)
Estrogen receptor modulator review.
Expert Opin. Ther. Pat., 16, 559–572.
35 Veeneman, G.H. (2005) Non-steroidal
subtype selective estrogens. Curr. Med.
Chem., 12, 1077–1136.
36 Richardson, D.N. (1988) The history of
Nolvadex. Drug Des. Deliv., 3, 1–14.
37 Bedford, G.R. and Richardson, D.N. (1966)
Preparation and identification of cis and
trans isomers of a substituted triarylethylene.
Nature (London), 212, 733–734.
38 Harper, M.J.K. and Walpole, A.L. (1966)
Contrasting endocrine activities of cis and
trans isomers in a series of substituted
triphenylethylenes. Nature, 212, 87.
39 Kilbourn, B.T. and Owston, P.G. (1970)
Crystal structure of the hydrobromide of
1-[p-[2-(dimethylamino)ethoxy]phenyl]-1,2cis-diphenyl-1-butene,
a compound with
estrogenic activity. J. Chem. Soc. B, 1, 1–5.
40 Holtkamp, D.E., Greslin, J.G., Root, C.A.,
and Lerner, L.J. (1960) Gonadotrophin
inhibiting and anti-fecundity effects of
chloramiphene. Proc. Soc. Exp. Biol. Med.,
105, 197–201.
41 Beatson, G.T. (1896) On the treatment of
inoperable cases of carcinoma of the
mamma: suggestions for a new method of
treatment, with illustrative cases. Lancet,
2, 104–107.
42 Huggins, C.B. and Bergenstal, D.M.
(1952) Inhibition of human mammary
and prostatic cancers by adrenalectomy.
Cancer Res., 12, 134–141.
43 Furr, B.J., Patterson, J.S., Richardson,
D.N., Slater, S.R., and Wakeling, A.E.
(1979) Tamoxifen. Pharmacol. Biochem.
Prop. Drug Subst., 2, 355–399.
44 Lunan, C.B. and Klopper, A. (1975)
Antioestrogens. A review. Clin.
Endocrinol., 4, 551–572.
45 Brzozowski, A.M., Pike, A.C., Dauter, Z.,
Hubbard, R.E. et al. (1997) Molecular
basis of agonism and antagonism in the
oestrogen receptor. Nature, 389, 753–758.
46 Shiau, A.K., Barstad, D., Loria, P.M.,
Cheng, L. et al. (1998) The structural basis
of estrogen receptor/coactivator recognition
and the antagonism of this interaction by
tamoxifen. Cell, 95, 927–937.
47 Pickar, J.H., MacNeil, T., and Ohleth, K.
(2010) SERMs: progress and future
perspectives. Maturitas, 67, 129–138.
48 Ahmad, A., Shahabuddin, S., Sheikh, S.,
Kale, P. et al. (2010) Endoxifen, a new
cornerstone of breast cancer therapy:
demonstration of safety, tolerability, and
systemic bioavailability in healthy human
182j7 Selective Estrogen Receptor Modulators
subjects. Clin. Pharmacol. Ther., 88,
814–817.
49 Beverage, J.N., Sissung, T.M., Sion, A.M.,
Danesi, R., and Figg, W.D. (2007) CYP2D6
polymorphisms and the impact on
tamoxifen therapy. J. Pharm. Sci., 96,
2224–2231.
50 Lim, Y.C., Desta, Z., Flockhart, D.A., and
Skaar, T.C. (2005) Endoxifen (4-hydroxy-Ndesmethyl-tamoxifen)
has anti-estrogenic
effects in breast cancer cells with potency
similar to 4-hydroxy-tamoxifen. Cancer
Chemother. Pharmacol., 55, 471–478.
51 Wu, X., Hawse, J.R., Subramaniam, M.,
Goetz, M.P. et al. (2009) The tamoxifen
metabolite, endoxifen, is a potent
antiestrogen that targets estrogen receptor
alpha for degradation in breast cancer
cells. Cancer Res., 69, 1722–1727.
52 Adam, H.K., Patterson, J.S., and Kemp,
J.V. (1980) Studies on the metabolism and
pharmacokinetics of tamoxifen in normal
volunteers. Cancer Treat. Rep., 64, 761–
764.
53 Desta, Z., Ward, B.A., Soukhova, N.V., and
Flockhart, D.A. (2004) Comprehensive
evaluation of tamoxifen sequential
biotransformation by the human
cytochrome P450 system in vitro:
prominent roles for CYP3A and CYP2D6.
J. Pharmacol. Exp. Ther., 310, 1062–1075.
54 Stearns, V., Johnson, M.D., Rae, J.M.,
Morocho, A. et al. (2003) Active tamoxifen
metabolite plasma concentrations after
coadministration of tamoxifen and the
selective serotonin reuptake inhibitor
paroxetine. J. Natl. Cancer Inst., 95,
1758–1764.
55 White, I.N. (2003) Tamoxifen: is it safe?
Comparison of activation and detoxication
mechanisms in rodents and in humans.
Curr. Drug Metab., 4, 223–239.
56 Kellen, J.A. (ed.) (1996) Tamoxifen: Beyond
the Antiestrogen, Birkhaeuser, Boston, MA.
57 Mani, C., Hodgson, E., and Kupfer, D.
(1993) Metabolism of the antimammary
cancer antiestrogenic agent tamoxifen. II.
Flavin-containing monooxygenasemediated
N-oxidation. Drug Metab.
Dispos., 21, 657–661.
58 Jordan, V.C. (1982) Metabolites of
tamoxifen in animals and man:
identification, pharmacology, and
significance. Breast Cancer Res. Treat., 2,
123–138.
59 Fornander, T., Rutqvist, L.E., Cedermark,
B., Glas, U. et al. (1989) Adjuvant
tamoxifen in early breast cancer:
occurrence of new primary cancers.
Lancet, 1, 117–120.
60 Han, X.L. and Liehr, J.G. (1992) Induction
of covalent DNA adducts in rodents by
tamoxifen. Cancer Res., 52, 1360–1363.
61 Nilsson, S., Koehler, K.F., and Gustafsson,
J.A. (2011) Development of subtypeselective
oestrogen receptor-based
therapeutics. Nat. Rev. Drug Discov., 10,
778–792.
62 Hard, G.C., Iatropoulos, M.J., Jordan, K.,
Radi, L. et al. (1993) Major difference in
the hepatocarcinogenicity and DNA
adduct forming ability between
toremifene and tamoxifen in female Crl:
CD(BR) rats. Cancer Res., 53, 4534–4541.
63 Mansel, R., Goyal, A., Nestour, E.L.,
Masini-Eteve, V., and O’Connell, K. (2007)
A phase II trial of Afimoxifene (4hydroxytamoxifen
gel) for cyclical
mastalgia in premenopausal women.
Breast Cancer Res. Treat., 106, 389–397.
64 Gennari, L., Merlotti, D., Valleggi, F., and
Nuti, R. (2009) Ospemifene use in
postmenopausal women. Expert Opin.
Invest. Drugs, 18, 839–849.
65 Jones, C.D., Jevnikar, M.G., Pike, A.J.,
Peters, M.K., Black, L.J., Thompson, A.R.,
Falcone, J.F., and Clemens, J.A. (1984)
Antiestrogens. 2. Structure–activity
studies in a series of 3-aroyl-2-arylbenzo[b]
thiophene derivatives leading to [6-
hydroxy-2-(4-hydroxyphenyl)benzo[b]
thien-3-yl][4-[2-(1-piperidiny1)ethoxyl]
phenyl]methanone hydrochloride (LY
156758), a remarkably effective estrogen
antagonist with only minimal intrinsic
estrogenicity. J. Med. Chem., 27,
1057–1066.
66 Shang, Y. (2006) Molecular mechanisms
of oestrogen and SERMs in endometrial
carcinogenesis. Nature, 6, 360–368.
67 Jordan, V.C. (2001) Selective estrogen
receptor modulation: a personal
perspective. Cancer Res., 61, 5683–5687.
68 Vogel, V.G., Costantino, J.P.,
Wickerham, D.L., Cronin, W.M. et al.
(2006) Effects of tamoxifen vs. raloxifene
References j183
on the risk of developing invasive breast
cancer and other disease outcomes: the
NSABP Study of Tamoxifen and
Raloxifene (STAR) P-2 trial. J. Am. Med.
Assoc., 295, 2727–2741.
69 Barrett-Connor, E., Mosca, L., Collins, P.,
Geiger, M.J. et al. (2006) Effects of
raloxifene on cardiovascular events and
breast cancer in postmenopausal women.
N. Engl. J. Med., 355, 125–137.
70 National Cancer Institute (2006) Study of
Tamoxifen and Raloxifene (STAR) Trial,
http://www.cancer.gov/clinicaltrials/
digestpage/STAR (accessed September 9,
2011).
71 Crenshaw, R.R., Jeffries, A.T., Luke, G.M.,
Cheney, L.C., and Bialy, G. (1971)
Potential antifertility agents. 1. Substituted
diaryl derivatives of benzo[b]thiophenes,
benzo[b]furans, 1H-2-benzothiapyrans,
and 2H-1-benzothiapyrans. J. Med. Chem.,
14, 1185–1190.
72 Jones, C.D., Suarez, T., Massey, E.H.,
Black, L.J., and Tinsley, F.C. (1979)
Synthesis and antiestrogenic activity of
[3,4-dihydro-2-(4-methoxyphenyl)-1-
naphthalenyl][4-[2-(1-pyrrolidinyl)ethoxy]phenyl]methanone,
methanesulfonic acid
salt. J. Med. Chem., 22, 962–966.
73 Black, L.J., Jones, C.D., and Falcone,
J.F. (1982) Antagonism of estrogen action
with a new benzothiophene derived
antiestrogen. Life Sci., 32, 1031–1036.
74 Gottardis, M. and Jordan, C. (1987)
Antitumor actions of keoxifene and
tamoxifen in the N-nitrosomethylureainduced
rat mammary carcinoma model.
Cancer Res., 47, 4020–4024.
75 Jordan, V.C. (2003) Tamoxifen: a most
unlikely pioneering medicine. Nat. Rev., 2,
205–213.
76 Lewis, J.S. and Jordan, V.C. (2005)
Selective estrogen receptor modulators
(SERMs): mechanism of
anticarcinogenesis and drug resistance.
Mutat. Res., 591, 247–263.
77 US Food and Drug Administration (2007)
FDA News Release.
78 Deroo, B.J. and Korach, K.S. (2006)
Estrogen receptors and human disease.
J. Clin. Invest., 116, 561–570.
79 Lui, H., Park, W.-C., Bentrem, D.J.,
McKain, K.P. et al. (2002) Structure–
function relationships of the raloxifene–
estrogen receptor-alpha complex for
regulating transforming growth factorsalpha
expression in breast cancer cells. J.
Biol. Chem., 277, 9189–9198.
80 Heringa, M. (2003) Review on raloxifene:
profile of a selective estrogen receptor
modulator. Int. J. Clin. Pharmacol. Ther.,
41, 331–345.
81 Jeong, E.J., Liu, Y., Lin, H., and Hu, M.
(2005) Species- and disposition modeldependent
metabolism of raloxifene in
gut and liver: role of UGT1A10. Drug
Metab. Dispos., 33, 785–784.
82 Lindstrom, T.D., Whitaker, N.G., and
Whitaker, G.W. (1984) Disposition and
metabolism of a new benzothiophene
antiestrogens in rats, dogs and monkey.
Xenobiotica, 14 (11), 841–847.
83 Miller, C.P., Harris, H.A., and Komm,
B.S. (2002) Bazedoxifene acetate. Drugs
Future, 27, 117–121.
84 Miller, C.P., Collini, M.D., Tran, B.D.,
Harris, H.A. et al. (2001) Design,
synthesis, and preclinical
characterization of novel, highly
selective indole estrogens. J. Med.
Chem., 44, 1654–1657.
184j7 Selective Estrogen Receptor Modulators
Paul W. Erhardt received a Ph.D. in Medicinal Chemistry
from the University of Minnesota and carried out postdoctoral
research at the University of Texas at Austin. He
then spent several years within the pharmaceutical industry,
where he is credited with the discovery and chemical development
of esmolol. For the past several years, he has been at the
University of Toledo (UT) where he is a Professor of Medicinal
and Biological Chemistry within the College of Pharmacy
and Pharmaceutical Sciences, a Joint Professor of Biochemistry
and Cancer Biology within the College of Medicine and Life Sciences,
and additionally serves as Director of UT’s Center for Drug Design and Development
(CD3).
Rachael Jetson received a Bachelor of Science in Chemistry
from The South Dakota School of Mines and Technology in
May 2005. She then received a Master of Science in Material
Science and Engineering in 2008 under the supervision of
Dr. Zhengato Zhu, in which she worked on polymer/ZnO
nanocomposites. In August 2008, Rachael started a Ph.D.
program in Medicinal and Biological Chemistry at the
University of Toledo. Since September 2009, Rachael has
been conducting thesis work under Dr. Paul W. Erhardt
within the Center for Drug Design and Development. Her thesis is directed toward
the discovery of novel SERM compounds.
Amarjit Luniwal obtained his Bachelor of Pharmacy degree
from Maharshi Dayanand University (MDU) and is a registered
pharmacist in India. After obtaining his Master of Pharmacy
degree from the State Technical University of Madhya
Pradesh, India, he joined the Center for Drug Design and
Development (CD3) at the University of Toledo to conduct
Ph.D. studies in medicinal chemistry. His Ph.D. work mainly
involved natural product synthesis and computer-aided
molecular modeling. He completed his Ph.D. dissertation in
November 2010 and presently holds a postdoctoral position in the Department of
Chemistry at the University of Toledo. His postdoctoral work involves design and
development of lead compounds for selective antibiotic development along with
research in molecular biology and protein chemistry.
j185
8
Discovery of Nonpeptide Vasopressin V2 Receptor Antagonists
Kazumi Kondo and Hidenori Ogawa
List of Abbreviations
ACTH adrenocorticotropic hormone
ADH antidiuretic hormone
AVP arginine vasopressin
cAMP cyclic adenosine monophosphate
EDG electron-donating group
ED3 the dose (mg/kg) required for a threefold increase in the 2 h urine volume
over the control rats
EWG electron-withdrawing group
FDA the US Food and Drug Administration
GPCR G-protein-coupled receptor
MHLW the Japanese Ministry of Health, Labour and Welfare
MOE molecular operating environment is a drug discovery software package
developed by CCG (Chemical Computing Group Inc., Canada)
8.1
Introduction
Arginine vasopressin (AVP, Figure 8.1), also called antidiuretic hormone (ADH), is
a cyclic nonapeptide hormone that is secreted from the posterior pituitary. AVP
production and secretion are initiated when plasma osmolality increases and/or
blood pressure decreases.
AVP mediates its action through three receptors: V1a, V1b, and V2. The V1a receptors
are mainly expressed in the liver, vascular smooth muscle cells, platelets,
uterus, and brain. The V1b receptors are mainly expressed in the anterior pituitary
as well as pancreatic b-cells. V2 receptors are exclusively expressed in the kidney
collecting ducts.
AVP receptors are members of the G-protein-coupled membrane receptor
(GPCR) superfamily. The V1a and V1b receptors are linked to the phospholipase C
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
j187
activation via Gq/11, which in turn stimulates phosphatidylinositol turnover,
thereby increasing the intracellular calcium ion level. The V2 receptor is linked to
the activation of adenylyl cyclase by interaction with Gs, which leads to an increase
in the intracellular cAMP.
AVP plays important roles in the cardiovascular system. Vasoconstriction is
mediated by the V1a receptor. A relatively large amount of AVP is necessary to raise
blood pressure. The activation of V1a receptors is also associated with myocyte
hypertrophy, glycogenolysis, and platelet aggregation.
The activation of the V2 receptor on renal collecting ducts induces an increase in
the osmotic water permeability of the apical membrane and causes water
reabsorption. This effect is known as the antidiuretic activity of AVP.
The cardiovascular and renal effects of AVP mediated by V1a as well as V2 receptors
are reasonably well characterized. On the other hand, the pharmacological profile
of the V1b receptor is somewhat complicated. The V1b receptor activation
stimulates the secretion of ACTH and b-endorphin from the anterior pituitary and
influences blood pressure, memory, body temperature control, and social behavior.
Insulin release also modulates V1b receptor activation.
8.2
Peptide AVP Agonists and Antagonists
Du Vigneaud et al. reported the original synthesis of oxytocin and AVP in
1954 [1, 2]. Numerous analogues of AVP have been synthesized and evaluated to
identify the structural features responsible for their respective biological features.
Diuretic vasopressin antagonists were first described in 1981 by Manning
et al. [3–5] and Sawyer et al. [6]. These compounds were reported to inhibit vasopressin
binding to renal receptors and vasopressin stimulation of renal adenylyl
cyclase in a competitive fashion. However, Stassen et al. [7–10] and Kinter
et al. [11, 12] reported that these compounds did not cause diuresis in dogs or squirrel
monkeys.
Further studies identified vasopressin antagonists with D-Tyr-O-alkyl residues at
position 2 as active water diuretic agents in rats, dogs, and squirrel monkeys [13, 14].
SK&F 101926 was identified as a potent vasopressin antagonist in human tissues
(Figure 8.2). However, Dubb et al. reported that SK&F 101926 did not show diuretic
activity or antagonism of the antidiuretic activity of AVP in humans, and it acted as a
potent full antidiuretic agonist [15]. Brooks et al. reported that i.v. administration of
SK&F 101926 to a hydrated rhesus monkey resulted in a full antidiuretic agonist
Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2
S S
Arginine vasopressin (AVP)
Figure 8.1 Amino acid sequence of arginine vasopressin.
188j8 Discovery of Nonpeptide Vasopressin V2 Receptor Antagonists
response, as observed in humans [16]. Modification of the terminal amino acids from
Pro7
, Arg8
to Arg7
, D-Arg8
led to a compound (SK&F 104146) that was a less potent
agonist than SK&F 101926. Further modification in SK&F 104146 of a disulfide group
with methylene groups in the peptide ring to form a “dicarba bridge” resulted in a
compound (SK&F 105494) that shows minimal agonist activity in hydrated monkeys.
Unfortunately, marked species differences and inconsistencies were revealed between
the in vivo and in vitro assay systems as reported in the case of SK&F 105494. This
compound again showed a full antidiuretic response in humans [17–19].
8.3
Lead Generation Strategies [20, 21]
Further investigations focused on the discovery of nonpeptide compounds with
structures distinct from AVP itself, because it is difficult to predict antagonistic
activity to the human vasopressin V2 receptor with peptide compounds. Another
potential drawback in the development of peptide compounds as V2 receptor antagonists
is the poor bioavailability of peptides by oral administration.
OPC-18549 was found during in-house compound library screening using
3
H-AVP as a natural ligand with rat V1a and rat V2 receptors (Figure 8.3). This compound
showed moderate affinity for the rat V1a receptor, IC50 ¼ 2.5 mM, while the
affinity to the rat V2 receptor was very weak, IC50 > 100 mM [20].
Initial SAR studies of OPC-18549 focused on the terminal thiophene carboxamide
moiety. Replacement of the thiophene ring with a phenyl ring (compound 1)
retained affinity for the V1a receptor. The insertion of a methylene group between
the carbonyl and the phenyl ring (compound 2) reduced V1a affinity. Replacement
of the thiophene ring with a furan ring (compound 3) also reduced binding affinity.
It was therefore very interesting to note that the carbonyl group on a piperidine
ring plays an important role in the affinity for the V1a receptor (compound 4).
CH2
CH2-CO-D-Tyr(Et)-Phe-Val-Asn-NHCHCO-Arg-D-Arg-NH2
CH2 CH2
SK&F 105494 (SmithKline Beecham)
S
CH2-CO-D-Tyr(Et)-Phe-Val-Asn-Cys-Pro-Arg-NH2
S
SK&F 101926 (SmithKline Beecham)
S
CH2-CO-D-Tyr(Et)-Phe-Val-Asn-Cys-Arg-D-Arg-NH2
S
SK&F 104146 (SmithKline Beecham)
Figure 8.2 Some efforts on peptide compounds.
8.3 Lead Generation Strategies j189
Further SAR evaluations investigated substituents on the terminal phenyl ring
of compound 1 (Table 8.1). Substitution of either an electron-donating group
(EDG) or electron-withdrawing group (EWG) was done on the phenyl ring.
There was no clear correlation between the presence of an EDG or EWG on the
binding affinity at the rat V1a receptor. Substituents including 4-methoxy (16),
4-methyl (10), and 4-chloro (7) enhanced the binding affinity for the V1a receptor,
while a para-nitro group (13) did not show any enhancement in binding
affinity. A methoxy group (14) only showed potent affinity for the V1a receptor
as a meta-substituent on the terminal phenyl ring and showed moderate affinity
for the V2 receptor.
Elongation on the terminal alkoxy group was examined because a para-methoxy
substituent (16) enhanced the binding affinity for the V1a receptor. An ethoxy compound
(17) showed the most potent affinity for the V1a receptor with moderate in
vivo efficacy by oral administration (30 mg/kg) (Table 8.2). Tolerability in binding
affinity of a substituent in the longitudinal direction looked very high (18–20).
These results were encouraging, hence a heteroatom was introduced at the end of
the alkoxy group to enhance oral efficacy.
OPC-21268 (Figure 8.4) was selected for further evaluation. This compound
showed binding affinity for rat V1a and rat V2 receptors with IC50 ¼ 0.44
N O
N
O
N O
N
O
N O
N
O
O
N O
N
1
IC50= 1.9 µM (rat, V1a)
2
IC50= 12 µM (rat, V1a)
4
IC50= >100 µM (rat, V1a)
3
IC50= 11 µM (rat, V1a)
N O
N
O
S
OPC-18549
IC50= 2.5 µM (rat, V1a)
Figure 8.3 Initial SAR on terminal thiophene carbonyl moiety.
190j8 Discovery of Nonpeptide Vasopressin V2 Receptor Antagonists
Table 8.1 SAR studies based on compound 1.
N
O
N CO
R
No. R Receptor affinity IC50 (mM) Antipressor activity
V1a V2 % Inhibitiona)
5 2-Cl 9.9 >100
6 3-Cl 4.4 >100
7 4-Cl 1.2 78
8 2-Me 8.4 >100
9 3-Me 1.3 >100
10 4-Me 0.5 >100 23 Æ 8.9 (100)
11 2-NO2 8.4 >100
12 3-NO2 3.1 >100
13 4-NO2 2.0 >100
14 2-OMe 0.65 36
15 3-OMe 2.6 >100
16 4-OMe 0.49 >100
a) The inhibition was expressed as percentage change in diastolic blood pressure increased by AVP
(30 mU/kg, i.v.) before and after test compounds p.o. administration. Except where indicated, the
number of determinations was four. Dosage in parentheses (mg/kg).
Table 8.2 SAR studies based on compound 16.
N
O
N CO R
No. R Receptor affinity IC50 (mM) Antipressor activity
V1a V2 % Inhibitiona)
17 OEt 0.21 >100 75 Æ 3.4 (30)
18 O-nPr 0.32 >100 65 Æ 6.1 (30)
19 O-nBu 0.42 >100
20 O-nHex 1.5 54
a) The inhibition was expressed as percentage change in diastolic blood pressure increased by AVP
(30 mU/kg, i.v.) before and after test compounds p.o. administration. Except where indicated, the
number of determinations was four. Dosage in parentheses (mg/kg).
8.3 Lead Generation Strategies j191
and >100 mM, respectively. Oral efficacy was confirmed to inhibit vasoconstriction
caused by AVP (30 mU/kg, i.v.) and ID50 ¼ 2.0 mg/kg [22].
OPC-21268 was reported to be an orally effective nonpeptide vasopressin V1a
receptor antagonist in 1991, and a marked species difference between rats and
humans was reported by several groups [23–27]. Molecular cloning of human V1a
receptor was reported in 1994.
8.4
Lead Generation Strategy-2, V2 Receptor Affinity [28]
Although the drug discovery program was initiated to find nonpeptide vasopressin
V2 receptor antagonists, binding affinity to the V2 receptor was not enhanced in
2-quinolinone derivatives by modifying the terminal phenyl ring.
Intensive efforts to enhance V2 receptor affinity were continued by replacement of
the piperidine ring of compound 7 with the 4-aminophenyl group, followed by
rearrangement of the carbonyl group of 2-quinolinone to the carbonyl between the
quinoline and the phenyl ring to create compound 21 (Figure 8.5) [28]. Compound 21
showed binding affinity for rat V1a and rat V2 with IC50 ¼ 5.1 and 1.9 mM, respectively.
Three-dimensional stable structures of compounds 7 and 21 are shown in
Figure 8.6. These structures were generated using MOE [29] as modeling software
N O
N
O
O(CH2)3NHAc
OPC-21268
IC50=0.44 µM (V1a, rat)
>100 µM (V2, rat)
ID50=2.0 mg/kg
Figure 8.4 Chemical structure of OPC-21268.
N O
N
O
Cl
N
O
N
H
O
Cl
7 21
IC50 = 1.2 µM, rat V1a
78 µM, rat V2
IC50 = 5.1 µM, rat V1a
1.9 µM, rat V2
N O
N
O
Cl
N
O
Figure 8.5 Structural modification of quinolinone.
192j8 Discovery of Nonpeptide Vasopressin V2 Receptor Antagonists
to examine the structure–activity relationships in a three-dimensional manner.
Structural alignment was also examined with 7 and 21 (Figure 8.6). The structures
of 7 and 21 (Figure 8.5) look quite different, but some pharmacophores such
as phenyl rings and carbonyl groups, shown in circles, are superposed in 3D
(Figure 8.6). These pharmacophores seemed to show affinity for the rat V1a receptor.
A small set of SAR on substituents at the terminal phenyl ring of the tetrahydroquinoline
derivative is shown in Table 8.3. A substitution at the 4-position reduced
the affinity for both V1a and V2 receptors (22 versus 21, 23, 24, and 25). While a
substitution at the 2-position did not alter the binding affinity for the V1a receptor,
2-Cl (26) and 2-Me (28) enhanced the binding affinity for the V2 receptor. A substitution
at the 3-position slightly reduced the binding affinity for the V1a receptor, but
it enhanced the binding affinity for the V2 receptor (29 and 30). Substitution of an
Figure 8.6 3D structures of 7 and 21.
Table 8.3 SAR studies based on compound 21.
N CO NHCO
R
2 3
4
No. R Receptor affinity IC50 (mM)
V1a V2
22 H 1.6 0.98
21 4-Cl 5.1 1.9
23 4-OMe 2.2 1.8
24 4-Me 10 1.4
25 4-NO2 >100 6.5
26 2-Cl 1.6 0.42
27 2-OMe 1.9 2.1
28 2-Me 1.4 0.20
29 3-Cl 6.4 0.20
30 3-OMe 2.8 0.40
31 3-Me 3.1 0.68
8.4 Lead Generation Strategy-2, V2 Receptor Affinity j193
EDG or an EWG did not affect the binding affinity, but the steric size seemed to
affect the binding affinity for the V2 receptor (26 versus 27) [28].
The SAR of substituents on the terminal phenyl ring of this series had a completely
different effect on V1a receptor affinity in comparison to those in Table 8.1.
It means that the binding mode of the terminal phenyl ring at the receptor is probably
different between each series of compounds.
The head group was also examined during the evaluation of substituents on the
terminal phenyl ring, shown in Table 8.3. Some examples are shown in Table 8.4.
The replacement of the tetrahydroquinoline ring (28) with a tetrahydrobenzazepine
ring (32) enhanced the binding affinity for both V1a and V2 receptors. There is
no substantial difference in the binding affinity between 2-Me and 2-Cl (32 and 33)
on the terminal phenyl ring. The introduction of a nitrogen atom at the 5-position
of the tetrahydrobenzazepine ring (34) did not alter the binding affinity for the V2
receptor, but slightly lowered that for the V1a receptor. The introduction of a nitrogen
atom at the 4-position (35) lowered the binding affinity for both V1a and V2
receptors. The difference in binding affinity by introducing a nitrogen atom on the
Table 8.4 SAR studies of the heteroaromatic ring of compound 28.
X CO NHCO
Y
No. X Y Receptor affinity IC50 (mM)
V1a V2
28
N
Me 1.4 0.20
32
N
Me 0.056 0.018
33
N
Cl 0.045 0.029
34
N
N
Me 0.38 0.014
35
N
N
Me 1.8 0.17
36
N
Me 0.41 0.028
194j8 Discovery of Nonpeptide Vasopressin V2 Receptor Antagonists
benzazepine ring either at the 4- or at the 5-position is due to the difference in steric
size of a substituent at the 4- or 5-position or as a difference in basicity of a nitrogen
atom. The replacement of the tetrahydroquinoline ring with a hexahydrobenzazocine
ring (36) enhanced the binding affinity for both V1a and V2 receptors [28].
3D structures of compounds 28, 32, and 36 were generated using MOE [29] as a
stable conformation with the default setting (Figure 8.7). The only difference among
these three structures was the dihedral angle of the amide linkage between the heteroaromatic
ring and the benzoyl C¼O moiety. Two phenyl rings beside the amide linkage
in 28 are in a trans conformation; however, those in 32 and 36 are in a cis
conformation. Therefore, this cis conformation of two phenyl rings seems to be important
to show potent affinity for both V1a and V2 receptors. Furthermore, the superposed
structure of compounds 7 and 21 (Figure 8.6) appeared to show a cis
conformation in the two phenyl rings of compound 21 and these are similar to the 3D
structure of compound 32.
Although compounds 32, 33, 34, and 36 showed potent binding affinity for the V2
receptor, these compounds failed to demonstrate oral diuretic activity. On the other
hand, at a dose of 100mg/kg compound 35 showed weak oral diuretic activity, with
a five- to sixfold increase in urine volume observed for 4 h in normally hydrated
conscious rats over the control rats, while the binding affinity for the V2 receptor
was moderate. These results suggested that a basic amino group at the 4-position in
the 1,4-benzodiazepine ring (35) helped to increase oral bioavailability [28].
Based on this finding, a series of compounds were designed with a basic amino
group at the 5-position of the benzazepine scaffold to enhance oral bioavailability
(Figure 8.8).
The introduction of an NH2 group at the 5-position (37) gave only a slight reduction
in the binding affinity for the V2 receptor, and the introduction of an
NH-methyl group (38) gave a binding affinity similar to that of compound 37
Figure 8.7 3D structures of 28, 32, and 36.
8.4 Lead Generation Strategy-2, V2 Receptor Affinity j195
(Table 8.5). This single methyl group dramatically enhanced in vivo efficacy, and an
eightfold increase in urinary output was observed relative to the control rats. NHethyl
(39) and NH-propyl (40) groups showed the same magnitude of binding affinity
for the V2 receptor with moderate diuretic activity by oral administration,
though these showed a slight reduction in the V1a binding affinity. While the introduction
of an N,N-dimethyl group (42) did not alter the binding affinity for V1a and
V2 receptors relative to NH-methyl (38), it provided very potent diuretic efficacy by
oral administration (ED3 ¼ 3.8 mg/kg). The in vivo efficacy and the binding affinity
N
N
O
N
H
O
N
O
N
H
O
R3
NR1
R2
35
Figure 8.8 Design of orally active compounds.
Table 8.5 SAR studies of the substituent at the 5-position.
N CO NHCO
X
Y
No. X Y IC50 (mM) In vivo
V1a V2 UV (ml)
37 NH2 2-Me 0.39 0.032
38 NHMe 2-Me 0.72 0.024 8.3 Æ 2.5 (4.6)
39 NHEt 2-Me 1.6 0.029 5.4 Æ 0.6 (8.0)
40 NHnPr 2-Me 0.89 0.050 5.6 Æ 1.0 (6.6)
41 NMe2 H 3.0 0.027 (>30)
42 NMe2 2-Me 1.4 0.012 12.3 Æ 2.3 (ED3 ¼ 3.8)
43 NMe2 3-Me 2.4 0.014 (>30)
44 NMe2 4-Me 26 0.044
45 NMe2 2-Cl 3.0 0.027 7.0 Æ 0.5 (4.2)
46 N(Me)nPr 2-Me 1.2 0.022 8.3 Æ 1.8 (4.9)
47 OH 2-Me 0.14 0.029 3.6 Æ 0.8
UV values mean 2 h urine volume (ml) when the test compounds were administrated orally at a dose
of 10 mg/kg and are expressed as mean Æ SEM (n ¼ 4). The mean value of 2 h urine volume of control
rats was 1.1 Æ 0.2 ml (n ¼ 4). Values in parentheses indicate the ED3 value. ED3 represents the dose
(mg/kg) required for a threefold increase in the 2 h urine volume over the control rats. Values such as
>30 designate that at this dose (mg/kg) a threefold increase in the 2 h urine volume was not observed
when compared with that of the control rats.
196j8 Discovery of Nonpeptide Vasopressin V2 Receptor Antagonists
for the V1a receptor were dramatically influenced by substitution on the terminal
phenyl ring (41, 43, 44, and 45). N-Methyl-N-propyl derivative (46) showed better in
vivo efficacy than NH-propyl derivative (40). The introduction of a hydroxyl group at
the 5-position (47) did not alter the binding affinity, but it did not provide the potent
in vivo efficacy of the 5-amino derivatives [28].
Compound 42 (Figure 8.9) was selected for further evaluation. This compound
increased urine flow and decreased urine osmolality after oral administration
(1–30 mg/kg) in normal conscious rats [30–32]. Compound 42 (mozavaptan,
OPC-31260) is a selective V2 receptor antagonist that acts as an aquaretic agent.
The binding affinities of 42 at V1a and V2 receptors are as follows: Ki ¼ 9.42 Æ 0.90
nM (human V2), 150 Æ 15 nM (human V1a), 6.36 Æ 1.56 nM (rat V2), and
524 Æ 119 nM (rat V1a). No meaningful differences were found in the binding
affinities between human and rat receptors.
Physuline1
(mozavaptan hydrochloride) was approved in Japan in October 2006
to treat paraneoplastic SIADH (the syndrome of inappropriate antidiuretic
hormone).
8.5
Lead Optimization [33]
Further optimization was performed on R1, R2, and R3 to enhance in vitro and
in vivo efficacy (Table 8.6). The introduction of a 7-Cl group (48 versus 42) enhanced
V1a affinity and also enhanced in vivo efficacy. Replacement of the 5-dimethylamino
group with a 5-monomethylamino group slightly enhanced the binding affinity for
both V1a and V2 receptors (48 versus 49). 5-OH (50) and 5-OMe (51) showed potent
affinity for both the V1a and V2 receptors. The steric size at the 5-position of the
benzazepine ring might affect binding affinity at V1a as well as V2 receptor.
Replacement of the 5-amino group with 5-OH reduced the in vivo efficacy.
It was very interesting that the introduction of a 2-Cl group as R3 potentiated the
oral efficacy (52), while water solubility was poor. The same enhancement was seen
with a 2-Me group (54) as well as a 2-OMe group (55) at R3. A 3-OMe group (56)
enhanced the binding affinity to both V1a and V2 receptors, but the oral efficacy
appeared to be weak [33].
N
N
CH3
H3C
O
N
H
OCH3
42
Physuline®
(mozavaptan hydrochloride)
(OPC-31260)
HCl
Figure 8.9 Chemical structure of physuline.
8.5 Lead Optimization j197
Compound 54 (tolvaptan, Figure 8.10) was selected for advanced in vivo studies
and clinical trials, and it showed a selective V2 receptor affinity relative to the V1a
receptor and a potent in vivo efficacy.
Binding affinities for both V1a and V2 receptors are as follows: Ki ¼ 0.43 Æ
0.06 nM (human V2), 12.3 Æ 0.8 nM (human V1a), 1.33 Æ 0.30 nM (rat V2), and
325 Æ 41 nM (rat V1a) [34]. Tolvaptan showed potent water diuretic activity in various
animal models as well as in clinical trials [35, 36].
The US Food and Drug Administration (FDA) approved Samsca1
(tolvaptan) as
the only oral selective vasopressin antagonist for the treatment of hypervolemic and
euvolemic hyponatremia on May 21, 2009, in patients with heart failure, cirrhosis,
and the syndrome of inappropriate antidiuretic hormone. The European
Table 8.6 Optimization of benzazepine analogues.
N
R2
CO NHCO
Me
R1
R3
2 3
No. R1 R2 R3 Receptor affinity
IC50 (mM)
In vivo
V1a V2 UV (ml) ED3
42 H NMe2 H 1.4 0.012 12.3 3.8
48 Cl NMe2 H 0.19 0.025 14.6 1.5
49 Cl NHMe H 0.064 0.007 15.5 1.4
50 Cl OH H 0.017 0.003 7.2
51 Cl OMe H 0.034 0.005 6.3
52 Cl OH 2-Cl 0.29 0.008 16.8 1.6
53 Cl OH 3-Cl 0.031 0.028 3.0
54 Cl OH 2-Me 0.58 0.003 17.3 0.54
55 Cl OH 2-OMe 0.039 0.013 15.2 1.4
56 Cl OH 3-OMe 0.007 0.005 3.5
N
O
NH
OCH3
54
Samsca®
(tolvaptan)
(OPC-41061)CH3
Cl
OH
Figure 8.10 Chemical structure of samsca.
198j8 Discovery of Nonpeptide Vasopressin V2 Receptor Antagonists
Commission approved the Marketing Authorization Application for the oral oncedaily
medication Samsca1
for the treatment of hyponatremia secondary to SIADH
in adults in August 2009, and the Japanese Ministry of Health, Labour and Welfare
(MHLW) approved Samsca1
for the treatment of excess water retention in patients
with cardiac failure, when the treatment by other diuretics including loop diuretics
is ineffective, in October 2010.
8.6
Reported Nonpeptide Vasopressin V2 Receptor Antagonist Compounds
8.6.1
Sanofi
Sanofi reported the nonpeptide V1a receptor selective antagonist, SR 49059 (relcovaptan,
Figure 8.11), in 1993 [37]. They intensively modified a dihydroindole as an
original scaffold and reported SR 121463 (satavaptan) as a nonpeptide V2 receptor
selective antagonist in 1996 [38], and SSR 149415 (nelivaptan) was reported as a
nonpeptide V1b receptor antagonist in 2002 [39].
8.6.2
Astellas (Yamanouchi)
OPC-31260 was reported to be an orally active nonpeptide V2 receptor antagonist
in 1992 and in 1996, and Yamanouchi reported nonselective V1a and V2
receptor antagonists with tetrahydrobenzazepine as a scaffold (Figure 8.12, 57)
in 1997 [40]. The benzodiazepine analogues (58 and 59) [41], 5-exo-olefinsubstituted
tetrahydrobenzazepine derivative (60) [42], 4,5-imidazole-substituted
analogue (61, YM 087, conivaptan) [43], and 4,4-difluoro-5-exo-olefin-
N
Cl
S OO
HO
H
Cl
O
N
O
NH2
O
O
N
O
S OO
O
O
N
O
O
O
N
H
N
Cl
SO O
O
Cl
N
O NH2
O
O
OH
Relcovaptan
(SR 49059)
Satavaptan
(SR 121463)
Nelivaptan
(SSR 149415)
Figure 8.11 Reported compounds from Sanofi.
8.6 Reported Nonpeptide Vasopressin V2 Receptor Antagonist Compounds j199
200j8 Discovery of Nonpeptide Vasopressin V2 Receptor Antagonists
N
CO NHCO
CH3
N
N
CO NHCO
N
N
N
CO NHCO
O
N N CH3
N
CO NHCO
O
N
H
CH3
N
CO NHCO
NHN
CH3
N
CH3
O
CO NHCO
N
H3C
N
O
N CH3
N
N
N
N
CH3
O
N
O
N
N
N
CH3
O
N
N
CH3
N
CO NHCO
O
O
N N
CH3
CH3F
F
N
CO NHCO
N
O
NH
F
F
N N
CO NHCO
O
N N
F
F
Cl
O
CH3
57 58 59 60
61 (Conivaptan, YM 087, Vaprisol®
)
63 64 65
66 67 68 (YM-218)
N
CO NHCO
O
N N
F
F
CH3
CH3
62
Figure 8.12 Reported compounds from Astellas.
substituted analogue (62) [44] were reported as nonselective antagonists. Noncyclized
benzamide derivative (63) [45] was derived from reported compounds
and reported to show potent V1a receptor affinity. Yamanouchi reported triazole
derivatives (64 and 65) [46, 47] to show potent V1a receptor affinity in 2002,
and these compounds were derived from a compound identified by internal
high-throughput screening efforts. Some modifications of the amino group at
the 5-position of the benzazepine ring as well as terminal benzoyl or heteroaromatic
carbonyl groups were reported to show potent V1a receptor antagonist
activity (66, 67, and 68) [48–50]. Vaprisol1
(conivaptan hydrochloride injection)
was approved by the FDA in 2005 for the treatment of euvolemic hyponatremia
in hospitalized patients.
8.6.3
Wyeth
Wyeth reported benzopyrrolo[1,4]diazepine derivatives as a nonpeptide V2 receptor
selective antagonist (Figure 8.13, 69, lixivaptan) in 1998 [51]. In addition, some
combinations of the end group, middle aromatic ring, and terminal phenyl ring
were reported (70, 71, and 72) [52–54]. Compound 73 (WAY-140288) was reported
in 2000 to show a good profile in parenteral formulation [55].
8.6.4
Johnson & Johnson
Johnson & Johnson also intensively reported modification of the top benzohetero
ring (Figure 8.14; 74, 75, and 76) [56–58]. An optically pure analogue with a substituted
oxazine ring was reported to show a potent affinity to the V2 receptor in 2004
(77, RWJ-351647) [59]. Another group in Johnson & Johnson reported compounds
N
N
CO NHCO
CH3
69 (VPA-985, Lixivaptan) 70 71 (CL-385004)
72 73 (WAY-140288)
FCl
N
CO NHCO
CH3
S
FCl
N
N
CO
N
NHCO
CH3
F
N
N
CO NHCO
Cl
N
N
CO NHCO
O
N
Figure 8.13 Reported compounds from Wyeth.
8.6 Reported Nonpeptide Vasopressin V2 Receptor Antagonist Compounds j201
having a spirocyclopentene ring at the 4-position of the benzazepine ring (78) in
2004 [60]. This compound showed potent affinity for both V1a and V2 receptors.
They reported the same class of compound in optically pure form in 2007 (79,
RWJ-676070) as a balanced vasopressin V1a/V2 antagonist [61].
8.6.5
Wakamoto Pharmaceutical Co. Ltd [62]
Some analogues with a benzohetero ring as an end group were reported in 1999 as
V2 receptor selective antagonists (Figure 8.15).
8.6.6
Japan Tobacco Inc.
Japan Tobacco reported a thienoazepine derivative (83, JTV-605) as a potent V1a and
V2 antagonist with a long duration of action in 2004 (Figure 8.16) [63].
N
N
CO NHCO
CH3
74 75 76
77 (RWJ-351647) 78
Cl
N
CO NHCO
N
S
CO NHCO
N
N
CO NHCO
Cl
N
CO NHCO
HN CO2H
O
H
N
CO NHCO
CH3O
H
N
O
N
F
CO2H
79 (RWJ-676070)
Figure 8.14 Reported compounds from Johnson & Johnson.
80 (VP-343)
N
N
CO NHCO
N
N
CO NHCO
O
N
CO NHCO
N
CH3
H
OH
H H
81 (VP-365) 82 (VP-339)
Figure 8.15 Reported compounds from Wakamoto.
202j8 Discovery of Nonpeptide Vasopressin V2 Receptor Antagonists
8.7
Conclusions
The discovery of aquaretics, water diuretics, to treat chronic heart failure has led to
numerous findings demonstrating that the biological effects of AVPs are mediated
through three receptor subtypes. A major contribution was the discovery of nonpeptide
compounds that show high selectivity to receptors, and good oral bioavailability
to permit repeated administration. In addition, the discovery of orally
effective nonpeptide AVP receptor antagonists led to the discovery of nonpeptide
oxytocin receptor antagonists and agonists [64].
X-ray analyses of some G-protein-coupled receptors have been reported [65–67].
More detailed consideration of the receptor–ligand interactions will be conducted
using the 3D structures of receptors, and this will lead to the discovery of ligands
with higher diversity in structure.
The efforts to discover nonpeptide vasopressin receptor modulators are still
not finished, because the physiological roles of AVP are still under investigation.
These efforts to find better compounds are therefore expected to be beneficial
for patients.
References
1 Du Vigneaud, V., Ressler, C., Swan, J.M.,
Roberts, C.W., and Katsoyannis, P.G.
(1954) The synthesis of oxytocin. J. Am.
Chem. Soc., 76, 3115–3121.
2 Du Vigneaud, V., Gish, D.T., and
Katsoyannis, P.G. (1954) A synthetic
preparation possessing biological
properties associated with argininevasopressin.
J. Am. Chem. Soc., 76,
4751–4752.
3 Manning, M., Lammek, B., Kolodziejczyk,
A.M., Seto, J., and Sawyer, W.H. (1981)
Synthetic antagonists of in vivo
antidiuretic and vasopressor responses to
arginine-vasopressin. J. Med. Chem., 24,
701–706.
4 Manning, M., Olma, A., Klis, W.A.,
Kolodziejczyk, A.M., Seto, J., and Sawyer,
W.H. (1982) Design of more potent
antagonists of the antidiuretic responses
to arginine-vasopressin. J. Med. Chem., 25,
45–50.
5 Manning, M., Klis, W.A., Olma, A., Seto,
J., and Sawyer, W.H. (1982) Design of
more potent and selective antagonists of
the antidiuretic responses to argininevasopressin
devoid of antidiuretic
agonism. J. Med. Chem., 25, 414–419.
N
CO NHCO
83 (JTV-605)
S
O
N
O
N
HCl
Figure 8.16 Reported compound from Japan Tabacco.
References j203
6 Sawyer, W.H., Pang, P.K.T., Seto, J.,
McEnroe, M., Lammek, B., and Manning,
M. (1981) Vasopressin analogs that
antagonize antidiuretic responses by rats
to the antidiuretic hormone. Science, 212,
49–51.
7 Stassen, F.L., Erickson, R.W., Huffman,
W.F., Stefankiewicz, J., Sulat, L., and
Wiebelhaus, V.D. (1982) Molecular
mechanisms of novel antidiuretic
antagonists: analysis of the effects on
vasopressin binding and adenylate cyclase
activation in animal and human kidney.
J. Pharmacol. Exp. Ther., 223, 50–54.
8 Stassen, F.L., Bryan, W., Gross, M.,
Kavanagh, B., Shue, D., Sulat, L.,
Wiebelhaus, V.D., Yim, N., and Kinter,
L.B. (1983) Critical differences between
species in in vivo and in vitro responses to
antidiuretic hormone antagonists. Prog.
Brain Res., 60, 395–403.
9 Stassen, F.L., Berkowitz, B.B., Huffman,
W.F., Wiebelhaus, V.D., and Kinter, L.B.
(1984) Molecular pharmacology and
mechanisms of action of aquaretic agents,
in Diuretics (ed. J.B. Puschett), Elsevier
Science Publishing Co., Inc., New York,
pp. 64–71.
10 Stassen, F.L., Heckman, G.D., Schmidt,
D.B., Stefankiewicz, J., Sulat, L.,
Huffman, W.F., Moore, M.M., and Kinter,
L.B. (1985) Actions of vasopressin
antagonists: molecular mechanisms, in
Vasopressin (ed. R.W. Schrier), Raven
Press, New York, pp. 145–154.
11 Kinter, L.B., Dubb, J., Huffman, F.L.,
Brennan, F., and Stassen, F.L. (1985)
Potential role of vasopressin antagonists
in treatment of water-retaining disorders,
in Vasopressin (ed. R.W. Schrier), Raven
Press, New York, pp. 553–561.
12 Kinter, L.B., Churchill, S., Stassen, F.L.,
Moore, M., and Huffman, W. (1987)
Characterization and mechanism of
action of vasopressin antagonists in
squirrel monkeys (Saimiri sciureus).
J. Pharmacol. Exp. Ther., 241, 797–803.
13 Manning, M., Olma, A., Klis, W.,
Kolodziejczyk, A.M., Nawrocka, E.,
Misicka, A., Seto, J., and Sawyer, W.H.
(1984) Carboxy terminus of vasopressin
required for activity but not binding.
Nature, 308, 652–653.
14 Kinter, L.B., Mann, W.A., Woodward, P.,
DePalma, D., and Brennan, F. (1987)
The vasopressin antagonist SK&F
101926 is a selective aquaretic agent in
dogs, in Diuretics: Basic,
Pharmacological, and Clinical Aspects
(eds V.E. Andreucci and A. Dal Canton),
Proceedings of the International
Meeting on Diuretics, Martinus Nijhoff,
Boston, MA, pp. 218–221.
15 Dubb, J., Allison, N., Tatoian, D.,
Blumberg, A., Lee, K., and Stote, R. (1987)
SK&F 101926 is antidiuretic in man.
Kidney Int., 31, 267.
16 Brooks, D.P., Koster, P.F., AlbrightsonWinslow,
C.R., Stassen, F.L., Huffman,
W.F., and Kinter, L.B. (1988) SK&F 105494
is a antidiuretic hormone antagonist in
the rhesus monkey (Macaca mulatta).
J. Pharmacol. Exp. Ther., 245, 211–215.
17 Caldwell, N., Brickson, B., Kinter, L.B.,
Brooks, D.P., Huffman., W.F., Stassen,
F.L., and Albrightson-Winslow, C. (1988)
SK&F 105494: a potent antidiuretic
hormone antagonist devoid of partial
agonist activity in dogs. J. Pharmacol. Exp.
Ther., 247, 897–901.
18 Brooks, D.P., Koster, P.F., Albrightson,
C.R., Huffman, W.F., Moore, M.L.,
Stassen, F.L., Schmidt, D.B., and Kinter,
L.B. (1989) Vasopressin receptor
antagonism in rhesus monkey and man:
stereochemical requirements. Eur. J.
Pharmacol., 160, 159–162.
19 Ruffolo, R.R., Jr., Brooks, D.P., Huffman,
W.F., and Poste, G. (1991) From
vasopressin antagonist to agonist: a saga
of surprise! Drug News Perspect., 4,
217–222.
20 Ogawa, H., Yamamura, Y., Miyamoto, H.,
Kondo, K., Yamashita, H., Nakaya, K.,
Chihara, T., Mori, T., Tominaga, M., and
Yabuuchi, Y. (1993) Orally active,
nonpeptide vasopressin V1 antagonists.
A novel series of 1-(1-substituted 4-
piperidyl)-3,4-dihydro-2(1H)-quinolinone.
J. Med. Chem., 36, 2011–2017.
21 Kondo, K., Ogawa, H., Nakaya, K.,
Tominaga, M., and Yabuuchi, Y. (1996)
Structure–activity relationships of nonpeptide
vasopressin V1a antagonists:
1-(1-multi-substituted benzoyl 4-
piperidyl)-3,4-dihydro-2(1H)-
204j8 Discovery of Nonpeptide Vasopressin V2 Receptor Antagonists
quinolinones. Chem. Pharm. Bull., 44,
725–733.
22 Yamamura, Y., Ogawa, H., Chihara, T.,
Kondo, K., Onogawa, T., Nakamura, S.,
Mori, T., Tominaga, M., and Yabuuchi, Y.
(1991) OPC-21268, an orally effective,
nonpeptide vasopressin V1 receptor
antagonist. Science, 252, 572–574.
23 Pettibone, D.J., Kishel, M.T., Woyden,
C.J., Clineschmidt, B.V., Bock, M.G.,
Freidinger, R.M., Veber, D.F., and
Williams, P.D. (1992) Radioligand
binding studies reveal marked species
differences in the vasopressin V1 receptor
of rat, rhesus and human tissues. Life Sci.,
50, 1953–1958.
24 Serradeil-Le Gal, C., Wagnon, J., Garcia,
C., Lacour, C., Guiraudou, P., Christophe,
B., Villanova, G., Nisato, D., Maffrand,
J.P., and Le Fur, G. (1993) Biochemical
and pharmacological properties of SR
49059, a new, potent, nonpeptide
antagonist of rat and human vasopressin
V1a receptors. J. Clin. Invest., 92, 224–231.
25 Serradeil-Le Gal, C., Raufaste, D., Marty,
E., Garcia, C., Maffrand, J.P., and Le Fur,
G. (1994) Binding of ½3
HŠ SR 49059, a
potent nonpeptide vasopressin V1a
antagonist, to rat and human liver
membranes. Biochem. Biophys. Res.
Commun., 199, 353–360.
26 Hirasawa, A., Shibata, K., Kotosai, K., and
Tsujimoto, G. (1994) Cloning, functional
expression and tissue distribution of
human cDNA for the vascular-type
vasopressin receptor. Biochem. Biophys.
Res. Commun., 203, 72–79.
27 Shinoura, H., Take, H., Itoh, S., Hirasawa,
A., Inoue, K., Ohno, Y., Hashimoto, K.,
and Tsujimoto, G. (2000) Key amino acids
of vasopressin V1a receptor responsible for
the species difference in the affinity of
OPC-21268. FEBS Lett., 466, 255–258.
Erratum in FEBS Lett., 474, 257 (2000).
28 Ogawa, H., Yamashita, H., Kondo, K.,
Yamamura, Y., Miyamoto, H., Kan, K.,
Kitano, K., Tanaka, M., Nakaya, K.,
Nakamura, S., Mori, T., Tominaga, M.,
and Yabuuchi, Y. (1996) Orally active,
nonpeptide vasopressin V2 receptor
antagonists: a novel series of 1-[4-
(benzoylamino)benzoyl]-2,3,4,5tetrahydro-1H-benzazepines
and related
compounds. J. Med. Chem., 39,
3547–3555.
29 Chemical Computing Group, MOE:
Molecular Operating Environment, Chemical
Computing Group, Montreal, Canada,
current version, MOE2011.10.
30 Yamamura, Y., Ogawa, H., Yamashita, H.,
Chihara, T., Miyamoto, H., Nakamura, S.,
Onogawa, T., Yamashita, T., Hosokawa, T.,
Mori, T., Tominaga, M., and Yabuuchi, Y.
(1992) Characterization of a novel aquaretic
agent, OPC-31260, as an orally effective,
nonpeptide vasopressin V2 receptor
antagonist. Br. J. Pharmacol., 105, 787–791.
31 Ohnishi, A., Orita, Y., Okahara, R.,
Fujihara, H., Inoue, T., Yamamura, Y.,
Yabuuchi, Y., and Tanaka, T. (1993)
Potent aquaretic agent. J. Clin. Invest.,
92, 2653–2659.
32 Ohnishi, A., Orita, Y., Takagi, N., Fujita, T.,
Toyoki, T., Ihara, Y., Yamamura, Y.,
Inoue, T., and Tanaka, T. (1995) Aquaretic
effect of a potent, orally active, nonpeptide
V2 antagonist in men. J. Pharmacol. Exp.
Ther., 272, 546–551.
33 Kondo, K., Ogawa, H., Yamashita, H.,
Miyamoto, H., Tanaka, M., Nakaya, K.,
Kitano, K., Yamamura, Y., Nakamura,
S., Onogawa, T., Mori, T., and
Tominaga, M. (1999) 7-Chloro-5-
hydroxy-1-[2-methyl-4-(2-
methylbenzoylamino)benzoyl]-2,3,4,5tetrahydro-1H-1-benzazepine
(OPC-
41061): a potent, orally active
nonpeptide arginine vasopressin V2
receptor antagonist. Bioorg. Med. Chem.,
7, 1743–1754.
34 Yamamura, Y., Nakamura, S., Ito, S.,
Hirano, T., Onogawa, T., Yamashita, T.,
Yamada, Y., Tsujimae, K., Aoyama, M.,
Kotosai, K., Ogawa, H., Yamashita, H.,
Kondo, K., Tominaga, M., Tsujimoto, G.,
and Mori, T. (1998) OPC-41061, a potent
human vasopressin V2-receptor
antagonist: pharmacological profile and
aquaretic effect by single and multiple oral
dosing in rats. J. Pharmacol. Exp. Ther.,
287, 860–867.
35 Ohnishi, A., Orita, Y., Okahara, R.,
Fujihara, H., Inoue, T., Yamamura, Y.,
Yabuuchi, Y., and Tanaka, T. (1993)
Potent aquaretic agent. J. Clin. Invest.,
92, 2653–2659.
References j205
36 Ohnishi, A., Orita, Y., Takagi, N., Fujita, T.,
Toyoki, T., Ihara, Y., Yamamura, Y., Inoue,
T., and Tanaka, T. (1995) Aquaretic effect
of a potent, orally active, nonpeptide V2
antagonist in men. J. Pharmacol. Exp.
Ther., 272, 546–551.
37 Serradeil-Le Gal, C., Wagnon, J., Garcia,
C., Lacour, C., Guiraudou, P., Christophe,
B., Villanova, G., Nisato, D., Maffrand,
J.P., Le Fur, G., Guillon, G., Cantau, B.,
Barberis, C., Trueba, M., Ala, Y., and Jard,
S. (1993) Biochemical and
pharmacological properties of SR 49059, a
new, potent, nonpeptide antagonist of rat
and human vasopressin V1a receptors.
J. Clin. Invest., 92, 224–231.
38 Serradeil-Le Gal, C., Lacour, C., Valette, G.,
Garcia, G., Foulon, L., Galindo, G., Bankir,
L., Pouzet, B., Guillon, G., Barberis, C.,
Chicot, D., Jard, S., Vilain, P., Garcia, C.,
Marty, E., Raufaste, D., Brossard, G.,
Nisato, D., Maffrand, J.P., and Le Fur, G.
(1996) Characterization of SR 121463A,
a highly potent and selective, orally active
vasopressin V2 receptor antagonist. J. Clin.
Invest., 98, 2729–2738.
39 Serradeil-Le Gal, C., Wagnon, J.,
Simiand, J., Griebel, G., Lacour, C.,
Guillon, G., Barberis, C., Brossard, G.,
Soubrie, P., Nisato, D., Pascal, M., Pruss,
R., Scatton, B., Maffrand, J.P., and Le Fur,
G. (2002) Characterization of (2S,4R)-1-[5-
chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-
3-(2-methoxy-phenyl)-2-oxo-2,3-dihydro-
1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2pyrrolidine
carboxamide (SSR149415), a
selective and orally active vasopressin V1b
receptor antagonist. J. Pharmacol. Exp.
Ther., 300, 1122–1130.
40 Matsuhisa, A., Tanaka, A., Kukuchi, K.,
Shimada, Y., Yatsu, T., and Yanagisawa, I.
(1997) Nonpeptide arginine vasopressin
antagonists for both V1a and V2
receptors: synthesis and pharmacological
properties of 2-phenyl-40
-[(2,3,4,5-
tetrahydro-1H-1-benzazepin-1-yl)
carbonyl]benzanilide derivatives. Chem.
Pharm. Bull., 45, 1870–1874.
41 Matsuhisa, A., Koshio, H., Sakamoto,
K., Taniguchi, N., Yatsu, T., and
Tanaka, A. (1998) Nonpeptide arginine
vasopressin antagonists for both V1a
and V2 receptors: synthesis and
pharmacological properties of 2-phenyl-
40
-(2,3,4,5-tetrahydro-1H-1,5-
benzodiazepine-1-carbonyl)benzanilide
derivatives. Chem. Pharm. Bull., 46,
1566–1579.
42 Matsuhisa, A., Kikuchi, K., Sakamoto, K.,
Yatsu, T., and Tanaka, A. (1999)
Nonpeptide arginine vasopressin
antagonists for both V1a and V2 receptors:
synthesis and pharmacological properties
of 40
-[5-(substituted methylidene)-2,3,4,5-
tetrahydro-1H-1-benzoazepine-1-carbonyl]
benzanilide and 40
-[5-(substituted methyl)-
2,3-dihydro-1H-1-benzoazepine-1carbonyl]benzanilide
derivatives. Chem.
Pharm. Bull., 47, 329–339.
43 Matsuhisa, A., Taniguchi, N., Koshio, H.,
Yatsu, T., and Tanaka, A. (2000)
Nonpeptide arginine vasopressin
antagonists for both V1a and V2 receptors:
synthesis and pharmacological properties
of 40
-(1,4,5,6-tetrahydroimidazo[4,5-d][1]
benzoazepine-6-carbonyl)benzanilide
derivatives and 40
-(5,6-dihydro-4H-
thiazolo[5,4-d][1]benzoazepine-6-carbonyl)
benzanilide derivatives. Chem. Pharm.
Bull., 48, 21–31.
44 Shimada, Y., Taniguchi, N., Matsuhisa, A.,
Sakamoto, K., Yatsu, T., and Tanaka, A.
(2000) Highly potent and orally active nonpeptide
arginine vasopressin antagonists
for both V1a and V2 receptors: synthesis
and pharmacological properties of 40
-[(4,4-
difluoro-5-methylidene-2,3,4,5-tetrahydro-
1H-1-benzoazepin-1-yl)carbonyl]-2phenylbenzanilide
derivatives. Chem.
Pharm. Bull., 48, 1644–1651.
45 Kakefuda, A., Tsukada, J., Kusayama, T.,
Tahara, A., and Tsukamoto, S. (2002)
N-Methylbenzanilide derivatives as a
novel class of selective V1a receptor
antagonists. Bioorg. Med. Chem. Lett., 12,
229–232.
46 Kakefuda, A., Suzuki, T., Tobe, T., Tahara,
A., Sakamoto, S., and Tsukamoto, S. (2002)
Discovery of 4,5-diphenyl-1,2,4-triazole
derivatives as a novel class of selective
antagonists for the human V1a receptor.
Bioorg. Med. Chem., 10, 1905–1912.
47 Kakefuda, A., Suzuki, T., Tobe, T.,
Tsukada, J., Tahara, A., Sakamoto, S., and
Tsukamoto, S. (2002) Synthesis and
pharmacological evaluation of 5-(4-
206j8 Discovery of Nonpeptide Vasopressin V2 Receptor Antagonists
biphenyl)-3-methyl-4-phenyl-1,2,4-triazole
derivatives as a novel class of selective
antagonists for the human vasopressin
V1a receptor. J. Med. Chem., 45,
2589–2598.
48 Shimada, Y., Taniguchi, N., Matsuhisa, A.,
Yatsu, T., Tahara, A., and Tanaka, A. (2003)
Preparation of non-peptide, highly potent
and selective antagonists of arginine
vasopressin V1a receptor by introduction
of alkoxy groups. Chem. Pharm. Bull., 51,
1075–1080.
49 Shimada, Y., Akane, H., Taniguchi, N.,
Matsuhisa, A., Kawano, N., Kikuchi, K.,
Yatsu, T., Tahara, A., Tomura, Y.,
Kusayama, T., Wada, K., Tsukada, J.,
Tsunoda, T., and Tanaka, A. (2005)
Preparation of highly potent and selective
non-peptide antagonists of the arginine
vasopressin V1a receptor by introduction
of a 2-ethyl-1H-1-imidazolyl group. Chem.
Pharm. Bull., 53, 764–769.
50 Shimada, Y., Taniguchi, N., Matsuhisa, A.,
Akane, H., Kawano, N., Suzuki, T., Tobe, T.,
Kakefuda, A., Yatsu, T., Tahara, A., Tomura,
Y., Kusayama, T., Wada, K., Tsukada, J.,
Orita, M., Tsunoda, T., and Tanaka, A.
(2006) Synthesis and biological activity of
novel 4,4-difluorobenzazepine derivatives
as non-peptide antagonists of the arginine
vasopressin V1a receptor. Bioorg. Med.
Chem., 14, 1827–1837.
51 Albright, J.D., Reich, M.F., Delos Santos,
E.G., Dusza, J.P., Sum, F.-W., Venkatesan,
A.M., Coupet, J., Chan, P.S., Ru, X.,
Mazandarani, H., and Bailey, T. (1998) 5-
Fluoro-2-methyl-N-[4-(5H-pyrrolo[2,1-c]-
[1,4]benzodiazepin-10(11H)-ylcarbonyl)-3chlorophenyl]benzamide
(VPA-985): an
orally active arginine vasopressin
antagonist with selectivity for V2
receptors. J. Med. Chem., 41, 2442–2444.
52 Aranapakam, V., Albright, J.D., Grosu, G.
T., Chan, P.S., Coupet, J., Saunders, T.,
Ru, X., and Mazandarani, H. (1999) 4,10-
Dihydro-5H-thieno[3,2-c][1]benzazepine
derivatives and 9,10-dihydro-4H-thieno
[2,3-c][1]benzazepine derivatives as orally
active arginine vasopressin receptor
antagonists. Bioorg. Med. Chem. Lett., 9,
1733–1736.
53 Aranapakam, V., Albright, J.D., Grosu,
G.T., Delos Santos, E.G., Chan, P.S.,
Coupet, J., Ru, X., Saunders, T., and
Mazandarani, H. (1999) 5-Fluoro-2-
methyl-N-[5-(5H-pyrrolo[2,1-c][1,4]
benzodiazepine-10(11H)-yl carbonyl)-2pyridinyl]benzamide
(CL-385004) and
analogs as orally active arginine
vasopressin receptor antagonists. Bioorg.
Med. Chem. Lett., 9, 1737–1740.
54 Albright, J.D., Delos Santos, E.G., Dusza,
J.P., Chan, P.S., Coupet, J., Ru, X., and
Mazandarani, H. (2000) The synthesis and
vasopressin (AVP) antagonist activity of a
novel series of N-aroyl-2,4,5,6-
tetrahydropyrazolo[3,4-d]thieno[3,2-b]
azepines. Bioorg. Med. Chem. Lett., 10,
695–698.
55 Ashwell, M.A., Bagli, J.F., Caggiano, T.J.,
Chan, P.S., Molinari, A.J., Palka, C., Park,
C.H., Rogers, J.F., Sherman, M.,
Trybulski, E.J., and Williams, D.K. (2000)
The design, synthesis and physical
chemical properties of novel human
vasopressin V2-receptor antagonists
optimized for parenteral delivery. Bioorg.
Med. Chem. Lett., 10, 783–786.
56 Dyatkin, A.B., Hoekstra, W.J., Hlasta, D.J.,
Andrade-Gordon, P., de Garavilla, L.,
Demarest, K.T., Gunnet, J.W., Hageman,
W., Look, R., and Maryanoff, B.E. (2002)
Bridged bicyclic vasopressin receptor
antagonists with V2-selective or dual
V1a/V2 activity. Bioorg. Med. Chem. Lett.,
12, 3081–3084.
57 Matthews, J.M., Greco, M.N., Hecker,
L.R., Hoekstra, W.J., Andrade-Gordon, P.,
de Garavilla, L., Demarest, K.T., Ericson,
E., Gunnet, J.W., Hageman, W., Look, R.,
Moore, J.B., and Maryanoff, B.E. (2003)
Synthesis and biological evaluation of
novel indoloazepine derivatives as nonpeptide
vasopressin V2 receptor
antagonists. Bioorg. Med. Chem. Lett., 13,
753–756.
58 Urbanski, M.J., Chen, R.H., Demarest,
K.T., Gunnet, J., Look, R., Ericson, E.,
Murray, W.V., Rybczynski, P.J., and
Zhang, X. (2003) 2,5-Disubstituted 3,4dihydro-2H-benzo[b][1,4]thiazepines
as
potent and selective V2 arginine
vasopressin receptor antagonists. Bioorg.
Med. Chem. Lett., 13, 4031–4034.
59 Matthews, J.M., Hoekstra, W.J., Dyatkin,
A.B., Hecker, L.R., Hlasta, D.J., Poulter,
References j207
B.L., Andrade-Gordon, P., de Garavilla, L.,
Demarest, K.T., Ericson, E., Gunnet, J.W.,
Hageman, W., Look, R., Moore, J.B.,
Reynolds, C.H., and Maryanoff, B.E.
(2004) Potent nonpeptide vasopressin
receptor antagonists based on oxazinoand
thiazinobenzodiazepine templates.
Bioorg. Med. Chem. Lett., 14, 2747–2752.
60 Xiang, M.A., Chen, R.H., Demarest, K.T.,
Gunnet, J., Look, R., Hageman, W.,
Murray, W.V., Combs, D.W., Rybczynski,
P.J., and Patel, M. (2004) Synthesis and
evaluation of nonpeptide substituted
spirobenzazepines as potent vasopressin
antagonists. Bioorg. Med. Chem. Lett., 14,
3143–3146.
61 Xiang, M.A., Rybczynski, P.J., Patel, M.,
Chen, R.H., McComsey, D.F., Zhang,
H.C., Gunnet, J.W., Look, R., Wang, Y.,
Minor, L.K., Zhong, H.M., Villani, F.J.,
Demarest, K.T., Damiano, B.P., and
Maryanoff, B.E. (2007) Next-generation
spirobenzazepines: identification of RWJ-
676070 as a balanced vasopressin V1a/V2
receptor antagonist for human clinical
studies. Bioorg. Med. Chem. Lett., 17,
6623–6628.
62 Ohtake, Y., Naito, A., Hasegawa, H.,
Kawano, K., Morizono, D., Taniguchi, M.,
Tanaka, Y., Matsukawa, H., Naito, K.,
Oguma, T., Ezure, Y., and Tsuriya, Y.
(1999) Novel vasopressin V2 receptorselective
antagonists, pyrrolo[2,1-a]
quinoxaline and pyrrolo[2,1-c][1,4]
benzodiazepine derivatives. Bioorg. Med.
Chem., 7, 247–254.
63 Cho, H., Murakami, K., Nakanishi, H.,
Fujisawa, A., Isoshima, H., Niwa, M.,
Hayakawa, K., Hase, Y., Uchida, I.,
Watanabe, H., Wakitani, K., and Aisaka, K.
(2004) Synthesis and structure–activity
relationships of 5,6,7,8-tetrahydro-4Hthieno[3,2-b]azepine
derivatives: novel
arginine vasopressin antagonists. J. Med.
Chem., 47, 101–109.
64 Bell, I.M., Erb, J.M., Freidinger, R.M.,
Gallicchio, S.N., Guare, J.P., Guidotti,
M.T., Halpin, R.A., Hobbs, D.W.,
Homnick, C.F., Kuo, M.S., Lis, E.V.,
Mathre, D.J., Michelson, S.R., Pawluczyk,
J.M., Pettibone, D.J., Reiss, D.R., Vickers,
S., Williams, P.D., and Woyden, C.J.
(1998) Development of orally active
oxytocin antagonists: studies on 1-(1-[4-[1-
(2-methyl-1-oxidopyridin-3-ylmethyl)
piperidin-4-yloxy]-2-methoxybenzoyl]
piperidin-4-yl)-1,4-dihydrobenz [d][1,3]
oxazin-2-one (L-372,662) and related
pyridines. J. Med. Chem., 41, 2146–2163.
65 Palczewski, K., Kumasaka, T., Hori, T.,
Behnke, C.A., Motoshima, H., Fox, B.A.,
Le Trong, I., Teller, D.C., Okada, T.,
Stenkamp, R.E., Yamamoto, M., and
Miyano, M. (2000) Crystal structure of
rhodopsin: a G protein-coupled receptor.
Science, 289, 739–745.
66 Rasmussen, S.G., Choi, H.J., Fung, J.J.,
Pardon, E., Casarosa, P., Chae, P.S.,
Devree, B.T., Rosenbaum, D.M., Thian,
F.S., Kobilka, T.S., Schnapp, A.,
Konetzki, I., Sunahara, R.K., Gellman,
S.H., Pautsch, A., Steyaert, J., Weis,
W.I., and Kobilka, B.K. (2011) Structure
of a nanobody-stabilized active state of the
b2 adrenoceptor. Nature, 469, 175–180.
67 Rosenbaum, D.M., Zhang, C., Lyons, J.A.,
Holl, R., Aragao, D., Arlow, D.H.,
Rasmussen, S.G., Choi, H.J., Devree, B.T.,
Sunahara, R.K., Chae, P.S., Gellman,
S.H., Dror, R.O., Shaw, D.E., Weis, W.I.,
Caffrey, M., Gmeiner, P., and Kobilka,
B.K. (2011) Structure and function of an
irreversible agonist–b(2) adrenoceptor
complex. Nature, 469, 236–240.
208j8 Discovery of Nonpeptide Vasopressin V2 Receptor Antagonists
Kazumi Kondo is a Senior Researcher at Otsuka Pharmaceutical
Co., Ltd. He was born in Ehime Prefecture (Japan) in 1959.
He obtained his B.S. degree (1982) and M.S. degree (1985) in
the field of Synthetic Organic Chemistry from Okayama University
under the supervision of Prof. Dr. Shigeru Torii. Before
his graduation from Okayama University, he spent 1 year
(1983–1984) at Lausanne University (Switzerland) in the laboratory
of Prof. Dr. Manfred Schlosser. In 1999, he received his
Ph.D. degree (Pharmaceutical Science) from the Tokyo University
under the supervision of Prof. Dr. Koichi Shudo. After he joined Otsuka in
1985, he contributed to a number of projects to discover cardiovascular drugs as
well as CNS drugs. He is an author of over 30 publications and a co-inventor on
over 20 small-molecule patents.
Hidenori Ogawa is an Associate Director for Qs’ Research
Institute of Otsuka Pharmaceutical Co., Ltd. He received his
B.Eng. degree (1976) in Chemistry from Okayama University.
He earned his Ph.D. (1988) in Medicinal Chemistry from
Osaka University, working on design and synthesis of positive
inotropic agents. He has worked at Otsuka for 32 years and
has been responsible for several inventions and publications
in the various therapeutic areas, including vasopressin receptor
V1a and/or V2 antagonists and V2 agonists.
References j209
9
The Development of Cysteinyl Leukotriene Receptor Antagonists
Peter R. Bernstein
List of Abbreviations
5-LO 5-lipoxygenase
b.i.d. dosed two times/day
CSS Churg–Strauss syndrome
CysLT cysteinyl leukotriene
CysLT1R cysteinyl leukotriene 1 receptor
CysLT2R cysteinyl leukotriene 2 receptor
CysLT3R cysteinyl leukotriene 3 receptor
DMPK distribution, metabolism, and pharmacokinetics
FLAP 5-lipoxygenase activating protein
gp guinea pig
HAP hydroxyacetophenone
HSA human serum albumin
ID intraduodenal
IP intraperitoneal
IV intravenous
LT leukotriene
MW molecular weight
PO per oral
q.d. dosed once/day
SAR structure–activity relationship
SRS-A slow-reacting substance of anaphylaxis
The development of the CysLT1R antagonists will be reviewed with a focus on the
three agents used clinically: montelukast, zafirlukast, and pranlukast. In addition to
describing the path that each company followed to their clinical agent, an effort will
be made to show how the interim results obtained by these and other groups (those
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
j211
that did not achieve a clinically available agent) influenced the competing teams’
efforts.
9.1
Introduction
At the time of its approval in 1996, zafirlukast represented the fruition of a decade’s
long effort and the dawning of a new treatment for asthma. As a selective CysLT1
receptor (CysLT1R) antagonist, it exemplified the first new class of medications
approved for treating asthma, in the United States in over 30 years [1]. Asthma is
one of the most common medical conditions that is, as defined by the Global Initiative
for Asthma, “a chronic inflammatory disorder of the airways in which many
cells and cellular elements play a role”.1)
It is characterized by variable and reversible
airway obstruction and hypersensitivity and had been treated by a wide variety
of agents including oral and inhaled corticosteroids, oral and inhaled b-agonists,
oral xanthines, and inhaled inhibitors of mediator release [2].
Due to asthma’s widespread nature and the fact that in spite of this broad array of
agents, it remained only partially controlled, there was a great deal of interest in the
development of new treatment paradigms. The clinical effectiveness of the
CysLT1R antagonists (e.g., zafirlukast, montelukast, and pranlukast) in preventing
asthmatic-type responses has been demonstrated in multiple challenge studies
involving allergens, aspirin, exercise, and cold air [3]. In asthmatic patients, the
response to these agents is variable. However, in those who respond these medicines
improve the patient’s general condition and reduce the need for other medications
such as b-agonists and steroids [4].
The path leading to the development of the CysLT1R antagonists as effective
treatments for asthma began during the period 1938–1940 when the slow-reacting
substance of anaphylaxis (SRS-A) was identified by Kellaway and Trethewie as a
potent smooth muscle constrictor [5]. SRS-A, unlike histamine, induced a slowly
developing and long-lasting contraction of guinea pig ileum. Initial efforts to
develop agents that would either block the formation of SRS-A (biosynthesis inhibitors)
or antagonize its actions (receptor antagonists) were hampered by the fact that
SRS-A was (1) very unstable, (2) only available in small amounts via a complicated
isolation following biological generation, and (3) its biosynthetic pathway, structure,
and biological target all remained unknown.
The next breakthrough in the development of CysLT1R antagonists came in 1973
with the discovery of FPL-55712, by Augstein et al. [6]. This archetypical antagonist
was discovered at Fisons by screening of compounds related to a chromone (cromolyn
sodium [7]), used to treat asthma, against extracts containing SRS-A.
Although FPL-55712 suffered from a short half-life and poor bioavailability [8], it
was very important both as a starting point from which other analogous antagonists
would be developed and because it played a key role in the structural elucidation of
1) http://www.ginasthma.org/ (accessed October 24, 2011).
212j9 The Development of Cysteinyl Leukotriene Receptor Antagonists
SRS-A. Using biological extracts isolated from multiple cell types and species,
research groups fractionated (purified) and concentrated the samples of SRS-A.
Antagonism by FPL-55712, of the contraction induced in a tissue strip by a fraction,
was the key assay to show which fractions were the ones containing SRS-A and also
that different biological systems were producing the same agonist [9].
The key discoveries that unlocked the floodgates of broad and sustained drug
discovery efforts occurred in 1979 and 1980. First, Bengt Samuelsson et al.
(Karolinska Institute) determined that SRS-A was comprised of a cysteinecontaining
derivative of 5-hydroxy-7,9,11,14-eicosatetraenoic acid [10]. This
compound was a product of the 5-lipoxygenase pathway of the arachidonic acid cascade
and he proposed the name leukotriene for this family of compounds, a name
that was derived from their cell source (leukocytes) and their unusual conjugated
triene chemical structure (Figure 9.1). Then working with E.J. Corey (Harvard
University), Samuelsson confirmed its exact structure and determined that three
members of the leukotriene family, known as LTC4, LTD4, and LTE4, and collectively
as cysteinyl leukotrienes (CysLTs, because although they contained different
peptides they all incorporated the amino acid cysteine), accounted for the original
biological activity of SRS-A [11].
The belief that modulators (antagonists or inhibitors) of this biological pathway
could become novel, effective antiasthma drugs was based on the finding that SRSA
and synthetic CysLTs were able to cause or potentiate many of the hallmarks of
asthma, specifically induce bronchoconstriction via contraction of airway smooth
muscle, enhance vascular permeability, induce migration of inflammatory cells,
and stimulate mucous secretion [12, 13].
LTA4-hydrolase
Phospholipids
PLA2
5-Lipoxygenase
and
5-Lipoxygenase
activating protein
FLAP
LTA-synthase
LTC4-synthase
γ-glutamyl
transpeptidase
amino
peptidase
arachidonic acid
5-HPETE
LTA4
LTC4
LTD4
LTE4
LTB4
O
OH
S
O
N
H
N
H
NH2
O
OH
S
O
N
H
NH2
OH
S
NH2
OHOH
C5H11
CO2H
C5H11
CO2H
OOH
CO2H
C5H11
CO2H
CO2H
CO2H
C5H11
CO2H
CO2H
C5H11
CO2H
CO2H
C5H11
C5H11
CO2H
Figure 9.1 Biosynthetic pathway for LT production.
9.1 Introduction j213
9.2
Scope of the Drug Discovery Effort on Leukotriene Modulators
Inspired by these discoveries, many pharmaceutical companies participated in the
race to develop specific leukotriene modulators. Also since the leukotrienes seem to
have limited, if any, physiological role and most of their pathophysiology appeared
restricted to the lung, it was the goal of most groups to develop orally effective
agents. The oral approach would provide a big advantage to patients over existing
asthma agents that were given by inhalation, to either limit systemic side effects or
because the compounds were not orally bioavailable. Oral dosing would eliminate
the complicated parallel development track for the devices (nebulizers or inhalers)
needed to deliver inhaled agents. In addition, an oral pill would alleviate the complicated
and difficult to learn (patients have to be trained not to swallow the dose)
regimens required for the inhaled agents (e.g., x number of puffs/y times per day).
Research focused on biosynthesis inhibitors (either 5-lipoxygenase inhibitors or
5-lipoxygenase activating protein inhibitors) and specific receptor antagonists
(either for the cysteinyl leukotrienes or for leukotriene B4) [14]. Just in the field of
cysteinyl leukotriene antagonists, those efforts led to the preparation of tens of
thousands of compounds and to dozens of clinical candidates [15–17]. Discussing
all of these efforts, even just all of the clinically evaluated antagonists, is beyond the
scope of this chapter. However, an excellent review on modulators of leukotriene
biosynthesis and receptor activation that had been under clinical evaluation was
published by Brooks and Summers [18], just before the approval of zafirlukast for
the US market.2)
It is noteworthy that in the area of cysteinyl (or peptidyl as they are
also known) leukotriene antagonists, of the 19 compounds for which clinical
results are reported in that review only 3 made it to the market. These three compounds,
pranlukast (1), zafirlukast (2), and montelukast (3), will be the focus of this
chapter (see Figure 9.2).
A cursory examination shows that these compounds do not look at all alike. They
were designed independently from each other, so they are not analogues in a chemical
sense. However, all of them have a lipophilic region, an acidic group, and have
been modeled back to a common cysteinyl leukotriene receptor pharmacophore.
2) NME approval date: September 26, 1996.
3: MK-0476, montelukast1: ONO-1078, pranlukast 2: ICI 204219, zafirlukast
Figure 9.2 Clinically registered CysLT1R antagonists.
214j9 The Development of Cysteinyl Leukotriene Receptor Antagonists
Since they also share a selective CysLT1R antagonist profile, they can be considered
as pharmacological analogues.
9.3
Synthetic Leukotriene Production and Benefits Derived from this Effort
Just as the effort to clarify the mysteries surrounding SRS-A (its structure, biosynthetic
pathway, and molecular targets) had required FPL-55712, the newer effort
to develop modulators that would be effective drugs required synthetic leukotrienes.
They were needed since preparation and isolation of SRS-A from biogenic
sources (primarily chopped sensitized guinea pig lung [19] or rat peritoneal cavity
[20]) was inadequate to support the drug discovery and development efforts. As
a result of this need, teams at many companies including Merck-Frosst [21],
SmithKline [22], Ciba-Geigy [23], Hoffmann-La Roche [24], and Fisons [25] reported
alternate syntheses to Corey’s original approach.
Some drug discovery teams, such as the ones at SmithKline, Merck-Frosst, and
Stuart Pharmaceuticals (Division of ICI Americas Inc.), in addition to developing
leukotriene syntheses leveraged their internal chemical success. The team at
Merck-Frosst provided many academic labs with synthetic leukotrienes, identified
and synthesized metabolites of leukotrienes [26], identified and characterized leukotriene
receptors [27], played a key role in the cloning, expression, and characterization
of the 5-lipoxygenase enzyme [28], and were the first to identify 5lipoxygenase
activating protein (FLAP) [29, 30], a cofactor necessary for leukotriene
synthesis in cells.
The group at ICI determined the relative potencies of leukotriene isomers [31],
deduced the solution conformation of the leukotrienes [32], broadly characterized
the nature of the leukotriene receptors in animals and humans [33, 34], and collaborated
with academic teams on clinical studies of the effects of synthetic leukotrienes
in humans [35, 36].
Although the overall scope of these synthetic efforts is not known, the team at
ICI alone prepared over 10 g of synthetic leukotrienes, and given the Merck team’s
provision of material to many labs they too may have prepared equally large or even
larger amounts. These stockpiles represented large resource commitment by their
companies. Also, since in the early 1980s the going rate to buy samples of the leukotrienes
from an academic group was $5000/mg, the commercial value of these
reserves was in the many millions of dollars. The per mg price is remarkably consistent
with the current commercial price of D3495/mg by Cayman Chemical
(quote on 12/04/11).
Utilization of synthetic leukotrienes in humans in the United States required
that they be prepared to exacting standards, and that detailed characterization, control,
and stability data be generated. In the case of ICI, the synthetic leukotrienes
were stored in liquid nitrogen cooled cryogenic storage systems. Although the
intermediate LTA4-methyl ester or protected CysLTs could be stored as oils or as
solutions, the final leukotrienes were best stored as lyophilized powders that were
9.3 Synthetic Leukotriene Production and Benefits Derived from this Effort j215
reconstituted into a standard strength solution prior to use. Since the CysLTs are
naturally amphiphilic substances and since their chemically unstable triene is in
the hydrophobic tail, it was critical that transformation of dilute solutions to dry
powder be done under conditions that minimized globular micelle formation.
Shipment of the powder to the various testing facilities was usually achieved under
dry ice cooling.
All of these data were supplied to the FDA in a “Drug Master File” that was submitted
by ICI on behalf of the clinical investigators who were doing these studies.
Initially, the leukotriene D4 produced by the chemistry team at ICI was the only
source covered by such a Drug Master File. This led to multiple requests for access
to their leukotrienes, to support other companies’ clinical studies in which LTD4
would be used as a specific challenge.3)
Although broadly supplying LTD4 was
beyond the ability of the ICI team, the sharing of some experimental protocols was
supported4)
and this eventually led to the commercial availability of high-quality
synthetic leukotrienes.
The sharing of protocols and data exemplified how, at the time this work was
done, the competition between the different research groups was relatively
“friendly.” Unlike now, when the value of multiple (“me too”) drugs working at the
same target has been called into question, in the early 1980s the view existed that
more than one group could be successful and that there was value to the patient
and the physician from them having multiple drugs from which to choose. The
apparent lead group (closest to a registered drug) was frequently changing and as
will be evident from the discovery story herein the different teams benefited from,
and were spurred on by the results published by their competitors. It is hoped that
the growing concept of “personalized medicine” that describes at a molecular
(genomic) level why patients respond differently to, for example, a specific receptor
antagonist will both inspire and support the value of developing multiple similarly
targeted drugs, each, however, focused to a more specific subpopulation.
9.4
Bioassays and General Drug Discovery Testing Cascade
The period during which the cysteinyl leukotriene antagonists were developed corresponds
to one during which drug discovery testing cascades were rapidly changing.
At the beginning of this era, cloned LT receptors (either human or animal) did
not exist and isolated tissue binding studies were just being introduced.
As their primary in vitro screen, some research teams initially relied on inhibiting
the contraction induced in isolated tissues (usually guinea pig ileum strips or
3) Peter Bernstein, Ph.D., personal communication, exemplified by letter from Dr. W.S.
Hitchings, Director Control & Manufacturing Data Registration, Merck Sharp & Dohme
Research Laboratories to Dr. Bernstein, Stuart Pharmaceuticals, dated June 12, 1985.
4) Tony Kreft, Ph.D., personal communication, exemplified by letter and experimental
procedures from Dr. P.R. Bernstein, Stuart Pharmaceuticals to Dr. Tony Kreft, Wyeth
Laboratories, dated July 19, 1982.
216j9 The Development of Cysteinyl Leukotriene Receptor Antagonists
guinea pig tracheal spirals) by a standardized unit of “SRS-A” that was isolated
from a biogenic source. After pure synthetic leukotrienes became available, assays
were developed that reported, for example, the % inhibition of the contraction
induced by 8 nM LTD4 on guinea pig tracheal spirals at a specific concentration of
drug. For compounds that warranted further evaluation, either IC50 values or the
calculated pKB values could be determined in an array of animal and/or human
tissues. Comparison of the compound’s ability to block the LTD4- or LTE4-induced
response and its ability to block the response in the same tissue of a different
spasmogen (e.g., 1.5 mM BaCl2 on guinea pig tracheal spirals) provided a measure
of its specificity as a LTantagonist.
Later, LTD4 binding assays were developed that measured the displacement of
[3
H]LTD4 from tissue membranes [37]. These assays were first done utilizing tissue
that naturally included the LT receptors (e.g., guinea pig lung). The Merck efforts
discussed in this chapter exemplify this approach as they report inhibition of
[3
H]LTD4 binding to guinea pig lung membranes. After cells expressing cloned
receptors [38] became available, those were utilized. Studies coincident with the
drug development efforts demonstrated the existence of two CysLT receptors designated
as CysLT1R and CysLT2R both in animals and in humans [39, 40]. The order
of binding affinity to the CysLT1R is LTD4 > LTC4 ) LTE4, whereas the rank order
to CysLT2R is LTD4 $ LTC4 ) LTE4. The two receptors are distinguished by the fact
that the actions of LTD4 and LTE4 on CysLT1R are blocked by FPL-55712, whereas
the effect of LTC4 on CysLT2R is not blocked by FPL-55712. The antagonists covered
in this chapter are all specific CysLT1R antagonist.
The complexity of this field is such that our understanding of the leukotriene
receptors, their distribution, and pathophysiological functions continues to
evolve [41]. Exemplifying this, a third CysLT receptor (CysLT3R) was recently proposed
[42], to explain biological effects by LTE4 that are not mediated via the two
well-characterized receptors.
The existence of multiple receptors and multiple ligands led to potentially confusing
screening cascades. Although all groups did not use the same cascade, the
group at ICI chose to use the effect of LTE4 on guinea pig trachea as their initial
primary screen. This was because LTE4 was selective for one of the two receptors
found in that tissue [43] and that receptor had high pharmacological similarity to
the single receptor (CysLT1R) found in human bronchus. The role of the human
CysLT2R remains undefined and awaits the clarifying impact that would occur
from discovery and development of a selective CysLT2R antagonist. That these compounds
are selective may in part be explained by the fact that there is only 38%
amino acid identity between the two receptors [44]. Furthermore, localization studies
of the hCysLT2R show that the highest receptor expression levels occur in adrenal
glands, heart, and placenta, indicating that whatever its role is, it is probably not
linked to asthma [45].
Initial in vivo evaluation of CysLT1R antagonists was most commonly done in
guinea pigs by evaluating their ability to block a leukotriene challenge. The leukotriene
could be administered either i.v. or by aerosol and pretreatment by the drug
(PO, IP, ID, or IV) would cause a delay in the bronchoconstriction induced by the
9.4 Bioassays and General Drug Discovery Testing Cascade j217
leukotriene. At first, pulmonary mechanics parameters were measured in anesthetized
animals. This model, while very precise, was very labor intensive and required
surgically prepared animals. Therefore, to create a higher throughput in vivo assay,
a “dyspnea” model was developed that measured the ability of compounds to block
the induction of “labored abdominal breathing” in conscious guinea pigs by
LTD4 [46].
While being very intensive in the use of both manpower and precious leukotrienes,
these in vivo assays proved invaluable for the progression of antagonist structure–activity
work. Also, as will be discussed in detail later, they helped to bridge
gaps in pharmacokinetic knowledge now routinely filled by in vitro and in vivo
DMPK assays.
9.5
Development of Antagonists – General Approaches
In the early 1980s when much of the discovery research on cysteinyl leukotriene
antagonists was done, there was little information on the structure of G-proteincoupled
receptors such as CysLT1R, the biological target of the cysteinyl leukotrienes.
As a result, some modern approaches, such as the building of a model of the
receptor and the de novo design of ligands to it, were not possible. Similarly, this
was before the advent of high-throughput screening or combinatorial (highthroughput
or robot-assisted) synthesis. This left chemistry teams with three starting
points: synthesizing analogues of FPL-55712, using the structure of the natural
leukotrienes as starting points, and the screening of selected compounds in a bioassay.
This could be either a binding assay or a functional assay formatted in a way
to provide higher throughput, for example, determining if 10 mM compound significantly
blocks the contraction induced by 8 nM LTE4 on guinea pig tracheal spirals.
Although all of these approaches were utilized, structural analogues of FPL-
55712 represented the greatest percentage of clinically evaluated compounds [18].
However, none of the three successful products discussed below has much structural
resemblance to FPL-55712 (see Figure 9.3).
9.6
Discovery of Zafirlukast
The discovery of zafirlukast has been described in many prior papers and presentations
that expand upon the summary given here [14, 47–49]. The team at ICI
explored all three possible starting points; however, their efforts on selected screening
did not yield useful leads, and it was the combination of output from making
analogues of FPL-55712 and of the natural leukotrienes that led to zafirlukast.
In the early 1980s, the newly reinvigorated efforts to prepare improved analogues
of FPL-55712 benefited from the researchers knowing the structure of the cysteinyl
leukotrienes. At ICI, the team viewed LTD4 as being made up of three regions:
218j9 The Development of Cysteinyl Leukotriene Receptor Antagonists
lipophilic lipid, polar C1 acid, and peptide areas, and that FPL-55712 incorporated
two regions: the chromone carboxylic acid and hydroxyacetophenone (HAP). They
hypothesized that FPL-55712 was mimicking two of the three regions of LTD4 with
the chromone being a surrogate for the peptide region and that the HAP was mimicking
either the lipid backbone or the C1 acid [50]. Early molecular modeling
efforts ($1981) were undertaken to illustrate these overlaps (Figure 9.3) [48].
This hypothesis led them to prepare a small set of compounds that linked either
the chromone carboxylic acid or the hydroxyacetophenone portions of FPL-55712
with fragments exemplifying the different regions of LTD4. That effort led to the
discovery that linkage of simple aliphatic acids of six to eight atoms in length to the
hydroxyacetophenone afforded leukotriene antagonists of modest potency, for
example, 4 (Figure 9.4). Given the well-exemplified path of improving potency by
introducing structural constraints that preorganize a compound into an orientation
preferred at a receptor [51], a series of aryl groups were explored as replacements
for the aliphatic chain. This led to the discovery that a para-toluic acid analogue 5
was slightly more potent. Further SAR exploration in this aryl series led to the discovery
that a 3-methoxy substituent 6 yielded greater in vitro potency and selectivity,
and demonstrated in vivo activity when administered i.p.
Simultaneously, the ICI team also explored preparation of antagonists using the
structure of LTC4 as their starting point (Figure 9.5). Support for this approach was
provided by the success of many efforts to prepare receptor antagonists by starting
Figure 9.3 Molecular modeling of FPL-55712 and LTD4.
9.6 Discovery of Zafirlukast j219
with the structure of their natural ligand, for example, H2 antagonists from histamine
or b-adrenergic blockers from epinephrine. However, unlike those earlier
examples the teams trying to develop CysLT receptor antagonists also had to
address the extreme biological and chemical lability of the leukotrienes.
Since 1
H-nuclear magnetic resonance studies done as part of their leukotriene
synthesis and characterization efforts [32] had indicated that the triene region of
the lipid backbone preferred a coplanar conformation, the ICI team explored
replacement of the unstable triene with a series of aryl groups. This led to the discovery
of potent (nM level) agonists, for example, 7, with much greater chemical
stability [52]. Detailed pharmacological studies showed that the effects induced by
these compounds were specifically antagonized by FPL-55712, affirming that they
were acting at the cysteinyl leukotriene receptor [53]. Replacement of the peptide
4: Inhibition 52% at 10 μM 6: Inhibition 57% at 1 μM
Selectivity vs. BaCl2: 50
Active in vivo: when administered
intraperitoneally 50 mg/kg, 60 min before
LTD4 challenge
5: Inhibition 100% at 10 μM
OHO
O
OHO
O
OHO
O
CO2H
CO2H CO2H
OMe
Figure 9.4 FPL-55712-derived compounds at ICI.
9: U19052 (ICI Am)
Inhibition 31% @ 5 µM
7: Agonist at 70 nM
10: Inhibition 40% @ 5 µM
selectivity versus BaCl2: 10
8: Inhibition 56% @ 50 µM
LTC4
OH
S
O
N
H
N
H
NH2
O
S
OH
S
O
CO2H
CO2H
CO2H
n-C7H15
MeO
CO2H
n-C7H15
CO2H
n-C6H13O
CO2H
n-C6H13O
CO2H
Figure 9.5 Leukotriene D4-derived compounds.
220j9 The Development of Cysteinyl Leukotriene Receptor Antagonists
region in these analogues with the aliphatic and aryl acidic groups that had been
found in the hydroxyacetophenone effort [50] led to compounds that had a range of
agonist and antagonist properties [54]. One of the more potent antagonists 9,
U19052, was broadly characterized as a specific antagonist of LTC4, LTD4, and
LTE4: it had a pKB value of 7.2 against LTD4- or LTE4-induced contraction of isolated
guinea pig ileum. However, these compounds demonstrated a very delicate balance
between agonist and antagonist activity. Minor changes in their structures, for
example, replacement of the n-C7H15-aryl substituent in U19052 with n-C6H13O-,
resulted in agonist activity. Weaker but consistent antagonist activity was found
upon removal of one of the two acidic regions [49]. The SAR of this simpler bisaromatic
series, exemplified by 8, was broadly explored and optimal potency was
found when the two ether groups were para to one another and the carboxylic acid
was ortho to the ether linkage, for example, 10. For example, moving the carboxylic
acid in 10 to the para-position resulted in $10-fold decrease in potency.
Although these bis-aryl antagonists represented great structural simplification
and improvement in stability compared to the starting leukotrienes, they were also
several orders of magnitude weaker at the leukotriene receptor than LTC4. As had
been demonstrated previously in the effort on FPL-55712-derived compounds, one
way to enhance potency is by decreasing flexibility and locking the compound into
an orientation preferred by the receptor. To achieve this in this series, various bicyclic
frameworks were considered, and due to a combination of synthetic considerations
and commercial availability nitroindazole was chosen as the starting material
(Figure 9.6). To explore this concept, eight regioisomeric indazoles 11 were prepared,
with the linkage positions indicated by the asterisks. Of these compounds
only one, the para-benzoic acid 12, was an antagonist at 10 mM. This was both surprising
and perplexing since the bis-aryl series that was the starting point for them
had demonstrated a clear preference for the ortho-benzoic acid. However, such
para-benzoic acid derivatives had been optimal both in the original leukotriene analogues
(those with three regions) and in the HAP series. Consideration of this fact
and comparison of potential overlaps between the series led to the suggestion to
add back in the methoxy substituent that had been beneficial in those earlier series.
Since only the N1-alkylated indazole had been active, this modification was done in
the corresponding indole series. This was to avoid having to separate the mixture
of N1- and N2-substituted compounds that were formed during the alkylation step
1110: R = nC6H13O-
Lipophilic
Amide
Core
Toluic Acid
12: X = N R = H Inhibition 60% @ 10 µM
13: X = CH R = OMe Inhibition 70% @ 5 µM
Selectivity vs. BaCl2: 100
*
*
*
*
*
*
N
X
N
H
O
O
R
N
H
O
CO2H
OMe
n-C5H11
CO2H
N1
N2
CO2H
n-C5H11
Figure 9.6 Discovery of the indazole/indole series by imposing structural rigidity.
9.6 Discovery of Zafirlukast j221
in the indazoles. The resultant compound 13, although slightly weaker than the
most potent HAP derivative 6, was surprisingly more selective for the leukotriene
receptor (100 versus 50).
At this point, the team concentrated its efforts on this indole series. In addition
to the improved selectivity of the indoles, efforts to improve potency in either of the
earlier series were not efficiently progressing and the indoles represented a novel
structural type of leukotriene antagonist. In contrast, both FPL-55712- and LTderived
antagonists were being worked on by many other groups.
Consideration of what needed to be achieved in their exploration of structure–
activity relationships, in combination with a synthetic analysis, led to the indole
antagonist being viewed as composed of three regions: the lipophilic amide, the
core heterocycle, and the toluic acid.
Exploration of the amide substituent (Figure 9.7) revealed that branching of the
aliphatic chain resulted in increased in vitro potency, with the a-ethyl-hexanamide
14 also demonstrating oral activity at 100 mg/kg in the guinea pig labored abdominal
breathing model [55]. However, b-branched amides with cyclic end cap groups
proved the most potent. The exploration also revealed that the carbon a to the
amide carbonyl could be replaced with either O or N resulting in carbamates or
ureas that retained excellent in vitro activity. Such b-cyclic compounds, for example,
16 and 17, were almost 100-fold more potent in vitro than the initial hexanamide. At
ICI Pharmaceuticals, this was an exciting find, breaking the plateau of 1 mM antagonism,
after having worked in the area for several years.
Simultaneous to the exploration of the amide substituents, a second set of explorations
was underway on the acid region of the molecule [56]. Those efforts were in
part inspired by efforts at Lilly that had shown in a set of HAP-derived leukotriene
3
14: pKB = 6.6, selectivity: 700
po: 100 mg/kg (233 µmol/kg) @ 1 h
16: X = CH2: pKB = 7.7
17: X = O: pKB = 7.8
15: pKB = 8.6, selectivity: > 10,000
po: 17 mg/kg (30 µmol/kg) @ 3 h
18: X = CH2: pKB = 9.6
po: 30 µmol/kg @ 3 h
19: X = O: pKB = 9.5
po: 30 µmol/kg @ 3 h
NN
H
O
O
N
H
S
O
O
NN
H
O
NN
H
X
O
O
N
H
S
O
O
NN
H
X
O
OMe
CO2H
n-C4H9
Et
OMen-C4H9
Et
OMe
CO2H
OMe
Figure 9.7 Exploration of the acid and amide regions.
222j9 The Development of Cysteinyl Leukotriene Receptor Antagonists
antagonists that replacement of a carboxylic acid with a tetrazole resulted in a 30fold
increase in potency, in vitro, and led to the clinical candidate LY-171883 [57].
The importance of the discovery and development of this compound by J. Fleisch,
W. Marshall, and their colleagues is hard to overstate. It was the first major
improvement over FPL-55712 and highlighted the importance of the nature of the
acid group to activity. The ICI team confirmed that the 3-methoxy-p-toluic acid was
the optimal aryl acid in this indole series and discovered that while the impact of
replacing the acid with tetrazole or most other carboxylic acid mimetics was small,
the use of phenylsulfonimide mimetics afforded an additional 100-fold increase in
potency, in vitro, for example, 15. The results also demonstrated a consistent crossover
of the impact on potency of changes in the different regions of the molecules.
Specifically, acid replacements that improved in vitro or in vivo potency in compounds
with one amide substituent had a similar effect with a different amide substituent,
18 and 19. Simultaneous to this, the team noted that these compounds
were only mimicking two of the three regions of the natural leukotrienes. They
postulated that addition of a second polar group would increase potency [58]. Substitution
at the 3-position of the 1,6-substituted indole of 18 did result in increased
in vitro potency, but not an equivalent increase in oral potency (compound not
shown). This provided support for how these compounds were being recognized
by the receptor, but was not a route to a better oral drug.
Turning back to the core heterocyclic structure, it was shown that variation of
the core resulted in significant changes in potency both in vitro and in vivo
(Figure 9.8) [59]. However, a disconnect existed between the results in that the
cores that were most potent in vitro, for example, 20 and 21, were not the most
potent when given orally, for example, 22. At the time, the reason for this disconnect
was not understood and an indazole 20 (ICI-198615) was initially considered
a potential clinical candidate because of its in vitro potency and its robust oral
efficacy. Subsequent detailed pharmacokinetic studies with ICI-198615 demonstrated
less than 0.3% oral bioavailability, demonstrating an issue in spite of its oral
efficacy. In the absence of routine DMPK assays, the team chose to use the PO/IV
ratio in the dyspnea model as a surrogate for oral bioavailability and focused on
obtaining compounds with low PO/IV ratios. Analysis of the data indicated that the
20: ICI 198615
X = CH, Q = N; pKB = 10.3
po: 10 µmol/kg @ 3 h; po/iv = 333
bioavailability: rat and dog < 1%
21:
X = NMe, Q = C pKB = 10.3
po: 10 µmol/kg @ 3 h; po/iv = 133
22: ICI 198707
pKB = 9.7
po: 3 µmol/kg @ 3 h, po/iv < 25
bioavailability: rat > 30%
O
N
H
S
O
O
Q
X
N
N
H
O
O
O
N
H
S
O
O
N
N
H
O
OOMe
OMe
Figure 9.8 Comparison of different core structures.
9.6 Discovery of Zafirlukast j223
“inverted” indole core was more effective than the other cores in translating in vitro
potency into in vivo efficacy.
Further studies on variation of the substitution pattern of the phenylsulfonimide
(Figure 9.9) showed that although potency in vitro was largely unchanged by substituents
on the phenyl ring, for example, 23, 24, and 25, there was a marked effect
on oral potency. In the amide-linked series, the 2-methylphenyl-substituted analogue
24 was approximately 30 times more potent orally than the parent phenyl 23
despite being less than twofold more potent in vitro. Application of this finding in
the carbamate-linked series led to 26 (ICI 204219, zafirlukast) [60]. Zafirlukast was
confirmed to have high bioavailability in the rats (68%) and dogs (67%).
Although the total number of compounds prepared and/or tested in the development
of zafirlukast is not available, some statistics have been reported. The ICI LT
antagonist effort started in September 1980 and ICI 204219 was nominated for
development in early 1986. The chemistry team that did both the LTsynthesis effort
and the LT antagonist SAR studies ranged from 3 to 10 chemists in size. During its
most rapid progress, the team increased potency by five orders of magnitude, from
the first indazole 12 to picomolar antagonists such as 20 and 22, in approximately
15 months. This too was exciting and quite remarkable, given the 1 mM potency
plateau that the team had struggled to surmount for several years!
9.7
Discovery of Montelukast
The discovery and development of montelukast sodium has been summarized in
many prior presentations and papers [14, 61, 62].5)
The Merck-Frosst team also
used all three of the previously listed approaches for lead generation. In their case,
in spite of their extensive synthetic leukotriene efforts, little is known about the
work they did to develop antagonists starting directly from the structure of the natural
leukotrienes. However, starting from FPL-77512 they developed several agents
23: R = H; pKB = 9.6, po: 30 µmol/kg @ 3 h
24: R = CH3: pKB = 9.9, po: 1 µmol/kg @ 3 h
25: R = Cl: pKB = 9.8, po: 3 µmol/kg @ 3 h
26: ICI 204219; zafirlukast
pKB = 9.7, po: 0.52 µmol/kg @ 3 h
bioavailability: rat (68%), dog (67%)
N
N
H
O
O
N
H
S
O
OO
N
N
H
O
N
H
S
R
O
OO
OMeOMe
Figure 9.9 Optimization of the sulfonamide and discovery of zafirlukast.
5) For a series of video presentations by Merck-Frosst scientists on the discovery and
development of montelukast sodium, see http://aboutdrugdiscovery.ca/en/main_ENG_short.
html (accessed October 29, 2011).
224j9 The Development of Cysteinyl Leukotriene Receptor Antagonists
(Figure 9.10) that entered development and were evaluated in humans, including
27 (L-649923) and 28 (L-648051) [63, 64]. Although progression of these compounds
was terminated and they do not appear to have provided SAR data used in
the montelukast series, they provided limited support to the hypothesis linking
leukotrienes to asthma. (Note: All Merck activity data are reported as binding of
[3
H]LTD4 to guinea pig lung membranes.)
As a second starting point (Figure 9.11), the team used a broad screening effort
that found a styryl quinoline 29 [65]. It is not clear how many compounds were
evaluated to come up with this lead, but it is safe to presume that this included
many of 15 000 compounds that were reported as being evaluated at Merck as
CysLT1R antagonists over the course of their research program.5)
In trying to
understand how this lead might be binding to the CysLT1 receptor, the Merck team
applied an analysis they had done of leukotriene receptor structure–activity studies.
That analysis had led to a hypothetical model for the LTD4 receptor that had three
regions: a planar hydrophilic region for the triene, an ionic hydrophilic site for one
of the carboxylates of LTD4, and a second hydrophilic (polar) binding site [66].
Fitting this compound 29 to this model implied that it was binding to the part of
the receptor that recognized the flat triene region of LTD4, and that polar substitution
at the 3-position would be beneficial. Independent support for the compound
binding in such a way was provided by the greater potency of a related compound,
30 (REV-5901) [67]. REV-5901 exemplified a second series of quinoline-based LTD4
antagonists [68] that had been independently discovered and that were eventually
abandoned. Furthermore, it is not known if the data on these quinolines played
any role in selecting what sets of compounds were included in the Merck-Frosst
screening efforts.
28: L-648051 Ki = 6200 nM vs LTD427: L-649923 Ki = 400 nM vs LTD4
O S
OH
OH
O
O
SO
O
OH
OO
COOH
CH3
COOH
Figure 9.10 FPL-derived clinical candidates developed at Merck-Frosst.
LTD4
30: REV-5901 IC50 0.51 µM [3
H] LTD429: IC50 6 µM [3
H] LTD4
S
OH
NH2
O
NN N
O
OH
HN CO2H
C5H11
CO2H
nC4H9
Figure 9.11 Quinolinyl hits and comparison to the structure of LTD4.
9.7 Discovery of Montelukast j225
As a polar substituent, the Merck team added two acidic groups to their styryl
quinoline core via a dithioacetal linker (Figure 9.12). This unit was based on SK&F
102922 (31), a compound that had been discovered at SmithKline during their development
of antagonists starting from the structure of LTD4 [69]. Extensive SAR studies
on this series at Merck-Frosst showed that the styryl linker [–CHÀÀÀÀCH–] could be
replaced with the CH2O linker found in the Revlon series with only a slight decrease
in potency and highlighted the importance of the quinoline group. Replacement of
the quinoline with a naphthalene led to almost complete loss of binding (not illustrated),
whereas incorporation of a 7-chloro substituent led to 32, a compound that
displayed a further 10-fold increase in activity in the in vitro binding assay.
Unfortunately, the increased in vivo efficacy of 32 did not equal the degree of
increase seen in vitro. The Merck team postulated that this was due to poor physicochemical
and DMPK properties by 32 and that this diacid might be too polar. Based
on work done on LTD4 that had shown that activity was retained if one of the acids
was replaced with an amide [70], they explored monoamide derivatives and discovered
33 (MK-0571), which was nominated for further development.
During toxicological studies, this racemic compound was found to be a
potent inducer of liver peroxisomal enzymes. It was then determined that its
R-enantiomer 34 (MK-0679, verlukast) retained most of the LT antagonist activity
(Figure 9.13) and had an improved safety profile (liver weight increase of
31: SK&F 102922
Ki= 225 nM vs. [3
H] LTD4
32: R = ONa IC50 = 3.0 ± 1.4 nM
33: MK-0571, R = NMe2 IC50 = 0.8 ± 0.6 nM
N S
S
Cl
S
S
CO2Na
COR
CO2Na
CO2Na
(CH2)7
Figure 9.12 Genesis of the aryl acid substituent.
35: L-695499
IC50 0.9 ±0.6 [3
H] gp lung
pA2 = 8.8 gp trachea
34: MK-0679, verlukast
IC50 3.9 ±1.1 [3
H] gp lung
pA2 = 8.8 gp trachea
36: MK-0476, montelukast sodium
pA2 = 9.3
N S
S
Cl
N
OH
S
Cl
S
OH
NCl
CON(CH3)2
CO2Na
CO2Na
CO2NaEt
Figure 9.13 Verlukast to montelukast.
226j9 The Development of Cysteinyl Leukotriene Receptor Antagonists
0% versus 38%) [71]. Development of MK-0679 was also terminated when
extended studies showed that it too induced liver function abnormalities.
Efforts to retain the leukotriene antagonist activity while separating out the
liver effects focused on replacement of one of the thioether chains. In addition,
since peroxisomes recognize and b-oxidize fatty acids, substituents blocking
the acid chain metabolism were explored and those studies led to 35 (L-
695499) [72]. This compound unfortunately had acute toxicity in mice at high
doses [73]. Further studies on the impact of the thioether stereochemistry to
both toxicity and LT efficacy along with additional acid chain substituents led
to the discovery that extending the aliphatic acid chain length and blocking
the carboxylate b-position via either dimethyl substitution or cyclopropanation
afforded potent orally active leukotriene D4 antagonists that were devoid of
peroxisomal enzyme induction. The choice of 36 (MK-0476) for clinical development
was due to its superior DMPK parameters [71, 74].
9.8
Discovery of Pranlukast
The effort to develop pranlukast has been reported to start with 37 (Figure 9.14)
[75, 76]. How this weak antagonist was found at Ono, whether it was specifically
prepared or was a hit in a screen, is not clear. The team has reported that they used
both the structure of the leukotrienes and results found in other series of leukotriene
antagonists in their effort to improve the potency of this lead. Notably, they felt that
both the cysteinyl leukotrienes and 37 each had hydrophobic and hydrophilic
regions. They postulated that in 37 the p-n-pentyl-cinnamyl and the anthranilate
regions were, respectively, mimicking the lipophilic triene and the combined C1
carboxylic acid–peptide region of the CysLTs.
Exploration of the carboxylate region (varying linker length and type) in 37 did
not yield significant improvements in activity until a carboxylate 38 was replaced
with tetrazole 39 (Figure 9.15). Then, as had been precedented by the work leading
to LY-171883 [57], potency of 39 versus 38 increased, both in vitro and in vivo. Simultaneous
to this (and similar to the reasoning at ICI), the team chose to preorganize
37: IC50 = 14 µM
Determined on guinea pig
ileum against LTC4
Hydrophobic
Hydrophilic
C5H11
CO2H
S
Peptide
OH
CO2H
C5H11
N
H
O
Figure 9.14 Ono screening hit and its comparison to the cysteinyl leukotrienes.
9.8 Discovery of Pranlukast j227
the orientation of the aliphatic acid chain by locking it into various ring systems.
This led to the discovery that a chromone ring (similar to that found in FPL-55712)
was the optimal group and that the increased potency of tetrazole versus carboxylic
acid carried over to the cyclic system, 41 versus 40. Given the multiple ring systems
explored by the Ono team, it is not clear whether the choice of a chromone was
inspired by FPL-55712.
Next variations of the lipophilic region were explored (Figure 9.16) and it was
confirmed that para-substitution of the aryl group was best, but that alkoxy chains
were more potent than the corresponding alkyl chain. Furthermore, deleting the
styryl link between the aryl group and the carboxamide combined with increasing
the length of the para chain resulted in maximum antagonism, 42 and 43. Then,
replacement of the terminal part of the chain with a phenyl ring combined with
varying the length of the linker to optimize activity afforded 44 (ON-RS 411, ONO-
1078, pranlukast).
Support for the original design hypothesis was provided by a detailed QSAR
analysis of the benzamide series (Figure 9.17) by an academic group [77]. Their
modeling suggested the overlap of LTE4 and ONO-1078 shown in Figure 9.1.
They suggested that much like had been found in the SKF (and ICI) work on
LT analogues that agonist activity required two acidic groups and that this was
why YM-17690 (45), a related compound reported by a team at Yamanouchi,
was an agonist.
38: X = CO2H IC50 = 9 µM
39: X = Tetrazole IC50 = 3 µM
40: X = CO2H IC50 = 0.1 µM
41: X = Tetrazole IC50 = 0.03 µM
O
X
N
H
O O
N
H
O
X
O
C5H11 C5H11
Figure 9.15 The impact of structural preorganization on potency for the Ono lead.
42: R = nC7H15- IC50 = 4.2 nM
43: R = nC7H15O- IC50 = 0.5 nM 44: ONO-1078, pranlukast IC50 = 0.044 nM
O
N
H
O
NN
N
O
O
N
N
N
O
O
N
H
R
O
NH
(CH2)4NH
Figure 9.16 Impact of varying the amide region.
228j9 The Development of Cysteinyl Leukotriene Receptor Antagonists
9.9
Comparative Analysis and Crossover Impact
All three of the marketed agents are potent, selective, orally active, CysLT1R antagonists
that, since they neither look like – nor were derived from – each other, are
pharmacological and not chemical analogues. However, they are all amphiphilic
compounds with distinct lipophilic and acidic regions.
Their rank order of potency (see Table 9.1) varies depending on which criteria are
used. For functional in vitro efficacy (inhibition of Ca2þ
mobilization), the order is
pranlukast > zafirlukast ) montelukast; for in vitro binding to the hCysLT1R, it is
zafirlukast ! pranlukast $ montelukast, whereas for clinical efficacy [daily dose]
it is montelukast > zafirlukast ) pranlukast. These results indicate that
Table 9.1 Comparison of clinical agents.
Montelukast sodium Zafirlukast Pranlukast
IC50 (nM) inh. of LTD4-induced Ca2þ
in
HEK-293-CysLTR cells [38]
2.3 0.26 0.1
IC50 (nM) inh. of LTD4 binding in HEK-
293-CysLTR cells [38]
4.9 Æ 1.2 1.8 Æ 0.7 4.4 Æ 0.9
Recommended adult dose 10 mg q.d. 20 mg b.i.d. 225 mg b.i.d.
Food interaction in label No Yes Yes
MW 585 (as free acid) 575 480
clogP (ACD) 7.8 6.15 3.77
Plasma protein binding >99 [80] >99 [80] >98.9 [81]
Rule of 5 violations 2 2 0
45: YM-17690
an agonist
Overlap of an exemplar
Ono antagonist (solid lines)
with LTE4 (dotted lines)
OH
OOH
S
NH2
O
OH
O
N
H
O
NN
N
O
O O
N
H
O
O
NH
CO2H
CO2H
Figure 9.17 Postulated overlap of the amino-chromone series and LTE4. Figure adapted from
illustration in Ref. [77].
9.9 Comparative Analysis and Crossover Impact j229
a disconnection exists between in vitro and in vivo potency. This can be fully
explained only by a detailed DMPK analysis that is beyond the scope of this
work. In part, this differential translation may be explained by their protein
binding. The impact of plasma protein binding (PPB) on relative in vivo efficacy
had been underappreciated at the time of the CysLT1R discovery effort,
but has since been highlighted [78]. Since all three are >$99% bound, differences
in the reported PPB values do not explain the divergence. However, it is
possible that M is $99.1% bound, whereas the other two agents are >99.9%
bound and this would then impact their relative potency. Support for this possibility
is provided by a second set of hCysLT1R binding studies that described
the impact of the addition of 0.06% human serum albumin (HSA) [79]. In
that study, the IC50 (nM) values for montelukast/pranlukast/zafirlukast change
upon addition of HSA from 3.3/5.6/2.2 to 1.9/72/5.4. The latter numbers
roughly approximate the ratios of their orally efficacious doses in humans.
In contrast to montelukast and zafirlukast, only pranlukast, which is by far the
least potent orally, meets Lipinski’s “rule of 5” for orally efficacious agents [82].
Both montelukast and zafirlukast fail due to their MW and extremely high lipophilicity.
Given that “rule of 5” violations do not preclude oral efficacy but rather indicate
it is less likely, and that most of the other clinically tested but failed CysLT1R
antagonists reported in the review by Brooks and Summers [18] do fail, these two
compounds are “exceptions” for unknown reasons.
Montelukast, unlike zafirlukast or pranlukast, is indicated for once-daily dosing.
However, it should be noted that the full prescribing information states “once daily
in the evening”.6)
This is an important distinction because asthma is a diurnal disease
and many individuals suffer from nocturnal asthma, or asthma worsening
overnight. The importance of the time of dose for asthma medications was first
demonstrated in a key study on oral corticosteroid treatment [83]. Oral corticosteroids
are considered the most effective antiasthmatic agents, used for the most
difficult cases. This study showed that oral prednisolone was only effective once
daily when taken at 15:00 h. When it was taken at either 08:00 or 20:00 h, it was not
effective once daily.
Although the three agents discussed in this chapter are pharmacological and
not chemical analogues, the efforts by each of the teams and also by groups that
did not produce a clinically registered compound did impact their development.
Following the limited clinical efficacy demonstrated by LY-171883 and L-648051
(28), doubts about the role of cysteinyl leukotrienes in asthma slowed the development
of ICI-204219 (26). Then, the clinical results with MK-0571 (33) showed
clear efficacy for a CysLT1R antagonist in asthma. Since by many measures ICI-
204219 was superior to MK-0571 (intrinsic potency and projected clinical dose),
this reinvigorated its development program. Similarly, when first MK-0571 and
6) Singulair1
(montelukast sodium) prescribing information. Available at http://www.
accessdata.fda.gov/drugsatfda_docs/label/2011/020829s057,020830s059,021409s034lbl.pdf
(accessed November 9, 2011).
230j9 The Development of Cysteinyl Leukotriene Receptor Antagonists
then its single isomer MK-0679 (34) both raised liver safety concerns in latestage
studies, there was concern at Merck over whether the “right compound”
could be found. Then, the results of ICI-204219 clinical studies became available
and they inspired Merck to continue to seek an agent that could be superior
by needing only once-daily dosing.
That only 3 of the 19 clinical candidates reported in the review by Brooks
made it to the market illustrates how difficult it was to successfully develop
CysLT1R antagonists. The lack of information on why many compounds were
stopped in development precludes a detailed analysis of the causes. However,
the fact that two of the three registered compounds violate the “rule of 5”
implies that competitive antagonists of the CysLT1R may populate the region of
medicinal chemistry that is relatively “unfriendly” to orally active agents. Also,
several of the compounds reported were being developed via aerosol administration,
so the success of the oral agents would have negatively impacted the commercial
viability of this subset.
9.10
Postmarketing Issues
In February 1998, a report described eosinophilia following corticosteroid withdrawal
in patients with asthma receiving zafirlukast [84]. The report proposed that
the eosinophilia represented a type of Churg–Strauss syndrome (CSS) that might
be linked to antileukotriene therapy and/or zafirlukast specifically. CSS is a rare
but serious vasculitis that is considered to develop in a phasic pattern over time,
which can initially present as asthma [85]. Because of the seriousness of this disease
and the potential for rare side effects to be uncovered only after a drug has
gone out to a larger population, significant effort has gone into clarifying the relationship
between CysLT1R antagonist therapy and CSS.7)
Following this initial
report, there have been numerous additional reports associating not only zafirlukast,
but also montelukast and pranlukast to CSS [86]. By 2002, the data appeared
to be most consistent with the association being the unmasking of a previously
contained condition, or that it was part of the natural progression of the disease
that had occurred because the use of CysLT1R antagonists allowed the avoidance of
systemic steroids [86]. This viewpoint was strengthened by the association of newer
high-potency inhaled corticosteroids (e.g., fluticasone), that also allowed for reduction
in systemic corticosteroids, with cases of CSS [86]. By 2005, the data had
swung back to being suggestive of a causal linkage, but not a definitive association,
between CysLT1R antagonist therapy and development of CSS [85]. The complexity
of the question is indicated by the fact that at this time, more than 10 years since
the first report, the potential association remains unclear [87]. FDA approved
7) A search of the PubMed database for “Churg–Strauss and leukotriene” on November 9, 2011,
resulted in 129 publications.
9.10 Postmarketing Issues j231
prescribing information for the two agents available in the United States, zafirlu-
kast8)
and montelukast;6
both cite the presentation of eosinophilia consistent with
Churg–Strauss in their “warnings” sections.
More perplexing, both zafirlukast and montelukast have been associated with
neuropsychiatric events (e.g., depression) and list this in the “warnings” sections
of their prescribing information. The linkage between CysLT1R antagonist therapy
and neuropsychiatric events is even less clear than that for CSS. This is because
neither agent is thought to cross the blood–brain barrier and therefore any effect
would need to be indirectly mediated. However, the potential linkage to leukotriene
modulation is indicated by the fact that when the FDA asked for this warning it also
asked for a similar change in the label for zileuton,9)
the only clinically approved LT
biosynthesis inhibitor (a 5-LO inhibitor).
While the labels for both zafirlukast and montelukast list liver injury in their
“adverse reactions” sections, zafirlukast also includes hepatoxicity in its “warnings”
section.
9.11
Conclusions
Multiple studies have concluded that introduction of the CysLT1R antagonist class
of medications has been of significant benefit to the treatment of asthma. Like all
other antiasthma treatments, there is variability of response to them, across broad
patient groups. Despite efforts to more accurately define good responders, the only
definitive conclusion is that individuals with aspirin-induced asthma tend to be
very strong responders. Exploration of the potential utility of these agents in other
diseases has led to the licensing of montelukast for the treatment of rhinitis. The
potential role for antagonists of the CysLT2R and the potential role for a CysLT3R
remain areas of active research.
Acknowledgment
The author thanks Dr. Fred J. Brown (AstraZeneca, retired) for his careful proofreading
of this chapter.
Disclaimer
The opinions expressed are those of the author alone based on the listed citations.
They do not represent the opinion of, or an endorsement by, his former employer,
AstraZeneca Pharmaceuticals.
8) Accolate1
(zafirlukast) prescribing information. Available at http://www.accessdata.fda.gov/
drugsatfda_docs/label/2011/020547s031lbl.pdf (accessed November, 2011).
9) FDA warning update. Available at http://www.fda.gov/drugs/drugsafety/postmarketdrug
safetyinformationforpatientsandproviders/drugsafetyinformationforheathcareprofessionals/
ucm165489.htm (accessed November 10, 2011).
232j9 The Development of Cysteinyl Leukotriene Receptor Antagonists
References
1 Jones, T.R. and Rodger, I.W. (1999) Role of
leukotrienes and leukotriene receptor
antagonists in asthma. Pulm. Pharmacol.
Ther., 12, 107–110.
2 Bernstein, P.R. (1992) Antiasthmatic
agents, in Kirk-Othmer’s Encyclopedia of
Chemical Technology, vol. 2, 4th edn, John
Wiley & Sons, Inc., New York, pp. 830–854.
3 Montuschi, P. (2008) Leukotrienes,
antileukotrienes and asthma. Mini-Rev.
Med. Chem., 8, 1–10.
4 Montuschi, P. and Peters-Golden, M.L.
(2010) Leukotriene modifiers for asthma
treatment. Clin. Exp. Allergy, 40,
1732–1741.
5 Kellaway, C.H. and Trethewie, E.R. (1940)
The liberation of a slow reacting
substance, smooth muscle stimulating
substance in anaphylaxis. Q. J. Exp.
Physiol., 30, 121–145.
6 Augstein, J., Farmer, J.B., Lee, T.B.,
Sheard, P., and Tattersall, M.L. (1973)
Selective inhibitor of slow-reacting
substance of anaphylaxis. Nat. New Biol.,
245, 215–217.
7 Shapiro, G.G. and Konig, P. (1985)
Cromolyn sodium: a review.
Pharmacotherapy, 5 (3), 156–170.
8 Sheard, P., Lee, T.B., and Tattersall, M.L.
(1977) Further studies on the SRS-A
antagonist FPL 55712. Monographs in
Allergy: Mediators of Immediate Type
Inflammatory Reaction, 11th Symposium of
the Collegium Internationale Allergologicum,
vol. 12, pp. 245–249.
9 Piper, P.J. (1984) Biological actions of the
leukotrienes, in The Leukotrienes Chemistry
and Biology (eds L.W. Chakrin and D.M.
Bailey), Academic Press, New York,
pp. 215–230.
10 Murphy, R.C., Hammarstr€om, S., and
Samuelsson, B. (1979) Leukotriene C: a
slow reacting substance from murine
mastocytoma cells. Proc. Natl. Acad. Sci.
USA, 76, 4275–4279.
11 Drazen, J.M., Austen, K.F., Lewis, R.A.,
Clark, D.A., Goto, G., Marfat, A., and
Corey, E.J. (1980) Comparative airway and
vascular activity of leukotriene C-1 and D
in vivo and in vitro. Proc. Natl. Acad. Sci.
USA, 77, 4345–4358.
12 Marom, Z., Shelhamer, J.H., Bach, M.K.,
Morton, D.R., and Kaliner, M. (1982)
Slow-reacting substances, leukotriene
C4and D4 increase the release of mucus
from human airways. Am. Rev. Respir. Dis.,
126, 449–451.
13 Barnes, N.C., Piper, P.J., and Costello, J.F.
(1984) Comparative actions of inhaled
leukotriene C4, leukotriene D4 and
histamine in normal human subjects.
Thorax, 39, 500–504.
14 Bernstein, P.R., Bird, T.G., and Brewster,
A.G. (1997) Agents affecting the actions of
leukotrienes and thromboxanes, in
Burger’s Medicinal Chemistry and Drug
Discovery, vol. 5: Therapeutic Agents, 5th
edn (ed. M.E. Woff), John Wiley & Sons,
Inc., New York, pp. 405–493.
15 Musser, J.H. (1989) Leukotriene D4
receptor antagonists. Drug News Perspect.,
2, 202–213.
16 von Sprecher, A., Beck, A., Gerspacher,
M., and Bray, M.A. (1992)
Peptidoleukotriene antagonists: state of
the art. Chimia, 46, 304–311.
17 Ford-Hutchison, A., Young, R., and
Gillard, J. (1991) Leukotriene blockers,
novel therapeutic strategies for the
treatment of asthma. Drug News Perspect.,
4, 264–271.
18 Brooks, C.D.W. and Summers, J.B. (1996)
Modulators of leukotriene biosynthesis
and receptor activation. J. Med. Chem., 39,
2629–2654.
19 Piper, P.J. and Seale, J.P. (1979) Nonimmunological
release of slow-reacting
substance from guinea-pig lungs. Br. J.
Pharmacol., 67, 67–72.
20 Orange, R.P., Murphy, R.C., Karnovsky,
M.L., and Austen, K.F. (1973) The
physicochemical characteristics and
purification of slow reacting substance of
anaphylaxis. J. Immunol., 110, 760–770.
21 Rokach, J., Zamboni, R., Lau, C.K., and
Guindon, Y. (1981) A C-gycloside route
to leukotrienes. Tetrahedron Lett., 22,
2759–2762.
22 Gleason, J.G., Bryan, D.B., and Kinzig, C.
M. (1980) Convergent synthesis of
leukotriene A methyl ester. Tetrahedron
Lett., 21, 1129–1132.
References j233
23 Ernest, I., Main, A.J., and Menasse, R.
(1982) Synthesis of the 7-cis isomer of the
natural leukotriene D4. Tetrahedron Lett.,
23, 167–170.
24 Cohen, N., Banner, B.L., and Lopresti,
R.C. (1980) Synthesis of optically active
leukotriene (SRS-A) intermediates.
Tetrahedron Lett., 21, 4163–4166.
25 Marriott, D.P. and Bantick, J.R. (1981)
5(S),6(R)-5,7-Dibenzoyloxy-6hydroxyheptanoate
ester: improved
synthesis of a leukotriene intermediate.
Tetrahedron Lett., 22, 3657–3658.
26 Foster, A., Fitzsimmons, B., Rokach, J.,
and Letts, L.G. (1987) Metabolism and
excretion of peptide leukotrienes in the
anesthetized rat. Biochim. Biophys. Acta,
921, 486–493.
27 Lynch, K.R., O’Neill, G.P., Liu, Q., Im,
D.S., Sawyer, N., Metters, K.M.,
Coulombe, N., Abramovitz, M., Figueroa,
D.J., Zeng, Z., Connolly, B.M., Bai, C.,
Austin, C.P., Chateauneu, A., Stocco, R.,
Greig, G.M., Kargman, S., Hooks, S.B.,
Hosfield, E., Williams, D.L., Jr., FordHutchinson,
A.W., Caskey, C.T., and
Evans, J.F. (1999) Characterization of the
human cysteinyl leukotriene CysLT1
receptor. Nature, 399 (6738), 789–793.
28 Dixon, R.A.F., Jones, R.E., Diehl, R.E.,
Bennett, C.D., Kargman, S., and Rouzer,
C.A. (1988) Cloning of the cDNA for
human 5-lipoxygenase. Proc. Natl. Acad.
Sci. USA, 85, 416–420.
29 Miller, D.K., Gillard, J.W., Vickers, P.J.,
Sadowski, S., Leveille, C., Mancini, J.A.,
Charleston, P., Dixon, R.A.F., FordHutchinson,
A.W., Fortin, R., Gauthier,
J.Y., Rodkey, J., Rosen, R., Rouzer, C.A.,
Sigal, I.S., Strader, C.D., and Evans, J.F.
(1990) Identification and isolation of a
membrane protein necessary for
leukotriene production. Nature, 343,
278–281.
30 Dixon, R.A.F., Diehl, R.E., Opas, E.,
Rands, E., Vickers, P.J., Evans, J.F.,
Gillard, J.W., and Miller, D.K. (1990)
Requirement of a 5-lipoxygenaseactivating
protein for leukotriene
synthesis. Nature, 343, 282–284.
31 Tsai, B.S., Bernstein, P.R., Macia, R.A.,
Conaty, J., and Krell, R.D. (1982)
Comparative potency and pharmacology
of isomers of leukotriene D4 on guinea
pig trachea: requirement for a 5(S),6(R)
configuration. Prostaglandins, 23, 489–506.
32 Loftus, P. and Bernstein, P.R. (1983) A
study of some leukotriene A4 and D4
analogues by proton NMR spectroscopy.
J. Org. Chem., 48, 40–44.
33 Patterson, R., Harris, K.E., Smith, L.J.,
Greenberger, P.A., Shaughnessy, M.A.,
Bernstein, P.R., and Krell, R.D. (1983)
Airway response to leukotriene D4 in
rhesus monkeys. Int. Arch. Allergy Appl.
Immunol., 71, 156–160.
34 Buckner, C.K., Krell, R.D., Laravuso, R.B.,
Coursin, D.B., Bernstein, P.R., and Will,
J.A. (1986) Pharmacological evidence that
human intralobar airways do not contain
different receptors that mediate
contractions to leukotriene C4 and
leukotriene D4. J. Pharmacol. Exp. Ther.,
237, 558–562.
35 Smith, L.J., Greenberger, P.A., Patterson,
R., Krell, R.D., and Bernstein, P.R. (1985)
The effect of inhaled leukotriene D4 in
humans. Am. Rev. Respir. Dis., 131,
368–372.
36 Kern, R., Smith, L.J., Patterson, R., Krell,
R.D., and Bernstein, P.R. (1986)
Characterization of the airway response to
inhaled LTD4 in normal subjects. Am. Rev.
Respir. Dis., 133, 1127–1132.
37 Aharony, D., Falcone, R.C., and Krell, R.D.
(1987) Inhibition of 3H-leukotriene D4
binding to guinea pig lung receptors by
the novel leukotriene antagonist ICI
198615. J. Pharmacol. Exp. Ther., 243,
921–926.
38 Sarau, H.M., Ames, R.S., Chambers, J.,
Ellis, C., Elshourbagy, N., Foley, J.J.,
Schmidt, D.B., Muccitelli, R.M., Jenkins,
O., Murdock, P.R., Herrity, N.C., Halsey,
W., Sathe, G., Muir, A.I., Nuthulaganti, P.,
Dytko, G.M., Buckley, P.T., Wilson, S.,
Bergsma, D.J., and Hay, D.W.P. (1999)
Identification, molecular cloning,
expression, and characterization of a
cysteinyl leukotriene receptor. Mol.
Pharmacol., 56, 657–663.
39 Krell, R.D., Tsai, B.-S., Berdoulay, A.,
Barone, M., and Giles, R.E. (1983)
Heterogeneity of leukotriene receptors in
guinea-pig trachea. Prostaglandins, 25,
171–178.
234j9 The Development of Cysteinyl Leukotriene Receptor Antagonists
40 Nicosia, S., Capra, V., Ravasi, S., and
Rovati, G.E. (2000) Binding to cysteinylleukotriene
receptors. Am. J. Respir. Crit.
Care Med., 161, S46–S50.
41 Back, M., Dahlen, S.-E., Drazen, J.M.,
Evans, J.F., Serhan, C.N., Shimizu, T., and
Yokomizo, T., and Rovati, G.E. (2011)
International union of basic and clinical
pharmacology. LXXXIV. Leukotriene
receptor nomenclature, distribution, and
pathophysiological functions. Pharmacol.
Rev., 63, 539–584.
42 Austen, K.F., Maekawa, A., Kanaoka, Y.,
and Boyce, J.A. (2009) The leukotriene E4
puzzle: finding the missing pieces and
revealing the pathobiologic implications.
J. Allergy Clin. Immunol., 124 (3), 406–414.
43 Snyder, D.W. and Krell, R.D. (1984)
Pharmacological evidence for a distinct
leukotriene C4 receptor in guinea-pig
trachea. J. Pharmacol. Exp. Ther., 231,
616–622.
44 Heise, C.E., O’Dowd, B.F., Figueroa, D.J.,
Sawyer, N., Nguyen, T., Im, D.S., Stocco,
R., Bellefeuille, J.N., Abramovitz, M.,
Cheng, R., Williams, D.L., Jr., Zeng, Z.,
Liu, Q., Ma, L., Clements, M.K.,
Coulombe, N., Liu, Y., Austin, C.P.,
George, S.R., O’Neill, G.P., Metters, K.M.,
Lynch, K.R., and Evans, J.F. (2000)
Characterization of the human cysteinyl
leukotriene 2 receptor. J. Biol. Chem., 275,
30531–30536.
45 Nothacker, H.P., Wang, Z., Zhu, Y.,
Reinscheid, R.K., Lin, S.H., and Civelli, O.
(2000) Molecular cloning and
characterization of a second human
cysteinyl leukotriene receptor: discovery of
a subtype selective agonist. Mol.
Pharmacol., 58 (6), 1601–1608.
46 Snyder, D.W., Liberati, N.J., and
McCarthy, M.M. (1988) Conscious guineapig
aerosol model for evaluation of
peptide leukotriene antagonists. J.
Pharmacol. Methods, 19 (3), 219–231.
47 Bernstein, P.R. (1997) The challenge of
drug discovery: developing leukotriene
antagonists, in SRS-A to Leukotrienes: The
Dawning of a new treatment (eds S. Holgate
and S.-E. Dahlen), Blackwell Science,
Oxford, UK, pp. 171–186.
48 Bernstein, P.R. (1998) The Development
of Accolate: A Peptide Leukotriene
Antagonist for the Treatment of Asthma.
Course Handouts, Residential School on
Medicinal Chemistry, Drew University,
Madison, NJ.
49 Brown, F.J. (1995) Discovery of
AccolateTM
(ICI 204, 219), a peptide
leukotriene antagonist for asthma, in The
Search for Anti-inflammatory Drugs (eds
V.J. Merluzzi and J. Adams), Birkhauser,
Boston, MA, pp. 161–189.
50 Brown, F.J., Bernstein, P.R., Cronk, L.A.,
Dosset, L.L., Hebbel, K.C., Maduskuie,
T.P., Shapiro, H.S., Vacek, E.P., Yee, Y.K.,
Willard, A.K., Krell, R.D., and Snyder,
D.W. (1989) Hydroxyacetophenone-derived
antagonists of the peptidoleukotrienes.
J. Med. Chem., 32, 807–826.
51 Mann., A. (2003) Conformational
restrictions and/or steric hindrance in
medicinal chemistry, in The Practice of
Medicinal Chemistry, 2nd edn (ed. C.G.
Wermuth), Academic Press, London, UK,
pp. 233–250.
52 Bernstein, P.R., Snyder, D.W., Adams,
E.J., Krell, R.D., Vacek, E.P., and Willard,
A.K. (1986) Chemically stable homocinnamyl
analogs of the leukotrienes;
synthesis and preliminary biological
evaluation. J. Med. Chem., 29, 2477–2483.
53 Snyder, D.W. and Bernstein, P.R. (1987)
Pharmacologic profile of chemically stable
analogs of peptide leukotrienes. Eur. J.
Pharm., 138, 397–405.
54 Bernstein, P.R., Vacek, E.P., Adams, E.J.,
Snyder, D.W., and Krell, R.D. (1988) The
synthesis and pharmacological
characterization of a series of leukotriene
analogues with antagonist and agonist
activities. J. Med. Chem., 31, 692–696.
55 Brown, F.J., Yee, Y.K., Hebbel, K.C.,
Cronk, L.A., Krell, R.D., and Snyder,
D.W. (1990) Evolution of a series of
peptidoleukotriene antagonists:
synthesis and structure–activity
relationships of 1,6-disubstituted
indoles and indazoles. J. Med. Chem.,
33, 2437–2351, 1771–1781.
56 Yee, Y.K., Bernstein, P.R., Adams, E.J.,
Brown, F.J., Cronk, L.A., Hebbel, K.C.,
Vacek, E.P., Krell, R.D., and Snyder, D.W.
(1990) A novel series of leukotriene
antagonists: exploration and optimization
of the acidic region in 1,6-disubstituted
References j235
indoles and indazoles. J. Med. Chem., 33,
2437–2351.
57 Fleisch, J.H., Rinkema, L.E., Haisch, K.D.,
Swanson-Bean, D., Goodson, T., Ho.,
P.P.K., and Marshall, W.S. (1985) 1-[2-
Hydroxy-3-propyl-4-([4-(1H-tetrazol-5-
ylmethyl)phenoxy]methyl)phenyl]
ethanone, an orally active leukotriene D4
antagonist. J. Pharmacol. Exp. Ther.,
233 (1), 148–157.
58 Brown, F.J., Cronk, L.A., Aharony, D., and
Snyder, D.W. (1992) 1,3,6-Trisubstituted
indoles as peptidoleukotriene antagonists:
benefits of a second, polar, pyrrole
substituent. J. Med. Chem., 35, 2419–2439.
59 Matassa, V.G., Brown, F.J., Bernstein,
P.R., Shapiro, H.S., Maduskuie, T.P., Jr.,
Cronk, L.A., Vacek, E.P., Yee, Y.K., Snyder,
D.W., Krell, R.D., Lerman, C.L., and
Maloney, J.J. (1990) Synthesis and in vitro
LTD4 antagonist activity of bicyclic and
monocyclic cyclopentylurethane and
cyclopentylacetamide N-arylsulfonyl
amides. J. Med. Chem., 33, 2621–2629.
60 Matassa, V.G., Maduskuie, T.P., Jr.,
Shapiro, H.S., Hesp, B., Snyder, D.W.,
Aharony, D., Krell, R.D., and Keith, R.A.
(1990) Evolution of a series of
peptidoleukotriene antagonists: synthesis
and structure/activity relationships of
1,3,5-substituted indoles and indazoles. J.
Med. Chem., 33, 1781–1790.
61 Young, R.N. (2007) Discovery of
Singulair1
and its impact on treatment of
asthma in children and adults. Book of
Abstracts. The 234th ACS National
Meeting, Boston, MA, August 19–23,
2007, MEDI 214.
62 Rodger, I.W. (2000) From bench to
bedside: the hurdles of discovering a new
leukotriene receptor antagonist. Am. J.
Respir. Crit. Care Med., 161, S7–S10.
63 Jones, T.R., Young, R., Champion, E.,
Charette, L., Denis, D., Ford-Hutchinson,
A.W., Frenette, R., Gauthier, J.Y.,
Guindon, Y., Kakushima, M., Masson, P.,
McFarlane, C., Piechuta, H., Rokach, J.,
and Zamboni, R. (1986) L-649,923,
sodium (beta SÃ
, gamma RÃ
)-4-(3-
(4-acetyl-3-hydroxy-2-propylphenoxy)
propylthio)-gamma-hydroxy-betamethylbenzenebutanoate,
a selective,
orally active leukotriene receptor
antagonist. Can. J. Physiol. Pharmacol., 64,
1068–1075.
64 Jones, T.R., Guindon, Y., Champion, E.,
Charette, L., De Haven, R.N., Denis, D.,
Ethier, D., Ford-Hutchinson, A.W., Fortin,
R., Frenette, R. et al. (1987) L-648,051: an
aerosol active leukotriene D4 receptor
antagonist. Adv. Prostaglandin
Thromboxane Res., 17, 1012–1017.
65 Zamboni, R., Belley, M., Champion, E.,
Charette, L., DeHaven, R., Frenette, R.,
Gauthier, J.Y., Jones, T.R., Leger, S.,
Masson, P., McFarlane, C.S., Metters, K.,
Pong, S.S., Piechuta, H., Rokach, J.,
Therien, M., Williams, H.W.R., and
Young, R.N. (1992) Development of a
novel series of styrylquinoline compounds
as high-affinity leukotriene D4 receptor
antagonists: synthetic and structure–
activity studies leading to the discovery of
(Æ)-3-[[[3[2-(7-chloro-2-quinolinyl)-(E)-
ethenyl]phenyl][[3-(dimethylamino)3oxopropyl]thio]methyl]thio]propionic
acid.
J. Med. Chem., 35, 3832–3844.
66 Young, R.N. (1989) The development of
new anti-leukotriene drugs L-648,501 and
L-649,923 specific leukotriene D4
antagonists. Drugs Future, 13, 745–759.
67 (a) Van Inwegen, R.G., Khandwala, A.,
Gordon, R., Sonnino, P., Coutta, S., and
Jolly, S., (1987) REV 5901: an orally
effective peptidoleukotriene antagonist,
detailed biochemical/pharmacological
profile. J. Pharmacol. Exp. Ther., 241,
117–124. (b) Musser, J.H., Kubrak, D.M.,
Chang, J., and Lewis, J. (1986) Synthesis
of [[(naphthalenylmethoxy)- and
[[(quinolinylmethoxy)phenyl]amino]
oxoalkanoic acid esters. A novel series of
leukotriene D4 antagonists and 5lipoxygenase
inhibitors. J. Med. Chem., 29,
1429–1435.
68 Musser, J.H., Chakraborty, U.R.,
Sciortino, S., Gordon, R.J., Khandwala, A.,
Neiss, E.S., Pruss, T.P., Van Inwegen, R.,
Weinryb, I., and Coutta, S.M. (1987)
Substituted arylmethyl phenyl ethers. 1. A
novel series of 5-lipoxygenase inhibitors
and leukotriene antagonists. J. Med.
Chem., 30, 96–104.
69 Perchonock, C.D., McCarthy, M.E.,
Erhard, K.F., Gleason, J.G., Wasserman,
M.A., Muccitelli, R.M., DeVan, J.F.,
236j9 The Development of Cysteinyl Leukotriene Receptor Antagonists
Tucker, S.S., Vickery, L.M., Kirchner, T.,
Weichman, B.M., Mong, S., Crooke, S.T.,
and Newton, J.F. (1986) Synthesis and
pharmacological characterization of 5-(2dodecylphenyl)-4,6-dithianonanedioic
acid
and 5-[2-(8-phenyloctyl)-phenyl-4,6dithianonanedioic
acid: prototypes of a
novel class of leukotriene antagonists. J.
Med. Chem., 28, 1145–1147.
70 Lewis, R.A., Drazen, J.M., Austen, K.F.,
Toda, M., Brion, F., Marfat, A., and Corey,
E.J. (1981) Contractile activities of
structural analogs of leukotriene C and D:
role of the polar substituents. Proc. Natl.
Acad. Sci. USA, 78, 4579–4583.
71 Labelle, M., Belley, M., Gareau, Y.,
Gauthier, J.Y., Guay, D., Gordon, R.,
Grossman, S.G., Jones, T.R., LeBlanc, Y.,
McAuliffe, M., McFarlane, C., Masson, P.,
Metters, K.M., Ouimet, N., Patrick, D.H.,
Piechuta, H., Rochette, C., Sawyer, N.,
Xiang, Y.B., Pickett, C.B., FordHutchinson,
A.W., Zamboni, R.J., and
Young, R.N. (1995) Discovery of MK-0476,
a potent and orally active leukotriene D4
receptor antagonist devoid of peroxisomal
enzyme induction. Bioorg. Med. Chem.
Lett., 5, 283–288.
72 Labelle, M., Prasit, P., Belley, M., Blouin,
M., Champion, E., Charette, L., DeLuca,
J.G., Dufresne, C., Frenette, R., Gauthier,
J.Y., Grimm, E., Grossman, S.G., Guay, D.,
Herold, E.G., Jones, T.R., Lau, C.K.,
Leblanc, Y., Leger, S., Lord, A., McAuliffe,
M., McFarlane, C., Masson, P., Metters,
K.M., Ouimet, N., Patrick, D.H., Perrier,
H., Pickett, C.B., Piechuta, H., Roy, P.,
Williams, H., Wang, Z., Xiang, Y.B.,
Zamboni, R.J., Ford-Hutchinson, A.W.,
and Young, R.N. (1992) The discovery of a
new structural class of potent orally active
leukotriene D4 antagonists. Bioorg. Med.
Chem. Lett., 2, 1141–1146.
73 Labelle, M., Belley, M., Champion, E.,
Gordon, R., Jones, T.R., Leblanc, Y.,
McAuliffe, M., McFarlane, C., Masson,
P., Metters, K.M., Nicoll-Griffith, D.,
Ouimet, N., Piechuta, H., Rochette, C.,
Sawyer, N., Xiang, Y.B., Yergey, J., FordHutchinson,
A.W., Pickett, C.B.,
Zamboni, R.J., and Young, R.N. (1994)
The discovery of L-699,392, a novel
potent and orally active leukotriene D4
receptor antagonist. Bioorg. Med. Chem.
Lett., 4, 463–468.
74 Jones, T.R., Labelle, M., Belley, M.,
Champion, E., Charette, L., Evans, J.,
Ford-Hutchinson, A.W., Gauthier, J.-Y.,
Lord, A., Masson, P., McAuliffe, M.,
McFarlane, C.S., Metters, K.M., Pickett,
C., Piechuta, H., Rochette, C., Rodger,
I.W., Sawyer, N., Young, R.N., Zamboni,
R., and Abraham, W.M. (1995)
Pharmacology of montelukast sodium
(SingulairTM
), a potent and selective
leukotriene D4 receptor antagonist.
Can. J. Physiol. Pharmacol., 73 (2), 191–201.
75 Toda, M., Nakai, H., Kosuge, S., Konno,
M., Arai, Y., Miyam-oto, T., Obata, T.,
Katsube, A., and Kawasaki, A. (1985) A
potent antagonist of the slow-reacting
substance of anaphylaxis. Adv.
Prostaglandin Thromboxane Leukot. Res.,
15, 307–308.
76 Nakai, H., Konno, M., Kosuge, S.,
Sakuyama, S., Toda, M., Arai, Y., Obata, T.,
Katsube, N., Miyamoto, T., Okegawa, T.,
and Kawasaki, A. (1988) New potent
antagonists of leukotriene C4 and D4 1.
Synthesis and structure–activity
relationships. J. Med. Chem., 31, 84–91.
77 Goto, S., Guo, Z., Futatsuishi, Y., Hori, H.,
Taira, Z., and Terada, H. (1992)
Quantitative structure–activity
relationships of benzamide derivatives for
anti-leukotriene activities. J. Med. Chem.,
35, 2440–2445.
78 Trainor, G.L. (2007) Plasma protein
binding and the free drug principle:
recent developments and applications.
Annu. Rep. Med. Chem., 42, 489–502.
79 Lynch, K.R., O’Neill, G.P., Liu, Q., Im,
D.S., Sawyer, N., Metters, K.M.,
Coulombe, N., Abramovitz, M., Figueroa,
D.J., Zeng, Z., Connolly, B.M., Bai, C.,
Austin, C.P., Chateauneuf, A., Stocco, R.,
Greig, G.M., Kargman, S., Hooks, S.B.,
Hosfield, E., Williams, D.L., Jr.,
Ford-Hutchinson, A.W., Caskey, C.T., and
Evans, J.F. (1999) Characterization of the
human cysteinyl leukotriene CysLT1
receptor. Nature, 399, 789–793.
80 Thummel, K.E. and Shen, D.D. (2001)
Plasma protein binding, in Goodman &
Gilman’s The Pharmacological Basis of
Therapeutics, 10th edn (eds J.G. Hardman,
References j237
L.E. Limbird, and A.G. Gilman), McGrawHill,
New York, pp. 1917–2023.
81 Ishido, M., Shibakawa, K., Takamoto, M.,
Kajiwara, I., Sawada, M., and Aishita, H.
(1993) Studies on the metabolic fate of
leukotriene antagonist ONO-1078 (4):
metabolism and protein binding.
Yakubutsudoutai, 8, 49–66.
82 Lipinski, C.A., Lombardo, F., Dominy,
B.W., and Feeney, P.J. (1997)
Experimental and computational
approaches to estimate solubility and
permeability in drug discovery and
development settings. Adv. Drug Deliv.
Rev., 23, 3–25.
83 Beam, W.R., Weiner, D.E., and Martin,
R.J. (1992) Timing of prednisone and
alterations of airways inflammation in
nocturnal asthma. Am. Rev. Respir. Dis.,
146 (6), 1524–1530.
84 Wechsler, M.E., Garpestad, E., Flier, S.R.,
Kocher, O., Weiland, D.A., Polito, A.J.,
Klinek, M.M., Bigby, T.D., Wong, G.A.,
Helmers, R.A., and Drazen, J.M. (1998)
Pulmonary infiltrates, eosinophilia, and
cardiomyopathy following corticosteroid
withdrawal in patients with asthma
receiving zafirlukast. J. Am. Med. Assoc.,
279 (6), 455–7.
85 McDanel, D.L. and Muller, B.A. (2005)
The linkage between Churg–Strauss
syndrome and leukotriene receptor
antagonists: fact or fiction? Ther. Clin. Risk
Manage., 1 (2), 125–40.
86 Lilly, C.M., Churg, A., Lazarovich, M.,
Pauwels, R., Hendeles, L., Rosenwasser,
L.J., Ledford, D., and Wechsler, M.E.
(2002) Asthma therapies and ChurgStrauss
syndrome. J. Allergy Clin.
Immunol., 109 (1), 1–19.
87 Bibby, S., Healy, B., Steele, R.,
Kumareswaran, K., Nelson, H., and
Beasley, R. (2010) Association between
leukotriene receptor antagonist therapy
and Churg–Strauss syndrome: an analysis
of the FDA AERS database. Thorax, 65 (2),
132–138.
238j9 The Development of Cysteinyl Leukotriene Receptor Antagonists
Peter R. Bernstein received a B.S. degree in Chemistry from
the University of Rochester, a Ph.D. degree in Organic
Chemistry from Columbia University (advisor Prof. Gilbert
Stork), and was a postdoctoral fellow at the University of
Wisconsin, Madison, with Prof. Barry Trost. He then joined
ICI Pharmaceuticals, Wilmington, DE, and worked, for over
30 years in the pharmaceutical industry, there and at its
successor companies, Zeneca and AstraZeneca. He is the
co-inventor of zafirlukast and is recognized for his work, on
many different targets, that has led to more than 10 clinical candidates. Upon his
retirement in 2010, he formed PhaRmaB LLC as a platform to provide consulting
and training in medicinal chemistry and drug discovery. He now holds several editorial
appointments including Digests Editor for Bioorganic Medicinal Chemistry
Letter and Section Editor for Topics in Drug Design and Discovery for Annual
Reports in Medicinal Chemistry. He has received several honors and awards and
most recently, in 2011, was inducted into the Hall of Fame of the ACS Division of
Medicinal Chemistry.
References j239
Part III
Case Studies
j241
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
10
The Discovery of Dabigatran Etexilate
Norbert Hauel, Andreas Clemens, Herbert Nar, Henning Priepke, Joanne van Ryn,
and Wolfgang Wienen
List of Abbreviations
aPTT activated partial thromboplastin time
ASA acetylsalicylic acid
AT antithrombin
bid bis in die (twice daily)
CrCl creatine clearance
DVT deep venous thrombosis
ECT ecarin clotting time
FDA US Food and Drug Administration
FXa coagulation factor Xa
INR international normalized ratio
LMWH low molecular weight heparin
PAR protease-activated receptor
Pgp permeability glycoprotein
PT prothrombin time
SPAF stroke prevention in patients with atrial fibrillation
TAFI thrombin activatable fibrinolysis inhibitor
THR total hip replacement
TKR total knee replacement
TM thrombomodulin
VKA vitamin K antagonist
VTE venous thromboembolism
10.1
Introduction
Thrombin is a serine protease that plays a central role in thrombosis and hemostasis.
It is a key enzyme in the blood coagulation cascade, exhibiting both pro- and
j243
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
anticoagulant properties [1, 2]. It is generated via proteolytic cleavage from inactive
prothrombin by the serine protease factor Xa (FXa, see Scheme 10.1) in the prothrombinase
complex. Thrombin plays a major role in the initiation and propagation
of thrombotic disease by catalyzing fibrinogen conversion into fibrin and
reinforcing the clot structure by FXIII activation. It also feeds back and activates
the factors V, VIII, and XI of the coagulation cascade, thereby amplifying the coagulation
response. Its activity is downregulated by inhibition via antithrombin (AT).
Thrombin is a very potent agonist of platelet activation and further promotes clot
stabilization by activating thrombin activatable fibrinolysis inhibitor (TAFI),
thereby reducing endogenous fibrinolytic capacities [3]. By activating protein C,
thrombin also prevents unnecessary progression of a thrombus once intact endothelium
in vessels is encountered [2].
In addition, thrombin induces many cellular effects, including cell proliferation,
cytokine release, and tissue remodeling [4–6]. These cellular effects of thrombin are
mediated via a family of G-protein-coupled protease-activated receptors (PARs)
PAR1, PAR3, and PAR4 [6–8]. The expression of PAR1 on the endothelium and
vasculature, in both physiological and disease states, suggests that thrombin may
play a role in these pathologies.
For more than 50 years, established anticoagulation therapy has also targeted
thrombin. Heparin has been the mainstay of anticoagulation in both the
prophylaxis and treatment of venous thromboembolism (VTE) [9]. It mainly
reduces thrombin activity by catalyzing the inhibition of thrombin by AT activity.
Though replaced by the low molecular weight heparins (LMWH) in many
indications today, both of these compounds are limited by their parenteral
route of administration for long-term therapy. The use of more targeted direct
Scheme 10.1 Simplified version of coagulation cascade.
244j10 The Discovery of Dabigatran Etexilate
thrombin inhibition has been approved with two other parenteral therapies,
both argatroban (1) for predominantly heparin-induced thrombocytopenia [10]
and the oligopeptide bivalirudin in percutaneous coronary interventions [11].
As the only available oral therapy, warfarin (2) and its derivatives have been
used for over 50 years in long-term anticoagulation treatment [12]. Warfarin, a
vitamin K antagonist (VKA), that blocks the biosynthesis of thrombin and
other coagulation factors has proven a very effective therapy in inhibiting
thrombosis. However, administration is problematic due to its slow onset and
offset of activity and the large number of drug–drug and drug–food interactions,
which require frequent monitoring and dose adjustment in patients to
ensure that appropriate anticoagulation is maintained [13].
Due to the central position of thrombin in the coagulation cascade, research
activities in many pharmaceutical companies have been focused on the identification
of inhibitors of this enzyme. In 2004, ximelagatran (3, AstraZeneca)
[14] gained approval in a number of countries for short-term
anticoagulation treatment after orthopedic surgery. However, in 2006 the drug
was withdrawn from the market due to reports of hepatotoxicity after longterm
application.
NH N
H
N
H
O
N
O
N
H
O
OCH3
OH
S
NH
NH NH2
N
H
O
N
CH3
O OH
NH O O
CH3
O O
O
CH3OH
21
3
Furthermore, a number of companies concentrated their research activities on
the inhibition of FXa [15], thereby blocking the conversion of prothrombin to
thrombin, and recently, three FXa inhibitors – rivaroxaban [16] (4), apixaban [17]
(5), and edoxaban [18] (6) – were approved in different countries.
10.1 Introduction j245
N
O
N
H
O
S
Cl
NO
O
O
N
N
N
O CH3
N
O
O
O
NH2
NH
O
O
N
H
N
N
H
O
N
S
N
O N
Cl
CH3
CH3
CH3
N
N
CH3
O
N
N
H
NH2
NH
N
O
OH
4 5
6
34
This chapter will focus on the design and development of dabigatran (34,
Table 10.3) [19], the first new oral anticoagulant that gained market approval for
long-term indications after introduction of warfarin 50 years ago. Dabigatran is a
potent, reversible, and direct inhibitor of thrombin, with a good efficacy, safety, and
tolerability profile as compared to both warfarin and low molecular weight heparin
[20]. We will discuss its in vitro and in vivo pharmacology and summarize the
clinical development. Dabigatran has now been approved in many countries
around the world, including the United States, Canada, Japan, and Europe, for
stroke prevention in patients with atrial fibrillation and in over 73 countries for the
prevention of thrombosis after orthopedic hip and knee surgery.
10.2
Dabigatran Design Story
In 1992, Brandstetter et al. published the X-ray crystal structure of a bovine thrombin
complex formed with the peptide-like benzamidine-based inhibitor NAPAP
(7) [21]. The analysis revealed, for the first time, the conformation of a noncovalent,
enzyme-bound thrombin inhibitor and its interactions with the residues of the
active site cleft. In this complex, the amidine group of the inhibitor forms a bidentate
salt bridge with the carboxylate of Asp189. Hydrophobic interactions are
formed by the piperidine ring and the naphthyl moiety with the S2 and S4 pockets
of the thrombin active site, respectively. In addition, the bridging glycine moiety of
NAPAP forms the canonical hydrogen bonding pattern with residues Trp215 and
Gly216 at the rim of the specificity (S1) pocket that was previously observed with
peptidic serine protease inhibitors.
246j10 The Discovery of Dabigatran Etexilate
Based on this structural information, we designed a very simple acylated diaminophenyl
scaffold (exemplified in compound 8) as a surrogate for the central
glycine in the NAPAP molecule. The aim was to avoid the peptidic
structure elements and eventually develop a new chemotype with improved
metabolic stability.
O
N
NH
NH2
N
H
O
N
H
S
O O
NH
NH2
S
O O
N
H
N
H
O
CH3
NAPAP IC50 = 3.3 µM7 8
Our efforts to improve the rather weak activity of 8 through variations of substituents
were not successful. With about 30 derivatives, no substantial improvement
in potency could be achieved (Hauel, N.H., unpublished work).
Further progress could only be achieved when we designed the bicyclic compound
9 that was derived from structure 8 by ring closure of the central part
(Hauel, N.H., unpublished work). This turned out to be the most important change
in the core structure.
NH
NH2
S
O O
N
H
N
H
O
CH3
NH
NH2
S
O O
N
H
N
N
CH3
9
IC50 = 1.5 µM
8
IC50 = 3.3 µM
Although only twofold more potent than compound 8, this new benzimidazole
core was a real breakthrough for the whole optimization program. In order to analyze
in detail the exact binding mode of inhibitor 9, we determined a crystal structure
of its complex with human a-thrombin. This analysis revealed that the
benzimidazole core appeared to be a most favorable template to place the three
essential substituents into the right positions (Figure 10.1).
Like with NAPAP, the amidine portion of compound 9 interacts via a salt bridge
with Asp189 in the thrombin active site. The terminal phenyl ring is bound by a
hydrophobic interaction in the S4 pocket, and the N-methyl group nicely fits into
the S2 pocket. There is no additional hydrogen bond between the benzimidazole
moiety of the inhibitor and enzyme, which in part may explain why in our test
10.2 Dabigatran Design Story j247
Figure 10.1 Evolution of thrombin inhibitor
structural classes and their binding modes
starting from NAPAP (compound 7).
Costructures of NAPAP (a), compound 8 (b),
compound 9 (c), and compound 26 (d) in
complex with thrombin. Thrombin is shown as
a surface representation; the compounds are
shown as stick models. The dominant common
motif of inhibitor binding is the interaction of
the conserved benzamidine moiety in the S1
pocket (lower right in each image) with the
Asp189 residue forming a bidentate salt bridge
(hidden). The hydrophobic S2 pocket (residues
Trp60D, His57, Tyr60B, and Leu99) is occupied
by methyl groups anchored at aromatic ring
systems in each of the three lead classes. The
S4 pocket above residue Trp215 (plus residues)
is filled by mono- or bicyclic (hetero-)aromatic
ring systems that are in edge-on position
relative to Trp215.
248j10 The Discovery of Dabigatran Etexilate
system compound 9 was about one order of magnitude less active (IC50 ¼ 1.5 mM)
than NAPAP (IC50 ¼ 0.2 mM).
Intrigued and stimulated by the binding mode of 9, we now focused our efforts
on the optimization of this new class of inhibitors, as described by the general formula
10.
NH
NH2
L
X
Y N
N
R
Aryl
10
Our goal was to refine this structure class in order to increase the inhibitory
potency and to achieve acceptable metabolic stability and pharmacokinetic
properties.
Table 10.1 shows a selection of sulfonamides with variations in three positions.
Starting from lead structure 9, we synthesized analogues 11–13 (Table 10.1) with
Table 10.1 Structural variations of lead compound 9 [19].
NH
NH2
N
N
N
R
1
R
2
S
OO
Ar
.
Compound R1
R2
Ar IC50 (mM)
9 ÀÀCH3 ÀÀH Phenyl 1.5
11 ÀÀH ÀÀH Phenyl 19
12 ÀÀEthyl ÀÀH Phenyl 2.4
13 ÀÀn-Propyl ÀÀH Phenyl 4.0
14 ÀÀCH3 ÀÀCH3 Phenyl 1.3
15 ÀÀCH3 ÀÀH 3-Pyridyl 1.6
16 ÀÀCH3 ÀÀH 1-Naphthyl 0.6
17 ÀÀCH3 ÀÀH 2-Naphthyl 24
18 ÀÀCH3 ÀÀH 5-Isoquinolyl 0.51
19 ÀÀCH3 ÀÀH 8-Quinolyl 0.26
20 ÀÀCH3 ÀÀCH2ÀÀCOOH 8-Quinolyl 0.12
21 ÀÀCH3 ÀÀ(CH2)3ÀÀCOOH 8-Quinolyl 0.13
10.2 Dabigatran Design Story j249
alkyl substituents R1
of different sizes at the benzimidazole N-1, in order to explore
their relative contributions to the binding energy and to identify the optimum alkyl
chain length. Compound 11 (R1
¼ H) turned out to be one order of magnitude less
potent than 9, which clearly demonstrates the importance of the lipophilic/hydrophobic
interaction of the methyl group of 9 with the lipophilic P pocket
formed by the residues His57, Tyr60A, Leu99, and Trp60D of thrombin. This finding
was not a surprise because it was part of our design concept to make use of this
hydrophobic interaction and because we had already information about the localization
of the methyl group in the enzyme active site from the X-ray structure
(Figure 10.1). We did, however, not expect that an increase in the alkyl chain length
(compounds 12 and 13) would result in a decrease in potency. We had hoped,
instead, that a larger buried hydrophobic surface area (and therefore a stronger van
der Waals interaction) would result in stronger ligand binding. The observed
decrease in affinity, albeit very small, may best be explained by a decrease of
entropy due to a loss of rotational degrees of freedom during formation of the
enzyme–inhibitor complex [22].
The X-ray structure (Figure 10.1) clearly shows that the sulfonamide hydrogen of
compound 9 does not take part in a hydrogen bond with the protein. The NÀÀH
bond is directed out of the binding site into the solvent phase; N-alkylation should
therefore have no influence on affinity, as was proven with the N-methyl derivative 14.
The inhibitor interaction with the so-called thrombin S4 pocket, formed by residues
Leu99, Ile174, and Trp215, was explored by variations of the terminal
aryl substituent. A selection of some derivatives is listed in Table 10.1 (compounds
15–19). Substitution of the phenyl group by 3-pyridyl (compound 15) did not influence
affinity. A small increase in potency compared to compound 9 was achieved
with the 1-naphthyl residue (compound 16), whereas activity dropped 40-fold with
the 2-naphthyl group (compound 17), probably caused by a repulsive interaction
with Asn98 at the back of the S4 pocket. The 5-isoquinolyl and 8-quinolyl groups
(compounds 18 and 19) were as effective as the 1-naphthyl.
As was reported by others with a different class of thrombin inhibitors [23], the
lipophilicity of the molecules influences their inhibitory activity when measured in
the presence of blood plasma: the more lipophilic the inhibitor, the more pronounced
the reduction in activity will be. The reason for this phenomenon most
probably is plasma protein binding that reduces the inhibitor free concentration.
We therefore planned to introduce polar groups into the molecules in order to
increase their hydrophilicity, thereby hopefully reducing protein binding and optimizing
in vivo activity. To this end, we synthesized the zwitterionic compounds 20
and 21 by adding acetic acid and butyric acid, respectively, to the sulfonamide nitrogen.
As inferred from the crystal structure of the thrombin–9 complex, the carboxylate
groups are located outside the active site in the solvent phase and do not
interfere with inhibitor binding. The inhibitory activity of both compounds was not
reduced in the presence of plasma proteins (data not shown).
From X-ray structure analyses of this class of inhibitors in complex with thrombin,
we reasoned that elongation of the linker L between the benzimidazole template
and the benzamidine moiety (general structure 4) by an additional atom
250j10 The Discovery of Dabigatran Etexilate
should generate molecules that fit equally well into the active site. This was proven
by compounds 22–26 (Table 10.2).
One of the diatomic linkers, the aminomethyl group, turned out to be especially
favorable: to our surprise, the potency of compound 26 was one order of magnitude
stronger than that of 20.
Based on various X-ray structures (one example shown in Figure 10.1d), our
hypothesis at this point was that the diatomic linker facilitates a better positioning
of the benzamidine group in the S1 pocket, whereas further elongation of the
linker (compound 27) resulted in a decrease of potency.
In the molecules of Tables 10.1 and 10.2, the position of the terminal aryl residue
Ar in relation to the central benzimidazole scaffold is determined by the torsion
angles of the sulfonamide spacer between them. Having obtained detailed information
about enzyme-bound conformations of a number of inhibitors, we reasoned
that utilizing a carboxamide instead of the sulfonamide spacer the aryl group
should also be placed in a favorable position for strong S4 pocket interaction. This
hypothesis was verified by the carboxamide analogues listed in Table 10.3.
From three variations of the linker ÀÀCH2ÀÀAÀÀ (compounds 28–30), again
the amino derivative 30 was the most potent, as was the case in the sulfonamide
series in Table 10.2. Compounds 30, 31, and 32 demonstrate that the chain length
n (up to three methylene groups) between the carboxylate and the amide nitrogen
has only a minor influence on the in vitro activity. Substitution of the terminal
phenyl by a 3-pyridyl residue (compound 33) reduced potency by a factor of 10,
whereas a 2-pyridyl group could be introduced without substantial loss of activity
(compound 34).
Table 10.2 Variations of the linker L in lead structure 10.
N
S
N
N
N
L
NH
NH2
OO
COOH
CH3
.
Compound L IC50 (mM)
22 ÀÀCH2ÀÀCH2ÀÀ 0.032a)
23 ÀÀSÀÀCH2ÀÀ 0.102b)
24 ÀÀCH2ÀÀSÀÀ 0.041b)
25 ÀÀCH2ÀÀOÀÀ 0.058a)
26 ÀÀCH2ÀÀNHÀÀ 0.011a)
27 ÀÀCH2ÀÀNHÀÀCH2- 1.31b)
a) Ref. [19].
b) Ref. (Hauel, N.H., unpublished work).
10.2 Dabigatran Design Story j251
The X-ray structure of 34 in complex with thrombin is shown in Figure 10.2. It
demonstrates that dabigatran (34) and our starting point, compound 9, share a similar
binding mode: the amidine group interacts with Asp189 via a bidentate salt
bridge, the central template is bound to thrombin by a hydrophobic interaction
with the S2 pocket, and the pyridine ring is positioned between Leu99 and Ile174
in the S4 pocket. As predicted, there is no interaction between the enzyme and the
carboxylate group that is directed out of the binding site into bulk solvent. It is one
of the most remarkable characteristics of this inhibitor that it does not make use of
the canonical hydrogen bonding pattern with the enzyme, which is a common feature
of most high-affinity thrombin inhibitors reported so far. Apart from the salt
bridge with Asp189, the binding energy of 34 solely results from hydrophobic interactions,
which underscores the general importance of this binding force in drug–
receptor interactions [24].
Compound 34 was investigated biologically in depth because of its strong in vitro
activity and its favorable selectivity profile versus related serine proteases (Table 10.4).
It turned out to be a very potent anticoagulant also in vivo. Among all inhibitors
of this structural class, it exhibited the strongest activity and the longest duration of
action in anaesthetized rats after i.v. administration. It was well tolerated in these
animals up to the highest given dose of 10 mg/kg. However, it was not orally active,
which is not a surprise considering that it is a very polar, permanently charged,
hydrophilic molecule (log P < À0.6 in n-octanol/buffer).
In principle, compounds with physicochemical characteristics like this can be
converted into orally active prodrugs by turning the carboxylate into an ester group
Table 10.3 Carboxamide analogues.
N
N
CH3
O
N
A
NH2
NH
X
Y (CH2
)
n
COOH
.
Compound A n X Y IC50 (mM)
28 ÀÀCH2ÀÀ 1 CH CH 0.054a
29 ÀÀOÀÀ 1 CH CH 0.33a
30 ÀÀNHÀÀ 1 CH CH 0.010a
31 ÀÀNHÀÀ 2 CH CH 0.0054a
32 ÀÀNHÀÀ 3 CH CH 0.010a
33 ÀÀNHÀÀ 2 CH N 0.054b
34 ÀÀNHÀÀ 2 N CH 0.0093a
a) Ref. [19].
b) Ref. (Hauel, N.H., unpublished work).
252j10 The Discovery of Dabigatran Etexilate
and by masking the amidinium moiety as a carbamate ester [25], provided that both
the carboxylate and the amidinium will be restored in vivo by hydrolytic cleavage of
the prodrug. Accordingly, we synthesized a series of double prodrugs of this type as
exemplified by 35 (Scheme 10.2), which is a rather lipophilic compound with a log
P value of > 2.5 (n-octanol/buffer).
Given orally to rhesus monkeys, this prodrug 35 exhibited strong and long-lasting
anticoagulant effects as measured by the activated partial thromboplastin time
(aPTT) ex vivo (Figure 10.3) [26].
Figure 10.2 X-ray crystal structure of
dabigatran (compound 34) in complex with
thrombin in a surface representation. The
ligand interacts in a similar fashion as
compound 8 with thrombin residues of the
specificity pocket and the S2 pocket. The S4
pocket is occupied by the pyridine ring, while
the propionic acid substituent on the amide
nitrogen projects into bulk solvent without
forming further interactions with the protein.
Table 10.4 Selectivity profile of dabigatran (34).
Human enzymes Ki (nM)
Thrombin 4.5
Factor Xa 3760
Trypsin 50
Plasmin 1695
tPA 45 360
Activated protein C 20 930
aPTT (human plasma): ED2 ¼ 230 Æ 21 nM
10.2 Dabigatran Design Story j253
Based on the overall profile and promising in vivo activity, compound 35 was
selected for clinical development.
10.3
Preclinical Pharmacology Molecular Mechanism of Action of Dabigatran
Dabigatran inhibits human thrombin in a concentration-dependent and competitive
fashion, with a Ki of 4.5 nM. This inhibition is rapid and reversible as shown
by real-time binding kinetics using surface plasmon resonance techniques. IC50
values for other proteases involved in the coagulation and fibrinolytic pathway
are >700-fold and up to >10,000-fold compared to thrombin, demonstrating the
high selectivity of dabigatran for thrombin (Table 10.4).
Thrombin generated on the platelet surface in response to a hemostatic challenge
also mediates platelet activation and aggregation as one of the most potent
Scheme 10.2 Synthesis of dabigatran and
dabigatran etexilate. (a) SOCl2/DMF, reflux; (b)
2-pyridylamino-propionic acid ethyl ester, THF,
triethylamine, rt; (c) H2, 10% Pd/C,
methanol/CH2Cl2, rt; (d) 4-cyanophenyl-glycine,
CDI, THF, 50 
C; (e) acetic acid, reflux; (f) HCl
(gas), ethanol 0 
C, (NH4)2CO3/ethanol, rt; (g)
NaOH, H2O/ethanol, rt; (h) n-hexyl
chloroformate, K2CO3, THF/H2O, rt.
254j10 The Discovery of Dabigatran Etexilate
physiological agonists. Dabigatran inhibits thrombin-induced platelet aggregation
with an IC50 of 10 nM, but has no inhibitory effect on platelet aggregation induced
by arachidonic acid, collagen, or adenosine 20
,50
-diphosphate at concentrations of
up to 1 Â 10À4
M [26].
Dabigatran also effectively inhibits tissue factor-induced thrombin generation in
human platelet poor plasma in a concentration-dependent manner with an IC50 of
0.56 mM as shown by the concentration-dependent decrease in the endogenous
thrombin potential. Thus, in addition to its direct inhibition of thrombin activity,
dabigatran is also able to inhibit thrombin generation.
Thrombin bound to fibrin, either in an existing thrombus or on an artificial
surface, is thought to be protected from inhibition by the high molecular weight
complex between heparin and antithrombin. This surface could therefore remain
“active” despite heparin therapy. Dabigatran inhibits both clot-bound and fluidphase
thrombin to a similar degree with IC50 values of 128 and 113 nM, respectively,
consistent with direct inhibition of the active site of thrombin by a small
molecule. This binding appears independent of whether thrombin is bound via the
exosite to fibrin or is present as free enzyme in plasma [27].
10.3.1
In Vitro Antihemostatic Effects of Dabigatran
Consistent and potent anticoagulant activity of dabigatran in vitro in plateletpoor
plasma has been demonstrated using common clotting assays across several
species (human, rhesus monkeys, dog, rabbit, and rat) [26]. A doubling of
the aPTT, prothrombin time (PT), and ecarin clotting time (ECT) is observed
Figure 10.3 The dose- and time-dependent effects on aPTT after oral administration of
compound 35 (dabigatran etexilate) to rhesus monkeys. Data represent mean Æ SEM.
10.3 Preclinical Pharmacology Molecular Mechanism of Action of Dabigatran j255
at concentrations ranging from as low as 0.1 up to 4.6 mM of dabigatran with
the ECT being the most sensitive parameter for anticoagulant activity.
10.3.2
Ex Vivo Antihemostatic Effects of Dabigatran/Dabigatran Etexilate
Dose- and time-dependent, significant anticoagulant efficacy ex vivo has also been
demonstrated after intravenous administration of dabigatran to rats and rhesus
monkeys. In rats, doses of 0.3, 1, and 3 mg/kg i.v. produce a maximum prolongation
of the ex vivo aPTT to 29, 159, and 582 s, respectively, 5 min after administration.
aPTT remains significantly elevated at all doses within the first hour after
administration and decreases gradually thereafter.
In rhesus monkeys, intravenous administration (0.15, 0.3, or 0.6 mg/kg) of dabigatran
prolongs the aPTT to 47.3, 70.1, and 98.9 s, respectively, 5 min after administration.
This effect is long lasting with a significant prolongation still observed at
>8 h after administration of the highest dose. Dabigatran etexilate also shows doseand
time-dependent anticoagulant effects in these species. Of note, single oral doses
of 1, 2.5, and 5 mg/kg, administered to conscious rhesus monkeys, revealed a significant
and long-lasting (>8 h) prolongation of the aPTT at all doses (Figure 10.3) [26].
10.3.3
Venous and Arterial Antithrombotic Effects of Dabigatran/Dabigatran Etexilate
The antithrombotic effects of dabigatran have been tested in a rat model of tissue
factor/stasis-induced venous thrombosis. Dabigatran, administered intravenously
as a slow bolus infusion, inhibited clot formation dose dependently and completely
with an ED50 of 0.033 mg/kg [28]. Of note, no significant increase in bleeding time
was observed at the maximum effective antithrombotic dose of 0.1 mg/kg. Using
the same model, the prodrug dabigatran etexilate was administered orally (5–
30 mg/kg) between 0.5 and 7 h prior to thrombus induction. Pretreatment resulted
in a dose- and time-dependent inhibition of thrombus formation with significant
inhibition lasting for >3 h for all doses tested.
Furthermore, both compounds have been examined in a rabbit arteriovenous
shunt model of thrombosis. Intravenous administration of dabigatran revealed a
dose-dependent inhibition of clot formation with a corresponding ED50 of
0.066 mg/kg [29]. Oral administration of dabigatran etexilate, in doses of 1–
20 mg/kg, inhibited clot formation dose dependently, and with the highest dose
completely. In both the rat and rabbit thrombosis models, the observed antithrombotic
effects were inversely correlated with a prolongation of the ex vivo aPTT in a
dose- and time-dependent manner.
Dabigatran, either alone or in combination with ASA, has also been tested in a
Folts model of arterial thrombosis in pigs. The model is characterized by acute and
severe arterial thrombus formation and a cyclic reduction of blood flow in response
to a mechanical injurious stimulus and a concomitant partial stenosis of the carotid
artery. ASA treatment alone reduced cyclic flow reductions by 40% and dabigatran
256j10 The Discovery of Dabigatran Etexilate
alone by 44%. When the two compounds were given together, closures were
reduced by 88%. In addition, a complete closure of the injured vessels was not
observed in the dabigatran-treated and the dabigatran–aspirin-treated animals.
Both compounds were shown to be effective and safe in this model of arterial
thrombosis. These effects were achieved at clinically relevant plasma levels and
show that dabigatran can effectively be used in an arterial thrombosis setting [30].
10.3.4
Mechanical Heart Valves
Thromboprophylaxis after mechanical heart valve implantation with warfarin therapy
is required for the lifetime of the patient; this may be an area where direct
thrombin inhibition could provide alternative therapy. This concept was tested in
several preclinical studies. In vitro, a thrombosis tester was used that circulates
human blood only anticoagulated with the agent being tested over a mechanical
heart valve in a closed circuit. Thrombus formation on the valve after adding various
antithrombotic regimens versus control was tested. In this study, it was shown
that dabigatran was an effective inhibitor of fibrin deposition in vitro [31].
In vivo, dabigatran was tested in an experimental model in pigs, with an aortic
heart valve placed as a graft in the descending aorta [32]. Dabigatran was administered
postimplantation in pigs for 30 days versus LMWH, and a significant reduction
in thrombus and platelet deposition onto the valve was seen.
10.3.5
Cancer
A link between cancer and thrombosis has long been recognized [33]. Whether this
is due to prothrombotic mediators from the tumor, chemotherapy interventions, or
both is unknown. In addition, there is a great deal of experimental evidence implicating
PAR1-mediated events with tumor growth and metastasis. There is a correlation
between expression of PAR1 on tumor cells and invasiveness of the
cancer [33]. Upon activation in tumor cells, PAR1 is not internalized as in normal
cells [34]. In addition, when metastatic breast cancer tumor cells (MDA-231) are
genetically modified to express an inactive form of PAR1, they were no longer as
tumorigenic as wild type in a xenograft model [35]. Studies testing the effects of
chronic dabigatran treatment (4 weeks) in a syngeneic mouse model of breast cancer
demonstrated a reduction in both primary tumor growth and metastasis in
dabigatran-treated mice as compared to control [36].
10.3.6
Fibrosis
One chronic hallmark of atrial fibrillation and many other degenerative diseases is
the extent of fibrosis, or the conversion of the normal cell type into a collagen
secreting, fibroblastic phenotype [4]. This is present in myocardium of patients
10.3 Preclinical Pharmacology Molecular Mechanism of Action of Dabigatran j257
with atrial fibrillation, in renal tissue of patients with renal impairment, and in pulmonary
tissue of patients with idiopathic pulmonary fibrosis. Activation of coagulation
following tissue injury is one of the earliest responses, resulting in the
deposition of fibrin. In addition, activation of PAR1 by thrombin can differentiate
normal lung fibroblasts into a myofibroblast phenotype and induces fibrogenic
cytokine release, such as transforming growth factor b, and increased expression of
extracellular matrix proteins, such as collagen [37]. These effects are all inhibited in
vitro by dabigatran. In addition, a recent study looking at chronic dabigatran etexilate
treatment in mice with bleomycin-induced pulmonary injury showed a reduction
in the extent of fibrosis and collagen release [38].
10.3.7
Atherosclerosis
There is increasing evidence in experimental atherosclerosis that inflammation
and coagulation may play an important role in atherosclerotic development [5, 39].
Thus, it was tested whether direct thrombin inhibition with dabigatran can mitigate
the progression of plaque formation. In an ApoEÀ/À
mouse model of atherosclerosis,
mice were fed a normal diet supplemented with dabigatran etexilate for
up to 20 weeks. Lesion size of forming but not advanced atherosclerotic plaque was
reduced in the dabigatran-fed mice and plaque stability, measured as fibrous cap
thickness, was also increased [40].
A separate study in a procoagulant/atherosclerotic mouse model (ApoEÀ/À
:
TMpro/pro
) also tested the effects of dabigatran etexilate-supplemented high-fat diet
versus placebo. These ApoEÀ/À
:TMpro/pro
mice have elevated plasma fibrinogen,
prothrombin, factor X, and thrombin–antithrombin levels and develop extensive
plaque formation due to their atherosclerotic backgrounds crossed with mice with
a mutation in thrombomodulin (TM), resulting in loss of activity. The administration
of dabigatran etexilate significantly reduced plaque area and the degree of
stenosis after a carotid cuff injury [41].
10.4
Clinical Studies and Indications
For over 50 years, vitamin K antagonists have been the gold standard in chronic
anticoagulation treatment. While they provide significant benefit when used properly
as compared to no treatment, there are also significant drawbacks (e.g., frequent
laboratory monitoring and dose adjustments, large number of drug
interactions, and risk of bleeding). This translates in clinical practice into an underuse
of treatment with VKAs in a substantial proportion of patients with atrial fibrillation.
Therefore, there is a significant unmet medical need to develop
anticoagulation treatment that is easy to use, predictable, effective, and safe. Dabigatran
etexilate represents the first broadly approved new oral anticoagulant with
these attributes.
258j10 The Discovery of Dabigatran Etexilate
Dabigatran has a fast onset of action (peak plasma concentrations 2–3 h after
ingestion of dabigatran etexilate). The half-life is 12–14 h [42]. It does not require
routine coagulation monitoring and has no drug–food interactions. Cytochrome
P450 enzymes are not involved in the metabolism, but it is a substrate for the Pgp
transporter, so there is the potential for drug interactions with agents using this
transporter that in some indications results in a dose adaptation. Dabigatran is
80% renally excreted; thus, caution needs to be executed when prescribing to
patients with kidney disease. In the United States, the FDA recommends a lower
therapeutic dose (75 mg bid) in patients with a creatine clearance (CrCl) between
15 and 30 ml/min and no use in patients with CrCl under 15 ml/min for the prevention
of strokes in patients with atrial fibrillation. In Europe, dabigatran is contraindicated
in patients with a CrCl below 30 ml/min. However, no dose
adjustment is needed for moderate liver disease [42, 43].
10.4.1
Prevention of Deep Venous Thrombosis
Dabigatran has been studied in more than 10 000 patients in four phase III trials of
DVT prophylaxis in lower extremity orthopedic surgery [44–48]. In a study in total
knee replacement (TKR), when compared to enoxaparin 30 mg bid, dabigatran etexilate
at 150 and 220 mg daily was inferior due to a greater number of distal
thromboses (dabigatran etexilate 220 mg – 27.6%, dabigatran etexilate 150 mg –
30.5%, and enoxaparin – 23.0%) [44]. In studies comparing dabigatran to 40 mg
enoxaparin (European dosing regimen), equivalence was shown for both thrombosis
prevention and bleeding in total hip replacement (THR) and TKR [45, 46]. The
result of the THR trial was reproduced in the RE-NOVATE II trial, where dabigatran
additionally showed a superior reduction of major venous thromboembolism
with a same rate of major bleeding [48]. A pooled analysis of the THR trials demonstrated
the superiority in the prevention of major VTE [49]. In the countries where
dabigatran etexilate is approved for DVT prophylaxis, it is recommended at a dose
of 220 mg daily (110 mg first dose after surgery) and at 150 mg (75 mg first dose
after surgery) daily for patients with renal impairment or concomitant treatment
with verapamil (a Pgp inhibitor).
10.4.2
Therapy of Venous Thromboembolism
In the treatment of VTE (includes DVT or pulmonary embolism), 150 mg dabigatran
etexilate twice daily was compared to standard warfarin (INR 2.0–3.0) treatment
[50]. All patients received heparin or low molecular weight heparin for a
median of 9 days following one of the oral anticoagulants. Dabigatran demonstrated
the same efficacy in the prevention of thrombosis with significantly less
minor bleeding and similar major bleeding. The extension study RE-MEDY (versus
warfarin) and RE-SONATE (versus placebo) showed in the warfarin comparison
again the same efficacy and a better bleeding profile and in the placebo comparison
10.4 Clinical Studies and Indications j259
a 92% relative risk reduction of recurrent VTE with statistically no difference in
major bleeding, but as to be expected against placebo, elevated clinical relevant
non-major bleeding (5.3% dabigatran versus 1.8% placebo) [51, 52]. These indications
have not yet been registered.
10.4.3
Stroke Prevention in Patients with Atrial Fibrillation
In the RE-LYstudy, 18 234 patients with atrial fibrillation and at least one risk factor
for stroke were randomized between warfarin INR 2–3, 110 mg of dabigatran twice
daily, and 150 mg of dabigatran twice daily [53, 54]. The RE-LY trial was the only
major phase 3 study that evaluated two different doses and had enough power for
each dose, to define the most appropriate patient population. This resulted in the
opportunity to tailor the dose according to the patient profile/population. The
150 mg bid dabigatran etexilate dose showed superior efficacy with the same major
bleeding rate as warfarin, and the 110 mg bid dose had a significantly lower major
bleeding rate and the same efficacy as warfarin. Both doses had a significant, up to
70%, reduction of intracranial hemorrhage and also a reduction in life-threatening
bleeding. There was a nonsignificant 12% mortality benefit (p ¼ 0.051) with the
150 mg bid dose [53, 54]. These results were consistent across all patient groups,
such as those with a previous history of stroke or transient ischemic attack [55], or
patients previously taking warfarin or those new to oral anticoagulation [56].
10.4.4
Prevention of Recurrent Myocardial Infarction in Patients with Acute Coronary
Syndrome
In a phase II dose identification study, RE-DEEM, with a treatment time of
6 months, dabigatran in addition to dual antiplatelet therapy showed an expected
dose-dependent increase in bleeding and significantly reduced coagulation activity.
In the dose groups of 150 and 110 mg bid, the composite of cardiovascular death,
nonfatal myocardial infarction, hemorrhagic stroke, cardiovascular death, and allcause
death was numerically lower compared to placebo (dual antiplatelet
therapy) [57].
10.5
Summary
Based on a NAPAP–thrombin X-ray crystal structure, we designed a new structural
class of inhibitors employing benzimidazole as a central scaffold. Supported
by a series of X-ray structure analyses, we optimized the potency of
these compounds in a number of iterative steps. During this process, enzyme
inhibition in the lower nanomolar range could be achieved by gaining binding
energy mainly from hydrophobic interactions. In order to reduce plasma
260j10 The Discovery of Dabigatran Etexilate
protein binding, we increased the hydrophilicity of the inhibitors by introducing
a carboxylate group at a position of the molecules, where it does not interfere
with the thrombin binding site interaction. One of these inhibitors,
dabigatran, showed very strong in vivo activity after i.v. administration; however,
due to its highly polar nature, its oral absorption was insufficient. We
therefore synthesized a number of prodrugs, from which dabigatran etexilate
emerged as a clinical candidate due to its strong and long-lasting oral activity.
In-depth preclinical pharmacological investigations illustrated that dabigatran is
an effective antithrombotic agent in both venous and arterial models. Furthermore,
since thrombin has been shown to play a central role in many disease processes,
dabigatran was investigated in additional biochemical pathways showing beneficial
effects, for example, in fibrotic and atherosclerotic animal models.
In four clinical studies, this new drug has been shown to be as effective as enoxaparin
in the prevention of venous thrombosis after orthopedic surgery, and to be
as effective as warfarin in the therapy of acute thrombosis with no increased risk of
bleeding. In stroke prevention in patients with atrial fibrillation, it was more effective
than warfarin with a lesser risk of intracranial hemorrhage. The prevention of
thromboembolic events in patients with mechanical heart valves is currently under
evaluation.
More than 50 years after the introduction of warfarin, the development of dabigatran
can be seen as a major breakthrough for the treatment of thrombosis.
References
1 DiCera, E. (2008) Thrombin. Mol. Aspects
Med., 29, 203–254.
2 Griffin, J.H. (1995) Blood coagulation.
The thrombin paradox. Nature, 378, 337–
338.
3 Lane, D.A., Philippou, H., and
Huntington, J.A. (2005) Directing
thrombin. Blood, 106, 2605–2612.
4 Tan, A.Y. and Zimetbaum, P. (2011) Atrial
fibrillation and atrial fibrosis.
J. Cardiovasc. Pharmacol., 57, 625–629.
5 Borissoff, J.I., Spronk, H.M.H.,
Heeneman, S., and ten Cate, H. (2009) Is
thrombin a key player in the “coagulation
atherogenesis” maze? Cardiovasc. Res., 82,
392–403.
6 Derian, C.K., Maryanoff, B.E., Zang, H.E.,
and Andrade-Gordon, P. (2003)
Therapeutic potential of protease receptor-
1 antagonists. Expert Opin. Invest. Drugs,
12, 209–221.
7 Martorell, L., Martínez-Gonzalez, J.,
Rodríguez, C., Gentile, M., Calvayrac, O.,
and Badimon, L. (2008) Thrombin and
protease-activated receptors (PARs) in
atherothrombosis. Thromb. Haemost., 99,
305–315.
8 Coughlin, S.R. (2005) Protease-activated
receptors in hemostasis, thrombosis and
vascular biology. J. Thromb. Haemost., 3,
1800–1814.
9 Hirsh, J., Bauer, K.A., Donati, M.B.,
Gould, M., Samama, M.M., and Weitz, J.I.
(2008) Parenteral anticoagulants:
American College of Chest Physicians
Evidence-Based Clinical Practice
Guidelines (8th edition). Chest, 133,
141S–159S.
10 Yeh, R.W. and Jang, I.-K. (2006)
Argatroban: update. Am. Heart J., 151,
1131–1138.
11 Warkentin, T.E., Greinacher, A., and
Koster, A. (2008) Bivalirudin. Thromb.
Haemost., 99, 830–839.
12 Hirsh, J., Dalen, J.E., Anderson, D.R.,
Poller, L., Bussey, H., Ansell, J., Deykin, D.,
References j261
and Brandt, J.T. (1998) Oral anticoagulants.
Mechanism of action, clinical effectiveness,
and optimal therapeutic range. Chest, 114,
445S–469S.
13 Mann, K.G. (2005) The challenge of
regulating anticoagulant drugs: focus on
warfarin. Am. Heart J., 149, S36–S42.
14 Gustafsson, D., Bylund, R., Antonsson, T.,
Nilsson, I., Nystroem, J.-E., Eriksson, U.,
Bredberg, U., and Teger-Nilsson, A.-C.
(2004) Case history: a new oral
anticoagulant: the 50-year challenge. Nat.
Rev. Drug Discov., 3, 649–659.
15 Pinto, D.J.P., Smallheer, J.M., Cheney,
D.L., Knabb, R.M., and Wexler, R.R.
(2010) Factor Xa inhibitors: nextgeneration
antithrombotic agents. J. Med.
Chem., 53, 6243–6274.
16 Perzborn, E., Roehrig, S., Straub, A.,
Kubitza, D., and Misselwitz, F. (2011) The
discovery and development of
rivaroxaban, an oral, direct factor Xa
inhibitor. Nat. Rev. Drug Discov., 10,
61–75.
17 Pinto, D.J.P., Orwat, M.J., Koch, S., Rossi,
K.A., Alexander, R.S., Smallwood, A.,
Wong, P.C., Rendina, A.R., Luettgen, J.M.,
Knabb, R.M., He, K., Xin, B., Wexler, R.R.,
and Lam, P.Y.S. (2007) Discovery of 1-(4-
methoxyphenyl)-7-oxo-6-(4-(2-
oxopiperidin-1-yl)phenyl)-4,5,6,7-
tetrahydro-1H-pyrazolo[3,4-c]pyridine-3carboxamide
(apixaban, BMS-562247), a
highly potent, selective, efficacious, and
orally bioavailable inhibitor of blood
coagulation factor Xa. J. Med. Chem., 50,
5339–5356.
18 Furugohri, T., Isobe, K., Honda, Y.,
Kamisato-Matsumoto, C., Sugiyama, N.,
Nagahara, T., Morishima, Y., and Shibano,
T. (2008) DU-176b, a potent and orally
active factor Xa inhibitor: in vitro and in
vivo pharmacological profiles. J. Thromb.
Haemost., 6, 1542–1549.
19 Hauel, N.H., Nar, H., Priepke, H., Ries, U.,
Stassen, J.M., and Wienen, W. (2002)
Structure-based design of novel potent
nonpeptide thrombin inhibitors. J. Med.
Chem., 45, 1757–1766.
20 Hankey, G.J. and Eikelboom, J.W. (2011)
Dabigatran etexilate: a new oral
thrombin inhibitor. Circulation, 123,
1436–1450.
21 Brandstetter, H., Turk, D., Hoeffken,
H.W., Grosse, D., Stuerzebecher, J.,
Martin, P.D., Edwards, B.F.P., and Bode, W.
(1992) Refined 2.3 A

X-ray crystal
structure of bovine thrombin complexes
formed with the benzamidine and
arginine-based thrombin inhibitors
NAPAP, 4-TAPAP and MQPA. J. Mol. Biol.,
226, 1085–1099.
22 Andrews, P.R., Craik, D.J., and Martin,
J.L. (1984) Functional group contributions
to drug–receptor interactions. J. Med.
Chem., 27, 1648–1657.
23 Tucker, T.J., Lumma, W.C., Lewis, S.D.,
Gardell, S.J., Lukas, B.J., Baskin, E.P.,
Woltmann, R., Lynch, J.J., Lyle, E.A.,
Appleby, S.D., Chen, I.-W., Dancheck,
K.B., and Vacca, J.P. (1997) Potent
noncovalent thrombin inhibitors that
utilize the unique amino acid Ddicyclohexylalanine
in the P3 position:
implications on oral bioavailability and
antithrombotic efficacy. J. Med. Chem., 40,
1565–1569.
24 Davis, A.M. and Teague, S.J. (1999)
Hydrogen bonding, hydrophobic
interactions, and failure of the rigid
receptor hypothesis. Angew. Chem., Int.
Ed., 38, 736–749.
25 (a) Himmelsbach, F., Austel, V., Guth, B.,
Linz, G., Mueller, T.H., Pieper, H.,
Seewaldt-Becker, E., and Weisenberger,
H., (1995) Design of potent nonpeptidic
fibrinogen receptor antagonists. Eur. J.
Med. Chem., 30, 243s–254s. (b) Mueller,
T.H., Weisenberger, H., Brickl, R., Narjes,
H., Himmelsbach, F., and Krause, J.
(1997) Profound and sustained inhibition
of platelet aggregation by fradafiban, a
nonpeptide platelet glycoprotein IIb/IIIa
antagonist, and its orally active prodrug,
lefradafiban, in men. Circulation, 96,
1130–1138.
26 Wienen, W., Stassen, J.-M., Priepke, H.,
Ries, U.-J., and Hauel, N. (2007) In-vitro
profile and ex-vivo anticoagulant activity
of the direct thrombin inhibitor
dabigatran and its orally active prodrug,
dabigatran etexilate. Thromb. Haemost.,
98, 155–162.
27 van Ryn, J., Hauel, N., Waldmann, L., and
Wienen, W. (2008) Dabigatran inhibits
both clot-bound and fluid-phase thrombin
262j10 The Discovery of Dabigatran Etexilate
in vitro: comparison to heparin and
hirudin. Arterioscler. Thromb. Vasc. Biol.,
28, e136–e137.
28 Wienen, W., Stassen, J.-M., Priepke, H.,
Ries, U.-J., and Hauel, N. (2007) Effects of
the direct thrombin inhibitor dabigatran
and its orally active prodrug, dabigatran
etexilate, on thrombus formation and
bleeding time in rats. Thromb. Haemost.,
98, 333–338.
29 Wienen, W., Stassen, J.-M., Priepke, H.,
Ries, U.-J., and Hauel, N. (2007)
Antithrombotic and anticoagulant effects
of the direct thrombin inhibitor
dabigatran, and its oral prodrug,
dabigatran etexilate, in a rabbit model of
venous thrombosis. J. Thromb. Haemost.,
5, 1237–1242.
30 van Ryn, J., Dietze, T., Kuritsch, I., KinkEiband,
M., and Wienen, W. (2009) Effect
of the direct thrombin inhibitor,
dabigatran, on arterial thrombosis when
given in combination with aspirin (ASA)
in a cyclic flow model in anesthetised pigs
(Abstract). J. Thromb. Haemost., 7 (Suppl.
2), 435.
31 Maegdefessel, L., Linde, T., Krapiec, F.,
Hamilton, K., Steinseifer, U., van Ryn, J.,
Raaz, U., Buerke, M., Werdan, K., and
Schlitt, A. (2010) In vitro comparison of
dabigatran, unfractionated heparin, and
low-molecular-weight heparin in
preventing thrombus formation on
mechanical heart valves. Thromb. Res.,
126, e196–e200.
32 McKellar, S.H., Abel, S., Camp, C.L., Suri,
R.M., Ereth, M.H., and Schaff, H.V. (2011)
Effectiveness of dabigatran etexilate for
thromboprophylaxis of mechanical heart
valves. J. Thorac. Cardiovasc. Surg., 141,
1410–1416.
33 Iodice, S., Gandini, S., Lohr, M.,
Lowenfels, A.B., and Maisonneuve, P.
(2008) Venous thromboembolic events
and organ-specific occult cancers: a review
and meta-analysis. J. Thromb. Haemost., 6,
781–788.
34 Booden, M.A., Eckert, L.B., Der, C.J.,
and Trejo, J.A. (2004) Persistent
signalling by dysregulated thrombin
receptor trafficking promotes breast
carcinoma cell invasion. Mol. Cell. Biol.,
24, 1990–1999.
35 Boire, A., Covic, L., Agarwal, A., Jacques,
S., Sherifi, S., and Kuliopulos, A. (2005)
PAR1 is a matrix metalloprotease-1
receptor that promotes invasion and
tumorigenesis of breast cancer cells. Cell,
120, 303–313.
36 DeFeo, K., Hayes, C., Chernick, M., van
Ryn, J., and Gilmour, S.K. (2010) Use of
dabigatran etexilate to reduce breast
cancer progression. Cancer Biol. Ther., 10,
1001–1008.
37 Bogatkevich, G.S. and Silver, R. (2009)
Dabigatran inhibits the thrombin-induced
differentiation of lung fibroblasts to a
myofibroblast phenotype. Arthritis
Rheum., 60, 3455–3464.
38 Bogatkevich, G.S., Ludwicka-Bradley, A.,
Nietert, P.J., van Ryn, J., and Silver, R.M.
(2011) Antifibrotic effects of the oral,
direct thrombin inhibitor dabigatran
etexilate on lung injury in mice model of
pulmonary fibrosis. Arthritis Rheum., 63,
1416–1425.
39 Borissoff, J.I., Spronk, H.M.H., and ten
Cate, H. (2011) The haemostatic system as
a modulator of atherosclerosis. N. Engl. J.
Med., 364, 1746–1760.
40 Preusch, M.R., Wijelath, E.S., Cabbage, S.,
Ieronimakis, N., Callegari, A., Bea, F.,
Blessing, E., Reyes, M., van Ryn, J., and
Rosenfeld, M.E. (2010) Dabigatran
etexilate, a new oral thrombin inhibitor,
retards the initiation and progression of
atherosclerotic lesions and inhibits the
expression of oncostatin M in
apolipoprotein E deficient mice (Abstract).
Arterioscler. Thromb. Vasc. Biol., 30, e185.
41 Borissoff, J.I., Loubele, S.T., Leenders, P.,
Soehnlein, O., Koenen, R.R., van Oerle,
R., Hamulyak, K., Heeneman, S.,
Daemen, M.J., Weiler, H., van Ryn, J.,
Spronk, H.M., and ten Cate, H. (2010)
Direct thrombin inhibition by dabigatran
protects against severe atherosclerosis
progression in prothrombotic mice
(Abstract). Circulation, 122 (Suppl. 1),
A19384.
42 Stangier, J. and Clemens, A. (2009)
Pharmacology, pharmacokinetics, and
pharmacodynamics of dabigatran
etexilate, an oral direct thrombin inhibitor.
Clin. Appl. Thromb. Hemost., 15 (Suppl 1),
9S–16S.
References j263
43 Garnock-Jones, K.P. (2011) Dabigatran
etexilate: a review of its use in the
prevention of stroke and systemic
embolism in patients with atrial
fibrillation. Am. J. Cardiovasc. Drugs, 11,
57–72.
44 Ginsberg, J.S., Davidson, B.L., Comp,
P.C., Francis, C.W., Friedman, R.J., Huo,
M.H., Lieberman, J.R., Muntz, J.E.,
Raskob, G.E., Clements, M.L., Hantel, S.,
Schnee, J.M., and Caprini, J.A. (2009) Oral
thrombin inhibitor dabigatran etexilate vs
North American enoxaparin regimen for
prevention of venous thromboembolism
after knee arthroplasty surgery. J.
Arthroplasty, 24, 1–9.
45 Eriksson, B.I., Dahl, O.E., Rosencher, N.,
Kurth, A.A., van Dijk, C.N., Frostick, S.P.,
Kalebo, P., Christiansen, A.V., Hantel, S.,
Hettiarachchi, R., Schnee, J., and Buller,
H.R. (2007) Oral dabigatran etexilate vs.
subcutaneous enoxaparin for the
prevention of venous thromboembolism
after total knee replacement: the REMODEL
randomized trial. J. Thromb.
Haemost., 5, 2178–2185.
46 Eriksson, B.I., Dahl, O.E., Rosencher, N.,
Kurth, A.A., van Dijk, C.N., Frostick, S.P.,
Prins, M.H., Hettiarachi, R., Hantel, S.,
Schnee, J., and Bueller, H.R. (2007)
Dabigatran etexilate versus enoxaparin for
prevention of venous thromboembolism
after total hip replacement: a randomised,
double-blind, non-inferiority trial. Lancet,
370, 949–956.
47 Friedman, R.J., Dahl, O.E., Rosencher,
N., Caprini, J.A., Kurth, A.A., Francis,
C.W., Clemens, A., Hantel, S., Schnee,
J.M., and Eriksson, B.I. (2010)
Dabigatran versus enoxaparin for
prevention of venous thromboembolism
after hip or knee arthroplasty: a pooled
analysis of three trials. Thromb. Res.,
126, 175–182.
48 Eriksson, B.I., Dahl, O.E., Huo, M.H.,
Kurth, A.A., Hantel, S., Hermansson, K.,
Schnee, J.M., and Friedman, R.J. (2011)
Oral dabigatran versus enoxaparin for
thromboprophylaxis after primary total
hip arthroplasty (RE-NOVATE II). Thromb.
Haemost., 105, 721–729.
49 Eriksson, B.I., Kurth, A.A., Dahl, O.E.,
Clemens, A., Schnee, J., Hantel, S.,
Feuring, M., Friedman, R.J., and Huo, M.
(2011) Oral dabigatran etexilate vs.
enoxaparin for prevention of venous
thromboembolism after total hip
arthroplasty: a pooled analysis of two
randomized trials. J. Thromb. Haemost., 9
(Suppl. 2), 856–857.
50 Schulman, S., Kearon, C., Kakkar, A.K.,
Mismetti, P., Schellong, S., Eriksson, H.,
Baanstra, D., Schnee, J., and Goldhaber, S.
Z. (2009) Dabigatran versus warfarin in
the treatment of acute venous
thromboembolism. N. Engl. J. Med., 361,
2342–2352.
51 Schulman, S., Eriksson, H., Goldhaber,
S.Z., Kakkar, A.K.L., Kearon, C., Kvamme,
A.M., Mismetti, P., Schellong, S., and
Schnee, J. (2011) Dabigatran or warfarin
for extended maintenance therapy of
venous thromboembolism. J. Thromb.
Haemost., 9 (Suppl. 2), 731–732.
52 Schulman, S., Baanstra, D., Eriksson, H.,
Goldhaber, S., Kakkar, A., Kearon, C.,
Mismetti, P., Schellong, S., and Schnee, J.
(2011) Dabigatran versus placebo for
extended maintenance therapy of venous
thromboembolism. J. Thromb. Haemost., 9
(Suppl. 2), 22.
53 Connolly, S.J., Ezekowitz, M.D., Yusuf,
S., Eikelboom, J., Oldgren, J., Parekh,
A., Pogue, J., Reilly, P.A., Themeles, E.,
Varrone, J., Wang, S., Alings, M.,
Xavier, D., Zhu, J., Diaz, R., Lewis,
B.S., Darius, H., Diener, H.C., Joyner,
C.D., and Wallentin, L. (2009)
Dabigatran versus warfarin in patients
with atrial fibrillation. N. Engl. J. Med.,
361, 1139–1151.
54 Connolly, S.J., Eszekowitz, M., Yusuf, S.,
Reilly, P.A., and Wallentin, L. (2010)
Newly Identified events in the RE-LY trial.
N. Engl. J. Med., 363, 1875–1876.
55 Diener, H.C., Connolly, S.J., Ezekowitz,
M.D., Wallentin, L., Reilly, P.A., Yang,
S., Xavier, D., di Pasquale, G., and
Yusuf, S. (2010) Dabigatran compared
with warfarin in patients with atrial
fibrillation and previous transient
ischaemic attack or stroke: a subgroup
analysis of the RE-LY trial. Lancet
Neurol., 9, 1157–1163.
56 Ezekowitz, M.D., Wallentin, L.,
Connolly, S.J., Parekh, A., Chernick,
264j10 The Discovery of Dabigatran Etexilate
M.R., Pogue, J., Aikens, T.H., Yang, S.,
Reilly, P.A., Lip, G.Y.H., and Yusuf,
S. (2010) Dabigatran and warfarin in
vitamin K antagonist-naive and
-experienced cohorts with atrial
fibrillation. Circulation, 122,
2246–2253.
57 Oldgren, J., Budaj, A., Granger, C.B.,
Khder, Y., Roberts, J., Siegbahn, A.,
Tijssen, J.G.P., van de Werf, F., and
Wallentin, L. (2011) Dabigatran vs.
placebo in patients with acute coronary
syndrome on dual antiplatelet therapy: a
randomised, double blind, phase II
trial. Eur. Heart J., 32, 2781–2789.
References j265
Andreas Clemens completed his graduation as Medical
Doctor, Internist, and Endocrinologist in 2002 and after
working for about 8 years in university hospitals from 2001
onward started his career in the pharmaceutical industry. He
joined Boehringer Ingelheim in 2005 in this global position
where he launched dabigatran worldwide for the primary prevention
of venous thromboembolic events in patients after
orthopedic surgery and recently for the prevention of stroke
in patients with atrial fibrillation. He is currently the Global
Senior Director Clinical Research & Medical Affairs and leads the group responsible
for the further development of dabigatran etexilate beyond the clinical indications
to date.
Norbert Hauel received his master’s degree in Organic
Chemistry and his Ph.D. from the University of Marburg,
Germany (Prof. R.W. Hoffmann). In October 1979, he joined
Boehringer Ingelheim and started to work as a lab head in
the Department of Medicinal Chemistry. In 1988, he was promoted
to become a Group Leader of four laboratories. At that
time, his main area of research was cardiovascular diseases,
which resulted in the invention of the angiotensin II antagonist
telmisartan (Micardis1
) in 1990 and dabigatran
(Pradaxa1
) in 1996. Since then, he has been working on numerous drug discovery
projects in oncology and CNS diseases.
Herbert Nar graduated from the Technical University
Munich in Chemistry and obtained his Ph.D. from the same
institute with structural studies on blue copper electron transfer
proteins. In his postdoctoral work with Robert Huber at
the Max-Planck-Institute f€ur Biochemie, he determined the
3D structures of proteins involved in pterin biosynthesis and
studied their enzymatic mechanisms. He joined Boehringer
Ingelheim in 1995 to establish a protein crystallography laboratory.
In 2000, he took over responsibility for the Structural
Research Group that comprises units for protein expression and purification, biophysics
of ligand binding, and NMR and protein crystallography.
266j10 The Discovery of Dabigatran Etexilate
Henning Priepke received his diploma degree from the University
of Marburg and his Ph.D. from the University of
W€urzburg (Prof. Reinhard Br€uckner). After two stimulating
postdoctoral stays at the University of Cambridge in Prof.
Steven Ley’s group and in Basel at the Ciba-Geigy Central
Research Unit with Prof. Beat Ernst, he joined Boehringer
Ingelheim in 1995 where he has worked in different therapeutic
areas including cardiovascular, CNS, and metabolic
diseases.
Joanne van Ryn graduated from McMaster University in
Hamilton, Canada, and also obtained an M.Sc. and Ph.D.
from this same institution in the Department of Medical
Sciences, specializing in thrombosis and fibrinolysis
research. She began her career at Boehringer in 1990,
first as a Post Doc and then as head of a laboratory, focusing
on translational aspects of thrombosis therapy and
detection. Currently, she is the Global Head of Scientific
Support for dabigatran, initiating studies investigating the
role of thrombin in other disease processes and proof-of-concept studies in
potential new indications.
Wolfgang Wienen received his master’s degree in Biology
from the Technical University of Aachen, Germany. He
joined the group of Professor H. Kammermeier in the
Department of Physiology in the Medical Faculty of the
RWTH Aachen, where he received a Ph.D. in Animal Physiology
and Pharmacology. In 1987, he started to work as lab
head in the Department of Cardiovascular Pharmacology at
Boehringer Ingelheim, Germany. As responsible pharmacologist,
he has supported the development of telmisartan
(Micardis1
), an angiotensin II receptor antagonist, and dabigatran etexilate
(Pradaxa1
). Since 2003, he has been working in various drug discovery research
projects in the Department of Respiratory Diseases Research.
References j267
11
The Discovery of Citalopram and Its Refinement to Escitalopram
Klaus P. Bøgesø and Connie Sanchez
List of Abbreviations
CHO Chinese hamster ovary
DA dopamine
DAT dopamine transporter
DMPK drug metabolism and pharmacokinetics
DTTA ditoluoyl tartaric acid
gSERT chicken (gallus) serotonin transporter
5-HIAA 5-hydroxyindoleacetic acid
HPLC high-pressure liquid chromatography
hSERT human serotonin transporter
5-HT 5-hydroxytryptamin
5-HT1A 5-hydroxytryptamin 1A (receptor)
5-HTP 5-hydroxytryptophan
LeuT leucine transporter
MAOI monoamine oxidase inhibitor
NE norepinephrine
NET norepinephrine transporter
NMR nuclear magnetic resonance
NRI norepinephrine reuptake inhibitor
PET positron emission tomography
PKC protein kinase C
QSAR quantitative structure–activity relationship
SAR structure–activity relationship
SERT serotonin transporter
SIP serotonin interaction protein
SLC6 solute carrier 6
SMB simulated moving bed
SNRI serotonin norepinephrine reuptake inhibitor
WT SERT wild type serotonin transporter
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
j269
SPECT single-photon emission computer tomography
SSRI selective serotonin reuptake inhibitor
TCA tricyclic antidepressants
11.1
Introduction
The serendipitous discoveries of the monoamine oxidase inhibitor (MAOI) iproniazid
and the tricyclic antidepressant (TCA) imipramine in the late 1950s were major
breakthroughs in the treatment of depression. The discovery of imipramine initiated
a search for new tricyclic antidepressants using analogue design. Among these
analogues were amitriptyline (1), nortriptyline (2), and melitracen (3) (Figure 11.1).
Lundbeck developed new patentable syntheses of these three drugs (at that time,
product patents were not obtainable, only process patents) and entered the market
with these products in the early 1960s. However, the use of TCAs is associated with
disturbing and serious side effects, such as dryness of the mouth, constipation,
confusion, dizziness, sedation, orthostatic hypotension, tachycardia, and/or
arrhythmia. Relatively narrow therapeutic indices limit the dose range in which
they can be used and pose a risk in the case of overdose.
As increasing knowledge was gained about the mechanism of action of the
TCAs and appropriate screening assays were developed, chemists started to
look for ways to improve them. TCAs either inhibit the reuptake of serotonin
1 Amitriptyline, R=CH3
2 Nortriptyline, R=H
3 Melitracen, R=H
4 R=CF3
5
N R
CH3
H
N CH3
H
CH3
OH
OH
N
CH3
R
CF3
CH3
6 Talopram, X=O, R=R1=H
7 X=O, R=CH3, R1=CF3
8 Talsupram, X=S, R=R1=H
X
N
R
CH3
R1
Figure 11.1 Lundbeck’s tricyclic antidepressants and the selective NE uptake inhibitors talopram
and talsupram.
270j11 The Discovery of Citalopram and Its Refinement to Escitalopram
(5-hydroxytryptamine, 5-HT) and norepinephrine (NE) (imipramine, amitriptyline),
or predominantly NE (desipramine, nortriptyline). But they also block a
number of postsynaptic receptors, notably those for NE, acetylcholine, and histamine.
The latter effects were mainly associated with the side effects, whereas
inhibition of the monoamine transporters was associated with the therapeutic
effects. Two major hypothesis of depression emerged from this research, the 5HT
and the NE hypotheses, where lowered 5-HT levels in the brain were associated
with lowered mood, and lowered NE levels were associated with lowered
psychomotor drive. The discovery of the selective serotonin reuptake inhibitors
(SSRIs) has already been described broadly in a previous volume of this
series [1]. Thus, the following will focus on the discovery of citalopram and escitalopram
[2–4].
11.2
Discovery of Talopram
The discoveries of both citalopram and escitalopram (S-citalopram) are good examples
of how analogue design can lead to drugs with either totally different or greatly
improved therapeutic profiles compared with the starting structure.
The discovery of citalopram started with the discovery of talopram (6)
(Figure 11.1) in 1965. In an attempt to make a trifluoromethyl-substituted derivative
of melitracen (4) using a precursor molecule (5) and reaction conditions (ring
closure in concentrated sulfuric acid) similar to those used in the production of
melitracen, a chemist at Lundbeck ended up with a different product, which after
meticulous structural elucidation proved to be the phthalane structure 7. This compound
proved to be a surprisingly selective NE uptake inhibitor. Analogue design
revealed that the unsubstituted N-des-methyl analogue 6 (talopram, Lu 03-010) was
a highly selective and very potent NE uptake inhibitor. The sulfur analogue of talopram,
called talsupram (8, Lu 05-003), was likewise a very potent, selective inhibitor
of the NE transporter (NET). Both compounds were therefore major improvements
with regard to selectivity compared with the nonselective TCAs, desipramine and
nortriptyline.
Both drugs entered early clinical testing in the late 1960s, but were stopped in
phase II for various reasons, among which was a rather activating profile. This
observation was supportive of the hypothesis proposed by Professor Arvid Carlsson
that NE uptake inhibition would mainly increase psychomotor drive. Carlsson had
noticed that the tertiary amine drugs, which were mixed 5-HT and NE reuptake
inhibitors, were “mood elevating,” while the secondary amines, being primarily NE
reuptake inhibitors, increased the psychomotor drive in the depressed patients [5].
Carlsson advocated the development of selective 5-HT uptake inhibitors to treat the
lowered mood state and avoid the potential suicide risk associated with an
increased psychomotor drive in a depressed patient. Carlsson presented his theory
at Lundbeck, and in 1971 it was decided to start a search for a selective serotonin
reuptake inhibitor.
11.2 Discovery of Talopram j271
Although it may seem paradoxical, it was decided to use talopram as template for
designing an SSRI. However, the reason was that there were already a few analogues
of talopram available in-house that had dual 5-HT/NE uptake inhibition [2].
In particular, derivatives lacking the 3,3-dimethyl substituents of talopram, but with
a chlorine either at the 5-position or at the 40
-position (see the structure in
Table 11.1), were more potent as 5-HT uptake inhibitors than NE uptake inhibitors.
Like their tricyclic dual-acting counterparts, these compounds also had a dimethylamino
group, instead of the monomethylamino group found in the selective NE
uptake inhibitors.
We, therefore, decided to use the structure at the top of Table 11.1 as the core
structure for a program investigating the structure–activity relationships (SARs) of
aromatic substitution in the two benzene rings.
11.3
Discovery of Citalopram
We published our initial SAR study (actually a quantitative structure–activity relationship
(QSAR) study) in 1977 [7]. At that time, in vitro assays measuring inhibition
of neuronal 5-HT and NE uptake were not available at Lundbeck, so 5-HT and
NE uptake inhibition was measured as inhibition of ½3
HŠ-5-HT uptake into rabbit
platelets and as inhibition of ½3
HŠ-NE uptake into the mouse heart ex vivo, respectively.
Although these models were not directly comparable, they were acceptable as
long as the goal was to develop selective compounds. Later on, assays based on
inhibition of ½3
HŠ-5-HT and ½3
HŠ-NE uptake into rat brain synaptosome preparations
as well as recombinant cell-based assays expressing the cloned human serotonin
transporter (hSERT) or NET were developed. Table 11.1 shows a number of
key citalopram derivatives (9–16) with the original blood platelet results as well as
data for inhibition of ½3
HŠ-5-HT uptake in recombinant cells expressing the cloned
hSERT. All citalopram derivatives are highly selective serotonin transporter (SERT)
inhibitors compared to inhibition of the NET and the dopamine transporters
(DATs). Thus, data for NETand DAT inhibition are not shown.
Applying the observations mentioned above regarding monosubstituted
chloro derivatives led to the synthesis of the 5,40
-dichloro derivative (10,
Table 11.1) that proved to be a very potent SSRI. SAR studies of inhibitors of
SERT, NET, and DAT very often show that optimal potency is found in 3,4dichlorophenyl
derivatives [8]. This derivative (11) also proved to be very
potent, whereas the corresponding 30
,50
-dichloro derivative (12) had 25–100
times weaker activity. Substitution with chlorine at the 6-, 4-, and 7-positions
(compounds 13, 14, and 15) was also allowed. The most potent derivatives
were generally found among the 40
,5-disubstituted derivatives substituted with
F, Cl, Br, or CF3. Electron-donating groups (30
-OCH3, 40
-OCH3, or 40
-isopropyl)
had very low activity [7].
At that time, a cyano group was not considered an obvious choice in systematic
aromatic substitution SAR investigations. One reason was that a nitrile
272j11 The Discovery of Citalopram and Its Refinement to Escitalopram
Table11.1Inhibitionof5-HTuptakeandallostericeffectinaseriesofselectedcitalopramderivatives.
Inhibitionofuptakeof½3
HŠ5-HT
Ratbloodpla
hSERThSERThSERTallostericb
CompoundR1R2IC50(nM)IC50(nM)IC50(nM)IC50(mM)
95-CN(citalopram)40
-F146.78.7
S-95-CN(escitalopram)40
-F2.15.1
R-95-CN(R-citalopram)40
-F17025
105-Cl40
-Cl207.412.1
11H30
,40
-Cl2171329.6
12H30
,50
-Cl2160020034015
12ac
H30
,50
-Cl23508.8
12bc
H30
,50
-Cl230020
136-Cl40
-Cl12023
144-Cl40
-Cl47339.26.1
157-Cl40
-Cl6326
165-Br40
-F221330.2
175-(CH3)2N(CH2)3CO40
-F1.6714
185-CN40
-(4-F-C6H4-S)20012
(continued)
O
N
CH3
CH3
R1
R2
2′
3′
4'
5′
6′
4
5
6
7
11.3 Discovery of Citalopram j273
Table11.1(Continued)
Inhibitionofuptakeof½3
HŠ5-HT
Ratbloodpla
hSERThSERThSERTallostericb
CompoundR1R2IC50(nM)IC50(nM)IC50(nM)IC50(mM)
18ac
5-CN40
-(4-F-C6H4-S)3108.3
18bc
5-CN40
-(4-F-C6H4-S)33022
195-CN[2-Naphthyl]3468
19ac
5-CN[2-Naphthyl]1041
19bc
5-CN[2-Naphthyl]15055
205-CN[1-Naphthyl]25019
20ac
5-CN[1-Naphthyl]74014
20bc
5-CN[1-Naphthyl]28019
Ifnotreferredtointhetext,dataareunpublisheddata,H.LundbeckA/S.Inhibitionof½3
HŠ-5-HTuptakeatthehSERTwasperformedinChinesehamsterovary(CHO)cells
stablyexpressingwild-typehSERT,asdescribedbyRef.[6].Thetwocolumnsrepresentessentiallysimilarassayconditions.
a)DatafromRef.[7].
b)DissociationbindingstudiesatthehSERTwereconductedasdescribed[6].
c)aandbrefertodifferentenantiomericforms.
Table11.1(Continued)
274j11 The Discovery of Citalopram and Its Refinement to Escitalopram
may be metabolically labile to be transformed to an amide or carboxylic acid.
However, one of the authors of this chapter (KPB) had previously worked on a
project in which nitriles were key intermediates and had used the relatively
new reaction where the aromatic halogen was reacted with cuprous cyanide to
produce the nitrile. In August 1972, having already synthesized the potent 5-
bromo-40
-fluoro-derivative 16, he used this reaction directly on 16 and made
the first sample of 9 (citalopram). The compound proved to be a very potent
SSRI. It was selected together with a few other potent SSRIs from the series
for further preclinical studies, proved overall to have the best safety profile,
and it was selected as a clinical development candidate. Interestingly, the
cyano group proved to be totally metabolically stable.
11.4
Synthesis and Production of Citalopram
The syntheses used for preparation of citalopram and derivatives are outlined
in Scheme 11.1. The starting materials were phthalides (I). These were either
made by methods published in the literature, or by improved or completely
new procedures. The majority of compounds could then be produced by a
“double Grignard” reaction, in which the phthalide was reacted with a substituted
phenyl magnesium bromide to give the benzophenone intermediate II,
which was further reacted with 3-(dimethylamino)propyl magnesium chloride
to afford the “dicarbinol” III. The dicarbinols were then ring closed in strong
acid to the final product VI.
O
O
X
X
MgBr
Y
O
OH
Y
X
OH
OH
Y
H3C
N MgCl
CH3
N
CH3
CH3
O
N
CH3
CH3
2′
3′
4′
5′
6′
4
5
6
7
X
Y
H+
IIII
O
H
X
Y
X
OH
OH
Y
H3C
N Cl
CH3
NaHH+
VIVIV
LiAlH4
II
(CuCN)[A]
(CuCN)[A]
Scheme 11.1 The syntheses used for preparation of citalopram and derivatives. [A] represents
extra step exchanging Br with CN using cuprous cyanide in DMF if the end product was a cyanosubstituted
derivative.
11.4 Synthesis and Production of Citalopram j275
The 5-bromo derivative 16 was made by this synthesis (from 5-bromophthalide)
and as mentioned above, citalopram (X ¼ 5-CN, Y ¼ 40
-F) was synthesized
for the first time by reacting the Br in 16 with cuprous cyanide. However, it
quickly turned out that this method was unsuited for the preparation of larger
quantities, so an alternative method was developed. Starting again with 5-bromophthalide,
the benzophenone II was reduced with LiAlH4 to the intermediate
IV, which was ring closed in strong acid to the phenylphthalane V (X ¼ 5Br,
Y ¼ 40
-F). By reaction with cuprous cyanide, a crystalline 5-cyano-1-(40
fluorophenyl)phthalane
was obtained. Citalopram was then obtained by metallation
with NaH in dimethyl sulfoxide and subsequent reaction with 3-(dimethylamino)propyl
chloride. This method was used to produce the first 5 kg.
Unfortunately, this method was also unsuited for large-scale production. This
was a critical point in the development of citalopram, but we made a very surprising
discovery. It turned out that the cyano group in 5-cyanophthalide did
not react with the Grignard reagents, contrary to expectation, and did not
hydrolyze in the strong acid used in the final ring closure. We also found that
the second “side chain” Grignard reagent could be added directly after the 4fluorophenyl
magnesium bromide without isolating the intermediate benzophenone.
This was a major improvement, and this synthesis proved to be an
excellent production method.
11.5
The Pharmacological Profile of Citalopram
The pharmacological characterization of citalopram showed that it potently inhibited
the uptake of ½3
HŠ-5-HT in rabbit and rat thrombocytes and rat brain synaptosomes
with IC50 values in the nanomolar range (14 and 1.7 nM, respectively) [9, 10]. Furthermore,
citalopram turned out to be the most selective 5-HT uptake inhibitor
among the SSRIs in clinical use at that time, with a selectivity ratio of 3400 relative
to the inhibition of ½3
HŠ-NE uptake in rat brain synaptosomes compared to selectivity
ratios of 840, 280, and 54 for sertraline, paroxetine, and fluoxetine, respectively,
and the selectivity relative to the inhibition of ½3
HŠ-dopamine (DA) uptake was even
greater, that is 22000 compared to selectivity ratios of 250, 18000, and 740 for sertraline,
paroxetine, and fluoxetine [11]. Citalopram had very low affinity for the
receptors studied, the highest affinity being for the s1 and histamine H1 receptors
(IC50 200 and 350 nM, respectively) [11]. Interestingly, early binding dissociation
studies of ½3
HŠ-imipramine, ½3
HŠ-paroxetine, and ½3
HŠ-citalopram suggested that
these drugs bind to different areas of the SERT [12], even though the implications
of this were unclear at that time.
The available mechanistic in vivo assays only provided indirect measures of
uptake inhibition and selectivity. For example, citalopram inhibited the uptake of
the 5-HT depleting agent, H75/12, into neurons with an ED50 of 0.80 mg/kg and
failed to inhibit NE depletion by the depleting agent H77/77 at doses as high as
160 mg/kg [9]. Similarly, studies of amine turnover showed that an acute dose of
276j11 The Discovery of Citalopram and Its Refinement to Escitalopram
citalopram reduced 5-HT turnover in the brain (i.e., [5-HT] was unchanged and the
metabolite, [5-hydroxyindoleacetic acid, 5-HIAA], was decreased) and had no effect
on NE synthesis. This suggested that citalopram was the most selective of the compounds
tested [13, 14]. Finally, simple behavioral screening assays, such as the
potentiation of 5-hydroxytryptophan (5-HTP)-induced 5-HT syndrome (5-HT
uptake inhibition), tetrabenazine-induced ptosis (NE uptake inhibition), and apomorphine-induced
gnawing (DA uptake inhibition), supported a selective 5-HT
uptake inhibition in vivo [15]. Citalopram, like other SSRIs, had limited and variable
effects in validated behavioral models predictive of antidepressant effect, such as
the forced swim test, which had been validated with TCAs and MAOIs [16]. It was
not until after the development of citalopram had been completed that more
advanced in vivo assays, such as microdialysis, in vivo electrophysiology, and more
complex behavioral models, became available.
11.6
Clinical Efficacy of Citalopram
Citalopram was first launched for the treatment of major depressive disorder in
1989 in Denmark as Cipramil1
and subsequently marketed worldwide, including
the United States in 1998 under the trade name Celexa1
. Following the approval
for major depressive disorder, citalopram was also approved for the treatment
of panic disorders. After only a few years on the market, the drug attained blockbuster
status.
A large number of clinical short- and long-term studies of patients with major
depressive disorder have been conducted with citalopram over the years, and different
subsets of the clinical data have been subjected to meta-analyses as well. In
general, citalopram was found to have an efficacy comparable to that of other SSRIs
and, in some studies, also like other SSRIs, to be slightly less efficacious than the
TCAs [17, 18]. Citalopram has also been shown to be efficacious in the treatment of
other conditions, such as social phobia, obsessive compulsive disorder, post-traumatic
stress disorder, mixed anxiety and depression, and poststroke depression
[17, 18]. Citalopram has a favorable pharmacokinetic profile with good
bioavailability and linear kinetics and a low potential for interactions with concomitant
medication [17]. It is generally well tolerated with a better tolerability than the
TCAs [19, 20]. These favorable properties made citalopram a good choice, particularly
for depressed patients who required continuation and long-term treatment, as
well as for elderly patients [17]. Overall, citalopram was on par with the other SSRIs
with respect to efficacy, but has more favorable drug metabolism and pharmacokinetics
(DMPK) properties (e.g., approximately 80% bioavailability and 80% protein
binding, dose-proportional linear pharmacokinetics, an elimination half-life of
1.5 days, negligible pharmacological activity of metabolites, and low drug–drug
interaction potential), which are the likely reasons why citalopram became such a
commercial success, even though it was the fifth SSRI introduced onto the US
market.
11.6 Clinical Efficacy of Citalopram j277
11.7
Synthesis and Production of Escitalopram
The preparation of the closest analogues of citalopram, its single enantiomers,
proved to be a major challenge. Direct resolution via diastereomeric salts failed
after many fruitless attempts. Different chiral acids, different solvents, and different
stoichiometric ratios of citalopram and acid were tried, but the major problem
was the lack of crystal formation in almost all cases. A crystalline (þ)-camphor sulfonate
was obtained, but no separation of enantiomers could be accomplished.
Chiral high-pressure liquid chromatography (HPLC) was in its infancy, and the
available analytical columns were tried with negative results. We wanted to avoid
acid ring closure of resolved intermediates due to the risk of racemization. Therefore,
we spent some time on various asymmetric syntheses focusing on derivatives
with a partially finished side chain that after potential resolution could be transformed
to the citalopram enantiomers without risk of racemization. However,
these attempts were also unsuccessful.
Finally, we decided to try to resolve the intermediate diol III (Scheme 11.1),
although we did not have a strategy for a subsequent stereoselective ring closure
at hand. We made the diasteromeric esters with the enantiomers of a-methoxya-trifluoromethyl
acetic acid (Mosher’s acid, known as a shift reagent for nuclear
magnetic resonance (NMR)) and tried to separate them on preparative (nonchiral)
HPLC. By repeated peak shaving, we obtained small samples of the pure diastereomers.
Importantly, we had noticed a seemingly spontaneous slow ring closure to
citalopram of the mixture of diastereomeric esters (in the presence of triethylamine)
during their synthesis. This encouraged us to try a stronger base (potassium
tert-butoxide), and to our great surprise, this resulted in a stereoselective ring
closure of the pure diastereomers to afford the very first small sample of the pure
citalopram enantiomers. Later we found that the diol III also could be resolved by
diastereomeric salt formation with di-p-toluoyl tartaric acid (DTTA) and, in this
way, it became possible to produce larger quantities of the enantiomers (using a
basic ring closure with a mesylate of the diol and triethylamine).
This method was possibly also suited for production scale, but as chromatographic
separation of the diols using simulated moving bed (SMB) technology in
the late 1990s proved very effective, two SMB plants (a pilot and a full scale) were
built for escitalopram production. Later, production became even more cost-effective
when development chemists discovered that acidic ring closure of the R-diol
led to a mixture of approximately 30% R-citalopram and 70% escitalopram, which
could subsequently be isolated as citalopram and pure escitalopram.
In recent years, many chemists outside Lundbeck have worked with alternative
syntheses (for a recent review, see Ref. [21]) of citalopram and escitalopram.
Of special interest was a publication in 2007 by researchers from Dr.
Reddy’s Laboratories who published a direct resolution of citalopram using
DTTA [22]. At that point, we had completed a systematic study with a large
number of chiral acids (including DTTA) without finding a single one that
worked. So we were not surprised to find that in our hands the Dr. Reddy
278j11 The Discovery of Citalopram and Its Refinement to Escitalopram
method did not work. Through a series of experiments, we showed that resolution
of citalopram was not possible by Dr. Reddy’s method [23]. In a subsequent
response, Dr. Reddy researchers admitted that the method did not work
as described. They then claimed success with a highly modified procedure, but
in our hands that did not work either [24, 25].
In conclusion, the preparation and production of citalopram and escitalopram
have been major challenges, but we finally succeeded in developing highly innovative
and effective syntheses that are still the best and most cost-effective production
methods.
11.8
The Pharmacological Profile of the Citalopram Enantiomers
Shortly after the citalopram enantiomers had been successfully produced in the
laboratory in 1988, they were characterized in vitro in the rat brain synaptosome
assay of ½3
HŠ-5-HT uptake inhibition and in vivo in the mouse 5-HTP potentiation
assay. Completely unexpectedly, the 5-HT uptake inhibition of citalopram turned
out to reside in the S-enantiomer, and R-citalopram was found to be practically
devoid of this activity in both the in vitro and in vivo assays. These initial findings
were reproduced and substantiated by studies of NE and DA uptake inhibitory
activity and published in 1992 [26]. Based on these studies and the fact that racemic
citalopram is a highly selective inhibitor of the SERT, R-citalopram was thought to
be pharmacologically inactive. Only very few pharmacological studies were conducted
with escitalopram in the next few years. It was not until new production
methods (see above) made it feasible to produce escitalopram at an industrial scale
and it was decided in 1997 to develop escitalopram for major depressive disorder
that there was a renewed interest in pharmacological studies of escitalopram. Based
on the knowledge available then, the original expectation was that escitalopram
would be comparable to citalopram with respect to efficacy, and that the tolerability
would be improved by removing the presumably pharmacologically inactive Renantiomer
and thereby minimizing the drug load in the body.
11.9
R-Citalopram’s Surprising Inhibition of Escitalopram
It was not until about a decade after the first characterization of the citalopram
enantiomers that observations from behavioral studies made us hypothesize
that R-citalopram counteracts the effect of escitalopram. A comparison of escitalopram
and citalopram in a simple rat model predictive of anxiolytic activity,
inhibition of footshock-induced ultrasonic vocalization, revealed that escitalopram
dose dependently and completely abolished the vocalization, whereas citalopram
only partially reduced the vocalization and reached a plateau of about
60% inhibition when the dose was increased [27]. These findings made us
11.9 R-Citalopram’s Surprising Inhibition of Escitalopram j279
hypothesize that R-citalopram might counteract the anxiolytic-like activity of
escitalopram in this model.
Knowing that the ultrasonic vocalization model is very sensitive to serotonergic
mechanisms, it was a logical next step to investigate the effect of R-citalopram
alone and in combination with escitalopram on the extracellular 5-HT
levels in relevant brain regions, such as the frontal cortex and the ventral hippocampus.
From pharmacokinetics studies in humans, we also knew that the
citalopram enantiomers are metabolized at a different rate, which meant that
the plasma level of R-citalopram is at least twofold higher than that of escitalopram
in individuals treated with racemic citalopram [28]. Thus, these first
behavioral observations were followed up by a series of microdialysis studies
in freely moving rats where the effects of escitalopram and citalopram as well
as ratios of escitalopram to R-citalopram of 1 : 1, 1 : 2, and 1 : 4 were studied.
The outcome of these studies was that escitalopram produced a significantly
higher increase in the level of extracellular 5-HT compared to equivalent doses
of citalopram, and that R-citalopram antagonized the effect of escitalopram in
a dose-dependent manner [29].
These observations triggered a series of experiments aimed at identifying the
mechanism by which R-citalopram counteracts the therapeutic effect of escitalopram.
Studies of brain exposure levels in rats treated with escitalopram alone versus
combinations of escitalopram and R-citalopram quickly ruled out a
pharmacokinetic interaction as the explanation [29]. A very broad receptor screening
including 144 targets did not reveal any obvious target through which R-citalopram
could exert its action [30]. A reverse dialysis study in rats dosed systemically
with escitalopram showed that R-citalopram could exert its inhibitory action when
administered at terminal areas of 5-HT neurons, such as the prefrontal cortex [29].
In vivo recordings of individual 5-HT neurons in the rat dorsal raphe nucleus demonstrated
that desensitization of somatodendritic 5-HT1A autoreceptors, a mechanism
believed to be associated with the delayed clinical response of SSRIs,
occurred after only 2 weeks’ treatment with escitalopram, whereas 3 weeks’ treatment
was required for an equivalent dose of citalopram [31]. In another study using
the same methodology, it was shown that R-citalopram delayed the recovery of 5HT
neuronal firing and the 5-HT1A receptor desensitization produced by escitalopram
[31–33]. The superior effect of escitalopram compared to citalopram and the
inhibitory action of R-citalopram on the pharmacological activities of escitalopram
have been confirmed in numerous animal models using acute or repeated dosing
[34–36] (Table 11.2).
The mechanistic in vivo studies all pointed toward the SERT as the target for
the interaction between escitalopram and R-citalopram. This and the original
in vitro reports from Plenge et al. [12] that SSRIs could bind differently to the
SERT made us decide to investigate the in vitro binding kinetics of the individual
citalopram enantiomers in further detail. Kinetic studies of ½3
HŠ-escitalopram
binding to the SERT demonstrated that cold escitalopram slows down
the dissociation rate of ½3
HŠ-escitalopram from the primary (inhibitory) site of
the SERT [6]. This is indicative of the existence of a secondary allosteric
280j11 The Discovery of Citalopram and Its Refinement to Escitalopram
Table 11.2 Superior effect of escitalopram over citalopram and inhibitory action of R-citalopram
on the pharmacological activities of escitalopram in nonclinical models after acute or prolonged
exposure.
Assay Outcome References
Mechanistic
Microdialysis
Extracellular 5-HT in
prefrontal cortex and ventral
hippocampus in rats
Escitalopram increases 5-HTmore than
citalopram
[29, 32, 37, 38]
R-Citalopram antagonizes escitalopraminduced
increase of 5-HT
Voltammetry in rats Escitalopram is more potent than
citalopram at inhibiting 5-HTclearance
[39]
R-Citalopram counteracts escitalopram
inhibition of 5-HTclearance
Electrophysiology .
5-HT neuronal firing in
dorsal raphe nucleus in rats
Escitalopram is four times more potent
than citalopram, acute dosing
[31–33]
R-Citalopram antagonizes escitalopraminduced
suppression of firing, acute
dosing
Escitalopram normalizes DRN firing
faster than citalopram, chronic dosing
R-Citalopram delays effect of
escitalopram on firing
DA neuron firing in ventral
tegmental area in rats
Escitalopram increases firing rate and
burst firing of DA neurons, acute dosing
[40]
R-Citalopram antagonizes the effect of
escitalopram on DA neuron firing
NMDA-induced currents in
hippocampal pyramidal
neurons in rats
Escitalopram but not citalopram
potentiates NMDA-induced currents in
pyramidal neurons
[40]
R-Citalopram antagonizes the effect of
escitalopram
LTP in rats R-Citalopram counteracts escitalopram
effect on LTP
[41]
Neurogenesis in rats R-Citalopram counteracts escitalopraminduced
cell proliferation in the dentate
gyrus
[33]
HPA axis
Plasma corticosterone in rat R-Citalopram antagonizes escitalopraminduced
increase of plasma
corticosterone
[42]
Behavior
Potentiating of 5-HTPinduced
behavior, rat,
mouse
R-Citalopram antagonizes the
potentiating effect of escitalopram
[42, 43]
(continued )
11.9 R-Citalopram’s Surprising Inhibition of Escitalopram j281
binding site that modulates the binding properties of the drug at the primary
site. In addition, we found that R-citalopram attenuated the decreased dissociation
rate of ½3
HŠ-escitalopram in the presence of cold escitalopram [6, 49].
Finally, we found that R-citalopram also slowed the association rate of
½3
HŠ-escitalopram to the SERT [32, 49]. These findings were supportive of the
notion that R-citalopram counteracts the effects of escitalopram at the SERT,
and that the interaction takes place at a secondary allosteric modulator site. As
described in further detail in the next section, species comparisons and sitedirected
mutagenesis studies have led to identification of domains on the
SERT that are critical for the delayed dissociation produced by escitalopram
and the negative effects of R-citalopram on the binding of escitalopram to the
SERT [49, 50].
Using brain imaging techniques, positron emission tomography (PET), or single-photon
emission computer tomography (SPECT) and appropriate radiolabeled
ligands, it was demonstrated that the occupancy at the SERT in the human brain is
significantly higher with escitalopram 10 mg than with the corresponding dose of
citalopram (20 mg, containing 10 mg of R-citalopram and 10 mg of S-citalopram) at
steady state. The mean occupancies at 6 and 54 h after last doses were 82 and 63%
for escitalopram, respectively, and 64 and 49% for citalopram, respectively [51]. The
results were confirmed in a pooled analysis, in which the authors concluded that
the buildup of the R-enantiomer after repeated citalopram dosing may lead to lower
SERToccupancy by the S-enantiomer [52].
Table 11.2 (Continued)
Assay Outcome References
Anxiolytic and antidepressant effects and cognitive function
Elevated plus maze, rat R-Citalopram antagonizes anxiolytic
effect of escitalopram
[44]
Vogel conflict test, rat R-Citalopram antagonizes anxiolytic
effect of escitalopram
[44]
Separation-induced
vocalization, mouse pup
R-Citalopram antagonizes anxiolytic
effect of escitalopram
[45]
Footshock-induced ultrasonic
vocalization, rat
R-Citalopram antagonizes anxiolytic
effect of escitalopram
[27]
Conditioned fear, rat R-Citalopram antagonizes anxiolytic
effect of escitalopram
[46]
Resident intruder, rat Escitalopram produces antidepressant
effect earlier than citalopram
[47]
R-Citalopram counteracts escitalopraminduced
antidepressant effect
Chronic mild stress, rat Escitalopram produces antidepressant
effect earlier than citalopram
[48]
R-Citalopram counteracts escitalopraminduced
antidepressant effect
Novel object recognition, rat Escitalopram but not citalopram
improved recognition memory
[40]
282j11 The Discovery of Citalopram and Its Refinement to Escitalopram
11.10
Binding Site(s) for Escitalopram on the Serotonin Transporter
Early on in the era of antidepressant drugs, medicinal chemists started to speculate
about the mode of binding of these drugs to their target, that is SERT and NET.
With respect to citalopram and escitalopram, as described above, we noticed that
relatively small changes in the structure could transform a selective NE uptake
inhibitor such as talopram into an SSRI such as citalopram. Eli Lilly made a similar
observation with the NRIs tomoxetine and nisoxetine, and the SSRI fluoxetine.
However, the chiral SSRIs differed with regard to stereoselectivity. While the
enantiomers of fluoxetine were equipotent as SSRIs, a high stereoselectivity was
observed with the enantiomers of citalopram and with paroxetine and its
enantiomers.
We worked in the 1980s with another structural class of antidepressants, the 3phenyl-1-indanamines
where the amine could be a dimethyl- or monomethylamine
or a piperazine [8, 53]. We observed a moderate stereoselectivity of the enantiomers
of trans-isomers with the small amines in favor of the 1R,3S conformation, but a
high stereoselectivity of the piperazine enantiomers with 1S,3R as the active stereoisomer.
We tried to rationalize this in a simple cartoon of a three-point binding
model [54]. However, this model was not useful for the prediction of the stereoselectivity
of citalopram.
Smith presented a three-dimensional model that he interpreted as predicting a
high stereoselectivity of the R-enantiomer of citalopram versus the S-enantiomer
(this was obviously later proved wrong) [55]. However, the author did not take the
semisymmetrical structure of citalopram into account. If one does that, both enantiomers
fit his model equally well, and the model could just as well predict no
stereoselectivity.
Later on when we established computational chemistry (and citalopram had been
resolved into its individual enantiomers), we developed a pharmacophore model
that was used to rationalize both the structural elements determining SERT or NET
selectivity and the observed stereoselectivity of citalopram and several other
SSRIs [56]. When fitting R- and S-citalopram into the model, we took advantage
of the semisymmetrical nature of the compound mentioned above, that is, the
p-fluorophenyl ring of one enantiomer was superimposed onto the phthalane ring
of the other enantiomer. Interestingly, these different binding modes were also the
outcome of a subsequent, more comprehensive computational study of the binding
of the enantiomers [57]. In order to further study the structural elements in talopram
and citalopram that were important for SERT and NET selectivity, respectively,
14 derivatives of the two compounds were either selected or synthesized [58]
and tested for 5-HT and NE uptake inhibition [59]. This study showed, in agreement
with our previous pharmacophore model study, that the phthalane 3,3dimethyl
substituents and 5-cyano group are key determinants for inhibitory activity
and selectivity toward NETand SERT, respectively.
However, the real breakthrough came with the publication of the X-ray structure
of the leucine transporter (LeuT), a bacterial homologue to the
11.10 Binding Site(s) for Escitalopram on the Serotonin Transporter j283
mammalian solute carrier 6 (SLC6) transporters [60]. We developed a SERT
homology model based on the LeuT structure with escitalopram bound to a
site equivalent to the leucine binding site in LeuT [61, 62]. Subsequently, several
other homology models supported by mutational studies strongly supported
this site as the escitalopram binding site, despite some differences in
the suggested escitalopram conformation and binding mode at the
site [57, 63, 64].
Interestingly, two X-ray structures of LeuT were then published with the
TCAs clomipramine and desipramine bound within the transporter [65, 66].
The TCAs bound (with low affinity) in a cavity (called the vestibule or the S2
site), located $15 A

above the S1 site (the substrate binding site). This site had
also been found in a study we made of the driving forces for migration of
leucine through LeuT [67]. Subsequently, X-ray structures of LeuT with the
SSRIs sertraline and R- and S-fluoxetine bound within the S2 site were published
[68]. The authors of this study speculated that the equivalent site in the
SERT might be the binding site for antidepressants. However, the mutational
studies mentioned above showed not only a strong influence on escitalopram
binding of mutations at the S1 site on escitalopram binding, but also that
mutations in the S2 pocket did not influence escitalopram binding [63, 64].
Moreover, we have recently shown that mutation of just five amino acids
within the S1 binding pocket of the NET to the corresponding residues in the
SERT conferred affinity equivalent to WT SERT for citalopram and its enantiomers
at the mutated NET. In contrast, the binding of talopram was almost
unaffected by all mutations at both the SERT and the NET. The binding mode
of talopram to NET is, therefore, not at the S1 site [59].
As mentioned above, escitalopram has an allosteric effect that we believe is
attained through a separate allosteric binding site on the SERT. Studies on
chicken SERT (gSERT) have demonstrated a low allosteric potency of escitalopram
in this species. However, mutation of nine residues in transmembrane
domains 10, 11, and 12 to their corresponding human residues restored the
allosteric effect [50]. The location of these nine residues did not seem to comprise
a cavity for a putative allosteric binding site. It has recently been proposed
that the SERT allosteric site could be identical to the S2 site [57].
Accordingly, the nine residues might have an indirect effect on escitalopram
binding at the S2 site (Figure 11.2).
A clear indication of the different nature of the S1 and S2 sites is that escitalopram
derivatives show a different SAR toward to two sites. In Table 11.1, the effect
on the hSERT S1 site (inhibition of ½3
HŠ-5-HT uptake) is shown, as well as the allosteric
effect (inhibition of ½3
HŠ-escitalopram dissociation) of a number of escitalopram
derivatives. While the 5-chloro (10), 5-bromo (16), and 30
,40
-dichloro (11)
derivatives retain their effect at both sites, the 30
,50
-dichloro analogues (12 and
enantiomers 12a and 12b), which lack an effect at the S1 site, are still potent allosteric
modulators. A study of naphthyl derivatives of citalopram was recently published
[69]. Here we found that while the 2-naphthyl derivative of citalopram (19
and enantiomers 19a and 19b) retained its effect at both sites, the 1-naphthyl
284j11 The Discovery of Citalopram and Its Refinement to Escitalopram
Figure 11.2 Escitalopram’s two binding sites
at SERT. (a) A schematic drawing of the
suggested positions of escitalopram’s primary
binding site (S1) and allosteric binding site
(S2). (b) The X-ray structure model of LeuT
taken from Ref. [68]. The leucine binding pocket
is encircled in green. The “vestibule” pocket is
encircled in red. The corresponding ligands
from the X-ray structure (leucine and sertraline,
respectively) are depicted in yellow.
Superimposed on these is the docked structure
of escitalopram, where the additional aromatic
ring of compound 20 has been sketched in.
11.10 Binding Site(s) for Escitalopram on the Serotonin Transporter j285
derivatives (20 and enantiomers 20a and 20b) were selective allosteric modulators
(Figure 11.3).
Compounds 17 and 18 were originally identified as impurities formed in escitalopram
production. Amazingly, testing showed that 17 (citalopram with an extra
side chain at the 5-position) has a very high affinity at the S1 site, but is devoid of
an allosteric effect. Conversely, 18 (and enantiomers 18a and 18b) are selective allosteric
modulators. The stereoselectivity at the S2 site is generally low as can be seen
from the data on the enantiomers of 9, 12, 18, 19, and 20.
In conclusion, analogue design of citalopram has turned out to be potentially
much more complicated than originally anticipated. The insight gained in recent
years into the dual effect of escitalopram and the different SARs of derivatives for
the S1 and S2 sites underlines how fortunate it is that citalopram was originally
selected for development. In principle, there was a risk, of which we were then
unaware, of selecting a derivative such as 17, which has no allosteric effect.
11.11
Future Perspectives on the Molecular Basis for Escitalopram’s Interaction
with the SERT
It has been shown that SSRIs in addition to blocking the reuptake of 5-HT also
cause an internalization of the SERT [70], and that the potency ranking of SSRIs to
cause internalization and to block the uptake of 5-HT appear to differ [71]. Recent
research shows that R-citalopram antagonizes escitalopram’s effect on the expression
of SERT in the cell membrane and that protein kinase C (PKC) appears to be
involved in this effect as the PKC inhibitor staurosporine abolished the effect of Rcitalopram
[72]. A putative interaction between the allosteric mechanism of the
SERT and the so-called serotonin interaction proteins (SIPs), of which PKC is one,
18 R=
19 R=
20 R=
O
R
N
CH3
CH3
NC
S
F
Figure 11.3 Citalopram derivatives with selective (S2 site) allosteric effect (18 and 20) and dual
(S1 and S2 sites) effect (19).
286j11 The Discovery of Citalopram and Its Refinement to Escitalopram
may potentially open up a new area of research into the function of the SERT and
eventually lead to new drug targets [73].
11.12
Clinical Efficacy of Escitalopram
In 2002, escitalopram was launched as Cipralex1
in Europe and as Lexapro1
in the
United States. Escitalopram is approved for the treatment of major depressive disorder
and social anxiety disorder in the United States, and major depressive disorder,
panic disorder, social anxiety disorder, generalized anxiety disorder, and
obsessive compulsive disorder in Europe and elsewhere.
Four pivotal randomized placebo-controlled short-term studies were conducted
for the registration file. Three of these studies had a citalopram group included as
active reference. Interestingly, and surprisingly, there was a signal that escitalopram
had a faster onset of acting and/or was more efficacious than an equivalent dose of
citalopram [74, 75]. These studies were not designed or powered to show differences
to an active reference, but subsequent studies with head-to-head comparisons
of citalopram and escitalopram confirmed the original observation that escitalopram
is superior to citalopram, particularly in patients with severe depression [76, 77]. In
a meta-analysis of pooled data at an individual patient level, escitalopram was statistically
significantly superior to citalopram, with response rates of 59.7 and 52.3%,
respectively, for the complete data set, and 61.2 and 49.9%, respectively, for the subset
of patients with severe depression [78]. In a meta-analysis of publicly available
data [79], statistically superior response rates of 72.3% for escitalopram versus
63.9% for citalopram were reported. The efficacy of escitalopram has been further
confirmed in a prospective, randomized, double-blind study against paroxetine [80],
and in a post-hoc analysis of the effect of anxiety symptoms on patients with severe
depression from the same study [81]. Data from this study and an analysis of data
from two studies comparing escitalopram with paroxetine [82] confirmed the superior
efficacy of escitalopram over another SSRI. Further comparisons of pooled data
from patients with severe depression treated with escitalopram versus other antidepressants
again confirm that escitalopram is superior to other comparators, including
the serotonin norepinephrine reuptake inhibitors (SNRIs) venlafaxine and
duloxetine [83–87]. In patients with generalized anxiety disorder, escitalopram has
also shown statistically significant superiority over the comparator, paroxetine
[88, 89]. Overall, the tolerability of escitalopram was very similar to that of citalopram
[34], whereas toxicity in relation to overdose has been reported to be more
serious for citalopram than escitalopram [90, 91]. Thus, the original expectation of
similar efficacy of escitalopram and citalopram and escitalopram being better tolerated
proved to be wrong, at least at therapeutic doses. Escitalopram not only
resulted in being clinically superior to citalopram, but has also proven to be superior
to other SSRIs and to SNRIs in several clinical studies. In 2010, it reached the
status of being the most prescribed branded antidepressant globally and, by mid-
2011, more than 265 million patients have been treated with the drug.
11.12 Clinical Efficacy of Escitalopram j287
11.13
Conclusions
The development of citalopram, and subsequently of escitalopram, has not been
trivial and was full of surprises, where time after time predictions based on the
established rules and perceptions have been proven wrong as the data emerged.
The discovery of citalopram and escitalopram underlines the point that analogue
design is not trivial and can lead to drugs with either totally different or greatly
improved profiles compared with the starting structure. Similarly, the complexity
of the biological mechanisms we are dealing with calls for caution when making
predictions and for an open mind to recognize and follow through on serendipitous
findings.
References
1 Childers, W.E., Jr., and Rotella, D.P. (2011)
Selective serotonin uptake inhibitors for
the treatment of depression, in AnalogueBased
Drug Discovery II (eds J. Fischer, and
C.R. Ganellin), Wiley-VCH Verlag GmbH,
Weinheim, pp. 269–295.
2 Bang-Andersen, B. and Bøgesø, K.P.
(2010) Dopamine and serotonin, in
Textbook of Drug Design and Discovery,
4th edn (eds P. Krogsgaard-Larsen, K.
Strømgaard, and U. Madsen), CRC Press,
Boca Raton, FL,, pp. 299–312.
3 H. Lundbeck A/S (2010) Cipralex1
:
Building a Success. H. Lundbeck A/S,
Valby, Denmark.
4 Pedersen, V. and Bøgesø, K.P. (1998) Drug
hunting, in The Psychopharmacologists II
(ed. D. Healy), Chapman & Hall, London,
UK, pp. 561–579.
5 Carlsson, A., Corrodi, H., Fuxe, K., and
H€okfelt, T. (1969) Effect of antidepressant
drugs on the depletion of intraneuronal
brain 5-hydroxytryptamine stores caused
by 4-methyl-a-ethyl-meta-tyramine. Eur. J.
Pharmacol., 5, 357–366.
6 Chen, F., Larsen, M.B., Neubauer, H.A.,
Sanchez, C., Plenge, P., and Wiborg,
O. (2005) Characterization of an
allosteric citalopram-binding site at the
serotonin transporter. J. Neurochem., 92,
21–28.
7 Bigler, A.J., Bøgesø, K.P., Toft, A., and
Hansen, V. (1977) Quantitative structure–
activity relationships in a series of
selective 5-HTuptake inhibitors. Eur. J.
Med. Chem., 12, 289–295.
8 Bøgesø, K.P., Christensen, A.V., Hyttel, J.,
and Liljefors, T. (1985) 3-Phenyl-1indanamines.
Potential antidepressant
activity and potent inhibition of
dopamine, norepinephrine, and serotonin
uptake. J. Med. Chem., 28, 1817–1828.
9 Hyttel, J. (1977) Neurochemical
characterization of a new potent and
selective serotonin uptake inhibitor: Lu
10-171. Psychopharmacology, 51, 225–233.
10 Hyttel, J. (1978) Effect of a specific 5-HT
uptake inhibitor, citalopram (Lu 10-171),
on ½3
HŠ-5-HTuptake in rat brain
synaptosomes in vitro.
Psychopharmacology, 60, 13–18.
11 Hyttel, J., Arnt, J., and Sanchez, C. (1995)
The pharmacology of citalopram. Rev.
Contemp. Pharmacother., 6, 271–285.
12 Plenge, P., Mellerup, E.T., and Laursen,
H. (1991) Affinity modulation of
½3
HŠimipramine, ½3
HŠparoxetine and
½3
HŠcitalopram binding to the 5-HT
transporter from brain and platelets. Eur.
J. Pharmacol., 206, 243–250.
13 Hyttel, J. (1977) Effect of a selective 5-HT
uptake inhibitor – Lu 10-171 – on rat brain
5-HT turnover. Acta Pharmacol. Toxicol.,
40, 439–446.
14 Carlsson, A. and Lindqvist, M. (1978)
Effects of antidepressant agents on the
synthesis of brain monoamines. J. Neural
Transm., 43, 73–91.
288j11 The Discovery of Citalopram and Its Refinement to Escitalopram
15 Christensen, A.V., Fjalland, B., Pedersen,
V., Danneskiold-Samsøe, P., and
Svendsen, O. (1977) Pharmacology of a
new phthalane (Lu 10-171), with specific
5-HTuptake inhibiting properties. Eur. J.
Pharmacol., 41, 153–162.
16 Porsolt, R.D., Bertin, A., Blavet, N.,
Deniel, M., and Jalfre, M. (1979)
Immobility induced by forced swimming
in rats: effects of agents which modify
central catecholamine and serotonin
activity. Eur. J. Pharmacol., 57, 201–210.
17 Joubert, A.F., Sanchez, C., and Larsen, F.
(2000) Citalopram. Hum.
Psychopharmacol., 15, 439–451.
18 Bezchlibnyk-Butler, K., Aleksic, I., and
Kennedy, S.H. (2000) Citalopram – a
review of pharmacological and clinical
effects. J. Psychiatry Neurosci., 25,
241–254.
19 Baldwin, D., and Johnson, F.N. (1995)
Tolerability and safety of citalopram. Rev.
Contemp. Pharmacother., 6, 315.
20 Muldoon, C. (1996) The safety and
tolerability of citalopram. Int. Clin.
Psychopharmacol., 11 (Suppl. 1), 35–40.
21 Harrington, P.J. (2011) Lexapro1
(escitalopram oxalate), in Pharmaceutical
Process Chemistry for Synthesis, Rethinking
the Routes to Scale-Up, John Wiley & Sons,
Inc., Hoboken, NJ, pp. 30–91.
22 Elati, C.R., Kolla, N., Vankawala, P.J.,
Gangula, S., Chalamala, S., Sundaram, V.,
Bhattacharya, A., Vurimidi, H., and
Mathad, V.T. (2007) Substrate
modification approach to achieve efficient
resolution: didesmethylcitalopram: a key
intermediate for escitalopram. Org. Process
Res. Dev., 11, 289–292.
23 Dancer, R.J., and de Diego, H.L. (2009)
Attempted resolution of citalopram using
(À)-O,O0
-di-p-toluoyl-(R,R)-tartaric acid,
and reflections on an alkylation reaction;
comment on an article by Elati et al. Org.
Process Res. Dev., 13, 23–33.
24 Dancer, R.J., and de Diego, H.L. (2009)
Response to the comments by Elati et al.
in response to our article examining one
of their previous papers. Org. Process Res.
Dev., 13, 38–43.
25 Elati, C.R., Kolla, N., and Mathad, V.T.
(2009) Response to Dancer’s comments
on our article “Substrate modification
approach to achieve efficient resolution:
didesmethylcitalopram: a key
intermediate for escitalopram.” Org.
Process Res. Dev., 13, 34–37.
26 Hyttel, J., Bogeso, K.P., Perregaard, J., and
Sanchez, C. (1992) The pharmacological
effect of citalopram residues in the (S)(þ)-enantiomer.
J. Neural Transm., 88,
157–160.
27 Sanchez, C. (2003) R-Citalopram
attenuates anxiolytic effects of
escitalopram in a rat ultrasonic
vocalisation model. Eur. J. Pharmacol.,
464, 155–158.
28 Sidhu, J., Priskorn, M., Poulsen, M.,
Segonzac, A., Grollier, G., and Larsen, F.
(1997) Steady-state pharmacokinetics of the
enantiomers of citalopram and its
metabolites in humans. Chirality, 9, 686–692.
29 Mørk, A., Kreilgaard, M., and Sanchez, C.
(2003) The R-enantiomer of citalopram
counteracts escitalopram-induced
increase in extracellular 5-HT in the
frontal cortex of freely moving rats.
Neuropharmacology, 45, 167–173.
30 Sanchez, C., Bergqvist, P.B., Brennum,
L.T., Gupta, S., Hogg, S., Larsen, A., and
Wiborg, O. (2003) Escitalopram, the
S-(þ)-enantiomer of citalopram, is a
selective serotonin reuptake inhibitor with
potent effects in animal models predictive
of antidepressant and anxiolytic activities.
Psychopharmacology, 167, 353–362.
31 El Mansari, M., Sanchez, C., Chouvet, G.,
Renaud, B., and Haddjeri, N. (2005)
Effects of acute and long-term
administration of escitalopram and
citalopram on serotonin
neurotransmission: an in vivo
electrophysiological study in rat brain.
Neuropsychopharmacology, 30, 1269–1277.
32 El Mansari, M., Wiborg, O., Mnie-Filali,
O., Benturquia, N., Sanchez, C., and
Haddjeri, N. (2007) Allosteric modulation
of the effect of escitalopram, paroxetine
and fluoxetine: in-vitro and in-vivo studies.
Int. J. Neuropsychopharmacol., 10, 31–40.
33 Mnie-Filali, O., Faure, C., Mansari, M.E.,
Lambas-Senas, L., Berod, A., Zimmer, L.,
Sanchez, C., and Haddjeri, N. (2007) RCitalopram
prevents the neuronal
adaptive changes induced by
escitalopram. Neuroreport, 18, 1553–1556.
References j289
34 Sanchez, C., Bogeso, K.P., Ebert, B.,
Reines, E.H., and Braestrup, C. (2004)
Escitalopram versus citalopram: the
surprising role of the R-enantiomer.
Psychopharmacology, 174, 163–176.
35 Sanchez, C. (2006) The pharmacology of
citalopram enantiomers: the antagonism
by R-citalopram on the effect of Scitalopram.
Basic Clin. Pharmacol. Toxicol.,
99, 91–95.
36 Zhong, H., Haddjeri, N., and Sanchez, C.
(2012) Escitalopram, an antidepressant
with an allosteric effect at the serotonin
transporter – a review of current
understanding of its mechanism of action.
Psychopharmacology, 219 (1), 1–13.
37 Cremers, T.I.F.H. and Westerink, B.H.C.
(2003) Pharmacological difference
between escitalopram and citalopram. Int.
J. Psychiatry Clin. Pract., 7, 306..
38 Lucki, I. and Brown, K. (2003) Different
roles of the enantiomers of citalopram on
serotonin transmission. Biol. Psychiatry,
53, 45S.
39 Altamirano, A.V., and Frazer, A. (2005)
Comparison of the effects of the
enantiomers of citalopram on the
clearance of exogenous serotonin. Poster
presented at the SoPB 2005.
40 Schilstrom, B., Konradsson-Geuken, A.,
Ivanov, V., Gertow, J., Feltmann, K.,
Marcus, M.M., Jardemark, K., and
Svensson, T.H. (2011) Effects of Scitalopram,
citalopram, and R-citalopram
on the firing patterns of dopamine
neurons in the ventral tegmental area, Nmethyl-D-aspartate
receptor-mediated
transmission in the medial prefrontal
cortex and cognitive function in the rat.
Synapse, 65, 357–367.
41 Mnie-Filali, O., El, M.M., Espana, A.,
Sanchez, C., and Haddjeri, N. (2006)
Allosteric modulation of the effects of the
5-HTreuptake inhibitor escitalopram on
the rat hippocampal synaptic plasticity.
Neurosci. Lett., 395, 23–27.
42 Sanchez, C., and Kreilgaard, M. (2004) RCitalopram
inhibits functional and 5HTP-evoked
behavioural responses to the
SSRI, escitalopram. Pharmacol. Biochem.
Behav., 77, 391–398.
43 Storustovu, S., Sanchez, C., Porzgen, P.,
Brennum, L.T., Larsen, A.K., Pulis, M.,
and Ebert, B. (2004) R-Citalopram
functionally antagonises escitalopram in
vivo and in vitro: evidence for kinetic
interaction at the serotonin transporter.
Br. J. Pharmacol., 142, 172–180.
44 Bien, E., Gruca, P., and Papp, M. (2003) RCitalopram
attenuates the anxiolytic-like
activity of escitalopram in two animal
models. Behav. Pharmacol., 14 (Suppl. 1),
S37.
45 Fish, E.W., Faccidomo, S., Gupta, S., and
Miczek, K.A. (2004) Anxiolytic-like effects
of escitalopram, citalopram, and Rcitalopram
in maternally separated mouse
pups. J. Pharmacol. Exp. Ther., 308,
474–480.
46 Sanchez, C., Gruca, P., Bien, E., and Papp,
M. (2003) R-Citalopram counteracts the
effect of escitalopram in a rat conditioned
fear stress model of anxiety. Pharmacol.
Biochem. Behav., 75, 903–907.
47 Mitchell, P.J., Hogg, S., and Sanchez, C.
(2004) Agonistic behaviour of resident rats
after acute and chronic treatment with S
(þ)- and R(À)-citalopram. Int. J.
Neuropsychopharmacol., 7, S187. Poster
presented at the 24th CINP, June 20–24,
2004, Paris, France, P01.206.
48 Sanchez, C., Gruca, P., and Papp, M.
(2003) R-Citalopram counteracts the
antidepressant-like effect of escitalopram
in a rat chronic mild stress model. Behav.
Pharmacol., 14, 465–470.
49 Zhong, H., Hansen, K.B., Boyle, N.J.,
Han, K., Muske, G., Huang, X., Egebjerg,
J., and Sanchez, C. (2009) An allosteric
binding site at the human serotonin
transporter mediates the inhibition of
escitalopram by R-citalopram: kinetic
binding studies with the ALI/VFL-SI/TT
mutant. Neurosci. Lett., 462, 207–212.
50 Neubauer, H.A., Hansen, C.G., and
Wiborg, O. (2006) Dissection of an
allosteric mechanism on the serotonin
transporter: a cross-species study. Mol.
Pharmacol., 69, 1242–1250.
51 Klein, N., Sacher, J., Geiss-Granadia, T.,
Mossaheb, N., Attarbaschi, T.,
Lanzenberger, R., Spindelegger, C., Holik,
A., Asenbaum, S., Dudczak, R., Tauscher,
J., and Kasper, S. (2007) Higher serotonin
transporter occupancy after multiple dose
administration of escitalopram compared
290j11 The Discovery of Citalopram and Its Refinement to Escitalopram
to citalopram: an 123
IADAM SPECTstudy.
Psychopharmacology, 191, 333–339.
52 Kasper, S., Sacher, J., Klein, N., Mossaheb,
N., Attarbaschi-Steiner, T., Lanzenberger,
R., Spindelegger, C., Asenbaum, S., Holik,
A., and Dudczak, R. (2009) Differences in
the dynamics of serotonin reuptake
transporter occupancy may explain
superior clinical efficacy of escitalopram
versus citalopram. Int. Clin.
Psychopharmacol., 24, 119–125.
53 Bøgesø, K.P. (1983) Neuroleptic activity
and dopamine-uptake inhibition in 1piperazino-3-phenylindans.
J. Med. Chem.,
26, 935–947.
54 Bøgesø, K.P., Hyttel, J., Christensen, A.V.,
and Arnt, J. (1986) Chirality as
determinant for neuroleptic or
antidepressant action of drugs, in
Innovative Approaches in Drug Research,
vol. 9 (ed. A.F. Harms), Elsevier,
Amsterdam, pp. 371–392.
55 Smith, D.F. (1986) The stereoselectivity of
serotonin uptake in brain tissue and blood
platelets: the topography of the serotonin
uptake area. Neurosci. Biobehav. Rev., 10,
37–46.
56 Gundertofte, K., Liljefors, T., and Bøgesø,
K.P. (1997) A stereoselective
pharmacophoric model of the serotonin
reuptake site, in Computer-Assisted Lead
Finding and Optimization (eds H. van de
Waterbeemd, B. Testa, and G. Folkers),
VHCA, Basel, pp. 443–459.
57 Koldso, H., Severinsen, K., Tran, T.T.,
Celik, L., Jensen, H.H., Wiborg, O.,
Schiott, B., and Sinning, S. (2010) The two
enantiomers of citalopram bind to the
human serotonin transporter in reversed
orientations. J. Am. Chem. Soc., 132,
1311–1322.
58 Eildal, J.N., Andersen, J., Kristensen, A.S.,
Jorgensen, A.M., Bang-Andersen, B.,
Jorgensen, M., and Stromgaard, K. (2008)
From the selective serotonin transporter
inhibitor citalopram to the selective
norepinephrine transporter inhibitor
talopram: synthesis and structure–activity
relationship studies. J. Med. Chem., 51,
3045–3048.
59 Andersen, J., Stuhr-Hansen, N.,
Zachariassen, L., Toubro, S., Hansen, S.
M., Eildal, J.N., Bond, A.D., Bogeso, K.P.,
Bang-Andersen, B., Kristensen, A.S., and
Stromgaard, K. (2011) Molecular
determinants for selective recognition of
antidepressants in the human serotonin
and norepinephrine transporters. Proc.
Natl. Acad. Sci. USA, 108, 12137–12142.
60 Yamashita, A., Singh, S.K., Kawate, T., Jin,
Y., and Gouaux, E. (2005) Crystal structure
of a bacterial homologue of Naþ
/ClÀ
dependent
neurotransmitter transporters.
Nature, 437, 215–223.
61 Jorgensen, A.M., Tagmose, L., Jorgensen,
A.M., Topiol, S., Sabio, M., Gundertofte,
K., Bogeso, K.P., and Peters, G.H. (2007)
Homology modeling of the serotonin
transporter: insights into the primary
escitalopram-binding site.
ChemMedChem, 2, 815–826.
62 Jorgensen, A.M., Tagmose, L.,
Jorgensen, A.M., Bogeso, K.P., and
Peters, G.H. (2007) Molecular dynamics
simulations of Naþ
/ClÀ
-dependent
neurotransmitter transporters in a
membrane-aqueous system.
ChemMedChem, 2, 827–840.
63 Andersen, J., Olsen, L., Hansen, K.B.,
Taboureau, O., Jorgensen, F.S., Jorgensen,
A.M., Bang-Andersen, B., Egebjerg, J.,
Stromgaard, K., and Kristensen, A.S.
(2010) Mutational mapping and modeling
of the binding site for (S)-citalopram in
the human serotonin transporter. J. Biol.
Chem., 285, 2051–2063.
64 Andersen, J., Taboureau, O., Hansen, K.
B., Olsen, L., Egebjerg, J., Stromgaard, K.,
and Kristensen, A.S. (2009) Location of
the antidepressant binding site in the
serotonin transporter: importance of Ser-
438 in recognition of citalopram and
tricyclic antidepressants. J. Biol. Chem.,
284, 10276–10284.
65 Singh, S.K., Yamashita, A., and Gouaux, E.
(2007) Antidepressant binding site in a
bacterial homologue of neurotransmitter
transporters. Nature, 448, 952–956.
66 Zhou, Z., Zhen, J., Karpowich, N.K.,
Goetz, R.M., Law, C.J., Reith, M.E., and
Wang, D.N. (2007) LeuT-desipramine
structure reveals how antidepressants
block neurotransmitter reuptake. Science,
317, 1390–1393.
67 Jorgensen, A.M., and Topiol, S. (2008)
Driving forces for ligand migration in the
References j291
leucine transporter. Chem. Biol. Drug Des.,
72, 265–272.
68 Zhou, Z., Zhen, J., Karpowich, N.K., Law,
C.J., Reith, M.E., and Wang, D.N. (2009)
Antidepressant specificity of serotonin
transporter suggested by three LeuT-SSRI
structures. Nat. Struct. Mol. Biol., 16,
652–657.
69 Bøgesø, K.P., Bregnedal, P., Juhl, K.,
Zhong, H., and Wiborg, O. (2010) New
naphthalene derivatives of citalopram
discriminate between the primary and the
allosteric binding sites at the human
serotonin transporter. Basic Clin.
Pharmacol. Toxicol., 107 (Suppl. 1), 210.
70 Lau, T., Horschitz, S., Berger, S., Bartsch,
D., and Schloss, P. (2008) Antidepressantinduced
internalization of the serotonin
transporter in serotonergic neurons.
FASEB J., 22, 1702–1714.
71 Kittler, K., Lau, T., and Schloss, P. (2010)
Antagonists and substrates differentially
regulate serotonin transporter cell surface
expression in serotonergic neurons. Eur. J.
Pharmacol., 629, 63–67.
72 Mnie-Filali, O., Mansari, M.E., Sanchez,
C., and Haddjeri, N. (2009) Therapeutic
relevance of the allosteric modulation of
the 5-HT transporter. Curr. Signal
Transduct. Ther., 4, 82–87.
73 Zhong, H., Sanchez, C., and Caron,
M.G. (2011) Consideration of
allosterism and interacting proteins in
the physiological functions of the
serotonin transporter. Biochem.
Pharmacol., 83 (4), 435–442.
74 Burke, W.J., Gergel, I., and Bose, A. (2002)
Fixed-dose trial of the single isomer SSRI
escitalopram in depressed outpatients J.
Clin. Psychiatry, 63, 331–336.
75 Lepola, U.M., Loft, H., and Reines, E.H.
(2003) Escitalopram (10–20mg/day) is
effective and well tolerated in a placebocontrolled
study in depression in primary
care. Int. Clin. Psychopharmacol., 18,
211–217.
76 Moore, N., Verdoux, H., and Fantino, B.
(2005) Prospective, multicentre,
randomized, double-blind study of the
efficacy of escitalopram versus citalopram
in outpatient treatment of major
depressive disorder. Int. Clin.
Psychopharmacol., 20, 131–137.
77 Yevtushenko, V.Y., Belous, A.I.,
Yevtushenko, Y.G., Gusinin, S.E., Buzik,
O.J., and Agibalova, T.V. (2007) Efficacy
and tolerability of escitalopram versus
citalopram in major depressive disorder: a
6-week, multicenter, prospective,
randomized, double-blind, activecontrolled
study in adult outpatients. Clin.
Ther., 29, 2319–2332.
78 Kennedy, S.H., Andersen, H.F., and
Thase, M.E. (2009) Escitalopram in the
treatment of major depressive disorder: a
meta-analysis. Curr. Med. Res. Opin., 25,
161–175.
79 Montgomery, S., Hansen, T., and Kasper,
S. (2011) Efficacy of escitalopram
compared to citalopram: a meta-analysis.
Int. J. Neuropsychopharmacol., 14,
261–268.
80 Boulenger, J.P., Huusom, A.K., Florea, I.,
Baekdal, T., and Sarchiapone, M. (2006) A
comparative study of the efficacy of longterm
treatment with escitalopram and
paroxetine in severely depressed patients.
Curr. Med. Res. Opin., 22, 1331–1341.
81 Boulenger, J.P., Hermes, A., Huusom,
A.K., and Weiller, E. (2010) Baseline
anxiety effect on outcome of SSRI
treatment in patients with severe
depression: escitalopram vs paroxetine.
Curr. Med. Res. Opin., 26, 605–614.
82 Kasper, S., Baldwin, D.S., Larsson, L.S.,
and Boulenger, J.P. (2009) Superiority of
escitalopram to paroxetine in the
treatment of depression. Eur.
Neuropsychopharmacol., 19, 229–237.
83 Khan, A., Bose, A., Alexopoulos, G.S.,
Gommoll, C., Li, D., and Gandhi, C.
(2007) Double-blind comparison of
escitalopram and duloxetine in the acute
treatment of major depressive disorder.
Clin. Drug Invest., 27, 481–492.
84 Montgomery, S.A., and Andersen, H.F.
(2006) Escitalopram versus venlafaxine
XR in the treatment of depression. Int.
Clin. Psychopharmacol., 21, 297–309.
85 Kennedy, S.H., Andersen, H.F., and Lam,
R.W. (2006) Efficacy of escitalopram in the
treatment of major depressive disorder
compared with conventional selective
serotonin reuptake inhibitors and
venlafaxine XR: a meta-analysis.
J. Psychiatry Neurosci., 31, 122–131.
292j11 The Discovery of Citalopram and Its Refinement to Escitalopram
86 Wade, A., Gembert, K., and Florea, I.
(2007) A comparative study of the efficacy
of acute and continuation treatment with
escitalopram versus duloxetine in patients
with major depressive disorder. Curr. Med.
Res. Opin., 23, 1605–1614.
87 Cipriani, A., Furukawa, T.A., Salanti, G.,
Geddes, J.R., Higgins, J.P., Churchill, R.,
Watanabe, N., Nakagawa, A., Omori, I.M.,
McGuire, H., Tansella, M., and Barbui, C.
(2009) Comparative efficacy and
acceptability of 12 new-generation
antidepressants: a multiple-treatments
meta-analysis. Lancet, 373, 746–758.
88 Baldwin, D.S., Huusom, A.K., and
Maehlum, E. (2006) Escitalopram and
paroxetine in the treatment of generalised
anxiety disorder: randomised, placebocontrolled,
double-blind study. Br. J.
Psychiatry, 189, 264–272.
89 Lader, M., Stender, K., Burger, V., and Nil,
R. (2004) Efficacy and tolerability of
escitalopram in 12- and 24-week treatment
of social anxiety disorder: randomised,
double-blind, placebo-controlled, fixeddose
study. Depress. Anxiety, 19, 241–248.
90 Hayes, B.D., Klein-Schwartz, W., Clark,
R.F., Muller, A.A., and Miloradovich, J.E.
(2010) Comparison of toxicity of acute
overdoses with citalopram and
escitalopram. J. Emerg. Med., 39, 44–48.
91 Yilmaz, Z., Ceschi, A., Rauber-Luthy, C.,
Sauer, O., Stedtler, U., Prasa, D., Seidel,
C., Hackl, E., Hoffmann-Walbeck, P.,
Gerber-Zupan, G., Bauer, K.,
Kupferschmidt, H., Kullak-Ublick, G.A.,
and Wilks, M. (2010) Escitalopram causes
fewer seizures in human overdose than
citalopram. Clin. Toxicol., 48, 207–212.
References j293
Klaus P. Bøgesø, B.Sc., D.Sc., has been working at H.
Lundbeck A/S with drug discovery within the CNS field for
more than 40 years. Klaus P. Bøgesø started at Lundbeck as a
Research Chemist in 1971. In 1986, he became Director of
Medicinal Chemistry and in 1999 he became Vice President
of Medicinal Chemistry Research. In 2006, he became Vice
President of Research, Denmark. In 2008, he became Vice
President of External Affairs, Lundbeck Research Management.
In 2011, he became Vice President, External Scientific
Affairs and Patents.
The research activities of Klaus P. Bøgesø have focused on drug design and
development within psychiatric and neurological diseases, such as schizophrenia,
depression, anxiety, Alzheimer’s disease, Parkinson’s disease, and epilepsy. Primary
targets have been serotonin, norepinephrine, and dopamine receptors and
transporters, muscarinic receptors, and glutamate receptors. Klaus P. Bøgesø is
inventor of the selective serotonin reuptake inhibitor citalopram (Cipramil1
, Cel-
exa1
) and co-inventor of its S-enantiomer, escitalopram (Cipralex1
, Lexapro1
).
Both drugs have attained blockbuster status. The differential action of stereoisomers
of chiral drugs has been a focus area in many of the research projects Klaus P.
Bøgesø has been involved in, and has been a major topic in several of his publications,
including his doctoral thesis.
Connie Sanchez, currently at Lundbeck Research USA and
before that at H. Lundbeck A/S, in Copenhagen, Denmark,
has more than 25 years of drug discovery experience. She
graduated from the Pharmaceutical University of Denmark
and has acquired a D.Sc. degree in Pharmacology at the
same institution. She has a broad experience within drug
discovery and drug development for neuropsychiatric and
neurological diseases, including depression, anxiety, schizophrenia,
Alzheimer’s disease, Parkinson’s disease, epilepsy,
and insomnia, and she has contributed to bringing various drug candidates into
clinical development, some of which have been brought to the market. Most notably,
she established and successfully led a research project aiming at uncovering
the mechanism of action of the antidepressant drug escitalopram (Cipralex1
). She
has authored approximately 100 scientific papers and more than 20 patent
publications.
294j11 The Discovery of Citalopram and Its Refinement to Escitalopram
12
Tapentadol – From Morphine and Tramadol to the Discovery
of Tapentadol
Helmut Buschmann
List of Abbreviations
5-HT 5-hydroxytryptamine (serotonin)
COX cyclooxygenases
CYP cytochrome P450
DOR delta-opioid receptor
ED50 dose that produces 50% effect (in vivo measure of potency)
ER extended release
FDA US Food and Drug Administration
i.c.v. intracerebroventricular
i.p. intraperitoneal
i.t. intrathecal
i.v. intravenous
IR immediate release
Ki dissociation constant (measure of affinity)
KOR kappa-opioid receptor
M-1 O-desmethyl metabolite of tramadol
MOR mu-opioid receptor
NE norepinephrine (noradrenaline)
NET norepinephrine (noradrenaline) transporter
NOP ORL1, or nociceptin receptor
NRI norepinephrine (noradrenaline) reuptake inhibitor
NSAID nonsteroidal anti-inflammatory drug
ORL1 NOP, or nociceptin receptor
p.o. per os, oral
SERT serotonin transporter
SNL spinal nerve ligation
SR sustained release
SRI serotonin reuptake inhibitor
STZ streptozotocin
THF tetrahydrofuran
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
j295
TMSCl trimethylsilyl chloride
WHO World Health Organization
12.1
Introduction
Tapentadol ((À)-(1R,2R)-3-(3-dimethylamino-1-ethyl-2-methylpropyl)phenol hydrochloride;
3-[(1R,2R)-3-(dimethylamino)-1-ethyl-2-methylpropyl]phenol hydrochloride),
discovered by Gr€unenthal (Germany) and shown in Figure 12.1 together
with morphine and tramadol, was approved in November 2008 by the US Food and
Drug Administration (FDA) for the treatment of moderate to severe acute pain in
adults as an immediate-release formulation (Nucynta1
, Johnson & Johnson).
Tapentadol is a novel, centrally acting analgesic with a dual mode of action: muopioid
receptor (MOR) agonism and norepinephrine reuptake inhibition. Tapentadol
is the first oral centrally acting analgesic to be approved in the United States
in more than a decade. In the United States, tapentadol has been placed into
schedule II of the Controlled Substances Act1)
. An extended-release formulation of
tapentadol (Nucynta ER1
) obtained FDA approval for the treatment of chronic pain
in August 2011.
1) The Controlled Substances Act (CSA) was
enacted into law by the Congress of the
United States as Title II of the
Comprehensive Drug Abuse Prevention and
Control Act of 1970. The CSA is the federal
US drug policy under which the
manufacture, importation, possession, use,
and distribution of certain substances is
regulated. Schedule II includes drugs that
may have a high potential for abuse. Further
examples of schedule II drugs are
morphine, oxycodone, fentanyl, cocaine,
methylphenidate, or methamphetamine.
O
H3C
N
H3C CH3
O H
OH
N
CH3H3C
OH
HO
N
H3C CH3
OH
N
CH3
H3C
CH3
H
OH
CH3
Metabolic Activation
Morphine Tramadol and Metabolites Tapentadol
O
HO
HO
N
H CH3
O
CH3
N
CH3H3C
OH
Morphine
(+)-(R,R)-Tramadol (-)-(S,S)-Tramadol
(+)-(R,R (-)-(1-M-) S,S)-M-1
Tapentadol
(-)-(R,R)
Figure 12.1 Chemical structure of tapentadol HCl, morphine, and tramadol (the absolute
stereochemistry of the (À)-(S,S)-M-1 metabolite of tramadol corresponds to the stereochemistry
of (À)-(R,R)-tapentadol).
296j12 Tapentadol – From Morphine and Tramadol to the Discovery of Tapentadol
On August 10, 2010, the European decentralized procedure regarding tapentadol
concluded positively and the granting of national marketing authorizations in 26
European markets is expected. Tapentadol will then be available as an oral, solid,
immediate-release formulation (tablets) for the relief of moderate to severe acute
pain in adults, and an oral, solid, prolonged-release formulation (tablets) for the
management of severe chronic pain in adults. Since October 2010, tapentadol is
available on the German market as Palexia1
retard for the treatment of nociceptive
and neuropathic pain types. Tapentadol has been clinically tested in multiple pain
models and found to combine opioid efficacy with favorable gastrointestinal tolerability.
Tapentadol as a centrally acting opioid analgesic has a potency between morphine
and tramadol.
12.1.1
Pain and Current Pain Treatment Options
Pain is defined as “an unpleasant sensory and emotional experience associated with
actual or potential tissue damage, or described in terms of such damage” [1]. Pain
is the most common reason that patients seek medical care. The experience of
physical pain is familiar to everyone; however, the intensity, character, and tolerability
of each person’s pain are subjective. How patients perceive and react to pain is
influenced by social, cultural, and psychological factors [2]. Thus, detailed examination
as to the character (e.g., dull, throbbing, and stabbing), location, and intensity
of pain, how and when it occurs, and the effects of pain is needed in order to recommend
appropriate interventions. Pain may be acute or chronic [3]. Acute pain is
generally associated with an identifiable cause, resolves after the removal of the
cause, responds to treatment, and lasts less than 1–3 months [4]. Acute pain is
defined as that associated with a sudden stimulus (e.g., accident or surgery [5]) and
is generally expected to resolve as the injury heals. Clinical examples of acute pain
include pain following surgery and pain associated with injury. Pain that does
not have a clear cause or that lasts longer than 1–3 months after the initial injury
heals is considered chronic pain. It is estimated that neuropathic pain affects over
6 million patients in the United States and Europe and over 26 million patients
worldwide.
Neuropathic pain is a pain associated with a functional abnormality of the nervous
system. Pain severity, however, is not correlated with the amount of damage
and symptoms can persist long after tissue damage from an antecedent injury
resolves [6]. Clinical features of neuropathic pain include the presence of an abnormal,
unpleasant sensation (dysesthesia) that frequently has a burning or electrical
quality with an occasional paroxysmal, brief, shooting, or stabbing quality. Pain
may be felt in a region of sensory deficit. Non-noxious stimuli may be painful
(allodynia). Noxious stimuli may produce a greater than normal response
(hyperalgesia).
Chronic noncancer pain can develop as a result of persistent stimulation of or
changes to nociceptors due to localized tissue damage from an acute injury or disease
(e.g., osteoarthritis), or damage to the peripheral or central nervous system, or
12.1 Introduction j297
both (e.g., painful diabetic neuropathy, poststroke pain, and spinal cord injury).
There is a wide range of treatments available to physicians for neuropathic pain,
some of which have specific indications, and some of which are used off-label.
However, currently available treatments provide modest improvements in pain and
minimum improvements in physical and emotional functioning.
Pain is further categorized according to the neural and biochemical mechanisms
at work. Nociceptive pain is the normal physiological response to a noxious stimulus,
such as injury or inflammation. The stimulus causes nociceptors to release the
neurotransmitters glutamate, calcitonin gene-related peptide, and substance P,
which transmit the signal to second-order neurons in the spinal cord. The secondorder
neurons form the ascending spinothalamic tract that leads to the thalamus,
O
HO
HO
N
H CH3
O
O
HO
N
H3C
H CH3
O
HO
O
H3C
HO CH3
H3C
CH3
CH3
N
Morphine Codeine Buprenorphine
O
O
O
N
OH CH3
H3C
N
O
O
CH3
CH3
N
CH3
N
O
H3C
OH
N
N
O
H3C
CH3
CH3
N
CH3
H3C
O
N
HO
Cl
N
O
CH3
CH3
Oxycodone Pethidine Ketobemidone
Fentanyl Levomethadone Loperamide
N
O
O CH3
CH3
CH3 HO O
O CH3
O OH
HN CH3
O
Tilidine Aspirin Paracetamol
Figure 12.2 The structural diversity of analgesics [8].
298j12 Tapentadol – From Morphine and Tramadol to the Discovery of Tapentadol
where they synapse with third-order neurons that project to the sensory cortex,
where the pain is perceived [7]. Pain signals are modulated by release of endorphins
and enkephalins in the midbrain. These neurotransmitters interact with specific
opioid receptors in descending modulatory neurons that synapse with the
primary or second-order pain transmission neurons. Descending neurons release
norepinephrine and serotonin, which directly inhibit the release of pain transmitters
and inhibit the activity of the second-order pain transmission neurons [7].
Pharmacological treatment for pain includes nonopioid analgesics, such as salicylates,
acetaminophen, and nonsteroidal anti-inflammatory drugs (NSAIDs),
opioids, and other drug classes, including antidepressants and anticonvulsants.
The structural diversity of analgesics is shown in Figure 12.2 [8].
NSAIDs produce pain relief by inhibition of cyclooxygenases (COX-1 and
COX-2), which catalyze the production of proinflammatory prostaglandins from
arachidonic acid. The traditional NSAIDs are nonselective inhibitors of COX-1 and
COX-2 [8]. Prostaglandins are important mediators of inflammation that increase
pain sensations through inflammatory mechanisms [9]. Nonopioid oral medications
are recommended for mild to moderate acute pain, and most are available
COOH
H
N
Cl
Cl COOH
CH3H3C
CH3
N
N
ON
CH3
HO3S
H3C
CH3
S
N
O O
CH3
OH
N
H
O
N
N
N CF3
H3C
S
O
O
H2N
N
NH3C
Cl
S
O
O
H3C
COOHH2N
Cl
N
OH2N
H
N
Cl
Cl
H
N
N
S
O N
H
CH3 NH2HOOC
NH
H
N O
O
Diclofenac Ibuprofen Metamizol (Dipyrone)
Piroxycam Celecoxib Etoricoxib
Baclofen Carbamazepine Clonidine
Duloxetine Gabapentin Phenytoin
Figure 12.2 (Continued)
12.1 Introduction j299
over the counter and by prescription. Potentially serious side effects of NSAIDs
include gastrointestinal effects, such as upsets, stomach ulcers, or bleeding, as well
as reduced blood clotting, decreased kidney function, and cardiovascular side
effects. The risk of these events is increased by higher doses and long-term use.
Opioid medications mimic the actions of endorphins by interacting with opioid
receptors in the brain and spinal cord. Activation of mu-opioid receptors in ascending
pathways may work to inhibit nociceptive signals [7]. An increase in the effect
on ascending pathways activity of opioid receptors may activate the modulating
descending neural pathways as detailed above and produce relief of pain [7].
Opioids are associated with the potential for abuse and addiction [2]. Opioids are
generally indicated for moderate to severe acute pain.
12.1.2
Pain Research Today
Already in 1999, it was stated by Butera in his review about treatment options for
neuropathic pain: “Despite an intensive research effort over the past two decades
involving many innovative approaches in the global academic community and by
the pharmaceutical industry, the latter representing an aggregate investment in
excess of $2.5 billion, the only new opioid-based pain medications either in clinical
development or on the market are alternative dosage forms of the classical opioids,
morphine, loperamide, and fentanyl, or compounds such as tramadol” (see
Figure 12.2) [10].
As physicians are faced with an increasing number of patients with numerous
neuropathic pain symptoms most likely stemming from multiple etiologies, they
are forced to resort to the polypharmacia approach as the mainstay therapy. Current
pharmacological treatment for neuropathic pain will typically include some combination
of agents from several of the following drug classes: opioids, tricyclic antidepressants,
anticonvulsant agents, or nonsteroidal anti-inflammatory drugs as
shown in Figure 1.2. Ironically, even with such an impressive arsenal of powerful
drugs, these approaches only provide an approximate 30–50% reduction in pain in
about 50% of patients. Coupled with this limited efficacy, there are low levels of
compliance due to intolerable side effect profiles associated with some of these
drugs. These results profoundly illustrate that treatment of neuropathic pain is a
hugely unmet medical need, and they underscore the importance of considering,
validating, and pursuing alternative targets to treat refractory neuropathic
pain [6, 11].
Research in understanding the underlying mechanisms behind people’s pain
has progressed rapidly, but despite these critical advances pain remains grossly
undertreated and is frequently mistreated. In the words of Jean-Marie Besson
(President of the International Association for the Study of Pain) at the 9th Congress
of the International Association for the Study of Pain in Vienna, Austria, in
August 1999, the current treatment options are summarized as follows: “Some
pain states can now be controlled, yet there are still others which are far from being
treatable” [12].
300j12 Tapentadol – From Morphine and Tramadol to the Discovery of Tapentadol
12.1.3
The Complex Mode of Action of Tramadol
Since the isolation of morphine two centuries ago, numerous derivatives have been
synthesized, with the primary aim of separating analgesic effects from unwanted
effects, such as nausea and emesis, constipation, respiratory depression, addiction,
and dependence. An alternative approach to improve the therapeutic usefulness of
opioid analgesics is to combine MOR activation with an additional mechanism of
action aimed at enhancing analgesic efficacy. Such an additional analgesic mechanism
may result in an opiate-sparing effect, thereby attenuating the unwanted
effects of MOR activation.
The concept of combined mechanisms of action was realized for the first time
with the introduction of tramadol, a compound that produces MOR activation and
inhibition of serotonin (5-HT) and norepinephrine reuptake.
Tramadol ((1R,2R)-rel-2-[(dimethylamino)methyl]-1-(3-methoxyphenyl)cyclohexanol)
is a centrally acting opioid analgesic, used in treating moderate to
severe pain. It is classified as a “step II” opioid analgesic in the WHO pain
treatment ladder.
Tramadol was first synthesized in 1962 by Flick at Gr€unenthal and has been available
for pain treatment in Germany since 1977. Immediate-release preparations of
tramadol were introduced in the United Kingdom in 1994, in the United States in
1995, and subsequently in many other countries. Nowadays, several once-daily, sustained-release
formulations of tramadol have been marketed in various countries.
The drug has a wide range of applications, including treatment for restless legs
syndrome and fibromyalgia.
Tramadol possesses weak agonist actions at the mu-opioid receptor, releases
serotonin, and inhibits the reuptake of norepinephrine [13]. The analgesic action of
tramadol has yet to be fully understood, but it is believed to work through modulation
of serotonin and norepinephrine in addition to its mild agonism of the muopioid
receptor. The contribution of nonopioid activity is demonstrated by the fact
that the analgesic effect of tramadol is not fully antagonized by the mu-opioid
receptor antagonist naloxone.
Tramadol is marketed as a racemic mixture of the (1R,2R)- and (1S,2S)-enantiomers
with a weak affinity for the mu-opioid receptor (the affinity for the mu-opioid
receptor is approximately 10 times lower than that of codeine and 6000 times lower
than that of morphine). The two enantiomers of racemic tramadol function in a
complementary manner to enhance the analgesic efficacy and improve the tolerability
profile of tramadol. This dual mechanism of action may be attributed to the
two enantiomers of racemic tramadol. The (þ)-enantiomer has a higher affinity for
the mu-receptor and is a more effective inhibitor of 5-HT reuptake, whereas the
(À)-enantiomer is a more effective inhibitor of norepinephrine reuptake and
increases its release by autoreceptor activation. The (1R,2R)-(þ)-enantiomer is
approximately four times more potent than the (1S,2S)-(À)-enantiomer in terms of
mu-opioid receptor affinity and 5-HTreuptake, whereas the (1S,2S)-(À)-enantiomer
is responsible for norepinephrine reuptake effects. These actions appear to produce
12.1 Introduction j301
a synergistic analgesic effect, with (1R,2R)-(þ)-tramadol exhibiting 10-fold higher
analgesic activity than (1S,2S)-(À)-tramadol [14].
Tramadol undergoes hepatic metabolism via the cytochrome P450 isozyme
CYP2B6, CYP2D6, and CYP3A4, being O- and N-demethylated to five different
metabolites. Of these, O-desmethyltramadol ((þ)-M-1) is the most significant since
it has 300 times the mu-affinity of (þ)-tramadol, and furthermore has an elimination
half-life of 9 h, compared with 6 h for tramadol itself [15]. Similarly to tramadol,
O-desmethyltramadol (M-1) has also been shown to be a norepinephrine
reuptake inhibitor [16].
The fact that these four compounds (the enantiomers of tramadol and the enantiomeric
metabolites M-1) have different pharmacological properties makes tramadol
an atypical opioid with a complex mechanism of action [17, 18]. This is the
reason why tramadol is used as a racemate, despite known different physiological
effects of the enantiomers, because the racemate has a better pharmacological profile
than the single enantiomers in terms of efficacy and tolerability in animals and
humans.
However, the relative contribution of the different mechanisms of action to the
overall analgesic effect changes over time. As the parent molecule is metabolized,
the contribution of 5-HT and NE reuptake inhibition is reduced, whereas the contribution
of MOR agonism increases, resulting in a complex time- and metabolismdependent
pattern of pharmacological activities. Because tramadol is mainly
metabolized via cytochrome P450 2D6 (CYP2D6), which is polymorphic in
humans, approximately 5–15% of the white population are “poor metabolizers” of
tramadol and do not experience satisfactory analgesia with standard doses. Furthermore,
preclinical and clinical evidence, with selective 5-HT reuptake inhibitors,
selective NE reuptake inhibitors, and mixed 5-HT/NE reuptake inhibitors, indicates
that analgesia is more readily obtained by NE reuptake inhibition than by
5-HT reuptake inhibition. Finally, a compound without serotonergic activity would
not bear the risk of producing or contributing to a serotonin syndrome.
12.2
The Discovery of Tapentadol
Based on elucidation of the favorable aspects of the multimodal analgesic
mechanism of action of tramadol, a research program was initiated in the late
1980s at Gr€unenthal to design a new class of opioid/nonopioid analgesics that
would combine MOR agonism and norepinephrine reuptake inhibition, with
minimal serotonergic activity. It was also desired that both activities would
reside in a single parent molecule that does not require metabolic activation
via the cytochrome P450 system. Increasing evidence demonstrates that many
pharmaceutically important compounds elicit their effects through binding to
multiple targets. Tapentadol is an example of a rationally designed multiple
ligand starting from the tramadol structure and its understanding of the complex
pharmacological activity.
302j12 Tapentadol – From Morphine and Tramadol to the Discovery of Tapentadol
12.2.1
From the Tramadol Structure to Tapentadol
The discovery of tapentadol involved multiple steps of chemical innovations to
become a unique drug, which may be summarizes as follows (Figure 12.3):
 The dimethylaminomethyl fragment: The dimethylaminomethyl moiety is a significant
part of the pharmacophore that is needed for interaction with mu-opioid
receptor and it seems to be relevant for interaction with the monoamine transporter
system [20]. Demethylation is a metabolic pathway leading to analgesic
inactive N-demethyl derivatives without opioid binding or transporter inhibition
effect [21–23]. Increasing the size of the nitrogen substituent (e.g., ethyl or benzyl
substituents) or cyclic substitution (piperidine or pyrrolidine substitution)
also leads to inactive compounds. The dimethylaminomethyl fragment, therefore,
appears to be essential for analgesic activity for both drugs keeping mu-opioid
receptor binding and norepinephrine reuptake activity.
 The role of absolute and relative stereochemistry: Tramadol and tapentadol contain
two chiral carbon atoms. Consequently, for both molecules, four stereoisomers
exist as two diasteromeric pairs of enantiomers. However, tramadol is a
racemate, whereas tapentadol is a pure enantiomer. The relative stereochemistry
is critical for biological activity. In both cases, only the (R,R) and (S,S) isomers
display analgesic activity, whereas the diastereomeric enantiomers are relatively
inactive. In past decades, the pharmacopeia was dominated by racemates
[24, 25, 23]; however, since the emergence in the 1980s of new technologies
that allowed the preparation of pure enantiomers in significant quantities,
the awareness and interest in the stereochemistry of drug action has increased.
These factors have led to an increasing preference for selection of single
Figure 12.3 Summary of chemical modifications leading to the discovery of tapentadol [19].
12.2 The Discovery of Tapentadol j303
enantiomers by both the pharmaceutical industry and regulatory authorities. As
a consequence of this changing climate toward drug chirality, it was decided to
use pure enantiomers for further optimization. The absolute stereochemistry of
tapentadol corresponds to the (À)-(1S,2S)-M-1 metabolite of tramadol (Figure
1.1). Subsequently, it was found that the MOR affinity of the enantiomer
acting on the norepinephrine transporter was increased, and in the following
steps, modifications were made to increase this MOR affinity. In the case of the
open-chain analogue, the MOR affinity is increased in comparison to the cyclic
analogue without affecting the transporter inhibition effect.
 Cyclic core structure versus open-chain molecule: Several drugs possess cyclic
aliphatic ring systems connected via linkers with other aromatic or heteroaromatic
groups [26]. However, in the lead optimization phase, a cyclic ring system
is typically modified by changing the ring size and introducing heteroatoms
(such as nitrogen, oxygen, or sulfur) as well as introducing additional substituents
in order to optimize desired hydrophobic or nonhydrophobic interactions
with the biological target, thereby leading to an increase in the potency and ligand
efficiency of a given lead structure. As a consequence, the cyclic structure is
retained in many lead optimization programs due to the entropic and enthalpic
balance of the thermodynamics of binding [27, 28]. Cyclic structures generally
have the advantage of decreasing the entropic proportion of the binding energy
compared with open-chain molecules, leading to more flexible derivatives with
greater ligand efficiency [29]. In addition, many examples are known wherein an
open-chain analogue of a given drug loses biological activity completely; for
instance, the open-chain analogue of diazepam is completely inactive [30]. In the
course of the development of tapentadol, it was found that opening the cyclohexane
ring system led to active compounds while conserving the relative stereochemistry.
However, the activity of the molecules was strongly dependent on the
length of the side chain. It was found that a methyl group and an ethyl group at
chiral carbon atoms were optimal in terms of analgesic potency and biochemical
binding profile.
 Replacement of the aromatic hydroxyl group: The introduction of a phenolic
hydroxy group instead of an aromatic methoxy group results in a dramatic
change in pharmacological activity due to an increase of binding affinity to muopioid
receptor [20], which is known for other opioids (e.g., morphine versus
codeine). The bioisoteric replacement of the phenolic hydroxyl group by CH2F
or CHF2 groups yielded in a significant decrease of activity. The enantiomers of
open-chain phenolic structures showed increased analgesic activity compared to
the cyclic tramadol-derived analogues.
 Replacement of the aliphatic hydroxyl group: It is known for many drugs that
their aliphatic hydroxyl group is essential for pharmacological activity [31, 23].
Derivatization by introducing more bulkier groups in this position (e.g., ether or
ester prodrugs) led to the complete loss of analgesic activity. However, replacement
of the tertiary hydroxyl group by either fluorine or hydrogen resulted in a
significant increase of MOR affinity for both enantiomers. The chlorine derivatives
were also analgesically active; however, the chemical stability was decreased
304j12 Tapentadol – From Morphine and Tramadol to the Discovery of Tapentadol
(loss of chlorine). However, the fluorine derivatives were not further developed
as well due to physicochemical stability issues. Critically, the hydrogen derivative
was found to have both activities in one molecule. While retaining the norepinephrine
reuptake inhibition (NRI) component, the MOR agonism component
was increased to achieve potent analgesic activity without the prominent
serotonin reuptake inhibition (SRI) effect present in tramadol.
In contrast to this serendipitous combination of mechanisms like in the tramadol
case, tapentadol was designed to rationally combine two analgesic mechanisms
(MOR agonism and NRI) within a single molecule. As part of this development,
tapentadol was designed to have less serotonergic activity. The result is an innovative
drug that retains the advantages of combining synergistic mechanisms of
action, but with a significantly greater “functional” opioid component and a relatively
greater inhibition of the norepinephrine reuptake than of the serotonin transporter,
all in a single molecule with no analgesically active metabolites. The
chemical innovations introduced in the development of tapentadol starting from
the tramadol structure are summarized in Figure 12.4.
N
CH3
CH3
CH3
OH
O
H3C
CH3
N
CH3
H3C
CH3
HO
O
CH3
CH3
N
CH3
CH3
CH3
OH
HO
CH3
N
CH3
H3C
CH3
HO
OH
CH3
Opening of
the
cyclohexane
ring
From
prodrug to
direct-acting drug
Racemate with
relative
stereochemistry
cis
Selecting of
one
enantiomer
O
CH3
N
CH3H3C
OH
O
CH3
N
CH3H3C
OH
Replacement
of tert.
OH group
Tramadol
Tapentadol
N
CH3
H3C
CH3
HO
OH
CH3
N
CH3
H3C
CH3
H
OH
CH3
Figure 12.4 Chemical innovations introduced in the development of tapentadol starting from the
tramadol structure [19].
12.2 The Discovery of Tapentadol j305
12.2.2
Synthetic Pathways to Tapentadol
Several syntheses of tapentadol have been reported [32–34]. In the original patent,
tapentadol hydrochloride is synthesized by ether cleavage (e.g., conc. HBr) from its
corresponding methyl ether 1. Compound 1 can be obtained by several methods,
for example, starting from diethyl ketone, which is transformed under standard
Mannich conditions to racemic 2. The Grignard reaction with 3-bromoanisole and
subsequent separation of diastereomers (crystallization as HCl salt from 2-butanone
with TMSCl/H2O) and enantiomers (HPLC) gives (À)-(S,S)- 3. Removal of
the hydroxy group can be achieved diastereoselectively (under retention) by chlorination
with thionyl chloride to yield 4, followed by treatment with zinc borohydride
(from ZnCl2 and NaBH4), leading to tapentadol hydrochloride (Figure 12.5).
The following route developed by Hell and coworkers is described in the recent
patent literature [35–37]. Ketone 5 was treated with Eschenmoser’s salt in acetonitrile
at room temperature and the resulting amine 6 was collected by adding ether
and crystallizing the product. Amine 6 was crystallized with L-(À)-dibenzoyl tartaric
acid monohydrate in ethanol at 6–8 
C to give the desired enantiomer as salt 7. The
free base of 7 was generated by reaction with aqueous sodium hydroxide and then
treated with ethyl magnesium bromide in THF at 10 
C followed by stirring at
H3C
O
CH3
H3C
NH
H3C
H Cl
(CH2O)n reflux
H3C
O
CH3
N
CH3
CH3
O
H3C
MgBr
THF
O
H3C N
CH3
CH3CH3
H3C
HO
1. Separation of diastereomers
via HCl formation
2. Racemic resolution
(e.g., p-toluyl tartaric acid)
O
H3C (S)
(S)
N
CH3
CH3CH3
H3C
HOSOCl2
reflux
O
H3C N
CH3
CH3CH3
H3C
Cl
O
H3C (R)
(R)
N
CH3
CH3CH3
CH3
Ph
1. conc. HBr, reflux
2. HCl salt preparation
a) CH2Cl2, aq. NaHCO3
b) Trimethylchlorosilane/H2O in 2-butanone
HO
N
CH3
CH3CH3
CH3
H Cl
Tapentadol hydrochloride
1
2
(-)-(S,S)-34
Figure 12.5 The synthesis of tapentadol hydrochloride as described in the first patent [32].
306j12 Tapentadol – From Morphine and Tramadol to the Discovery of Tapentadol
room temperature overnight to give tertiary alcohol 8. Alcohol 8 was treated with
trifluoroacetic anhydride in 2-methyl THF at 40–45 
C to give the corresponding
trifluoromethyl ester that was then treated with 10% Pd/C and hydrogenated at
3 bar at ambient temperature. These hydrogenolytic conditions effected reductive
cleavage of the trifluoromethyl ester and removal of the benzyl protecting group
with retention of stereochemistry. Filtration of the catalyst followed by the addition
of water and trimethylchlorosilane to generate HCl in situ and allowing the product
to crystallize out at 5–8 
C gave the desired tapentadol hydrochloride with 99%
purity and 96.9% ee (Figure 12.6).
Another synthetic route starts from 1-(3-methoxy-phenyl)-propan-1-one 9[38],
which reacts in a Mannich reaction to the corresponding racemic Mannich base
10. The diastereomeric salt formation with (R)-(À) mandelic acid give the desired
enantiomer as the mandelic acid salt in high chemical yields, because the
undesired enantiomer is transformed via a keto–enol equilibrium to the desired
one. Grignard reaction of the free base with ethyl magnesium bromide in tetrahydrofuran
yields the corresponding tertiary alcohol 11. Alcohol 11 was treated with
trifluoroacetic anhydride in 2-methyl THF to give the corresponding trifluoromethyl
ester that was then treated with 10% Pd/C and hydrogenated at ambient
O
O
CH3
H3C
N
H2C
CH2 Cl
Acetonitrile, RT,
50%
O
O
CH3
N
CH3
CH3
L-(-) Dibenzoyl tartaric acid H2O
Ethanol, 5oC, 65%
O
O
CH3
N
CH3
CH3
(-)-L-DBTA
O
(S)
(S)
N
CH3
CH3CH3
H3C
HO
1. NaOH, pentanone, 87%
2. EtMgBr, THF, 10oC - RT, 89%
1. (CF3CO)2O, 2-Me-THF
2. 10% Pd/C, H2, RT
3. Trimethylchlorosilane, H2O, 5oC, 89%, 96.9% ee
HO
N
CH3
CH3CH3
CH3
H Cl
Tapentadol hydrochloride
Figure 12.6 The synthesis of tapentadol hydrochloride according to Refs [35–37].
12.2 The Discovery of Tapentadol j307
temperature. These hydrogenolytic conditions effected reductive cleavage of the trifluoromethyl
ester and removal of the N-benzyl protecting group with retention of
stereochemistry. Crystallization of the hydrochloride salt 12 and reductive methylation
with formaldehyde in formic acid leads to the dimethyl amino derivative 13.
The methoxy ether is cleaved by methionine and methanesulfonic acid. Working
up with NaOH and extraction with ethyl acetate yields the free base of tapentadol
(Figure 12.7).
O
CH3 H3C
NH H Cl
(CH2O)n
O
H3C
O
O
H3C
CH3
N
CH3
10
OH
O
OH
OH
O
H3C
CH3
N
CH3
OH
O
OH
NaIOH/toluene
O
O
H3C
CH3
N
CH3
EtMgBr, THF
O
H3C
CH3
N
CH3
H3C
HO
11
1. (CF3CO)2O, 2-Me-THF
2. 10% Pd/C, H2, RT
3. HCl O
H3C
CH3
NH
CH3
12
CH3
H Cl
HCOOH
HCHO
O
H3C
CH3
N
CH3
CH3
CH3
1. Methanesulfonic acid, methionine
2. NaOH, ethyl acetateHO
CH3
N
CH3
CH3
CH3
Tapentadol
13
Figure 12.7 The synthesis of tapentadol hydrochloride according to Ref. [38].
308j12 Tapentadol – From Morphine and Tramadol to the Discovery of Tapentadol
Starting from N,N-dimethylpropionamide, a synthesis for racemic tapentadol
without a Mannich reaction is described [39] as shown in Figure 12.8. N,NDimethylpropionamide
is transformed to derivatives 14 and 15. A Grignard
reaction with 3-anisole magnesium bromide and ethyl magnesium bromide,
CH3
N
H3C
O
CH3
CH3
N
H3C
O
CH3
O
CH3
POCl3
CH3
N
H3C
O
CH3
O
H3C O
O
CH3
CH3
N
H3C
O
CH3
O
O
CH3
+
2,2,6,6-Tetramethylpiperidine, THF
n-BuLi
O
H3C
MgBr
THF EtMgBr, THF, CeCl3
CH3CH3
O
CH3
O
N
CH3
CH3
CH3CH3
O
CH3
O
N
CH3
CH3
OH OH
CH3CH3
O
CH3
O
N
CH3
CH3
CH3CH3
O
CH3
O
N
CH3
CH3
OH OH
+ +
CH3CH3
O
CH3
O
N
CH3
CH3
CH3CH3
O
CH3
O
N
CH3
CH3
1. HBr
2. NaOH, ethyl acetate
1. HBr
2. NaOH, ethyl acetate
+
KOtBu
H2, Pd/C
CH3
O
N
CH3
CH3
1. Methanesulfonic acid, methionine
2. NaOH, ethyl acetateO
H3C
H3C
CH3
O
N
CH3
CH3HO
H3C
CH3
N
CH3
CH3HO
H3C
LiAlH4
rac-Tapentadol
14 15
16
ent-16
17a 17b
18 19
Figure 12.8 The synthesis of tapentadol hydrochloride according to Ref. [39].
12.2 The Discovery of Tapentadol j309
respectively, gives the diasteromeric alcohols 3-hydroxy-3-(3-methoxyphenyl)-2methylpentanoic
acid dimethylamide 16 and ent-16 as racemates, respectively. By
elimination of the hydroxyl group with HBr, the olefins 17a and 17b are obtained.
With a strong base such as KOtBu, olefin 17b could be isomerized to 17a, which is
hydrogenated by Pd/C to compound 18. The methoxy ether is cleaved by methionine
and methanesulfonic acid to compound 19. The final reduction by LiAlH4
gives racemic tapentadol (Figure 12.8).
Alternatively to the dehydroxylation sequence as shown in Figure 1.5, the tertiary
alcohol (À)-(S,S)-3 could be treated by formic acid to yield the olefin 20 as a diasteromeric
mixture (Figure 12.9). Isolation of the E-olefin as hydrochloride salt and
the hydrogenation of the free base yield the corresponding methoxy derivative. Chiral
induction of the present chiral center creates the desired chirality of the second
asymmetry carbon atom. Final ether cleavage by methionine and methanesulfonic
followed by hydrochloride formation gives tapentadol hydrochloride [40].
12.3
The Preclinical and Clinical Profile of Tapentadol
Given this background, a molecule combining MOR affinity with NE reuptake inhibition
in one chemical entity would be a major improvement of the combined
mechanism of action approach because the full availability of MOR activity would
no longer be dependent on metabolic activation, overall drug exposure would be
reduced, and pharmacological activity would be limited to the two most relevant
mechanisms of action. Moreover, because these two mechanisms would reside in a
nonracemic molecule that does not form active metabolites, the relative contribution
of the different mechanisms would not change over the course of metabolic
transformation.
O
H3C (S)
(S)
N
CH3
CH3CH3
H3C
HO
HO
N
CH3
CH3CH3
CH3
H Cl
Tapentadol hydrochloride
(-)-(S,S)-3
HCOOH
reflux
O
H3C
(R)
N
CH3
CH3CH3
CH3H
O
H3C N
CH3
CH3CH3
CH3
HCl
H Cl
1. NaOH
2. 10% Pd/C, H2
3. 1. Methanesulfonic acid, methionine
4. NaOH, ethyl acetate
5. HCl, acetone
20
Figure 12.9 The synthesis of tapentadol hydrochloride according to Ref. [40].
310j12 Tapentadol – From Morphine and Tramadol to the Discovery of Tapentadol
12.3.1
Preclinical Pharmacology of Tapentadol
Tapentadol is a novel analgesic that combines MOR agonism and norepinephrine
reuptake inhibition in a single molecule and that shows potent and efficacious analgesia
in various rodent models of nociceptive and neuropathic pain [34, 41, 42].
Tapentadol binds to mu, delta, and kappa opiate receptors. The compound
showed Ki values of 0.1 and 0.5 mM in a rat MOR binding assay and a rat synaptosomal
NE reuptake assay, respectively. Affinity for the other opioid receptor subtypes
(i.e., kappa-opioid receptor (KOR), delta-opioid receptor (DOR), and ORL1 receptor
(NOP)) was at least approximately one order of magnitude lower (Table 12.1). The
affinity of morphine at the MOR was about 50-fold higher than that of tapentadol.
At the recombinant human MOR, both tapentadol and morphine had a somewhat
lower affinity than at the rat MOR (Ki ¼ 0.54 and 0.023 mM, respectively). MOR agonism
was confirmed in a guinea pig ileum assay [34].
Dose-dependent inhibition of norepinephrine reuptake was observed, resulting
in large increases in extracellular norepinephrine levels and only a moderate
increase in serotonin levels. This indicated that tapentadol’s serotonergic activity
was much lower than its norepinephrinergic activity. Other uptake systems, such
as dopamine, adenosine, choline, and c-aminobutyric acid, did not appear to be
affected by tapentadol (Table 12.2) [34, 42].
In summary, tapentadol’s MOR agonist activity is several-fold greater than tramadol’s,
with prominent norepinephrine reuptake inhibition and minimal serotonin
effect. Preclinical studies with MOR and a2-adrenoceptor antagonists have confirmed
that both mechanisms effectively contribute to tapentadol-induced
analgesia [33, 34, 42].
The two mechanisms interact in a synergistic way [21], resulting in a broad therapeutic
range and high efficacy in neuropathic pain models. In non-neuropathic
pain models, morphine was found only to be two to three times more potent than
tapentadol, although the MOR affinity of tapentadol is 50-fold lower than that of
morphine [34, 42].
Table 12.1 Comparison of the affinity of tapentadol and morphine at various opioid receptor
subtypes, as assessed in binding assays using rat brain membranes (MOR, KOR, and DOR) or
human recombinant receptors (NOP) [34, 42].
Compound Ki value (mM)
MOR KOR DOR NOP
Tapentadol 0.1 0.9 1.9 >100
Morphine 0.002 0.17 0.08 >100
MOR, mu-opioid receptor; KOR, kappa-opioid receptor; DOR, delta-opioid receptor; NOP or ORL1,
nociceptin receptor.
12.3 The Preclinical and Clinical Profile of Tapentadol j311
In a neuropathic pain model, tapentadol is even more potent than morphine in
STZ-induced heat hyperalgesia2)
, suggesting that the NRI component of tapentadol
contributes significantly to its activity in this model [3].
The analgesic properties of tapentadol were investigated in various acute and
chronic pain models in mice, rats, rabbits, and dogs to demonstrate the analgesic,
antihyperalgesic, and antiallodynic properties of tapentadol. These experiments
were performed with different routes of administration, including intravenous (i.
v.), intraperitoneal (i.p.), oral (p.o.), intracerebroventricular (i.c.v.), and intrathecal
(i.t.) routes, and employed a wide range of pain stimuli, including thermal, tactile,
chemical, and electrical modalities. The pain tests consisted of models of acute
nociceptive pain (tail flick, hot plate, tooth pulp stimulation, and phenylquinoneinduced
writhing models), acute and persistent inflammatory pain (yeast and formalin
models), visceral pain (colorectal distension and mustard oil-induced visceral
pain), and chronic mononeuropathic pain (chronic constriction injury (CCI)). The
results for tapentadol in comparison to morphine and tramadol are summarized in
Table 12.3[34, 42].
The animal models of pain demonstrate the substantial potency difference
between tapentadol and tramadol. Across a variety of assays, tapentadol is consistently
two to five times more potent than tramadol [19].
12.3.2
Clinical Trials
The pharmacological and functional differences of tramadol and tapentadol that are
observed in vitro and in vivo translate into significant clinical differences. Tapentadol
has demonstrated in clinical trials efficacy in both neuropathic and nociceptive
pain conditions. As a result, it is expected to be prescribed for conditions involving
both pain types, such as chronic lower back pain [41]. These published phase II and
phase III studies used active control medications including oxycodone, morphine,
or NSAIDs such as ibuprofen.
Table 12.2 Comparison of tapentadol and desipramine with respect to neurotransmitter reuptake
inhibition, as assessed in rat brain synaptosomes.
Transmitter Ki value (mM)
Tapentadol Desipramine
NE (norepinephrine) 0.5 0.001
5-HT (serotonin) 2.4 1.4
Dopamine NE ND
Choline 39 ND
NE, no effect <5% inhibition; ND, not determined.
2) The streptozotocin (STZ)-induced diabetic rat is the most commonly employed animal model
used to study mechanisms of painful diabetic neuropathy and to evaluate potential therapies.
312j12 Tapentadol – From Morphine and Tramadol to the Discovery of Tapentadol
Clinical trials have demonstrated that in patients with moderate to severe chronic
pain, tapentadol as a controlled-release formulation resulted in significant pain
reduction compared to placebo. Nausea, dizziness, constipation, and CNS sedation
are common side effects of opioid pain medications. However, tapentadol demonstrated
a marked reduction in gastrointestinal (GI) side effects such as nausea,
vomiting, and constipation, and CNS side effects such as dizziness and somnolence,
compared to other opioids such as oxycodone. In addition, the long-term
safety and efficacy of tapentadol has been shown in two separate studies over a
12-month and 2-year period of continuous treatment.
Table 12.3 Overview of the analgesic activity of tapentadol, tramadol, and morphine in various
animal models of acute and chronic paina)
[34, 42].
Pain model Route of
administration
ED50 value (mg/kg)
Tapentadol Morphine Tramadol
Hot plate 58 
C (mouse) i.p. 27.7 8.5 28.7
Hot plate 48 
C (mouse) i.v. 3.3 1.3 NA
Tail flick (mouse) i.v. 4.2 1.4 13.7
Tail flick (mouse) p.o. 53.4 18.9 23.8
Tail flick (mouse) i.c.v.b)
65.0 0.4 NA
Tail flick (rat) i.v. 2.2 1.1 9.6
Tail flick (rat) i.p. 10.0 5.8 10.0
Tail flick (rat) p.o. 121 55.7 16.6
Tail flick (dog) i.v. 4.3 0.7 NA
Phenylquinone-induced writhing
(mouse)
i.v. 0.7 0.4 3.6
Phenylquinone-induced writhing
(mouse)
p.o. 31.3 4.7 4.5
Phenylquinone-induced writhing
(mouse)
i.c.v.b)
18.4 0.08 NA
Yeast model (rat) i.v. 2.0 0.9 9.3
Yeast model (rat) i.p. 10.1 5.6 8.6
Yeast model (rat) i.t.b
56.8 1.9 NA
Formalin, phase II (rat) i.p. 3.8 0.8 5.9
Tooth pulp stimulation (rabbit) i.v. 3.1 2.3 NA
Colorectal distension-induced visceral
pain (rat)
i.v. 5.5 3.5 18 (ND)
Spinal nerve ligation neuropathy (rat) i.p. 8.2 2.9 4.6
Chronic constriction injury neuropathy
(CCI) (rat)
i.p. 13 13.8 7.1
Vincristine polyneuropathy (rat) i.p. 5.1 3.0 7.2
Diabetic polyneuropathy (rat) i.p. 8.9 3.0 9.2
Mustard oil visceral pain (rat) i.v. 1.5 1.0 9.0
ND, not determined; NA, not available.
a) All drug doses for preclinical and clinical testing are for the hydrochloride salt.
b) Dose in mg/animal.
12.3 The Preclinical and Clinical Profile of Tapentadol j313
The advantages of tapentadol over current treatment options using NSAIDs and
opioids may be summarized as follows: The two categories of analgesics, that is,
opioids and NSAIDs, are both effective but are associated with potentially serious
side effects. Concerns regarding tolerance and dependence minimize the use of
narcotics such as morphine and codeine for the treatment of acute or chronic pain.
Patients on chronic NSAID therapy risk severe gastrointestinal symptoms, including
ulceration and bleeding. An alternative to this dilemma is tapentadol, indicated
for the management of moderate to severe pain. Its opioid-sparing effects and
reduced potential for abuse will be key clinical advantages for tapentadol in neuropathic
pain conditions requiring long-term management. The anticipated uptake of
tapentadol controlled-release formulations in the neuropathic lower back pain market
is expected to drive market growth as there are currently no treatments indicated
for this prevalent condition [41].
12.3.3
Pharmacokinetics and Drug–Drug Interactions of Tapentadol
After oral administration, 32% of the drug is absorbed. It is widely distributed in
the body. The plasma protein binding is low and amounts to approximately 20%.
The activity of tapentadol is independent of metabolic activation and resides in a
single enantiomer that readily crosses the blood–brain barrier; hence, tapentadol
displays a rapid onset of action after administration. The half-life is 4 h and the
peak effect is attained after 1 h. The duration of action is 4–6 h. The drug undergoes
extensive first-pass hepatic metabolism, that is, 97%.
Its metabolites have no analgesic activity. The biotransformation of tapentadol by
metabolic enzymes results in deactivation, that is, tapentadol has no active metabolites,
and the main metabolic pathway for elimination is phase II glucuronidation.
Phase I biotransformations such as hydroxylation and N-demethylation play only a
minor role in the metabolic fate of tapentadol. Owing to the minor involvement of
phase I metabolic pathways, tapentadol has a low potential for drug–drug interactions
and interindividual variability. Ninety-nine percent of the drug is excreted
through renal route [42].
Drug–drug interaction studies to date have documented no induction of
CYP1A2, CYP2C9, or CYP3A4 activity; in addition, these studies found no significant
inhibition of CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2E1, or CYP3A4
activity. CYP2D6 activity was inhibited, but to a degree that did not appear to be
clinically relevant. Tapentadol did not undergo significant protein binding. Based
on these findings, it is unlikely that tapentadol will produce clinically important
drug–drug interactions with agents affected by CYP activity or plasma protein
binding [42].
The differences in the central nervous system functional activity of tapentadol
and tramadol (based on the differences in their pharmacodynamic, pharmacokinetic,
and functional activity characteristics) correlate with their clinical features
and common uses.
314j12 Tapentadol – From Morphine and Tramadol to the Discovery of Tapentadol
12.4
Summary
Tapentadol is a novel centrally acting synthetic analgesic with a dual mechanism of
action in one single molecule, mu-opioid agonist and norepinephrine reuptake
inhibitor. It is much times less potent than morphine in binding to the human
mu-opioid receptor and is two to three times less potent in producing analgesia in
animal models. Tapentadol exerts its analgesic effects without a pharmacologically
active metabolite. Tapentadol is effective in treating moderate to severe pain (WHO
step 3). In clinical trials, tapentadol has been shown to be equiefficacious to oxycodone
with fewer gastrointestinal adverse effects [19].
References
1 Merskey, H. and Bogduk, N. (eds) (2004)
Part III: Pain terms, a current list with
definitions and notes on usage, in
Classification of Chronic Pain, 2nd edn,
IASP Press, Seattle, WA, pp. 209–214.
Available at www.iasp-pain.org (accessed
February 21, 2012).
2 Waldmann, S.D. (2007) Pain Management,
Saunders Elsevier, Philadelphia, PA.
3 Anonymous (2007) Drugs for pain. Treat.
Guidel. Med. Lett., 5 (56), 23–32.
4 Barkin, R.L. and Barkin, D. (2001)
Pharmacologic management of acute and
chronic pain: focus on drug interactions
and patient-specific pharmacotherapeutic
selection. South Med. J., 94 (8), 756–770.
5 Wu, C.L. and Raja, S.N. (2011) Treatment
of acute postoperative pain. Lancet, 377,
2215–2225.
6 Turk, D.C., Wilson, H.D., and Cahana, A.
(2011) Treatment of chronic non-cancer
pain. Lancet, 377, 2226–2235.
7 Vanderah, T.W. (2007) Pathophysiology of
pain. Med. Clin. North Am., 91, 1–12.
8 Friderichs, E., Christoph, T., and
Buschmann, H.H. (2007) Analgesics and
antipyretics, in Ullmann’s Encyclopedia of
Industrial Chemistry, Wiley-VCH Verlag
GmbH, Weinheim, pp. 1–97.
9 Rao, P.N.P. and Knaus, E.E. (2008)
Evolution of nonsteroidal antiinflammatory
drugs (NSAIDs):
cyclooxygenase (COX) inhibition and
beyond. J. Pharm. Pharm. Sci., 11, 81–110.
10 Williams, M. et al. (1999) Emerging
molecular approaches to pain therapy.
J. Med. Chem., 42, 1481–1500.
11 Datamonitor (2007) Pipeline Insight:
Neuropathic Pain.
12 Butera, J.A. (2007) Current and emerging
targets to treat neuropathic pain. J. Med.
Chem., 50, 2543–2596.
13 Reimann, W. and Schneider, F. (1998)
Induction of 5-hydroxytryptamine release
by tramadol. Eur. J. Pharmacol., 349 (2–3),
199–203.
14 Minami, K., Uezono, Y., and Ueta, Y.
(2007) Pharmacological aspects of the
effects of tramadol on G-protein coupled
receptors. J. Pharmacol. Sci., 103, 253–260.
15 Grond, S. and Sablotzki, A. (2004) Clinical
pharmacology of tramadol. Clin.
Pharmacokinet., 43 (13), 879–923.
16 Valle, M., Garrido, M.J., Pavo, J.M., Calvo,
R., and Troconiz, I.A. (2000)
Pharmacokinetic pharmacodynamic
modeling of the antinociceptive effects of
main active metabolites of tramadol, (1)O-desmethyltramadol
and (2)-Odesmethyltramadol,
in rats. J. Pharmacol.
Exp. Ther., 293, 646–653.
17 Raffa, R.B., Friderichs, E., Reimann, W.,
Shank, R.P., Codd, E.E., and Vaught, J.L.
(1992) Opioid and nonopioid components
independently contribute to the
mechanism of action of tramadol, an
“atypical” opioid analgesic. J. Pharmacol.
Exp. Ther., 260, 275–285.
References j315
18 Raffa, R.B., Friderichs, E., Reimann, W.,
Shank, R.P., Codd, E.E., Vaught, J.L.,
Jacoby, H.I., and Selve, N. (1993)
Complementary and synergistic
antinociceptive interaction between the
enantiomers of tramadol. J. Pharmacol.
Exp. Ther., 267, 331–340.
19 Raffa, R.B., Buschmann, H., Christoph,
T., Eichenbaum, G., Englberger, W.,
Flores, C.W., Hertrampf, T., K€ogel, B.,
Schiene, K., Straburger, W., Terlinden,
R., and Tzschentke, T.M. (2012)
Mechanistic and functional differentiation
of tapentadol and tramadol. Expert Opin.
Pharmacother., 13 (10), 1437–1449.
20 Casy, A.F. and Parfitt, R.T. (1986) Opioid
Analgesics – Chemistry and Receptors,
Plenum Press, New York.
21 Friderichs, E. and Buschmann, H. (2002)
Opioids with clinical relevance, in
Analgesics (eds H. Buschmann, T.
Christoph, E. Friderichs, C. Maul, and B.
Sundermann), Wiley-VCH Verlag GmbH,
Weinheim, pp. 171–245.
22 Maul, C., Buschmann, H., and
Sundermann, B. (2002) Synthetic opioids,
in Analgesics (eds H. Buschmann, T.
Christoph, E. Friderichs, C. Maul, and B.
Sundermann), Wiley-VCH Verlag GmbH,
Weinheim, pp. 159–169.
23 McCurdy, C.R. (2006) Development of
opioid receptor ligands, in Analogue-based
Drug Discovery (eds J. Fischer and C.R.
Ganellin), John Wiley & Sons, Ltd,
Chichester, pp. 259–276.
24 Agranat, I., Caner, H., and Caldwell, J.
(2002) Putting chirality to work: the
strategy of chiral switches. Nat. Rev. Drug
Discov., 1, 753–768.
25 (a) Aboul-Enein, H.Y. and Wainer, I.W.
(1997) The Impact of Stereochemistry on
Drug Development and Use, John Wiley &
Sons, Ltd, Chichester. (b)Francotte, E. and
Lindner, W. (2006) Chirality in Drug
Research, Methods and Principles in
Medicinal Chemistry, vol. 33, John Wiley
& Sons, Ltd, Chichester.
26 Hu, Y., Stumpfe, D., and Bajorath, J.
(2011) Lessons learned from molecular
scaffold analysis. J. Chem. Inf. Model., 51,
1742–1753.
27 Ferenczy, G.G. and Keser€u, G.M. (2010)
Thermodynamics guided lead discovery
and optimization. Drug Discov. Today, 15,
919–932.
28 Gleeson, M.P., Hersey, A., Montanari, D.,
and Overington, J. (2011) Probing the
links between in vitro potency, ADMET
and physicochemical parameters. Nat.
Rev. Drug Discov., 10, 197–208.
29 B€ohm, H.J., Klebe, G., and Kubinyi, H.
(1996) Wirkstoffdesign – Der Weg zum
Arzneimittel, Spektrum Akademischer
Verlag.
30 Wermuth, C.G. (2008) The Practice of
Medicinal Chemistry, 3rd edn, Academic
Press.
31 Buschmann, H., Strassburger, W., and
Friderichs, E. (1996) 1-Phenyl-3dimethylaminopropane
compounds with
a pharmacological effect. EP 693475 A1,
US Patent 6,248,737 B1.
32 Hell, W., Kegel, M., Akteries, B.,
Buschmann, H., Holenz, J., Loebermann,
H., Heller, D., Drexler, H., and Gladow,
S. (2004) Method for the preparation of 3substituted
3-aryl-butyl amine
compounds. WO 2004108658 A1.
33 Tzschentke, T.M., De Vry, J., Terlinden, R.,
Hennies, H.H., Lange, C., Strassburger, W.,
Haurand, M., Kolb, J., Schneider, J.,
Buschmann, H., Finkam, M., Jahnel, U.,
and Friderichs, E. (2006) Tapentadol
hydrochloride. Drugs Future, 31, 1053–1061.
34 Hell, W. (2008) Preparation of 3-[(1R,2R)-
3-(dimethylamino)-1-ethyl-2-methylpropyl]phenol.
WO 2008012046 A1.
35 Hell, W., Zimmer, O., Buschmann, H.H.,
Holenz, J., and Gladow, S. (2008) Process
for the preparation of (1R,2R)-3-(3-
dimethylamino-1-ethyl-2-methyl-propyl)phenol.
WO 2008012047A1.
36 Liu, K.K.C., Subas Sakya, M., O’Donnell,
C.J., Flick, A.C., and Li, J. (2011)
Synthetic approaches to the 2009
new drugs. Bioorg. Med. Chem., 19,
1136–1154.
37 Motta, G., Vergani, D., and Bertolini, G.
(2012) New process for the synthesis of
tapentadol and intermediates thereof. WO
2012/001571 A1.
38 Reynolds, C.H. and Holloway, C.K. (2011)
Thermodynamics of ligand binding and
efficiency. ACS Med. Chem. Lett., 2, 433–437.
39 Buschmann, H.H. and Holenz, J. (2011)
Process for the preparation of substituted
316j12 Tapentadol – From Morphine and Tramadol to the Discovery of Tapentadol
3-(1-amino-2-methylpetane-3-yl)phenyl
compounds. WO 2011/157390 A2.
40 Hell, W., Kegel, M., Akteries, B.,
Buschmann, H.H., Holenz, J.,
L€obermann, H., Heller, D., and Drexler,
H.J. (2004) Method for the production of
substituted 3-aryl-butyl amine
compounds. WO 2004/108658 A1.
41 Nightingale, S. (2012) The neuropathic
pain market. Nat. Rev. Drug Discov., 11,
101–102.
42 Wade, W.E. and Spruill, W.J. (2009)
Tapentadol hydrochloride: a centrally
acting oral analgesic. Clin. Ther., 31,
2804–2818.
References j317
Helmut Buschmann studied chemistry at RWTH Aachen
and received his Ph.D. in the group of Prof. Scharf (Isoinversion
Principle). As a chemist, he started his career in
pharmaceutical industry in 1992 at Gr€unenthal GmbH. In
his final position, he was responsible for chemical research
(combinatorial and medicinal chemistry, process development,
and pilot plant). From 2002 to 2008, he worked as
Research Director at Laboratorios Dr. Esteve (Barcelona).
In this position, he managed a staff of 120 employees
(more than 60 persons with university degrees) and was
responsible for chemistry, pharmacology, toxicology, pharmacokinetic, and intellectual
property departments. In 2009, he joined Pharma-Consulting Aachen. As a
result of his research activities, more than 15 NCEs within different therapeutic
areas have entered the clinical phase (e.g., rosonabant, cizolirtine, dulopetine, axomadol).
Some of them have reached clinical phase III or are marketed (e.g., tapentadol:
Nucynta1
, Palexia1
). Besides this, he is a member of several scientific
advisory boards of public research institutions in Europe (Leibniz Institut f€ur Katalyse,
Rostock; Institut Catala d’Investigacio Quõmica, Tarragona) and gives lectures
at the Universities of Aachen and Rostock. He led or coauthored more than
100 publications in major scientific journals and is listed as inventor on more than
200 patent application families. As author and editor, he has also published three
books in the field of stereochemistry, analgesics, and antidepressants.
318j12 Tapentadol – From Morphine and Tramadol to the Discovery of Tapentadol
13
Novel Taxanes: Cabazitaxel Case Study
Herve Bouchard, Dorothee Semiond, Marie-Laure Risse, and Patricia Vrignaud
List of Abbreviations
10-DAB 10-deacetylbaccatin III
AUC area under the curve
bid twice daily
BSA body surface area
CAP capecitabine
CBZ cabazitaxel
CI confidence interval
CL clearance
CR complete response
CRPC castration-resistant prostate cancer
DLT dose-limiting toxicity
FN febrile neutropenia
G-CSF granulocyte colony-stimulating factor
HNTD highest nontoxic dose
HR hazard ratio
IV intravenous
mCRPC metastatic castration-resistant prostate cancer
MDR1 multidrug resistance gene 1
mHRPC hormone-refractory metastatic prostate cancer
MTD maximum tolerated dose
nd not determined
OS overall survival
PFS progression-free survival
P-gp P-glycoprotein
PK pharmacokinetics
POMA 2-(4-methoxyphenyl)-4-phenyloxazolidine-3,5-dicarboxylic acid-3-tertbutyl
ester
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
j319
PR partial response
PSA prostate-specific antigen
q3w every 3 weeks
RD recommended dose
SC subcutaneous
SD stable disease
TES-Cl triethylsilyl chloride
TFS tumor-free survivors
TTP time to tumor progression
Vss volume of distribution at steady state
13.1
Introduction
Previous volumes in this series have highlighted ways in which analogue-based
drug discovery (ABDD) has contributed to the discovery of new chemical entities
that ultimately proved to be useful clinically [1, 2]. The application of
ABDD to the modification of candidate drugs derived from natural products is
an extension of this approach that was illustrated in the chapter on paclitaxel
and epothilone analogues published in Volume II of this series [3]. The current
chapter focuses on a novel taxane compound that was developed using ABDD
from an established marketed drug that was itself derived from a natural
product.
Taxoids are diterpene molecules found mainly in various yew tree species
(Taxus spp.). They are characterized by the presence of a 20-carbon taxane core
ring structure. Since the isolation and purification of the first taxane-containing
molecule in the 1960s, taxanes have become an important class of cancer
chemotherapeutic drugs, with proven efficacy in a wide range of human
cancers.
Taxanes inhibit cell division by binding to microtubules, which are key components
of the cytoskeleton and critical regulators of cell division [4]. At the
onset of mitosis, microtubules assemble into a structure known as the mitotic
spindle. The spindle serves as an attachment point for chromosomes prior to
their segregation into the resultant daughter cells. The cycle of microtubule
assembly and disassembly during cell division involves a dynamic equilibrium
between microtubule polymers and a pool of intracellular tubulin, consisting of
a- and b-tubulin heterodimers [5]. Research by Horwitz and colleagues [6] demonstrated
that Taxol, the first taxane to be isolated, stabilizes microtubules by
binding to the microtubule polymer, thereby shifting the equilibrium in favor
of the microtubule and preventing depolymerization. In turn, this prevents cell
division and leads to programmed cell death. The mechanism of action of taxanes
is distinct from that of other microtubule inhibitors such as the vinca alkaloids,
which act by inhibiting tubulin polymerization and microtubule
assembly [7].
320j13 Novel Taxanes: Cabazitaxel Case Study
13.1.1
Isolation and Chemical Synthesis of Taxanes
Taxol (generic name paclitaxel; marketed as Taxol1
(1 in Figure 13.1)) was originally
extracted from the bark of the Pacific yew tree, Taxus brevifolia [8]. However,
purification of taxol with sufficient yield to allow testing in cancer patients required
vast quantities of bark from the extremely slow-growing Pacific yew tree. As a
result, significant efforts focused on finding a sustainable source of taxol and an
efficient process for its isolation. Although the total synthesis of taxol has been
achieved [9–11], the process, involving 40 steps with only 2% yield, is not commercially
viable. An advance came with the development of a process to synthesize
taxol from 10-deacetylbaccatin III (10-DAB in Figure 13.1), a natural precursor molecule
that could be extracted easily and sustainably from the needles of the
Docetaxel [2]
O
OCOC6
H5
HO O
OH
HO
H O
OCOCH3
O
O
NH
O
OH
Larotaxel [5]Cabazitaxel [4]
10-deacetylbaccatin III [10-DAB]
O
OCOC6
H5
O
OH
HO
H O
OCOCH3
Paclitaxel [1]
NH
O
OH
CH3
COOO
Tesetaxel [3]
HO
OCOC6
H5
HO O
10
OH
13
HO
H O
OCOCH3
Side chain
attachment
at C-13
O O
H
N
N
F
O
OCOC6
H5
HO
H O
OCOCH3
O
O
NH
O
OH
10
7
O
OCOC6
H5
O
HO
H O
OCOCH3
O
O
NH
O
OH
O
O
H3
C CH3
10
7
O
O
OCOC6
H5
O
HO
H
OCOCH3
O
O
NH
O
OH
CH3
COO
Figure 13.1 Chemical structures of 10-deacetylbaccatin III and taxane compounds.
13.1 Introduction j321
European yew, Taxus baccata [12, 13]. Taxol can now also be produced using plant
cell fermentation technology [14]. A specific paclitaxel-producing Taxus cell line is
cultured on a large scale. The paclitaxel is then extracted, purified by chromatography,
and isolated by crystallization.
Other synthetic taxanes include docetaxel (Taxotere1
; 2 in Figure 13.1), which is
an esterified product of 10-DAB. Docetaxel differs from paclitaxel at two positions
in its chemical structure: a hydroxyl functional group at C-10 (paclitaxel has an acetate
ester) and a tert-butyl carbamate on the phenyl propionate side chain (paclitaxel
has a benzyl amide) (Figure 13.1). The change at C-10 confers greater water solubility
for docetaxel compared with paclitaxel [15].
13.1.2
Drug Resistance and Novel Taxanes
Paclitaxel and docetaxel are approved for the treatment of a range of human cancers.
Paclitaxel is indicated for first-line treatment of ovarian, breast, and lung cancer
and for second-line treatment of AIDS-related Kaposi’s sarcoma. Docetaxel is
indicated for first-line treatment of breast, head and neck, gastric, lung, and prostate
cancer and for second-line treatment of breast cancer. However, the utility of
both paclitaxel and docetaxel as anticancer drugs is limited by the frequent development
of tumor resistance [16]. Resistance to taxanes is frequently described as
resulting from expression of the P-glycoprotein (P-gp) efflux pump encoded by the
multidrug resistance gene MDR1, which leads to decreased intracellular drug levels.
This mechanism of resistance was mainly observed in cell lines in which resistance
was induced in vitro; however, the clinical significance of P-gp overexpression
has yet to be validated in patients progressing after taxane therapy. Other postulated
mechanisms of resistance involve changes in tubulin structure and altered intracellular
signaling [7, 17].
Considerable efforts have been made toward developing novel taxanes capable of
overcoming resistance to paclitaxel and docetaxel. Among several second-generation
taxanes that have been developed, the two most advanced are tesetaxel (3 in
Figure 13.1) and cabazitaxel (4 in Figure 13.1).
Tesetaxel is an orally active taxane with a similar mechanism of action to paclitaxel
and docetaxel. Like docetaxel, tesetaxel has a tert-butyl carbamate on the propionate
side chain. However, tesetaxel differs from paclitaxel and docetaxel as it
includes in the propionate side chain a 3-fluoropyridine aromatic ring in place of a
phenyl group. Other specific features of tesetaxel include reduced moieties at positions
C-7 (CH2) and C-9 (CH–O). Tesetaxel is currently under investigation in a
variety of different human cancers [18–20].
Cabazitaxel (XRP6258, RPR 116258A, TXD258, Jevtana1
) is a novel secondgeneration,
semisynthetic taxane rationally designed to be an active anticancer
treatment in both taxane-naïve and taxane-resistant tumors. Clinical activity of
cabazitaxel was observed in the phase I setting and proof of concept was first
achieved in a phase II study with responses obtained in patients with metastatic
breast tumors resistant to previous docetaxel therapy [21–23] (see below). Clinical
322j13 Novel Taxanes: Cabazitaxel Case Study
activity post-taxane failure was confirmed and demonstrated in a pivotal phase III
study in patients with metastatic castration-resistant prostate cancer (mCRPC, also
known as hormone-refractory metastatic prostate cancer (mHRPC)) previously
treated with a docetaxel-containing regimen, where cabazitaxel in combination
with prednisone/prednisolone (6 and 7 in Figure 13.2) demonstrated a statistically
significant benefit in overall survival compared with mitoxantrone (8 in Figure
13.2) [24]. Cabazitaxel in combination with prednisone received regulatory
approval by the US Food and Drug Administration (in 2010) and the European
Medicines Agency (in 2011) for the treatment of mHRPC previously treated with a
docetaxel-containing regimen. This chapter focuses on the chemical synthesis of
cabazitaxel and its structure–activity relationships, including a summary of the preclinical
and clinical testing of cabazitaxel.
13.2
Cabazitaxel Structure–Activity Relationships and Chemical Synthesis
13.2.1
Chemical and Physical Properties
The International Union of Pure and Applied Chemistry chemical name of cabazitaxel
is (2a,5b,7b,10b,13a)-4-(acetoxy)-13-({(2R,3S)-3-[(tert-butoxycarbonyl)amino]-
CapecitabineMitoxantrone
PrednisolonePrednisone
O OHHNOH
H
N
O OHHNOH
N
H
NHCOO(CH2)4CH3
O
H3C
F
OH
OH
N
N
O
O
O
O
OH
OH
HH
H
O
HH
HO
O
OH
OH
H
Figure 13.2 Chemical structures of nontaxane drugs.
13.2 Cabazitaxel Structure–Activity Relationships and Chemical Synthesis j323
2-hydroxy-3-phenylpropanoyl}oxy)-1-hydroxy-7,10-dimethoxy-9-oxo-5,20-epoxytax-
11-en-2-yl-benzoate-propan-2-one (1 : 1). Physical and chemical properties are summarized
in Table 13.1.
13.2.2
Structure–Activity Relationships of Cabazitaxel
Cabazitaxel is a semisynthetic derivative of 10-DAB and was identified through a
preclinical screening process that involved the testing of approximately 450 candidate
molecules. The primary objective of the screen was to select a compound as
potent as docetaxel in tumor models sensitive to docetaxel and more potent than
docetaxel in tumors resistant to chemotherapy, including taxanes. The main structure–activity
relationships for cabazitaxel are depicted in Figure 13.3.
With initial preclinical comparative structure–activity relationships for paclitaxel
and docetaxel in mind, our initial efforts with cabazitaxel were focused on changes
in the side chain as it was considered that this region would be critical for potency.
Attempts to replace the C-20
-Boc group of the side chain (tert-butyl carbamate) by
other functions (e.g., ureas) or by substituted benzyl amides generally resulted in
decreased in vitro potency when tested on cancer cell lines [25, 26]. Modulation of
Table 13.1 Physical and chemical properties of cabazitaxel (4).
Molecular formula C45H57NO14ÁC3H6O
Relative molecular
mass
894.01 g/mol for the acetone solvate
835.93 g/mol for the solvate-free molecule
Appearance White to almost white solid
Solubility Lipophilic, soluble in dehydrated alcohol and insoluble in water at
25 
C
C2'-Boc replacement
Activity
C10-ethers most favorable on RCL
Longer alkyl ether Toxicity
C7-ethers most favorable on RCL
C7-esters less active than docetaxel
Oxetane modification
Activity
C2-benzoate/C4-acetate modification
Activity modulation with no gain on RCL
C3'-phenyl replacement
Activity modulation
with no gain on RCL
O
OO
O
RCL: Resistant cell line
O
O
HO
H O
O
O
O
O
NH O
OH
CH3
CH3
Figure 13.3 Structure–activity relationships for cabazitaxel.
324j13 Novel Taxanes: Cabazitaxel Case Study
activity was observed when the C-30
phenyl group of the propionate side chain was
replaced, as exemplified by the structure of the tesetaxel analogue (3 in Figure 13.1).
Substitution of this phenyl group or replacement by alkylidene groups or heteroaromatic
rings (e.g., furan or thiophene) improved the in vitro activity but, in our
hands, resulted in no relative improvement in activity against cell lines resistant to
docetaxel [27].
The role of the benzoate group at position C-2 was also studied. Some ortho/metamethoxy-substituted
benzoates proved to be highly cytotoxic in vitro, although no
significant impact was observed on docetaxel-resistant cell lines [28]. Removal of the
C-4 acetate resulted in C-4 hydroxy analogues that demonstrated a large decrease
in potency. Replacement of the C-4 acetate by other analogues such as propionate
or methoxyacetate produced more potent analogues in in vitro assays, but the therapeutic
index was reduced in in vivo models due to dose-limiting toxicities.
The oxetane ring was found to be crucial for maintaining overall activity; opening
of the ring or replacement of the oxygen atom reduced in vitro potency.
Our efforts to better understand the structure–activity relationship in the C-7/C-10
region focused on modifications at C-7 aimed at generating new analogues of
docetaxel by nucleophilic substitution at this position. We initially planned to
treat a C-7 trifluoromethanesulfonate-activated derivative with different nucleophilic
agents and found that 7,8,19-cyclopropanation could occur under these
conditions [29]. This chemical modification in the C-7/C-10 region resulted in
the larotaxel structure (5 in Figure 13.1) and we discovered that this lipophilic
modification addressed in vitro and in vivo resistance to docetaxel and issues
related to P-gp. Further investigation of structure–activity relationship in the
C-7/C-10 region found that C-7/C-10 ether analogues present in cabazitaxel and
the 7,8,19-cyclopropane modification present in larotaxel were the most favorable
modifications of the upper part of the baccatin moiety in terms of in vitro potency
in docetaxel-sensitive and docetaxel-resistant cell lines. When compared with C-7
ethers, C-7 esters were found to be less active than docetaxel in in vivo models.
Some longer C-10 alkyl ethers were evaluated in vivo; however, drug-related
deaths were observed at doses equal to or lower than the maximum tolerated
doses of docetaxel, thereby limiting the antitumor activity of these compounds.
13.2.3
Chemical Synthesis of Cabazitaxel
Cabazitaxel is structurally distinct from docetaxel due to the presence of two
methoxy groups in positions C-7 and C-10 of the baccatin moiety (Figure 13.1). In
the course of structure–activity relationship studies on analogues of docetaxel, a
regioselective synthesis of C-7/C-10 ether derivatives was developed. This initial
synthetic pathway allowed for the introduction of selectivity as well as for the
sequential addition of substituents at C-7 and C-10. From the naturally occurring
10-DAB, selective protection of the C-7 and C-13 hydroxy functions using triethylsilyl
chloride (TES-Cl) in pyridine produced intermediate 1 (Scheme 13.1a). At this
stage, only the hydroxy function at C-10 could be easily modified as the tertiary
13.2 Cabazitaxel Structure–Activity Relationships and Chemical Synthesis j325
alcohol at C-1 was unreactive under the same conditions. Following treatment with
sodium hydride and methyl iodide, intermediate 1 was converted into its methyl
ether analogue. The triethylsilyl groups were then removed using hydrofluoric
acid–triethylamine to give C-10 monomethoxylated baccatin (intermediate 2). As
the hydroxy function at position C-7 of the baccatin core is generally more reactive
HO
(a)
10
OH
13
HO
H O
Intermediate 1
Intermediate 2Intermediate 3
1) NaH, methyl iodide
2) HF, triethylamine
TESO
HO
OTES
HO
H O
10-DAB
Triethylsilyl chloride
Pyridine
NaH, methyl iodide
H3CO
10
OH
13
HO
H O
HO
HO
HO
OCOC6
H5
HO
H O
OCOCH3
OCOC6
H5
OCOCH3
OCOC6
H5
OCOCH3
OCOC6H5
OCOCH3
1) DCC, DMAP
2) HCI, ethanol
Cabazitaxel
O
OH
O
OMe
O
N
tBuO
POMA
OCH3
H3CO
O O
O
O
O
OCOC6
H5
O
HO
H O
OCOCH3
tBuO
O
NH
O
OH
H3CO
OCH3
DCC:N,N'-dicyclohexylcarbodiimide
DMAP: 4-dimethylaminopyridine
POMA: 2-(4-methoxyphenyl)-4-phenyl-oxazolidine-3,5-dicarboxylic acid 3-tert-butyl ester
Scheme 13.1 (a) First synthetic pathway to cabazitaxel chemical entity. (b) Cabazitaxel alternative
synthesis.
326j13 Novel Taxanes: Cabazitaxel Case Study
than the one at position C-13, methylation of intermediate 2 with sodium hydride
and methyl iodide resulted in the dimethyl ether (intermediate 3). Esterification of
the hydroxy group at position C-13 with a protected form of the side chain 2-(4methoxyphenyl)-4-phenyloxazolidine-3,5-dicarboxylic
acid-3-tert-butyl ester (POMA)
under standard conditions (N,N0
-dicyclohexylcarbodiimide, 4-dimethylaminopyridine)
[28], followed by cleavage of the para-methoxybenzylidene protecting group
under acidic conditions, allowed completion of the synthesis of cabazitaxel.
Although this first synthetic scheme proved to be useful and efficient for the
preparation of various regioselective C-7 and C-10 taxane analogues, it was not a
dedicated straightforward synthesis for cabazitaxel. Therefore, an alternative synthesis
was developed, minimizing the number of protection/deprotection steps
and resulting in simultaneous methylation of the hydroxy functions at positions
C-7 and C-10 (Scheme 13.1b). Under standard conditions, esterification of the
POMA-protected side chain with 7,10-di-Troc-baccatin III (intermediate 4) followed
by cleavage of the Troc groups (2,2,2-trichloroethoxycarbonyl) with zinc in acetic
acid produced the 7,10-dihydroxy taxoid (intermediate 5). Pummerer reaction conditions
(dimethyl sulfoxide, acetic anhydride) applied to intermediate 5 permitted
HO
HO(b)
10
OH
13
HO
H O
OCOC6
H5
TrocO
OTroc
HO
HO
H O
POMA
1) DCC, DMAP
2) Zinc, acetic acid
OCOCH3
10-DAB
TrocCl
Pyridine
O
O
O
O
N
O
O
S
S
tBuO
HO
H
5etaidemretnI6etaidemretnI
Intermediate 4
10
7
O
OH
HO
HO
H
10
7
O
Cabazitaxel
1) Nickel Raney
2) HCl, ethanol
O
O
O
Dimethyl sulfoxide
Acetic anhydride
Pummerer
N
tBuO
OCH3OCH3
OCOC6H5
COCH3
O
OCOC6H5
OCOCH3
OCOC6
H5
OCOCH3
O
O
O
O
O
O
OCOC6
H5
O
HO
H O
OCOCH3
tBuO
O
NH
O
OH
H3CO
OCH3
DCC:N,N'-dicyclohexylcarbodiimide
DMAP: 4-dimethylaminopyridine
POMA: 2-(4-methoxy-phenyl)-4-phenyl-oxazolidine-3,5-dicarboxylic acid 3-tert-butyl ester
Scheme 13.1 (Continued )
13.2 Cabazitaxel Structure–Activity Relationships and Chemical Synthesis j327
the simultaneous functionalization of the C-7 and C-10 positions as methylthiomethyl
ethers resulting in intermediate 6. Reduction of the methylthioether groups
of intermediate 6 with Raney nickel produced the dimethoxy analogue that was
then deprotected on its side chain under acidic conditions, as previously described,
to complete the alternative synthesis of cabazitaxel.
13.3
Cabazitaxel Preclinical and Clinical Development
13.3.1
Preclinical Development
Cabazitaxel was selected for preclinical testing based on its enhanced antiproliferative
activity in chemotherapy-resistant cell lines compared with docetaxel. In addition,
cabazitaxel was shown to promote tubulin assembly in vitro and to stabilize
microtubules against cold-induced depolymerization as efficiently as docetaxel [30].
Using tumor cell lines with acquired resistance to doxorubicin, vincristine, vinblastine,
paclitaxel, and docetaxel, resistance factors (calculated by dividing the IC50
in resistant cells by the IC50 in sensitive cells) ranged from 1.8 to 10 for cabazitaxel,
compared with 4.8–59 for docetaxel [30]. In Caco-2 TC7 cells (a cell line that
expresses high levels of the P-gp efflux pump), cabazitaxel was shown to be a highly
permeable compound (as expected, based on its lipophilicity (log P ¼ 3.9)) despite
being a substrate of P-gp (unpublished observations).
Like docetaxel, cabazitaxel exhibits very limited solubility in aqueous solution,
and was formulated using a docetaxel-like formulation containing ethanol and polysorbate
80. In murine and human tumor xenograft models, cabazitaxel exhibited a
broad spectrum of antitumor activity, not only in docetaxel-sensitive tumors, but
also in tumors with little or no sensitivity to chemotherapy, including taxanes
[31, 32]. In docetaxel-sensitive tumors, cabazitaxel was found to exhibit a similar
potency to docetaxel, inducing complete regressions in advanced stage disease
leading to tumor-free survivors in 8/16 mouse tumor models, including human
prostate DU145, murine colon C38, human colon HCT 116, murine pancreas P03,
human pancreas MIAPaCa-2, human gastric N87, human breast Calc 18, and
human head and neck SR 475 (Table 13.2).
The pharmacokinetics (PK) and tumor distribution of cabazitaxel were evaluated in
mice bearing advanced stage mammary adenocarcinoma MA16/C [33, 34]. At the
highest nontoxic dose (HNTD) of 40 mg/kg, drug uptake into the tumor was both
rapid (tmax occurred 2 min postdosing) and sustained (concentration of cabazitaxel
was 40-fold higher in the tumor versus plasma at 48 h postdosing). Exposure to cabazitaxel
was 2.9 times higher in the tumor than in plasma (Figure 13.4). Perhaps
more interestingly, both the tumor and plasma concentrations were higher than the
cellular antiproliferative IC50 for up to 24h in plasma and up to 96h in the tumor [34].
Cabazitaxel was selected for further clinical evaluation based on its activity in
a tumor model rendered resistant to docetaxel in vivo in an attempt to mimic
328j13 Novel Taxanes: Cabazitaxel Case Study
Table 13.2 Preclinical antitumor activity of cabazitaxel and docetaxel in mice bearing docetaxelsensitive
solid tumors.
Subcutaneous tumora)
IV cabazitaxel IV docetaxel
CR TFS SRI scoreb)
CR TFS SRI scoreb)
Advanced human prostate DU145 6/6 5/6 þþþþ nd nd nd
Early murine colon C51 À À þþþ À À þþþþ
Advanced murine colon C38 5/5 5/5 þþþþ 0/5 À þþþþ
Advanced human colon HCT 116 7/7 2/7 þþþþ nd nd nd
Advanced human colon HT-29 6/6 À þþþ 6/6 À þþþþ
Advanced murine pancreas P03 5/5 4/5 þþþþ nd nd nd
Advanced human pancreas MIA PaCa-2 6/6 6/6 þþþþ 6/6 6/6 þþþþ
Early murine breast MA16/C À À þþþþ À À þþþþ
Early murine breast MA17/A À À þþþþ À nd nd
Early human breast Calc18 À 5/8 þþþþ À nd nd
Advanced human gastric N87 À 1/8 þþþþ À 1/8 þþþþ
Advanced human lung NCI-H460 2/6 À þþþ nd nd nd
Advanced human lung A549 2/6 À þþþ 0/5 À þþ
Early murine B16 melanoma À À þþþþ À À þþ
Advanced human head and neck SR475 6/6 6/6 þþþþ 1/6 À þþþ
Advanced human kidney Caki-1 5/6 À þþ 2/5 À þþ
CR, complete regression; IV, intravenous; nd, not determined in the same study; SC, subcutaneous;
TFS, tumor-free survivors.
a) Advanced stage disease defined for median tumor burden >100 mg.
b) SRI (Southern Research Institute) score criteria: À (inactive) ¼ log cell kill total <0.7; þ ¼ 0.7–1.2;
þþ ¼ 1.3–1.9; þþþ ¼ 2.0–2.8; þþþþ (highly active) ¼ >2.8.
0 6 12 18 24 30
0.01
0.1
1
10
100
Time (h)
Cabazitaxelconcentration(µg/mlorµg/g)
40 mg/kg
(120 mg/m2
)
Tumor
Plasma
Cellular IC50
Figure 13.4 Plasma pharmacokinetics and tumor distribution of cabazitaxel in MA16/C-bearing
mice.
13.3 Cabazitaxel Preclinical and Clinical Development j329
the clinical situation. Mice bearing docetaxel-sensitive B16 tumors were treated
at the HNTD of docetaxel for 17 months to achieve full resistance to docetaxel
[30]. In addition, cabazitaxel was more active than docetaxel in tumor models
that were less sensitive or insensitive to docetaxel, such as murine P388
leukemia, human gastric GXF-209, murine lung 3LL, and human breast carcinoma
UISO BCA-1. The latter HER2-positive breast tumor was obtained from a
patient never treated with taxanes [35]. Further, docetaxel at its HNTD of
21.7 mg/kg per injection was not able to delay UISO BCA-1 tumor growth
whereas cabazitaxel induced a significant delay in tumor growth (75 days) at its
HNTD of 12.4 mg/kg per injection and also at the dose below the HNTD
(7.7 mg/kg per injection) (Figure 13.5).
Another feature that distinguishes cabazitaxel from docetaxel is the ability of cabazitaxel
to penetrate the blood–brain barrier in rodents [36]. In addition, antitumor
activity of cabazitaxel has been observed in mice bearing intracranial glioblastoma
xenografts [37].
13.3.2
Clinical Studies
The safety, efficacy, and PK of cabazitaxel have been evaluated in a series of studies
involving patients with a number of advanced solid tumors. A selection of these
studies is summarized in Table 13.3.
0 10 30 40 50 60
0
500
1000
1500
Time postimplantation (days)
Tumorvolume(mm3
) 2000
0702 80 90
Control IV treatments (days 13, 16, 19)
Docetaxel (21.7 mg/kg/injection) HNTD
Cabazitaxel (7.7 mg/kg/injection)
Cabazitaxel (4.8 mg/kg/injection)
Cabazitaxel (12.4 mg/kg/injection) HNTD
HNTD: highest nontoxic dose
IV: intravenous
Figure 13.5 Antitumor activity of cabazitaxel in human breast UISO BCA-1 xenografts.
330j13 Novel Taxanes: Cabazitaxel Case Study
Table 13.3 Summary of selected cabazitaxel clinical studies.
Study Tumor type (no. of
patients)
Treatment Key results
Phase I [21] Advanced solid
tumors previously
treated with <2
chemotherapy
regimens (n ¼ 25)
CBZ 10–25 mg/m2
q3w
RD: 20 mg/m2
q3w
DLTs: hematologic
toxicity and diarrhea
Phase I [38, 39] Advanced solid
tumors previously
treated with
chemotherapy
(n ¼ 21)
CBZ 10–30 mg/m2
q3w
RD: 25 mg/m2
q3w
DLTs: hematologic
toxicity and diarrhea
Phase I [40] Advanced solid
tumors previously
treated with
chemotherapy
(n ¼ 31a
)
CBZ 1.5–12 mg/m2
weekly (4/5 weeks)
RD: 10 mg/m2
weekly
DLTs: diarrhea
Phase I/II [23] Metastatic breast
cancer previously
treated with taxane
therapy (n ¼ 33)
CBZ (20 mg/m2
) on
day 1 þ CAP
(825 mg/m2
) bid on
days 1–12 q3w
MTD: CBZ
20 mg/m2
plus
capecitabine
1000 mg/m2
Phase II [22] Metastatic breast
cancer previously
treated with taxane
therapy (n ¼ 71)
CBZ 20 mg/m2
(cycle
1) and 25 mg/m2
from
cycle 2 in the absence
of cycle 1 toxicity; q3w
Efficacy: CR (n ¼ 2);
PR (n ¼ 8); SD
(n ¼ 18)
Safety: grade 3/4
neutropenia 73%;
febrile neutropenia
3%; neutropenia
infection 4%
Phase III (TROPIC;
NCT00417079) [24]1)
Metastatic CRPC
previously treated
with docetaxel
(n ¼ 755)
CBZ 25 mg/m2
q3w þ prednisone
10 mg/day or
mitoxantrone
12 mg/m2
q3w þ prednisone
10 mg/day
Efficacy: median OS
15.1 months (CBZ)
versus 12.7 months
(mitoxantrone);
hazard ratio of death
for CBZ relative to
mitoxantrone 0.70
(95% CI: 0.59–0.83;
p < 0.0001)
Safety: grade 3/4
neutropenia CBZ
82%, mitoxantrone
58%; febrile
neutropenia CBZ
7.5%, mitoxantrone
1.3%
(continued )
13.3 Cabazitaxel Preclinical and Clinical Development j331
13.3.2.1 Phase I and II Studies
Phase I/II studies including patients with a variety of solid tumors indicated that
cabazitaxel had an acceptable safety profile and also, similar to other taxanes, evidence
of antitumor activity. Two dose-ranging phase I studies investigated cabazitaxel
administered as a 1 h infusion every 3 weeks. In one of these studies [21],
neutropenia was identified as main dose-limiting toxicity and the recommended
dose of cabazitaxel was 20 mg/m2
. Objective clinical responses included partial
responses (PR) in two patients with metastatic prostate cancer, including one with
docetaxel-refractory disease, and 12 patients had stable disease (SD). In the other
phase I study, neutropenia was also the primary dose-limiting toxicity [38, 39]; however,
the recommended dose of cabazitaxel was identified as 25 mg/m2
. Among the
21 patients who participated, one achieved an unconfirmed PR (lung cancer) and
six had SD.
Another phase I study investigated a weekly dosing schedule of cabazitaxel in 31
patients with advanced solid tumors ([40]; unpublished observations). Diarrhea was
the main dose-limiting toxicity and the recommended dose for weekly dosing studies
was 10 mg/m2
administered for 4 out of 5 weeks. Two patients with breast
cancer achieved PR and 13 patients had SD.
A phase II study in patients with metastatic breast cancer resistant to prior
taxane therapy reported two complete responses (CR) and eight PR among the
Table 13.3 (Continued)
Study Tumor type (no. of
patients)
Treatment Key results
Phase III
(PROSELICA;
NCT01308580)2)
Metastatic CRPC CBZ 20 mg/m2
q3w þ prednisone
10 mg/day or CBZ
25 mg/m2
q3w þ prednisone
10 mg/day
Study ongoing
Phase III
(FIRSTANA;
NCT01308567)3)
Metastatic CRPC
chemonaive
CBZ 20 mg/m2
q3w þ prednisone
10 mg/day or CBZ
25 mg/m2
q3w þ prednisone
10 mg/day or docetaxel
75 mg/m2
q3w þ prednisone
10 mg/day
Study ongoing
bid, twice daily; CAP, capecitabine; CBZ, cabazitaxel; CI, confidence interval; CR, complete response;
CRPC, castration-resistant prostate cancer; DLT, dose-limiting toxicity; mg/m2
, milligrams per square
meter of body surface area; MTD, maximum tolerated dose; PR, partial response; q3w, every 3 weeks;
RD, recommended dose; SD, stable disease.
a) Unpublished observations.
332j13 Novel Taxanes: Cabazitaxel Case Study
71 patients who received treatment with cabazitaxel; a further 18 patients had
SD for at least 3 months [22]. Median time to tumor progression was 2.7
months and median overall survival was 12.3 months. A subsequent phase I/II
study evaluated cabazitaxel in combination with capecitabine (9 in Figure 13.2)
in patients with metastatic breast cancer whose disease had progressed after
taxane therapy [23]. The recommended doses were cabazitaxel 20 mg/m2
every
3 weeks plus capecitabine 1000 mg/m2
twice daily from day 1 to day 14. PK
analysis revealed no apparent drug–drug interaction. Promising clinical activity
was observed with 2 CR, 5 PR, and 20 SD among the 33 patients. The overall
response rate was 23.8%. The safety profile was consistent with what would be
expected with the combination of a taxane plus capecitabine. At the recommended
dose (received by 21 patients), the main grade 3–4 adverse events were
neutropenia (57%), asthenia (10%), dyspnea (10%), hand–foot syndrome (10%),
anorexia (5%), and febrile neutropenia (5%).
13.3.2.2 Clinical Pharmacokinetics
The PK profile of cabazitaxel is similar to that of docetaxel. Cabazitaxel exhibits
dose proportionality across a dose range of 10–25 mg/m2
[21]. Like docetaxel, the
elimination profile of cabazitaxel is triphasic; however, the terminal phase t1/2 is
longer and the plasma clearance (CL) is higher than that of docetaxel. In addition,
the mean volume of distribution at steady state (Vss) of cabazitaxel is substantially
greater than that of docetaxel.
A population PK model for cabazitaxel was developed and validated using data
obtained from 170 patients (unpublished observations) with advanced solid
tumors included in five phase I–III studies [21, 22, 24, 38, 40]. In agreement
with data previously reported in a single phase I study [21], the PK profile of
cabazitaxel was consistent with a three-compartment model characterized by
rapid initial and intermediate phases (population half-lives ¼ 4.4 min and 1.6 h,
respectively) and a long terminal phase (half-life ¼ 95.1 h). Cabazitaxel exhibited
a very large Vss (population PK estimate 4870 l; 2640 l/m2
for a median body
surface area (BSA) of 1.84 m2
) and a relatively high plasma CL (population PK
estimate 48.5 l/h; 26.4 l/h/m2
for a median BSA of 1.84 m2
). The model showed
that the impact of intrinsic and extrinsic factors on the PK behavior of cabazitaxel
was limited to BSA, with no effect of gender, age, race, and mild to moderate
renal impairment observed. The PK data did not suggest a need for dose
adjustment in these special populations. In addition, the concomitant administration
of the weak CYP3A inducers prednisone or prednisolone (6 and 7 in
Figure 13.2) in the phase III study (1)
; unpublished observations) or the
concomitant administration of capecitabine [23] did not modify the PK profile
of cabazitaxel.
1) ClinicalTrials.gov. XRP6258 plus prednisone compared to mitoxantrone plus prednisone in
hormone refractory metastatic prostate cancer (TROPIC). NCT00417079. Available at http://
clinicaltrials.gov/ct2/results?term¼00417079.
13.3 Cabazitaxel Preclinical and Clinical Development j333
Another phase I study investigated the disposition of [14
C]-cabazitaxel administered
at a dose of 25 mg/m2
[41]. In plasma, the parent drug was the main circulating
compound, representing approximately 70% of the radioactivity area under the
curve (AUC). No relevant metabolites were observed in plasma (i.e., AUC metabolite
5% of the parent drug AUC), indicating that cabazitaxel is the only entity
responsible for the pharmacological activity in humans. Approximately 80% of the
dose was recovered in the excreta within 2 weeks. Cabazitaxel was largely excreted
in the feces (76% of the dose) as numerous metabolites. The urinary route contributed
markedly less to the overall excretion (3.7% of the dose with 2.3% excreted as
unchanged drug). These results indicate that cabazitaxel is extensively metabolized
in the liver. The main metabolites corresponded to combined mono- or di-Odemethyl
derivatives on the taxane ring with hydroxyl or cyclized derivatives on the
lateral chain. Docetaxel (di-O-demethyl cabazitaxel) was only detected as traces in
plasma and excreta.
13.3.2.3 Phase III Trial
Based on clinical activity observed in patients with tumors resistant to taxanes
including patients with prostate cancer in the phase I trial [20] and its manageable
safety profile, cabazitaxel was further evaluated in a large, multinational,
randomized, controlled trial, known as TROPIC (NCT00417079).1)
This study
was conducted in 755 patients with mCRPC who had disease progression following
previous docetaxel-based therapy. TROPIC compared the efficacy of
cabazitaxel with the active agent mitoxantrone (8 in Figure 13.2). Cabazitaxel
was administered intravenously every 3 weeks at a dose of 25 mg/m2
, with the
comparator group receiving intravenous mitoxantrone at a dose of 12 mg/m2
every 3 weeks. Patients in both groups received oral prednisone (6 in Figure
13.2) 10 mg/day.
Patients who received cabazitaxel had a 30% reduction in the risk of death compared
with those who received mitoxantrone (hazard ratio 0.70 (95% confidence
interval (CI): 0.59; 0.83); p < 0.0001) [24]. Median overall survival was 15.1 months
(95% CI: 14.1; 16.3) for cabazitaxel versus 12.7 months (95% CI: 11.6; 13.7) for
mitoxantrone. Cabazitaxel was the first drug to demonstrate a significant improvement
in survival in patients with mCRPC who had progressed following previous
docetaxel-based therapy.
Other clinical end points measured in the TROPIC trial (progression-free survival,
tumor response rate, prostate-specific antigen response rate, and time to
tumor progression) were also improved following treatment with cabazitaxel compared
with mitoxantrone (Table 13.4).
With regard to side effects, more patients in the cabazitaxel group experienced
neutropenia of at least grade 3 severity (82% for cabazitaxel; 58% for
mitoxantrone). Febrile neutropenia (FN) was reported in 7.5% of the cabazitaxel
patients versus 1.3% of the mitoxantrone patients. The incidence of grade
!3 diarrhea was also higher in the cabazitaxel group (6.2% versus 0.3%). Proactive
management of these potential side effects of cabazitaxel treatment is
highly recommended. Neutropenia may be effectively managed with the
334j13 Novel Taxanes: Cabazitaxel Case Study
prophylactic use of granulocyte colony-stimulating factor (G-CSF), in particular
in patients with high-risk clinical features (including those aged >65 years and
those with advanced disease, comorbidities, prior episodes of FN, extensive
prior radiotherapy, etc.). Primary G-CSF prophylaxis was not permitted in the
TROPIC trial.
Based on the positive results from the TROPIC trial, cabazitaxel has been
approved by the US Food and Drug Administration, the European Medicines
Agency, and other national health authorities worldwide for the treatment of
humans with mHRPC previously treated with a docetaxel-containing treatment
regimen.
13.3.3
Other Ongoing Trials
Among other strategies available to alleviate the risk of neutropenic complications
with cabazitaxel treatment is the implementation of a dose reduction. This strategy
is being formally investigated in an ongoing phase III trial, PROSELICA,2)
which is
comparing the standard 25 mg/m2
dose with a reduced dose of 20 mg/m2
. Another
ongoing phase III trial is evaluating these two doses of cabazitaxel versus docetaxel
Table 13.4 Summary of efficacy results in the TROPIC triala)
[42].
End point Mitoxantrone
(n ¼ 377)
Cabazitaxel
(n ¼ 378)
HR
(95% CI)
p-Value
Median OS (months) 12.7 15.1 0.70 (0.59–0.83) <0.0001
Median PFS (months) 1.4 2.8 0.74 (0.64–0.86) <0.0001
Tumor response rate (%)b)
4.4 14.4 — 0.0005
PSA response rate (%)c)
17.8 39.2 — 0.0002
Pain response rate (%)d)
7.7 9.2 — 0.63
Median TTP (months) 5.4 8.8 0.61 (0.49–0.76) <0.0001
Median time to PSA
progression (months)
3.1 6.4 0.75 (0.63–0.90) 0.001
Median time to pain
progression (months)
Not reached 11.1 0.91 (0.69–1.19) 0.52
CI, confidence interval; HR, hazard ratio; OS, overall survival; PFS, progression-free survival; PSA,
prostate-specific antigen; TTP, time to tumor progression. Reproduced from Ref. [42] with permission
of Future Medicine Ltd.
a) From final analysis, cutoff date September 25, 2009 [18].
b) Number of evaluable patients: 204 (mitoxantrone); 201 (cabazitaxel).
c) Number of evaluable patients: 325 (mitoxantrone); 329 (cabazitaxel).
d) Number of evaluable patients: 168 (mitoxantrone); 174 (cabazitaxel).
2) ClinicalTrials.gov. Cabazitaxel at 20 mg/m2
compared to 25 mg/m2
with prednisone for the
treatment of metastatic castration resistant prostate cancer (PROSELICA). NCT01308580.
Available at http://clinicaltrials.gov/ct2/results?term¼NCT01308580.
13.3 Cabazitaxel Preclinical and Clinical Development j335
in patients with mCRPC who have not previously received chemotherapy.3)
Other
early-phase clinical trials are investigating cabazitaxel in combination with various
chemotherapy agents and hormonal therapies.
13.4
Summary
Cabazitaxel is a novel semisynthetic taxane that promotes tubulin assembly and
stabilizes microtubules as efficiently as docetaxel. Cabazitaxel is structurally distinct
from docetaxel due to the presence of two methoxy groups at positions C-7
and C-10. Two chemical syntheses for cabazitaxel have been developed that involve
modification of positions C-7 and C-10 in the naturally occurring 10-DAB precursor
molecule and esterification of a POMA moiety at position C-13.
The selection of cabazitaxel for clinical development was based on its superior
antiproliferative activity compared with docetaxel when tested against chemotherapy-resistant
tumor cell lines as well as its broad spectrum of in vivo antitumor
activity. The activity of cabazitaxel has been demonstrated in docetaxel-sensitive
tumors including prostate, colon, lung, pancreas, head and neck, gastric, skin, and
kidney. Cabazitaxel also has activity in tumor models in which docetaxel was either
poorly active or inactive, including B16/TXTmelanoma, a tumor model with in vivo
acquired resistance to docetaxel. Further, unlike docetaxel, cabazitaxel is able to
cross the blood–brain barrier, and displays better antitumor activity than docetaxel
against intracranial glioblastomas. The PK profile of cabazitaxel is similar to that of
docetaxel; both molecules show a triphasic elimination profile, although the terminal
phase of cabazitaxel is longer than that of docetaxel. The plasma clearance of
cabazitaxel is also higher than that of docetaxel.
The clinical development of cabazitaxel is being conducted worldwide and is
focused on solid tumors. Based on the results of studies that have been completed,
cabazitaxel has a manageable safety profile consistent with that seen with other taxanes.
The clinical activity of cabazitaxel was observed in phase I studies. Proof of
concept was first achieved in a phase II study with responses obtained in patients
with metastatic breast tumors resistant to previous docetaxel therapy. Clinical activity
was then confirmed in the pivotal phase III study in mCRPC patients previously
treated with docetaxel, where cabazitaxel in combination with prednisone demonstrated
a statistically significant overall survival benefit when compared with mitoxantrone
plus prednisone. Cabazitaxel in combination with prednisone is currently
approved in the United States, the European Union, Canada, Switzerland, and several
other countries in Latin America, Asia, and the Middle East for the treatment
of patients with mHRPC previously treated with a docetaxel-containing treatment
regimen.
3) ClinicalTrials.gov. Cabazitaxel versus docetaxel both with prednisone in patients with
metastatic castration resistant prostate cancer (FIRSTANA). NCT01308567. Available at
http://clinicaltrials.gov/ct2/results?term¼01308567.
336j13 Novel Taxanes: Cabazitaxel Case Study
Acknowledgments
The authors gratefully acknowledge FranScois Lavelle, Alain CommerScon, MarieChristine
Bissery, Cecile Combeau, Jean-FranScois Riou, and Jean-Dominique Bourzat
for their contribution to the discovery of cabazitaxel, and Michele Besenval,
Sunil Gupta, Sylvie Assadourian, Emmanuelle Magherini, Gerard Sanderink, Eric
Beys, Veronique Benning, Yves Archimbaud, Serge Sable, Laurence Ridoux, Olivier
Pasquier, and Marie-Helene Pascual for their contribution to the preclinical and
clinical development of cabazitaxel. Editorial assistance for preparing this chapter
was supported by Sanofi.
References
1 Fischer, J. and Ganellin, C.R. (eds) (2006)
Analogue-Based Drug Discovery, WileyVCH
Verlag GmbH, Weinheim.
2 Fischer, J. and Ganellin, C.R. (eds) (2010)
Analogue-Based Drug Discovery II, WileyVCH
Verlag GmbH, Weinheim.
3 Erhardt, P.W. and El-Dakdouki, M. (2010)
Paclitaxel and epothilone analogues,
anticancer drugs, in Analogue-Based Drug
Discovery II (eds J. Fischer and C.R.
Ganellin), Wiley-VCH Verlag GmbH,
Weinheim, pp. 243–268.
4 Abal, M., Andreu, J.M., and Barasoain, I.
(2003) Taxanes: microtubule and
centrosome targets, and cell cycle
dependent mechanisms of action. Curr.
Cancer Drug Targets, 3, 193–203.
5 Jordan, M.A. and Wilson, L. (2004)
Microtubules as a target for anticancer
drugs. Nat. Rev. Cancer, 4, 253–265.
6 Schiff, P.B., Fant, J., and Horwitz, S.B.
(1979) Promotion of microtubule
assembly in vitro by taxol. Nature, 277,
665–667.
7 Perez, E.A. (2009) Microtubule inhibitors:
differentiating tubulin-inhibiting agents
based on mechanisms of action, clinical
activity, and resistance. Mol. Cancer Ther.,
8, 2086–2095.
8 Wani, M., Taylor, H., Wall, M., Coggon, P.,
and McPhail, A. (1971) Plant antitumor
agents. VI. The isolation and structure of
taxol, a novel antileukemic and antitumor
agent from Taxus brevifolia. J. Am. Chem.
Soc., 93, 2325–2327.
9 Holton, R.A., Somoza, C., Kim, H.-B.,
Liang, F., Biediger, R.J., Boatman, P.D.,
Shindo, M., Smith, C.C., and Kim, S.
(1994) First total synthesis of taxol. 1.
Functionalization of the B ring. J. Am.
Chem. Soc., 116, 1597–1598.
10 Holton, R.A., Somoza, C., Kim, H.-B.,
Liang, F., Biediger, R.J., Boatman, P.D.,
Shindo, M., Smith, C.C., and Kim, S.
(1994) First total synthesis of taxol. 2.
Completion of the C and D rings. J. Am.
Chem. Soc., 116, 1599–1600.
11 Nicolaou, K.C., Yang, Z., Liu, J.J., Ueno,
H., Nantermet, P.G., Guy, R.K.,
Claiborne, C.F., Renaud, J.,
Couladouros, E.A., Paulvannan, K. et al.
(1994) Total synthesis of taxol. Nature,
367, 630–634.
12 Denis, J.N., Greene, A.E., Guenard, D.,
Gueritte-Voegelein, F., Mangatal, L., and
Potier, P. (1988) Highly efficient, practical
approach to natural taxol. J. Am. Chem.
Soc., 110, 5917.
13 Holton, R.A., Biediger, R.J., and Boatman,
P.D. (1995) Taxol: Science and Applications
(ed. M. Suffness), CRC Press, Boca Raton,
FL, pp. 97–121.
14 Venkat, K. (1998) Paclitaxel production
through plant cell culture: an exciting
approach to harnessing biodiversity. Pure
Appl. Chem., 70, 2127.
15 Clarke, S.J. and Rivory, L.P. (1999) Clinical
pharmacokinetics of docetaxel. Clin.
Pharmacokinet., 36, 99–114.
16 Chien, A.J. and Moasser, M.M. (2008)
Cellular mechanisms of resistance to
anthracyclines and taxanes in cancer:
intrinsic and acquired. Semin. Oncol., 35
(2 Suppl. 2), S1–S14.
References j337
17 Fojo, T. and Menefee, M. (2007)
Mechanisms of multidrug resistance: the
potential role of microtubule stabilizing
agents. Ann. Oncol., 18 (Suppl. 5), v3–v8.
18 Baas, P., Szczesna, A., Albert, I.,
Milanowski, J., Juhasz, E., Sztancsik, Z.
et al. (2008) Phase I/II study of a 3 weekly
oral taxane (DJ-927) in patients with
recurrent, advanced non-small cell lung
cancer. J. Thorac. Oncol., 3, 745–750.
19 Roche, M., Kyriakou, H., and Seiden, M.
(2006) Drug evaluation: tesetaxel – an oral
semisynthetic taxane derivative. Curr.
Opin. Invest. Drugs, 7, 1092–1099.
20 Saif, M.W., Sarantopoulos, J., Patnaik, A.,
Tolcher, A.W., Takimoto, C., and Beeram,
M. (2011) Tesetaxel, a new oral taxane, in
combination with capecitabine: a phase I,
dose-escalation study in patients with
advanced solid tumors. Cancer Chemother.
Pharmacol., 68 (6), 1565–1573.
21 Mita, A.C., Denis, L.J., Rowinsky, E.K.,
Debono, J.S., Goetz, A.D., Ochoa, L.,
Forouzesh, B., Beeram, M., Patnaik, A.,
Molpus, K., Semiond, D., Besenval, M.,
and Tolcher, A.W. (2009) Phase I and
pharmacokinetic study of XRP6258 (RPR
116258A), a novel taxane, administered as
a 1-hour infusion every 3 weeks in
patients with advanced solid tumors. Clin.
Cancer Res., 15, 723–730.
22 Pivot, X., Koralewski, P., Hidalgo, J.L.,
Chan, A., GonScalves, A., Schwartsmann,
G., Assadourian, S., and Lotz, J.P. (2008)
A multicenter phase II study of XRP6258
administered as a 1-h i.v. infusion every 3
weeks in taxane-resistant metastatic breast
cancer patients. Ann. Oncol., 19, 1547–
1552.
23 Villanueva, C., Awada, A., Campone, M.,
Machiels, J.P., Besse, T., Magherini, E.,
Dubin, F., Semiond, D., and Pivot, X.
(2011) A multicentre dose-escalating study
of cabazitaxel (XRP6258) in combination
with capecitabine in patients with
metastatic breast cancer progressing after
anthracycline and taxane treatment: a
phase I/II study. Eur. J. Cancer, 47, 1037–
1045.
24 de Bono, J.S., Oudard, S., Ozguroglu, M.,
Hansen, S., Machiels, J.-P., Kocak, I.,
Gravis, G., Bodrogi, I., Mackenzie, M.J.,
Shen, L., Roessner, M., Gupta, S., and
Sartor, A.O. (the TROPIC Investigators)
(2010) Prednisone plus cabazitaxel or
mitoxantrone for metastatic castrationresistant
prostate cancer progressing after
docetaxel treatment: a randomised openlabel
trial. Lancet, 3761147–1154.
25 Bourzat, J.D., Bouchard, H., CommerScon,
A., Bissery, M.C., Combeau, C., Vrignaud,
P., Riou, J.F., and Lavelle, F. (1994)
Preparation and biological evaluation of
new docetaxel analogs modified at the 30
position of the side chain. Proceedings of
16th International Cancer Congress, vol.
4, pp. 2751–2755.
26 Bourzat, J.D., Lavelle, F., and
CommerScon, A. (1995) Synthesis and
biological activity of para-substituted 30
phenyl
docetaxel analogs. Bioorg. Med.
Chem. Lett., 5, 809–814.
27 Georg, G.I., Harriman, G.C.B., Hepperle,
M., and Himes, R.H. (1994)
Heteroaromatic taxol analogs: the
chemistry and biological activities of 30
furyl
and 30
-pyridyl substituted taxanes.
Bioorg. Med. Chem. Lett., 4, 1381–1384.
28 Pulicani, J.-P., Bezard, D., Bourzat, J.D.,
Bouchard, H., Zucco, M., Deprez,
D., and CommerScon, A. (1994) Direct
access to 2-debenzoyl taxoids by
electrochemistry, synthesis of 2modified
docetaxel analogs. Tetrahedron
Lett., 35, 9717–9720.
29 Bouchard, H., Pulicani, J.-P., Vuilhorgne,
M., Bourzat, J.D., and CommerScon, A.
(1994) Improved access to 19-nor-7b,8bmethylene-taxoids
and formation of a 7membered
C-ring analog of docetaxel by
electrochemistry. Tetrahedron Lett., 35,
9713–9716.
30 Bissery, M.C., Bouchard, H., Riou, J.F.,
Vrignaud, P., Combeau, C., Bourzat, J.D.
et al. (2000) Preclinical evaluation of
TXD258, a new taxoid. Proc. Am. Assoc.
Cancer Res., 41, abstract no. 1364.
31 Aller, A.W., Kraus, L.A., and Bissery, M.C.
(2000) In vitro activity of TXD258 in
chemotherapeutic resistant tumor cell
lines. Proc. Am. Assoc. Cancer Res., 41,
abstract no. 1923.
32 Vrignaud, P., Lejeune, P., Chaplin, D.,
Lavelle, F., and Bissery, M.C. (2000) In
vivo efficacy of TXD258, a new taxoid,
against human tumor xenografts. Proc.
338j13 Novel Taxanes: Cabazitaxel Case Study
Am. Assoc. Cancer Res., 41, abstract no.
1365.
33 Corbett, T.H., Griswold, D.P., Jr., Roberts,
B.J., Peckham, J.C., and Schabel, F.M., Jr.
(1978) Biology and therapeutic response
of a mouse mammary adenocarcinoma
(16/C) and its potential as a model for
surgical adjuvant chemotherapy. Cancer
Treat. Rep., 62, 1471–1499.
34 Archimbaud, Y., Gires, P., Pellerin, R.,
Ridoux, L., Vrignaud, P., Bissery, M.C.,
Semiond, D., and Marietta, M. (2000)
Pharmacokinetics and tissue distribution
of a new taxoid antitumor agent, TXD258,
in tumor-bearing mice. Proc. Am. Assoc.
Cancer Res., 41, abstract no.1375.
35 Mehta, R.R., Bratescu, L., Graves, J.M.,
Hart, G.D., Shilkaitis, A., Green, A. et al.
(1992) Human breast carcinoma cell lines:
ultrastructural, genotypic, and
immunocytochemical characterization.
Anticancer Res., 12, 683–692.
36 Cisternino, S., Bourasset, F., Archimbaud,
Y., Semiond, D., Sanderink, G., and
Scherrmann, J.M. (2003) Nonlinear
accumulation in the brain of the new
taxoid TXD258 following saturation of Pglycoprotein
at the blood–brain barrier in
mice and rats. Br. J. Pharmacol., 138,
1367–1375.
37 Dykes, D.J., Sarsat, J.P., and Bissery, M.C.
(2000) Efficacy evaluation of TXD258, a
taxoid compound, against orthotopic and
subcutaneous glioblastomas. Proc. Am.
Assoc. Cancer Res., 41, abstract no. 1916.
38 Lorthoraly, A., Pierga, J.Y., Delva, R.,
Girre, V., Gamelin, E., Terpereau, A.,
Crespel, G., Pouillard, P., Fontaine, H.,
Semiond, D., Perard, D., Besenval, M.,
and Dieras, V. (2000) Phase I and
pharmacokinetics study of RPR116258A
given as 1-hour infusion in patients with
advanced solid tumors. Clin. Cancer Res., 6
(Suppl.), abstract no. 569.
39 Dieras, V, Lortholary, A, Laurence, V,
Delva, R, Girre, V, Livartowski, A,
Assadourian, S, Semiond, D, and Pierga,
J.Y. (2011) Cabazitaxel as a 1-hour infusion
every 3 weeks in patients with advanced
solid tumours: a phase 1 and
pharmacokinetic study. Accepted for
publication to the European Journal of
Cancer.
40 Fumoleau, P, Trigo, J, Campone, M,
Baselga, J, Sistac, F, Giminez, P, Manos,
L, Fontaine, H, Semiond, D, Sanderink,
G, Perard, D, and Besenval, M. (2001)
Phase I and pharmacokinetic study of
RPR 116258A given as a weekly 1-hour
infusion at day 1, day 8, day 15, day 22
every 5 weeks in patients with advanced
solid tumors. Clin. Cancer Res., 7 (Suppl.),
Abstract no. 282.
41 Ridoux, L, Semiond, D, Vincent, C,
Fontaine, H, Mauriac, C, Sanderink, G,
Oprea, C, and Clive, S. (2012.) A phase I
open-label study investigating the
disposition of [14
C] cabazitaxel in patients
with advanced solid tumors. Annual
Meeting of the American Association of
Cancer Research, abstract no. 749.
42 Oudard, S. (2011) TROPIC: phase III trial
of cabazitaxel for the treatment of
metastatic castration-resistant prostate
cancer. Future Oncol., 7, 497–506.
References j339
Herve Bouchard completed his Ph.D. on natural product synthesis
under the supervision of Professor J.-Y. Lallemand at
the University Pierre et Marie Curie, Paris, in 1990 before
conducting postdoctoral research on the total synthesis of
reserpine in the laboratory of Professor S. Hanessian. In
1991, Dr. Bouchard joined the Medicinal Chemistry Department
of Rh^one-Poulenc Rorer (Vitry, France). He initiated
total and hemisynthesis studies of natural products in oncology,
antibiotic and antidiabetic therapeutic areas, and contributed
to the discovery of several new chemical entities. These efforts led to the
development of two new taxanes, namely, larotaxel (RPR109881) and cabazitaxel
(RPR116258), which resulted in the recent approval by FDA and EMA of cabazitaxel
in metastatic castration-resistant prostate cancer. After driving research activities
in combinatorial chemistry, CB1 antagonism, and kinase inhibition pathways
in oncology, Dr. Bouchard’s current interests are focussed on immunoconjugate
biotherapeutics.
Marie-Laure Risse obtained her medical degree from
Paris VI University (Paris, France) before completing
postgraduate courses in clinical oncology (Paris VI
University), palliative care and chemotherapy (both
Gustave Roussy Institute, Villejuif, France), and HIV
infection (Paris VI University). She was Resident at the
Gustave Roussy Institute for 1 year (1996–1997) before
taking up a position as Oncology Clinical Project
Leader in the Department of Clinical Development and Medical Communication at
Rh^one-Poulenc Rorer French affiliate (later Aventis Pharma). Dr. Risse became
Clinical Study Director in 2002 (Aventis) and then Clinical Research Director in
2008 (Sanofi-Aventis) in Research and Development. Her current position is Cabazitaxel
Medical Lead in the Global Oncology Division at Sanofi.
Dorothee Semiond received two master’s degrees, one in
Analytical Chemistry (Claude Bernard University, Lyon,
France) and the other in Clinical Pharmacokinetics (Institute
of Pharmaceutical and Biological Sciences of Lyon (ISPBL),
Lyon, France), and completed her Ph.D. at the University of
Pharmacy in Paris under the supervision of Dr. J.M. Schermann
on the subject of “Add on of pharmacokinetic modeling
in the development of new taxoids.” She also received her
Pharm.D. degree from the Institute of Pharmaceutical and
Biological Sciences of Lyon (ISPBL). From 1996 to 2006, Dr. Semiond served as a
clinical pharmacokineticist and drug disposition project team representative at
Rh^one-Poulenc Rorer (later Aventis Pharma, then Sanofi-Aventis), and from 2006
340j13 Novel Taxanes: Cabazitaxel Case Study
to 2010 she managed the Pharmacokinetic Unit at Sanofi-Aventis. Since 1997, Dr.
Semiond has served as the clinical pharmacokineticist and project team representative
for drug disposition in the development of two novel taxanes larotaxel
(RPR109881) and cabazitaxel (RPR116258). Dr. Semiond is currently the dedicated
project expert for drug disposition in the Department of Disposition, Safety and
Animal Research at Sanofi, where she focuses on oncology projects. She has authored
8 papers and 20 congress abstracts.
Patricia Vrignaud received her Ph.D. in Nutrition and Metabolism
from Bordeaux I University. Her thesis “Growth rates
of chemically induced sarcomas in mice: influence of age,
sex and number of primotransplants” was written under the
directorship of Dr. M. Denechaud. Following her Ph.D., Dr.
Vrignaud worked as a Research Scientist at the Fondation
Bergonie (Cancer Hospital), Bordeaux, where she studied the
pharmacokinetics and metabolism of anthracyclines and the
cellular pharmacology of anthracyclines and multidrug resistance.
From 1991 to 2002, she served as Head of the Laboratory in Experimental
Therapeutics in Oncology at Rh^one-Poulenc Rorer. She then went on to become
the Head of Section evaluating anticancer agents in experimental models at Aventis
Pharma (later Sanofi-Aventis), where she participated in the registration processes
for docetaxel and irinotecan and served as the preclinical lead for cabazitaxel. Dr.
Vrignaud is currently the Head of Late Development – discovery liaison in Translational
& Experimental Medicine at Sanofi. She has authored 46 papers, 20 patents,
and 155 congress abstracts.
References j341
14
Discovery of Boceprevir and Narlaprevir: A Case Study for Role
of Structure-Based Drug Design
Srikanth Venkatraman, Andrew Prongay, and George F. Njoroge
List of Abbreviations
Boc tert-butyl carbamate
FDA Food and Drug Administration
HCV hepatitis C virus
HNE human neutrophil elastase
NS3 nonstructural protein 3
SAR structure–activity relationship
SVR sustained virologic response
The hepatitis C epidemic rightly termed as a silent killer has attracted major attention
in recent years. The identification of hepatitis C virus (HCV) by Choo et al. as
the primary causative agent for non-A, non-B hepatitis resulted in explosive growth
in the number of publication describing the viral life cycle and new targets for drug
intervention [1, 2]. HCV is a positive strand RNA virus of the Flaviviridae family,
whose members include dengue, west Nile, and Japanese encephalitis. Unscreened
blood transfusions in the 1960s and 1970s resulted in many new infections [3]. A
vast majority of these infections turn chronic resulting in liver cirrhosis and hepatocellular
carcinoma [4]. The slow progression of the disease and lack of symptoms
has made early detection difficult. HCV infections are the primary causes for latestage
liver transplantations in the United States. Recent advances in detection techniques
allow early diagnosis of patients by screening for HCV antibodies. Prior to
the development of Victrelis (boceprevir) and Incivek (telaprevir), the standard of
care for treatment was a combination of peg-interferon and ribavirin [5]. This combination
though effective in genotype 2 is suboptimal for treatment of genotype 1.
Only approximately 40% of genotype 1-treated patients achieve sustained virologic
response (SVR) defined as undetectable viral RNA after 6 months of completion of
therapy [6]. Recent advances in virology have identified many proteins vital for
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
j343
HCV viral replication [7, 8]. Development of inhibitors targeting these enzymes
could potentially develop new direct-acting antiviral.
HCV is a bloodborne pathogen, which primarily infects hepatocytes. Most of
these infections turn chronic. After infecting hepatocytes, the virus codes a single
polypeptide of $3000 amino acids that contains all the core and functional proteins.
This polypeptide containing proteins C-E1-E2-P7-NS2-NS3-NS4A-NS4BNS5A-NS5B
undergoes a cis-cleavage at the NS3-NS4A junction followed by transcleavage
at NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions catalyzed by a serine
enzyme NS3 protease. This central role of NS3 protease in the life cycle of the
enzyme makes it an important target for drug intervention.
HCV NS3 protease is a serine enzyme belonging to the chymotrypsin superfamily
[9–12]. Its functionality is formed by the N-terminal third of the NS3
bifunctional protein. The N-terminal 181 residues fold into two b-barrel subdomains.
The active site of the enzyme is located on the surface and is a shallow,
featureless cleft that is solvent exposed. The catalytic triad (His57, Asp81, and
Ser139) is located between the two b-barrel subdomains. There is a zinc ion at a
distance of $20 A

from the catalytic site that is coordinated by Cys97, Cys99,
Cys145, and a water molecule that is hydrogen bonded to His149. It plays a critical
role in maintaining structural stability and integrity of the protein. The substrate of
the protease is long and recognizes the enzyme with a series of hydrogen bonds.
The P1 and P10
residues of the natural substrate of NS3 protease are highly conserved
[13–15]. Cysteine is retained at the P1 position in all three trans-cleavage
sites (NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B) and is replaced by threonine in
cis-cleavage event (NS3/NS4A).
NS4A, a 54 amino acid cofactor, enhances catalytic activity and the stability of the
protease. This protein binds to NS3 with residues 21–32 forming a b-strand that is
situated between the first (A0) and second (A1) strands of the N-terminal b-barrel
subdomain and with the main chain amide and carbonyl groups of these three
chains sharing hydrogen bonds. The binding of the NS4A cofactor organizes the
catalytic triad in a preferred conformation by orienting His57 to facilitate deprotonation
of Ser139 and hydrogen bond to Asp81. Thus, in the absence of NS4A
cofactor the enzymatic activity of the protease is greatly diminished making its
binding a prerequisite for efficient catalytic turnover.
The quintessential role of HCV NS3 protease in the life cycle of the virus makes
it an attractive target for drug intervention. We initiated our search for inhibitors by
screening compound libraries to identify potential leads. It was very disheartening
to find that even with new advances in high-throughput screening and evaluation of
many compound libraries, we could not identify any viable lead. We therefore
decided to pursue a structure-based drug design. This required the development of
a continuous spectrophotometric assay [16] to facilitate assessing the kinetic parameters
of slow tight binding inhibitors [17]. An undecapeptide 1 with a ketoamide
electrophilic trap was identified that inhibited the enzyme with a KÃ
i ¼ 1:9 nM
(Figure 14.1). With this as our starting point, we explored various modifications to
engineer desirable pharmaceutical properties. Our initial goal for the modification
of 1 was to reduce its molecular weight and identify the minimal fragment that
344j14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role of Structure-Based Drug Design
retained potent binding. It was also decided to replace various proteinogenic amino
acids with modified synthetic amino acids to enhance metabolic stability and
improve ligand efficiency.
Synthesized compounds were evaluated in a continuous spectrophotometric
assay for the inhibition of hydrolysis of chromogenic ester from the acetylDTEDVVP(norvaline)-O-phenylazophenol
substrate. Progression curves were fit
into a two-step slow tight binding model and a composite inhibition constant KÃ
i
was derived, which allowing rank ordering of compounds. Compounds that displayed
acceptable binding were evaluated in a replicon-based cell assay. Key compounds
that had excellent enzyme inhibition and acceptable cellular activity were
dosed in rats in a rapid rat assay. After a single oral dose, blood was periodically
withdrawn and concentration of the parent compound was analyzed in the plasma
generating AUC for exposure and other pharmacokinetic parameters.
Some interesting compounds were soaked with HCV NS3 protease and bound
structure was solved by X-ray diffraction. The crystals of the HCV NS3 protease
domain (residues 1–181) complexed with a peptide containing the NS4A sequences
spanning residues 21–39 were crystallized by vapor diffusion using sodium chloride
as the precipitant. The structures of the inhibitor compounds were obtained
by soaking the crystals in a solution of the inhibitor at 10–100 mM concentration
overnight at 12 
C. The crystals were transferred to a cryoprotectant solution containing
sucrose prior to super cooling in liquid nitrogen. The diffraction data were
collected as previously described [18].
Thus, replacement of P10
–P50
segment of inhibitor 1 with glycine allyl
ester resulted in compound 2 with a KÃ
i ¼ 57 nM (Figure 14.1). Similarly,
N
H
N
H
N
N
H
H
N
N
H
Me
O
O
H
N
N
H
O
O
O
SMe
O
OH
O
OH
H
N
NH2
O
OH
O
MeMe
O
Me Me
H
N
N
H
Me
O
COOH
O
COOH
O
MW = 1265
Ki
*
= 1.9 nM
HNE/HCV = 6.8
1
P2
P1
P3
P4
P5
P6
P'1
P'2
P'3
P'4
P'5
N
H
N
H
N
R
O
O
H
N
N
H
O
O
O
H
N
N
H
Me
O
COOH
O
COOH
O
O
O
CH2
CH2NH2
O
2
3 4 5
Ki
*
= 57 nM Ki
*
= 270 nM Ki
*
= 43 nM
Figure 14.1 X-ray structure of undecapeptide 1 bound to HCV NS3 protease.
14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role of Structure-Based Drug Design j345
introduction of glycine primary amide and allyl amide in the place of P10
–P50
was also well tolerated resulting in analogues 4 (KÃ
i ¼ 270 nM) and 5
(KÃ
i ¼ 43 nM). Even though these modifications resulted in analogues with
diminished potency compared to 1, it was reassuring to note that the compounds
were still potent allowing us to make a decision to limit our future
analogues at P20
, thus reducing molecular weights.
The X-ray structure of 3 bound to NS3 protease revealed the propyl chain of P1
norvaline that was buried into the S1 pocket. However, the side chain did not completely
fill the S1 pocket and the van der Waals contacts were not optimized. As
seen in Figure 14.1, the pucker of the P2 proline ring was oriented toward the solvent
and away from the protein surface resulting in a suboptimal interaction. It was
also clear that the remaining binding sites were shallow and other residues made
only partial contact to the protease. We decided to reduce polarity of analogues of
type 2 by capping our inhibitors at the P3 position removing acidic amino acids at
P5 and P6. We hypothesized that this transformation would facilitate absorption
and permeability into cells of resulting analogues (Figure 14.2).
Boc-capped P3 truncated inhibitor 6 had modest activity in inhibition of HCV
NS3 protease. Even though this activity was much reduced compared to inhibitors
spanning from P6 to P20
, it was clear that one could retain potency with truncated
inhibitors. The X-ray structure of 6 bound to the protease revealed various sites for
modification to improve activity. From the structure, it was once again clear that the
P1 norvaline occupied the S1 pocket effectively, albeit incompletely. The P20
allyl
ester made lipophilic interaction with the butyl side chain of Lys136. This stabilized
N
H
N
H
N
CH3
O
O
H
NO
O
O
O
6
Ala-156
Cys-159
Arg-155
Ser-139
Lys-136
S3
S1
S4
S2
O
O
Figure 14.2 X-ray structure of P3–P10
truncated analogue 6 in the active site of HCV NS3.
346j14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role of Structure-Based Drug Design
the side chain of Lys136 in an extended conformation as the P20
allyl and P1 norvaline
side chains packed against it. The binding of the allyl group projected toward a
pocket that could potentially provide additional binding energy. As with compound
3, the pucker of the P2 proline projects the Cc atom toward the solvent,
leaving the S2 pocket incompletely filled. The His57 and Arg155 side chains
formed a U-shaped wall on either side of the proline ring. The P3 cyclohexylglycine
made surface contact as its side chain laid above the shallow S3 pocket with its Cb,
Cd1, and Cd2 atoms projecting closest to the pocket. The closest contact is between
Cd2 and Cys159Sc at $3.3A

, while Cd1 and Cd2 are each $3.9 A

from Ile132Cb.
These contacts are the result of the side chains of Cys159 and Ile132 adopting alternate
rotamer positions to optimize the van der Waals interactions. The tert-butyl
group of the Boc cap occupied the S4 pocket, which had undergone a conformational
change with the rotation of R123 and Asp168 side chains. One tert-butyl
methyl group projects toward the bottom of the pocket resulting in a fairly snug
packing.
To improve potency and depeptidize inhibitor 6, various modifications were evaluated
based on the structural information. It was clear from the X-ray structure that
the P1 norvaline and P3 cyclohexylglycine were in close proximity. We therefore
envisioned syntheses of P1–P3 macrocylic inhibitors that linked these groups by a
suitable linker. Similarly, the proximity of P2 proline to the P4 Boc group resulted
in novel ideas of linking these groups to synthesize many P2–P4 macrocylic inhibitors.
It was also clear from the X-ray structure that the P2 proline made inefficient
contact at the S2 site. We therefore decided to evaluate many modifications at proline
from the 3, 4, and 5 positions to maximize interactions that could be beneficial
to binding. Therefore, a large number of modified proline derivatives were synthesized
and incorporated into 6. We also decided to modify the P20
allyl ester in an
effort to gain improvement in binding. The results of these modifications are discussed
below (Figure 14.3).
The incorporation of 4-tert-butoxy proline at P2 and phenylglycine at P20
resulted
in marked improvement in binding ( 7, KÃ
i ¼ 19 nM) and HNE/HCV selectivity
(HNE/HCV ¼ 161) [19]. Inhibitor 7 also showed cellular activity in a replicon-based
assay [20] (EC90 ¼ 2000 nM). Introduction of bicyclic P2 (3aR,4S,6aR)-2,2-dimethylhexahydro-2H-furo[2,3-c]pyrrole-4-carboxylic
acid resulted in analogue 8 with
KÃ
i ¼ 5 nM and EC90 ¼ 400 nM. Similarly, replacement of P2 proline with
(1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid [21] resulted in
analogue 9 (KÃ
i ¼ 10 nM, HNE/HCV ¼ 964, EC90 ¼ 400 nM). It was clear that modification
of P2 proline had a positive impact on binding and cellular activity. The
introduction of modified proline analogues reduced molecular weight of resultant
inhibitors compared to the undecapeptide 1, retained enzyme inhibition, imparted
cellular activity, and improved metabolic stability. Rat pharmacokinetic studies of 9
showed modest oral exposure with an AUC ¼ 1.1 mM h when dosed at 10 mg/kg
and a low bioavailability of 6%. The X-ray structure of 9 bound to the enzyme
revealed that the P10
–P20
glycine–phenylglycine amide made a “C-clamp” structure
around Lys136 restricting its mobility. The aryl group of phenylglycine made van
der Waals contact with the protruding amino butyl chain contributing to enhanced
14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role of Structure-Based Drug Design j347
binding. As with compound 6, this position of the Lys136 side chain provided an
additional surface in the S1 pocket for the P1 side chain to pack against. The additional
methylene group of the P1 norleucine side chain extends deeper into the S1
pocket nearly filling it completely. In contrast to the cyclohexyl P3 group of compound
6, the P3 tert-butyl projects down into the S3 dimple, making close contacts
and more completely filling the space. The methyl group Cc3 packs at distances of
3.7, 4.0, and 4.3 A

from Cys159Sc, Ala157Cb, and Ile132Cd1, respectively, while
Cys159Sc is 4.1 A

from the methyl group Cc2. Unlike the pucker of the proline
ring, the dimethylcyclopropyl ring of the proline analogue projects toward the
Arg155 side chain, fitting snugly against the amidino group of the arginine side
chain. With the Ng2 of Arg155 at 3.6 A

from the Cf1 of P2 and Ng1 of Arg155 at
3.4 A

from Cf2, and the Cb at distances of 3.3 and 3.4 A

from His57Ne2 and Ce1,
the S2 pocket is more snugly filled than with leucine or proline at P2. As a result of
this packing, the side chain of His57 is swung in toward the protein surface (“In”
position). The amidino group of Arg155 was also rotated to make room for the gemdimethyls
and remove steric clash between these groups.
The incorporation of modified proline resulted in analogues with desirable binding
and cellular activity. However, these analogues spanning from P3 to P20
were
poorly absorbed and displayed low bioavailability. We therefore investigated syntheses
of macrocyclic compounds (Figure 14.4).
N
H
N
H
N
N
H
Me
O
O
H
NO
O
O
O
O
Me
Me
NMe2
O
7
Ki
*
= 19 nM
HNE/HCV = 161
EC90 = 2000 nM
O
N
H
N
H
N
N
H
Me
O
O
H
NO
O
O
O
O
NMe2
O
8
Ki
*
= 5 nM
HNE/HCV = 50
EC90 = 400 nM
O
N
H
N
H
N
N
H
O
O
H
NO
O
O
O
O
Me
NMe2
O
9
Me
Me
Ki
*
= 10 nM
HCV/HNE = 964
EC90 = 400 nM
AUC (rat)0-24h = 1.1 µM h
%F = 6.4
Lys136
His57Arg155
Lys136
His57Arg155
Lys136
His57Arg155
Figure 14.3 X-ray structure of norleucine derivative 9 bound to HCV protease. Key interactions to
Arg155 and Lys136.
348j14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role of Structure-Based Drug Design
Biaryl P2–P4 macrocyclic inhibitor 10 derived from phenylalanine P2 and P4
phenyl propionic acid was one of the first macrocyclic compounds potent in inhibition
of the HCV protease assay with a KÃ
i ¼ 110 nM [22]. However, this compound
did not show any cellular activity. From previous SAR studies in the acyclic analogues,
we had established that P2 proline compounds demonstrated good cellular
potency. We therefore synthesized other macrocycles containing P2 proline modifications.
This resulted in compounds such as the acetal analogue 11 that had a
potent inhibition of KÃ
i ¼ 38 nM, a threefold improved binding compared to biaryl
analogue 10 [23]. Similarly, 4-hydroxyproline-derived macrocyclic analogue 12 displayed
excellent enzyme inhibition (KÃ
i ¼ 8 nM) and replicon-based cellular activity
(EC90 ¼ 1.5 mM) [24]. We also synthesized P1–P3 macrocylic inhibitors of type 13.
These compounds were more potent than the P2–P4 analogues and had better cellular
potency. Thus, the 16-membered P1–P3 macrocyclic analogue 13 had excellent
binding KÃ
i ¼ 6 nM with acceptable cellular activity of EC90 ¼ 600 nM [25].
Both P2–P4 and P1–P3 macrocyclic analogues were well tolerated and improved
enzyme potency in some cases compared to the linear analogues. However, these
compounds did not have markedly improved pharmacokinetics compared to
acyclic derivatives. Macrocycle 12 was only partially absorbed in rats with a bioavailability
of 3% that was no better than derivative 8. These compounds were also
poorly absorbed in monkeys and dogs without any plasma exposure.
H
N
H
N
N
H
H
N
O
O
O O O
O
NMe2
CH3
N
O
O O
Ki* = 8 nM
HCV/HNE = 46
EC90 = 1500 nM
AUC rat 0-24h =1.5 µM h
%F =3
12
N
O
BocHN
H
N
O
O
H
N
O
N
H
O
NMe2
O
Ki
*
= 6 nM
HCV/HNE = 780
EC90 = 600 nM
13
H
N
H
N
N
H
H
N
O
O
O O O
O
OH
CH3
N
O
O
Ki* = 38 nM
OO
H
H
N
N
H
O
H
N
H
N
O
CH3
O
O
N
H
OH
O
O
Ki
*
=110 nM
O
O
1101
Figure 14.4
14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role of Structure-Based Drug Design j349
It was clear from these studies that potent inhibitors of NS3 protease could be
synthesized that also showed acceptable cellular activity in the replicon-based cellular
assay. This was itself a substantial progress compared to the initial lead 1 that
lacked any of these desired properties. From a point of inhibitor design, the structure-based
hypothesis was successfully utilized for design and refinement resulting
in analogues that differed structurally from the initial lead with many drug-like
properties. However, to develop a drug candidate, incorporation of acceptable pharmacokinetics
is paramount. Thus, we decided to focus on further modifying analogues
of type 9.
It was clear that inhibitors spanning from P3 to P20
contained many amide bonds
with multiple hydrogen bond donors and acceptors. We reasoned that the poor bioavailability
of these analogues was due to high molecular weights and peptidic
nature. At this point, we decided to truncate the molecules by reducing the number
of amide bonds. Concurrently, we also explored different P1, P3, and capping residues
to improve ligand efficiency (Figure 14.5).
Replacement of dimethylcarboxamide of P2 phenylglycine with a methyl group
resulted in analogue 14 (KÃ
i ¼ 55 nM, AUC ¼ 2.66 mM h), with improved exposure
in rats compared to compounds of type 9. It was very encouraging to notice that
removal of a single amide group had profound impact on rat PK. Further truncation
at P10
with allyl amide resulted in analogue 15 that had excellent oral exposure
in rats with AUC ¼ 26 mM h when dosed at 10 mg/kg. The introduction of P10
primary
amide group resulted in compound 16 with a plasma exposure of 2.52 mM h.
N
H
N
H
N
N
H
Me
O
O
H
NO
O
O
O
O
14
Me
Ki
* = 55 nM
AUC (rat)0-6h = 2.66 µM h
N
H
N
H
N
Me
O
O
H
NO
O
O
O
Me
Me
Me
Ki
*
= 280 nM
AUC (rat)0-6h = 26 µM h
Ki
*
=100 nM
AUC (rat)0-6h = 2.52 µM h
15
16
N
H
N NH2
Me
O
O
H
NO
O
O
O
Me
Me
Me
Figure 14.5
350j14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role of Structure-Based Drug Design
It was clear that improvement in plasma exposure could be achieved by truncation,
but it was achieved at the expense of potency. With the knowledge of improving
exposure by truncating analogues at the P10
position, we focused our attention in
modification of P1 residue to improve potency.
We synthesized a series of small cyclic P1 alanine amino acids and incorporated
them into the inhibitors (Figure 14.6). Thus, replacement of norvaline in 16 with
isoleucine resulted in compound 17 (KÃ
i ¼ 400 nM), a fourfold loss in potency.
However, incorporation of cyclopropyl alanine at this site was well tolerated resulting
in 18 (KÃ
i ¼ 25 nM, EC90 ¼ 400 nM) with a fourfold improvement in binding
and good cellular activity compared to 17. Homologation of cyclopropyl alanine
to cyclobutyl alanine resulted in compound 19 with further improved potency
(KÃ
i ¼ 8 nM) and comparable cellular activity to 18. The cyclobutyl analogue 19 had
acceptable oral exposure and bioavailability warranting further investigation in this
series. Structurally, the cyclopropyl alanine side chain resembled a branched norvaline
side chain that filled the S1 pocket and introduced rotational and packing constraints.
Similarly, cyclobutyl alanine side chain resembled a branched norleucine
side chain that almost completely filled the S1 pocket and introduced further rotational
and packing constraints. These constraints resulted in Ile132 rotating to
make room for the cyclobutyl ring, while Lys136 rotates to make van der Waals
contacts between its side chain and the cyclobutyl ring. The rotation of Lys136 to
this new orientation is facilitated by the absence of the P10
and P20
moieties of previous
compounds.
We further explored the modification of P1 cyclic alanine-derived compounds of
type 18 and 19 by changing P3 amino acid and P3 cap. The SAR of these modifications
are summarized in the cyclobutyl series in Figure 14.7.
N
H
N NH2
R1O
O
H
NO
O
O
O
Me
Me
Me
Me
Me
191817 20
Ki
*
= 25 nM
HNE/HCV = 23
EC90 = 400 nM
AUC (rat) =1.3 µM h
%F=3
Ki
*
= 400 nM
HNE/HCV = 9
Ki
*
= 8 nM
HNE/HCV = 138
EC90 = 700 nM
AUC (rat)=1.5 µM h
%F=28
Ki
*
= 150 nM
HNE/HCV = 370
Figure 14.6
14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role of Structure-Based Drug Design j351
The replacement of P3 cyclohexylglycine (21, KÃ
i ¼ 8:0 nM) with P3 tert-butylglycine
(23, KÃ
i ¼ 76 nM) resulted in a marked loss in enzyme activity. However, this
modification improved HNE/HCV selectivity (138 versus 684). Similarly, the
replacement of the Boc group in analogue 21 with tert-butyl urea 22 resulted in a
loss in enzyme activity as well as HNE/HCV selectivity. Even though both of these
modifications independently were detrimental to potency and selectivity, in combination
they proved to be very beneficial. The tert-butyl urea analogue of 23 resulted
in 24 (boceprevir) with a KÃ
i ¼ 14 nM and HNE/HCV selectivity of 2200. Boceprevir
(24) had good activity in the replicon-based cellular assay (EC90 ¼ 350 nM) [26, 18].
Based on these and additional studies [27], it was progressed to clinical trials
[28–30]. It was approved for treatment of genotype 1-infected patients by the FDA
in May 2011 and is marketed with the trade name Victrelis.
We later initiated a backup program with the goal of identifying a more potent
compound with improved PK in monkeys. For ease of development, we decided to
develop a backup candidate that was single diastereomer at P1. To achieve these
lofty goals, we once again visited the X-ray structure of inhibitors bound to protease
to capitalize on additional interactions. It was clear from our previous studies that
P10
secondary amide analogues displayed better pharmacokinetics than primary
ketoamide (example 15). We therefore decided to retain allyl amide at P10
in our
initial screen of second-generation compounds.
As part of SAR investigation at the P4 region in the discovery of boceprevir, we
replaced the tert-butyl urea cap of 24 with various carbamate and urea caps. Of
H
N
O
N
O
H
N NH2
O
OH
N
O
H
N
O
N
O
H
N NH2
O
OH
N
O
Ki
*
= 8 nM
HNE/HCV= 138
Ki
*
= 50 nM
HNE/HCV= 90
Ki
*
= 76 nM
HNE/HCV= 684
Ki
*
= 14 nM
HNE/HCV= 2200
EC90 = 350 nM
H
N
O
N
O
H
N NH2
O
OO
O
H
N
O
N
O
H
N NH2
O
OO
O
2212
4232
Figure 14.7
352j14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role of Structure-Based Drug Design
these modifications, two classes of compounds, represented by 25 and 26, demonstrated
marginal improvement in cellular activity (Figure 14.8).
The incorporation of methylcyclohexyl urea cap at P3 in 24 resulted in analogue
25 (KÃ
i ¼ 8 nM; EC90 ¼ 100 nM), a threefold improvement in cellular activity
compared to boceprevir. Similarly, introduction of tert-butyl glycine tert-butyl ester
cap in 24 yielded compound 26 (KÃ
i ¼ 6 nM; EC90 ¼ 200 nM). Improved cellular
activities of these analogues clearly showed that modifications at P4 could be beneficial
in improving cellular potencies. We therefore further modified these analogues
by introducing other functionalities in place of the methyl group in 25 and
the ester moiety in 26. After many modifications, four new series of analogues were
identified that had improved cellular activities (Figure 14.9).
The incorporation of a tert-butyl sulfone group in the place of the methyl substituent
on the P4 cap in 25 resulted in compound 27 with excellent enzyme binding
and cellular potency (KÃ
i ¼ 4 nM; EC90 ¼ 100 nM) [31]. However, it was poorly
absorbed in rats with low plasma exposure. Analysis of X-ray structure of compounds
of type 27 bound to the enzyme showed that the sulfone group was
H
N
H
N
O
N
H
N NH2
O
O
O
O
H
N
H
N
O
N
H
N NH2
O
O
O
O
O
O
t
Bu
25
26
Ki
* = 8 nM
EC90 = 100 nM
Ki
* =6 nM
EC90 = 200 nM
Figure 14.8
H
N
H
N
O
N
H
N NH2
O
O
O
O
S
O O
Ki
* =4nM
EC90 =100nM
AUC (Rat)0-6h =0.002 µM h
AUC (Mky)0-24h =3.18 µM h
27
Figure 14.9 Hydrogen bonding of P4 sulfone oxygen to Cys159 of HCV NS3 protease.
14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role of Structure-Based Drug Design j353
involved in a hydrogen bond interaction with the backbone amide nitrogen of
Cys159. Interestingly, the structural constraints to minimize the binding energy
placed one of the terminal tert-butyl methyl groups at a very close distance ($3.1 A

)
to the backbone carbonyl oxygen of Cys159. This close contact allows the tert-butyl
group to remain in a gauche dihedral orientation with the sulfone oxygens. Similarly,
modifications in the P4 tert-butyl glycine series also resulted in identification
of many potent analogues that capitalized on this interaction with Cys159
(Figure 14.10).
Replacement of the ester at P4 in analogue of type 26 with a reverse sulfonamide
group resulted in compound 28 [32] (KÃ
i ¼ 6 nM; EC90 ¼ 60 nM), a sevenfold
improvement in cellular activity compared to 24. Similarly, introduction of dimethylcyclohexyl
imide cap and reverse carbamate resulted in compounds of type
29 [33] (EC90 ¼ 45 nM) and 30 (EC90 ¼ 150 nM) with up to 10-fold improvement in
cellular potency. Once again, the P4 functionalities were within hydrogen bonding
distance to Cys159 amide nitrogen on the enzyme. It was very interesting to note
that these compounds were allyl amides at P10
and retained majority of the activity
that of the primary ketoamide. We next designed new secondary amide analogues
in the P4 spirocyclohexyl series of inhibitors 27 (Figure 14.11).
H
N
H
N
O
N
H
N NH
O
O
O
O
N
S
OO
H
N
H
N
O
N
H
N NH
O
O
O
O
N
O
O
H
N
H
N
O
N
H
N NH
O
O
O
O
ON
H
O
9282
30
Ki
*
= 6 nM
EC90 = 60 nM
AUC (Rat)0-6h = 1.2 µM h
AUC (Mky)0-24h = 0.69 µM h
Ki
* = 4 nM
EC90 = 45 nM
AUC (Rat)0-6h = 1.58 µM h
AUC (Mky)0-24h = 0.1 µM h
Ki
* = 9 nM
EC90 = 150 nM
Figure 14.10 X-ray structure of interaction between P4 imide and Cys159.
354j14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role of Structure-Based Drug Design
Modification of P10
primary ketoamide in 27 with a cyclopropyl amide resulted in
inhibitor 31 with poorer binding and cellular activity (KÃ
i ¼ 20 nM; EC90 ¼ 250nM)
compared to 27. However, it had good oral exposure in rats with AUC ¼ 2.8 mM h,
when dosed at 10 mg/kg. From our previous SAR studies in the identification of boceprevir,
it was clear that P1 cyclobutyl alanine was less tolerated in the P10
secondary
amide series. P1 norvaline and norleucine were more preferred. We therefore synthesized
analogues 32 and 33 containing P1 norvaline and norleucine moieties. In line
with our expectations, compound 32 had excellent binding (KÃ
i ¼ 8:0 nM) and
improved cellular activity (EC90 ¼ 40 nM). This cellular activity was about 10-fold better
than boceprevir. In addition, the norvaline derivative had good PK in rats. It had a
good oral exposure of 7.9mM h when dosed at 10 mg/kg in rats and 1.1 mM h in monkeys
at 3 mg/kg. The incorporation of P1 norleucine resulted in inhibitor 33 that had
similar characteristics to norvaline analogues, with a much improved HNE/HCV
selectivity. Thus, compound 33 had an enzyme inhibition KÃ
i ¼ 6 nM and a cellular
potency EC90 ¼ 40nM. It was selective against HNE with a HNE/HCV¼ 600 and
well absorbed in animals. In rats, compound 33 had a good bioavailability (F¼ 46%)
and an exposure of 6.5mM h when dosed at 10 mg/kg. It had a much improved PK in
monkeys compared to boceprevir with an AUC ¼ 1.1mM h at 3 mg/kg and oral bioavailability
of 46%. Based on these desirable properties, compound 33 (narlaprevir) [34]
was chosen for further investigation and progressed to clinical trials.
H
N
H
N
O
N
H
N
H
N
O
O
O
O
S
O O
Ki
* = 20nM
HNE/HCV = 1600
EC90 = 250 nM
AUC (Rat)0-6h = 2.8 µM h
31
H
N
H
N
O
N
H
N
H
N
O
O
O
O
S
O O
Ki
*
= 8 nM
HNE/HCV = 8
EC90 = 40 nM
AUC (Rat)0-24h = 7.9 µM h
AUC (Mky)0-24h = 1.1 µM h
32
H
N
H
N
O
N
H
N
H
N
O
O
O
O
S
O O
Ki
* =6 nM
HNE/HCV= 600
EC90 = 40 nM
AUC (Rat)0-24h = 6.5 µM h (F=46%)
AUC (Mky)0-24h = 1.1 µM h (F=46%)
33
Figure 14.11
14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role of Structure-Based Drug Design j355
Figure 14.12 shows the X-ray structures of boceprevir (24) and narlaprevir (33)
bound to the active site of the HCV NS3 protease. Ser139 makes a covalent interaction
with the ketoamide group in both compounds. While the absence of a prime
side moiety in boceprevir allows the side chain of Lys136 to pack against the P1
cyclobutyl ring, thereby resulting in snug van der Waals interactions throughout
the S1 pocket, the P10
cyclopropyl cap causes the Lys136 side chain to rotate away
from the S1 pocket. In addition, the more flexible linear chain of the P1 norleucine
versus the constrained cyclobutyl ring pack in such a way that the Ile132 side chain
adopts different rotamers in the two structures. While this alters the surface of the
S1–S3 pockets somewhat, the P3 tert-butyl group binds in the same orientation in
both structures. The P2 dimethylcyclopropyl-derived proline makes the same key
interactions with the S2 pocket in both structures. The side chains of His57,
Asp81, and Arg155 are in the same positions in both structures and make identical
contacts with P2. Interestingly, the S4 pocket undergoes little change with the presence
of the cyclohexyl ring – only the Arg123 side chain is different in the two
structures. The reason is presumably that the binding orientation of the tert-butyl
group of boceprevir has pushed the side chain of Asp168 into a rotamer position
that enlarges the pocket. This new position of Asp168 upon binding either of these
compounds combined with the rotations of Arg155 and His57 results in the formation
of an ion-pair/H-bonding network that extends from His57–Asp81–Arg155–
Asp168–Arg123. These side chains do not project face to face, but are aligned side
by side. Interestingly, the binding of the P4 cyclohexyl ring in narlaprevir brings
Arg123 in a bit closer to Asp168 as the readjusted pocket is tightened around
the ring.
In addition to the previously described lipophilic interactions, the main chain
atoms of boceprevir and narlaprevir make key hydrogen bonding interactions to
the enzyme (Figure 14.13). The P1 ketoamide nitrogen donated a hydrogen bond
to the side chain oxygen of Gln41, while the ketoamide oxygen projected into the
oxyanion hole and accepted hydrogen bonds from the amide nitrogens of Ser139
Ala-156
Cys-159
Arg-155
His-57
Lys-136
Ala-156
Cys-159
Arg-155
His-57
Lys-136
Arg-155
Ala-156
Lys-136
His-57
Arg-155
Ala-156
Lys-136
His-57
(a) (b)
Figure 14.12 X-ray structures of boceprevir (24) (a) and narlaprevir (33) (b) bound to HCV
protease.
356j14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role of Structure-Based Drug Design
and Gly137. Similarly, the amide nitrogen of the P1 moiety donated a hydrogen
bond to the carbonyl oxygen of Arg155. The P3 urea nitrogen made a pair of hydrogen
bonds to the backbone carbonyl oxygen of Ala157. These interactions make the
binding of boceprevir potent and selective. In addition to these hydrogen bonds,
narlaprevir also made an additional hydrogen bond with the oxygen of the P4 sulfone
that is in hydrogen bonding distance to the nitrogen of Cys159.
Computational analysis estimated the contribution of various ligand components
toward the overall binding of the inhibitor to the enzyme. It revealed substantial
proportions of binding correlates well with the contact surface area. About 50% of
the surface area of boceprevir made contact to the enzyme contributing 105
orders
of magnitude toward the binding potency. The remaining potency was achieved
through hydrogen bonds and covalent interactions.
The use of structure-based design facilitated our understanding of binding interactions
of inhibitors to the protease. It allowed new modifications to be conceived
and synthesized. Not all these modifications resulted in improvement in activity.
There were many instances into which the modeled compound fit very well in the
active site but resulted in analogues with much diminished activity. Figure 14.14
Me N N
N
N N
H
O
O
O O
O
H
Gln41 N
H
NH2
O
O
Me
Me
H
Me Me
Me
HH
OMe
Me
H H
N N
Ser139 Gly137
Ser139
H
N
H
N His57
HN
O
H
N
N
H
N
Arg155
OAla156
O
H
Ala157
O
O
N
H
Figure 14.13 Hydrogen bonding network of boceprevir with protease backbone.
S
O
O
H
N NH
O
O
NH
N
O
O
O
H
N
OHO
Ki
*
= 890 nM
N
O
NH
OO
H
O
H
N
H
N
O
O
Ki
* = 8100 nM
H
N
N
O
NH
O
HN
O
NH
O
O
Ki
* = 14400 nM
733635
Figure 14.14
14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role of Structure-Based Drug Design j357
outlines three examples of these modifications that were acceptable by modeling,
but had poor activity.
As previously mentioned, the X-ray structure of boceprevir indicated the methyl
of the P2 proline group interacted with Arg155. Based on this and related compounds,
we synthesized analogues of type 35 that contained a carboxylic acid on
the proline. We modeled this analogue to make an ionic interaction with Arg155
and thereby improve potency. However, this effort did not bear fruit because the
side chain of arginine was flexible and solvent exposed allowing it to occupy alternate
rotamers. Other attempts to capture this interaction proved equally futile. We
also explored many novel ideas to depeptidize the inhibitors in the acyclic series.
Analogues 36 and 37 are representative compounds that modeled very well in the
structure of the enzyme. Bicyclic analogues of 36 were synthesized in different ring
sizes. These analogues were considerably less potent with analogue 36 having a
KÃ
i ¼ 8100 nM. We could not completely rationalize this loss based on structure.
Similarly, modeled analogue 37 made an excellent fit to the protease active site
based on the structure of the enzyme. However, the synthesis of this analogue
resulted in compound that lost significant activity with a KÃ
i ¼ 14 400 nM. The loss
in activity may possibly be due to the quaternization of P1, which hinders nucleophilic
attack of serine to the ketoamide.
In the absence of a viable lead for modification, we embarked on a structurebased
design to develop novel inhibitors for the HCV NS3 protease. The starting
lead 1 was initially truncated at P10
with an allyl group to identify compounds that
retained most of the activity of 1. From the X-ray structure of truncated compounds
5 and 6, we identified various sites for modification that could potentially
improve activity and depeptidize resultant analogues. Capping of inhibitors at P3
followed by modification at P2 prolines resulted in the discovery of novel analogues
that were very potent in the enzyme and cellular assay. (1R,2S,5S)-6,6-Dimethyl-3azabicyclo[3.1.0]hexane-2-carboxylic
acid moiety was identified as an excellent P2
residue that allowed generation of potent compounds of type 9 spanning from P3
to P20
. These compounds though very potent in in vitro assays had poor PK in animals.
Various depeptidization strategies were explored to improve PK in animals.
P2–P4 and P1–P3 macrocyclic inhibitors were synthesized and evaluated resulting
in many potent compounds. These analogues though retained and improved
potency in some cases did not improve PK in animals.
In an effort to improve PK, we further truncated analogues to identify a primary
ketoamide derivative 16 that retained significant activity of the initial leads. SAR
studies on primary ketoamide compound 16 at the P1, P3, and P3 capping site
resulted in the identification of cyclobutyl alanine, tert-butyl glycine, and tert-butyl
urea as desirable moieties. This led to the discovery of boceprevir 24, the first
direct-acting HCV NS3 protease approved by FDA for the treatment of genotype 1
infections.
Toward the identification of a backup compound, we revisited the X-ray structure
of inhibitors bound to the protease and identified modifications at P3 capping
resulted in analogues with improved cellular activity. Four series of P3 modifications
were discovered that yielded compounds with up to 10-fold improvement in
358j14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role of Structure-Based Drug Design
cellular potencies. A tert-butyl sulfone group interacted with Cys159 was discovered
resulting in analogue 33 (narlaprevir) that had excellent potency in enzyme and
cellular assay. It was progressed to clinical trials.
The role of structure in modification of HCV NS3 protease inhibitors from 1 to
boceprevir and narlaprevir is that of a successful journey. Based on the structure of
inhibitors bound to the enzyme, new designs to depeptidize analogues and
improve potency were framed. Many of these ideas resulted in compounds with
improved activity, while others resulted in loss in potency. Structure-based design
in combination with structure–activity relationship is a powerful tool to design
inhibitors for enzymes where initial leads are not readily available. It is a tool that
can facilitate improvement in enzyme activity of inhibitors allowing researchers to
capitalize novel interactions with the protein. However, optimization of other properties
such as cellular potency, PK, and selectivity needed for a successful drug
development still requires traditional medicinal chemistry approaches such as SAR
investigation.
References
1 Choo, Q.L., Kuo, G., Weiner, A.J., Overby,
L.R., Bradley, D.W., and Houghton, M.
(1989) Isolation of a cDNA clone derived
from a blood-borne non-A, non-B viral
hepatitis genome. Science, 244, 359–362.
2 Kuo, G., Choo, Q.L., Alter, H.J., Gitnick,
G.L., Redeker, A.G., Purcell, R.H.,
Miyamura, T., Dienstag, J.L., Alter, M.J.,
Stevens, C.E., Tegtmeier, G.E., Bonino, F.,
Colombo, M., Lee, W.-S., Kuo, C., Berger,
K., Shuster, J.R., Overby, L.R., Bradley, W.,
and Houghton, M. (1989) An assay for
circulating antibodies to a major etiologic
virus of human non-A, non-B hepatitis.
Science, 244, 362–364.
3 Brown, R.S., Jr. and Gaglio, P. (2003)
Scope of worldwide hepatitis C problem.
Liver Transpl., 9 (11), S10–S13.
4 Freeman, A.J., Dore, G.J., Law, M.G.,
Thorpe, M., Overbeck, J.V., Llyods, A.R.,
Marinos, G., and Kaldor, J.M. (2001)
Estimating progression to cirrhosis in
chronic hepatitis C infection. Hepatology,
34, 809–816.
5 Zeuzem, S., Feinman, S.V., Rasenack, J.,
Heathcote, E.J., Lai, M.-Y., Gane, E.,
O’Grady, J., Reichen, J., Diago, M., Lin, A.,
Hoffman, J., and Brunda, M.J. (2000)
Peginterferon alfa-2a in patients with
chronic hepatitis C. N. Engl. J. Med., 343
(23), 1666–1172.
6 Manns, M.P., McHutchison, J.G.,
Gordon, S.C., Rustgi, V.K., Shiffman, M.,
Reindollar, R., Goodman, Z.D., Koury, K.,
Ling, M.-H., and Albrecht, J.K.
(International Hepatitis Interventional
Therapy Group) (2001) Peginterferon alfa-
2b plus ribavirin compared with
interferon alfa-2b plus ribavirin for initial
treatment of chronic hepatitis C: a
randomized trial. Lancet, 358, 958–965.
7 Bartenschlager, R. (1999) The NS3/4A
proteinase of the hepatitis C virus:
unraveling structure and function of an
unusual enzyme and a prime target
for antiviral therapy. J. Virol. Hepat., 6,
165–181.
8 Bartenschlager, R., Ahlborn-Laake, L.,
Mous, J., and Jacobsen, H. (1993)
Nonstructural protein 3 of the hepatitis C
virus encodes a serine-type proteinase
required for cleavage at the NS3/4 and
NS4/5 junctions. J. Virol., 67, 3835–3844.
9 Yan, Y., Li, Y., Munshi, S., Sardana, V.,
Cole, J.L., Sardana, M., Steinkuehler, C.,
Tomei, L., De-Francesco, R., Kuo, L.C.,
and Chen, Z. (1998) Complex of NS3
protease and NS4A peptide of BK strain
hepatitis C virus: a 2.2 A

resolution
structure in a hexagonal crystal form.
Protein Sci., 7, 837–847.
References j359
10 Love, R.A., Parge, H.E., Wickersham, J.A.,
Hostomsky, Z., Habuka, N., Moomaw,
E.W., Adachi, T., and Hostomska, Z.
(1996) The crystal structure of hepatitis C
virus NS3 proteinase reveals a trypsin-like
fold and a structural zinc binding site.
Cell, 87, 331–342.
11 Kim, J.L., Morgenstern, K.A., Griffith, J.
P., Dweyer, M.D., Thomson, J.A., Murcko,
M.A., Lin, C., and Caron, P.R. (1998)
Hepatitis C virus NS3 RNA helicase
domain with a bound oligonucleotide: the
crystal structure provides insights into the
mode of unwinding. Structure, 6, 89–100.
12 Kwong, A.D., Kim, J.L., Rao, G., Lipovsek,
D., and Raybuck, S.A. (1998) Hepatitis C
virus NS3/4A protease. Antiviral Res., 40,
1–18.
13 Steinkuhler, C., Urbani, A., Tomei, L.,
Biasiol, G., Sandana, M., Bianchi, E.,
Pessi, A., and DeFrancesco, R. (1996)
Activity of purified hepatitis C virus
protease NS3 on peptide substrates.
J. Virol., 70, 6694–6700.
14 Urbani, A., Bianchi, E., Narjes, F.,
Tramontano, A., De Francesco, R.,
Steinkuhler, C., and Pessi, A. (1997)
Substrate specificity of the hepatitis C
virus serine protease NS3. J. Biol. Chem.,
272, 9204–9209.
15 Kolykhalov, A.A., Agapov, E.V., and Rice,
C.M. (1994) Specificity of the hepatitis C
virus NS3 serine protease: effects of
substitutions at the 3/4A, 4A/4B, 4B/5A,
and 5A/5B cleavage sites on polyprotein
processing. J. Virol., 68, 7525–7533.
16 Zhang, R., Beyer, B.M., Durkin, J.,
Ingram, R., Njoroge, F.G., Windsor, W.T.,
and Malcolm, B.A. (1999) A continuous
spectrophotometric assay for the hepatitis
C virus serine protease. Anal. Biochem.,
270, 268–275.
17 Morrison, J.F. and Walsh, C. (1988) The
behavior and significance of slow binding
enzyme inhibitors, in Advances in
Enzymology, vol. 61 (ed. A. Meister), John
Wiley & Sons, Inc., New York, pp. 201–301.
18 Prongay, A.J., Guo, Z., Yao, N., Pichardo,
J., Fischmann, T., Strickland, C., Myers,
J., Jr., Weber, P.C., Beyer, B.M., Ingram,
R., Hong, Z., Prosise, W.W., Ramanathan,
L., Taremi, S.S., Yarosh-Tomaine, T.,
Zhang, R., Senior, M., Yang, R.S.,
Malcolm, B., Arasappan, A., Bennett, F.,
Bogen, S.L., Chen, K., Jao, E., Liu, Y.-T.,
Lovey, R.G., Saksena, A.K., Venkatraman,
S., Girijavallabhan, V., Njoroge, F.G., and
Madison, V. (2007) Discovery of the HCV
NS3/4A protease inhibitor (1R,5S)-N-[3-
amino-1-(cyclobutylmethyl)-2,3-
dioxopropyl]-3-[2(S)-[[[(1,1-dimethylethyl)
amino]carbonyl]amino]-3,3-dimethyl-1-
oxobutyl]-6,6-dimethyl-3-azabicyclo[3.1.0]
hexan-2(S)-carboxamide (Sch 503034). Key
steps in structure-based optimization.
J. Med. Chem., 50, 2310–2318.
19 Bogen, S.L., Arasappan, A., Bennett, F.,
Chen, K., Jao, E., Liu, Y.-T., Lovey, R.G.,
Venkatraman, S., Pan, W., Parekh, T.,
Pike, R.E., Ruan, S., Liu, R., Baroudy, B.,
Agrawal, S., Chase, R., Ingravallo, P.,
Pichardo, J., Prongay, A., Brisson, J.-M.,
Hsieh, T.Y., Cheng, K.-C., Kemp, S.J.,
Levy, O.E., Lim-Wilby, M., Tamura, S.Y.,
Saksena, A.K., Girijavallabhan, V., and
Njoroge, F.G. (2006) Discovery of
SCH446211 (SCH6): a new ketoamide
inhibitor of the HCV NS3 serine protease
and HCV subgenomic RNA replication.
J. Med. Chem., 49, 2750–2757.
20 Lohmann, V., K€orner, F., Koch, J.-O.,
Herian, U., Theilmann, L., and
Bartenschlager, R. (1999) Replication of
subgenomic hepatitis C virus RNAs in a
hepatoma cell line. Science, 285, 110–113.
21 Mamai, A., Zhang, R., Natarajan, A., and
Madalengoitia, J.S. (2001) Poly-L-proline
type-II peptide mimics based on the 3azabicyclo[3.1.0]hexane
system. J. Org.
Chem., 66, 455–460.
22 Venkatraman, S., Njoroge, F.G.,
Girijavallabhan, V.M., Madison, V.S., Yao,
N.H., Prongay, A.J., Butkiewicz, N., and
Pichardo, J. (2005) Design and synthesis
of depeptidized macrocyclic inhibitors of
hepatitis C NS3-4A protease using
structure-based drug design. J. Med
Chem., 48, 5088–5091.
23 Chen, K.X., Njoroge, F.G., Vibulbhan, B.,
Prongay, A., Pichardo, J., Madison, V.,
Buevich, A., and Chan, T.-M. (2005)
Proline-based macrocyclic inhibitors of
the hepatitis C virus: stereoselective
synthesis and biological activity.
Angew. Chem., Int. Ed., 44,
7024–7028.
360j14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role of Structure-Based Drug Design
24 Chen, K., Njoroge, F.G., Arasappan, A.,
Venkatraman, S., Vibulbhan, B., Yang, W.,
Parekh, T.N., Pichardo, J., Prongay, A.,
Cheng, K.-C., Butkiewicz, N., Yao, N.,
Madison, V., and Girijavallabhan, V.
(2006) Novel potent hepatitis C virus NS3
serine protease inhibitors derived from
proline-based macrocycle. J. Med. Chem.,
49, 995–1005.
25 Venkatraman, S., Velazquez, F., Wu, W.,
Blackman, M., Chen, K.X., Bogen, S.,
Nair, L., Tong, X., Chase, R., Hart, A.,
Agrawal, S., Pichardo, J., Cheng, K.-C.,
Girijavallabhan, V., Piwinski, J., Shih, N.
Y., and Njoroge, F.G. (2009) Discovery and
structure–activity relationship of P1–P3
ketoamide derived macrocyclic inhibitors
of hepatitis C virus NS3 protease. J. Med.
Chem., 52, 336–346.
26 Venkatraman, S., Bogen, S.L., Arasappan,
A., Bennett, F., Chen, K., Jao, E., Liu, Y.-T.,
Lovey, R., Hendrata, S., Huang, Y., Pan,
W., Parekh, T., Pinto, P., Popov, V., Pike,
R., Ruan, S., Santhanam, B., Vibulbhan,
B., Wu, W., Yang, W., Kong, J., Liang, X.,
Wong, J., Liu, R., Butkiewicz, N., Chase,
R., Hart, A., Agarwal, S., Ingravallo, P.,
Pichardo, J., Kong, R., Baroudy, B.,
Malcolm, B., Guo, Z., Prongay, A.,
Madision, B.L., Cui, X., Cheng, K.-C.,
Hsieh, T.Y., Brisson, J.-M., Prelusky, D.,
Kormacher, W., White, R., BogonowichKnipp,
S., Pavlovsky, A., Prudence, B.,
Saksena, A.K., Ganguly, A., Piwinski, J.,
Girijavallabhan, V., and Njoroge, F.G.
(2006) Discovery of (1R,5S)-N-[3-amino-1-
(cyclobutylmethyl)-2-3-dioxopropyl]-3-[2
(S)-[[[(1,1-dimethylethyl)-amino]carbonyl]
amio]-3,3-dimethyl-1-oxobutyl]-6,6-
dimethyl-3-azabicyclo[3.1.0]hexan-2(S)carboxamide
(SCH 503034), a selective,
potent, orally bioavailable, hepatitis C
virus NS3 protease inhibitor: a potential
therapeutic agent for the treatment of
hepatitis C infection. J. Med. Chem., 49,
6074–6086.
27 Cheng, K.-C., Li, C., Liu, T., Wang, G.,
Hsieh, Y., Pavlovsky, A., Broske, L.,
Prelusky, D., Chen, J., Liu, R., Uss, A.S.,
White, R.E., Gupta, S., and Njoroge, F.G.
(2009) Use of preclinical in vitro and
in vivo pharmacokinetics for selection
of potent hepatitis C protease inhibitor,
boceprevir for clinical development. Lett.
Drug Des. Discov., 6, 312–218.
28 Kwo, P.Y., Lawitz, E.J., McCone, J., Schiff,
E.R., Vierling, J.M., Pound, D., Davis, M.
N., Galati, J.S., Gordon, S.C., Ravendhran,
N., Rossaro, L., Anderson, F.H., Jacobson,
I.M., Rubin, R., Koury, K., Pedicone, L.D.,
Brass, C.A., Chaudhri, E., and Albrecht,
J.K. (the SPRINT-1 Investigators) (2010)
Efficacy of boceprevir, an NS3 protease
inhibitor, in combination with
peginterferon alfa-2b and ribavirin in
treatment-naive patients with genotype 1
hepatitis C infection (SPRINT-1): an openlabel,
randomised, multicentre phase 2
trial. Lancet, 376, 705–716.
29 Poordad, F., McCone JJr., J., Bacon, B.R.,
Bruno, S., Manns, M.P., Sulkowski, M.S.,
Jacobson, I.M., Reddy, K.R., Goodman, Z.D.,
Boparai, N., DiNubile, M.J., Sniukiene, V.,
Brass, C.A., Albrecht, J.K., and Bronowicki,
J.-P. (the SPRINT-2 Investigators) (2011)
Boceprevir for untreated chronic HCV
genotype 1 infection. N. Engl. J. Med., 364
(13), 1195–1206.
30 Bacon, B., Gordon, S.C., Lawitz, E.,
Marcellin, P., Vierling, J.M., Zeuzem, S.,
Poordad, F., Goodman, Z.D., Sings, H.L.,
Boparai, N., Burroughs, M., Brass, C.A.,
Albrecht, J.K., and Esteban, R. (the HCV
RESPOND-2 Investigators) (2011)
Boceprevir for previously treated chronic
HCV genotype 1 infection. N. Engl. J.
Med., 364 (13), 1207–1217.
31 Bennett, F., Huang, Y., Hendrata, S., Lovey,
R., Bogen, S., Pan, W., Guo, Z., Prongay,
A., Chen, K.X., Arasappan, A.,
Venkatraman, S., Velazquez, F., Nair, L.,
Sannigrahi, M., Tong, X., Pichardo, J.,
Cheng, K.-C., Girijavallabhan, V.M.,
Saksena, A.K., and Njoroge, F.G. (2010)
The introduction of P4 substituted 1methylcyclohexyl
groups into Boceprevir1
:
a change in direction in the search for a
second generation HCV NS3 protease.
Bioorg. Med. Chem. Lett., 20, 2617–2621.
32 Venkatraman, S., Blackman, M., Wu, W.,
Nair, L., Arasappan, A., Padilla, A., Bogen,
S., Bennett, F., Chen, K., Pichardo, J., Tong,
X., Prongay, A., Cheng, K.-C.,
Girijavallabhan, V., and Njoroge, F.G.
(2009) Discovery of novel P3 sulfonamidecapped
inhibitors of HCV NS3 protease.
References j361
Inhibitors with improved cellular potencies.
Bioorg. Med. Chem., 17, 4486–449.
33 Arasappan, A, Padilla, A.I., Jao, E.,
Bennett, F., Bogen, S., Chen, K.X., Pike,
R.E., Sannigrahi, M., Soares, J.,
Venkatraman, S., Vibulbhan, B., Saksena,
A.K., Girijavallabhan, V., Tong, X., Cheng,
K.C., and Njoroge, F.G. (2009) Discovery
of novel P4 modified analogues with
improved potency and pharmacokinetic
profile. J. Med. Chem., 52, 2806–2817.
34 Arasappan, A., Bennett, F., Bogen, S.,
Venkatraman, S., Blackman, M., Chen, K.
X., Hendrata, S., Huang, Y., Huelgas, R.M.,
Nair, L., Padilla, A.I., Pan, W., Pike, R.,
Pinto, P., Ruan, S., Sannigrahi, M.,
Velazquez, F., Vibulbhan, B, Wu, W., Yang,
W., Saksena, A.K., Girijavallabhan, V., Shih,
N.-Y., Kong, J., Meng, T., Jin, Y., Wong, J.,
McNamara, P., Prongay, A., Madison, V.,
Piwinski, J.J., Cheng, K.C., Morrison, R.,
Malcolm, B., Tong, X., Ralston, R., and
Njoroge, F.G. (2010) Discovery of
narlaprevir (SCH 900518): a potent, second
generation HCV NS3 protease inhibitor.
ACS Med. Chem. Lett., 1, 64–69.
362j14 Discovery of Boceprevir and Narlaprevir: A Case Study for Role of Structure-Based Drug Design
George F. Njoroge obtained his B.S. degree in Chemistry
from University of Nairobi, Kenya, in 1979 and Ph.D. in
Organic Chemistry from Case Western Reserve University
(CWRU) in 1985. He was a postdoctoral fellow in
the Institute of Pathology at CWRU working with Professor
Vincent Monnier. He joined the former ScheringPlough
(now Merck) as a Senior Research Scientist and
rose through the ranks to the position of Director of
Medicinal Chemistry. He is currently a Senior Research
Fellow at Eli Lilly and Company.
Andrew J. Prongay was born and raised in New Jersey. He
earned a B.S. degree in Biology in 1982 from Cooks College,
Rutgers University, New Brunswick, New Jersey. He earned a
Ph.D. in Biological Chemistry in 1989 from The University
of Michigan, Ann Arbor, Michigan, in the laboratory of Professor
Charles H. Williams, Jr. He was a Scholar of the American
Foundation for AIDS Research and performed
postdoctoral studies in the laboratory of Professor Michael Rossmann of the Biology
Department at Purdue University, West Lafayette, Indiana. In 1993, he began
his industrial career that included research efforts in “protein science” and “structural
chemistry”.
Srikanth Venkatraman obtained his B.Sc. degree in Chemistry
from the University of Madras, India, and M.Sc. degree
from the Indian Institute of Technology, Chennai. He later
joined University of Pittsburgh and worked on azoles and
azole natural products working with Dr. Peter Wipf to
earn his Ph.D. After working for 2 years with Dr. Scott E.
Denmark at the University of Illinois at Urbana Champaign,
he joined Schering-Plough now Merck. He has extensively
worked in many antiviral projects relating to hepatitis C.
References j363
15
A New-Generation Uric Acid Production Inhibitor: Febuxostat
Ken Okamoto, Shiro Kondo, and Takeshi Nishino
List of Abbreviations
AMP adenosine monophosphate
ADP adenosine diphosphate
ATP adenosine triphosphate
FAD flavin adenine dinucleotide
GMP guanosine monophosphate
GDP guanosine diphosphate
GTP guanosine triphosphate
HGPRT hypoxanthine guanine phosphoribosyltransferase
IMP inosine monophosphate
NAD nicotinamide adenine dinucleotide
PDB ID protein data bank identification code
XDH xanthine dehydrogenase
XO xanthine oxidase
XOR xanthine oxidoreductase
15.1
Introduction
Purine bases, which are components of nucleic acids and nucleotides, are catabolized
in tissues and ultimately excreted into the urine as uric acid in humans. Xanthine
oxidoreductase (XOR) catalyzes the last two steps in the purine catabolic
pathway, that is, the oxidations of hypoxanthine to xanthine and xanthine to uric
acid (Figure 15.1). Gout is a disease in which crystals of uric acid collect in tissues,
causing painful inflammation. The factors that lead to gout are not fully understood,
but include high levels of uric acid in the blood. As XOR inhibitors significantly
lower uric acid production and concentration in the blood, they can be used
to treat gout. Based on this strategy, Elion and coworkers of Burroughs Wellcome
developed an XOR inhibitor, allopurinol (Figure 15.2), as a therapeutic drug for
Analogue-based Drug Discovery III, First Edition. Edited by Janos Fischer, C. Robin Ganellin, and
David P. Rotella.
# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
j365
Molybdopterin
Glu802
Glu1261
Arg880
Phe914
Phe1009
Xanthine
(a)
(b)
(c)
N
N
H
N
H
N
O
HN
N
H
N
H
N
O
O
HN
N
H
N
H
H
N
O
O
O
GMP
IMP
Guanine
Hypoxanthine
Xanthine Uric Acid
AMP
N
H
NH
H
N
N
O
O
Mo
O
S
OH
S
S
C
O
O-
C
O
OH
C+
H2
N
NH2HN
N
H
NH
H
N
N
O
O
Mo
O
S
O-
S
S
C
O
OH
C
O
OH
C+
H2
N
NH2HN
N
H
NH
H
N
N
O
O
Mo
O
SH
O
S
S
C
O
OH
C+
H2
N
NH2HN
C
O
OH
VI VI IV
H2
O 2e-,
2H+
Urate
Figure 15.1 Catalytic roles of XOR in purine
metabolism. (a) XOR catalyzes two steps of
reaction (red arrows), that is, hypoxanthine to
xanthine and xanthine to uric acid.
Hypoxanthine is the metabolic product of
inosine monophosphate (IMP) and adenine
nucleotides (AMP, ADP, and ATP). Xanthine is
also formed from guanine in the catabolism of
guanine nucleotides (GMP, GDP, and GTP). (b)
Proposed reaction mechanism of the
molybdenum cofactor with xanthine as a
substrate. Nucleophilic attack of Mo(VI)–OÀ
occurs at the C8 atom of xanthine to form the
reaction intermediate (Mo(IV)–O–C) as
illustrated. The two electrons are transferred to
the other redox centers (the iron sulfur clusters
and FAD) accompanied by breakdown of the
reaction intermediate by hydrolysis with water
to give urate. (c) The crystal structure of the
reaction intermediate formed by the reduced
Mo(IV) bound with excess urate under
anaerobic conditions (PDB ID: 3AMZ).
366j15 A New-Generation Uric Acid Production Inhibitor: Febuxostat
gout [1]. It has been on the market for over 40 years, and the efficacy of XOR inhibition
for treatment of gout is well established. However, in some cases allopurinol
causes serious adverse effects [2, 3], and so new uric acid inhibitors have been
sought to replace it. Many XOR inhibitors have been reported [4], but none of them
has been found to be superior to allopurinol with regard to efficacy and safety; consequently,
allopurinol is still the only XOR inhibitor in clinical use. Although allopurinol
was first reported to be a competitive inhibitor [5], further characterization
showed that it is a kind of suicide inhibitor, as described below, and this seems to
be one of the reasons why many inhibitors having apparently higher affinity than
allopurinol do not show greater efficacy than allopurinol. In recent years, however,
Japanese pharmaceutical companies have developed several very effective inhibitors
[6–9], including febuxostat, and these have attracted great interest as candidate
therapeutic agents [10, 11]. Clinical trials indicate that febuxostat is superior to allopurinol
in lowering uric acid production. By means of enzymatic, spectroscopic,
and structural–biological analyses of the inhibition mechanism, the present
authors have shown that febuxostat binds tightly to both the oxidized and reduced
forms of XOR in a highly structure-specific manner [7].
15.2
Xanthine Oxidoreductase – Target Protein for Gout Treatment
Mammalian xanthine oxidoreductase is a homodimeric enzyme with a molecular
mass of 290 kDa; each subunit contains one molybdenum cofactor, two [2Fe–2S]
centers, and one FAD center [12]. It catalyzes the oxidative hydroxylation of purine
substrates that takes place at the molybdenum center. The electrons are then transferred
via the two [2Fe–2S] centers to the FAD center, where reduction of the physiological
electron acceptor, NADþ
or O2, occurs. Mammalian XORs exist as two
Glu802
Allopurinol Oxipurinol
(a) (b)
NNN
N
N
H
N
HNHN
OO
O
Figure 15.2 Inhibition of XOR by allopurinol.
(a) Allopurinol is hydroxylated by XOR to give
oxipurinol (alloxanthine). (b) The crystal
structure of oxypurinol bound to the reduced
Mo(IV) (PDB ID: 3BDJ). Two glutamines
(Glu802 and Glu1261) and Arg880 form
hydrogen bonds with oxipurinol, similarly to the
urate-bound form (Figure 15.1).
15.2 Xanthine Oxidoreductase – Target Protein for Gout Treatment j367
forms of the same gene product: xanthine dehydrogenase (XDH) and xanthine oxidase
(XO). XDH prefers NADþ
as an electron acceptor, whereas XO prefers molecular
oxygen. In normal cells, XOR exists as its XDH form, but can be converted to
XO reversibly by formation of disulfide bridges or irreversibly by limited proteolysis
[12]. XO generates reactive oxygen species (H2O2 and O2
À
), which are associated
with various diseases, such as reperfusion injury [13]. As XOR is the only enzyme
that produces uric acid and patients with XO deficiency do not show serious disease
symptoms [14], XOR is an excellent target for designing drugs to control uric
acid production.
The active center of XOR for the production of uric acid is molybdopterin, where
the pterin molecule is complexed with a molybdenum atom. X-ray crystallographic
studies have revealed the structure in the vicinity of the cofactor [9, 15, 16]. Molybdopterin
binds in a narrow groove formed by two phenylalanines, two glutamates,
an arginine, and a threonine residue (Figure 15.1). Five atoms coordinate to the
molybdenum atom: a water-exchangeable hydroxo ligand (ÀÀOH), a sulfide (ÀÀÀÀS)
and two dithiolene sulfurs (ÀÀS) linked to the pterin moiety at equatorial positions,
and the oxo (ÀÀÀÀO) group at an apical position [9]. The active site residues and the
coordinating atoms in XOR are completely conserved among species from bacteria
to humans [17]. The water-exchangeable oxygen atom is transferred from the
molybdenum atom to the substrate as a hydroxyl group and then the active center
is regenerated from oxygen derived from a water molecule during catalysis
(Figure 15.1b). Two glutamic acids and an arginine form hydrogen bonds with substrate
xanthine (Figure 15.1c).
15.3
Mechanism of XOR Inhibition by Allopurinol
Allopurinol is an isomer of hypoxanthine, in which the N7 nitrogen is replaced
with carbon and the C8 carbon is replaced with nitrogen (Figure 15.2). The mechanism
of XOR inhibition by allopurinol is complex, and allopurinol proved to be a
more potent inhibitor than had originally been expected [18–20]. Allopurinol is converted
into oxipurinol (4,6-dihydroxypyrazolopyrimidine) (Figure 15.2) by XOR and
oxipurinol covalently binds to the reduced molybdenum (Mo4þ
) (Figure 15.2); this
is analogous to the binding mode of the reduced molybdenum with urate
(Figure 15.1c) [16]. Formation of this oxipurinol–molybdenum complex is the reason
for the inhibition by allopurinol. But, Mo4þ
is gradually oxidized to Mo6þ
with
a half-life of 300 min at 25 
C [20] and the covalent bond of molybdenum with oxipurinol
is broken. Different from the case of febuxostat, described later, the interaction
of oxipurinol and oxidized XOR is weak in the absence of the covalent bond,
and oxipurinol acts only as a weak competitive inhibitor with a Ki value of about
1.7 Â 10À6
M [5]. Thus, the inhibition by allopurinol strongly depends on the oxidation
or reduction state of the enzyme, and this may be the reason why allopurinol
must be administered frequently (three times a day) to be effective, that is, to maintain
a sufficient level of the oxipurinol-reduced molybdenum complex in the body.
368j15 A New-Generation Uric Acid Production Inhibitor: Febuxostat
15.4
Development of Nonpurine Analogue Inhibitor of XOR: Febuxostat
Analysis of the structure–activity relationship of various drug candidates suggested
that a purine structure was not necessary for inhibitory activity [21–25], and that a
five-membered heterocycle with a phenyl ring would be a suitable inhibitor template,
as shown in Figure 15.3. Several scaffolds matching this template were synthesized
and 2-phenyl-4-methyl-5-thiazolecarboxylic acid was found to have weak
inhibitory activity with an IC50 value of 700 nM. This compound was selected as a
lead compound. Preliminary modification of the lead compound revealed that the
introduction of an electron-withdrawing substituent and hydrogen bond acceptor
such as NO2 or CF3 at the 3-position of the phenyl ring or an electron-donating
substituent such as OR or COOH at the 4-position led to an increase of the inhibitory
activity. The NO2-substituted derivative at high blood concentration showed
prolonged hypouricemic action, so lead optimization was carried out using mainly
nitro derivatives. The above substituent effects could also be seen in disubstituted
compounds containing NO2 at the 3-position of the phenyl ring and an alkoxy or
alkylamino group at the 4-position of the thiazole ring. Ten or more compounds
showed potent inhibitory activity with IC50 values of about 1 nM, and they reduced
serum uric acid levels more effectively than did allopurinol in mice. However,
almost all of the nitro derivatives showed mutagenic activity in the Ames test, even
at low concentrations, as expected. Attempts to replace the nitro group with other
electron-withdrawing substituents at the 3-position resulted in identification of the
X
OH
R
N
N
N N
N N
N N
N
N
N
N
NH
N
N
O
H2
N
N
S
OH
HO
OH
H2
N
CH3
NHCOCH3
OH
N
N
SOPh
OCH3
HO
CF3
CH3
HO
N
O
5
3′
5′ 4′
6′
2′1′
S
2
1
3
4
54321
IC50; 25nM IC50; 25nMIC50; 10nM IC50; 40nM IC50; 38nM
Breakaway from purine structure
Common template Thiazole derivatives
Figure 15.3 Lead compound of febuxostat. Top: Chemical structures and the IC50 values of
various inhibitors reported in the literature [20–24]. Bottom: Lead compound for development of
higher affinity inhibitors.
15.4 Development of Nonpurine Analogue Inhibitor of XOR: Febuxostat j369
cyano group as the best substituent among those examined. Finally, 2-(3-cyano-4isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic
acid (febuxostat) (Figure 15.4a) was
selected as a candidate for clinical development. The suitability of these substituents
at the 3- and 4-positions is clearly supported by the structure of febuxostatbound
bovine XOR, as described below [7].
15.5
Mechanism of XOR Inhibition by Febuxostat
Febuxostat contains a thiazole ring with a methyl group at the 4-position and a benzene
ring with a CN group at the 3-position (Figure 15.4a). These two rings can
rotate freely. The structural similarity with purine is low, and the molecular weight
Figure 15.4 (a) Crystal structure of the
complex of bovine XOR and febuxostat (PDB
ID; 1N5X). (b) Amino acid residues of XOR
located close to febuxostat are drawn using the
van der Waals radius. (c) Hydrogen bonds are
drawn with dotted red lines. Ionic bonds are
drawn with dotted blue lines. Distances are
taken from unpublished data of febuxostatbound
bovine XOR at 2.1 A

resolution. (d)
Possible p–p interactions of amino acids with
febuxostat are indicated with dotted blue lines
and possible hydrophobic interactions with
dotted orange lines.
370j15 A New-Generation Uric Acid Production Inhibitor: Febuxostat
is much larger than that of purine. The crystal structure of the complex of milk
XOR with febuxostat shows features that account for the tight binding of febuxostat
[7]. Febuxostat fits well into the substrate binding channel of XOR, and no
open space remains in the channel after binding (Figure 15.4b). In contrast to allopurinol,
febuxostat does not form a covalent bond with molybdenum. However,
although no covalent bond is formed between the enzyme and the inhibitor, multiple
weak interactions such as ionic and hydrogen bonds, p–p interactions between
the thiazole ring and nearby phenylalanine residues, van der Waals interactions,
and hydrophobic interactions were found in the structure (Figure 15.4c and d). The
combination of these interactions results in tight binding; the dissociation constant
determined for the desulfo form was 2 Æ 0.03 Â 10À9
M, and for the active form,
the corresponding number was too low to allow accurate measurements [7]. Moreover,
the fit of febuxostat to the enzyme’s active site structure is enhanced by rotation
of the region between the thiazole ring and the benzene ring. Thus, febuxostat
efficiently matches the structure of the substrate binding region of the enzyme. A
hydrogen bonding interaction of the CN group of the inhibitor with an asparagine
residue of the enzyme should be noted. In the crystal structure, the side chain
amide of Asn768 and the cyano group of 3-position are 2.9 A

apart, forming a
hydrogen bond (Figure 15.4c). Although this asparagine residue (Asn768 in the
bovine enzyme) is located too far from the active center for direct involvement in
purine substrate recognition or catalytic activity, the CN group of febuxostat is necessary
for potent enzyme inhibitory activity. When the 3-cyano moiety of febuxostat
was replaced by hydrogen, the binding affinity was significantly decreased
(S. Kondo, unpublished data). In contrast, when the cyano group of the inhibitor
was substituted with a nitro ligand, which is also capable of accepting a hydrogen
bond, the resulting derivative showed a binding affinity very similar to that of
febuxostat. A bulky hydrophobic moiety at the 4-position is also essential for tight
binding. The 4-isobutoxy group is surrounded by hydrophobic amino acids, with
distances ranging from 3.7 to 4.2 A

(Figure 15.4d). Although some of these crystallographically
determined values are too large to be consistent with a role of direct
van der Waals binding, they construct a suitable pocket for bulky hydrophobic
moieties, which might interact with the 4-isobutoxy tail.
The catalytic reaction of XOR involves a ping-pong mechanism alternating
between Mo6þ
and Mo4þ
states, and the inhibition constants for the two states are
different, so that febuxostat exhibits mixed-type inhibition in a Lineweaver–Burk
plot [7]. However, the inhibition constants are extremely low in both cases, Ki ¼ 1.2
 10À10
M and Ki ¼ 9 Â 10À10
M for the oxidized and reduced forms of molybdenum,
respectively. Thus, unlike allopurinol, the inhibitory activity of febuxostat is
not much influenced by the valency of molybdenum. This indicates that febuxostat
can bind to the enzyme irrespective of its redox state. That is to say, febuxostat
binds stably to XOR throughout the in vivo lifetime of XOR (about 36 h from synthesis
to degradation). It was expected from these crystallographic and enzymological
analyses that a low administration frequency and low dose of febuxostat would
be suitable for the treatment of gout. Indeed, clinical studies conducted in the
United States and Japan supported this prediction (as described below).
15.5 Mechanism of XOR Inhibition by Febuxostat j371
Allopurinol contains a purine-like structure, and might cause adverse effects by
interacting with purine metabolic enzymes other than XOR. Both allopurinol and
oxypurinol are structurally similar to purines. As a result, they are substrates for
certain purine and pyrimidine metabolic enzymes, such as hypoxanthineguanine
phosphoribosyltransferase, or orotate phosphoribosyltransferase (HGPRT), which
convert them to the corresponding ribonucleotides, that is, allopurinol-10
-ribonucleotide,
oxypurinol-10
-ribonucleotide, and oxypurinol-70
-ribonucleotide [26–29].
These ribonucleotides are quite potent inhibitors of the pyrimidine biosynthesis
enzyme orotidine-50
-monophosphate decarboxylase, with Ki values of 10À10
M
order of magnitude [30–32]. On the other hand, febuxostat does not have a structural
similarity to purine, and does not inhibit enzymes such as guanine deaminase,
purine nucleoside phosphorylase, and HGPRT at concentrations lower than
10À4
M [33].
15.6
Excretion of XOR Inhibitors
Oxipurinol, an oxidation product of allopurinol, is excreted from the kidney into the
urine, as is uric acid [34], so patients with renal dysfunction tend to have delayed
excretion of oxipurinol as well as a high blood concentration of oxipurinol. An
increased blood concentration of oxipurinol is correlated with the occurrence of
adverse side effects; therefore, the administration of allopurinol must be adjusted
with respect to the level of kidney function of the patients [2]. In contrast to allopurinol,
febuxostat has multiple excretion routes, being excreted into urine and feces
in a well-balanced manner after conjugation with glucuronic acid in the liver.
Therefore, it was expected that febuxostat could be administered to patients who
have a mild to moderate decrease in kidney function without the need to adjust the
dose. Clinical trials confirmed that febuxostat could be used safely in patients with
renal impairment [35–37].
15.7
Results of Clinical Trials of Febuxostat in Patients with Hyperuricemia and Gout
In a phase III placebo-controlled double-blind comparative study conducted among
Japanese patients with hyperuricemia (including gout patients), administration of
20 or 40 mg of febuxostat once a day reduced serum uric acid concentration to
below the target value of 6.0 mg/dl [38]. The ratio of patients who achieved a uric
acid concentration of lower than 6 mg/dl (i.e., the achievement ratio) was 47 or
83% for patients who received 20 or 40 mg of febuxostat, respectively [39]. Notably,
the results in the group given 40 mg of febuxostat, in terms of both the ratio of
change in serum uric acid concentration and the achievement rate of serum uric
acid concentration below 6 mg/dl, were significantly superior to those in the group
given 200 mg allopurinol [40]. Moreover, in a phase III allopurinol-controlled
372j15 A New-Generation Uric Acid Production Inhibitor: Febuxostat
clinical trial conducted in the United States, the ratio of patients achieving uric acid
concentrations below 6 mg/dl was significantly higher in the 80 or 120mg febuxostat
group, as compared with the 300 mg allopurinol group [10]. There were no severe
adverse effects that developed into clinically significant problems in either of the
febuxostat groups, and there was no dose-dependent increase of adverse effects.
15.8
Summary
Febuxostat binds tightly to XOR, the target enzyme, via multiple weak interactions.
The inhibition mechanism of febuxostat is completely different from that of allopurinol;
in particular, its inhibitory effect on XOR remains almost constant over the
in vivo lifetime of the XOR molecule, and it does not inhibit other enzymes involved
in nucleic acid metabolism. In these respects, febuxostat is a more effective inhibitor
of uric acid production than allopurinol, and its safety and efficacy for treatment of
hyperuricemia (including gout) have been confirmed in phase III clinical trials.
15.9
Added in proof
Febuxostat inhibits bovine XOR with a Ki in the picomolar-order, but it is a much
weaker inhibitor of Rhodobacter capsulatus XOR with a high Ki value (17.5 mM),
even though the three-dimensional structures of the substrate-binding pockets are
well-conserved and both enzymes permit the inhibitor to be accommodated in the
active site as indicated by computational docking studies [41]. Molecular dynamics
simulations indicate that differences in mobility of hydrophobic residues that do
not directly interact with the substrate account for establishing a pocket well suited
to accommodate bulky hydrophobic moieties of febuxostat.
References
1 Elion, G.B. (1966) Enzymatic and
metabolic studies with allopurinol. Ann.
Rheum. Dis., 25, 608–614.
2 Hande, K.R., Noone, R.M., and Stone, W.
J. (1984) Severe allopurinol toxicity.
Description and guidelines for prevention
in patients with renal insufficiency. Am. J.
Med., 76, 47–56.
3 Singer, S., Elion, G.B., and Hitchings, G.
H. (1966) Resistance to inhibitors of
dihydrofolate reductase in strains of
Lactobacillus casei and Proteus vulgaris. J.
Gen. Microbiol., 42, 185–196.
4 Borges, F., Fernandes, E., and Roleira, F.
(2002) Progress towards the discovery of
xanthine oxidase inhibitors. Curr. Med.
Chem., 9, 195–217.
5 Elion, G.B., Kovensky, A., and Hitchings,
G.H. (1966) Metabolic studies of
allopurinol, an inhibitor of xanthine
oxidase. Biochem. Pharmacol., 15, 863–880.
6 Okamoto, K. and Nishino, T. (1995)
Mechanism of inhibition of xanthine
oxidase with a new tight binding inhibitor.
J. Biol. Chem., 270, 7816–7821.
References j373
7 Okamoto, K., Eger, B.T., Nishino, T.,
Kondo, S., Pai, E.F., and Nishino, T. (2003)
An extremely potent inhibitor of xanthine
oxidoreductase. Crystal structure of the
enzyme–inhibitor complex and
mechanism of inhibition. J. Biol. Chem.,
278, 1848–1855.
8 Fukunari, A., Okamoto, K., Nishino, T.,
Eger, B.T., Pai, E.F., Kamezawa, M.,
Yamada, I., and Kato, N. (2004) Y-700 [1-[3-
cyano-4-(2,2-dimethylpropoxy)phenyl]-1Hpyrazole-4-carboxylic
acid]: a potent
xanthine oxidoreductase inhibitor with
hepatic excretion. J. Pharmacol. Exp. Ther.,
311, 519–528.
9 Okamoto, K., Matsumoto, K., Hille, R.,
Eger, B.T., Pai, E.F., and Nishino, T. (2004)
The crystal structure of xanthine
oxidoreductase during catalysis:
implications for reaction mechanism and
enzyme inhibition. Proc. Natl. Acad. Sci.
USA, 101, 7931–7936.
10 Becker, M.A., Schumacher, H.R., Jr.,
Wortmann, R.L., MacDonald, P.A.,
Eustace, D., Palo, W.A., Streit, J., and
Joseph-Ridge, N. (2005) Febuxostat
compared with allopurinol in patients
with hyperuricemia and gout. N. Engl. J.
Med., 353, 2450–2461.
11 Bruce, S.P. (2006) Febuxostat: a selective
xanthine oxidase inhibitor for the
treatment of hyperuricemia and gout.
Ann. Pharmacother., 40, 2187–2194.
12 Nishino, T., Okamoto, K., Eger, B.T., Pai,
E.F., and Nishino, T. (2008) Mammalian
xanthine oxidoreductase – mechanism of
transition from xanthine dehydrogenase
to xanthine oxidase. FEBS J., 275 (13),
3278–3289.
13 Nishino, T. (1994) The conversion of
xanthine dehydrogenase to xanthine
oxidase and the role of the enzyme in
reperfusion injury. J. Biochem., 116 (1), 1–6.
14 Simmonds, H.A., Reiter, S., and Nishino,
T. (1995) Hereditary xanthinuria, in The
Metabolic and Molecular Basis of Inherited
Disease, 7th edn, vol. II (eds C.R. Scriver,
A.L. Beaudet, W.S. Sly, and D. Valle),
McGraw-Hill, New York, pp. 1781–1797.
15 Enroth, C., Eger, B.T., Okamoto, K.,
Nishino, T., Nishino, T., and Pai, E.F.
(2000) Crystal structures of bovine milk
xanthine dehydrogenase and xanthine
oxidase: structure-based mechanism of
conversion. Proc. Natl. Acad. Sci. USA, 97
(20), 10723–10728.
16 Okamoto, K., Kawaguchi, Y., Eger, B.T.,
Pai, E.F., and Nishino, T. (2010) Crystal
structures of urate bound form of
xanthine oxidoreductase: substrate
orientation and structure of the key
reaction intermediate. J. Am. Chem. Soc.,
132 (48), 17080–17083.
17 Hille, R. (1996) The mononuclear
molybdenum enzymes. Chem. Rev., 96 (7),
2757–2816.
18 Spector, T. and Johns, D.G. (1968)
Oxidation of 4-hydroxypyrazolo(3,4-d)
pyrimidine by xanthine oxidase, the route
of electron transfer from substrate to
acceptor dyes. Biochem. Biophys. Res.
Commun., 32 (6), 1039–1044.
19 Spector, T. and Johns, D.G. (1970) 4Hydroxypyrazolo(3,4-d)pyrimidine
as a
substrate for xanthine oxidase: loss of
conventional substrate activity with
catalytic cycling of the enzyme. Biochem.
Biophys. Res. Commun., 38 (4),
583–589.
20 Massey, V., Komai, H., Palmer, G., and
Elion, G.B. (1970) The existence of
nonfunctional active sites in milk
xanthine oxidase: reaction with functional
active site inhibitors. Vitam. Horm., 28,
505–531.
21 Springer, R.H., Dimmitt, M.K.,
Novinson, T., O’Brien, D.E., Robins, R.
K., Simon, L.N., and Miller, J.P. (1976)
Synthesis and enzymic activity of some
novel xanthine oxidase inhibitors. J.
Med. Chem., 19, 291–296.
22 Silipo, C. and Hansch, C. (1976) Correlation
analysis of Baker’s studies on enzyme
inhibition. J. Med. Chem., 19, 62–71.
23 Fujii, S., Kawamura, H., Kiyokawa, H.,
and Yamada, S. (2012) Pyrazolotriazine
compounds. EP 269859, JP 89079184, US
Patent 4,824,834.
24 Baldwin, J.J., Kasinger, P.A., Novello, F.C.,
and Sprague, J.M. (1975)
4-Trifluoromethylimidazoles and
5-(4-pyridyl)-1,2,4-triazoles, new classes of
xanthine oxidase inhibitors. J. Med.
Chem., 18, 895–900.
25 Wortmann, R.L., Ridolfo, A.S., Lightfoot, R.
W., Jr., and Fox, I.H. (1985)
374j15 A New-Generation Uric Acid Production Inhibitor: Febuxostat
Antihyperuricemic properties of amflutizole
in gout. J. Rheum., 12, 540–543.
26 Reiter, S., Loffler, W., Grobner, W., and
Zollner, N. (1986) Urinary oxipurinol-1riboside
excretion and allopurinol-induced
oroticaciduria. Adv. Exp. Med. Biol., 195
(Part A), 453–460.
27 Simmonds, H.A., Reiter, S., Davies, P.M.,
and Cameron, J.S. (1991) Orotidine
accumulation in human erythrocytes
during allopurinol therapy: association
with high urinary oxypurinol-7-riboside
concentrations in renal failure and in the
Lesch–Nyhan syndrome. Clin. Sci., 80,
191–197.
28 Nelson, D.J. and Elion, G.B. (1984)
Metabolic studies of high doses of
allopurinol in humans. Adv. Exp. Med.
Biol., 165Pt (A), 167–170.
29 Krenitsky, T.A., Elion, G.B., Strelitz, R.
A., and Hitchings, G.H. (1967)
Ribonucleosides of allopurinol and
oxoallopurinol. Isolation from human
urine, enzymatic synthesis, and
characterization. J. Biol. Chem., 242,
2675–2682.
30 Kelley, W.N. and Beardmore, T.D. (1970)
Allopurinol: alteration in pyrimidine
metabolism in man. Science, 169, 388–390.
31 Fyfe, J.A., Miller, R.L., and Krenitsky, T.A.
(1973) Kinetic properties and inhibition of
orotidine 50
-phosphate decarboxylase.
Effects of some allopurinol metabolites on
the enzyme. J. Biol. Chem., 248, 3801–3809.
32 Brown, G.K. and O’Sullivan, W.J. (1977)
Inhibition of human erythrocyte
orotidylate decarboxylase. Biochem.
Pharmacol., 26, 1947–1950.
33 Takano, Y., Hase-Aoki, K., Horiuchi, H.,
Zhao, L., Kasahara, Y., Kondo, S., and
Becker, M.A. (2005) Selectivity of
febuxostat, a novel non-purine inhibitor of
xanthine oxidase/xanthine
dehydrogenase. Life Sci., 76, 1835–1847.
34 Elion, G.B., Yu, T.F., Gutman, A.B., and
Hitchings, G.H. (1968) Renal clearance of
oxipurinol, the chief metabolite of
allopurinol. Am. J. Med., 45, 69–77.
35 Hoshide, S., Takahashi, Y., Ishikawa, T.,
Kubo, J., Tsuchimoto, M., Komoriya, K.,
Ohno, I., and Hosoya, T. (2004) PK/PD
and safety of a single dose of TMX-67
(febuxostat) in subjects with mild and
moderate renal impairment. Nucleosides
Nucleotides Nucleic Acids, 23, 1117–1118.
36 Mayer, M.D., Khosravan, R., Vernillet, L.,
Wu, J.T., Joseph-Ridge, N., and Mulford,
D.J. (2005) Pharmacokinetics and
pharmacodynamics of febuxostat, a new
non-purine selective inhibitor of xanthine
oxidase in subjects with renal
impairment. Am. J. Ther., 12, 22–34.
37 Becker, M.A., Schumacher, H.R.,
Espinoza, L.R., Wells, A.F., MacDonald,
P., Lloyd, E., and Lademacher, C. (2010)
The urate-lowering efficacy and safety of
febuxostat in the treatment of the
hyperuricemia of gout: the CONFIRMS
trial. Arthritis Res. Ther., 12 (2), R63.
38 Shoji, A., Yamanaka, H., and Kamatani, N.
(2004) A retrospective study of the
relationship between serum urate level
and recurrent attacks of gouty arthritis:
evidence for reduction of recurrent gouty
arthritis with antihyperuricemic therapy.
Arthritis Rheum., 51, 321–325.
39 Naoyuki, K., Shin, F., Toshikazu, H.,
Tatsuo, H., Kenjiro, K., Toshitaka, N.,
Takanori, U., Tetsuya. Y., Hisashi, Y., and
Yuji, M. (2011) Placebo-controlled doubleblind
dose-response study of the nonpurine-selective
xanthine oxidase inhibitor
febuxostat (TMX-67) in patients with
hyperuricemia (including gout patients) in
japan: late phase 2 clinical study. J. Clin.
Rheumatol. 17(Suppl 2), S35–43.
40 Kamatani, N., Fujimori, S., Hada, T.,
Hosoya, T., Kohri, K., Nakamura, T.,
Ueda, T., Yamamoto, T., Yamanaka, H.,
and Matsuzawa, Y. (2011) An
allopurinol-controlled, randomized,
double-dummy, double-blind, parallel
between-group, comparative study of
febuxostat (TMX-67), a non-purineselective
inhibitor of xanthine oxidase,
in patients with hyperuricemia
including those with gout in Japan:
phase 3 clinical study. J. Clin
Rheumatol. 17(Suppl 2), S13–18.
41 Kikuchi, H., Fujisaki, H., Furuta, T.,
Okamoto, K., Leimkühler, S., and
Nishino, T. (2012) Different inhibitory
potency of febuxostat towards mammalian
and bacterial xanthine oxidoreductases:
insight from molecular dynamics. Sci Rep.
2, 331.
References j375
Ken Okamoto studied medicine at Yamanashi Medical College,
receiving an M.D. in 1990. He studied biochemistry at
Yokohama City University, receiving a Ph.D. in 1994 under
Professor Takeshi Nishino, and was a Research Associate at
Nippon Medical School with Professor Taro Okazaki and
Takeshi Nishino in 1994. In 2000 and 2001, he learned X-ray
crystallography at the University of Toronto under Professor
Emil Pai. In 2005, he was an Associate Professor at Department
of Biochemistry and Molecular Biology at Nippon Medical
School.
Shiro Kondo studied chemistry at Tokyo Institute of Technology
in Japan and finished the master’s course in 1978. He
then joined Teijin Ltd. and mainly worked in the drug discovery
section. He successfully launched the new XO inhibitor
project and was the main inventor of febuxostat. He also contributed
to the creation of some clinical candidate compounds
for the treatment of metabolic syndrome and
cardiovascular diseases. He was a Director of Pharmaceutical
Development Research Laboratories in 2004 and Discovery
Research Laboratories in 2006. He was awarded the title of Fellow of Teijin Ltd. in
2009 and is currently Head of the Kondo Research Laboratory of Teijin.
Takeshi Nishino studied medicine at Yokohama City University
School of Medicine receiving an M.D. in 1970 and then
studied biochemistry at Department of Biochemistry, Graduate
School of Yokohama City University, under Professor
Keizo Tsushima receiving Ph.D. in 1975. He was a Postdoctoral
Fellow in 1976–1978 at Department of Biological
Chemistry of The University of Michigan, Ann Arbor, with
Professor Vincent Massey. He was an Assistant Professor
and then an Associate Professor at Department of Biochemistry
at Yokohama City University School of Medicine in 1976–1991. In 1992,
he moved to Department of Biochemistry and Molecular Biology at Nippon Medical
School in Tokyo, where he was appointed Professor and Chairman and then
Professor Emeritus of Nippon Medical School in 2009. He was appointed the Distinguished
Visiting Scholar of Biochemistry in University of California, Riverside,
in 2009–2010. In 2011, he was appointed Specially Employed Professor of The
Graduate School of Agricultural and Life Science at The University of Tokyo. He
has been studying the various aspects of xanthine oxidoreductase and its related
enzymes and has authored over 150 scientific publications. He was the Vice President
of the Japanese Biochemical Society Meeting and was the Chairman of the
International Symposium on Flavins and Flavoproteins in 2005.
376j15 A New-Generation Uric Acid Production Inhibitor: Febuxostat
Index
a
absorption 8
acetaminophen (paracetamol) 61, 298, 299
acetylcholine 271
active metabolite (AM) 143–145, 147,
150, 173
acute coronary syndrome 142, 150, 153, 157
acute myelogenous leukemia (AML) 119, 125
acute nociceptive pain models See also
pain 312
acute pain See also pain 297
adenosine 5’-diphosphate (ADP) 142
adenosine triphosphate (ATP) 152
ADME (absorption, distribution, metabolism
and excretion) 38, 39, 92, 104
ADP-induced platelet aggregation 144,
145, 152
afimoxifene 168, 171, 175
agranulocytosis 12, 61, 65
aldehyde oxidase (AO) 38, 46–48
allodynia 297
allopurinol 365, 367, 368
almotriptan 48, 49
alogliptin 8
Ames test 54, 369
AMG458 60
aminodiaquine 65–66
amitriptyline 270, 271
amoxapine 12, 13
analogue-based drug discovery (ABDD) 12,
16, 21, 22, 25–27, 167, 176, 180,
288, 320
analogue drug 3, 16, 22, 25
anastrozole 24, 25
angina pectoris 10
angiotensin-converting enzyme (ACE) 6
anovulation 170, 171
antiandrogen effect 6
anticancer therapy 116
anticholinergic effect 14
anticoagulation and treatment 8, 244, 245, 258
anticonvulsants 299, 300
antidepressant drug 13, 299, 300
antidiuretic hormone (ADH) 187
antiepileptic drug 66
antiestrogens 176
antihypertensive therapy 4
antimalarial agent 65
antimetabolite 118
antimigraine drugs 48, 49
anti-obesity agent 31, 59, 66, 67
antiplatelet agents 142, 144
antithrombin (AT) 244
apixaban 245
aplastic anemia 61
apparent intrinsic clearance (CLint,app) 39, 40
aquaretics 197, 203
arachidonic acid 213, 299
argatroban 8, 10, 245
arginine vasopressin (AVP) 187, 188, 203
ArimidexTM
24
aromatase inhibitor (AI) 24, 25
aromatic cyano group 272
arrhythmias 10
aspirin (ASA) 142, 149, 256, 298
aspirin-induced asthma 232
asthma 51, 212, 230
atenolol 11
atherosclerosis 258
atherothrombosis 142
atrial fibrillation 246, 258–260
atypical antipsychotic agents 12
5-azacytidine (5-azaC) 118, 119
– clinical trials 119, 120
– derivatives 121
– mechanism of action 119, 120
– prodrug 120
– synthesis 120
j377
Analogue-based Drug Discovery III, First Edition. Edited by János Fischer, C. Robin Ganellin, and
David P. Rotella.
Ó 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
5-aza-2’-deoxycytidine (5-aza-dC) 118, 119
– mechanism of action 121
azumamide E 132
b
baclofen 299
bazedoxifene 168, 171, 179
b adrenergic receptor antagonists 10
b1- and b2 adrenergic receptors 11
b-adrenergic receptor agonists 212
17b-estradiol 166, 167
bioavailability 9, 99, 122, 179, 195, 223,
224, 277
bioisosteric replacement 64
bivalirudin 8, 245
bleeding liability 143, 144, 150, 156, 259,
260, 300
blood-brain barrier 314, 330
blood coagulation cascade 243, 244
blood pressure 188
boceprevir (VictrelisTM
) 343
– backup research 352
– tert-butyl urea analogue (boceprevir) 352
– discovery 344–352, 358
– hydrogen bonding network with the
enzyme 357
– macrocyclic lead molecule 349
– optimization of the PK properties by
analogues 350
– truncated analogue of the lead molecule 346
– undecapeptide lead molecule 344–345
– X-ray studies 345, 346, 348
breast cancer 24, 172, 175–178, 322
– prevention 179
bromodomain inhibitor 133
bronchial asthma 11
bronchoconstriction 213
buprenorphine 298
burimamide 5
burkholdac B (thailandepsin A) 131
c
cabazitaxel (JevtanaTM
) 319, 322
– chemical and physical properties 323
– clinical studies 330–336
– preclinical development 328–330
– structure-activity relationships 323
– synthesis 325–327
cachexia 78
caco-2 assay 97
calcitonin gene-related peptide 298
calcium channel blocker 41
cancer chemotherapeutic drugs 320
cangrelor 153, 154
cannabinoid receptor 1 antagonists (CB1
antagonists) 28–32, 66, 67
capecitabine 333
captopril 6, 7
carbamazepine 299
celecoxib 45, 299
CelexaTM
see citalopram 272
chemical named entity recognition (NER) 25
chlorothiazide 4
chlorotrianisene (TACE) 167
chlorpromazine 12–14
chromatine 114, 116, 117
Chromobacterium violaceum 128
chromosome 114
chronic constriction injury neuropathy pain
(CCI) See also pain 313
chronic heart failure 203
chronic lower back pain See also pain 312
chronic neuropathic pain models
See also pain 312
chronic pain See also pain 297
Churg-Strauss syndrome (CSS) 231
cimetidine 5, 6
CipralexTM
see escitalopram
CipramilTM
see citalopram
cisapride 79
citalopram 14, 15, 269, 271, 288
– binding of the enantiomers to the SERT 280
– clinical efficacy 277
– discovery 272–277
– metabolism of the enantiomers 280
– pharmacological profile 276
– R-citalopram’s inhibition of
escitalopram 279, 280
– synthesis 275
clearance 45, 48, 58, 92, 94
clogP 32, 89, 103
clomiphene 167–171, 180
clomipramine 284
clonidine 299
clopidogrel 143, 145, 149, 154
clorgyline 48
clozapine 12, 13, 62, 63
cocaine 296
codeine 298, 301
colorectal distension-induced visceral pain
model (rat) See also pain 313
combinatorial chemistry 79
competition 21, 22, 25, 26, 216
conivaptan 199–201
constipation 12, 78
contraceptives See non-steroidal
contraceptives 171, 172
controlled-release formulations 314
378j Index
Controlled Substances Act (CSA) 296
coronary artery disease (CAD) 155
corticosteroids 212
corticotropin-releasing factor-1
antagonists 62–64
covalent adduct 123
cyclic adenosine monophosphate (cAMP) 188
CTP-347 56–58
cutaneous T-cell lymphoma (CTCL) 133
cyclooxygenases (COX-1 and COX-2) 299
CYP1A2 48, 147, 314
CYP3A 147
CYP2B6 145, 147, 302
CYP2C9 147, 314
CYP2C19 145, 147, 150
CYP2D6 46, 48, 56, 174, 302, 314
CYP3A4 39–41, 45, 47, 48, 54, 58–60, 155, 302,
314
cysteinyl leukotrienes (CysLTs, LTC4, LTD4 and
LTE4) 213, 215
– See also leukotrienes
cysteinyl leukotriene 1 receptor (CysLT1R)
antagonists 211, 212, 217, 225, 229
cytochrome P450 (CYP) enzymes 6, 38, 42, 46,
174
– mechanism-based inactivation 38
– time-dependent inactivation 56, 58
cytosine 114, 116, 117
cytosine phosphate guanine dinucleotide
(CpG) 116
cytotoxicity 82, 121, 122
d
dabigatran etexilate 9, 10, 243, 246–264
– clinical studies and indications 258
– double prodrug 254
– in vitro and in vivo activity 252
– log P 252
– preclinical pharmacology 254
– selectivity profile 253
10-deacetylbaccatin III (10-DAB) 321, 325
deep venous thrombosis (DVT)
prophylaxis 259
delta opioid receptor (DOR) 311
depression treatment 270
desipramine 271, 284, 312
N-desmethyltamoxifen 174
O-desmethyltramadol 302
deuterated paroxetine analogue 56, 57
deuteration 58
dextromethorphan 58
diabetes, type 2 7
diabetic gastroparesis 92
diabetic neuropathy 298, 312
diabetic polyneuropathy pain model (rat) 313
diazepam 6
diclofenac 299
diethylstilbestrol (DES) 167
dihydro-5-azacytidine 122
diltiazem 41
dipeptidyl peptidase IV (DPP IV) inhibitor 7
diphenhydramine 14, 15
direct-acting P2Y12 antagonists 152
direct analogue 4
direct thrombininhibitors See also thrombin 8,
9, 245
– bivalent 8
– univalent 8
DMPK (distribution, metabolism, and
phamacokinetics) 230, 277
DNA 52, 114
DNA methyltransferases (DNMTs) 116, 117
DNMT inhibitors 118, 121–124
docetaxel (TaxotereTM
) 321, 322
DOPA decarboxylase 4
dopamine 78, 311
dopamine transporter (DAT) inhibition 272
double prodrug See also prodrug 9, 253
drug class 3, 16, 22
drug-drug interactions (DDIs) 51, 54, 56, 58,
59, 61, 99, 245, 277, 314, 333
“drug-like” molecules 38, 79, 103
drug metabolizing enzymes (DMEs) 38
drug types 3
dual-acting drugs 10, 11, 301, 311, 315
dual antiplatelet therapy 142, 145
duloxetine 287, 299
duration of action 41
dyspnea 154, 156, 158
e
early-phase analogues 8, 16
edoxaban 245
electrophilic compounds 60
eletriptan 48, 49
elinogrel 157
enalapril 7
enclomiphene 169
endogenous estrogen 167
endorphins 299, 300
endoxifen 173
enkephalins 299
enoxaparin 259
eosinophilia 231
epigallocatechin gallate 123
epigenetic code 115
epigenetic modulation 116
epigenetics 114, 133
Index j379
epigenome 114, 115
epothilone 320
eraser enzymes 115
escitalopram (CipralexTM
, LexaproTM
) 15, 269,
271, 288
– allosteric binding site at SERT 273, 274, 280,
282, 284–286
– anxiolytic-like activity 280
– clinical efficacy 287
– derivatives 284
– pharmacological profile 279, 281–285
– positron emission tomography (PET) 282
– synthesis 278
estrogen receptor (ER) 166
ethamoxytriphetol (MER-26) 167
– antifertility action in animals 167
etoricoxib 299
euchromatin 114
eukaryots 114
extended-release formulation 296
extrapyramidal side effects (EPS) 12
ezetimibe 44, 45
f
factor Xa (FXa) 244, 245
fadrozole 24
famotidine 6
fazarabine 122
febuxostat 365
– discovery 369–373
– lead optimization 369, 370
– mixed-type inhibition 371
– 2-phenyl-4-methyl-5-thiazolecarboxylic
(lead compound) 369
– selectivity 372
– use in patients with renal impairment 372
– X-ray of febuxostat-bound bovine XOR 370
felbamate 66, 67
FemaraTM
24
fentanyl 296, 298, 300
fibrin 244, 258
fibrinogen 244, 258
fibromyalgia 301
FK228 (romidepsin, IstodaxTM
) 129
– bacterial fermentation 129
– total synthesis 130
flavin-containing monooxygenase (FMO) 174
fluorine substitution 42–44
fluorofelbamate 67
5-fluoro-2’-deoxycytidine 122
5-fluorouracil 118
fluoxetine 14, 15, 276, 283
focal adhesion kinase inhibitors 58
formalin model 313
FPL-55712 212, 213, 215, 219, 220, 228
frovatriptan 48, 49
Fukuyama-Mitsunobu reaction 81
fulvestrant 166, 167, 171
functional dyspepsia 78
g
gabapentin 299
gastric cancer 322
gastric hyperacidity 5
gastrointestinal (GI) motility 77–79, 100
gastroparesis 78
gene expression 114, 116
gene mutation 114
genetic polymorphism 147, 156, 174
genome 114, 124
genotoxicity 38, 51, 52, 54, 61
genotype 114
GERD (gastroesophageal reflux disease) 79
ghrelin 77, 78
ghrelin agonists 77
ghrelin receptor (GRLN) 82
GI (gastrointestinal) prokinetic agent 79
glucuronidation 50, 51
glutamate 298
glutathione (GSH) 51, 52, 58, 60–67, 77, 78
gonadotropin releasing hormone
(GnRH) 169
gout 365, 371–373
G-protein-coupled receptors (GPCRs) 25,
26, 187
granulocytopenia 5
green tea 123
growth hormone (GH) 77
growth hormone secretagogues (GHS) 77, 78
guinea-pig ileum strips 216
guinea-pig labored abdominal breathing
model 222
guinea-pig tracheal spirals 216
gynecomastia 6
h
half-life 8, 45, 46, 48, 51, 92, 94, 179, 277,
302, 314
haloperidol 12
HCV NS3 protease inhibitors 344–359
head cancer 322
heat shock protein (HSP) 166, 178
heparin 244, 259
– See also low molecular weight heparin
heparin-induced thrombocytopenia 245
hepatic side effects 9
– alanine aminotransferase 9
– bilirubin 9
380j Index
hepatitis C virus (HCV) 343
– bloodborne pathogen 344
– epidemic 343
– viral life cycle 343
hepatocellular carcinoma 343
hepatocytes 51
hepatotoxicity 61, 62, 65, 66, 232, 245
heterochromatin 114
hexamethylene bisacetamide 125, 126
high-throughput screening (HTS) 82
hirudin 8
histamine 212, 271
histamine H1-receptor antagonist 14
histamine H2-receptor antagonist 5
histone deacetylase (HDAC) 124
– catalytic reaction mechanism 125
– selectivity 125
– X-ray structure 124
– zinc-dependent and inhibitors 124–132
histone proteins 114, 115
HIV-1 protease inhibitor 41
hormone refractory metastatic prostate
cancer (mHRPC) 323, 336
hormone replacement therapy (HRT) 180
hot plate model (mouse) 313
HPLC (high-pressure liquid
chromatography) 278
human carboxyl esterases (hCEs) 147, 150
human immunodefficiency virus (HIV) 40
human liver cytosol 47, 51
human liver microsomes (HLMs) 37–40, 42,
58, 64, 97
hydralazine 123
hydrochlorothiazide 4
5-hydroxyindoleacetic acid (5-HIAA) 277
5-hydroxytryptamine 2C receptor agonist 52
hyperalgesia 297
hypertension 10
hyperuricemia 157, 372, 373
hyponatremia 198, 201
i
ibuprofen 299, 312
ICI 198615 223
ICI 198707 223
idiosyncratic toxicity 38, 56, 61, 63
idoxifene 175
imipramine 13–15, 270, 271
immature rat uterotrophic assay 176
immediate-release formulation 296, 301
indinavir 40
indoleacetic acid metabolite 48
inhalers 214
intracranial glioblastomas 336
intraocular pressure (IOP) 23
intravenous ( i.v.) administration 92
iproniazid 270
irritable bowel syndrome (IBS) 78, 79
j
JevtanaTM
see cabazitaxel
k
Kaposi’s sarcoma 322
kappa opioid receptor (KOR) 311
ketobemidone 298
l
L-648051 225, 230
L-649923 225
L-695499 226
largazole 131
larotaxel 322
lasofoxifene 168, 171
latanoprost 23
lead compound 14, 15, 24, 50, 125, 225, 227,
249, 369
lead optimization 197, 344, 345, 369
letrozole 24, 25
leukotrienes See also cysteinyl
leukotrienes 213, 215, 216
levodopa 3
levomethadone 298
LexaproTM
see escitalopram
ligand efficiency 304, 345, 350
linagliptin 8
lipophilicity 39, 48
5-lipoxygenase (5-LO) 50, 51, 213, 214
5-lipoxygenase activating protein (FLAP) 215
liver cirrhosis 343
liver transplantation 343
lixivaptan 201
loop diuretics 199
loperamide 298, 300
loxapine 62, 63
low molecular weight heparin (LMWH) 244,
246, 259
LTD4 binding assays 217
LTD4-derived compounds 220
LTD4 molecular modeling 219
lung cancer 322
LY-171883 223, 227, 230
m
macrocycles in drug design 77, 79, 80
major depressive disorder 277, 287
MAO-A 48, 49
MAO-B 48
Index j381
matrix metalloproteinases 124
mechanism-based CYP inactivation 38
melagatran 9
melanocortin-4 receptor 59, 60
melitracen 270, 271
meloxicam 62, 63
mercaptopurine 118
metabolic soft spots 44
metabolic stability 97, 345
metabolism sites 43
metamizol (dipyrone) 299
metastatic breast cancer 333, 336
metastatic castration-resistant prostate
cancer (mCRPC) 323, 336
methamphetamine 296
N-methylacetamide 125, 126
methyldopa 3, 4
methylphenidate 296
metiamide 5
metoclopramide 79
5-methylcytosine 114, 118
methyl binding domains (MBDs) 116
MI complex (metabolite-intermediate
complex) 56, 59
mibefradil 54
Michael addition 122, 123
microdialysis 280
microtubules 320
midazolam 58
mitoxantrone 323, 334, 336
MK-0571 226, 230
MK-0679 231
mode of action (MoA) 22, 23, 142, 152
– dual mode of action 296
MOE (molecular operating environment) drug
discovery software 192, 194
molecular weight (MW) 103
molibdopterin 368
monoamine oxidases (MAO) See also MAO-A
and MAO-II 38, 46, 48
monoamine oxidase inhibitor (MAOI) 270
monotarget drugs 5
montelukast sodium (MK-0476) 211, 212, 214,
229, 232
– discovery 224–227
morphine 11, 296, 297, 300, 301, 311,
312, 314
mosavaptan (OPC-31260, PhysulineTM
) 197
mucous secretion 213
multitarget drugs 12
mu (m) opioid receptor (MOP) agonists 11,
296, 300, 301, 315
mustard oil visceral pain model (rat)
See also pain 313
mutagenicity 51, 369
myelodysplastic syndromes (MDS) 119–121,
125
n
nafoxidine 168
naloxone 301
nanaomycin A 123
NAPAP and analogues 246–248
NAPQI (N-acetyl-p-benzoquinone imine) 62
naratriptan 48, 49
narlaprevir 343
– boceprevir analogues 352
– discovery of narleprevir 355–359
– improving cellular activities of
analogues 353
– X-ray studies 353, 356
natural products 119, 123, 126, 131, 132, 320
nebulizers 214
neck cancer 322
nelivaptan (SSR 149415) 199
neuropathic lower back pain 314
neuropathic pain 297, 298, 300
neutropenia 144, 332, 334
neutrophils 51
nisoxetine 283
nociceptive pain see also pain 297, 298
nonsteroidal anti-inflammatory drugs
(NSAIDs) 299, 300, 312, 314
nonsteroidal contraceptives 171, 172
nonsteroidal estrogen 167
norepinephrine (NE) 271, 299, 311
norepinephrine transporter (NET)
inhibition 272
norepinephrine reuptake inhibitor 296, 301,
311, 315
nortriptyline 270, 271
nucleosome 114
nutraceuticals 133
o
obsessive compulsive disorder 277, 287
olanzapine 12
onset time 314
OPC-18549 189
OPC-21268 192
ophena 168
opioid drugs 11, 299, 300
opioid receptors 311
oral bioavailability 92, 122
ORL1 (NOP, nociceptinreceptor) receptor 311
ormeloxifene 168, 171
orthopedic surgery 245, 246, 259
ospemifene 171, 175
382j Index
osteoarthritis 297
osteoporosis 45, 58, 175
ovarian cancer 58, 322
ovulation induction 169
oxipurinol 367, 372
2-oxo-clopidogrel 146, 147
2-oxo-ticlodipine 145
oxycodone 12, 296, 298, 312, 313
oxytocin 188
p
paclitaxel (TaxolTM
) 320–322
pain 11, 296, 297, 299, 300
– acute noniceptive pain models 312
– acute pain 297
– chronic constriction injury neuropathic
pain (CCI) 313
– chronic lower back pain 312
– chronic pain 297
– colorectal distension-induced visceral pain
model (rat) 313
– mustard oil visceral pain model (rat) 313
– visceral pain model 312
panic disorders 277, 287
paracetamol see acetaminophen
Parkinson’s disease 42
paroxetine 56, 57, 276, 283, 287
patents 21
peg-interferon 343
peptic ulcer 5
percutaneous coronary artery intervention
(PCI) 144, 150, 153, 157
peroxisome proliferator-activated receptor-c
(PPAR-c) 49
“personalized medicine” 216
pethidine 298
PF-562271 58
permeability glycoprotein (P-gp) 32, 97, 129
– inhibitor 259
– overexpression 322
pharmacological analogue 6, 215, 229, 230
phenotype 114
phenylquinone-induced writhing model 313
phenytoin 299
pioglitazone 49, 50
pioneer drug 3, 4, 6, 8, 10, 13, 16, 21, 22, 25
piroxycam 299
plasma protein binding see PPB
platelet 142
polar surface area (PSA) 103
polycystic ovarian syndrome (PCOS) 170
polypharmacia 300
postoperative ileus (POI) 78
poststroke depression 277
poststroke pain 298
posttranslational modifications 115
post-traumatic stress disorder 277
PPB (plasma protein binding) 230, 250,
277, 314
pranlukast (ONO-1078) 211, 212, 214
– discovery 227–229
prasugrel 143, 145, 147–149, 152
prednisolone 323, 333
prednisone 323, 333
procainamide 123
prodrug 24, 123, 131, 143, 172, 174, 252
– See also double prodrug
profertility drugs 169
proinflammatory prostaglandins 299
pronethalol 10
propranolol 6, 10, 11
prostaglandin F2a (PGF2a) 23, 24
prostate cancer 322
protease-activated receptors (PARs) 244
protein kinases 25, 286
prothrombin 244
purinergic receptors (P2Y1 and P2Y12) 142,
143
P2Y12 receptor antagonists 142
q
quetiapine 12, 13, 62, 63
r
raloxifene (keoxifene, EvistaTM
) 168, 171,
175–180
ranitidine 6
rat arteriovenous shunt thrombosis model 148
reactive metabolite (RM) 38, 51, 52, 54, 60, 62,
64–68
reactive oxygen species 368
reader enzymes 115
regulatory agencies 52
relcovaptan (SR 49059) 199
reperfusion injury 368
ResculaTM
23
respiratory depression 12
restless legs syndrome 301
REV-5901 225
reversible inhibitor 8
RG108 123
rhinitis 232
ribavirin 343
rimonabant 28–32
rivaroxaban 245
rizatriptan 48, 49
RNA methyltransferases 117
romidepsin see FK228
Index j383
rosiglitazone 49, 50
rotable bond count 103
rule breaker 89
“rule of 5” 230, 231
s
S-adenosyl-methonine (SAM) 116–118
salicylates 299
Salmonella reverse mutation assay 52
satavaptan (SR 121463) 199
saxagliptin 8
sedative effect 14
selective estrogen receptor modulator
(SERM) 166, 167
selective norepinephrine reuptake inhibitor
(SNRI) 271, 287
selective serotonin reuptake inhibitors
(SSRI) 13, 14, 56, 271, 272
serendipity 12, 13, 288, 303
serotonin (5-hydroxytryptamine, 5-HT) 78,
270, 271, 299, 311
serotonin reuptake inhibitor 301
serotonin transporter (SERT) 276
sertraline 276
SGI-1027 123
simulated moving bed (SMB) technology 278
sirtuins (SIRTs) 124
sitagliptin 7, 8
SK&F 102922 226
skin rashes 61
slow onset time 245
social anxiety disorder 287
social phobia 277
solid-phase synthesis 83
solid tumors 332
solution-phase synthesis 87
spinal cord injury 298
spinal nerve ligation neuropathic pain
model (rat) 313
SRS-A 212, 213, 215, 217
stability 122
stand-alone drug 4
steady-state platelet inhibition 144
stent placement 144
Streptoverticillium ladakanus 119
streptozotocin (STZ) – induced heat
hyperalgesia 312
stroke prevention 144, 246, 259, 260
structure-activity relationship (SAR) 38, 44, 46,
50, 58, 68, 83–92, 144, 167, 190, 193, 194,
219, 250, 251, 272, 273–69, 303, 304,
323, 351
structure-based drug design 343, 344
– limitations 357, 358
suberoylanilide hydroxamic acid (SAHA)
125, 126
substance P 298
substrate analogue 7, 118
sudoxicam 62, 63
sulfotransferases (SULTs) 49
sumatriptan 48, 49
sustained-release formulations 301
syndrome of inappropriate antidiuretic
hormone (SIADH) 197
synergistic analgesic effect 302
t
tacedinaline 132
tail flick model (rat, mouse, dog) 313
talopram 14, 15, 271
talsupram (Lu 05-003) 271
tamoxifen 168, 171, 172–175, 178, 180
– metabolic pathways 173
tapentadol (PalexiaTM
) 11, 12, 295, 297
– chemical structure 296
– discovery 296–318
– pharmacokinetics and drug-drug
interactions 314
– preclinical and clinical profile 310–314
– reducedgastrointestinal (GI)sideeffects 313
– synegisitic dual mechanism of action 311
– synthesis 306–310
taranabant 66, 67
tardive dyskinesia 79
taxoids 320
TaxolTM
see paclitaxel
Taxus baccata (European yew) 322
Taxus brevifolia (Pacific yee tree) 321
tegaserod 79
telaprevir (IncivekTM
) 343
teprotide 6, 7
tesetaxel 321, 322
“tether” 80, 92, 93
theophylline 6
thienopyridines 143
thrombin 243–245
– X-ray crystal structure of thrombindabigatran-complex
252–253
– X-ray crystal structure of thrombinNAPAP-complex
246
thrombin inhibitors 8, 43
thrombopoietin receptor agonists 64
thrombosis 8, 243
thrombosis prevention 246
thrombotic thrombocytopenic purpura 144
thromboxane A2 (TXA2) 142
ticagrelor 154–157
ticlopidine 143, 144, 149
384j Index
tilidine 298
tolvaptan (SamscaTM
) 198
tomoxetine 283
tooth pulp stimulation (rabbit) 313
toremifene 168, 171, 175
tramadol 11, 296, 297, 300–303
transcription 114, 116
trichostatin A 126
tricyclic antidepressant (TCA) 270, 277, 284
triptan class 48, 49
troglizazone 49, 50
tumor resistance 322
TZP-102 92
u
U19052 220, 221
ulimorelin 77, 93, 94
– C14
-labaled 98
– clinical investigations 101–103
– conformational rigidity 95
– gastric emptying data 100
– human cytochrome P450 profile 99, 100
– intramolecular H-bond 95
– NMR spectroscopy 95
– pharmacokinteic data 99
– protein binding 98
– synthesis 96
– X-ray crystallography 95
ultrasonic vocalization model 280
unoprostone 23
uric acid 365
uridine glucuronosyl transferases
(UGTs) 38, 49
v
validated hits 82
van der Waals interaction 250, 346, 347, 351
vascular endothelial growth factor-2
receptor 50
vasopressin V1a receptor binding
affinities 190–193
vasopressin V1a receptor cloning 192
vasopressin V2 receptor antagonists 187, 188,
193–203
venlafaxine 287
venous thromboembolism (VTE) 244, 259
verapamil 259
verlukast (MK-0679) 226
vildagliptin 7, 8
vincristine polyneuropathy model (rat) 313
visceral pain model See also pain 312
vitamin K antagonist (VKA) 245, 258
vorinostat (SAHA, ZolinzaTM
) 127
– analogues 127, 128
– synthesis 127
w
warfarin 6, 245, 246, 259, 260
writer enzymes 115
x
XalatanTM
23
xanthines 212
xanthine dehydrogenase (XDH) 368
xanthine oxidase (XO) 368
xanthine oxidoreductase (XOR) 365
xenobiotics 51, 54, 147
ximelagatran 9, 10, 245
y
YM-17690 229
yeast model (rat) 313
yew tree species (Taxus spp.) 320
z
zafirlukast(ICI 204219) 211,212,214,231,232
– discovery 218–224, 229, 230
zeburaline 122
zileuton 51, 232
zimelidine 14, 15
zolmitriptan 48, 49
zuclomiphene 169
Index j385