Combinatorial Chemistry As a Part of
Drug Discovery Process
Drug Discovery Process
Target molecule
eg. Enzymes
High-throughput
Screening (HTS)
Combinatorial Chemistry
Natural products
Lead &
Drug
optimization
N
N
R2
R3
R4
N
N
X N
H
R1
R1
R1
- R2 : diverse substituent
X= O, S
NH
HO
HO
R2
R1
OH
Toxicity Clinical trials
Combinatorial Chemistry
• Definition: the synthesis of chemical compounds
as ensembles (libraries) and the screening of
those libraries for compounds with desirable
properties
• Potentially speedy route to new catalysts,
materials and namely drugs
• Technique invented in the late 1980s and early
1990s to enable tasks to be applied to many
molecules simultaneously
Combichem Techniques
• Tools
– Solid-phase synthesis – some reagents are
anchored on resins
– Sets of reagents (Monomers)
– Linkers
– Screening methods, preferably HTS (highthroughput
screening)
Combichem Methods
• Use of solid supports originally for peptide
synthesis led to wider applications
• Products from one reaction are divided
and reacted with other reagents in
succession
– Split-mix scheme: library size increases
exponentially
combinatorial synthesis approach
Ilustration of a difference between classical and combinatorial
synthetic approaches
Classical and combinatorial synthesis
1 Reactant * 1 Reactant = 1 Product
Combinatorial synthesis:
CH3
NH2
Cl
O
OMe CH3
NH
O
OMe
+
Classical sythesis:
R1 NH2
Cl
O
R2 N
H
O
R2
R1
+
Eg.: 50 Reactants * 20 Reactants = 1000 Products
In more stages eg.: 50 * 20 * 20 = 20 000 Products
Synthetic methods for reaching of
combinatorial libraries
N N
S
R1
R2
H
H
R3
O
Cl
H
R4 N
S
N
R1
R2
R4
R3+
H2O, HCl+
The goal is to enable to get many compounds by the same
chemical reactions
Parallel Synthesis (diversity orientiented)
Problem: Many reaction vessels are necessary mainly if a
synthesis has many stages.
Solution proposal: multi-component reactions
Historic Milestones
A.Strecker (1850)
A.R. Hantzsch (1890)
P. Biginelli (1893)
R
C
NH2
N
NH3 + HCN + RCHO
R1
Cl
O
R2
H OR
O O N
H
R1
R2 COOR
NH3 + +
NH2
O
NH2
R2
O
R3
N
NH
O
R1
R2 R3
H
R1-CHO + +
Historic Milestones
C. Mannich (1912)
M. Passerini (1921)
H.T. Bucherer (1934)
R1
NH
R2
R3
R4
O
H N H
O
R4R3
R1
R2
CH2O + +
NH NH
O
O R
HCN + NH3 + CO2 + RCHO
R3 O
O R2
N
O
H
R1R1NC + R2CHO + R3COOH
Historic Milestones (III)
F. Asinger (1958)
I. Ugi (1959)
R1
O
R2 R3
O
SH
R4
N
S
R2
R1R4
R3
NH3 + +
R4 N
O R2
N
O
H
R3
R1
R1NH2 + R2CHO + R3NC + R4COOH
Synthetic methods for reaching of
combinatorial libraries (II)
N N
S
R1
R2
H
H
R3
O
Cl
H
R4 N
S
N
R1
R2
R4
R3+
H2O, HCl+
The goal is to enable to get many compounds by the same
chemical reactions
Parallel Synthesis (diversity orientiented)
One of the biggest problems is the isolation of a product from
the reaction mixture mainly in non-crystalline compounds and
small amounts.
Proposal of a solution: Temporary fixing to a solid holder
Solid phase synthesis
Anchoring on a polymer resin with suitable functional groups
small polystyrene balls = beads
Crosslinked co-polymer of polystyrene with 1-1.5% divinylbenzene
Ph Ph Ph Ph
PhPh
Cl
Cl
Cl
=
Solid phase synthesis
Originally developed for synthesis of long chain polypeptides.
The peptide remains anchored on polystyrene beads.
Lit. R. B. Merrifield J.Am.Chem.Soc. 85 (1963) 2149.
An example of Merrifield:
NH2
R1
O
O
Cl
O
NH2
R1
O
+
H2O
-Cl-
1. C-terminal amino acid
O
NH2
R1
O
N
R2
O
OH
O
O
H
N C N
O
N
R1
O
N
R2
O
O
O
H
H
+
2. N-Boc protected amino acid (with benzyloxycarbonyl)
Linkers for synthesis on solid phase
Merrifield resin
Wang resin
Cl
O
OH
Rink acid linker
OH
OMe
OMe
NHFmoc
OMe
OMe
OMe
OMe
NH
O
O
= Fmoc-protected Rink amine linker
„Split-and-pool strategy“
If we start from one
scaffold we can modify
the substitition pattern in
every new pool by
splitting
Possible technical
solution: magnetic
beads
Synthesis of Peptide Libraries
The division of the product enables an efficient parallel
synthesis for example for the construction of orthogonal
libraries:
Systematic vary of the amino acid at the Xth position of a
protein. Required for the epitop-mapping on antibodies
A successful restriction of the most active amino acid
sequence (split-and-mix)
Ac-DVXXXX-NH2 204
peptides
Ac-DVPXXX-NH2 203
peptides
Ac-DVPDYA-NH2
Solid phase synthesis
split-and-mix strategy for the construction of peptide libraries
The formation of peptides with the same end in one mixture
A B C
A
B
C
split
combine
AA
A
BA
CA
B
C
split
AB CA
BB
CB
CB
CC
Solid phase synthesis of peptides (continued)
The formation of peptides with the same end in one mixture
AA
combine
A
BAA
CAA
A
ABA
BBA
CBA
CAA
CBA
CCA
AAB AAC
B
C
split
BAB
CAB
ABB
BBB
CBB
CAB
CBB
CCB
BAC
CAC
ABC
BBC
CBC
CAC
CBC
CCC
Synthetic methods for generation of combinatorial libraries
The reliable synthetic steps are required for the parallel
synthesis for the construction of compound libraries, which are
possible to perform also by means of synthetic robots, e.g.
reactions such as
●
reductive amination
●
acylation
●
Hantzsch synthesis of 2-aminothiazols
●
Suzuki coupling (building of a C-C bond)
●
Ugi condensation (dipetides)
Synthesis of peptides / Anchored on a solid phase
Ugi 4-components condensation
The reaction of an isonitrile with a carboxyl group and an aldehyde leads to
a -acyloxy-amide (Passerini reaction). If an amine is then added the
appropriate bisamide is formed:
R1 COOH
R2 NH2
R3 CHO
R4 N C
R1 N
CH*
O
R2
R3
O
N
H
R4-H2O
Disadvantage: only few isonitrils are commercilally available; their smell is
very unpleasant
Solutions:
●
Usage of an universal isonitrile which serves as a precursor for further
products
●
Formation of an isonitrile in situ
Synthesis of peptides / Anchored on a solid phase
1-isocyanocyclohexene as a versatile convertible isonitrile for Ugi-
condensations
Enables a series of of products and also capture to a resin :
Keating & Armstrong J.Am.Chem.Soc. 118 (1996) 2574
O
C N
N H
O
H
N
C
NaCN / NH4Cl
Et2O /H2O
HCOOH
Ac2O
61% 44%
tBuOK
THF
Triphosgen
DABCO, CH2Cl2
50% in 2 steps
N
R1 N
O
R2
R3
O
H
R1 N
O
R2
R3
O
OMe
R1 N
O
R2
R3
O
O
R1 N
O
R2
R3
O
OH
OH
AcCl
MeOH
HCl / THF CH2Cl2
20% TFA
1-isocyanocyclohexene as a versatile convertible isonitrile for Ugi-
condensations
Possibilities of further reactions of Ugi Products
Keating & Armstrong J.Am.Chem.Soc. 118 (1996) 2574
N
R1 N
O
R2
R3
O
H
R5 R6
N
R5 R6
R2
R3R1HCl / Toluene
-CO2
N
N
O
R2
R3
O
H
NH2
R1 R1
N
O
R2
N
H
O
R3
1,4-Benzodiazepine-2,5-dione
3 components Ugi condensation
Paralell synthesis of local anesthetics of anilide type
(Morphochem, Munich)
N
C
H H
O
N
H N
H
O
N
+ +
Lidocaine
N
H
O
N
COOH
COOH
N
H
O
N
N
Cl H
O
N
H
N
Cl H
O
N
N
COOMe
N
H
O
N
N
H
O
N
Lidofenin Pyrrocaine
Butanilicaine Clodacaine
Tolycaine
Trimecaine
Combinatorial dihydropyridine library
Lit: K.C. Nicolaou et al. in Handbook of Combinatorial
Chemistry, VCH Wiley (2000) pp. 659-660
NH2
split
10 lots
R2
O
O O
R1 R2
O
O R1
N
H10 synthons
combine
pool of 10 enamines
R3
O O
R4split
30 lots 3 synthons +
Ar-CHO (10 synthons)
Ar
O
R3
O
R4
O
OR2
R1
NH
Ar O
R3
R4
O
O
R2
R1 N
H
TFA
100 products
COOMe
Me
O
Me N
H
FO
O
iPr
screening
deconvolution
IC50 (calcium channel) = 14 nM
80.77% similarity with felodipine
Angiotensin converting enzyme (ACE)-Inhibitors Library
Lit: M.M. Murphy et al. J.Am.Chem.Soc, 117 (1995) 7029
CH2
Cl
O
O
R
NH2
AAs:
Gly
Ala
Leu
Phe
+
R1
O
H
O
O
R
N Ar
+
Ar-CHO:
R1:
H
Me
OMe
OSiMe2tBu
Cl Thio
O
O
O
R
N Ar
Y
Thio
O
+ Thio:
CH2SAc
CH2CH2SAc
CH(Me)CH2SAc
N
O
CH3
OH
O
SH
COOMeKi (ACE) = 160 pM
CN
COOMe
COMe
COOtBu
COOMe
O
O
R
N
H
Ar
Y
Y:
+
Multireactor vessels
Problems with Early
Combichem Libraries
• Many compounds had undesirable
properties:
– Size (molecular weight)
– Solubility
– Inappropriate functional groups
Criticism of the Technique
• Early libraries were often based on a single
skeleton (scaffold - basic structure)
• Limited number of skeletons accessible
• Individual library members were structurally
similar
• Compounds tended to be achiral or racemic
• Initial emphasis on creating mixtures of very
large numbers of compounds is now out of favor
DIVERSE AND FOCUSED
LIBRARIES
• Many early disappointments led to:
– Design of smaller, more focused libraries with much
information about the target
• May concentrate on a family of targets (e.g., proteases or
kinases)
– Use of more diverse libraries when little is known
about the target
• “Primary screening” libraries
• Give broad coverage of chemistry space
– Selection of compounds with “drug-like”
physicochemical properties
Multistage screening
High-throughput Screening (HTS)
 A process of assaying a large number of compounds against biological
targets.
 Up to 100,000 compounds can be analyzed in a day.
 Robots can usually prepare and analyze many plates simultaneously.
http://www.metprog.org.uk/images/manufacturing_icon.jpg
Applications
Whole cell fluorescence assays
Cell viability, cell differentiation, cell
proliferation, cytotoxicity, apoptosis,
transporter phenomena
Cell signaling assays
Calcium flux, second messengers, ion channels,
membrane potential
Gene expression assays
Expression of house-keeping and reporter
genes, gene activity and protein regulation,
RNAi
Membrane receptor assays
Ligand binding, receptor activation and
desensitization, translocation and endocytosis,
recruitment of signaling molecules
Translocation assays
Target molecule redistribution
Morphological assays
Neurite outgrowth, cell differentiation, cell
adhesion and spreading
Opera Imaging Reader
>50,000 multi-color data
points/24 hours
High-Throughput Screening at the University of Cincinnati
Compound Repository
Haystack Neat Compound Storage
• Capacity = 200,000 bottles
• Current = 207,000 bottles
• Freezer storage when appropriate
Solar (Solution Archive) – DMSO solutions
• Capacity = 1.8 million tubes, 10,000 deep well
• (96) plates, 13,600 shallow well (384) plates
• Current = 338,000 compounds in 383,400
• tubes, 1862 deep wells and 2332 shallow wells
Compound handling and dissolution instruments
Housed at P&G’s Mason Business Center (~3500 sq. ft.)
GRI Compound Library
• It’s NOT just a numbers game –
compound selection can greatly
enhance screening efficiency
• Originally from P&G Pharma and
represents a $22M investment
• Selected based on drug-like
properties and to maximize
structural diversity within a
6-dimensional “drug-like” space
• Both external (commercial suppliers)
and internal discovery and
combinatorial chemistry programs
used as sources
• ~250,000 compounds
• Software to rapidly expand around
structural leads identified
Vendor
Database
Remove duplicates
Remove reactives,
Unusual groups,
& toxicophores
(80 substructures)
MW filter
Solubility Filter
Lipinski Rule of Five
•> 5 H-bond donors
•MW > 500
•c log P  5
 N's + O's > 10
“Cleaned”
database
26 databases
>4 million structures
0
100
200
300
400
500
600
Potency
RelativeMolecularMass
1µM 10 nM1mM
HTS
hits
Drugs
Lead optimization
Drug
Candidates
HTS Library