New organic compounds with
targeted biological activity
Kamil Paruch
Brno 2016
Contents
Aims and scope of the work 1
Part 1
1a. Versatile templates for the development of novel kinase inhibitors:
discovery of novel CDK inhibitors 20
1b. Pyrazolo[1,5-a]pyrimidines as orally available inhibitors
of cyclin-dependent kinase 2 31
1c. Discovery of dinaciclib (SCH 727965): a potent and selective
inhibitor of cyclin-dependent kinases 38
1d. Dinaciclib (SCH 727965), a novel and potent cyclin-dependent
kinase inhibitor 50
Part 2
2a. Discovery of pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors:
a template-based approach-part 1 69
2b. Discovery of pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors:
a template-based approach-part 2 80
2c. Targeting the replication checkpoint using SCH 900776, a potent and
functionally selective CHK1 inhibitor identified via high content screening 90
2d. Discovery of pyrazolo[1,5-a]pyrimidine-based Pim inhibitors:
a template-based approach 113
Part 3
3a. Cyclin-dependent kinase inhibitors inspired by roscovitine:
purine bioisosteres 125
3b. Furopyridines as inhibitors of protein
kinases 141
Part 4
4a. Syntheses of 5´-amino-2´,5´-dideoxy-2´, 2´-difluorocytidine derivatives
as novel anticancer nucleoside analogs 243
4b. New carbocyclic nucleosides: synthesis of carbocyclic pseudoisocytidine
and its analogs 253
4c. Highly diastereoselective flexible synthesis of new
carbocyclic C-nucleosides 284
Part 5
Synthesis of carbocyclic analogs of dehydroaltenusin: identification
of a stable inhibitor of calf DNA polymerase  353
1
Aims and scope of the work
Small organic molecules, i.e. compounds with molecular weight less than 1000 g/mol, are ubiquitous
in nature. Their biological activity can be multifaceted: e. g., they can bind to receptors, modulate
activity of enzymes, get incorporated into membranes etc.1
Despite the rapid development of
therapeutics based on biomolecules, small-molecule organic compounds still represent the majority of
clinically used substances.2
In addition to their medicinal use, selective organic compounds can be utilized as chemical biology
probes that enable elucidation of fundamental biological processes.3
Information obtained by the
chemical biology approach can be of crucial importance, given the recent concerns of possibly
misleading phenotypes upon elimination of a protein (e. g., by RNAi-mediated knock-down), rather
than just inhibiting its function while preserving the protein’s abundance, interactions and
compartmentalization, thereby avoiding the otherwise potentially non-physiological or compensatory
substitutions by other proteins.4
Protein kinases form a large family of ATP-dependent phosphotransferases, encoded by over 500
genes.5
Protein kinases catalyze reversible phosphorylation on the hydroxyl group of tyrosine, serine,
or threonine residues of protein substrates. Reversible phosphorylation is a major post-translational
signaling mechanism which is involved in many regulatory pathways that control a diverse set of
cellular processes. Modulation of activity of some protein kinases can be in principle utilized in
modern anti-cancer therapy as well as for treatment of other diseases such as rheumatoid arthritis,
cardiovascular diseases, diabetes, diabetic complications and Alzheimer’s disease.6
It has been estimated that protein kinases represent ca. 20 % of the druggable genome.7
Accordingly,
development of new protein kinase inhibitors has been one of the most active fields in the academic
as well as in the industrial sector. Massive effort in the last two decades has enabled clinical
development of new substances for therapeutical use.8
Table 1 summarizes all current clinically used protein kinase inhibitors; including recently approved
palbociclib (February 2015) and lenvatinib (February 2015).9
Except for the Janus kinase inhibitor
tofacitinib, all inhibitors have been approved for treatment of different malignancies. It should be
noted that in some cases inhibition of several targets is necessary in order to achieve optimal
therapeutic outcome; therefore, some inhibitors are relatively non-selective.10
In other cases, however,
inhibition of a particular pathway is sufficient and high selectivity is required to minimize side
effects.11
Typically, protein kinase inhibitors are often well tolerated and possess overall better safety
2
profiles than classical cytotoxic chemotherapies, with toxicities and side effects that are generally
more manageable and reversible.
Table 1. Clinically used protein kinase inhibitors, year of their approval and their known biological
targets.9
Structure Year Known targets
afatinib
2013 EGFR
axitinib
2012 VEGFR1/2/3
bosutinib
2012
BCR-Abl, Src, Lyn,
and Hck
3
cabozantinib
2012
RET, Met,
VEGFR1/2/3, Kit,
TrkB, Flt3, Axl, Tie2
ceritinib
2014
ALK, IGF-1R, InsR,
ROS1
crizotinib
2011
ALK, c-Met (HGFR),
and Ros
dabrafenib
2013 B-raf
4
dasatinib
2006
BCR-Abl, Src, Lck,
Yes, Fyn, Kit, EphA2,
and PDGFRβ
erlotinib
2004 EGFR
everolimus
2009 FKBP12/mTOR
gefitinib
2003 EGFR
5
imatinib
2001
BCR-Abl, Kit, and
PDGFR
lapatinib
2007 EGFR and ErbB2
ibrutinib
2013 Bruton's kinase
lenvatinib
2015
VEGFRs/FGFRs/
PDGFR/Kit/RET
6
nilotinib
2007 BCR-Abl, PDGFR
nintedanib
2014
VEGFR, FGFR,
PDGFR
palbociclib
2015 CDK4/6
pazopanib
2009
VEGFR1/2/3,
PDGFRα/β, FGFR1/3,
Kit, Lck, Fms, and Itk
7
ponatinib
2012
BCR-Abl, BCR-Abl
T315I, VEGFR,
PDGFR, FGFR, Eph,
Src family kinases,
Kit, RET, Tie2, and
Flt3
regorafenib
2012
VEGFR1/2/3, BCRAbl,
B-Raf, BRaf(V600E),
Kit,
PDGFRα/β, RET,
FGFR1/2, Tie2
ruxolitinib
2011 JAK1/2
sirolimus
1999 FKBP12/mTOR
sorafenib
2005
C-Raf, B-Raf, B-Raf
(V600E), Kit, Flt3,
RET, VEGFR1/2/3,
and PDGFRα/β
8
sunitinib
2006
PDGFRα/β,
VEGFR1/2/3, Kit,
Flt3, CSF-1R, and
RET
tofacitinib
2012 JAK3
temsirolimus
2007 FKBP12/mTOR
trametinib
2013 MEK1/2
vandetanib
2011
EGFRs, VEGFRs,
RET, Brk, Tie2,
EphRs, and Src family
kinases
9
vemurafenib
2011
A/B/C-Raf and B-Raf
(V600E)
The overarching theme of this work is the search for new organic compounds with defined biological
activity that can be used in modern oncology either as single agents or in combination with other
therapeutics.
Part 1 of the work describes discovery of potent cyclin-dependent kinase (CDK) inhibitors with the
pyrazolo[1,5-a]pyrimidine core. First, the pyrazolo[1,5-a]pyrimidine motif, exemplified by
compound 1, was identified among several purine bioisosteres as optimal scaffold (Part 1a). Early
SAR development around the core led to identification of potent bioavailable inhibitors of CDKs (e.
g., compound 2) that were used in proof-of-concept studies in vivo (Part 1b). Further optimization of
the substituents enabled discovery of extremely potent sub-series with aminoalcohols at position 5 of
the pyrazolo[1,5-a]pyrimidines core (Part 1c), which included SCH727965 (dinaciclib).
Based on optimal therapeutic index, dinaciclib was chosen for further preclinical progression (Part
1d) and later entered clinical trials. Currently, dinaciclib is profiled in Phase III clinical trials as an
agent against chronic lymphocytic leukemia (CLL).12
10
Part 2 describes discovery of potent and selective inhibitors of kinases CHK1 and PIM. Due to the
enzyme’s central role in the cell cycle, inhibition of CHK1 was identified as an attractive component
for therapeutic regimes that utilize the concept of synthetic lethality, where the required phenotype
(apoptosis and cell death of tumor cells) is caused by a synergic modulation of two or more biological
processes.13
High throughput screening of differently substituted pyrazolo[1,5-a]pyrimidines revealed
hits; e. g., compound 3. Modification of the substitution pattern at positions 3, 5, 6, and 7 of the
central pyrazolo[1,5-a]pyrimidine core yielded the sub-series (exemplified by compound 4) with
significantly reduced activity towards CDKs and single-digit nanomolar activity against CHK1 (Part
2a and Part 2b). High content cell-based screening then allowed identification of SCH900776, which
was further preclinically profiled (Part 2c). Since it showed encouraging efficacy in in vivo models,
SCH900776 later entered clinical trials.
Similarly, exploration of the SAR around the positions 3 and 5 of the pyrazolo[1,5-a]pyrimidine core
yielded a series of potent and selective inhibitors of PIM kinases; e. g., compound 5 (Part 2d).
The strategy utilized the atypical cavity present in the ATP-binding site of PIM kinases; the inhibitors
possessing large hydrophobic motifs at position 3 bind to the enzymes via an alternative mode, which
is different from the “canonical” one observed for the inhibitors of CDKs and CHK1 described above
(Figure 1).
11
Figure 1. Binding modes of pyrazolo[1,5-a]pyrimidine-based inhibitors of CDK (left), CHK1 (middle), and
PIM (right); the “hinge” regions of the kinases are in compatible orientation.
Part 3 covers our efforts to identify new central pharmacophores as alternatives to the pyrazolo[1,5a]pyrimidine
scaffold. Analysis of various bioisosteres of the purine-based CDK inhibitor roscovitine
revealed the fact that only certain bicyclic condensed heterocycles are suitable to replace the central
purine scaffold (Part 3a).
The main aim of the project described in Part 3b was to find out whether the furo[3,2-b]pyridine
scaffold can be used as a central pharmacophore for inhibitors of protein kinases. In order to prepare a
sufficiently diverse set of compounds for initial “unbiased” screen, we optimized two known methods
(Route I and Route II in Scheme 1) to assemble the furo[3,2-b]pyridine core and developed one new
annulation methodology (Route III).
Scheme 1: Three different strategies for the construction of the furo[3,2-b]pyridine scaffold.
12
Thorough profiling of the starting set our heretofore unknown furo[3,2-b]pyridines against 206
human kinases (in the Merck Millipore KinaseProfiler) revealed early leads. We further elaborated
the sub-series with proper substituents at positions 3 and 5 of the furo[3,2-b]pyridine scaffold,
utilizing especially our newly developed annulation methodology to flexibly install a variety of
substituents at the two positions. We prepared heretofore unknown compound 6, which proved to be a
valuable intermediate as it could be used for orthogonal manipulation of position 5 and (then) position
3 via sequential Suzuki couplings. This enabled us to identify some very potent (IC50 < 50 nM) and
selective inhibitors of CLK and HIPK kinases, exemplified by compound 7.
CLK and HIPK kinases have emerged only recently as possible therapeutic targets and in terms of
activity, some of our compounds are at least comparable to the known inhibitors of these enzymes.
Part 4 is focused on synthesis and biological profiling of new nucleoside analogs. Due to their
multifaceted function nucleosides and their analogs remain attractive molecules to scientist across life
sciences.14
Currently, 29 nucleoside analogs are used as medicaments and numerous others are in
clinical trials; especially as antivirals (Figure 2) and chemotherapeutic agents in oncology, including
in synthetic lethal combinations with inhibitors of CHK1 kinase (Figure 3).
13
Figure 2. Antiviral nucleoside analogs.
14
Figure 3. Anticancer nucleoside analogs.
However, the biology and the corresponding potential use of nucleosides in therapy are usually not
straightforward due to several factors: (i) many nucleoside analogs are not effectively phosphorylated
by the cellular kinases; (ii) metabolic stability toward deaminases, hydrolysis of nucleoside-base
linkage and sugar ring opening are also limiting for many of analogs; and (iii) the resulting
metabolites of mostly unidentified structures can be potentially toxic. These factors often complicate
the testing and interpretation of nucleosides’ biological activity.15
On the other hand, the biological
activity of nucleoside analogs with non-phosphorylatable substituents at position 5´ should be
significantly easier to decipher as the biological effects of the compounds would not rely on the full
complex scenario described above.
Along this line, Part 4a describes identification of analogs of gemcitabine with non-hydroxylic
substituents at position 5´ of the ribose scaffold; exemplified by compound 8. Interestingly, despite
the fact that the compounds cannot undergo metabolic phosphorylation, they are capable of inhibition
of ribonucleotide reductase (RNR) in the cell16
and could be therefore used as potential partners for
synthetic lethal treatment in combination with CHK1 inhibitors.17
15
Classical nucleosides (structure A in Figure 1) possess the hemiaminal motif; their chemical and
metabolic stability is therefore often limited and the resulting metabolites can be a source of
undesired side effects.18
Significant effort has thus been invested into identification of more stable
analogs while preserving the biological activity. Two main strategies involve replacement of the C-N
bond between sugar and base by the more stable C-C bond (C-nucleosides, structure B in Figure 1)19
and replacement of the tetrahydrofuran motif by a carbocyclic ring (e. g., cyclopentane), which leads
to carbocyclic N-nucleosides (structure C in Figure 1).20
Figure 4. Generic structures of natural nucleosides (A), C-nucleosides (B), carbocyclic N-nucleosides (C), and
carbocyclic C-nucleosides (D).
Structure D in Figure 4 combines the stabilizing elements of structures B and C (i. e. C-C connection
between the (heterocyclic) base and the carbocyclic scaffold) and represents carbocyclic Cnucleosides,
which are only sporadically documented in the literature. It is conceivable that, at least
in some cases, those compounds might be more robust versions of nucleoside analogs B and C. In
addition, installation of certain substituents (e. g. R1´
= OH) is meaningful only in this series, as this
would lead to chemically unstable ketals and aminals in the other analog series.
Part 4b includes our target-oriented synthesis of racemic carbocyclic pseudoisocytidine (10) and its
analogs, which were prepared in 13 steps from cyclopentadiene and methyl propiolate via ketoester 9.
16
We also prepared versatile cyclopentanone intermediate 11, which we converted into novel
carbocyclic nucleosides via highly stereoselective addition of organometallic nucleophiles. Reaction
with phenyllithium led to target compound 12 (the stereochemistry was confirmed by X-ray
crystallography), which inhibits human glycosylase NEIL1 in a dose-dependent manner.
Part 4c describes our modular and highly diastereoselective synthesis of three classes of new
carbocyclic C-nucleosides, represented by generic structures D1, D2 and D3.
Our route enables flexible preparation of three classes of these nucleoside analogs from common
precursors – properly substituted cyclopentanones 13a and 13b, which can be prepared racemic (in
six steps) or optically pure (in ten steps) from inexpensive norbornadiene. The methodology allows
flexible manipulation of individual positions around the cyclopentane ring, namely highly
diastereoselective installation of carbo- and heterocyclic substituents at position 1´, orthogonal
functionalization of position 5´, and efficient inversion of stereochemistry at position 2´. The
methodology represents, to our knowledge, first sufficiently flexible synthetic approach to carbocyclic
C-nucleosides.
17
Part 5 includes synthesis and biological profiling of heretofore unknown carbocyclic analogs of
dehydroaltenusin. The project was part of our efforts to identify suitable new substances for synthetic
lethal treatments in combination with CHK1 inhibitors. Dehydroaltenusin is one of the very few
known inhibitors of DNA polymerase , whose depletion has been shown to afford the synthetic
lethal phenotype in combination with co-depletion or pharmacological inhibition of CHK1 kinase.21
However, chemical stability of dehydroaltenusin is limited, as it undergoes a rearrangement in
aqueous solutions to give a mixture of the spirocyclic and non-spirocyclic forms (structures DHAs
and DHA in Scheme 1; absolute configurations are not known).22
It is not clear which of these forms
is active and it has been suggested that the rearrangement o-quinone intermediate might be also
responsible for the inhibitory activity.23
In order to help elucidate this issue, we decided to carry out
bioisosteric replacement of the lactone ring oxygen by methylene group and prepare the carbocyclic
analogs of the spirocyclic and non-spirocyclic forms of dehydroaltenusin - cDHAs and cDHA,
respectively (Scheme 1). These compounds were envisioned to be significantly more stable and
resistant to the rearrangement.
Scheme 1. Design and synthesis of carbocycylic analogs of both forms of dehydroaltenusin.
The target compounds cDHA and cDHAs were prepared from 3,5-dimethoxybenzaldehyde in 11 and
13 steps, respectively. Unlike dehydroaltenusin, both cDHA and cDHAs are stable and their
structures were confirmed by X-ray crystallography. Compound cDHA was found to be active
against calf DNA polymerase  but not related isozymes, while the spirocyclic analog cDHAs was
inactive.
18
Bioisosteric replacement of oxygen atom by the methylene group is commonly used in medicinal
chemistry;24
however, in the context of this project, it enabled not only identification of more stable
analogs, but also provided significant (albeit indirect) information on which of the dehydroaltenusin
isomers is responsible for its biological activity. Our observation of the dramatic difference in
activities of cDHA and cDHAs strongly suggests that dehydroaltenusin’s inhibitory activity toward
polymerases is due to its non-spirocyclic form and while the presence reactive o-quinone intermediate
may contribute to dehydroaltenusin’s inhibitory activity, it may not be absolutely required.
Our results could be used to guide further development of potent and selective inhibitors of
mammalian polymerases, namely DNA polymerase , exploiting the recently published crystal
structure.25
References and notes
1. Ganellin, C. R.; Jefferis, R.; Roberts, S. M. Introduction to Biological and Small Molecule Drug
Research and Development: Theory and Case Studies; Elsevier Science, 2013.
2. (a) Chem. Eng. News 2016, 94, 12. (b) http://www.economist.com/news/business/21637387-wave-
new-medicines-known-biologics-will-be-good-drugmakers-may-not-be-so-good
3. (a) Schreiber, S. L.; Kapoor, T. M.; Wess, G. Chemical Biology: From Small Molecules to Systems
Biology and Drug Design; Ed.: Wiley-VCH, 2007. (b) Waldmann, H.; Janning, P. Chemical Biology;
Ed.: Wiley-VCH, 2009.
4. Weiss, W. A; Taylor, S. S.; Shokat, K. M. Nat. Chem. Biol. 2007, 3, 739.
5. Grant, S. K. Cell. Mol. Life Sci. 2009, 66, 1163.
6. Saitoh, M.; Kunitomo, J.; Kimura, E.; Iwashita, H.; Uno, Y.; Onishi, T.; Uchiyama, N.; Kawamoto,
T.; Tanaka, T.; Mol, C. D.; Dougan, D. R.; Textor, G. P.; Snell, G. P.; Takizawa, M.; Itoh, F.; Kori,
M. J. Med. Chem. 2009, 52, 6270.
7. Lim, F. P.; Dolzhenko, A. V. Eur. J. Med. Chem. 2014, 85, 371.
8. Matthews, D. J.; Gerritsen, M. E. Targeting Protein Kinases for Cancer Therapy; Wiley, 2010.
9. http://www.brimr.org/PKI/PKIs.htm
10. Legraverend, M.; Grierson, D. S. Bioorg. Med. Chem. 2006, 14, 3987.
11. Koch, P.; Gehringer, M.; Laufer, S. A. J. Med. Chem. 2015, 58, 72.
12. https://ash.confex.com/ash/2015/webprogram/Paper79907.html
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13. (a) Kaelin, W. G. Nat. Rev. Cancer 2005, 5, 689. (b) Benson, J. D.; Chen, Y.-N. P.; CornellKennon,
S. A.; Dorsch, M.; Kim, S.; Leszcznicka, M.; Sellers, W. R.; Lengauer, C. Nature, 2006,
441, 451. (c) Arlander, S. J. H.; Greene, B. T.; Innes, C. L.; Paules, R. S. Cancer Res. 2008, 68, 89.
(d) Banerjee, S.; Kaye, S. B.; Ashworth, A. Nat. Rev. Clin. Oncol. 2010, 7, 508.
14. Herdewijn, P. Modified Nucleosides in Biochemistry, Biotechnology and Medicine; Ed.: WileyVCH,
2008.
15. Park, W. B. Chem. Rev. 2009, 109, 2880.
16. Labroli, M. A; Dwyer, M. P.; Shen, R.; Popovici-Muller, J.; Pu, Q.; Wyss, D.; McCoy, M.;
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Parry, D.; Guzi, T. J. Bioorg. Med. Chem. 2014, 22, 2303.
17. Taricani, L.; Shanahan, F.; Malinao, M.-C.; Beaumont, M.; Parry, D. PLoS ONE 2014, 9:
e111714.
18. (a) Kawaguchi, T.; Fukushima, S.; Ohmura, M.; Mishima, M.; Nakano, M. Chem. Pharm. Bull.
1989, 37, 1944. b) Azuma, A.; Hanaoka, K.; Kurihara, A.; Kobayashi, T.; Miyauchi, S.; Kamo, N.;
Tanaka, M.; Sasaki, T.; Matsuda, A. J. Med. Chem. 1995, 38, 3391.
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Rev. 1995, 24, 169. c) Agrofoglio, L. A.; Suhas, E.; Farese, A.; Condom, R.; Challand, S. R.; Earl, R.
A.; Guedj, R. Tetrahedron, 1994, 50, 10611. d) Zhu, X. F. Nucleos. Nucleot. Nucl. 2000, 19, 651. e)
Agrofoglio, L. A. Curr. Org. Chem. 2006, 10, 333. f) Ichikawa, E.; Kato, K. Curr. Med. Chem. 2001,
8, 385. g) Schneller, S. W. Curr. Top. Med. Chem. 2002, 2, 1087. Boutureira, O.; Matheu, M. I.;
Diáz, Y.; Castillón, S. Chem. Soc. Rev. 2013, 42, 5056.
21. Taricani, L.; Shanahan, F.; Parry, D. Cell Cycle 2009, 8, 482-489.
22. Kamisuki, S.; Takahashi, S.; Mizushina, Y.; Sakaguchi, K.; Nakata, T.; Sugawara, F. Bioorg.
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Acids Res. 2014, 42, 14013.
20
Part 1a
Versatile templates for the development of novel kinase
inhibitors: discovery of novel CDK inhibitors*
*published as:
Dwyer, M. P.;*
Paruch, K.;*
Alvarez, C.; Doll, R. J.; Keertikar, K.; Duca, J.; Fischmann, T.; Hruza, A.;
Madison, V.; Lees, E.; Parry, D.; Sgambellone, N.; Seghezzi, W.; Shanahan, F.; Wiswell, D.; Guzi, T. J.
Versatile Templates for the Development of Novel Kinase Inhibitors: Discovery of Novel CDK Inhibitors.
Bioorg. Med. Chem. Lett. 2007, 17, 6216.
The cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases that function as critical
regulators of the mammalian cell cycle which integrates extracellular signaling, DNA synthesis, and
mitosis.1
Dysregulation of cell cycle control is a hallmark of all human cancers and is frequently
associated with aberrant activation/regulation of cyclin-dependent kinases (CDKs).2
While
coordinated CDK2/CDK1 activity is required for appropriate regulation of S-phase entry (DNA
synthesis), suppression of apoptosis in late S-phase, S-phase exit and entry into mitosis, it has been
illustrated that inhibition of CDK2/CDK1 in tumors provokes cell cycle arrest and apoptosis.3
Hence,
inhibition of the essential, rate-limiting activities of CDK2 and CDK1 represents an attractive
therapeutic strategy for oncology indications.4
Several CDK inhibitors are currently under clinical evaluation including flavopiridol ( 1),5
roscovitine
( 2),6
BMS
387032 (3),7
R547 (4),8
and PD0332991 (5) (Fig. 1).9
However, opportunities exist to identify and
develop additional novel CDK inhibitors that may possess superior biological profiles to current
candidates. One considerable challenge that exists in this area is the identification of novel core
structures for the development of selective kinase inhibitors. Toward this end, the targeting of ATP
competitive inhibitors of the CDKs has emerged as the mainstay of this area with several classes of
compounds having been developed.10
21
Figure 1. CDK inhibitors currently under clinical evaluation.
Herein, we report our initial efforts exploring the utility of several bicyclic cores toward the
development of novel ATP-competitive kinase inhibitors of the CDKs. In the early course of our
CDK program, two lead classes of compounds emerged from our in-house screening: the
pyrazolo[1,5-a]pyrimidine 611
and the imidazo[1,2-a]pyrazine 7 both of which possessed
submicromolar potency in a cyclin A/CDK2 kinase biochemical assay12
(Fig. 2).
Figure 2. Pyrazolo[1,5-a]pyrimidine 6 and imidazo[1,2-a]pyrazine 7 lead structures from library screening.
22
Owing to the structural similarities of 6 with the purine CDK2 inhibitor roscovitine (2 ) (Fig. 1),
initial C3 bromide substitution of 8 yielded compound 9 which demonstrated over 10-fold
improvement of potency in the biochemical assay12
(Fig. 3).
Figure 3. Four bicyclic cores of interest as potential CDK inhibitors.
Having achieved reasonable potency in compound 9 , efforts were undertaken to explore the potential
utility of other bicyclic cores outside those represented by 6 and 7 . Toward this end, we targetted
two additional cores besides the pyrazolo- [1,5-a]pyrimidine derivative 9 and the imidazo[1,2a]pyrazine
10 to include: pyrazolo[1,5-a]pyridine 11 ; and imidazo[1,2-a]pyridine 12 (Fig. 3). Each
of these compounds was prepared bearing identical substitution and subsequent biological evaluation
would allow for the identification of the best CDK bicyclic scaffold.
The preparation of the individual core molecules 8-12 is shown in Schemes 1–4.
23
Preparation of the pyrazolo[1,5-a]pyrimidine derivatives 8 and 9 was achieved by treatment of 3aminopyrazole
with ethyl benzoylacetate under acid conditions followed by chlorination under
standard conditions to afford compound 13 (Scheme 1 ).13
Regioselective bromination followed by
displacement with 4-pyridylmethylamine afforded the title compound 9 while treatment with the
amine directly on 13 afforded compound 8.
Scheme 1. Reagents and condition: (a) ethyl benzoylacetate, AcOH, 100 °C; (b) POCl3, pyridine, DMAP; (c)
NBS, CH3CN; (d) 4-pyridylmethylamine, DIPEA, dioxane.
Preparation of the imidazo[1,2-a]pyrazine analog 10 was carried out in an analogous fashion to the
protocol depicted in Scheme 1. Treatment of 1-methyl-1H-imidazole- 2-carboxamide 1414
with –
bromomethylphenyl ketone followed by dealkylation of the resultant quaternary salt with imidazole
afforded compound 15 (Scheme 2 ).14
Conversion to chloride 16 followed by regioselective
bromination and amine displacement yielded title compound 10.
24
Scheme 2. Reagents and condition: (a) -bromomethylphenyl ketone, CH3CN; (b) imidazole, heat; (c) POCl3,
pyridine, DMAP; (d) NBS, CH3CN; (e) 4-pyridylmethylamine, DIPEA, dioxane.
Preparation of the pyrazolo[1,5-a]pyridine analog 11 began with cyclization of the known 1aminopyridinium
adduct 1715
with ethyl propiolate under basic conditions followed by acid-catalyzed
decarboxylation to afford cycloadduct 18 (Scheme 3 ).16
Regioselective iodination17
followed by Pdcatalyzed
amination afforded imine 19. Regioselective bromination, imine deprotection, followed by
ZnCl2-promoted reductive amination afforded the title compound 11.
25
Scheme 3. Reagents and conditions: (a) ethyl propiolate, K2CO3, air; (b) H2SO4, heat; (c) n-BuLi, diiodoethane;
(d) Ph2CNH, Pd(OAc)2, BINAP, Cs2CO3, toluene; (e) NBS, CH3CN; (f) NH2OH, NaOAc; (g) 4pyridinecarboxaldehyde,
ZnCl2 then NaBH3CN.
Preparation of the imidazo[1,2-a]pyridine adduct 12 began with treatment of nitro adduct 20 under
reductive conditions18
followed by cyclization with in-situ generated bromoacetaldehyde to afford
21.19
Suzuki coupling with phenyl boronic acid followed by acetylation afforded 22. Regioselective
bromination, deprotection, and reductive amination afforded the title compound 12.
26
Scheme 4. Reagents: (a) SnCl2, H2O, EtOH; (b) BrCH2CHO, K2CO3; (c) PhB(OH)2, Pd(PPh3)4, K3PO4,
DME/H2O; (d) AcCl, pyridine; (e) NBS, CH3CN; (f) aq. HCl, EtOH; (g) 4-pyridinecarboxaldehyde, ZnCl2 then
NaBH3CN.
The targets described above were assayed in a biochemical assay for the inhibition of cyclin
A/CDK2.12
Analogs that demonstrated reasonable potency in the biochemical assay were advanced
into a thymidine incorporation assay20
which was used to measure the ability of the compounds to
inhibit asynchronously growing A2780 ovarian carcinoma cells. The data generated for compounds 9-
12 and flavopiridol are summarized in Table 1.
Table 1. Cyclin A/CDK2 assay12
and thymidine incorporation assay20
for analogs 9–12 and flavopiridol.
a
All IC50 values are means of at least two determinations.
b
Assay conditions listed in Ref. 20.
In Table 1, incorporation of a C3 bromide into imidazo[1,2-a]pyrazine series (7-10) showed a clear
improvement in in-vitro potency (twofold) but to a lesser extent as was observed in the pyrazolo[1,5a]pyrimidine
series (represented by 9). Modification of the bicyclic core had a much more dramatic
effect on the CDK2 in-vitro potency as displayed in Table 1. Deletion of the N6 nitrogen in the
imidazo[1,2-a]pyrazine adduct 10 yielded imidazo[1,2-a]pyridine 12 which led to a twofold loss in
in vitro potency. More noticeably, the deletion of the N4 nitrogen of pyrazolo[1,5-a]pyrimidine 9
afforded pyrazolo[1,5-a]pyridine 11 which suffered a 100-fold loss in in vitro potency. The great
disparity in potency based upon the nature of the bicyclic core is suggestive that the placement of N
atoms in cores such as 9 and 10 is imperative. This functionality may play a role in mediating the Hbond
donor/acceptor capability of the core or possibly pick up additional interactions with the protein.
Our in house X-ray data21
for compound 9 bound to CDK2 elucidated several key polar interactions
27
between the 7-NH and Leu83 backbone carbonyl and the pyrazole N and Leu83 backbone NH in a
purine-like binding mode (Figure 4).
Figure 4. X-ray structure of 9 bound to CDK2.21
Red dotted lines represent key polar interactions.
Recent X-ray data of related pyrazolo[1,5-a]pyrimidine analogs11
are consistent with these results
while additional computational and X-ray structural data rationalizing the dramatic potency
differences between the core structures
9–12 will be reported in due course.22
The two most potent analogs (9 and 10) were further evaluated
for their ability to alter uptake and incorporation of radioactively labeled thymidine by living cells. In
line with the observed in vitro potency trends in Table 1, pyrazolo[1,5-a]pyrimidine adduct 9
possessed superior activity in the thymidine incorporation assay with an IC50 = 0.6 M versus the
comparable imidazo[1,2-a]pyrazine adduct 10. As summarized in Table 1, pyrazolo[ 1,5a]pyrimidine
adduct 9 displayed comparable in vitro potency in the cyclin A/CDK2 assay to the
known CDK inhibitor flavopiridol (1) shown in Figure 1. In summary, four bicyclic cores were
designed and prepared bearing identical functionality based upon early screening hits. Based upon
both in vitro and cell-based data, the pyrazolo[1,5-a]pyrimidine core (represented by 9) emerged from
these efforts as the preferred bicyclic motif for our CDK2 program. Further optimization of the
pyrazolo[1,5-a]pyrimidine series as CDK2 inhibitors appears in the accompanying paper.23
28
References and notes
1. Murray, A. W. Cell 2004, 116, 221.
2. (a) Sherr, C. J. Cancer Res. 2000, 60, 3689; (b) Nevins, J. R. Hum. Mol. Genet. 2001, 10, 699.
3. Webster, K. R.; Kimball, D. Emerging Drugs 2000, 5, 45.
4. (a) Thomas, M. P.; McInnes, C. IDrugs 2006, 9, 273; (b) Shapiro, G. I. J. Clin. Oncol. 2006, 24,
1770; (c) Fischer, P.
M. Drugs Future 2005, 30, 911; (d) Fischer, P. M. Cell Cycle 2004, 3, 742.
5. Bible, K. C.; Lensing, J. L.; Nelson, S. A.; Lee, Y. K.; Reid, J. M.; Ames, M. M.; Isham, C. R.;
Piens, J.; Rubin, S. L.; Rubin, J.; Kaufmann, S. H.; Atherton, P. J.; Sloan, J. A.; Daiss, M. K.; Adjei,
A. A.; Erlichman, C. Clin. Cancer Res. 2005, 11, 5935.
6. McClue, S. J.; Blake, D.; Clarke, R.; Cowan, A.; Cummings, L.; Fischer, P. M.; MacKenzie, M.;
Melville, J.; Stewart, K.; Wang, S.; Zhelev, N.; Zheleva, D.; Lane, D. P. Int. J. Cancer 2002, 102,
463.
7. Misra, R. N.; Xiao, H.; Kim, K. S.; Lu, S.; Han, W.-C.; Barbosa, S. A.; Hunt, J. T.; Rawlins, D. B.;
Shan, W.; Ahmed, S. Z.; Qian, L.; Chen, B.-C.; Zhao, R.; Bednarz, M. S.; Kellar, K. A.; Mulheron, J.
G.; Batorsky, R.; Roongta, U.; Kamath, A.; Marathe, P.; Ranadive, S. A.; Sack, J. S.; Tokarski, J. S.;
Pavletich, N. P.; Lee, F. Y. F.; Webster, K. R.; Kimball, S. D. J. Med. Chem. 2004, 47, 1719.
8. Chu, X.-J.; DePinto, W.; Bartkovitz, D.; Sung-Sau, S.; Vu, B. T.; Packman, K.; Luckacs, C.; Ding,
Q.; Jiang, N.; Wang, K.; Goelzer, P.; Yin, X.; Smith, M. A.; Higgins, B. X.; Chen, Y.; Xiang, Q.;
Moliterni, J.; Kaplan, G.; Graves, B.; Lovey, A.; Fotouhi, N. J. Med. Chem. 2006, 49, 6549.
9. Toogood, P. L.; Harvey, P. J.; Repine, J. T.; Sheehan, D. J.; VanderWel, S. N.; Zhou, H.; Keller, P.
R.; McNamara, D. J.; Fry, D. W. J. Med. Chem. 2005, 48, 2388.
10. (a) Hirai, H.; Kawanishi, N.; Iwasawa, Y. Curr. Top. Med. Chem. 2005, 5, 167; (b) McInnes, C.;
Fischer, P. M. Curr. Pharm. Des. 2005, 11, 1845; (c) Fischer, P. M. Curr. Med. Chem. 2004, 11,
1563.
11. For a recent report on pyrazolo[1,5-a]pyrimidine-based CDK2 inhibitors, see: Williamson, D. S.;
Parratt, M. J.; Bower, J. F.; Moore, J. D.; Richardson, C. M.; Dokurno, P.; Cansfield, A. D.; Francis,
G. L.; Hebdon, R. J.; Howes, R.; Jackson, P. S.; Lockie, A. M.; Murray, J. B.; Nunns, C. L.; Powles,
J.; Robertson, A.; Surgenor, A. E.; Torrance, C. J. Bioorg. Med. Chem. Lett. 2005, 15, 863.
12. Cyclin A/CDK2 kinase assay. Recombinant baculoviruses were purified from Sf9 cells
engineered to express cyclin A and CDK2. Cyclin A/CDK2 enzyme was diluted to a final
concentration of 50 g/mL in kinase buffer containing 50 mM Tris, pH 8.0, 10 mM MgCl2, 1 mM
DTT, and 0.1 mM sodium orthovanadate. For each kinase reaction, 1 g of enzyme and 20 L of 2
29
M substrate solution (a biotinylated peptide derived from Histone H1; Amersham, UK) were mixed
and combined with 10 L of diluted compound. The reaction was started by addition of 50 L of 2
M ATP and 0.1 Ci of 33P-ATP (Amersham, UK). Kinase reactions ran for 1 h at room temperature
and were stopped by the addition of 0.1% Triton X-100, 1 mMATP, 5 mM EDTA, and 5 mg/mL
streptavidin-coated SPA beads (Amersham, UK). SPA beads were captured using a 96-well GF/B
filter plate (Packard/Perkin Elmer Life Sciences) and a Filtermate universal harvester (Packard/
Perkin-Elmer Life Sciences.) Beads were washed twice with 2M NaCl and twice with 2 M NaCl with
1% phosphoric acid. Signal was then assayed using a TopCount 96-well liquid scintillation counter
(Packard/Perkin-Elmer Life Sciences). Dose–response curves were generated from duplicate 8 point
serial dilutions of inhibitory compounds. IC50 values were derived by nonlinear regression analysis.
13. Senga, K.; Novinson, T.; Wilson, H. R.; Robins, R. K. J. Med. Chem. 1981, 24, 610.
14. Davey, D. D. J. Org. Chem. 1987, 57, 4379.
15. Larson, S. D.; Spilman, C. H. WO 1993/25553.
16. Lober, S.; Huber, H.; Utz, W.; Gmeiner, P. J. Med. Chem. 2001, 44, 2941.
17. Aboul-Fadl, T.; Lober, S.;Gmeiner, P. Synthesis 2000, 1727.
18. Cai, S. X.; Huang, J.-C.; Espitia, S. A.; Tran, M.; Ilyin, V. I.; Hawkinson, J. E.; Woodward, R.
M.; Weber, E.; Keana, J. F. W. J. Med. Chem. 1997, 40, 3679.
19. Hand, E. S.; Paudler, W. W. J. Org. Chem. 1978, 43, 2906.
20. Thymidine uptake growth inhibition assay. The thymidine incorporation assay was used to
measure inhibition of asynchronously growing A2780 ovarian carcinoma cells exposed to test
compound. These cells were maintained in DMEM (Cellgro) plus 10% fetal bovine serum (HyClone)
and passaged twice a week by detaching the monolayer with Trypsin-EDTA (Gibco). One hundred
microliters of A2780 cells (5000 cells/well) was added per well to a 96 wellCytostar Tplate
(Amersham,UK) and incubated for 16–24 h at 37 °C. Compounds were serially diluted in complete
media plus 2% 14C-labeled thymidine (Amersham, UK). Media were removed from Cytostar T plate,
200 L of compound dilution was added in quadruplicate and incubated for 24 h at 37 °C.
Accumulated incorporation of radiolabel was assayed using scintillation proximity and measured on
TopCount (Packard/Perkin-ElmerLife Sciences).Percentage inhibitions, relative to vehicle controls,
were calculated and plotted on log-linear plots to allow derivation of IC50 values.
21. The X-ray coordinates for compound 9 bound to CDK2 have been deposited in the
ProteinDataBank (PDB ID code: 2R3R).
22. Duca, J.; Fischmann, T. O.; Hruza, A.; Madison, V. Unpublished results.
23. Paruch, K.; Dwyer, M. P.; Alvarez, C.; Brown, C.; Chan, T.-Y.; Doll, R. J.; Keertikar, K.;
Knutson, C.; McKittrick, B.; Rivera, J.; Rossman, R.; Tucker, G.; Fischmann, T. O.; Hruza, A.;
30
Madison, V.; Nomeir, A. A.; Wang, Y.; Lees, E.; Parry, D.; Sgambellone, N.; Seghezzi, W.; Schultz,
L.; Shanahan, F.; Wiswell, D.; Xu, X.; Zhou, Q.; James, R. A.; Paradkar, V. M.; Park, H.; Rokosz, L.
R.; Stauffer, T. M.; Guzi, T. J. Bioorg. Med. Chem. Lett. 2007. doi:10.1016/j.bmcl.2007.09.017,
subsequent paper.
Note: Experimental details can be found in our publicly available patents WO 2004/022062 A1, WO
2004/026310 A1, WO 2004/026867 A1, WO 2004/026872 A1, and WO 2004/026877 A1.
31
Part 1b
Pyrazolo[1,5-a]pyrimidines as orally available inhibitors
of cyclin-dependent kinase 2*
*published as:
Paruch, K.;*
Dwyer, M. P.; *
Alvarez, C.; Brown, C.; Chan, T.-Y.; Doll, R. J.; Keertikar, K.; Knutson, C.;
McKittrick, B.; Rivera, J.; Rossman, R.; Tucker, G.; Fischmann, T.; Hruza, A.; Madison, V.; Nomeir, A. A.;
Wang, Y.; Lees, E.; Parry, D.; Sgambellone, N.; Seghezzi, W.; Schultz, L.; Shanahan, F.; Wiswell, D.; Xu, X.;
Zhou, Q.; James, R. A.; Paradkar, V. M.; Park, H.; Rokosz, L. R.; Stauffer, T. M.; Guzi, T. J. Pyrazolo[1,5a]pyrimidines
as Orally Available Inhibitors of Cyclin-Dependent Kinase 2. Bioorg. Med. Chem. Lett. 2007,
17, 6220.
One of the characteristics of cancer is uncontrolled cell growth and proliferation. Cyclin-dependent
kinases (CDKs) are key regulators of the cell cycle1
and the proper regulation of CDK activity is
crucial for the ordered execution of the phases of the cycle. A large number of human neoplasias
show overexpression of positive regulators of CDKs and/or decrease in negative regulators.2
Abnormal expression of CDK2/cyclin E has been detected in colorectal, ovarian, breast, and prostate
cancers.3
CDK inhibitors have been shown to induce apoptosis in different tumor cell lines.4
Therefore, CDK inhibitors have the potential to enlarge the group of anticancer agents. A number of
more or less selective CDK inhibitors have been described in the literature5
; those undergoing clinical
trials are flavopiridol (1),6
roscovitine (2),7
and BMS 387032 (3).8
Recently, an article on a series of
pyrazolo[ 1,5-a]pyrimidines (4) (with amines linked through NH or O at the 5-position and
arylsulfones at the 7-position NH) possessing CDK2 inhibitory activity has been published.9
Herein,
pyrazolo[1,5-a]pyrimidines with benzylic substituents at the 7-position are described. The selectivity
and pharmacokinetic profiles of these compounds are significantly different from those with N-aryl
substitution at the 7-position.9
32
Our effort had started by identification of a relatively weak inhibitor 5 (CDK2/cyclinA IC50 = 500
nM). A number of pyrazolo[1,5-a]pyrimidines were synthesized from appropriately substituted
acetonitriles and -ketoesters as shown in Scheme 1. Desired substitution at the 3-position was
achieved by choosing properly substituted acetonitriles 6 or via Pd-catalyzed coupling of
intermediate 10.
Scheme 1. Reagents: (a) HCO2Et, t-BuOK, THF; (b) N2H4, AcOH, EtOH; (c) R5
COCH2CO2Me, PhCH3; (d)
POCl3, N,N-dimethylaniline; (e) R7
NH2, DIPEA, dioxane; (f) Boc2O, DMAP, CH2Cl2; (g) NBS, CH3CN; (h)
R3
B(OH)2, Pd[PPh3]4, Na2CO3, DME, H2O or R3
SnBu3, Pd[PPh3]4, dioxane; (i) TFA, CH2Cl2.
33
Incorporation of halogens and other small substituents at the 3-position resulted in significant
improvement of potency (Table 1). Most active compounds were selective against GSK3 and
MAPK kinases. 12b exhibited activity in cells (measured by incorporation of radiolabeled thymidine)
with IC50 = 350 nM. The X-ray crystal structure of 13 in CDK2 (without cyclin) given in Figure 1 is
consistent with the observed SAR-only a relatively small cavity occupied by 3-substituents is
available in the vicinity of Phe 80; (13 CDK2/cyclinA IC50 = 49 nM). Thus, only small non-polar
substituents (H, Br, Me, Et, c-Pr, and SCH3 ) were tolerated; incorporation of large (Ph, Bn) or polar
(NO2 , CH2OH) motifs resulted in a sharp drop of activity.
Table 1. CDK2 inhibitory activity of pyrazolo[1,5-a]pyrimidines 12a–12y.
34
Figure 1. X-ray of crystal structure of 13 in CDK2.
Exploration of the 5-position led to a variety of inhibitors whose IC50s were below 50 nM (Table 2).
A somewhat greater differentiation was noted in the cell-based assay, where the compounds with
relatively non-polar substituents showed best potency. Notable exceptions are piperidine-containing
compounds 14l and 14m; the presence of the piperidine moiety, however, resulted in somewhat
inconsistent SAR across the series. 14g, 14h, 14i, and 14n were prepared from 5,7-dichloro[1,5a]pyrimidine
by sequential displacements at the 7-position with 3-(aminomethyl)pyridine and at the
5-position with the corresponding nucleophiles followed by bromination with NBS.
Table 2. CDK2 inhibitory activity of pyrazolo[1,5-a]pyrimidines 14a–14p.
35
A variety of substituents were tolerated at the 7-position (Table 3), which is close to the solventexposed
part of the enzyme. The best cell activity (measured by radiolabeled thymidine uptake) was
noted for the subclass containing pyridines and pyridine-N-oxides. In addition, unlike the aniline
series, those compounds exhibited good oral PK profile.
Table 3. CDK2 inhibitory activity of pyrazolo[1,5-a]pyrimidines 15a–15l.
Compound 15j was profiled further: it was screened against a panel of 50 kinases (e.g. cSRC, JNK1,
PDK1, PKB, ROCK-II) without observing any non-CDK cross-reactivity. The compound is
moderately protein-bound (mouse: 90%, rat: 85%, monkey: 89%, dog: 93%, human: 95%). 15j was
active against a panel of 17 different tumor cell lines in the clonogenicity assay with IC50s in the
range of 120–390 nM. The compound is orally available and its PK parameters are summarized in
Table 4.
36
Table 4. Pharmacokinetic parameters of 15j.
Compound 15j demonstrated efficacy in a staged A2780 tumor xenograft model in the mouse (Fig.
2). The dose of 40 mpk, qd, PO for 10 days caused 96% tumor growth inhibition with observed tumor
regression in 9 of 10 animals. The compound was well tolerated and only a moderate and reversible
decrease of white blood cells was observed.
Figure 2. Efficacy of 15j in A2780 xenograft model (mouse).
In conclusion, we demonstrated that properly substituted pyrazolo[1,5-a]pyrimidines can serve as
potent, selective, and efficacious orally available CDK2 inhibitors.
37
References and notes
1. Murray, A. W. Cell 2004, 116, 221.
2. Carnero, A. Br. J. Cancer 2002, 87, 129.
3. Webster, K. R.; Kimball, D. Emerging Drugs 2000, 5, 45.
4. Cai, D.; Byth, K. F.; Shapiro, G. I. Cancer Res. 2006, 66, 435.
5. Kong, N.; Fotouhi, N.; Wovkulich, P. M.; Roberts, J. Drugs Fut. 2003, 28, 881.
6. Bible, K. C.; Lensing, J. L.; Nelson, S. A.; Lee, Y. K.; Reid, J. M.; Ames, M. M.; Isham, C. R.;
Piens, J.; Rubin, S. L.; Rubin, J.; Kaufmann, S. H.; Atherton, P. J.; Sloan, J. A.; Daiss, M. K.; Adjei,
A. A.; Erlichman, C. Clin. Cancer Res. 2005, 11, 5935.
7. McClue, S. J.; Blake, D.; Clarke, R.; Cowan, A.; Cummings, L.; Fischer, P. M.; MacKenzie, M.;
Melville, J.; Stewart, K.; Wang, S.; Zhelev, N.; Zheleva, D.; Lane, D. P. Int. J. Cancer. 2002, 102,
463.
8. Misra, R. N.; Xiao, H.; Kim, K. S.; Lu, S.; Han, W.-C.; Barbosa, S. A.; Hunt, J. T.; Rawlins, D. B.;
Shan, W.; Ahmed, S. Z.; Qian, L.; Chen, B.-C.; Zhao, R.; Bednarz, M. S.; Kellar, K. A.; Mulheron, J.
G.; Batorsky, R.; Roongta, U.; Kamath, A.; Marathe, P.; Ranadive, S. A.; Sack, J. S.; Tokarski, J. S.;
Pavletich, N. P.; Lee, F. Y. F.; Webster, K. R.; Kimball, S. D. J. Med. Chem. 2004, 47, 1719.
9. Williamson, D. S.; Parratt, M. J.; Bower, J. F.; Moore, J. D.; Richardson, C. M.; Dokurno, P.;
Cansfield, A. D.; Francis, G. L.; Hebdon, R. J.; Howes, R.; Jackson, P. S.; Lockie, A. M.; Murray, J.
B.; Nunns, C. L.; Powles, J.; Robertson, A.; Surgenor, A. E.; Torrance, C. J. Bioorg. Med. Chem.
Lett. 2005, 15, 863.
Note: Experimental details can be found in our publicly available patents WO 2004/022062 A1, WO
2004/022559 A1, WO 2004/022560 A1, WO 2004/022561 A1, and WO 2004/026229 A1.
38
Part 1c
Discovery of dinaciclib (SCH 727965): a potent and
selective inhibitor of cyclin-dependent kinases*
*published as:
Paruch, K.; Dwyer, M. P.; Alvarez, C.; Brown, C.; Chan T.-Y.; Doll, R. J.; Keertikar K.; Knutson, C.;
McKittrick, B.; Rivera, J.; Rossman, R.; Tucker, G.; Fishmann, T.; Hruza, A.; Madison, V.; Nomeir, A. A.;
Wang, Y.; Kirschmeier, P.; Lees, E.; Parry, D.; Sgambellone, N.; Seghezzi, W.; Schultz, L.; Shanahan, F.;
Wiswell, D.; Xu, X.; Zhou, Q.; James, R. A.; Paradkar, V. M.; Park, H.; Rokosz, L. R.; Stauffer, T. M.; Guzi, T.
J.*
Discovery of Dinaciclib (SCH 727965): A Potent and Selective Inhibitor of Cyclin-Dependent Kinases.
ACS Med. Chem. Lett. 2010, 1, 204.
The mammalian cell cycle is a nonredundant proces that integrates extracellular signaling, DNA
synthesis, and mitosis.1
Loss of cell cycle control is a hallmark of all human cancers and is frequently
associated with aberrant activation of cyclin-dependent kinases (CDKs).2,3
The CDKs are a wellcharacterized
family of serine/threonine kinases that regulate cell cycle progression by
phosphorylation and inactivation of the retinoblastoma tumor suppressor gene product (Rb)
throughout late-G1, S, and G2/M phases as well as playing a role in the G2/M checkpoint and
progression through mitosis.4-9
Other members of the CDK family have also been shown to have
functions beyond cell cycle regulation. For example, CDK5 is involved in neuronal function and τ
phosphorylation,10
and CDK7, CDK8, and CDK9 have been implicated in transcriptional
regulation.11,12
Inhibition of multiple members of the CDK family has been shown to induce
therapeutically desirable phenotypes such as inhibition of proliferation and apoptosis. For example,
expression of dominant negative forms of CDK2 and combinatorial silencing of CDK1 and CDK2 via
siRNA generates cell cycle arrest and apoptosis.13-17
Likewise, inhibition of the noncell cycle-related
CDKs, CDK7 and CDK9, depress transcriptional regulation a variety of targets including several
antiapoptotic proteins.8,17
Moreover, inhibition of CDK8 modulates B-catenin function, resulting in
decreased proliferation of colon cancer cells.18,19
Thus, inhibition of the essential, rate-limiting
activities of multiple members of the CDK family represents an attractive therapeutic strategy for
oncology indications.
A number of diverse CDK inhibitors have progressed into clinical development (Figure 1).20-23
Flavopiridol has been the most clinically studied CDK inhibitor, and several phase II trials targeting a
39
variety of indications have been completed. Thus far, the most significant activity has been reported
in chronic lymphocytic leukemia.24
Figure 1. Representative CDK inhibitors in clinical trials.
Efforts in applying an in vivo screening system to select candidate CDK inhibitors with the optimal
combination of potency, pharmacokinetic, efficacy, and safety parameters are herein described. This
functional approach allowed rapid differentiation within a discrete series of compounds as well as
benchmarking against flavopiridol. Ultimately, this approach led to the identification of SCH 727965,
which is currently in phase II clinical trials.
The utility of the pyrazolo[1,5-a]pyrimidine scaffold as a basis for the development of novel CDK
inhibitors has previously been described.25,26
From these efforts, compound 6 was identified as a
novel CDK inhibitor, which was effective at reducing tumor burden in vivo upon oral delivery. In an
effort to further expand the understanding of the impact of positional substitution on activity,
structure-activity relationship development was expanded around each position in the pyrazolo[1,5a]pyrimidine
core. The preparation of these novel pyrazolo[1,5-a]pyrimidine CDK inhibitors is
outlined in Schemes 1 and 2. The synthesis of these analogues for the 3-bromo series began with
treatment of 3-aminopyrazole with diethylmalonate under basic conditions followed by chlorination
to afford 16. Treatment with 3-aminomethylpyridine followed by Boc protection afforded
intermediate 17. Bromination with NBS followed by oxidation yielded 18, which was treated with
commercially available amines and deprotected under basic conditions to afford the title compounds
7-11.
40
Scheme 1. Preparation of 3-Bromo Derivatives 7-11.a
a
Reagents and conditions: (a) Na, EtOH, diethylmalonate, reflux. (b) POCl3, dimethylaniline, 60 °C. (c) 3aminomethyl
pyridine, DIPEA, dioxane. (d) Boc2O, DMAP, CH2Cl2. (e) NBS, CH3CN, 0 °C. (f) MCPBA,
CH2Cl2. (g) HNR1
R2
, DIPEA, dioxane, 90 °C. (h) KOH, EtOH/H2O, 100 °C.
The corresponding 3-ethyl derivatives were prepared by cyclization of 3-amino-4-ethyl pyrazole with
dimethylmalonate under basic conditions followed by chlorination, which afforded 19 (Scheme 2).
Treatment of 19 with 3-(aminomethyl) pyridine N-oxide monohydrochloride under basic conditions
yielded 20 followed by treatment with known amino alcohols, which afforded title compounds 13 and
14. The preparation of 6 and 12 has been previously described.26,27
41
Scheme 2. Preparation of 3-Ethyl Aminoalcohol Derivatives 13 and 14.a
a
Reagents and conditions: (a) Dimethylmalonate, reflux. (b) NaOMe, MeOH, reflux. (c) POCl3,
dimethylaniline, reflux. (d) 3-Aminomethyl pyridine N-oxide hydrochloride, NaHCO3, CH3CN, reflux. (e)
HNR1
R2
, NaHCO3
, NMP, 150 °C.
As illustrated in Table 1, a key observation made in the 3-bromo series was the marked improvement
in potency by replacement of the aryl functionality at the 5-position of 6 with N-linked motifs bearing
hydroxy substitution such as 8. Furthermore, with correct positioning of this functionality, additional
improvements in both kinase activity as well as inhibition of cell growth were observed in 9-11.
42
Table 1. Optimization in the 5-Position.a
a
Dose-response curves were generated from duplicate 8-point serial dilutions of inhibitory compounds. IC50
values were derived by nonlinear regression analysis.
b
Thymidine uptake growth inhibition assay in A2780 cells. Percentage inhibitions, relative to vehicle controls,
were calculated and plotted on log-linear plots to allow derivation of IC50 values.25,26
The structural basis for these improvements in activity can be readily explained by interactions
revealed in the X-ray crystal structure of this series of compounds bound to CDK2 and the
CDK2/cyclin A complex.28
A detailed description of these findings will be published in due course.
The initial goal of these effortswas to identify a novel inhibitor of CDK1 and CDK2. Because of the
high degree of homology within the CDK family, inhibition of additional CDKs within this series of
compound was also observed (data not shown). Thus, there exists a realistic likelihood that these
small molecule inhibitors of the CDKs would exert their effects via compound-specific, combinatorial
inhibition of multiple members of the family. This expected multitargeted nature of CDK inhibitors
places a premium on maintaining an adequate therapeutic index in vivo. In this context, a critical issue
in the development of CDK inhibitors is the relationship between desirable on-target effects and the
43
onset of off-target tolerability issues that might adversely affect clinical dose escalation. Ultimately, a
screening system reminiscent of the historical standard paradigm for cytotoxic drug discovery, which
evaluated the tolerability versus activity in vivo, was implemented.
Simply, this in vivo system sought to establish the ratio of maximally tolerated and minimally
effective doses in the A2780 ovarian carcinoma xenograft model. It was reasoned that through the
application of this screening system, a compound could be identified that might give the best
opportunity to achieve pharmacologically relevant doses clinically before the manifestation of
toxicity. Hence, the maximally tolerated dose (MTD) was determined by intraperitoneal
administration of compound to nude mice on a fixed, QDx7 schedule at varying dose levels. In these
experiments, the MTD was defined as the dose on the QDx7 schedule required to reduce body weight
by 20%. In parallel, the minimum effective doses (MEDs) were established on the same schedule.
The MED was defined as the dose given QDx7 that was associated with >50% tumor growth
inhibition. Pharmacokinetic parameters were also established to enable calibration of effects on the
basis of systemic exposure. Thus, a series of compounds comprised of diverse structures, potency,
and pharmacokinetic parameters were selected in an attempt to identify the optimal profile for a CDK
inhibitor within this class of compound.
Through the application of this in vivo screen, clear trends emerged among compounds of diverse
structures and ranges of potency (Table 2).
44
Table 2. Relative Therapeutic Indices from the in Vivo Screening Paradigm.
a
Dose-response curves were generated from duplicate 8-point serial dilutions of inhibitory compounds. IC50
values were derived by nonlinear regression analysis.
b
Thymidine uptake growth inhibition assay in A2780 cells. Percentage inhibitions, relative to vehicle controls,
were calculated and plotted on log-linear plots to allow derivation of IC50 values.
c
Relative therapeutic indices following intraperitoneal (IP) dosing using the in vivo screening paradigm
(MTD=20% weight loss; MED = 50% inhibition of tumor growth; and QDx7 = once a day dosing for 7
consecutive days).25,26
45
Compound 12, an analog with improved in-cell activity relative to the previously described 6, was
found to have an MED of 5 mpk with an MTD of 5 mpk, giving a putative therapeutic index of 1. In
contrast, the more highly potent compound 10 displayed a relative therapeutic index of 5.
Interestingly, simple modification of substitution within this highly potent series of inhibitor induced
significant differences in relative therapeutic indices. For example, incorporation of a 3-ethyl
substituent as in 13 resulted in an improved overall profile relative to 10. Conversely, the
aminocyclohexyl methyl alcohol 14 gave a decreased tolerability profile relative to the piperidine
ethanol derivative 13. Notably, these compounds displayed no significant differences in
pharmacokinetic profile (Table 3) or kinase cross reactivity across a set of diverse kinases (data not
shown).
Table 3. Exposure parameters.a
a
Plasma samples from mice were collected at various times after intraperitoneal administration at 5 mg/kg and
analyzed by liquid chromatography-tandem mass spectrometry. Pharmacokinetic variables were estimated from
the plasma concentration data.
b
The area under the plasma concentration vs time curve (AUC) was calculated using the linear trapezoidal rule.
c
Cmax values (maximum plasma concentration) were taken directly from the plasma concentration-time
profiles.26,29
We also benchmarked flavopiridol 15. The ratio of MTD to MED for flavopiridol was <1, indicating
that in this screening system minimal antitumor efficacy was attained before the onset of toxicity.
While ramifications of these findings are unknown, it is compelling to hypothesize that a compound
such as 13 may have increased potential to achieve a more robustly active dose range prior to the
manifestation of dose-limiting toxicities. Several unexpected trends emerged from the utilization of
46
this screening system. Namely, compounds with very high levels of potency tended to give improved
therapeutic indices relative to those with lower affinity. Interestingly, these compounds displayed
lower exposure and higher clearance rates relative to those with lower therapeutic indices. Taken
together, short exposures to highly potent CDK inhibitors appear to induce long-lasting effects and
have the potential to do so with improved
tolerability. This stands in contrast to efforts in earlier stages of the program, which had been more
focused on chronic exposure to induce continual CDK inhibition. Additionally, within this series of
compounds, small changes in substitution had a large impact on overall activity and tolerability
relationships that were not predictable from simple in vitro and pharmacokinetic profiling.
In summary, a series of pyrazolo[1,5-a]pyrimidine CDK inhibitors, which display a range of potency
and pharmacokinetic parameters, were identified. Utilizing a functional in vivo screen, compounds
were readily differentiated on the basis of efficacy versus tolerability profiles even in those structures
with remarkably similar substitution and in vitro and pharmacokinetic profiles. A key observation
arising from the application of this approach was that highly potent, rapidly cleared CDK inhibitors
appear to give an optimal balance between efficacy and tolerability. Within compounds of this
profile, 13 was selected as a candidate CDK inhibitor suitable for progression. In-depth evaluation of
the in vitro and in vivo properties further supported the conclusion that 13 had the appropriate
qualities for a development candidate.29
Compound 13 (SCH 727965) is currently undergoing phase
II clinical trials.
47
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O'Brien, M. A.; O'Reilly, M.; Reule, M.; Saxty, G.; Seavers, L. C. A.; Smith, D.-M.; Squires, M. S.;
Trewartha, G.; Walker, M. T.; Woolford, A. J.-A. Identification of N-(4-piperidinyl)- 4-(2,6dichlorobenzoylamino)-1H-pyrazole-3-carboxamide
(AT7519), a novel cyclin dependent kinase
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Tokarski, J. S.; Pavletich, N. P.; Lee, F. Y. F.; Webster, K. R.; Kimball, S. D. N(Cycloalkylamino)acyl-2-aminothiazole
inhibitors of cyclin- dependent kinase 2. N-[5-[[[5-(1,1Dimethylethyl)-2-
oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide (BMS-387032), a
highly efficacious and selective antitumor agent. J. Med. Chem. 2004, 47, 1719–1728.
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(25) Dwyer, M. P.; Paruch, P.; Alvarez, C.; Doll, R. J.; Keertikar, K.; Duca, J.; Fischmann, T. O.;
Hruza, A.; Madison, V.; Lees, E.; Parry, D.; Seghezzi, W.; Sgambellone, N.; Shanahan, F.; Wiswell,
D.; Guzi, T. J. Versatile templates for the development of novel kinase inhibitors: discovery of novel
CDK inhibitors. Bioorg. Med. Chem. Lett. 2007, 17, 6216–6219.
(26) Paruch, P.; Dwyer, M. P.; Alvarez, C.; Brown, C.; Chan, T.-Y.; Doll, R. J.; Keertikar, K.;
Knutson, C.; McKittrick, B.; Rivera, J.; Rossman, R.; Tucker, G.; Fischmann, T.O.; Hruza, A.;
Madison, V.; Nomeir, A.; Wang, Y.; Lees, E.; Parry, D.; Sgambellone, N.; Seghezzi, W.; Schultz, L.;
Shanahan, F.; Wiswell, D.; Xu, X.; Zho, Q.; James, R. A.; Paradkar, V.; Park, H.; Roskosz, L.;
Stauffer, T.; Guzi, T. J. Pyrazolo[1,5-a]pyrimidines as orally available inhibitors of cyclin-dependent
kinase 2. Biorg. Med. Chem. Lett. 2007, 17, 6220–6223.
(27) Full experimental details for the preparation of compounds 6 and 12 can be found in the
Supporting Information.
(28) Fischmann, T. O.; Hruza, A.; Duca, J. S.; Ramanathan, L.; Mayhood, T.; Windsor,W. T.; Le, H.
V.; Guzi, T. J.; Dwyer, M. P.; Paruch, K.; Doll, R. J.; Lees, E.; Parry, D; Seghezzi, W.; Madison, V.
Structure-guided discovery of cyclin-dependent kinase inhibitors. Biopolymers 2008, 89, 372–379.
(29) Parry, D.; Guzi, T.; Shanahan, F.; Davis, N.; Prabhavalkar, D.; Wiswell, D.; Seghezzi, W.;
Paruch, K.; Dwyer, M. P.; Doll, R.; Nomeir, A.; Windsor, W.; Fischmann, T.; Wang, Y.; Oft, M.;
Chen, T.; Kirschmeier, P.; Lees, E. SCH 727965, a novel and potent cyclin-dependent kinase
inhibitor. Mol. Cancer Ther. Submitted for publication.
50
Part 1d
Dinaciclib (SCH 727965), a novel and potent cyclin-dependent kinase inhibitor*
*published as:
Parry, D.;*
Guzi, T. J.; Shanahan, F.; Davis, N.; Prabhavalkar, D.; Wiswell, D; Seghezzi, W.; Paruch, K.;
Dwyer, M. P.; Doll, R. J.; Nomeir, A. A.; Windsor, W.; Fischmann, T.; Wang, Y.; Oft, M.; Chen, T.;
Kirschmeier, P.; Lees, E. M. Dinaciclib (SCH 727965), a Novel and Potent Cyclin-Dependent Kinase Inhibitor.
Mol. Cancer Ther. 2010, 9, 2344.
The mammalian cell cycle is a nonredundant process that integrates extracellular signaling, DNA
synthesis, and mitosis.1, 2
Dysregulation of cell cycle control is a hallmark of all human cancers and is
frequently associated with selective, aberrant activation of cyclin-dependent kinases (CDK).3,4
Several members of the CDK family are critical regulators of cell cycle progression. CDK2 and
CDK1 are two closely related kinases that play overlapping roles during cell division, contributing to
the phosphorylation and inactivation of the retinoblastoma (Rb) tumor suppressor gene product
throughout late G1, S, and G2-M phases.5-7
Other CDK family members play important roles outside
of direct regulation of cell cycle progression. For instance, CDK7, CDK8, and CDK9 contribute to
the regulation of RNA polymerase II and the control of cellular transcription.8,9
Inhibition of CDK activities represents an attractive therapeutic strategy in oncology. Indeed,
expression of dominant-negative forms of CDK2 or combinatorial silencing of CDK1 and CDK2
through small interfering RNA generates therapeutically relevant phenotypes, such as cell cycle
arrest.10-12
Suppression of CDK9 using RNAi has desirable therapeutic effects in vitro.13
Similarly,
inhibition of CDK9 and the subsequent suppression of MCL1 transcription are proposed as a potential
mechanism-of-action for the pan-CDK inhibitor flavopiridol in chronic lymphocytic leukemia.14,15
Finally, CDK8 is a key modulator of β-catenin function, and the CDK8 gene is amplified in some
human colorectal cancers.16,17
Furthermore, the lack of appropriate cell cycle regulation in tumor cells
predicts their increased propensity for apoptosis, compared with normal tissue.18
Based on the intriguing and multifaceted attributes of the CDK target class, several small-molecule
CDK inhibitors have entered clinical development.19
However, due to the high degree of structural
homology within the CDK protein family, putative small-molecule CDK inhibitors may exert their
effects through combinatorial inhibition of multiple CDKs and other closely related serine/threonine
kinases. Hence, the almost inevitable multitargeted nature of CDK inhibitors places a high premium
51
on maintaining an adequate therapeutic index in vivo. A critical issue for the successful development
of CDK inhibitors (and indeed the majority of cytoreductive agents) is, therefore, the relationship
between desirable, target-specific effects and the onset of nonspecific adverse events that might
negatively influence clinical dose escalation.20
Selection of dinaciclib (SCH 727965; Fig. 1A) for
clinical development was facilitated by a range of diverse assays and included an in vivo screening
strategy that identified candidate compounds with the optimal combination of pharmacokinetic,
efficacy, and safety characteristics.21
This highly effective, functional approach allowed for rapid
benchmarking against known CDK inhibitors with undesirable side effects, such as flavopiridol (Fig.
1B), and showed SCH 727965 to be a compound with a significantly superior therapeutic profile.
Figure 1. Structures of SCH 727965 (A) and flavopiridol (B).
Materials and Methods
Experimental and control agents
SCH 727965 ((S)-(−)-2-(1-{3-ethyl-7-[(1-oxy-pyridin-3-ylmethyl)amino] pyrazolo[1,5-a]pyrimidin-
5-yl}piperidin-2-yl)ethanol) and flavopiridol (2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3hydroxy-1-methyl-4-piperidinyl]-4-chromenone)
were supplied by Schering-Plough Research
Institute. Paclitaxel was purchased from Sigma. The vehicle for these agents, except paclitaxel, was
20% hydroxypropyl-β-cyclodextrin. The vehicle for paclitaxel was Cremophor. Dosing volume was
0.2 mL per injection.
Cyclin/CDK kinase assay
Recombinant cyclin/CDK holoenzymes were purified from Sf9 cells engineered to produce
baculoviruses that express a specific cyclin or CDK. Cyclin/CDK complexes were typically diluted to
a final concentration of 50 μg/mL in a kinase reaction buffer containing 50 mmol/L Tris-HCl (pH
52
8.0), 10 mmol/L MgCl2, 1 mmol/L DTT, and 0.1 mmol/L sodium orthovanadate. For each kinase
reaction, 1 μg of enzyme and 20 μL of a 2-μmol/L substrate solution (a biotinylated peptide derived
from histone H1; Amersham) were mixed and combined with 10 μL of diluted SCH 727965. The
reaction was started by the addition of 50 μL of 2 μmol/L ATP and 0.1 μCi of 33
P-ATP (Amersham).
Kinase reactions were incubated for 1 hour at room temperature and were stopped by the addition of
0.1% Triton X-100, 1 mmol/L ATP, 5 mmol/L EDTA, and 5 mg/mL streptavidin-coated SPA beads
(Amersham). SPA beads were captured using a 96-well GF/B filter plate (Packard/Perkin-Elmer Life
Sciences) and a Filtermate universal harvester (Packard/Perkin-Elmer Life Sciences.) Beads were
washed twice with 2 mol/L NaCl and twice with 2 mol/L NaCl containing 1% phosphoric acid. The
signal was then assayed using a TopCount 96-well liquid scintillation counter (Packard/Perkin-Elmer
Life Sciences). Dose-response curves were generated from duplicate, eight-point serial dilutions of
inhibitory compounds. IC50 values were derived by nonlinear regression analysis.
Kinase counter-screening
SCH 727965 and flavopiridol were counter-screened using the Millipore Kinase Profiler service. Both
compounds were tested at 1.0 and 10.0 μmol/L against a panel of diverse kinases, using a fixed (10
μmol/L) concentration of ATP.
Cell lines and cell culture
The majority of the tumor cell lines were obtained from the American Type Culture Collection and
propagated under the suggested growth conditions. Mantle cell lymphoma cell lines were generously
provided by Dr. Geoffrey Shapiro (Dana-Farber Cancer Institute, Boston, MA).
dThd uptake growth inhibition assay
A2780 cells were maintained in DMEM (Cellgro) plus 10% fetal bovine serum (HyClone) and
passaged twice weekly by detaching the monolayer with trypsin-EDTA (Life Technologies). One
hundred microliters of A2780 cells (5 × 103
cells) were added per well to a 96-well Cytostar-T plate
(Amersham) and incubated for 16 to 24 hours at 37 °C. Compounds were serially diluted in complete
media plus 2% 14
C-labeled dThd (Amersham). Media were removed from the Cytostar T plate; 200
μL of various compound dilutions were added in quadruplicate; and the cells were incubated for 24
hours at 37 °C. Accumulated incorporation of radiolabel was assayed using scintillation proximity
and measured on a TopCount (Packard/Perkin-Elmer Life Sciences). The percentage of dThd uptake
inhibition, relative to a vehicle control, was calculated and plotted on log-linear plots to allow
derivation of IC50 values.
53
Bromodeoxyuridine incorporation assay
A2780 cells were plated into six-well tissue culture dishes and allowed to adhere. Cells were then
exposed to differing concentrations of SCH 727965 or a DMSO control vehicle for 24 hours,
followed by a brief (30 min) pulsed exposure to bromodeoxyuridine (BrdUrd). Cells were then
harvested, immunostained using FITC-conjugated antibodies specific for BrdUrd (BD Biosciences),
counter-stained with propidium iodide/RNase A solution (BD Biosciences), and analyzed using flow
cytometry. Fluorescence-activated cell sorting analyses were done on a FACSCalibur instrument
(Becton Dickinson). FITC-positive BrdUrd staining and propidium iodide signal allowed assessment
of ongoing DNA replication and the cell cycle stage. Percentages of the cell population in each cell
cycle stage were plotted for each test article concentration.
Immunoblotting
Asynchronously growing tumor cell lines were exposed to differing concentrations of SCH 727965.
Subsequently, cells were harvested and lysed in a 50 mmol/L Tris-HCl buffer containing 350 mmol/L
NaCl, 0.1% NP40, 1 mmol/L DTT, and a cocktail of protease and phosphatase inhibitors
(Calbiochem). Following protein concentration determination (Bio-Rad), cell lysates were separated
on reducing SDS-PAGE gels and immunoblotted with antisera specific for Rb phosphorylated on
serines 807, 811 (Cell Signaling), hypophosphorylated Rb (Cell Signaling), or the p85 poly ADP
ribose polymerase (PARP) caspase cleavage product (Promega).
Induction of apoptosis assessed by activated caspase
Assays of caspase activation were done using the Beckman Coulter CellProbe HT Caspase-3/7 Whole
Cell Assay system. Asynchronously growing cells were plated into 96-well plates and allowed to
adhere. Cells were exposed to differing concentrations of SCH 727965 or vehicle for 24 hours. Cells
were subsequently incubated with a fluorescently labeled caspase substrate (CellProbe); uptake and
fluorescence of the substrate within cells correlate with the level of activated caspases. The caspase
activity was determined using an Analyst AD 96.384 fluorometer (485 nm excitation and 530 nm
emission).
Clonogenicity and alamarBlue viability assays
Cells were plated onto tissue culture dishes and propagated with the appropriate growth media.
Growing cultures were exposed to increasing concentrations of SCH 727965 or a vehicle control,
typically for 7 days. After removing the medium, cells were fixed with 50% methanol/50% acetone
54
for 5 minutes and stained with 0.2% crystal violet (Fisher) in 2% ethanol for 5 minutes. Following
staining, cells were washed with 5 to 10 mL of water. Stained cells were solubilized in 1%
deoxycholic acid (Sigma), and the absorbance of the resulting solution was measured at 600 nm using
a SOFTmax PRO 4.3 plate reader (Molecular Devices). Absorbance of SCH 727965–treated samples
was plotted as a percent of that of a vehicle-treated control, and data were reported as an IC50 value
relative to these controls. For suspension cell lines, assessments of cell viability were obtained using
the alamarBlue Cell Viability Assay kit (Biotium), using the manufacturers' recommended protocol.
Experimental mice
All mouse strains were obtained from Charles River Laboratories. Female nude mice age 9 to 14
week or female BALB/c mice age 6 to 14 weeks were used. Animals were housed in an Association
for Assessment and Accreditation of Laboratory Animal Care-accredited animal facility at ScheringPlough
Biopharma, Palo Alto, CA, and Schering-Plough Research Institute, Kenilworth, NJ.
Conventional animal chow and water were provided ad libitum. All protocols related to experiments
using animals have been approved by the appropriate Institutional Animal Care and Use Committee.
In vivo tumor growth assessments
For tumor implantation, specific cell lines were grown in vitro, washed once with PBS, and
resuspended in 50% Matrigel (BD Biosciences) in PBS to a final concentration of 4 × 107
to 5 × 107
cells per milliliter. Nude mice were injected with 0.1 mL of this suspension s.c. in the flank region.
Tumor length (L), width (W), and height (H) were measured by a caliper twice weekly on each mouse
and then used to calculate tumor volume using the formula (L × W × H)/2. When the tumor volume
reached ∼100 mm3
, the animals were randomized to treatment groups (10 mice/group) and treated i.p.
with either SCH 727965 or individual chemotherapeutic agents according to the dosing schedule
indicated in table and figure legends. Tumor volumes and body weights were measured during and
after the treatment periods. Data were expressed as means ± SEM. Animals were euthanized
according to the Institutional Animal Care and Use Committee guidelines.
Immunohistochemical staining of phospho-Rb protein
Samples were harvested from the nude mice skin at various time points following administration of a
single 40-mg/kg dose of SCH 727965. The samples were fixed overnight in 10% formaldehyde,
washed in 70% ethanol, and processed in a tissue processor (Thermo Electronic Co.); the tissues were
dehydrated in graded ethanol solutions, cleared in three changes of xylene, and penetrated in heated
paraffin (at 56–58 °C). The tissues were embedded in paraffin, cut into 4- to 6-μm sections, and
55
placed onto slides. Before staining, the slides were placed in a chamber containing 1× Reveal
Solution (BioCare Medical) for deparaffinization and antigen retrieval. The slides were rinsed in hot
water, placed in PBS for 15 minutes at room temperature, and loaded onto a DAKO automated
immunostainer. Endogenous hydrogen peroxidase activity was blocked with hydrogen peroxide for
10 minutes followed by rinsing with a Tris-HCl buffered saline solution containing 0.5% Tween 20
(TBST; Sigma). The slides were then sequentially incubated with avidin, rinsed in TBST, treated with
biotin, and rinsed again in TBST (avidin/biotin block kit; Vector Laboratories). Nonspecific binding
sites were blocked with 1× blocking buffer (Sigma) for 20 minutes. The slides were then incubated
with anti-phospho Rb 807/811 (Cell Signaling) diluted 1:75 or rabbit control antisera at 1 μg/mL for
45 minutes, rinsed with TBST, and incubated with biotinylated goat anti-rabbit IgG (Elite ABC kit;
Vector Laboratories) for 30 minutes. The slides were rinsed again with TBST and treated with ABC
complex (Elite ABC kit; Vector Laboratories) for 30 minutes, followed by another TBST rinse. The
slides were developed in 3,3′-diaminobenzidine (Dakocytomation) for 5 minutes, rinsed with TBST,
and incubated for 2 minutes with hematoxylin (Dakocytomation). Finally, the slides were rinsed in
distilled water, dehydrated in graded ethanol solutions, cleared in xylene using a Leica autostainer,
and cover slipped in a Leica Cover slipper.
Assessments of SCH 727965 effects on hematologic parameters
A daily dose of SCH 727965 (40 mg/kg) was administered i.p. to BALB/c mice for 5 days. Blood
was collected on day 6 and day 13 (1st and 7th day after the final dose, respectively), diluted 1:5 in
PBS, and immediately analyzed on an Advia 120 hematology analyzer (with differential).
Pharmacokinetic determinations
Plasma samples from mice were collected at various times after i.p. administration of SCH 727965.
At each time point, blood samples from three animals were combined and analyzed for SCH 727965
by liquid chromatography-tandem mass spectrometry. Pharmacokinetic variables were estimated
from the plasma concentration data. Maximum plasma concentration values were taken directly from
the plasma concentration time profiles, and the area under the plasma concentration versus time curve
(0–24 h) was calculated using the linear trapezoidal rule.
56
Results
SCH 727965 selection using a mouse tumor xenograft model
SCH 727965 was selected as the optimal drug candidate for clinical development by screening
individual, diversely substituted compounds against the A2780 ovarian carcinoma mouse xenograft
model, using flavopiridol as a benchmark control agent. This system established the ratio of
maximum tolerated dose (MTD) and minimum effective dose (MED) for each tested compound.
MTD was determined following i.p. administration of each compound to nude mice at varying dose
levels once daily for 7 days and defined as the dose associated with a body weight reduction of 20%.
In parallel, MED was defined as the dose, given by the same schedule, associated with >50% tumor
growth inhibition. Promising compounds were further profiled in rats and dogs (21). The screening
data pertinent to SCH 727965 are outlined in Table 1. Thus, MTD and MED of SCH 727965 were
>60 and 5 mg/kg, respectively; in contrast, MTD and MED of flavopiridol were <10 and 10 mg/kg,
respectively. Therefore, a screening therapeutic index (MTD/MED ratio) of SCH 727965 was >10,
whereas the index of flavopiridol was <1, indicating that minimal antitumor efficacy was not attained
with flavopiridol before the onset of dose-limiting toxicity. These data indicated that SCH 727965 has
an attractive in vivo profile that is superior to flavopiridol.
Table 1. Comparison between SCH 727965 and flavopiridol: inhibition of CDKs, dThd incorporation, and
activity in a mouse tumor xenograft.
SCH 727965 is a potent and selective inhibitor of CDKs
Inhibition of the kinase activity of various cyclin/CDKs was examined using isolated, baculovirusexpressed
holoenzymes in vitro. SCH 727965 inhibits CDK2, CDK5, CDK1, and CDK9 with IC50
57
values of 1, 1, 3, and 4 nmol/L, respectively. Compared with flavopiridol and assayed under identical
conditions, SCH 727965 is an equally potent inhibitor of CDK1 and CDK9 but a 12- and 14-fold
stronger inhibitor of CDK2 and CDK5, respectively (Table 1). SCH 727965 was found to be a potent
DNA replication inhibitor that blocked thymidine (dThd) DNA incorporation in A2780 cells with an
IC50 of 4 nmol/L. In contrast, flavopiridol had an ∼16-fold lesser potency in that assay. These data
show that SCH 727965's stronger and more selective inhibition of CDKs translates into its more
potent inhibition of DNA synthesis compared with flavopiridol in cell-based assays (Table 1).
SCH 727965 is not a general kinase inhibitor (Supplementary Table S1) and, in a series of additional
kinase counter-screens (Table 2), was shown to be more selective or the CDK family, compared with
flavopiridol. These data showed that flavopiridol affects a broader range of serine/threonine and
tyrosine kinases (e.g., c-Src), which may contribute to its poor screening therapeutic index compared
with that of SCH 727965.
Table 2. Millipore kinase profiler counter-screening for SCH 727965 and flavopiridol.
58
SCH 727965 inhibits phosphorylation of the Rb tumor suppressor protein and induces
accumulation of the p85 PARP caspase cleavage product
Inhibition of CDK-specific serine 807 and 811 (Ser 807/811) phosphorylation on the Rb tumor
suppressor protein and accumulation of p85 PARP caspase cleavage product (p85 PARP) were
selected as mechanism-based markers. These were used to explore the mechanism of dThd uptake
inhibition observed upon cell exposure to SCH 727965 or flavopiridol, and correlate onset of
apoptosis with inhibition of CDKs (Fig. 2). Lysates from asynchronously growing A2780 cells
treated with increasing concentrations of SCH 727965 or flavopiridol for 16 hours were analyzed on
SDS-PAGE and immunoblotted with Ser 807/811 Rb–specific antisera or with p85 PARP-specific
antisera.
59
Figure 2. Mechanism-based marker analyses in human cancer cells.
A, asynchronously growing A2780 cells were treated for 16 h with increasing concentrations of SCH 727965
(left) or flavopiridol (right), as indicated. Cell lysates were immunoblotted with anti-phospho Rb 807/811, a
marker of cellular CDK activity, anti-p85 PARP caspase cleavage product, a marker indicating activation of
caspases, and anti-tubulin, a loading control.
B, short treatment with SCH 727965 induces mechanism-based effects in human cancer cells. Lysates from
asynchronously growing A2780 cells exposed to 100 nmol/L SCH 727965 for 2 h were separated on PAGESDS
and immunoblotted with antisera specific for hypophosphorylated Rb, p85 PARP caspase cleavage
product, and tubulin as a control.
C, caspase activation analysis using a quantitative fluorometric analysis. A2780 cells were exposed to
increasing concentrations of SCH 727965, as indicated, for 2 h, and analyzed 6 h after the drug washout.
Caspase activation was observed following exposure to 50 nmol/L of SCH 727965 and did not increase with
elevated levels of SCH 727965 (up to 5 μmol/L).
60
D, cell cycle distribution following short treatment with SCH 727965. Asynchronously growing A2780 cells
were exposed to increasing concentrations of SCH 727965 for 2 h, cultured for an additional 24 h in drug-free
medium, and then pulsed for 30 min with BrdUrd. A 2-h exposure to ≤500 μmol/L SCH 727965 was sufficient
to suppress >90% of BrdUrd incorporation 24 h later.
SCH 727965 strongly suppressed phosphorylation of Rb on Ser 807/811 at concentrations >6.25
nmol/L, which is in accord with the observation that 4 nmol/L concentrations are required for 50%
inhibition of dThd DNA incorporation in the same cell model as previously described. Significantly,
complete suppression of Rb phosphorylation was correlated with the onset of apoptosis, as indicated
by the appearance of the p85 PARP cleavage product in cells exposed to >6.25 nmol/L SCH 727965
(Fig. 2A, left). No evidence of apoptosis induction was detectable before the complete suppression of
Rb phosphorylation. In contrast, flavopiridol was less effective at inducing significant suppression of
Rb phosphorylation, and concentrations approaching 1 μmol/L were required to induce detectable
effects on phospho-Rb Ser 807/811 levels (Fig. 2A, right). Of note, flavopiridol stimulated the
accumulation of the p85 PARP cleavage product concentrations otherwise insufficient to inhibit Rb
phosphorylation, suggesting a poor correlation between flavopiridol-induced apoptosis and Rb
phosphorylation status. In contrast, the cytotoxic activity of SCH 727965 and its effect on Rb
phosphorylation correlate with its proposed mechanism of action based on selective inhibition of
CDKs.
Mechanism of action-based effects following a short cellular SCH 727965 exposure
To determine whether short exposures to SCH 727965 were able to induce responses similar to those
following 16- to 24-hour exposures described above, A2780 cells were treated with 100 nmol/L SCH
727965 for 2 hours, washed out and supplied with a drug-free medium. Cell lysates from a time
course of 2-hour intervals were then separated on SDS-PAGE and immunoblotted with antisera
specific for hypophosphorylated Rb and p85 PARP; this allowed the assessment of CDK inhibition
and apoptosis activation. A 2-hour exposure to 100 nmol/L SCH 727965 was sufficient to induce
suppression of Rb phosphorylation and caspase activation, detectable up to 6 hours later (Fig. 2B).
Induction of apoptosis was confirmed using a quantitative, fluorometric cell-based assay of caspase
activation following short exposures to as little as 50 nmol/L of SCH 727965 (Fig. 2C).
To examine the effects of a short SCH 727965 exposure on cell cycle, asynchronously growing
A2780 cells treated with increasing concentrations of SCH 727965 for 2 hours were maintained for
61
24 hours in a drug-free medium, then pulse labeled with BrdUrd to establish the percentage of cells
undergoing active DNA replication, before analysis by fluorescence-activated cell sorting. Under
these conditions, cells exposed to 125 to 250 nmol/L SCH 727965 for 2 hours partially suppressed
DNA synthesis and BrdUrd incorporation for 24 hours (Fig. 2D). Higher exposures (≤500 nmol/L)
were sufficient to completely suppress DNA synthesis for 24 hours and were correlated with the
accumulation of apoptotic (sub-G1) cells. Overall, these data show that short exposures to SCH
727965 can induce long-lasting effects in target cells and imply that continuous exposure to SCH
727965 may not be required for sustained activity in vivo.
SCH 727965 activity in a panel of tumor cell lines
CDK inhibitors are expected to have broad antiproliferative activity against a wide range of tumor
cells. SCH 727965 antiproliferative activity (clonogenicity, alamarBlue viability assay, dThd and
BrdUrd incorporation, and p85 PARP marker analysis) was examined in an extended panel of human
tumor cell lines that included the full NCI-60 screening set and an additional 47 cell lines procured to
expand representation of small-cell lung cancer, lymphoma, leukemia, prostate, and pancreatic
cancers. The broad range of transformed cellular backgrounds (p53, pRB, p16, c-Myc, K-Ras, etc.)
allowed SCH 727965 testing in diverse settings. Tumor cell line origin and response characteristics
are summarized in Table 3.
Table 3. SCH 727965 is active against a broad spectrum of human tumor cell lines.
62
The mean and median IC50 values across this cell line panel were 10 and 11 nmol/L, respectively.
Complete suppression of BrdUrd incorporation in the tested cell lines was typically apparent at
concentrations that induce inhibition of clonogenicity by 90% (20–25 nmol/L; data not shown).
Following exposure to SCH 727965, all tested human tumor cell lines underwent cell cycle arrest, and
no selectivity toward a specific tumor type was observed. Furthermore, apoptosis, which was
determined by the appearance of the p85 PARP cleavage product on Western blots following a single
exposure to SCH 727965 at concentrations <100 nmol/L, was detected in >85% of the cell lines
tested. Approximately 50% of cell lines from the NCI-60 screening panel express multiple drug
resistance gene 1, and it is noteworthy that multiple drug resistance gene 1 status did not significantly
influence sensitivity to SCH 727965. Overall, these data suggest that SCH 727965 has
antiproliferative activity across a broad range of tumor types and genetic backgrounds.
SCH 727965 efficacy and tolerability in vivo
The previously described A2780 ovarian cancer mouse xenograft model developed for initial
selection of active agents was used for further assessment of SCH 727965 efficacy and tolerability;
paclitaxel was a positive control. Nude mice with ∼100 mm3
A2780 tumors were randomized into
groups of 10 animals ∼7 days after initial s.c. cell inoculation and assigned to each SCH 727965
dosage group, paclitaxel, or vehicle control. SCH 727965 i.p. administration at 8, 16, 32, and 48
mg/kg daily for 10 days resulted in tumor inhibition by 70%, 70%, 89%, and 96%, respectively;
paclitaxel i.p. administration at 20 mg/kg twice weekly inhibited tumor growth by 63% (Fig. 3A).
Consistent with earlier in vivo screening data, SCH 727965 MED appears to be <8 mg/kg. SCH
727965 was well tolerated, and the maximum body weight loss in the highest dosage group was 5%
(data not shown). This is well below the MTD defined as 20% loss of body weight over the course of
this experiment. Taken together, the data show that SCH 727965 has dose-dependent antitumor
activity in vivo, and that nearly complete inhibition of tumor growth occurs at a dose level below the
MTD (Table 4; Fig. 3A).
63
Figure 3.
A, SCH 727965 efficacy in a mouse xenograft model. Nude mice inoculated s.c. with A2780 cells were treated
with SCH 727965, paclitaxel, or vehicle control when tumor volume reached ∼100 mm3
, ∼7 d after inoculation.
SCH 727965 was administered at 8, 16, 32, and 48 mg/kg i.p. daily for 10 d; paclitaxel was administered at 20
mg/kg i.p. twice weekly. Left, growth curves; right, percent tumor growth inhibition (%TGI) and percent of
maximally tolerated dose (%MTD).
B, SCH 727965 is active using intermittent dosing schedules in mice. A total dose equivalent to 20 mg/kg daily
for 13 days (260 mg/kg) was fractionated over several diverse schedules and administered to nude mice bearing
established A549 xenografts. SCH 727965 (87 mg/kg) exceeded the MTD (>20% body weight loss) when given
on a once-weekly schedule. Similar regressions were observed on all schedules.
C, SCH 727965 induces mechanism-based systemic effects in vivo. SCH 727965 induced rapid and sustained
suppression of phospho-Rb within the proliferating epithelial cells of the basal epithelium and hair follicles.
Skin samples were obtained from mice before treatment (T0, predose) or 2 and 4 h following administration of
64
40 mg/kg SCH 727965 i.p. (T2 and T4 postdose). Samples were stained with phospho-Rb Ser 807/811 specific
antisera.
D, SCH 727965 induces reversible hematologic effects. BALB/c mice were treated with SCH 727965 at 40
mg/kg on a dose-intense, daily for 5 days schedule. To determine blood cell count nadirs and reversibility,
blood samples were obtained from mice on the day after the last SCH 727965 dose (day 6) and then 7 d later
(day 13). Absolute neutrophil (left) and reticulocyte responses (right) are shown compared with BALB/c mice
that were administered vehicle (20% hydroxypropyl-β-cyclodextrin) or left nontreated. Neutrophil and
reticulocyte nadirs were observed on day 6. By day 13, counts neutrophil and reticulocyte counts in SCH
727965–treated animals had returned to levels similar to those observed in vehicle-treated or nontreated mice.
There were no detectable effects on platelets or RBC during the duration of this time course (data not shown).
Table 4. Dose dependent antitumor activity of SCH 727965.
SCH 7279765 has antitumor activity on various intermittent schedules
Pharmacokinetic studies showed that SCH 727965 has a short plasma half-life in mouse. Thus, a dose
of 5 mg/kg SCH 727965 given i.p. in mice was associated with a plasma half-life of ∼0.25 hour
(Supplementary Table S2), perhaps suggesting a need for frequent dosing. However, previous results
imply that continuous SCH 727965 exposure may not be a prerequisite for antiproliferative activity
because short treatments with the drug induce long-term effects in vitro and in vivo. To test that
hypothesis, a total SCH 727965 dose of 260 mg/kg, equivalent to 20 mg/kg once daily for 13 days,
was fractionated over several diverse schedules and administered to nude mice bearing established
65
(>100 mm3
) A549 tumor xenografts (Fig. 3B; dosing days are indicated by arrows). Primary end
points for this study were tumor volume/mass and body weight. SCH 727965 dosing of 87 mg/kg
given once weekly exceeded the MTD and was terminated early. Similar tumor regressions were
observed for all schedules. These data agree with earlier observations and indicate that similar in vivo
responses can be generated on a wide range of intermittent SCH 727965 dosing schedules.
Mechanism-based systemic effects in vivo following exposure to SCH 727965
In this study, phospho-Rb 807/811 has been used as a surrogate marker of CDK engagement and
mechanism-based SCH 727965 activity. To show that 20-mg/kg to 60-mg/kg doses of SCH 727965
previously exhibiting significant antitumor activity in a mouse xenograft model are associated with
modulation of the CDK mechanism, the expression of phospho-Rb 807/811 was analyzed in skin
samples taken from SCH 727965–treated, tumor-naïve nude mice. Skin and hair follicles are an
excellent peripheral source of surrogate proliferating (nontumor) tissue. Skin punch biopsies were
harvested at various time points following the administration of SCH 727965. Immunohistochemical
staining of murine skin indicates that a single 40-mg/kg SCH 727965 dose induces rapid and
sustained suppression of phospho-Rb 807/811 within the proliferating epithelial cells of the basal
epithelium and hair follicles (Fig. 3C). These observations suggest that doses of SCH 727965
associated with regressions in the A549 xenograft model (Fig. 3B) are correlated with the modulation
of a mechanism-based marker in proliferating surrogate tissues. These data are consistent with the
hypothesis that inhibition of CDKs can induce inhibition of cell cycle within proliferating normal
tissues and suggest that proliferating compartments will likely be sensitive to SCH 727965.
To assess effects of SCH 727965 on hematologic parameters, BALB/c mice were i.p. dosed daily
with 40 mg/kg for 5 days, with controls nontreated or dosed with a vehicle, 20% hydroxypropyl-βcyclodextran.
Blood samples were obtained 1 day after administration of the last dose (day 6) as well
as 7 days later (day 13). Blood cells were counted (with differential) to examine nadir and rebound
kinetics of several hematologic parameters. Neutrophils and reticulocytes were most sensitive to SCH
727965, and nadirs in their absolute counts were detected on day 6 (Fig. 3D). Significantly, absolute
neutrophil counts (Fig. 3D, left) and reticulocyte counts (Fig. 3D, right) returned to normal levels by
day 13. No detectable effects on platelets or RBC over this time course were observed (data not
shown). These results are consistent with the mechanism-based activity of SCH 727965 within the
examined dose range and suggest that cell cycle inhibition effects are transient and reversible in
proliferating normal cell compartments.
66
Discussion
SCH 727965 was selected for clinical development following a functional in vivo screen that
integrated both efficacy and tolerability of tested compounds. This rapid and discriminatory approach
identified a candidate for clinical development that was significantly more effective and better
tolerated than flavopiridol. SCH 727965 has several distinct in vitro properties consistent with an
improved in vivo therapeutic index. Notably, the compound exhibits strong selectivity for the CDK
family. These data suggest the activated CDK conformation has unique structural aspects, not present
in closely related serine/threonine kinases (such as the extracellular signal-regulated kinase and GSK3
families), thus providing a potential explanation for the observed excellent selectivity and tolerability
profiles of SCH 727965.
In vitro and in vivo analyses presented in this study support the conclusion that SCH 727965 has the
potential to inhibit the growth of a broad spectrum of human cancers. SCH 727965 induced
mechanism-based apoptosis in the vast majority of tested human tumor cell lines of diverse origin,
following a single exposure. In agreement, SCH 727965 was effective at doses below the MTD level
in multiple in vivo models and induced regression in several xenografts using continuous or
intermittent schedules. Under similar conditions, the observed xenograft efficacy profiles of SCH
727965 were consistently superior to those achieved using approved benchmark agents, such as
taxanes. Moreover, in mechanism-based biomarker studies, effective doses of the drug were sufficient
to suppress phosphorylated Rb levels in surrogate tissues, such as skin and hair follicles. Likewise,
active dose levels in the mouse were also associated with reversible effects on hematologic
parameters. Quantitative tracking of leukocyte cell counts may offer an additional approach for
tracking mechanism-based pharmacodynamic effects of SCH 727965.
Interestingly, the in vivo activity of SCH 727965 observed in murine systems was readily detectable
despite rapid clearance of the parent compound from mouse plasma, indicating that continual
exposure to SCH 727965 was not necessary for activity in vivo. Consistent with this, short exposure
to SCH 727965 can induce long-lasting pharmacodynamic effects in vitro. Thus, a 2-hour exposure to
≤500 nmol/L SCH 727965 was sufficient to suppress BrdUrd incorporation 24 hours later. Similarly,
transient in vitro exposure to SCH 727965 induced suppression of Rb phosphorylation that was
correlated with induction of apoptosis. Significantly, escalation of SCH 727965 exposure (≤30
μmol/L) did not augment apoptotic phenotypes, suggesting a relative lack of nonspecific or off-target
cytotoxicity. Taken together, the available in vitro and in vivo data show that long-lasting therapeutic
effects can be induced within sensitive cells following short exposures to SCH 727965. It is possible
67
that selecting compounds for further development solely on the basis of pharmacokinetic parameters
would not have facilitated selection of SCH 727965 for clinical development. In summary, the
approach of in vivo screening in mice ultimately led to the selection of a compound with attractive
biochemical and pharmacologic properties.
Inhibitors of the CDK family have been proposed as attractive drug targets and pursued for oncology
indications for several years, and several candidate molecules have entered clinical studies. In the
case of flavopiridol, a combination of suboptimal selectivity, poor drug-like qualities, and adverse
side effects may ultimately obscure any potentially desirable mechanism-based activities of this
agent. In this study, we have described the novel pharmacologic properties of SCH 727965, a highly
potent and selective CDK inhibitor that is differentiated from first generation CDK inhibitor
compounds, such as flavopiridol. SCH 727965 is currently undergoing clinical testing against a range
of solid and hematologic malignancies. The overall excellent profile of SCH 727965 suggests this
molecule has the necessary properties to allow further pharmacologic exploration of the cell cycle
mechanism in oncology.
References
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2. Pines, J. Protein kinases and cell cycle control. Semin. Cell Biol. 1994, 5, 399–408.
3. Hall, M.; Peters, G. Genetic alterations of cyclins, cyclin-dependent kinases, and Cdk inhibitors in
human cancer. Adv. Cancer Res. 1996, 68, 67–108.
4. Sherr, C. J. Cancer cell cycles. Science 1996, 274, 1672–7.
5. Hunter, T.; Pines, J. Cyclins and cancer II: cyclin D and CDK inhibitors come of age. Cell 1994,
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6. Ewen, M. E. The cell cycle and the retinoblastoma protein family. Cancer Metastasis Rev. 1994,
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7. Ewen, M. E. Regulation of the cell cycle by the Rb tumor suppressor family. Results Probl. Cell
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9. Oelgeschlager, T. Regulation of RNA polymerase II activity by CTD phosphorylation and cell
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10. van den Heuvel, S.; Harlow, E. Distinct roles for cyclin-dependent kinases in cell cycle control.
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inducible expression of a dominant-negative mutant in human cells. Mol. Cell. Biol. 2001, 21,
2755–66.
12. L'Italien, L.; Tanudji, M.; Russell, L.; Schebye, X. M. Unmasking the redundancy between Cdk1
and Cdk2 at G2 phase in human cancer cell lines. Cell Cycle 2006, 5, 984–93.
13. Cai, D.; Latham, V. M Jr.; Zhang, X. Shapiro, G. I. Combined depletion of cell cycle and
transcriptional cyclin-dependent kinase activities induces apoptosis in cancer cells
10.1158/0008-5472.CAN-06-1758. Cancer Res. 2006, 66, 9270–80.
14. Gojo, I.; Zhang, B.; Fenton, R. G. The cyclin-dependent kinase inhibitor flavopiridol induces
apoptosis in multiple myeloma cells through transcriptional repression and down-regulation
of Mcl-1. Clin. Cancer Res. 2002, 8, 3527–38.
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mechanism of chronic lymphocytic leukemia cell death 10.1182/blood-2005-04-1678. Blood
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16. Firestein, R.; Bass, A. J.; Kim, S. Y. et al. CDK8 is a colorectal cancer oncogene that regulates
[bgr]-catenin activity. 2008 2008/09/14/online.
17. Morris, E. J.; Ji, J.-Y.; Yang, F. et al. E2F1 represses [bgr]-catenin transcription and is
antagonized by both pRB and CDK8. 2008 2008/09/14/online.
18. Chen, Y. N.; Sharma, S. K.; Ramsey, T. M. et al. Selective killing of transformed cells by
cyclin/cyclin-dependent kinase 2 antagonists. Proc. Natl. Acad. Sci. U S A 1999, 96, 4325–9.
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69
Part 2a
Discovery of pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors:
a template-based approach-part 1*
*published as:
Dwyer, M. P.;*
Paruch, K.; Labroli, M.; Alvarez, C.; Keertikar, K. M;. Poker, C.; Rossman, R.; Fischmann, T.
O.; Duca, J. S.; Madison, V.; Parry, D.; Davis, N.; Seghezzi, W.; Wiswell, D.; Guzi, T. J. Discovery of
pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors: A template-based approach-Part 1. Bioorg. Med. Chem. Lett.
2011, 21, 467.
Checkpoint kinase 1 (CHK1) is a serine/threonine kinase that controls the cellular response to DNA
damage. In response to a DNA-damaging agent, CHK1 is activated by phosphorylation via ATR and
ATM to arrest cells at various cell-cycle checkpoints (G1, S and G2) in order to initiate the DNA
repair process.1
Inhibition of CHK1 abrogates cell-cycle arrest resulting in genomic instability and
ultimately progression into mitosis and cell death.2
The inhibition of CHK1 creates a ‘synthetic lethal’
response by which aberrant cells cannot replicate which should impede the progression of cancer. In
contrast, normal cells still arrest at the G1 checkpoint, via p53, to repair the DNA damage caused by
these agents. Due to the fact that inhibition of CHK1 represents a targeted approach to enhance the
cytotoxicity of DNA-damaging agents toward tumor cells while having a lesser effect on normal
cells, it has been an attractive target in the oncology field.3
A number of small-molecule CHK1
inhibitors have been described recently and several comprehensive reviews have provided overviews
of the emerging CHK1 small-molecule chemotypes.4
In addition, several checkpoint kinase
inhibitors, such as PF-00477736 (1)5
and AZD7762 (2)6
(Fig. 1), have recently entered the clinic in
combination with various DNA-damaging agents. Due to the therapeutic value of a CHK1 inhibitor as
a chemopotentiator,7
efforts were directed towards the identification of additional, novel CHK1
inhibitors.
Figure 1. CHK1 inhibitors currently under clinical evaluation.
70
Screening of an internal compound library identified compounds 3 and 4, as early CHK1 program hits
(Fig. 2). While these initial hits possessed better in vitro potency versus CDK2 than CHK1, it was
rationalized that proper substitution around the pyrazolo[1,5-a]pyrimidine core might improve the
potency and selectivity of this series for CHK1. Previously, the pyrazolo[1,5-a]pyrimidine core was
shown to be a viable template for the preparation of CDK2 inhibitors such as compound 5 which was
orally bioavailable and found to be efficacious in a mouse tumor xenograft model (Fig. 2).8
Figure 2. Initial pyrazolo[1,5-a]pyrimidine CHK1 Hits.
With compound 4 as a starting point, we focused upon making systematic modifications around the
pyrazolo[1,5-a] pyrimidine core in order to enhance the in vitro potency for CHK1 while monitoring
the selectivity versus CDK2. It was rationalized that selectivity for CHK1 over CDK2 was required
since the inhibition of the CDK function may antagonize CHK1 ablation/inhibition phenotypes.9
Since a limited set of substituents had been tolerated at the C3 position in the pyrazolo[1,5a]pyrimidine
core in our previous CDK2 program,8
initial synthetic efforts focused upon
modifications of the 3-position of compound 4 to further explore potential differences in this region
for CHK1. The preparation of C3 analogs of compound 4 is illustrated in Schemes 1 and 2.10
71
Scheme 1. Reagents and conditions: (a) 3-aminopyrazole, PhCH3, 73%; (b) POCl3, N,N-dimethylaniline, 71%;
(c) NH3, 2-propanol, H2O, 98%; (d) SEMCl, DIPEA, (CH2Cl)2, 76%; (e) NBS, CH3CN or NIS, CH3CN, 92%;
(f) RB(OH)2, PdCl2dppf, K3PO4, DME/H2O or Bu3SnR, Pd[PPh3]4, dioxane, 29–89%; (g) 3 N HCl/EtOH, 23–
78%.
Cyclization of -keto ester 6 with 3-aminopyrazole followed by chlorination and amination afforded
7. Diprotection of the resultant C7 amino group11
followed by introduction of either the C3 iodide via
NIS treatment or C3 bromide via NBS treatment afforded intermediates 8a or 8b. Treatment of 8a, b
under either Suzuki or Stille coupling conditions followed by global deprotection with 3 N HCl in
EtOH afforded the C3 analogs 9a–b, e–r which are summarized in Table 1.
72
Table 1. CHK1 and CDK2 inhibitory activity of C3 substituted pyrazolo[1,5-a]pyrimidines 9a–r.
nt = not tested; values are means of two experiments.
a
Assay conditions can be found in Ref. 12.
b
Assay conditions can be found in Ref. 8a.
73
Alternatively in Scheme 2, cyclization of the Cbz-protected -keto ester with either the cyano or ethyl
ester substituted pyrazole followed by subsequent chlorination under standard conditions afforded 11.
Treatment with NH3 in isopropanol followed by Cbz deprotection with TMSI afforded the final
adducts 9c and 9d.
Scheme 2. Reagents and conditions: (a) 3-aminopyrazole-4-carbonitrile or 3- amino-4-carboethoxypyrazole,
PhCH3, 41–61%; (b) POCl3, N,N-dimethylaniline, 91–98%; (c) NH3, 2-propanol, H2O, 82–90%; (d) TMSI,
MeOH, 31–45%.
As shown in Table 1, small linear substituents (9b–f) at the C3 position improved the CHK1 potency
versus the 3-H adduct (9a) with the exception of ester 9c. Despite the potency improvements for
CHK1, these analogs also retained good potency for CDK2. Incorporation of aryl (9g, h) and
heteroaryl substituents into the C3 position substituents (9i–n) resulted in marked improvement in
CHK1 potency and selectivity versus CDK2 in the biochemical assay. Additionally, several
heteroaryl derivatives in Table 1 (9o, p) showed weaker CHK1 activity which suggested that the
proper placement of heteroatoms in the heteroaryl ring at C3 is critical to retain CHK1 potency. From
this SAR survey of the C3 position, compound 9q emerged as a key compound which demonstrated
good CHK1 potency (IC50 = 60 nM) and nearly 100-fold selectivity for CHK1 over CDK2.
Compound 9q was selected as a lead structure for further SAR optimization efforts.
With compound 9q in hand, attention turned toward the exploration of additional substitution of the
primary amine located at the C7 position of this compound. The preparation of these analogs is
outlined in Scheme 3.10
74
Scheme 3. Reagents and conditions: (a) NIS, CH3CN, 97%; (b) NaOMe or NaSMe, THF, 85–97%; (c) 1methylpyrazole-4-boronic
acid pinacol ester, PdCl2dppf, K3PO4, DME/H2O, 52–75%; (d) H2NR, NaH, DMF,
38–98%; (e) TFA, CH2Cl2, 13–98%; (f) MCPBA, CH2Cl2, 85%; (g) H2NR, n-BuOH, 100 °
C, 21–85%.
Iodination of 12 followed by treatment with either sodium methoxide or sodium thiomethoxide in
THF afforded compounds 13 or 14, respectively. Treatment of 13 or 14 with 1-methylpyrazole-4boronic
acid pinacol ester under Suzuki coupling conditions afforded the corresponding coupled
products 15 and 16, respectively. For the preparation of C7 amino derivatives bearing either aryl or
heteroaryl functionality, compound 15 was treated with the anion of the anilinic/heteroaryl coupling
partners using NaH followed by TFA treatment to afford the title compounds 17i–r (Table 2).
75
Table 2. CHK1 and CDK2 inhibitory activity of C7 substituted amino pyrazolo[1,5-a]pyrimidines.
na = not active up to >50 M; nt = not tested.
a
Assay conditions can be found in Ref. 12.
b
Assay conditions can be found in Ref. 8a.
76
Surprisingly, treatment of 15 with simple alkyl and benzyl amines with heating yielded none of the
desired addition product but only the demethylated (7-OH) adduct. This issue was circumvented by
utilization of the corresponding thiomethyl adduct 16. Oxidation of the thiomethyl group with
mCPBA afforded a mixture of sulfoxide/sulfone which was treated with either alkyl or branched alkyl
amine derivatives in hot n-BuOH in the presence of Et3N to afford the desired addition products.
Deprotection of the intermediate Boc adducts with TFA afforded the C7 substituted amino derivatives
17a–h listed in Table 2. As summarized in Table 2, incorporation of simple alkyl, branched alkyl, and
cycloalkyl groups at the C7 amino group (17a–e) resulted in poorer in vitro potency for CHK1 versus
the parent NH2 derivative (9q). Simple alkyl modifications with pendant functionality (17f–h)
resulted in derivatives with modest CHK1 activity. All of the C7 substituted amino derivatives in
Table 2 demonstrated improved selectivity versus CDK2 (IC50 >30 M). While simple aryl
derivatives or heteroaryl derivatives demonstrated good CHK1 activity (17i–j, o–p), the proper
placement of heteroatom functionality in these motifs was important for retaining CHK1 potency as
illustrated by 17k–n. Aminoisothiazole 17r emerged from the SAR work at the C7 position with
excellent potency for CHK1 (<10 nM) and very good selectivity over CDK2. In order to better
understand the SAR trends observed in Tables 1 and 2, a single crystal X-ray structure of 17r bound
to CHK1 (shown in Fig. 3) was obtained.13
Figure 3. X-ray of crystal structure of 17r in CHK1.13
77
In the X-ray structure, three key interactions were observed between 17r and the CHK1 protein. First,
the N1 moiety and C7 NH of the pyrazolo[1,5-a]pyrimidine core bind to the peptide backbone in the
hinge area. Secondly, the 1-methylpyrazole moiety at the C3 position interacts with an array of
ordered water molecules in the kinase specificity domain of CHK1. The SAR observed for the C3
heterocyclic derivatives shown in Table 1 may be explained in part by the propensity of these motifs
to interact favorably with these water molecules which may mediate interactions with other amino
acids. Lastly, the C5 piperidine nitrogen interacts with the carboxylate of Glu 91 as well as the amide
carbonyl of Glu 134. Interestingly, the SAR observed at the C7 amino group (Table 2) is difficult to
rationalize since this substitution projects into the solvent-exposed region based upon the X-ray
structure depicted in Figure 3. Additional SAR efforts directed toward trying to elucidate the
structural/electronic requirements in this region of this class of CHK1 inhibitors will be discussed in
the accompanying manuscript.14
In summary, systematic optimization of both the C3 and C7 positions of pyrazolo[1,5-a]pyrimidine
CHK1 hit 4 led to the discovery of potent, selective CHK1 inhibitors represented by 17r. Single Xray
crystallography of 17r in CHK1 elucidated several key interactions with the protein that appear to
be critical to the improvement of CHK1 potency and selectivity versus CDK2 for this class of
compounds. Additional SAR development and further analysis of this novel class of CHK1 inhibitors
is found in the accompanying paper.14
78
References and notes
1. (a) Sancar, A.; Lindsey-Boltz, L. A.; Unsal-Kacmaz, K.; Linn, S. Annu. Rev. Biochem. 2004, 73,
39; (b) Kastan, M. B.; Bartek, J. Nature 2004, 432, 316.
2. (a) Bucher, N.; Britten, C. D. Br. J. Cancer 2008, 98, 523; (b) Luo, Y.; Leverson, J. D. Expert Rev.
Anticancer Ther. 2005, 5, 333.
3. (a) Powell, S. N.; DeFrank, J. S.; Connell, P.; Eogan, M.; Preffer, F.; Dombkowski, D.; Tang, W.;
Friend, S. Cancer Res. 1995, 55, 1643; (b) Zhou, B.-B. S.; Elledge, S. J. Nature 2000, 408, 433; (c)
Li, Q.; Zhu, G.-D. Curr. Top. Med. Chem. 2002, 2, 939.
4. (a) Matthews, T. P.; Klair, S.; Burns, S.; Boxall, K.; Cherry, M.; Fisher, M.; Westwood, I. M.;
Walton, M. I.; McHardy, T.; Cheung, K.-M. J.; Van Montfort, R.; Williams, D.; Aherne, G. W.;
Garrett, M. D.; Reader, J.; Collins, I. J. Med. Chem. 2009, 52, 4810. and references cited therein; (b)
Janetka, J. W.; Ashwell, S. Expert Opin. Ther. Patents 2009, 19, 165; (c) Janetka, J. W.; Ashwell, S.;
Zabludoff, S.; Lyne, P. Curr. Opin. Drug Discov. Dev. 2007, 10, 473; (d) Arrington, K. L.; Dudkin,
V. Y. ChemMedChem 2007, 2, 1571.
5. Blasina, A.; Hallin, J.; Chen, E.; Arango, M. E.; Kraynov, E.; Register, J.; Gant, S.; Ninkovic, S.;
Chen, P.; Nichols, T.; O’Connor, P.; Anderas, K. Mol. Cancer Ther. 2008, 7, 2394.
6. Zabludoff, S. D.; Deng, C.; Grondine, M. R.; Sheehy, A. M.; Ashwell, S.; Caleb, B. L.; Green, S.;
Haye, H. R.; Horn, C. L.; Janetka, J. W.; Liu, D.; Mouchet, E.; Ready, S.; Rosenthal, J. L.; Queva, C.;
Schwartz, G. K.; Taylor, K. J.; Tse, A. N.; Walker, G. E.; White, A. M. Mol. Cancer Ther. 2008, 7,
2955.
7. (a) Tao, Z.-F.; Lin, N.-H. Anti-Cancer Agents Med. Chem. 2006, 6, 377; (b) Prudhomme, M.
Recent Patents Anti-Cancer Drug Discov. 2006, 11, 55.
8. (a) Dwyer, M. P.; Paruch, K.; Alvarez, C.; Doll, R. J.; Keertikar, K.; Duca, J.; Fischmann, T. O.;
Hruza, A.; Madison, V.; Lees, E.; Parry, D.; Seghezzi, W.; Sgambellone, N.; Shanahan, F.; Wiswell,
D.; Guzi, T. J. Bioorg. Med. Chem. Lett. 2007, 17, 6216; (b) Paruch, K.; Dwyer, M. P.; Alvarez, C.;
Brown, C.; Chan, T.-Y.; Doll, R. J.; Keertikar, K.; Knutson, C.; McKittrick, B.; Rivera, J.; Rossman,
R.; Tucker, G.; Fischmann, T.; Hruza, A.; Madison, V.; Nomeir, A. A.; Wang, Y.; Lees, E.; Parry, D.;
Sgambellone, N.; Seghezzi, W.; Schultz, L.; Shanahan, F.; Wiswell, D.; Xu, X.; Zhou, Q.; James, R.
A.; Paradkar, V. M.; Park, H.; Rokosz, L. R.; Stauffer, T. M.; Guzi, T. J. Bioorg. Med. Chem. Lett.
2007, 17, 6220; (c) Paruch, K.; Dwyer, M. P.; Alvarez, C.; Brown, C.; Chan, T.-Y.; Doll, R. J.;
Keertikar, K.; Knutson, C.; McKittrick, B.; Rivera, J.; Rossman, R.; Tucker, G.; Fischmann, T.;
Hruza, A.; Madison, V.; Nomeir, A. A.; Wang, Y.; Kirschmeier, P.; Lees, E.; Parry, D.; Sgambellone,
N.; Seghezzi, W.; Schultz, L.; Shanahan, F.; Wiswell, D.; Xu, X.; Zhou, Q.; James, R. A.; Paradkar,
V. M.; Park, H.; Rokosz, L. R.; Stauffer, T. M.; Guzi, T. J. ACS Med. Chem. Lett. 2010, 1, 204.
79
9. Walton, M. I.; Eve, P. D.; Hayes, A.; Valenti, M.; De Haven Brandon, A.; Box, G.; Boxall, K. J.;
Aherne, G. W.; Eccles, S. A.; Raynaud, F. I.; Williams, D. H.; Reader, J. C.; Collins, I.; Garrett, M.
M. D. Mol. Cancer Ther. 2010, 9, 89.
10. Full experimental details have appeared elsewhere: Guzi, T. J.; Paruch, K.; Dwyer, M. P.; Parry,
D. A. US 2007/0082900.
11. Protection of the C7 amino group as the di-SEM analog proved to be optimal for efficient
coupling reactions using either the Suzuki or Stille coupling protocols.
12. CHK1 SPA assay. An in vitro assay utilizing recombinant His-CHK1 expressed in the
baculovirus expression system as an enzyme source and biotinylated peptide based upon CDC25C as
substrate. His-CHK1 was diluted to 32 nM in kinase buffer containing 50 mM Tris pH 8.0, 10 mM
MgCl2, and 1 mM DTT. CDC25C (CDC25 Ser216 C-term biotinylated peptide, Research Genetics)
peptide was diluted to 1.93 M in kinase buffer. For each kinase reaction, 20 L of 32 nM CHK1
enzyme solution and 20 L of 1.926 M substrate solution were mixed and combined with 10 L of
compound diluted in 10% DMSO, making final reaction concentrations of 6.2 nM CHK1, 385 nM
CDC25C and 1% DMSO after addition of start solution. The reaction was started by addition of 50
L of start solution consisting of 2 M ATP and 0.2 Ci of 33
PATP (Amersham, UK), making a final
reaction concentration of 1 M ATP, with 0.2 Ci of 33
P-ATP per reaction. Kinase reactions ran for 2
h at room temperature and were stopped by the addition of 100 L of stop solution consisting of 2 M
NaCl, 1% H3PO4, and 5 mg/mL Streptavidin-coated SPA beads (Amersham, UK). SPA beads were
captured using a 96-well GF/B filter plate (Packard/Perkin Elmer Life Sciences) and a Filtermate
universal harvester (Packard/Perkin Elmer Life Sciences). Beads were washed twice with 2 M NaCl
and twice with 2 M NaCl with 1% phosphoric acid. Signal was then assayed using a TopCount 96well
liquid scintillation counter (Packard/Perkin Elmer Life Sciences). Dose–response curves were
generated from duplicate 8 point serial dilutions of inhibitory compounds. IC50 values were derived
by nonlinear regression analysis.
13. The coordinates of compound 17r bound to CHK1 have been deposited in the Protein Databank:
pdb ID 3OT8.
14. Labroli, M.; Paruch, K.; Dwyer, M. P.; Alvarez, C.; Keertikar, K.; Poker, C.; Rossman, R.;
Fischmann, T. O.; Duca, J. A.; Madison, V.; Parry, D.; Davis, N.; Seghezzi, W.; Wiswell, D.; Guzi,
T. J. Bioorg. Med. Chem. Lett. 2010, 21, 471.
Note: Experimental details can be found in our publicly available patents WO 2007/041712 A1 and
WO 2008/153870 A1.
80
Part 2b
Discovery of pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors:
a template-based approach-part 2*
*published as:
Labroli, M.;*
Paruch, K..; Dwyer, M. P.; Alvarez, C.; Keertikar, K. M;. Poker, C.; Rossman, R.; Fischmann, T.
O.; Duca, J. S.; Madison, V.; Parry, D.; Davis, N.; Seghezzi, W.; Wiswell, D.; Guzi, T. J. Discovery of
pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors: A template-based approach-Part 2. Bioorg. Med. Chem. Lett.
2011, 21, 471.
As one of the key regulators of the cell cycle progression, CHK1 is a serine/threonine kinase which
has been an attractive target in oncology.1
A number of small-molecule ATP-competitive CHK1
inhibitors have been described2
and several compounds have recently entered the clinic including PF-
00477736 (1)3
and AZD7762 (2)4
(Fig. 1).
Figure 1. CHK1 inhibitors currently under clinical evaluation.
Herein, we describe our continued SAR development of the pyrazolo[1,5-a]pyrimidine core to
develop potent and selective CHK1 inhibitors. Initial screening of our internal compound library
identified compound 3 as a CHK1 program hit which was derived from an earlier CDK program (Fig.
2).
81
Figure 2. Pyrazolo[1,5-a]pyrimidine hits for CHK1 program.
Initial SAR optimization work on the pyrazolo[1,5-a]pyrimidine scaffold at both the C3 and C7–NH2
positions led to several promising targets, including 4 and 5, which displayed improved in vitro
potency for CHK1 and increased selectivity against CDK2 versus the early hit 3 (Fig. 2).5
As
discussed in the preceding Letter,5
the SAR study of the C3 position demonstrated that the 4-N
methylpyrazole moiety conferred optimal CHK1 potency while maintaining selectivity against
CDK2. Additionally, structural modifications of the C7–NH2 position were extremely challenging as
a majority of substituents incorporated in this region displayed a loss in CHK1 potency versus the
C7–NH2 analog with the exception of compound 5. These observations could, in part, be attributed to
the deleterious effect of the C7–NH2 substituents on the crucial H-bonding motif in the hinge region.5
In light of these observations, we felt there might be additional opportunities to explore the solvent
exposed region using C6 substitution. Initial synthetic efforts focused upon the preparation of the 6halo
compounds 9a–c which maintained key functionality at C3 (N-methyl pyrazole) and C5 (3piperidine)
that was previously noted for CHK1 potency.5
It was envisioned that the C6 halo
functionality could serve as handles for further elaboration at the C6 position. The preparation of
these analogs was discussed previously and shown in Scheme 1.5,6
82
Scheme 1. Reagents: (a) 3-aminopyrazole, PhCH3, 75%; (b) POCl3, N,N-dimethylaniline, 71%; (c) NH3, 2propanol,
H2O, 98%; (d) SEMCl, DIPEA (CH2Cl)2, 76%; (e) NIS, CH3CN, 92%; (f) 1-methyl-4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole,
PdCl2dppf, K3PO4, DME, 81%; (g) HCl, EtOH, 90%; (h)
NCS, CH3CN, 72% or NBS or Br2, t-BuNH2, CH2Cl2, 73% or NIS, CH3CN, 83%.
Interestingly, the 6-halo compounds 9a–c demonstrated a 20-fold improvement in CHK1 activity
relative to the parent compound 4 (Table 1).
Table 1. CHK1 and CDK2 inhibitory activity of pyrazolo[1,5-a]pyrimidines 4 and 9a–p.
Values reported are means of two experiments.
a
Assay conditions can be found in Ref. 5.
b
Assay conditions can be found in Ref. 10.
83
Unfortunately, all attempts to install additional functionality at the C6 position via the 6-halo
precursors using either Suzuki or Stille coupling protocols were unsuccessful (not shown). While the
limitations of using these coupling protocols in sterically congested systems has been documented,7
the initial synthetic route had to be modified to allow for more facile incorporation of substituents at
the C6 position which is shown in Scheme 2. Treatment of 7 with bromine in t-butylamine8
yielded
the corresponding 6-bromo derivative 10 for further elaboration (Scheme 2). Bisprotection of 10 with
SEMCl9
followed by Pd-mediated couplings introduced desired substitution at the C6 position to
yield compound 11. Functionalization of the C3 position was achieved by bromination, Suzuki or
Stille coupling with the appropriate heteroaromatic group, and global deprotection to afford targets
9d–q.
Scheme 2. Reagents: (a) Br2, t-BuNH2, CH2Cl2, 79%; (b) SEMCl, DIPEA (CH2Cl)2, 39%; (c) R1
B(OH)2,
PdCl2dppf, K3PO4, DME, H2O or Bu3SnR1
, Pd(PPh3)4, dioxane, 27–83%; (d) NBS, CH3CN, 66–92%; (e)
R2
B(OH)2, PdCl2dppf, K3PO4, DME, H2O or Bu3SnR2
, Pd[PPh3]4, dioxane, 41–90%; (f) HCl, EtOH, 17–80%.
The biochemical assay results for both CHK1 and CDK2 are summarized in Table 1 for compounds 4
and 9a–q. As evident from Table 1, small alkyl or cycloalkyl substituents at the C6 position retained
reasonable CHK1 potency with varying levels of CDK2 selectivity (9d–i) similar to the 6-halo
compounds 9a–c. Incorporation of either aryl or heteroaryl derivatives at the C6 position generally
led to a loss of CHK1 potency versus the smaller substituents while maintaining some selectivity over
84
CDK2 (9j–p). Although it appears that the solvent exposed region would be more accommodating to
a variety of substituents, the SAR observed at the C6 position may be a combination of both size and
polarity requirements of the substituents. Additionally, the electron withdrawing substituents such as
the 6-halo derivatives 9a–c seem to be preferred at this position and may in fact play a role in
modulating the acidity of the adjacent C7–NH2 group.
Based upon the SAR observed in Table 1, we decided to briefly investigate a series of C3
heteroaromatic groups bearing the C6–Br to determine if we were maintaining optimal potency and
selectivity among the C3, C6, and C7 substituents. Representative examples are depicted in Table 2.
Table 2. CHK1 and CDK2 inhibitory activity of C3-substituted pyrazolo[1,5-a]pyrimidines 9b and 12a–c.
Values reported are means of two experiments.
a Assay conditions can be found in Ref. 5.
b Assay conditions can be found in Ref. 10.
Although compound 9b remained the most potent analog, other C3 heteroaromatic groups, for
example, 12c, demonstrated comparable CHK1 potency with a comparable selectivity profile against
CDK2. With the initial SAR investigations at the C3, C6, and C7–NH2 positions of the pyrazolo[1,5a]pyrimidine
core complete, attention was turned toward exploration of the C5 position. From the Xray
structure of compound 5 bound to CHK1,5
the NH of the C5 3-piperidinyl group was observed to
form several key hydrogen bond interactions with several acidic residues as well as a conserved water
molecule in this region. Initial SAR efforts at the C5 position focused upon further optimization of
this key H-bonding interaction. In order to more rapidly examine the SAR in this region, an
alternative synthesis was developed taking into account the optimal substituents at the C3, C6, and C7
positions of the pyrazolo[1,5-a]pyrimidine core. Retrosynthetically, we envisioned a more convergent
85
assembly of the pyrazolo[1,5-a]pyrimidine core (e. g., 4) via the cyclocondensation of -keto nitrile
13 and 3-amino-1-methyl-1H-1‘H-4,4‘-bispyrazole 14 (Fig. 3).
Figure 3. Retrosynthetic analysis of 4.
The preparation of aminopyrazole 1411
began with Vilsmeier- Haack formylation of N-methyl-1Hpyrazole
15 to afford aldehyde 16 (Scheme 3). Treatment of 16 with tosyl methyl isocyanide12
(TosMIC) in the presence of potassium t-butoxide in DME resulted in the one-step homologation and
cyanation process to yield the acetonitrile 17. -Formylation, followed by cyclization with hydrazine
monohydrochloride in ethanol yielded the final bispyrazole 14.
Scheme 3. Reagents: (a) POCl3, DMF, 50%; (b) KO-t-Bu, TosMIC, DME, 65%; (c) ethyl formate, KO-t-Bu,
DME, 82%; (d) N2H4–HCl, EtOH, 90%.
It should be noted that this preparation of bispyrazole 14 was amenable to large-scale synthesis and
the use of TosMIC avoids the need for toxic cyanide reagents commonly employed in other cyanation
protocols.
With the bispyrazole 14 in hand, the syntheses of the C5 targets 22a–k were accomplished utilizing
the route displayed in Scheme 4. Treatment of acids 19 with 1,1-carbonyldiimidazole followed by
86
addition of the anion of acetonitrile provided the substituted -keto nitriles 20 in good yield.
Cyclocondensation of 20 with bispyrazole 14 from Scheme 4 in EtOH or toluene provided the core
21. Regioselective bromination with NBS followed by acid deprotection of Boc intermediates, when
necessary, provided the desired targets 22a–k listed in Table 2.
Scheme 4. Reagents and conditions: (a) CDI, THF; (b) LiHMDS, CH3CN, THF, -78 o
C, 22–78% overall for
two steps; (c) 14, EtOH, 45 °C, or toluene, 115 °C, 37–87%; (d) NBS, DCM, 71–89%; (e) TFA, DCM, 39–
93%.
As summarized in Table 3, the SAR at C5 clearly demonstrates the necessity of both the proper
positioning and basicity of the piperidine NH for optimal CHK1 potency and selectivity against other
kinases, specifically CDK2. Loss of the piperdine NH (e.g., 22a) as well as different positional
isomers (22b, c) led to a loss of CHK1 potency. Additional heteroatoms are tolerated with varying
degrees of CHK1 potency (e.g., 22d, e) while ring-contracted (22f) or the acyclic amine derivative
(22g) generally showed reduced CHK1 potency. Several homologated analogs (22i, j) as well as
exocyclic amine analog 22k demonstrated comparable CHK1 potency to 9b with a comparable or
improved selectivity profile against CDK2 (Table 3).
87
Table 3. CHK1 and CDK2 inhibitory activity of C5-substituted pyrazolo[1,5-a]pyrimidines 9b and 22a–k.
Values reported are means of two experiments.
a
Assay conditions can be found in Ref. 5.
b
Assay conditions can be found in Ref. 10.
88
A single X-ray crystal structure of 22k bound to CHK1 (Fig. 4) was obtained which confirmed a
similar binding mode to that observed for compound 5.5
From Figure 4, the key H-bond network from
the C3 pyrazole in 22k with ordered waters is maintained while the C7–NH2 maintains a key hinge
contact with the C6–Br group projecting into the solvent exposed region as expected. Interestingly,
the exocyclic NH2 of 22k occupies a similar position as the piperidine NH of 5 to maintain the key
interactions with an acidic group and a conserved water molecule which is imperative for CHK1
potency as demonstrated in Table 3.
Figure 4. X-ray of crystal structure of 22k in CHK1.13
Starting with a CDK2 selective pyrazolo[1,5-a]pyrimidine CHK1 lead 3, systematic SAR studies of
the C3, C5, C6, and C7–NH2 positions of the core led to the identification of potent and selective
CHK1 inhibitors, for example, 5, 9a–c, 12c, and 22i–k.5
Interestingly, potent CHK1 inhibition can be
retained through the appropriate combination of C6 substitution with a primary amine at C7.
Approaches to differentiate between this novel class of pyrazolo[1,5-a]pyrimidines and those
identified in the preceding Letter5
as well as to calibrate the extent of CDK2 selectivity required in a
CHK1 inhibitor utilizing a mechanistically based in-cell evaluation assay will be the subject of a
future Letter.
89
References and notes
1. Zhou, B.-B. S.; Elledge, S. J. Nature 2000, 408, 433.
2. (a) Janetka, J. W.; Ashwell, S.; Zabludoff, S.; Lyne, P. Curr. Opin. Drug Discov. Devel. 2007, 10,
473; (b) Matthews, T. P.; Klair, S.; Burns, S.; Boxall, K.; Cherry, M.; Fisher, M.; Westwood, I. M.;
Walton, M. I.; McHardy, T.; Cheung, K.-M. J.; Van Montfort, R.; Williams, D.; Aherne, G. W.;
Garrett, M. D.; Reader, J.; Collins, I. J. Med. Chem. 2009, 52, 4810. and references cited therein.
3. Blasina, A.; Hallin, J.; Chen, E.; Arango, M. E.; Kraynov, E.; Register, J.; Gant, S.; Ninkovic, S.;
Chen, P.; Nichols, T.; O’Connor, P.; Anderas, K. Mol. Cancer Ther. 2008, 7, 2394.
4. Zabludoff, S. D.; Deng, C.; Grondine, M. R.; Sheehy, A. M.; Ashwell, S.; Caleb, B. L.; Green, S.;
Haye, H. R.; Horn, C. L.; Janetka, J. W.; Liu, D.; Mouchet, E.; Ready, S.; Rosenthal, J. L.; Queva, C.;
Schwartz, G. K.; Taylor, K. J.; Tse, A. N.; Walker, G. E.; White, A. M. Mol. Cancer Ther. 2008, 7,
2955.
5. Dwyer, M. P.; Paruch, K.; Labroli, M. A.; Alvarez, C.; Keertikar, K.; Poker, C.; Rossman, R.;
Duca, J. A.; Madison, V.; Parry, D.; Davis, N.; Seghezzi, W.; Wiswell, D.; Guzi, T. J. Bioorg. Med.
Chem. Lett. 2010, 21, 467.
6. Full experimental details have appeared elsewhere: Guzi, T. J.; Paruch, K.; Dwyer, M. P.; Parry, D.
A. US 2007/0082900.
7. (a) Mei, X.; Martin, R. M.; Wolf, C. J. Org. Chem. 2006, 71, 2854; (b) Johnson, M. G.; Foglesong,
R. J. Tetrahedron Lett. 1997, 38, 7001.
8. (a) Pearson, D. E.; Wysong, R. D.; Breder, C. V. J. Org. Chem. 1967, 32, 2358; (b) Ishizaki, M.;
Ozaki, K.; Kanematsu, A.; Isoda, T.; Hoshino, O. J. Org. Chem. 1993, 58, 3877.
9. Protection of the 7-amino group as the di-SEM analog proved to be optimal as it was shown that
this protection was imperative for efficient coupling reactions via either the Suzuki or Stille
couplings.
10. Dwyer, M. P.; Paruch, K.; Alvarez, C.; Doll, R. J.; Keertikar, K.; Duca, J.; Fischmann, T. O.;
Hruza, A.; Madison, V.; Lees, E.; Parry, D.; Seghezzi, W.; Sgambellone, N.; Shanahan, F.; Wiswell,
D.; Guzi, T. J. Bioorg. Med. Chem. Lett. 2007, 17, 6216.
11. Labroli, M. A.; Poker, C.; Guzi, T. J.; Paruch, K.; Dwyer, M. P.; Keertikar, K. M.; Alvarez, C. S.
Process for preparation of substituted 3-aminopyrazoles via formylation, cyanation, and
cyclocondensation reactions. PCT Int. Appl. WO 2008153873.
12. van Leusen, A. M.; Oomkes, P. G. Synth. Commun. 1980, 10, 399.
13. The co-ordinates of 22k bound to CHK1 have been deposited in the Protein Databank pdb 3OT3.
Note: Experimental details can be found in our publicly available patents WO 2007/041712 A1 and
WO 2008/153870 A1.
90
Part 2c
Targeting the replication checkpoint using SCH 900776, a
potent and functionally selective CHK1 inhibitor identified
via high content screening*
*published as:
Guzi, T.; Paruch, K.; Dwyer, M.; Labroli, M.; Shanahan, F.; Davis, N.; Taricani, L.; Wiswell, D.; Seghezzi, W.;
Penaflor, E.; Bhagwat, B.; Wang, W.; Gu, D.; Hsieh, Y.; Lee, S.; Liu, M.; Parry, D.*
Targeting the Replication
Checkpoint Using SCH 900776, a Potent and Functionally Selective CHK1 Inhibitor Identified Via High
Content Screening. Mol. Cancer Ther. 2011, 10, 591.
Introduction
DNA antimetabolite drugs are used extensively in modern clinical oncology.1
A primary mechanism
of action of DNA antimetabolite drugs is to suppress DNA synthesis, which leads to stalled
replication forks and activation of the replication checkpoint.2, 3
This checkpoint is critical for
maintaining viability, acting to stabilize and preserve replication fork complexes.4–6
Replication fork
collapse is an irretrievable and catastrophic event and the serine/threonine kinase checkpoint kinase 1
(CHK1) is an essential mediator of the mammalian replication checkpoint.5
Thus, CHK1 is associated
with key mediators of DNA replication and, following exposure to hydroxyurea, is activated in this
context in a manner that requires TopBP1 and ataxia telangiectasia-related protein.7
Activation of
CHK1 ultimately causes inactivation of cyclin-dependent kinases (CDK) leading to appropriately
controlled delays in downstream cell cycle progression.8–10
Ablation of CHK1 using short interfering RNA (siRNA) during hydroxyurea exposure led to rapid
generation of double-strand DNA breaks and subsequent cell death. In addition, tumor cells lacking
CHK1 were unable to resume DNA synthesis following withdrawal of hydroxyurea and underwent
apoptosis in a manner independent of CHK2 or p53 status. Hence, CHK1 appears essential for
suppression of DNA damage and maintains viability during replication stress.5
By extension, the
nonredundant function of CHK1 at the replication checkpoint appears mechanistically distinct from
the previously characterized role at the G2-M or DNA damage checkpoint.9, 11
Significantly, similar
phenotypes were not observed following depletion of CHK1 in nontransformed, diploid fibroblasts.5
In this study, we once more focus on the role of CHK1 at the replication checkpoint and describe the
use of mechanism-based phenotypic screening to identify and discriminate between potent CHK1
91
inhibitor compounds. The cellular γ-H2AX biomarker of double-strand DNA break accumulation was
a key component of this highly functional approach.12
Using this marker, the relative contributions of
CHK1, CHK2, and the CDKs to the replication checkpoint were assessed.13
These experiments
prompted a deeper understanding of the inhibitory profile required in a fully functional CHK1
inhibitor and led to the identification of SCH 900776.
Materials and Methods
Experimental compounds and siRNAs
Experimental compounds A, B, C, D, E, SCH 900776, and SCH 727965 were synthesized and
purified as described previously.14–17
Clinical formulations of gemcitabine and pemetrexed were
obtained from Eli Lilly (Gemzar, Alimta, Eli Lilly). SN38, the active metabolite of CPT-11, was
purchased from Tocris Biosciences. Other reagents were obtained from Sigma-Aldrich Chemical
Company. Characterization of CHK1, CHK2, CDK siRNAs, and transfection conditions have been
described previously.5, 7, 18
Cell lines and cell culture
Tumor cell lines were obtained from the American Type Culture Collection and the Schering Plough
cell line repository (no authentication was done by the authors).
Immunoblotting
Cells were harvested and lysed in a 50 mmol/L Tris-HCl buffer containing 350 mmol/L NaCl, 0.1%
NP40, 1 mmol/L dithiothreitol, and a cocktail of protease and phosphatase inhibitors (Calbiochem).
Following protein concentration determination (Biorad), cell lysates were separated on reducing SDSPAGE
gels and immunoblotted with antisera specific for CDK1 (cell signaling), CDK2 (cell
signaling), CHK1 pS345 (cell signaling), CHK1 pS296 (cell signaling), total CHK1 (7), and phosphoreplication
protein A (RPA) (Bethel Labs).
Kinase assays
CHK1, CHK2, and CDK kinase assays have been described previously.14–17, 19, 20
The Millipore
Kinase Profiler service was used to generate general selectivity data for SCH 900776 against a broad
range of serine/threonine and tyrosine kinases. Assays were typically run at two concentrations of
92
SCH 900776 (0.5 and 5 μmol/L), at a fixed (10 μmol/L) concentration of ATP. Data were provided as
percent activity remaining, relative to uninhibited controls.
Affinity assessment using temperature-dependent fluorescence
An amount of 1 μmol/L CHK1 recombinant kinase domain protein (amino acid residues 2–274) was
mixed with micromolar concentrations (usually 1–50 μmol/L) of compounds in 20 μL of assay buffer
(25 mmol/L HEPES, pH 7.4, 300 mmol/L NaCl, 5 mmol/L dithiothreitol, 2% dimethyl sulfoxide,
Sypro Orange 5x) in a white 96-well PCR plate. The plate was sealed by clear strips and placed in a
thermocycler (Chromo4, BioRad). The fluorescence intensities were monitored at every 0.5 °C
increment during melting from 25 °C to 95 °C. The data were exported into Excel and were subject to
proprietary custom curve fitting algorithm (unpublished) to derive temperature-dependent
fluorescence (TdF) Kd values. For CHK1 TdF data, a two-state binding model (compound binding to
both the native and thermally unfolded molten globule state) is routinely used. Compound binding to
the molten globule state of the target kinase is usually over 1,000-fold weaker than to the native state.
All TdF Kd values have an error margin of ∼50% due to uncertainty with the enthalpy change of
binding.
γ-H2AX assay
Briefly, cells were exposed to an antimetabolite to induce the activation of CHK1. Control
populations were left untreated. SCH 900776 was then titrated onto cells over a 2-hour exposure
window (in the presence of the antimetabolite). Following the 2-hour coexposure to SCH 900776,
cells were fixed and permeabilized (70% ethanol) before staining with a fluorescein isothiocyanate
(FITC)-conjugated anti-γ-H2AX monoclonal antibody (cell signaling). Cells were counterstained
with propidium iodide and subsequently analyzed using flow cytometry (Becton Dickinson LSR II)
or the Discovery 1 immunofluorescence platform (Molecular Devices). Experiments were typically
done in triplicate and data are presented as the percentage of γ-H2AX positive cells, and thus reflect
the overall penetrance of the γ-H2AX phenotype.
Induction of apoptosis assessed by active caspase
Assays of caspase activation were done using the Beckman Coulter CellProbe HT Caspase 3/7 Whole
Cell Assay system. Briefly, cells were exposed to an antimetabolite (hydroxyurea) overnight and then
differing concentrations of SCH 900776 over a 2-hour exposure window. Cells were then washed to
remove all antimetabolite and SCH 900776. Caspase activity was assessed at this point (T0, or
release) and further assays were done at T + 24 and T + 48 hours. Cells were subsequently incubated
93
with a fluorescently labeled caspase substrate (CellProbe); uptake and fluorescence of the substrate
within cells correlate with the level of activated caspases. The percentage of cells expressing activated
caspases was then determined by flow cytometry.
Bromodeoxyuridine incorporation assay
Cells were plated into 10 cm tissue culture dishes and allowed to adhere. Cells were exposed over 2
hours to differing concentrations of SCH 900776 either with, or without, prior antimetabolite
exposure. Cells were then washed and allowed to attempt resumption of S-phase for 24 hours. This
was followed by a brief (30 minute) exposure to bromodeoxyuridine (BrdU) to assess the percentage
of cells that were capable of re-entering the cell cycle in a viable manner. Cells were then harvested,
fixed, and permeabilized. This was followed by an acid denaturation step to expose incorporated
BrdU epitopes within the genomic DNA, after which samples were immunostained with a FITCconjugated
monoclonal antibody specific for BrdU (BD Biosciences). Cells were then counterstained
with propidium iodide to allow assessment of DNA content and analyzed using flow cytometry.
Bivariant analysis of positive BrdU staining and propidium iodide signal allowed assessment of the
number of cells undergoing DNA synthesis and the overall cell cycle distribution of the cell line (G1,
S, G2-M, and sub-G1). Percentages of each population at each concentration of the test article were
plotted.
Experimental animals
Strains used were typically 6- to 8-week old, female nude mice, Sprague-Dawley rats and beagle
dogs. Animals were housed in an Association Assessment and Accreditation of Laboratory Animal
Care accredited animal facilities (Merck Research Laboratories; Xenometrics). All protocols using
animals were approved by the relevant Institutional Animal Care and Use Committee.
In vivo tumor growth assessments, sampling, and skin biopsies
For tumor implantation, specific cell lines were grown in vitro, washed once with PBS and
resuspended in 50% Matrigel (BD Biosciences) in PBS to a final concentration of 4 × 107
to 5 × 107
cells per mL. Nude mice were injected with 0.1 mL of this suspension subcutaneously in the flank
region. Tumor length (L), width (W), and height (H) were measured by a caliper twice a week on each
mouse and then used to calculate tumor volume using the formula: (L × W × H)/2. Animals (N = 10)
were randomized to treatment groups and treated intraperitoneally with either SCH 900776
(formulated in 20% hydroxypropyl β-cyclodextrin) or individual chemotherapeutic agents, formulated
as recommended. Tumor volumes and body weights were measured during and after the treatment
94
periods. Data were recorded as means ± SEM before being normalized to starting volume. Time to
progression to 10x starting volume (TTP 10x) was monitored in some experiments. Animals were
euthanized according to Institutional Animal Care and Use Committee guidelines. For
pharmacodynamic marker analyses in mice, tumors and adjacent skin were collected at necropsy,
fixed overnight in 10% formalin, and washed/stored in 70% ethanol. For skin punch biopsies, an area
of approximately 4 square inches was shaved. Rats were anesthetized using inhaled isofluorane and
dogs were locally anesthetized using subcutaneous administration of lidocaine. Samples were
collected using a 4 mm biopsy punch. Skin punches were fixed in 10% formalin overnight before
washing/storage in 70% ethanol.
Immunohistochemistry
Fixed samples were processed in a tissue processor (Thermo Electronic Co.). Tissues were
dehydrated in graded ethanol solutions, cleared in 3 changes of xylene, and penetrated in heated
paraffin (at 56 °C–58 °C). The tissues were embedded in paraffin, cut into 4- to –6-μm sections, and
placed onto slides. Before staining, deparaffinization and rehydration was done in a Leica autostainer
(65 °C, 20′; xylene 5′, x3; 100% ethanol 1′, x3; 95% ethanol 1′, x2; 70% ethanol 1′; distilled water,
5′). Antigen retrieval was done via pressure cooker. Slides were incubated in 1x target retrieval
solution (Dako) at 120°C for 4 minutes at 18 to 20 psi. The pressure cooker was returned to 0 PSI and
89°C before opening. Slides were then rinsed in water and PBS (5′ each). Slides were stained using
polymer detection (Envision, Dako) on a Dako automated immunostainer. Endogenous hydrogen
peroxidase activity was blocked with hydrogen peroxide for 10 minutes followed by rinsing with
wash buffer (Dako). Slides were incubated with antibodies (e.g., γ-H2AX clone 20E3 and CHK1
pS345 clone 133D3; cell signaling) diluted 1:250 in wash buffer for 60′. Alternatively, slides were
incubated with appropriate isotype controls, diluted similarly. Slides were washed and incubated with
anti-rabbit horseradish peroxidase polymer for 30′, followed by a further wash. Slides were developed
using 3,3′-diaminobenzidine (DAB)+
chromogen (Dako) for 10′ and washed with water. After
staining, slides were counterstained, dehydrated, and cleared using a Leica autostainer (Dako
hematoxylin, 5′; distilled water, 1′; Richard Allen Blueing Reagent, 1′; distilled water, 1′; 95%
ethanol, 1′ x1′ 100% ethanol, 1′ x2; xylene, 1′ x3). Finally, slides were cover-slipped with mounting
reagent (Permount, Fisher).
Peripheral hematological parameters
Blood samples were obtained from mice, diluted 1 to 5 in PBS, and immediately analyzed on an
Advia 120 hematology analyzer. A full differential blood count was done, in particular red blood cell
95
analysis (including reticulocyte, variant count, and hemoglobin analyses), white blood cell analysis
(including differential lineage counts and peroxidase staining), and a thrombopoiesis analysis.
Pharmacokinetic determinations
Plasma samples from test species were collected at various times after administration of SCH 900776.
At each time-point, blood samples from 3 animals were combined and analyzed for SCH 900776 by
LC/MS. Pharmacokinetic variables were estimated from the plasma concentration data. Cmax values
(maximum plasma concentration) were taken directly from the plasma concentration-time profiles,
and the area under the plasma concentration versus time curve area under curve (AUC) was
calculated using the linear trapezoidal rule.21
Results
Contributions of CHK1, CHK2, and CDKs to replication checkpoint override phenotypes
Exposure to hydroxyurea induced activation of CHK1 in U2OS cells and depletion of CHK1 in this
context led to accumulation of γ-H2AX signal in ∼62% of the cell culture population (refs. 5; Table 1
and Supplementary Fig. S1A and B). In contrast, depletion of CHK2 did not significantly enhance the
hydroxyurea phenotype and combinatorial depletion of CHK1 and CHK2 was not beneficial,
appearing inferior to single CHK1 ablation (Table 1). This led to an examination of other possible
antagonistic mechanisms, in particular the CDKs. A consequence of checkpoint activation is
suppression of downstream CDK activity.8,9,22
Therefore, inhibition of CDK function might
antagonize CHK1 ablation/inhibition phenotypes. Indeed, codepletion of CDK2 or CDK1 with CHK1
suppressed γ-H2AX signals and combined ablation of CDK2 and CDK1 further exacerbated
suppression (Table 1). Knockdowns in each case were confirmed by Western blotting (data not
shown). Additionally, simultaneous addition of SCH 727965 (a potent CDK inhibitor; refs. 15, 20)
during hydroxyurea exposure also suppressed accumulation of γ-H2AX, in a dose-dependent manner
(Table 1). Representative fluorescence-activated cell sorter plots stemming from these experiments
are shown in Supplementary Fig. S1C to E. Taken together, these data suggested a requirement for
sufficient CHK1 versus CDK selectivity, whilst the CHK2 observations implied the existence of
additional antagonistic pathways. Global counter screening for kinase cross-reactivity is impractical
and the degree of selectivity required in each case is inherently unpredictable. To circumvent this, we
devised a high content/high throughput, single-cell assay to track anticipated mechanism-based
effects following override of the hydroxyurea-mediated replication checkpoint.
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Table 1. Contributions of CHK1, CHK2, and the CDKs to replication checkpoint control assessed using
siRNAs directed against CHK1, CHK2, CDK1, and CDK2 or pharmacological inhibition of the CDKs using
SCH 727965.
SAR trends and selection of SCH 900776 using the Discovery 1 γ-H2AX assay
Pyrazolo[1,5-a]pyrimidines have been established as a viable core for the development of potent and
selective CDK inhibitors.14, 19, 20, 23
Through a focused medicinal chemistry effort, substitution patterns
were identified that showed improved CHK1 inhibition in vitro.16, 17
To calibrate this in vitro activity
and determine if this series was able to reveal the desired mechanism-based effects, the γ-H2AX
fluorescence-activated cell sorter based assay was adapted for quantitative, high throughput
immunofluorescence (Fig. 1A). A functionally selective CHK1 inhibitor would not be expected to
suppress γ-H2AX accumulation at higher concentrations. In agreement with the hypothesis that
individual compounds have varying degrees of on-target and off-target (antagonistic) properties, bellshaped
responses were observed within this series of compounds (Fig. 1). Ultimately, SCH 900776
(Fig. 1G) was identified as a candidate for additional evaluation.
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Figure 1.
Discovery 1 γ-H2AX assay and structure-activity-relationships within the pyrazolopyrimidine lead series. A,
Discovery 1 immunofluorescence images of U2OS cells stained with PI and γ-H2AX following transfection
98
with luciferase or CHK1 siRNAs and hydroxyurea exposure, as indicated. HU, hydroxyurea. B–G, structures of
compounds A, B, C, D, E, and SCH 900776 with respective CHK1, CHK2, CDK2 kinase IC50s, and Discovery
1 γ-H2AX EC50s. Graphical representations of the Discovery 1 titration profiles, assessed in the presence or
absence of hydroxyurea (blue and red bars, respectively), are shown on the right of each compound. HU,
hydroxyurea.
SCH 900776 (Fig. 1G) is a potent and functionally selective inhibitor of CHK1. In direct binding studies, the Kd
(TdF methodology) of SCH 900776 for the CHK1 kinase domain was determined to be 2 nmol/L, in agreement
with the enzymatically determined IC50. SCH 900776 is not a potent inhibitor of CHK2 and is a weak inhibitor
of CDK2 (Fig. 1F). The overall kinase selectivity profile of SCH 900776 was further characterized via the
Millipore Kinase Profiling service (Supplementary Table S1). SCH 900776 is not highly protein bound
(Supplementary Table S2) and showed no significant inhibition of cytochrome P450 human liver microsomal
isoforms 1A2, 2C9, 2C19, 2D6, and 3A4 (Supplementary Table S3). The solubility of SCH 900776 in buffers
representing the physiologically acceptable pH range indicates suitability for aqueous based formulation
(Supplementary Table S4).
In-cell profile of SCH 900776
Checkpoint override phenotypes following hydroxyurea exposure were confirmed using γ-H2AX
staining and flow cytometry. SCH 900776 exhibits an approximate EC50 of 60 nmol/L under these
conditions, in good agreement with those obtained via Discovery 1 (Fig. 2A). Next, to assess
potential off-target activities in a functional manner, BrdU incorporation following SCH 900776
exposure was measured. CHK1 siRNA treatment does not suppress entry into DNA synthesis
(Supplementary Fig. 2A). In agreement, treatment of U2OS cells with increasing concentrations of
SCH 900776 did not decrease BrdU incorporation (Supplementary Fig. 2B).
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Figure 2.
In-cell activities of SCH 900776. A, confirmation of SCH 900776 γ-H2AX profile using flow cytometry in the
presence and absence of hydroxyurea (blue and red bars, as indicated). B, SCH 900776 rapidly suppresses
accumulation of the CHK1 p296 auto-phosphorylation epitope (top) with concomitant accumulation of DNA
damage (RPA pS33; center). C, short-term exposure to SCH 900776 following hydroxyurea treatment induces
long-term loss of BrdU incorporation capacity (red bars) and leads to accumulation of cells with a sub-G1 DNA
content (light blue bars). D, short-term exposure to SCH 900776 following hydroxyurea treatement induces
caspase activation. U2OS cells were exposed to hydroxyurea overnight and then to increasing concentrations of
SCH 900776 for 2 hours. Cells were harvested immediately (T0; red bars) or cultured for an additional 24 or 48
hours in drug-free medium before being assayed for activated caspases (T24 and T48; yellow and blue bars,
respectively). HU, hydroxyurea.
Serine 296 of CHK1 has been proposed as a site of CHK1 autophosphorylation.13, 24, 25
Following
exposure to hydroxyurea, U2OS cells accumulate CHK1 pS296, and 2 hour exposures to SCH
900776 induced dose-dependent suppression of CHK1 pS296 and concomitant accumulation of
phospho-RPA signal (Fig. 2B), suggestive of DNA damage.
Cells lacking CHK1 following siRNA treatment cannot efficiently resume DNA synthesis. Rather,
these cells accumulate double-strand DNA breaks and undergo cell death.5
SCH 900776 induced a
100
dose-dependent loss of DNA replication capability 24 hours after hydroxyurea exposure (Fig. 2C, red
bars). An increase in the sub-G1 population was also observed, suggestive of cell death within the
culture (Fig. 2C, light blue bars). In agreement, SCH 900776 exposure enhanced apoptosis for at least
48 hours following release from hydroxyurea blockade (Fig. 2D). These data are consistent with the
observed increase in the sub-G1 populations and are suggestive of cell death within the culture.
In vivo modeling of SCH 900776: gemcitabine combinations
CHK1 ablation can phenotypically enhance hydroxyurea, 5-fluoruracil, and cytarabine γ-H2AX
profiles.5, 26
To extend these observations pharmacologically, SCH 900776 was used in combination
with a range of diverse agents. SCH 900776 enhanced the γ-H2AX response of all the agents tested
(Supplementary Fig. S3A-I). Strong combination responses were observed following exposure to
nucleoside DNA antimetabolites and antifolates. Gemcitabine (like hydroxyurea) is an inhibitor of
ribonucleotide reductase (27) that activates CHK1 (Supplementary Fig. S4A), and was selected as the
partner chemotherapy during in vivo modeling of SCH 900776 activities.
To confirm activation of CHK1 in vivo, gemcitabine (25, 75, and 150 mg/kg) was used in the A2780
xenograft model. Immunohistochemical staining revealed dose-dependent activation of the CHK1
pS345 marker within 2 hours (Supplementary Fig. S4B to E). The threshold dose of SCH 900776
associated with intratumoral induction of γ-H2AX was then determined in established A2780
xenografts (∼250 mm3
), in combination with 150 mg/kg gemcitabine. SCH 900776 was administered
30 minutes after gemcitabine. Animals were scheduled to receive 3 cycles of treatment on an every
fifth day regimen before cessation of dosing and monitoring of regression response. Satellite animals
were sacrificed 2 hours after the first dosing cycle for γ-H2AX marker analyses. Thus, 4 mg/kg SCH
900776 was sufficient to induce the γ-H2AX biomarker (Fig. 3D) while 8 mg/kg led to enhanced
tumor pharmacodynamic and regression responses relative to gemcitabine or SCH 900776 alone (Fig.
3E and H). Dose escalation of SCH 900776 (16 and 32 mg/kg) induced incremental improvements in
tumor response (Fig. 3F to H). The approximate Cmax plasma concentration in mice following IP
administration of 10 mg/kg SCH 900776 was ∼0.6 μmol/L and the plasma AUC was ∼0.9 μmol/L.h
(Supplementary Table S5). Therefore, rapid and pronounced modulation of the CHK1 mechanism in
vivo is associated with low doses and exposures of SCH 900776.
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Figure 3.
Active dose threshold of SCH 900776 in combination with gemcitabine (A2780). Representative images from
tumor sections stained for γ-H2AX 2 hours after dosing. A, vehicle. B, 8 mg/kg SCH 900776. C, 150 mg/kg
gemcitabine. D, 150 mg/kg gemcitabine with 4 mg/kg SCH 900776. E, 150 mg/kg gemcitabine with 8 mg/kg
SCH 900776. F, 150 mg/kg gemcitabine with 16 mg/kg SCH 900776. G, 150 mg/kg gemcitabine with 32 mg/kg
SCH 900776. Scale bars represent 100 μm. H, tumor regression responses after 3 cycles of treatment (dose
groups as indicated).
To test the hypothesis that SCH 900776 effects in vivo are also dependent on the dose of CHK1activating
partner chemotherapy, 25, 75, and 150 mg/kg doses of gemcitabine were combined with a
fixed dose of SCH 900776 (50 mg/kg). A2780 xenografts were staged at ∼50 mm3
. Mice were given
two cycles of treatment, with pharmacodynamic sampling done 2 hours after the first cycle. Exposure
to single agent SCH 900776 or gemcitabine induced minor γ-H2AX responses (Supplementary Fig.
S5B and C). In contrast, combination of 50 mg/kg SCH 900776 with 25, 75, or 150 mg/kg
gemcitabine was sufficient to enhance the γ-H2AX staining pattern at 2 hours post dose
102
(Supplementary Fig. S5D to F). Mice were given a second and final cycle of treatment 3 days later
and TTP 10x was monitored (Supplementary Fig. S5G). Improvements in progression kinetics driven
by SCH 900776 were clearly dependent on the dose of gemcitabine and likely reflect the overall
penetrance of the initial CHK1 activation within these xenografts.
MiaPaca2 is a slow growing pancreatic xenograft that progresses during gemcitabine treatment,
implying a degree of resistance to the cytotoxic effects of this agent.28
However, gemcitabine can
suppress BrdU incorporation in MiaPaCa2 cells (Supplementary Fig. S6A). Furthermore,
combination with SCH 900776 induces γ-H2AX in MiaPaCa2 cells at concentrations of gemcitabine
associated with suppression of BrdU incorporation (Supplementary Fig. S6B), consistent with an
active replication checkpoint. Gemcitabine (150 mg/kg) was administered to staged (∼50 mm3
)
MiaPaCa2 tumors followed by escalating doses of SCH 900776 (8, 20, and 50 mg/kg). Satellite
animals were again used for pharmacodynamic marker analyses following the first cycle of dosing
and TTP 10x was followed after 4 cycles of dosing. Gemcitabine retains activity in MiaPaCa2, as
CHK1 pS345 was readily detectable within 2 hours of dosing (Supplementary Fig. S7B). SCH
900776 or gemcitabine dosed as monotherapy induced minimal γ-H2AX signal as a monotherapy in
MiaPaCa2 tumors. However, administration of 8, 20, or 50 mg/kg SCH 900776 to animals previously
dosed with gemcitabine augmented the γ-H2AX response (Supplementary Fig. S7D to H). TTP 10x
was then tracked in each dose cohort. Single agent SCH 900776 (50 mg/kg) was nonefficacious on
this schedule. Administration of gemcitabine or the combination of 150 mg/kg of gemcitabine with 8
mg/kg SCH 900776 both induced a similar TTP 10x benefit of ∼9 days. Escalation of SCH 900776
dose to 20 and 50 mg/kg in combination with gemcitabine led to improvements in TTP 10x
(Supplementary Fig. S7I). Summaries of the preclinical xenograft modeling are provided in Table 2
and Supplementary Tables S6 to 8.
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Table 2. Summary of in vivo studies utilizing SCH 900776 and gemcitabine in the A2780 and MiaPaCa2
xenograft systems.
Replication checkpoint override phenotypes in nontransformed cells
SCH 900776 in combination with hydroxyurea did not lead to a dramatic increase in γ-H2AX signal
in WS1 diploid fibroblasts (ref. 5; Supplementary Fig. S8), consistent with earlier data using CHK1
siRNA. Furthermore, in a survey of several diverse hematological parameters in BALB/c mice
(neutrophils, lymphocytes, red blood cells, and platelets), SCH 900776 did not exacerbate the
myelosuppressive effects of gemcitabine (Table 3). Three days post dosing, gemcitabine (400 mg/kg;
104
1200 mg/m2
) rapidly induced nadirs in total white cell, absolute neutrophil, and absolute lymphocyte
counts. Counts typically rebounded to within the normal range over 7 days and consistently attained
control levels by day 14. Administration of SCH 900776 to animals previously exposed to
gemcitabine did not adversely alter the severity of the nadirs or subsequent rebound kinetics. Platelet
and red blood cell counts were not significantly affected by any dose level of gemcitabine, SCH
900776, or the combination. In summary, combination of gemcitabine at clinically relevant levels
with active doses of SCH 900776 was not associated with synergistic myelosuppression in BALB/c
mice.
Table 3. Effects of SCH 900776, gemcitabine, and the combination on hematological parameters in BALB/c
mice.
In vivo assessment of SCH 900776 active dose range skin biopsies in toxicology species
During clinical trials, it will be important to demonstrate active doses of SCH 900776 can be safely
attained in combination with partner chemotherapy. A biomarker strategy that indicates engagement
105
of CHK1 is therefore critical. CHK1 is an essential kinase and exposure to CHK1 siRNA or SCH
900776 as monotherapy induces intra-S phase DNA damage (ref. 5; Supplementary Fig. S9A and B).
Hence, DNA damage biomarkers (e.g., γ-H2AX and CHK1 pS345) may be useful readouts for SCH
900776-driven target engagement.29
Indeed, SCH 900776 induces dose-dependent accumulation of
CHK1 pS345 in proliferating WS1 cells (Supplementary Fig. S9C). Furthermore, CHK1 pS345
positive cells were detected in skin punch biopsies taken from mice at SCH 900776 doses ≤25 mg/kg
(75 mg/m2
), in rats dosed IV at 5 and 10 mg/kg (30 and 60 mg/m2
) and from dogs dosed IV at 2.5 and
5 mg/kg (45 and 89 mg/m2
; Supplementary Fig. S10A to C). These data and the associated plasma
exposures are summarized in Table 4 and comprise a pharmacological audit trail (30) of SCH 900776
activity in three relevant preclinical species.
Table 4. Dose, pharmacodynamic, pharmacokinetic, and tolerability relationships of SCH 900776 in mouse, rat,
and dog.
106
Discussion
CHK1 preserves tumor cell viability by suppressing the catastrophic accumulation of DNA damage
that would ensue following replication fork collapse.4–6, 31, 32
This is in contrast to the role of CHK1 in
the DNA damage checkpoint, wherein CHK1 is activated in response to pre-existing DNA lesions.33
Hence, dramatic accumulation of DNA damage is predicted to be a signature phenotype of CHK1
inhibition during replication checkpoint override. SCH 900776 is a potent and functionally selective
CHK1 inhibitor currently in clinical development. This molecule was identified using a mechanismof-action
related biomarker (γ-H2AX) in a functional screen that was highly discriminatory.
Moreover, this assay allowed a functional assessment of the CHK1 pathway and other, potentially
antagonistic, mechanisms. Thus, via a combination of siRNA and medicinal chemistry approaches,
the relative contributions of CHK1, CHK2, and CDKs to the replication checkpoint were assessed.
These experiments revealed absolute antagonism following CDK inhibition and suggested CHK2
inhibition to be neither necessary nor desirable. Intriguingly, the functional approach highlighted a
dilemma often faced during the discovery of targeted kinase inhibitors. Prospective reliance on
comprehensive in vitro kinase counter screening may not have identified a CHK1 inhibitor with the
mechanism-based characteristics exhibited by SCH 900776. In contrast, the high content functional
approach identified molecules with the necessary selectivity characteristics against all kinases (more
accurately, those expressed within the screening cell line), as well as other potential nonkinase
antagonistic mechanisms; in vitro kinase selectivity was then determined post hoc. Taken together, it
is clear CHK1 selectivity is an important component in clinical compounds targeting this mechanism.
SCH 900776 is also of low molecular weight (<300 Da), is not highly protein bound (∼50% protein
bound in human plasma), is highly soluble in aqueous buffers at neutral pH, and does not
significantly inhibit a diverse range of P450 enzymes. In summary, SCH 900776 is a drug-like
compound with the key characteristics required for replication checkpoint override.
SCH 900776 recapitulates the key replication checkpoint override phenotypes described following
CHK1 ablation with siRNA. Thus, in combination with an antimetabolite, SCH 900776 induces
accumulation of γ-H2AX within 2 hours, indicative of replication fork collapse and double stranded
DNA breaks. Additionally, SCH 900776 suppressed accumulation of the CHK1 pS296
autophosphorylation epitope in a dose-dependent manner, once more within a 2 hour exposure
window. The rapid onset of these phenotypes was intimately linked to a long-term loss of DNA
synthetic capacity and cytotoxicity, suggesting little need for continual exposure when used in
combination. This was confirmed in a series of in vivo studies wherein SCH 900776 activity appeared
correlated with the penetrance of CHK1 activation driven by gemcitabine. Moreover, intermittent
107
schedules, low doses, and low exposures of SCH 900776 were associated with modulation of
mechanism-based biomarkers and enhancement of gemcitabine response. Importantly, similar
biomarker activation and enhancement of gemcitabine response were observed in gemcitabine
sensitive (A2780) and gemcitabine refractory (MiaPaCa2) models. The scheduling strategy used was
designed to target the replication checkpoint. Thus, SCH 900776 was administered within 30 minutes
of gemcitabine during the window of CHK1 activation induced by replication fork stalling. This is in
contrast to checkpoint inhibition strategies that target the role of CHK1 at the G2-M DNA damage
checkpoint in p53 mutant tumor cells. In this setting, delayed administration of the CHK1 inhibitor is
necessary to allow accumulation of cells at the G2-M boundary.34–36
Targeting the replication
checkpoint represents a mechanistically distinct approach to CHK1 inhibition. Moreover, this strategy
offers several advantages; notably lack of dependence on p53 status5
and, importantly, patient
convenience.
Target organs of DNA antimetabolites include the blood and immune systems. These effects are
generally reversible, clinically manageable mechanism-based toxicities. Importantly, doses of SCH
900776 associated with robust biomarker activation and improved tumor response were not
associated with enhanced toxicity of gemcitabine on hematological parameters in BALB/c mice.
These data (and those derived in vitro using WS1 cells) imply an interesting difference between SCH
900776 responses in normal and transformed backgrounds, in the context of partner chemotherapy
combinations.
Interestingly, exposure of proliferating WS1 cells to SCH 900776 as a single agent was associated
with rapid, dose-dependent accumulation of CHK1 pS345. These data raised the possibility that
cycling populations of normal cells induce CHK1 pS345 following exposure to SCH 900776 as part
of a futile cycle, perhaps driven by AT-family kinases and DNA-PK (refs. 6, 29, 32, 37–40;
Supplementary Fig. S9D). During these experiments, we noted that monotherapy doses of SCH
900776 associated with detection of CHK1 pS345 in skin were moderately in excess of those
associated with γ-H2AX modulation and enhanced response within xenografts (Table 4), suggesting
doses of SCH 900776 sufficient to induce CHK1 pS345 in skin punch biopsies are likely to be equal
to or greater than those required in combination with agents such as gemcitabine. Significantly,
monotherapy doses of SCH 900776 sufficient to induce biomarker responses in skin punch biopsies
taken from rats and dogs were achieved with no dose limiting toxicities. Hence, active doses of SCH
900776 are readily attainable in relevant toxicology species. These observations led to a clinically
tractable biomarker strategy and prompted the design of a 2-stage phase 1 protocol to establish the
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safety and activity of SCH 900776, initially as a lead-in monotherapy, before determining safety of
the same SCH 900776 dose in combination with gemcitabine.41
Multiple chemotherapeutic agents that impact DNA replication provoke synergistic accumulation of
γ-H2AX when combined with SCH 900776. These agents include nucleoside antimetabolites,
antifolates, and alkylators. Interestingly, combination with topoisomerase I inhibitors such as SN38,
did not induce γ-H2AX to a similar extent. These studies are far from comprehensive but they
illuminate a number of other potential SCH 900776 combination strategies. Importantly, under some
circumstances tumors described as nonresponsive to antimetabolites may retain the ability to cease
DNA synthesis following rechallenge. 42–44
This suggests such tumors still exhibit the primary
response to these agents (suppression of S-phase) but are perhaps better able to tolerate long-term
intra-S phase arrest, thus avoiding cell death. Hence, the lack of overall response to agents like
cytarabine or gemcitabine in a resistant setting may not necessarily be a result of the
chemotherapeutic being inactive. Rather, this may reflect selection of cells more tolerant of the
primary mechanistic effect (e.g., stalled replication forks; ref. 45). CHK1 inhibition may represent a
novel opportunity to regenerate meaningful responses on repeat antimetabolite therapy within this
target patient population, by redirecting the mechanism of action of this successful class of drugs
toward tumor-targeted cytotoxicity.
109
References
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3. Zhang, Y. W.; Hunter, T.; Abraham, R. T. Turning the replication checkpoint on and off. Cell
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4. Zachos, G.; Rainey, M. D.; Gillespie, D. A. Chk1-deficient tumour cells are viable but exhibit
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5.Cho, S. H.; Toouli, C. D.; Fujii, G. H.; Crain, C.; Parry, D. Chk1 is essential for tumor cell
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6. Syljuasen, R. G.; Sorensen, C. S.; Hansen, L. T.; Fugger, K.; Lundin, C.; Johansson, F. et al.
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11. Liu, Q.; Guntuku, S.; Cui, X. S.; Matsuoka, S.; Cortez, D.; Tamai, K. et al. Chk1 is an essential
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110
14. Dwyer, M. P.; Paruch, K.; Alvarez, C.; Doll, R. J.; Keertikar, K.; Duca, J. et al. Versatile
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113
Part 2d
Discovery of pyrazolo[1,5-a]pyrimidine-based Pim inhibitors:
a template-based approach*
*published as:
Dwyer, M. P.;*
Keertikar, K.;*
Paruch, K.; Alvarez, C.; Labroli, M.; Poker, C.; Fischmann, T. O.; Mayer-Ezell,
R.; Bond, R.; Wang, Y.; Azevedo, R.; Guzi, T. J. Discovery of Pyrazolo[1,5- a]pyrimidine-based Pim
Inhibitors: A Template-Based Approach. Bioorg. Med. Chem. Lett. 2013, 23, 6178.
The Pim kinases are a family of serine/threonine kinases in the CAMK (calmodulin-dependent
protein kinase) family consisting of three members Pim-1, Pim-2, and Pim-3.1
There is a high level of
sequence homology between these family members which is suggestive of functional redundancy.2
Both Pim-1 and Pim-2 have been shown to be overexpressed in a number of human hematopoietic
malignancies such as myeloid leukemia and lymphomas.3
In addition, DNA microarray analyses
demonstrated that Pim-1 is overexpressed in human prostate cancer and correlates with disease
outcome.4
In animal models, overexpression of Pim-1 and Pim-2 in human prostate cancer cells
enhances their growth in nude mice.5
These observations suggest that Pim-1 and/or Pim-2 inhibitor
may be effective in the treatment for hematologic malignancies.
In 2005, Knapp and co-workers described the first small molecule Pim inhibitors6
represented by 1
(Fig. 1). Since these initial reports, several additional small molecule Pim inhibitor classes7–13
have
been reported and several excellent reviews of recent Pim inhibitor chemotypes have appeared.14
114
Figure 1. Representative Pim inhibitors 1–4.
In addition, several X-ray structures of Pim-1 co-crystallized with inhibitors point to some very
unique structural features for this family of kinases: the hinge region. The unique hinge region for
Pim-1 contains an extra residue, proline-123, which allows for the formation of only a single
hydrogen bond with either ATP or ATP mimetics. With this information in hand, we set about
identifying novel Pim inhibitors that may serve as effective agents for the treatment of hematologic
malignancies.
Initial HTS screening of our internal compound collection identified pyrazolo[1,5-a]pyrimidines 5
and 615
as early Pim program hits (Figure 2). These initial hits possessed sub-micromolar potency for
Pim-1, and 5 showed good selectivity versus both CDK2 and CHK1. We found this chemotype was
of particular interest due to our long standing efforts utilizing the pyrazolo[1,5-a]pyrimidine core for
the identification of selective cell-cycle kinase inhibitors of CDK (dinaciclib)16
and CHK1 (SCH
900776)17
which are currently under clinical evaluation.
115
Figure 2. Pyrazolo[1,5-a]pyrimidine-based Pim hits and pyrazolo[1,5-a]pyrimidine-based cell-cycle kinase
inhibitors.
With these initial screening hits in hand (Fig. 2), efforts were initiated to conduct systematic
modifications around the pyrazolo[1,5-a]pyrimidine core to enhance in-vitro potency against Pim-1
as well as other family members such as Pim-2.
It was not clear at the outset of the program whether a Pim isoform selective inhibitor or a pan–Pim
inhibitor would be more desireable. The ability to access compounds that possessed either profile was
of interest for biology validation studies. Initial chemistry efforts focused upon structural
modifications of the C5 position of naphthyl derivative 5 to explore the SAR around this region. The
synthetic preparation of the C5 derivatives is depicted in Scheme 1. Condensation of 3-aminopyrazole
with 1,3-dimethyl uracil18,19
followed by treatment with POCl3 and iodination afforded compound 7.
Treatment with sodium thiomethoxide followed by Suzuki coupling with 2-naphthyl boronic acid
under standard conditions afforded 8. Oxidation of the thiomethyl moiety with mCPBA afforded
roughly a 1:1 mixture of the intermediate sulfoxide/sulfone which was treated directly with amines in
NMP to afford the title compounds 9a–e, k, m–n found in Table 1. The Boc protected diamine
intermediates were treated with TFA to afford final compounds 9f–j, l (Scheme 1).
116
Scheme 1. (a) 1,3-dimethyluracil, NaOMe; (b) POCl3; (c) NIS; 55% (3 steps) (d) NaSMe, THF; (e) 2-naphthyl
boronic acid, PdCl2(dppf), K3PO4, DME/H2O 65% (2 steps) (f) mCPBA, CH2Cl2; (g) HNRR1
, NMP, 20–62%
(two steps): (h) TFA, CH2Cl2; 85– 96%.
As depicted in Table 1, simple shortening of the chain or elimination of the pyridyl functionality from
compound 5 resulted in a dramatic loss in both Pim-1 and Pim-2 potency. While simple alkyl or
cycloalkyl functionality (9c and d) did not retain potency versus either Pim isoform, incorporation of
polar functionality particularly a primary amine moiety resulted in a dramatic improvement in
potency for both Pim-1 and Pim-2 as demonstrated by compound 9f. While the pendant amino
functionality could be moved around and retain reasonable Pim-1 activity, the Pim-2 activity seemed
to be very sensitive towards the steric environment. This modification resulted in the identification of
extremely selective Pim-1 compounds such as compounds 9m and 9n. Owing to our interest in
obtaining a pan–Pim inhibitor for a tool compound for biological studies, we chose to further explore
SAR around compound 9i which represented the best pan–Pim profile in Table 1 as measured by the
Pim-1 and Pim-2 in-vitro assays.
117
Table 1. Pim-1 and Pim-2 in vitro activity of N5 pyrazolo[1,5-a]pyrimidines 5, 9a–n.
a
Values are the mean of two (n = 2) runs. See Ref. 20 for assay details.
b
na = Not active at >5000 nM.
With compound 9i in hand, efforts were directed towards the exploration of additional C3
modifications, particularly around bicyclic and biaryl motifs.21
The preparation of these C3 analogs is
shown in Scheme 2. Treatment of iodide 7 with tert-butyl(2-aminoethyl) carbamate followed by
subsequent Boc protection afforded 10. Using 10 as a common starting material, Suzuki coupling
118
with various boronic acids/boronates under standard conditions followed by TFA-mediated Boc
deprotection afforded the title compounds 11a–n (Table 2).
Scheme 2. (a) tert-Butyl(2-aminoethyl)carbamate, NMP; (b) Boc2O, DMAP; 60% (two steps) (c) RB(OH)2,
PdCl2(dppf), K3PO4, DME/H2O; (d) TFA, CH2Cl2, 40–65% (two steps).
As summarized in Table 2, simple replacement of the naphthyl motif with either a benzothiophene
(11a) or benzothiazole (11b) resulted in very comparable Pim in vitro profiles to the compound 9i.
However, incorporation of polar functionality such as a benzimidazolone (11c) or indazole (11d)
resulted in 3-4 fold improvement in Pim-2 activity while retaining good Pim-1 potency. Systematic
exploration of placement of biaryl functionality demonstrated that meta-substituted derivatives (11f)
were far superior to both ortho and para-substituted derivatives. The pendant ring of the biaryl motif
of 11f was found to be tolerant of certain heterocyclic rings such as a 1,2,4-oxadiazole (11j) and furan
(11i) while the 2-imidazole derivative (11k) was completely devoid of Pim activity, illustrating the
stringent structural requirements for this ring. In this vein, attempted replacement of the pendant ring
with heteroatom linked rings or O-tethered functionality led to a loss in Pim potency while
replacement of the proximal ring of 11f was generally not well tolerated as illustrated by compounds
11m and 11n.
119
Table 2. Pim-1 and Pim-2 in vitro activity of C3 pyrazolo[1,5-a]pyrimidines 9i, 11a–n.
a
Values are the mean of two (n = 2) runs. See Ref.20 for assay details.
120
b
na = Not active at >5000 nM.
As depicted in Table 2, compound 11j emerged as a lead pan– Pim inhibitor based upon the single
digit nanomolar potency it demonstrated versus both Pim-1 and Pim-2. Further evaluation of this
compound across a battery of kinases summarized in Table 3 reinforces the pan Pim activity
(including Pim-3) of 11j while demonstrating excellent selectivity versus several other kinases.
Table 3. Kinase selectivity of compound 11j.
This compound was found to be clean in both CYP P450 evaluation (3A4, 2D6 and 2C9 > 20 10 M
and hERG ion channel evaluation (11% inhibition @ 10 M)
In order to better understand the SAR trends observed in Tables 1 and 2 for this class of compounds,
X-ray co-crystal structures of two compounds (11o22
and 9g) bearing different functionality at C3 and
C5 bound to Pim-1 (shown in Figs. 3 and 4) were obtained.
Figure 3. X-ray of crystal structure of 11o bound to Pim-1.23
Figure 4. X-ray of crystal structure of 9g in Pim-
1.24
121
In Figure 3, compound 11o does not make contact with the hinge area of the Pim-1 protein but makes
only a single hydrogen bond to a water molecule found in the kinase specificity pocket. The canonical
binding mode observed for 11o in Pim-1 is consistent with the orientation observed for most other
pyrazolo[1,5- a]pyrimidine derivatives from both our CDK and CHK1 programs.16,17
In Figure 4, the larger C3 naphthyl group in 9g induces a flip from the canonical binding mode
observed for 11o shown in Figure 3. The N1 moiety of the pyrazolo[1,5-a]pyrimidine acts as a Hbond
acceptor for Lys57 with the C3 napthyl group oriented parallel to the hinge region of the
protein. In addition, the primary amine on the cyclohexyl ring of 9g makes a H-bond contact with the
oxygen of Glu171. The binding mode observed for 9g is consistent with an X-ray structure for a
structurally related imidazo[1,2-b]pyridazine bound to Pim-1.6
While a X-ray co-crystal structure of
compound 11j bound to Pim-1 could not be obtained, it can be rationalized this compound might bind
in a similar fashion to 9g with the large C3 group oriented along the hinge region.
In summary, systematic optimization of both the C3 and C5 position of pyrazolo[1,5-a]pyrimidine
Pim hit 5 led to the discovery of potent, pan Pim inhibitors represented by 11j. Modifications of
either the C3 or C5 functionality of this series were shown to play a critical role in determining the
ultimate selectivity versus the various Pim isoforms. In addition, single X-ray crystallography of
several members of the pyrazolo[1,5-a]pyrimidine series bound to Pim-1 illustrated several unique
binding modes which seem to be driven by the size of the C3 substituent. This work further validates
the utility of the pyrazolo[1,5-a]pyrimidine core toward the development of additional classes of
kinase inhibitors. Additional evaluation of this class of Pim inhibitors25
including compound 11j will
be reported in due course.
122
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Fischmann, T. O.; Duca, J. S.; Madison, V.; Parry, D.; Davis, N.; Seghezzi, W.; Wiswell, D.; Guzi, T.
J. Bioorg. Med. Chem. Lett. 2011, 21, 467; (b) Labroli, M.; Paruch, K.; Dwyer, M. P.; Alvarez, C.;
Keertikar, K.; Poker, C.; Rossman, R.; Duca, J. S.; Fischmann, T. O.; Madison, V.; Parry, D.; Davis,
N.; Seghezzi, W.; Wiswell, D.; Guzi, T. J. Bioorg. Med. Chem. Lett. 2011, 21, 471–474; (c) Guzi, T.
J.; Paruch, K.; Dwyer, M. P.; Labroli, M.; Shanahan, F.; Davis, N.; Taricani, L.; Wiswell, D.;
Seghezzi, W.; Penaflor, E.; Bhagwat, B.; Wang, W.; Gu, D.; Hsieh, Y.; Lee, S.; Liu, M.; Parry, D.
Mol. Cancer Ther. 2011, 10, 591.
18. It should be noted that this original paper describing the regiochemical outcome of the cyclization
of 3-aminopyrazole with 1,3-dimethyl uracil assigned the product as the 7-OH pyrazolo[1,5a]pyrimidine.
More recent examination of this reaction by Gavrin and coworkers (Ref.19) found this
cyclization in fact affords the 5-OH pyrazolo[1,5-a]pyrimidine instead of the 7-OH analog first
reported by Cutler and coworkers. Independent X-ray analysis conducted in our research laboratories
of a derivative prepared via this method (unpublished results) supports the revised assignment as the
5-OH pyrazolo[1,5-a]pyrimidine core as proposed by Gavrin and coworkers.
Chu, C. K.; Suh, J. J.; Mesbah, M.; Cutler, S. J. J. Heterocycl. Chem. 1986, 23, 349.
19. Gavrin, L. K.; Lee, A.; Provencher, B. A.; Massefski, W. W.; Huhn, S. D.; Ciszewski, G. M.;
Cole, D. C.; McKew, J. C. J. Org. Chem. 2007, 72, 1043.
20. Pim-1, -2, -3 kinase assays: Pim1, 2, and 3 activity were measured using CisBio KinEASE HTRF
assays (catalog # 62ST3PEC) with substrates STK3 (Pim1) or STK1 (Pim2 & 3), anti-phospho
Ser/Thr Cryptate and streptavidin-XL-665 in Corning black LVP 384 well plates (Fisher Catalog #
3676). (Pim 1 and 3 are from Millipore catalog #s 14–573 and 14–738, Pim 2 is from Invitrogen
catalog # PV4036). Briefly, 0.75 ng/well enzyme was added in 7 l kinase buffer (10 mM HEPES pH
7, 0.02% sodium azide, 0.01% BSA, 0.1 mM orthovanidate, 1 mM DTT, 10 mM MgCl2) followed by
124
3 l of compound diluted from DMSO 1:6 with kinase buffer. After a 30 min preincubation, the
reaction was started by adding 2 l of 6ATP/STK mix (final concentrations for Pim 1: 25 M ATP
and 200 nM STK3; Pim 2: 5 M ATP and 700 nM STK1; Pim 3: 10 M ATP and 300 nM STK1).
Reactions were run at room temperature for 45 min (Pim 1) or 60 min (Pim 2 and 3) and stopped by
addition of 10 l detection buffer containing 50 nM, 175 nM, or 75 nM SAXL665 for Pims 1, 2, and
3 respectively and anti-phospho Ser/Thr Cryptate at 1:100. Fluorescense at 620 nM (Cryptate) and
665 nM (XL665) were measured on a Pherastar (BMG Labtech) and the 665/620 ratio x 10,000 was
used for data analysis (ABASE). Dose–response curves were generated from duplicate 8 point serial
dilutions of inhibitory compounds. IC50 values were derived by nonlinear regression analysis.
21. Early SAR efforts around the C3 monocyclic aryl motifs as found in compound 6 did not yield
significant improvements in the in vitro potency (unpublished results).
22. Compound 11o was prepared according to the synthetic route outlined in Scheme 2 by
substituting dimethylethylamine and Bocpyrazole boronic acid to afford the final compound. The
binding assay data for 11o is Pim-1 IC50 = 7 nM and Pim-2 IC50 = 185 nM.
23. The X-ray coordinates for compound 11o bound to Pim-1 have been deposited in the
ProteinDataBank (PDB ID code: 4MBI).
24. The X-ray coordinates for compound 9g bound to Pim-1 have been deposited in the
ProteinDataBank (PDB ID code: 4MBL).
25. A series of potent 3-carboxamido pyrazolo[1,5-a]pyrimidine Pim inhibitors has been recently
disclosed: Wang, X.; Magnuson, S.; Pastor, R.; Fan, E.; Hu, H.; Tsui, V.; Deng, W.; Murray, J.;
Steffek, M.; Wallweber, H.; Moffat, J.; Drummond, J.; Chan, G.; Harstad, E.; Ebens, A. J. Bioorg.
Med. Chem. Lett. 2013, 23, 3149.
125
Part 3a
Cyclin-dependent kinase inhibitors inspired by roscovitine:
purine bioisosteres*
*published as:
Jorda, R.; Paruch, K.; Krystof, V.*
Cyclin-dependent Kinase Inhibitors Inspired by Roscovitine: Purine
Bioisosteres. Curr. Pharm. Design 2012, 18, 2974.
Introduction
Since their discovery as key elements of the cell cycle regulatory machinery, cyclin-dependent
kinases (CDKs) have been considered to be potential targets for drugs against proliferative diseases.1
Indeed, the first small molecule inhibitors of CDKs were found to block proliferation in a variety of
cellular models and induce cell death in transformed cell lines.2,3
Moreover, several cyclins and CDKs
were shown to be oncogenes, while their natural peptide inhibitors (and some of their substrates)
proved to be tumour suppressors.4,5
Taken together, these findings prompted a number of extensive
research programs focused on identifying novel CDK inhibitors as drug candidates for oncology.
Historically, many inhibitors were discovered during random screening programs. Notable examples
include flavopiridol and roscovitine, two of the most well-known first-generation CDK inhibitors to
have undergone clinical trials.6-8
Other compounds were developed via structure-based design using a
number of three dimensional structures of individual CDKs, with or without ligands.9,10
To the best of
our knowledge, only one successful compound has been developed by fragment-based inhibitor
discovery the aminopyrazole derivative AT7519.11
The most extensively used approach is ligandbased
rational design and synthesis of different analogs based on targeted modifications of early leads
and bioisosteric replacements of their functional groups. This approach has yielded structurally
diverse CDK inhibitors that have successfully passed through preclinical testing, such as P276-00 (a
derivative of flavopiridol12
) and ZK 304709 (which is based on the scaffold of the indigoid dye
indirubin13
). In addition, PHA-848125 could be regarded as a bioisostere of PD-0332991, although
both drugs were apparently developed independently.14,15
This review focuses specifically on CDK
inhibitors developed as bioisosteres of roscovitine.
Roscovitine and its analogues
Systematic structural modifications of the 2,6,9-trisubstituted purine derivative olomoucine, which
was identified during random screening, lead to the development of roscovitine16-18
, one of the first
126
CDK inhibitors to enter clinical trials.19,20
Roscovitine is a pan-selective inhibitor of CDK
1/2/5/7/921,22
whose antiproliferative activities correlate with dephosphorylation of the retinoblastoma
protein and the down-regulation of CDKs and cyclins.23-27
It also influences global transcription by
inhibiting CDK7 and CDK9 and thereby inhibiting the activity of RNA polymerase II (RNAP II).24,28
This causes down-regulation of proteins with short half-lives, including several anti-apoptotic
proteins. The reduced abundance of anti-apoptotic proteins alters the balance between cell survival
and apoptosis.
Roscovitine is currently undergoing Phase 2 clinical trials as a single agent against non-small cell
lung carcinoma and nasopharyngeal cancer. It is also being used in combination with other drugs in
two Phase I trials. In the first, it is being evaluated with sapacitabine to treat patients with advanced
solid tumours, while in the second it is being tested in combination with liposomal doxorubicin to
treat patients with metastatic triple negative breast cancer.29,30
The success of roscovitine has prompted attempts to develop related CDK inhibitors by
i) optimizing the substituents of the purine,
ii) changing the positions and ratios of nitrogen and carbon atoms in the heterocyclic core,
iii) using a combination of the two approaches discussed above.
The first approach resulted in the development of many highly potent purine CDK inhibitors (Fig. 1),
including H71731
, purvalanol A32,33
, MDLI0852234
, 3-chloranilino derivatives35
, the
cyclohexylmethoxy compounds NU2058 and NU61029,36
, CR837
, and other biaryl derivatives38-40
(Fig. 1). With the exception of the NU-series, all of these compounds retain similar C2-, C6-, and
N9substituents, i.e. a small hydrophobic chain (isopropyl or cyclopentyl) at N9, an aromatic side
chain coupled through the secondary amino group at C6, and a polar alkyl- or cycloalkylamine at C2.
Many of these compounds are at least a hundred-fold more potent CDK inhibitors than roscovitine.
127
Figure 1. Roscovitine and related purine inhibitors of CDK2. This selection of compounds summarizes
structure-activity relationships within the class a nd demonstrates the diversity of acceptable substitutions.
More synthetically challenging modifications of the purine core have led to the discovery of several
groups of purine bioisosteres (Fig. 2). Purine isomers retaining all four nitrogens (4N) comprise the
largest group, but several types of bioisosteres with two (2N) and three nitrogens (3N) have also been
developed, along with one group having 5 nitrogens (SN). Many of these bioisosteres can replace
purine without sacrificing activity, including imidazo[2, 1-ƒ]-1,2,4-triazines41,42
, pyrrolo[3,2-
d]pyrimidines43
, triazolo[l,5-a]pyrirnidines44,45
, imidazo[4,5-d]pyridines46
, imidazo[1,2-a]pyrazines47
and imidazo[l,2-a]pyridines48,49
. However, the use of pyrazolo[3,4-d]pyrirnidines50
, triazolo[4,5d]pyrimidines
(8-azapurines)51
and benzo[d]imidazoles52
resulted in the loss of CDK inhibitory
potential. Notably, four classes of bioisosteres that yield improved potency relative to purine have
been described: pyrazolo[l,5-a]-1,3,5-triazines41,42,53
, pyrazolo[1,5-a]pyrimidines54-60
, pyrazolo[l,5-
a]pyridines48
and pyrazolo[4,3-d]pyrimidines.61-63
Unfortunately, because the structure-activity
relationships within each group of purine bioisosteres have been studied in different levels of detail, it
is difficult to directly compare their activity. Specifically, only six types of direct roscovitine
analogues have been prepared and evaluated biochemically side by side with roscovitine (Table 1).
128
Figure 2. Structural motifs of known purine bioisosteres primarily designed as CDK inhibitors.
129
Table 1. CDK Inhibitory and Antiproliferative Activity of Selected Roscovitine Bioisosteres.
Type of Bioisostere Compound· CDK2/CYCA I C50
(pM)
CDK21CYCE
ICso(pM)
Average of t he growth
inhibition {ltM)I Number of
tested cancer cell lines Refs.
purine R-roscovitine 0.22 0.15 19.3 /.60 41,42
pyrazolo[l,5-a]- 1,3,5-triazine 7a§
0.04 0.026 1.41 / 60 41,42
imidazo[2,1-ƒ]-1,2,4-triazine 13§
0.22 0.16 25.0 / 6 41
pyrazolo[3,4-d]pyrimidine 33a 0.5 n.a. 76.3 / 3 50
pyrazolo[l,5-a]pyrimidine BS193§
> 1 n.a. >100 / 1 59
pyrazolo[l ,5-a]py rimidine BS181 n.a. 0.88 19.3 / 18 54
pyrazolo[l,5-a]pyrimidine 13 (SCH727965) 0.001 n.a. 0.01 / 13 64,65
imidazo[4,5-d]pyridine Ia§
0.3 0.18 16.1 / 5 46
triazolo[ ,5-a]pyrimidine 79§
5.05 n.a. n.a. / n.a. 44
triazolo[l,5-a]pyrimidine 6 0.35 0.35 25 / 1 45
triazolo[4,5-d]pyrimidine 4§
n.a. 4.1 82.75 / 4 51
triazolo[4,5-d]pyrimidine 19 n.a. 1.1 7.6 / 17 51
imidazo[l,2-a]pyridine 105 n.a. 0.12 n.a. / n.a. 48,49
imidazo[l,2-a]pyrazine 2 n.a. 0.8 n.a. / n.a. 48
pyrazolo[4,3-d]pyrimidine 7§
n.a. 0.04 10.2 / 60 61
Pyrazolo[4,3-d]pyrimidine LGRI406 1.0 0.6 n.a. / n .a. 63
*compound identifiers refer to those used in original publications; §
direct analogue of roscovitine (all side
chains identical); * compound closely related to roscovitine (at least two side chains identical).
Two-nitrogen purine bioisosteres (2N)
While there are many possible two-nitrogen purine bioisosteres, only three groups have been prepared
and described to date: imidazo[l,2-a]pyridines, pyrazolo[l,5-a)pyridines and benzo[d]
imidazoles.49,52,66
The CDK inhibitory activity of imidazo[l,2-a]pyridines and pyrazolo[l,5-a]pyridines
is worse than that of pyrazolo[l,5-a]pyrimidines and imidazo[l,2-a]pyrazines despite the fact that their
modes of binding to CDK2 are identical.48
The 6-O- linked series of benzo[d]imidazoles were
130
designed as potential CDK5 inhibitors, but the direct analogue of roscovitine from this series (4) is
less potent than the parent compound.52
Three-nitrogen purine bioisosteres (3N)
Pyrazolo[l,5-a]pyrimidines
Numerous pyrazolo[l,5-a]pyrimidines with nanomolar aktivity against CDK2 have been synthesized
to date. 56,58,60,67
The most potent pyrazolo[1,5-a]pyrimidines l5j58
and 4k (Fig. 3)56
were tested on
different tumour cell lines (average IC50 ͂ 250 nM). Their mode of binding to CDK2 was studied with
several compounds, including 4k (PDB: 3NS9)56
and the related 9a (PDB: 1Y91)60
, 13 (PDB:
2R3Q)58
and 9 (PDB: 2R3R).67
In order to differentiate between series with different pharmacokinetic
profiles and in vitro activities, an in vivo screening approach with integrated efficacy and tolerability
parameters was adopted. SCH727965 (Dinaciclib) (Fig. 3) had the best therapeutic index of the tested
compounds and was therefore selected for clinical progression.58,64
A computer-aided approach yielded a series of pyrazolo[l,5-a)pyrimidine-based CDK7 inhibitors.54,59
The most potent compound was BS-181 (Fig. 3), which strongly inhibits CDK7 (IC50 = 21 nM),
weakly inhibits CDK2 (IC50 = 0.88 µM), and has no effect on 69 other kinases.54
It is considered to be
the first potent and selective CDK7 inhibitor.54
The roscovitine analog for this series, BS193, was
synthesized but unfortunately did not exhibit any significant selective CDK inhibition.54
Figure 3. Examples of interesting pyrazolo[1,5-a]pyrimidine CDK inhibitors.
131
Imidazo[4,5-d]pyridines
Elimination of the nitrogen atom from position 1 of the purine skeleton (purine numbering) generates
the imidazo[4,5-d]pyridines. CDK inhibitors of this type have been described in a patent.46
In general,
the activity and selectivity of these compounds is similar to that of the analogous purines, including
both enantiomers of roscovitine.
Pyrrolo[3,2-d]pyrimidines
Removal of the nitrogen in position 9 (purine numbering) yielded the pyrrolo[3,2-d]pyrimidines (9deazapurines)
prepared by Capek et al.43
The olomoucine isostere 1 was synthesized, but did not
significantly affect cell growth in a primary biological activity screen.
Imidazo[1,2-a]pyrazines
Only a little information is available about the imidazo[l,2-a]pyrazines.47
CDK inhibition data for
several derivatives sug­ gest that imidazo[l,2-a]pyrazines do not have greater activity than purines
even though ab initio results indicated that the scaffold would bind more tightly to the hinge region
than pyrazolo[l,5-a]pyrimidines.48
The binding mode of some of these compounds to CDK2 (PDB:
2R3G, 2R3H) was recently studied alongside that of other purine bioisosteres such as pyrazolo[l,5a]pyrimidines,
pyrazolo[1,5-a]pyridines and imidazo[l,2-a]pyridines48
; compound 2 was suggested to
have an unusual mode of binding due to the interaction of the fluorophenyl group with the hinge
region (Fig. 4).
132
Figure 4. Binding modes of roscovitine and some purine bioisosteres in the active site ofCDK2 Lines represent
amino acid residues ofCDK2 with a distance of 4 Å from the ligand. Ligands are shown in a ball and stick
representation, with all heteroatoms shown in black.
Four-nitrogen purine bioisosteres (4N)
Pyrazolo[1,5-a]-1,3,5-triazines
Shifting the nitrogen atom at position 9 of the purine skeleton to position 5 yields the pyrazolo[l,5-a]-
1,3,5-triazines. A large number of those derivatives have been prepared, including analogs of
roscovitine and purvalanol.42
The roscovitine bioisostere 7a (N­&-Nl, GP0210, NSC 743927) was
reported to be a pan-selective CDK inhibitor with 2-3 times more activity than roscovitine. However,
both 7a and roscovitine appear to have very similar modes of binding to CDK2 as judged by a
superimposition of their conformations in the active site.41
On average, compound 7a is 14 times
more potent than roscovitine against the NCI panel of 60 tumour cell lines and does not appear to
have any bias towards specific types of tumour.42
Its pharmacokinetic profile is similar to that of
roscovitine.41
133
Imidazo[2,1-f]-1,2,4-triazines
A series of imidazo[2,1-f]-1,2,4-triazines was prepared, including roscovitine analog 13.
Unfortunately, 13 is a less effective CDK inhibitor than roscovitine41,42
: it was only observed to have
activity against CDKs or to induce antiproliferative effects (as judged by the dephosphorylation of the
retinoblastoma protein and changes in the expression of the anti-apoptotic protein Mel-1) at midmicromolar
concentrations.
Pyrazolo[3,4-d]pyrimidines
Replacing the pyrazole-like part of the roscovitine purine skeleton with an imidazole yields
trisubstituted pyrazolo[3,4-d]pyrimidines; a series of olomoucine analogues with this skel eton have
been prepared.50
Most of these compounds did not show any kinase inhibitory activity. This is
probably due to the absence of a nitrogen atom at the 7 position (purine numbering), which is crucial
for binding to the CDK active site.
Triazolo[1,5-a]pyrimidines
The successful identification of potent pyrazolo[l,5-a]pyrimidine-based CDK2 inhibitors60
probably
inspired the investigation of the closely related triazolo[l,5-a]pyrimidine series.44,45
A docking study
on the pyrazolo[1,5-a]pyrimidines suggested that replacing the ligand C3 atom with nitrogen might
increase the compounds' potency.45
Compounds 5 and 6 are analogues of purines NU6102 and
H717.45
Importantly, compound 6 (Fig. 4) showed improved potency for CDK2 inhibition (32-fold)
and also showed good activity against CDKI (IC50 = 140 nM). These findings were supported by Xray
structures of the compound bound to CDK2 (PDB: 2C6M).
Pyrazolo[4,3-d]pyrimidines
The first series of pyrazolo[4,3-d]pyrimidines prepared contained only two substituents, at the 3- and
7-positions.68
As those compounds were more potent than the corresponding purines, 3,5,7trisubstituted
derivatives were subsequently synthesized.69
While comprehensive data on the
134
structure-activity relationships of these purine bioisosteres have not been published, the anticancer/anti­kinase
activities of some compounds from this family have been described.61,63
Compound LGR1406 was examined as a potential inhibitor of abnormal vascular smooth muscle cell
proliferation, which contributes to the pathogenesis of restenosis. Compared to roscovitine, LGR1406
is not a more potent CDK inhibitor but it arrested smooth muscle cell proliferation at one fifth of the
dosage.63
The protein kinase selectivity profile and anti-cancer activity of the pyrazolo[4,3d]pyrimidine-based
analogue of roscovitine are better than those of roscovitine itself.61
An X-ray
crystal structure of compound 7 bound to CDK2 (PDB: 3PJ8) revealed that its binding mode
resembles that of roscovitine.
Five-nitrogen purine bioisosteres (5N)
To date, only one group of purine bioisosteres containing five nitrogen atoms has been reported in the
literature: the 1,2,3- triazolo[4,5-d]pyrimidines.51
All of the compounds in this class that have been
prepared (including the roscovitine analogue) showed significantly reduced CDK inhibitory
activity.51
Apparently, the nitrogen atom at the 2-position interacts unfavourably with the Glu81
residue of the CDK2 active site.
Conclusions
Of the purine bioisosteres described above, only four classes exhibited better CDK inhibition and/or
cytotoxicity than the corresponding purines: the pyrazolo[l,5-a] -1,3,5-triazines, pyrazolo[4,3d]pyrimidines,
pyrazolo[l,5-a]pyrimidines, and pyrazolo[l,5-a]pyridines. The greater potency of those
analogues is apparently due to the arrangement and number of nitrogen atoms in the five-membered
ring that makes direct contact with the hinge region of CDK2:
• The importance of the nitrogen atom at position 7 (purine numbering) is evident from the
binding modes of roscovitine and other purine inhibitors in the active site of CDK2.9
This atom
participates in a hydrogen bond with the amino group of Leu83 and, together with the hydrogen at N6
that interacts with carbonyl group of Leu83, creates an optimal donor-acceptor motif in the hinge
region of CDK2. Replacing this nitrogen with carbon (to give pyrazolo[3,4-d]pyrimidines) yields
significantly less effective CDK inhibitors.50
• The position of the second nitrogen in the five-membered ring is apparently also important in
determining the CDK affinity of purine bioisosteres. The presence of a second nitrogen adjacent to
135
the 7 position in the heterocyclic core (i.e. at position 5 or 8 of the purine skeleton) has a modest
effect on the compounds' electrostatic potential41
and markedly increases the CDK2 affinity of all four
listed groups of bioisosteres.
• The number of nitrogen atoms in the five-membered ring seems to be an important variable in
determining inhibitory activity: all active inhibitors have only 2 nitrogens here. As demonstrated by
the triazolo[l,5-a]pyrimidines and triazolo[4,5-d]pyrimidines, adding a third nitrogen to ring generates
less effective inhibitors.45,51
In sum, ligand-based design has yielded new structurally diverse CDK inhibitors with improved
biochemical and biological properties in some cases. As demonstrated for the heterocyclic skeleton of
the purine-related CDK inhibitors, bioisosteric replacement represents a valid strategy for innovating
from the structures of known drugs. The structure-activity relationships summarized in this review
suggest that the pyrazolo[l,5-a]pyrimidine derivative dinaciclib, which is currently going through
phase II clinical trials, can be considered a bioisostere of roscovitine, one of the best known CDK
inhibitors.
136
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1.
50. Kim, D. C.; Lee, Y. R.; Yang, B. S.; Shin, K. J.; Kim, O. J.; Chung, B. Y.; Yoo, K. H. Synthesis
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benzimidazole based inhibitors of CDK5/p25. Bioorg. Med. Chem. 2011, 19, 359-73.
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56. Heathcote, D. A.; Patel, H.; Kroll, S. H. et al. A novel pyrazolo[1,5-a]pyrimidine is a potent
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human tumor xenografts following oral administration. J. Med. Chem. 2010, 53, 8508-22.
57. Parratt, M. J.; Bower, J. F.; Williams, J. W.; Cansfield, A. D. inventors; Pyrazolopyrimidine
compounds a their use in medicine.WO 2004/087707. 2004 Oct 14.
58. Paruch, K.; Dwyer, M. P.; Alvarez, C. et al. Pyrazolo[l,5-a]pyrimidines as orally available
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60. Williamson, O. S.; Parratt, M. J.; Bower, J. F. et al. Structure-guided design of pyrazolo[l,5a]pyrimidines
as inhibitors of human cyclin­ dependent kinase 2. Bioorg. Med. Chem. Lett. 2005, 15,
863-7.
61. Jorda, R.; Havlicek L.; McNae, I. W. et al. Pyrazolo[4,3-d]pyrimidine bioisostere of roscovitine:
evaluation of a novel selective inhibitor of cyclin-dependent kinases with antiproliferative activity. J.
Med. Chem. 2011, 54, 2980-93.
62. Krystof, V.; Moravcova, D.; Paprskarova, M. et al. Synthesis and biological activity of 8azapurine
and pyrazolo[4,3-d]pyrimidine analogues of myoseverin. Eur. J. Med. Chem. 2006, 41,
1405-11.
63. Sroka, I. M.; Heiss, E. H.; Havlicek, L. et al. A novel roscovitine derivative potently induces G1phase
arrest in platelet-derived growth factor-BB-activated vascular smooth muscle cells. Mol.
Pharmacol. 2010, 77, 255-61.
64. Parry, D.; Guzi, T.; Shanahan, F. et al. Dinaciclib (SCH 727965), a novel and potent cyclin-d
ependent kinase inhibitor. Mol. Cancer Ther. 2010, 9, 2344-53.
65. Paruch, K.; Dwyer, M. P.; Alvarez, C. et al. Discovery of Dinaciclib (SCH 727965): A Potent and
Selective Inhibitor of Cyclin­ Dependent Kinases. ACS Med. Chem. Lett. 2010, 1, 204-8.
66. Dwyer, M. P.; Guzi, T. J.; Paruch, K.; Doll, R. J.; Keertikar, K. M.; Girijavallabhan, V. M.
inventors; Pyrazolopyridines as cyclin­dependent kinase inhibitors. WO 2004/026872. 2004 Apr I.
67. Dwyer, M. P.; Paruch, K.; Alvarez, C. et al. Versatile templates for the development of novel
kinase inhibitors: Discovery of novel CDK inhibitors. Bioorg. Med. Chem. Lett. 2007, 17, 6216-9.
68. Moravcova, D.; Krystof, V.; Havlicek, L.; Moravec, J.; Lenobel, R.; Strnad, M . Pyrazolo[4,3d]pyrimidines
as new generation of cyclin­ dependent kinase inhibitors. Bioorg. Med. Chem. Lett.
2003, 13, 2989-92.
69. Moravcova, D.; Havlicek, L.; Krystof, V.; Lenobel, R.; Strnad, M. inventors; Novel pyrazolo[4,3d]pyrimidines,
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141
Part 3b
Furopyridines as inhibitors of protein kinases*
*published as:
Paruch, K.;*
Petrůjová, M.; Němec, V. Furopyridines as inhibitors of protein kinases PCT Int. Appl. 2015, WO
2015165428 A1.
Field of the Invention
The present invention relates to substituted furo[3,2-b]pyridines as inhibitors of various protein
kinases, regulators or modulators, pharmaceutical compositions containing the compounds, and
pharmaceutical use of the compounds and compositions in the treatment of the diseases such as, for
example, cancer, inflammation, pain, neurodegenerative diseases or viral infections.
Background
Protein kinases are involved in regulation of practically all processes that are central to the growth,
development, and homeostasis of eukaryotic cells. In addition, some protein kinases have an
important role in oncogenesis and tumor progression and several kinase inhibitors are now approved
for the treatment of cancer (D. J. Matthews and M. E. Gerritsen: Targeting protein kinases for cancer
therapy, Wiley, 2010).
Examples of kinase inhibitors that are used in modern oncology include: imatinib (treatment of
CML); dasatinib (CML with resistance to prior treatment, including imatinib); nilotinib (CML);
bosutinib (CML); gefitinib (non-small cell lung cancer); erlotinib (non-small cell lung cancer and
pancreatic cancer); lapatinib (breast cancer); sorafenib (metastatic renal cell carcinoma, hepatocellular
cancer); vandetanib (metastatic medullary thyroid cancer); vemurafenib (inoperable or metastatic
melanoma); crizotinib (non-small cell lung cancer); sunitinib (metastatic renal cell carcinoma,
gastrointestinal stromal tumor that is not responding to imatinib, or pancreatic neuroendocrine
tumors); pazopanib (renal cell carcinoma and advanced soft tissue sarcoma); regorafenib (metastatic
colorectal cancer); cabozantinib (metastatic medullary thyroid cancer); dabrafenib (BRAF V600E
mutation-positive advanced melanoma); and trametinib (in combination with dabrafenib for the
treatment of BRAF V600E/K-mutant metastatic melanoma).
142
Various kinases are regarded as good targets for pharmacological inhibition in order to treat
proliferative and/or neurodegenerative diseases. Biological and potential therapeutic significance of
some selected kinases is briefly summarized below.
The regulation of splice site usage provides a versatile mechanism for controlling gene expression
and for the generation of proteome diversity, playing an essential role in many biological processes.
The importance of alternative splicing is further illustrated by the increasing number of human
diseases that have been attributed to mis-splicing events. Appropriate spatial and temporal generation
of splicing variants demands that alternative splicing be subjected to extensive regulation, similar to
transcriptional control. The CLK (Cdc2-like kinase) family has been implicated in splicing control
(Experimental Cell Research 1998, 241, 300.). Pharmacological inhibition of CLK1/Sty results in
blockage of SF2/ASF-dependent splicing of beta-globin pre-mRNA in vitro by suppression of CLKmediated
phosphorylation. It also suppresses dissociation of nuclear speckles as well as CLK1/Stydependent
alternative splicing in mammalian cells and was shown to rescue the embryonic defects
induced by excessive CLK activity in Xenopus (Journal of Biological Chemistry 2004, 279, 24246.).
Alternative mRNA splicing is a mechanism to regulate protein isoform expression and is regulated by
alternative splicing factors. The alternative splicing factor 45 (SPF45) is overexpressed in cancer and
its overexpression enhances two processes that are important for metastasis, i.e. cell migration and
invasion, dependent on biochemical regulation by CLK1 (Nucleic Acids Research 2013, 41, 4949.).
CLK1 phosphorylates SPF45 on eight serine residues. CLK1 expression enhances, whereas CLK1
inhibition reduces, SPF45-induced exon 6 exclusion from Fas mRNA. Inhibition of CLK1 increases
SPF45 degradation through a proteasome-dependent pathway. In addition, small-molecule inhibitors
of specific CLKs can suppress HIV-1 gene expression and replication (Retrovirology 2011, 8, 47.),
which could be used in concert with current drug combinations to achieve more efficient treatment of
the infection. Inhibition of CLK1 can be applicable in the treatment of Alzmeimer’s disease (Current
Drug Targets 2014, 15, 539.).
DYRK (dual specifity tyrosine phosphorylation-regulated kinase) family enzymes are essential
components of important signaling cascades in the pathophysiology of cancer and Alzheimer’s
disease and their biological expression levels regulate key signaling processes in these diseases. In
particular, DYRK2 is over-expressed in adenocarcinomas of the esophagus and lung (Cancer
Research 2003, 63, 4136.) and DYRK1A in glioblastoma where its inhibition compromised
tumors‘survival and produced a profound decrease in tumor burden (Journal of Clinical Investigation
2013, 123, 2475.). DYRK1B activation that is induced by microtubule damage triggers microtubule
143
stabilization and promotes the mitochondrial translocation of p21Cip1/waf1 to suppress apoptosis. Its
inhibition caused reduced viability of cancer cells (ACS Chemical Biology 2014, 9, 731.).
Correspondingly, it has been understood that inhibition of DYRK kinases alone or in combination
with other chemotherapeutic drugs may have tumor suppression effect and the enzymes are therefore
appropriate targets for pharmacological inhibition (Bioorganic & Medicinal Chemistry Letters 2013,
23, 6610.; Medicinal Chemistry Research 2014, 23, 1925.).
In addition, DYRK kinases are also over-expressed in neurodegenerative diseases such as
Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and Pick disease (Neurobiology of
Disease 2005, 20, 392.; Cellular and Molecular Life Sciences 2009, 66, 3235.).
HIPK2 (homeodomain-interacting protein kinase) is a tumor suppressor and functions as an
evolutionary conserved regulator of signaling and gene expression. This kinase regulates a vast array
of biological processes that range from the DNA damage response and apoptosis to hypoxia signaling
and cell proliferation. Recent studies showed the tight control of HIPK2 by hierarchically occurring
posttranslational modifications such as phosphorylation, small ubiquitin-like modifier modification,
acetylation, and ubiquitination. Dysregulation of HIPK2 can result in increased proliferation of cell
populations as it occurs in cancer or fibrosis. Inappropriate expression, modification, or localization
of HIPK2 can be a driver for these proliferative diseases (Journal of Molecular Medicine 2013, 91,
1051.).
FMS-like tyrosine kinase 3 (FLT3), a receptor tyrosine kinase (RTK), is a membrane-bound receptor
with an intrinsic tyrosine kinase domain. Its activation regulates a number of cellular processes (e.g.
phospholipid metabolism, transcription, proliferation, and apoptosis), and through these processes,
FLT3 activation plays a critical role in governing normal hematopoiesis and cellular growth
Expression of FLT3 has been evaluated in hematologic malignancies. The majority of B-cell acute
lymphocytic leukemia (ALL) and acute myeloid leukemia (AML) blasts (> 90%) express FLT3 at
various levels (Clinical Cancer Research 2009, 15, 4263.). Overexpression or/and activating mutation
of FLT3 kinase play a major driving role in the pathogenesis of acute myeloid leukemia (AML).
Hence, pharmacologic inhibitors of FLT3 are of therapeutic potential for AML treatment (Oncologist
2011, 16, 1162.; PLoS One 2014, 9, e83160/1.; Leukemia Lymphoma 2014, 55, 243.).
Tropomyosin-related kinase (TRK) is a family of three RTKs (TRK-A, TRK-B, TRK-C) regulating
several signaling pathways that are important for survival and differentiation of neurons. TRK-A
regulates proliferation and is important for development and maturation of the nervous system,
promotes survival of cells from death. Point mutations, deletions and chromosomal rearrangements
144
cause ligand-independent receptor dimerization and activation of TRK-A. In mutated version of TRK,
abnormal function will render cells unable to undergo differentiation in response to ligand in their
microenvironment, so they would continue to grow when they should differentiate, and survive when
they should die.
Activated TRK-A oncogenes have been associated with several human malignancies, e.g., breast,
colon, prostate, thyroid carcinomas and AML (Cell Cycle 2005, 4, 8.; Cancer Letters 2006, 232, 90.).
In addition, inhibition of TRK can be relevant for the treatment of inflammation (PLoS One 2013, 8,
e83380.) and pain (Expert Opinion on Therapeutic Patents 2009, 19, 305.).
In summary, there is a need for inhibitors of different protein kinases in order to treat or prevent
disease states associated with abnormal regulation of the kinases-mediated biological processes.
Disclosure of the Invention
The present invention provides substituted furo[3,2-b]pyridine compounds, methods of preparing
such compounds, pharmaceutical compositions comprising one or more of such compounds, and
their use in the treatment, prevention, inhibition or amelioration of one or more diseases associated
with protein kinases using such compounds or pharmaceutical compositions.
The present invention provides compounds represented by the structural formula (I):
N
O
L
3
R
3
L
2
R
2
L
7
R
7
L
6
R
6
L
5
R
5
(I)
or a pharmaceutically acceptable salt, solvate or a prodrug thereof, wherein:
145
L5
is selected from the group consisting of a bond, -N(R11
)-;
L2
is selected from the group consisting of a bond, -O-;
L3
is selected from the group consisting of a bond, -N(R11
)-, -O-;
L6
is selected from the group consisting of a bond, -O-;
L7
is selected from the group consisting of a bond, -N(R11
)-;
R5
is selected from the group consisting of C1-C6 alkyl; aryl; heteroaryl; biaryl; bi(heteroaryl);
cycloalkylaryl; heterocyclylaryl; heteroarylaryl; arylheteroaryl; cycloalkylheteroaryl;
heterocyclylheteroaryl; wherein each of the substituent moieties can be unsubstituted or optionally
substituted;
R2
is selected from the group consisting of H; -CF3; NH2; -Cl; -Br; -F; C1-C6 alkyl;
R3
is selected from the group consisting of H; C1-C6 alkyl; aryl; cycloalkyl; heteroaryl; biaryl;
heteroarylaryl; arylheteroaryl; wherein each of the substituent moieties can be unsubstituted or
optionally substituted;
R6
is selected from the group consisting of H; -CF3; NH2; -Cl; -Br; -F; C1-C6 alkyl; aryl;
heteroaryl; wherein each of the substituent moieties can be unsubstituted or optionally substituted;
R7
is selected from the group consisting of H; C1-C6 alkyl; aryl; cycloalkyl; heteroaryl; biaryl;
heteroarylaryl; arylheteroaryl; wherein each of the substituent moieties can be unsubstituted or
optionally substituted;
146
R11
is selected from the group consisting of H, C1-C6 alkyl;
provided that the substituent in position 5 (L5-R5) is not oxadiazolyl or methyl-oxadiazolyl.
As used in this disclosure, the following terms, unless otherwise indicated, have the following
meanings:
“alkyl” means an aliphatic hydrocarbon group which may be straight or branched and contains 1
to 6 carbon atoms, more preferably 1 to 4 carbon atoms in the chain. Examples of suitable alkyls
are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl. The alkyl
can be unsubstituted or optionally substituted by one or more substituents which can be the same
or different, each substituent being independently selected from the group consisting of F, Cl, Br,
CF3, OCF3, OR9
, SR9
, SOH, SO2H, SO2N(H, C1-C4 alkyl)2, CHO, COO(H, C1-C4 alkyl), COH,
C(O)N(H, C1-C4 alkyl), O(CH2)pN(CH3)2 and NR9
R10
;
“aryl” means an aromatic monocyclic or polycyclic ring system containing 6 to 14 carbon atoms,
preferably 6 to 10 carbon atoms. Examples of suitable aryls are phenyl, naphthyl. The aryl can be
unsubstituted or optionally substituted by one or more substituents which can be the same or
different, each substituent being independently selected from the group consisting of F, Cl, Br,
CF3, OCF3, OR9
, SR9
, SOH, SO2H, SO2N(H, C1-C4 alkyl)2, CHO, COO(H, C1-C4 alkyl), COH,
C(O)N(H, C1-C4 alkyl), NR9
R10
, -(CR9
R10
)pR9a
, O(CH2)pN(CH3)2 and –(CR9
R10
)pOR9a
;
“cycloalkyl” means an aliphatic monocyclic or bicyclic ring system comprising 3 to 10 carbon
atoms, preferably 5 to 7 carbon atoms. Suitable examples include cyclopentyl, cyclohexyl,
cycloheptyl, 1-decalinyl, norbornyl, adamantyl. The cycloalkyl can be unsubstituted or optionally
substituted by one or more substituents which can be the same or different, each substituent being
independently selected from the group consisting of F, Cl, Br, CF3, OCF3, OR9
, SR9
, SOH, SO2H,
SO2N(H, C1-C4 alkyl)2, CHO, COO(H, C1-C4 alkyl), COH, C(O)N(H, C1-C4 alkyl), NR9
R10
, -
(CR9
R10
)pR9a
, O(CH2)pN(CH3)2 and –(CR9
R10
)pOR9a
;
“heterocyclyl” means an aliphatic monocyclic or bicyclic ring system containing 3 to 10 carbon
atoms, preferably 4 to 8 carbon atoms, and at least one heteroatom selected from the group
consisting of nitrogen, oxygen and sulfur. Suitable examples include piperazinyl and morpholinyl.
Preferably, heterocyclyl is not a bicyclic ring system containing only N heteroatoms. The
heterocyclyl can be unsubstituted or optionally substituted by one or more substituents which can
147
be the same or different, each substituent being independently selected from the group consisting
of F, Cl, Br, CF3, OCF3, OR9
, SR9
, SOH, SO2H, SO2N(H, C1-C4 alkyl)2, CHO, COO(H, C1-C4
alkyl), COH, C(O)N(H, C1-C4 alkyl), NR9
R10
, -(CR9
R10
)pR9a
, O(CH2)pN(CH3)2 and –
(CR9
R10
)pOR9a
;
“heteroaryl” means an aromatic monocyclic or bicyclic ring system containing 1 to 14 carbon
atoms, preferably 3 to 7 carbon atoms, most preferably 3 to 5 carbon atoms, and at least one
heteroatom selected from the group consisting of nitrogen, oxygen and sulfur. Examples of
suitable heteroaryls are pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, furanyl, thienyl, oxazolyl,
thiazolyl, isothiazolyl, isoxazolyl, pyrrolyl, imidazolyl. Preferably, heteroaryl is not indolyl,
indolinolyl or imidazopyridazinyl. The heteroaryl can be unsubstituted or optionally substituted by
one or more substituents which can be the same or different, each substituent being independently
selected from the group consisting of F, Cl, Br, CF3, OCF3, OR9
, SR9
, SOH, SO2H, SO2N(H, C1C4
alkyl)2, CHO, COO(H, C1-C4 alkyl), COH, C(O)N(H, C1-C4 alkyl), NR9
R10
, -(CR9
R10
)pR9a
,
O(CH2)pN(CH3)2 and –(CR9
R10
)pOR9a
;
“biaryl” means an aryl-aryl- group in which each of the aryls is independently as previously
described. An example is biphenyl;
“bi(heteroaryl)” means an heteroaryl-heteroaryl- group in which each of the heteroaryls is
independently as previously described;
“cycloalkylaryl” means a cycloalkyl-aryl- group in which the cycloalkyl and aryl are as previously
described;
“heterocyclylaryl” means a heterocyclyl-aryl- group in which the heterocyclyl and aryl are as
previously described;
“heteroarylaryl” means a heteroaryl-aryl- group in which the heteroaryl and aryl are as previously
described;
“arylheteroaryl” means a aryl-heteroaryl- group in which the aryl and heteroaryl are as previously
described;
“cycloalkylheteroaryl” means a cycloalkyl-heteroaryl- group in which the heteroaryl and
cycloalkyl are as previously described;
“heterocyclylheteroaryl” means a heterocyclyl-heteroaryl- group in which the heterocyclyl and
heteroaryl are as previously described;
wherein
148
each of aryl, cycloalkyl, heterocyclyl, heteroaryl, biaryl, bi(heteroaryl), cycloalkylaryl,
heterocyclylaryl, heteroarylaryl, arylheteroaryl, cycloalkylheteroaryl, and heterocyclylheteroaryl
can be bound directly or via a methylene or ethylene spacer;
p is an integer in the range of from 1 to 7, more preferably from 1 to 5, even more preferably 1 to
3;
R9
is H or C1-C6 alkyl, unsubstituted or optionally substituted by -OH, -NH2, -N(CH3)2;
R9a
is H or C1-C6 alkyl, unsubstituted or optionally substituted by -OH, -NH2, -N(CH3)2;
R10
is H or C1-C6 alkyl, unsubstituted or optionally substituted by -OH, -NH2, -N(CH3)2.
In a preferred embodiment, R5
is selected from the group consisting of aryl; heteroaryl;
heterocyclylaryl; heteroarylaryl; arylheteroaryl; heterocyclylheteroaryl; wherein each of the
substituent moieties can be unsubstituted or optionally substituted, preferably by at least one
substituent selected from the group consisting of F, Cl, Br, methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, OH, NH2, N(CH3)2, O(CH2)pN(CH3)2.
More preferably, R5
is selected from the group consisting of aryl; heteroaryl; wherein each of the
substituent moieties can be unsubstituted or optionally substituted, preferably by at least one
substituent selected from the group consisting of F, Cl, Br, methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, OH, NH2, N(CH3)2, O(CH2)pN(CH3)2.
Even more preferably, the heteroaryl in R5
is pyrazolyl.
In a preferred embodiment, any of L5
, L7
is independently selected from the group consisting of a
bond, -NH-.
In another preferred embodiment, any of L2
, L6
is a bond.
In yet another preferred embodiment, L3
is a bond or -O-.
In a preferred embodiment, R3
is selected from the group consisting of aryl; heteroaryl; biaryl;
wherein each of the substituent moieties can be unsubstituted or optionally substituted, preferably
by at least one substituent selected from the group consisting of F, Cl, Br, methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, OH, NH2, N(CH3)2,
149
O(CH2)pN(CH3)2. Even more preferably, the aryl in R3
is phenyl, naphthyl (e.g., 2-naphthyl) and
the biaryl in R3
is biphenyl (e.g., 3-biphenyl).
In a preferred embodiment, R6
is selected from the group consisting of H; -Cl; -Br; -F; -OH; -NH2;
or methyl.
In a preferred embodiment, R2
is selected from the group consisting of H; -Cl; -Br; -F; -OH; -NH2;
or methyl.
In a preferred embodiment, R7
is selected from the group consisting of H; C1-C6 alkyl; aryl;
heteroaryl; wherein each of the substituent moieties can be unsubstituted or optionally substituted,
preferably by at least one substituent selected from the group consisting of F, Cl, Br, methyl, ethyl,
propyl, isopropyl, butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, OH, NH2,
N(CH3)2, O(CH2)pN(CH3)2.
Preferably, at least one of R3
and R7
is not H when the corresponding L (i.e., L3
or L7
,
respectively) is a bond.
In a preferred embodiment, -L2
-R2
, -L6
-R6
, -L7
-R7
are hydrogens and -L3,
-R3
is not hydrogen.
In one preferred embodiment, -L2
-R2
, -L6
-R6
, -L7
-R7
are hydrogens, -L3
-R3
is aryl or biaryl
(optionally substituted) and -L5
-R5
is heteroaryl (optionally substituted).
In a preferred embodiment, any of aryl; cycloalkyl; heterocyclyl; heteroaryl; biaryl; bi(heteroaryl);
cycloalkylaryl; heterocyclylaryl; heteroarylaryl; arylheteroaryl; cycloalkylheteroaryl;
heterocyclylheteroaryl is unsubstituted or substituted with at least one substituent selected from
the group consisting of NH2, N(CH3)2, OH, methoxy, ethoxy, propoxy, isopropoxy, methyl, ethyl,
propyl, isopropyl, butyl, isobutyl and tert-butyl.
150
In another preferred embodiment, any of -L2
-R2
, -L3
-R3
, -L6
-R6
, -L7
-R7
, –L5
-R5
can be hydroxy(C1C6)alkylamino,
amino(C1-C6)alkylamino or dimethylamino(C1-C6)alkylamino.
Pharmaceutically acceptable salts are salts with acids or bases, or acid addition salts. The acids
and bases can be inorganic or organic acids and bases commonly used in the art of formulation,
such as hydrochloride, hydrobromide, sulfate, bisulfate, phosphate, hydrogen phosphate, acetate,
benzoate, succinate, fumarate, maleate, lactate, citrate, tartarate, gluconate, methanesulfonate,
benzenesulfonate, para-toluenesulfonate, primary, secondary and tertiary amides, ammonia.
In general, the compounds described in this invention can be prepared through the general routes
described below in Schemes 1-6.
Pd-catalyzed coupling of 6-chloro-2-iodopyridin-3-ol with vinyl boronates provides intermediate 1
(as shown in Scheme 1), whose copper-mediated closure provides the furopyridine system in
intermediate 2. Subsequent Pd-catalyzed coupling of intermediate 2 with proper C-nucleophiles leads
to compounds 3 with R5
substituent attached via C-C bond.
Scheme 1
Alternatively, intermediate 2 can be subjected to amination to yield amine-containing compounds 4
depicted in Scheme 2.
151
Scheme 2
Also, 5-chlorofuro[3,2-b]pyridine can be converted into iodide 5, which can be brominated to
yield intermediate 6 (Scheme 3). Sequential chemoselective Pd-catalyzed couplings provide target
compounds 3 where R5
and R3
are diferent aryls or heteroaryls (Scheme 3).
Scheme 3
Reaction of 6-chloro-2-iodopyridin-3-ol with trimethylsilylacetylene gives the furopyridine
intermediate 8, which can be subjected to a Pd-catalyzed coupling (e.g. Suzuki reaction) and
subsequent N-oxidation followed by the treatment with POCl3 to yield chlorinated intermediate
10, as illustrated in Scheme 4.
152
Scheme 4
The TMS group in 10 can be removed by KF in methanol to yield intermediate 11, which can be
subjected to Pd-catalyzed C-C bond formation or amination (indicated in Scheme 5) to yield
compounds 12 and 13, respectively.
Scheme 5
153
As depicted in Scheme 6, 6-chloro-2-iodopyridin-3-ol can be allylated to give intermediate 14,
which can be cyclized to furopyridine intermediate 15, which upon Pd-catalyzed C-C bond
formation or amination yields compounds 16 and 17, respectively.
Scheme 6
Compounds 16 can be further elaborated (shown in Scheme 7) by N-oxidation-chlorination
sequence to yield chlorinated intermediate 18, which can be subjected to Pd-catalyzed C-C bond
formation or amination to yield compounds 19 and 20, respectively.
Scheme 7
154
In addition, iodination of 5-bromopyridin-3-ol provides intermediate 21, which can be converted into
compound 22. Subsequent Pd-catalyzed coupling followed by N-oxidation and regioselective
chlorination yield chloride 24 (Scheme 8).
Scheme 8
Another Pd-catalyzed coupling on intermediate 24, followed by the by N-oxidation-chlorination
sequence and another Pd-catalyzed coupling provide intermediate 26. Removal of the TMS group,
followed by final Pd-catalyzed coupling provide target compounds 27 with substituents at positions
7- and 5, respectively (Scheme 9).
Scheme 9
155
The compounds of formula (I) can be useful as protein kinase inhibitors and can be useful in the
treatment and prevention of proliferative diseases, e.g. cancer, inflammation and arthritis,
neurodegenerative diseases such as Alzheimer’s disease, cardiovascular diseases, viral diseases, and
fungal diseases. In one preferred embodiment, the protein kinase is not GSK3. In another preferred
embodiment, the protein kinase is selected from CLK2, CLK4, HIPK1, HIPK2, HIPK3, FLT3,
TRKA and DYRK2.
The present invention thus provides the compounds of formula (I) for use as medicaments. More
specifically, it provides the compounds of formula (I) for use in the treatment and prevention of
conditions selected from proliferative diseases, neurodegenerative diseases, cardiovascular
diseases, pain, viral diseases, and fungal diseases.
The present invention also provides a method for treatment, inhibition, amelioration or prevention
of a condition selected from proliferative diseases, neurodegenerative diseases, cardiovascular
diseases, pain, viral diseases, and fungal diseases, in a patient suffering from such condition,
comprising the step of administering at least one compound of formula (I) to said patient.
The present invention further includes pharmaceutical compositions comprising at least one
compound of formula (I) and at least one pharmaceutically acceptable auxiliary compound. The
auxiliary compounds may include, e.g., carriers, diluents, fillers, preservatives, stabilisers, binders,
wetting agents, emulsifiers, buffers, etc. Suitable auxiliary compounds are well known to those
skilled in the art of formulation. The pharmaceutical compositions are prepared by known
methods, e.g., mixing, dissolving etc.
156
Examples of carrying out the Invention
Preparative Example 1
To a solution of (PhO)3P (2.09 g; 6.75 mmol) in anhydrous CH2Cl2 (10 mL) was added Br2 (0.380
mL; 7.38 mmol) dropwise under Ar atmosphere at -60 °C. Then triethylamine (1.10 mL; 7.89 mmol)
and a solution of 2-acetonaphthone (1.03 g; 6.05 mmol) in anhydrous CH2Cl2 (5 mL) were added.
The resulting reaction mixture was stirred under Ar for 18 h, while warming to 25 °C, and then heated
to reflux for additional 2 h. Then, the CH2Cl2 and excess of triethylamine and Br2 were evaporated
and the residue was purified by column chromatography on silica gel (eluent: hexane/CH2Cl2 – 2:1).
The product was obtained as a pale orange solid (0.947 g; 67 %).
1
H NMR (500 MHz, CDCl3) δ 8.07 (d, J = 1.55 Hz, 1H); 7.87-7.77 (m, 3H); 7.69-7.66 (m, 1H);
7.52-7.46 (m, 2H); 6.25 (d, J = 2.10 Hz, 1H); 5.87 (d, J = 2.10 Hz, 1H).
13
C NMR (126 MHz, CDCl3) δ 135.9, 133.7, 133.2, 131.3, 128.8, 128.1, 127.8, 127.1, 126.9, 124.4,
118.2.
HRMS (APCI): calcd. for C12H10Br [M+H]+
= 232.9960; found [M+H]+
= 232.9958.
Preparative Example 2A
A mixture of vinyl bromide from Preparative Example 1 (0.947 g; 4.06 mmol),
bis(pinacolato)diboron (1.140 g; 4.49 mmol), PPh3 (0.066 g; 0.25 mmol), potassium phenolate (0.809
g; 6.12 mmol) and PdCl2(PPh3)2 (0.089 g; 0.13 mmol) in anhydrous toluene (20 mL) was stirred
under N2 at 50 °C for 24 h. The crude mixture was then cooled to 25 °C, poured into water (100 mL)
157
and extracted with EtOAc (3×80 mL). The organic extracts were washed with brine (80 mL), dried
over Na2SO4, filtered, and the solvent was evaporated. The obtained oil was purified by column
chromatography on silica gel (eluent: hexane/CH2Cl2 – 2:1) to yield the product as a pale orange solid
(0.460 g; 40 %).
1
H NMR (500 MHz, CDCl3) δ 7.94 (s, 1H); 7.84-7.75 (m, 3H); 7.61 (dd, J = 1.50 Hz, 8.54 Hz, 1H);
7.46-7.38 (m, 2H); 6.20 (d, J = 2.29 Hz, 1H); 6.14 (d, J = 2.73 Hz, 1H); 1.35 (s, 12H).
13
C NMR (126 MHz, CDCl3) δ 139.1, 133.8, 132.9, 131.4, 128.5, 127.8, 127.7, 126.4, 126.0, 125.8,
84.1, 25.1.
HRMS (APCI): calcd. for C18H22BO2 [M+H]+
= 281.1711; found [M+H]+
= 281.1708.
Preparative Example 2B
A heatgun-dried round bottom flask containing Ni(dppp)Cl2 (0.251 g; 0.46 mmol) was flushed with
N2, anhydrous THF (24 mL) was added, followed by dropwise addition of DIBAL-H (1.0 M solution
in heptane; 20 mL; 20 mmol) at 25 °C. The mixture was cooled to 0 °C and 4-ethynylanisole (2.0 mL;
15.4 mmol) was added slowly over 5 min. The resulting black solution was allowed to warm to 25 °C
and stirred for additional 2 h. Then, 2-methoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (7.6 mL; 46.4
mmol) was added dropwise at 0 °C and the resulting reaction mixture was stirred under N2 at 80 °C
for 15 h. The reaction was then quenched by dropwise addition of water (50 mL) at 0 °C, allowed to
warm to 25 °C and stirred for additional 1 h. The mixture was poured into saturated aqueous solution
of potassium sodium tartarate (200 mL) and extracted with Et2O (3×150 mL). The extracts were
washed with brine (200 mL), dried over MgSO4, filtered and the solvent was evaporated. The
resulting oil was purified by column chromatography on silica gel (eluent: hexane/EtOAc – 10:1) to
yield the product as a pale yellow solid (3.22 g; 80 %).
1
H NMR (500 MHz, CDCl3) δ 7.45-7.41 (m, 2H); 6.87-6.82 (m, 2H); 5.99 (d, J = 2.65 Hz, 1H); 5.94
(d, J = 2.82 Hz, 1H); 3.79 (s, 3H); 1.31 (s, 12H).
13
C NMR (126 MHz, CDCl3) δ 159.1, 134.2, 129.2, 128.5, 113.9, 84.0, 77.5, 77.2, 77.0, 55.5, 25.0.
158
HRMS (APCI): calcd. for C15H22BO3 [M+H]+
= 260.1693; found [M+H]+
= 260.1696.
Preparative Example 2C
By essentially same procedure set forth in Preparative Example 2B, using 1-(tert-butyl)-4ethynylbenzene,
the compound given below was prepared.
White solid.
1
H NMR (500 MHz, CDCl3) δ 7.44 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 6.08 (d, J = 2.8 Hz,
1H), 6.02 (d, J = 3.0 Hz, 1H), 1.36 – 1.30 (m, 21H).
13
C NMR (126 MHz, CDCl3) δ 150.0, 138.5, 130.2, 126.9, 125.2, 83.8, 34.6, 31.5, 25.0.
HRMS (APCI): calcd. for C18H27BO2 [M+H]+
= 286.2213; found [M+H]+
= 286.2213.
Preparative Example 3
To a stirred solution of 2-chloro-5-hydroxypyridine (6.0 g, 46.3 mmol) in H2O (80 mL) were added
Na2CO3 (10.3 g, 97.3 mmol) and I2 (11.8 g, 46.3 mmol). The resulting mixture was stirred at 25 °C
under N2 for 2 h. Then it was neutralized by HCl (1M, aprox. 50 mL) to pH=7 and extracted with
EtOAc (3×110 mL). The organic extracts were washed with brine (150 mL), dried over Na2SO4,
filtered, and the solvent was evaporated. The product was obtained as a pale yellow solid (11.1 g, 94
%).
1
H NMR (500 MHz, DMSO-d6) δ 11.10 (s, 1H); 7.30 (d, J = 8.40 Hz, 1H); 7.18 (d, J = 8.40 Hz, 1H).
13
C NMR (126 MHz, DMSO-d6) δ 153.9, 138.1, 124.0, 123.9, 107.7.
HRMS (APCI): calcd. for C5H4ClINO [M+H]+
= 255.9021; found [M+H]+
= 255.9018.
159
Preparative Example 4A
To a mixture of vinyl boronate from Preparative Example 2A (0.460 g; 1.64 mmol), pyridinol from
Preparative Example 3 (0.349 g; 1.37 mmol), K3PO4 (1.196 g; 5.64 mmol) and PdCl2.dppf (0.063 g;
0.068 mmol) were added under N2 1,2-dimethoxyethane (8 mL) and water (2 mL). The resulting
reaction mixture was refluxed for 15 h. Then it was cooled to 25 °C, poured into brine (80 mL) and
extracted with CH2Cl2 (3×60 mL). The organic extracts were dried over Na2SO4, filtered, and the
solvent was evaporated. The residue was purified by column chromatography on silica gel (eluent:
CH2Cl2) to yield the product as a pale yellow solid (0.105 g; 27 %).
1
H NMR (500 MHz, CDCl3) δ 7.85-7.75 (m, 3H); 7.72 (d, J = 1.33 Hz, 1H); 7.52 (dd, J = 1.86 Hz,
8.57 Hz, 1H); 7.50-7.45 (m, 2H); 7.26-7.22 (m, 2H); 6.06 (s, 1H); 5.79 (s, 1H); 5.07 (s, 1H).
13
C NMR (126 MHz, CDCl3) δ 149.6, 145.6, 143.8, 142.3, 134.9, 133.7, 133.6, 129.1, 128.7, 127.9,
127.4, 127.0, 126.9, 126.8, 125.0, 124.5, 120.9.
Preparative Example 4B
By essentially same procedure set forth in Preparative Example 4A, using 1-phenylvinylboronic acid
pinacol ester instead of vinyl boronate from Preparative Example 2A, the compound given below was
prepared.
160
1
H NMR (500 MHz, CDCl3) δ 7.39-7.31 (m, 5H); 7.23-7.18 (m, 2H); 5.93 (d, J = 0.73 Hz, 1H); 5.71
(d, J = 0.68 Hz, 1H); 5.00 (brs, 1H).
13
C NMR (126 MHz, CDCl3) δ 149.5, 145.5, 143.8, 142.2, 137.6, 129.3, 129.3, 127.3, 127.2, 125.0,
120.1.
HRMS (APCI): calcd. for C13H11ClNO [M+H]+
= 232.0524; found [M+H]+
= 232.0525.
Preparative Example 4C
By essentially same procedure set forth in Preparative Example 4A, using the vinyl boronate from
Preparative Example 2B, the compound given below was prepared.
1
H NMR (500 MHz, CDCl3) δ 7.30-7.25 (m, 2H); 7.23-7.17 (m, 2H); 6.89-6.85 (m, 2H); 5.83 (d, J =
0.65 Hz, 1H); 5.59 (d, J = 0.52 Hz, 1H); 5.10 (brs, 1H); 3.80 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 160.6, 149.5, 145.8, 143.1, 142.1, 129.8, 128.6, 127.2, 124.9, 118.3,
114.7, 55.6.
HRMS (APCI): calcd. for C14H13ClNO2 [M+H]+
= 262.0629; found [M+H]+
= 262.0631.
Preparative Example 4D
161
The product from Preparative Example 3 (1.16 g, 4.53 mmol), the product from Preparative Example
2C (1.18 g, 4.12 mmol), K3PO4 (3.5 g, 16.5 mmol), DMF (5.5 mL) and PdCl2(dppf) (150 mg, 0.206
mmol) were placed into a 50 mL round bottom flask and the mixture was stirred under N2 at 80 °C for
14 h. The solvent was evaporated and the residue was loaded on silica gel and purified by column
chromatography (CH2Cl2/hexane; 4:1). The product was obtained as a white solid (492 mg, 41 %).
1
H NMR (500 MHz, CDCl3) δ 7.42 – 7.37 (m, 2H), 7.32 – 7.28 (m, 2H), 7.24 – 7.20 (m, 2H), 5.92 (d,
J = 0.8 Hz, 1H), 5.69 (d, J = 0.7 Hz, 1H), 5.04 (s, 1H), 1.32 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 152.5, 149.4, 145.5, 143.4, 142.0, 134.3, 127.1, 126.8, 126.2, 124.7,
119.3, 34.8, 31.4.
HRMS (APCI): calcd. for C17H18ClNO [M+H]+
= 288.1150; found [M+H]+
= 288.1148.
Preparative Example 4E
2-(3,3-dimethylbut-1-en-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (400 mg, 1.90 mmol), DMF (7
mL), K3PO4 (1.2 g, 5.72 mmol), the product from Preparative Example 3 (584 mg, 2.28 mmol) and
PdCl2(dppf) (69 mg, 95 µmol) were placed into a 25 mL round bottom flask. The mixture was stirred
under N2 at 80 °C for 9 h. Then, additional PdCl2(dppf) (47 mg, 64 µmol) was added and the mixture
was stirred at 90 °C for additional 45 h. The solvent was evaporated and the residue was loaded on
silica gel and purified by column chromatography (from EtOAc/hexane; 1:20 to EtOAc). The product
was obtained as a white solid (40 mg, 10 %) of limited stability.
1
H NMR (300 MHz, CDCl3) δ 7.22 (d, J = 8.5 Hz, 1H), 7.12 (d, J = 8.5 Hz, 1H), 5.62 (s, 1H), 5.35 (s,
1H), 5.15 (s, 1H), 1.20 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 153.4, 148.6, 147.7, 140.8, 125.8, 123.8, 116.0, 37.2, 29.6.
HRMS (APCI): calcd. for C11H14ClNO [M+H]+
= 212.0837; found [M+H]+
= 212.0835.
162
Preparative Example 5A
A mixture of the product from Preparative Example 4A (0.105 g; 0.37 mmol), copper(I) acetate
(0.029 g; 0.24 mmol), 8-hydroxyquinoline (0.037 g; 0.25 mmol) and K2CO3 (0.067 g; 0.48 mmol) in
anhydrous N,N-dimethylacetamide (1.5 mL) was stirred under O2 at 140 °C for 18 h. Then the
reaction mixture was concentrated under reduce pressure, the residual oil was poured into water (50
mL) and extracted with EtOAc (3×30 mL). The organic extracts were washed with brine (40 mL),
dried over MgSO4, filtered and the solvent was evaporated. The resulting residue was purified by
column chromatography on silica gel (eluent: hexane/CH2Cl2 – 1:1) to yield the product as a pale
solid product (0.037 g; 36 %).
1
H NMR (500 MHz, CDCl3) δ 8.67 (s, 1H); 8.23 (s, 1H); 8.01-7.93 (m, 2H); 7.90 (d, J = 8.53 Hz,
1H); 7.85-7.82 (m, 1H); 7.75 (d, J = 8.60 Hz, 1H); 7.53-7.45 (m, 2H); 7.29 (d, J = 8.59 Hz, 1H).
13
C NMR (126 MHz, CDCl3) δ 148.0, 147.3, 146.4, 146.0, 133.9, 133.2, 128.7, 128.7, 127.9, 127.3,
126.5, 126.4, 126.4, 124.9, 121.7, 121.3, 119.9.
HRMS (APCI): calcd. for C17H10 ClNO [M+H]+
= 280.0524; found [M+H]+
= 280.0526.
Preparative Example 5B
By essentially same procedure set forth in Preparative Example 5A, using product from Preparative
Example 4B, the compound given below was prepared.
1
H NMR (500 MHz, CDCl3) δ 8.12 (s, 1H); 8.04-7.99 (m, 2H); 7.73 (d, J = 8.61 Hz, 1H); 7.49-7.43
(m, 2H); 7.37-7.32 (m, 1H); 7.27 (d, J = 8.60 Hz, 1H).
163
13
C NMR (126 MHz, CDCl3) δ 147.9, 147.3, 146.1, 145.9, 129.9, 129.1, 128.2, 127.3, 121.8, 121.3,
119.8.
HRMS (APCI): calcd. for C13H9ClNO [M+H]+
= 230.0367; found [M+H]+
= 230.0365.
Preparative Example 5C
By essentially same procedure set forth in Preparative Example 5A, using the product from
Preparative Example 4C, the compound given below was prepared.
1
H NMR (500 MHz, CDCl3) δ 8.05 (s, 1H); 7.97-7.93 (m, 2H); 7.71 (d, J = 8.59 Hz, 1H); 2.25 (d, J =
8.59 Hz, 1H); 7.01-6.97 (m, 2H); 3.84 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 159.7, 147.9, 147.1, 146.0, 145.2, 128.5, 122.4, 121.5, 121.2, 119.6,
114.6, 55.6.
HRMS (APCI): calcd. for C14H11ClNO2 [M+H]+
= 260.0473; found [M+H]+
= 260.0469.
Preparative Example 5D
The product from Preparative Example 4D (470 mg, 1.63 mmol), Cu(OAc)2 (148 mg, 0.816 mmol),
quinolin-8-ol (118 mg, 0.816 mmol), K2CO3 (248 mg, 1.79 mmol) were placed into a 50 mL round
bottom flask. The flask was filled with O2. Then, N, N-dimethylacetamide (4 mL) was added and the
mixture was stirred at 140 °C for 75 min. The solvent was evaporated and the residue was loaded on
silica gel and purified by column chromatography (CH2Cl2/hexane; 1:1). The product was obtained as
an orange solid (378 mg, 74 %).
164
1
H NMR (500 MHz, CDCl3) δ 8.11 (s, 1H), 7.94 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 8.6 Hz, 1H), 7.51 (d,
J = 8.5 Hz, 2H), 7.28 (d, J = 8.6 Hz, 1H), 1.36 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 151.2, 147.8, 147.1, 146.0, 145.7, 127.0, 126.8, 126.0, 121.7, 121.1,
119.6, 34.8, 31.5.
HRMS (APCI): calcd. for C17H16ClNO [M+H]+
= 286.0993; found [M+H]+
= 286.0991.
Preparative Example 5E
By essentially same procedure set forth in Preparative Example 5D, using the product from
Preparative Example 4E, the compound given below was prepared.
White solid.
1
H NMR (500 MHz, CDCl3) δ 7.63 (d, J = 8.6 Hz, 1H), 7.56 (s, 1H), 7.17 (d, J = 8.6 Hz, 1H), 1.48 (s,
9H).
13
C NMR (126 MHz, CDCl3) δ 147.7, 147.0, 145.8, 144.3, 131.0, 120.6, 118.8, 31.0, 29.6.
HRMS (APCI): calcd. for [M+H]+
= 210.0680; found [M+H]+
= 210.0682.
Preparative Example 6A
To a mixture of the product from Preparative Example 5B (0.052 g; 0.23 mmol), 1-methylpyrazole-4boronic
acid pinacol ester (0.059 g; 0.28 mmol), K3PO4 (0.227 g; 1.07 mmol) and PdCl2(dppf) (0.011
g; 0.015 mmol) were added under N2 1,2-dimethoxyethane (2 mL) and water (0.5 mL). The resulting
reaction mixture was refluxed for 18 h. Then it was cooled to 25 °C, diluted with EtOAc (10 mL),
165
poured into brine (25 mL) and extracted with EtOAc (3×10 mL). The organic extracts were dried
over Na2SO4, filtered, and the solvent was evaporated. The residue was purified by column
chromatography on silica gel (eluent: CH2Cl2/EtOAc – 2:1) to yield the product as a pale orange solid
(0.051 g; 81 %).
1
H NMR (500 MHz, CDCl3) δ 8.17-8.13 (m, 2H); 8.09 (s, 1H); 7.99 (d, J = 5.58 Hz, 2H); 7.72 (d, J =
8.61 Hz, 1H); 7.50-7.7.44 (m, 2H); 7.42 (d, J = 8.60 Hz; 1H); 7.37-7.32 (m, 1H); 3.96 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 148.9, 147.7, 145.9, 145.2, 137.7, 131.0, 129.1, 128.9, 127.8, 127.3,
124.4, 121.7, 119.2, 115.8, 39.4.
HRMS (APCI): calcd. for C17H14N3O [M+H]+
= 276.1131; found [M+H]+
= 276.1128.
Preparative Example 6B
To a mixture of the product from Preparative Example 5B (0.311 g; 1.36 mmol), phenylboronic acid
pinacol ester (0.225 g; 1.85 mmol), K3PO4 (1.20 g; 5.64 mmol) and PdCl2.dppf (0.062 g; 0.084 mmol)
were added under N2 1,2-dimethoxyethane (8 mL) and water (2 mL). The reaction mixture was
refluxed under N2 for 19 h. Then it was cooled to 25 °C, diluted with EtOAc (40 mL), poured into
brine (50 mL) and extracted with EtOAc (3×40 mL). The organic extracts were dried over Na2SO4,
filtered, and the solvent was evaporated. The residue was purified by column chromatography on
silica gel (eluent: hexane/EtOAc – 5:1) to yield the product as a pale yellow wax (0.262 g; 71 %).
1
H NMR (500 MHz, CDCl3) δ 8.26-8.19 (m, 2H); 8.17-8.10 (m, 3H); 7.83 (d, J = 8.66 Hz, 1H); 7.73
(d, J = 8.67 Hz, 1H); 7.54-7.46 (m, 4H); 7.45-7.33 (m, 2H).
13
C NMR (126 MHz, CDCl3) δ 154.3, 148.3, 146.1, 145.4, 140.0, 130.9, 129.0, 128.9, 128.8, 127.9,
127.4, 127.3, 122.0, 119.2, 116.8.
HRMS (APCI): calcd. for C19H14NO [M+H]+
= 272.1070; found [M+H]+
= 272.1074.
166
Preparative Example 6C
To a mixture of the product from Preparative Example 5B (0.078 g; 0.34 mmol), 1-Boc-pyrazole-4boronic
acid pinacol ester (0.122 g; 0.42 mmol), K3PO4 (0.282 g; 1.33 mmol) and palladium catalyst
PdCl2.dppf (0.018 g; 0.024 mmol) were added under N2 1,2-dimethoxyethane (2 mL) and water (0.5
mL). The reaction mixture was refluxed under N2 for 15 h. Then it was cooled to 25 °C, diluted with
EtOAc (15 mL), poured into brine (25 mL) and extracted with EtOAc (3×15 mL). The organic
extracts were dried over Na2SO4, filtered, and the solvent was evaporated. The residue was purified
by column chromatography on silica gel (eluent: hexane/EtOAc – 2:1) to yield the product as a pale
yellow solid (0.047 g; 38 %).
1
H NMR (300 MHz, CDCl3) δ 8.27 (s, 1H); 8.18-8.11 (m, 3H); 7.78 (d, J = 8.59 Hz, 1H); 7.53-7.44
(m, 3H); 7.40-7.32 (m, 1H); 1.68 (s, 9H).
Preparative Example 6D
To a mixture of the product from Preparative Example 5B (0.088 g; 0.38 mmol), 3-pyridineboronic
acid pinacol ester (0.098 g; 0.48 mmol), K3PO4 (0.336 g; 1.58 mmol) and palladium catalyst
PdCl2.dppf (0.019 g; 0.026 mmol) were added under N2 1,2-dimethoxyethane (2 mL) and water (0.5
mL). The reaction mixture was refluxed under N2 for 19 h. Then it was cooled to 25 °C, diluted with
EtOAc (15 mL), poured into brine (25 mL) and extracted with EtOAc (3×15 mL). The organic
extracts were dried over Na2SO4, filtered, and the solvent was evaporated. The residue was purified
by column chromatography on silica gel (eluent: CH2Cl2/MeOH – 15:1) to yield the product as a pale
brown solid (0.087 g; 83 %).
167
1
H NMR (500 MHz, CDCl3) δ 9.43 (brs, 1H); 8.73 (brs, 1H); 8.44 (d, J = 7.88 Hz, 1H); 8.23-8.15 (m,
3H); 7.87 (d, J = 8.57 Hz, 1H); 7.74 (d, J = 8.61 Hz, 1H); 7.53-7.34 (m, 4H).
13
C NMR (126 MHz, CDCl3) δ 151.5, 149.6, 148.7, 148.6, 146.6, 145.8, 1358,; 134.8, 130.6, 129.1,
128.1, 127.3, 124.2, 122.0, 119.5, 116.7.
HRMS (APCI): calcd. for C18H13N2O [M+H]+
= 273.1022; found [M+H]+
= 273.1022.
Preparative Example 7A
To a mixture of Preparative Example 5A (0.053 g; 0.19 mmol), 1-methylpyrazole-4-boronic acid
pinacol ester (0.048 g; 0.23 mmol), K3PO4 (0.177 g; 0.83 mmol) and PdCl2.dppf (0.010 g; 0.014
mmol) were added under N2 1,2-dimethoxyethane (2 mL) and water (0.5 mL). The reaction mixture
was refluxed for 19 h. Then it was cooled to 25 °C, diluted with EtOAc (10 mL), poured into brine
(25 mL) and extracted with EtOAc (3×10 mL). The organic extracts were dried over Na2SO4, filtered
and the solvent was evaporated. The residue was purified by column chromatography on silica gel
(eluent: EtOAc/MeOH – 20:1) to yield the product as a pale yellow solid (0.049 g; 79 %).
1
H NMR (500 MHz, CDCl3) δ 8.86 (s, 1H); 8.21 (s, 1H); 8.11-7.99 (m, 3H); 7.97-7.89 (m, 2H); 7.85
(d, J = 7.82 Hz, 1H); 7.76 (d, J = 8.26 Hz, 1H); 7.54-7.42 (m, 3H); 3.99 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 148.9, 147.8, 145.9, 145.6, 137.8, 133.9, 133.1, 129.2, 128.6, 128.5,
128.3, 128.0, 126.5, 126.2, 125.1, 124.4, 121.6, 119.4, 116.0, 39.5.
HRMS (APCI): calcd. for C21H16N3O [M+H]+
= 326.1288; found [M+H]+
= 326.1284.
168
Preparative example 7B
Tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate (77 mg, 0.26
mmol), the product from Preparative Example 5A (61 mg, 0.22 mmol), K3PO4 (180 mg, 0.85 mmol),
1,2-dimethoxyethane (2 mL), H2O (0.5 mL) and PdCl2(dppf) (1.8 mg, 0.008 mmol) were added into a
25 mL round bottom flask. The mixture was refluxed under N2 for 18 h, then saturated aqueous
solution of NH4Cl (15 mL) was added, the mixture was extracted with EtOAc (10 mL) and then with
CH2Cl2 (2×20 mL). The organic extracts were dried over Na2SO4, filtered, and the solvents were
evaporated. The residue was loaded on silica gel and purified by column chromatography
(EtOAc/hexane; 5:4) to afford the product as a light yellow solid (47 mg, 69 %).
1
H NMR (500 MHz, CDCl3) δ 8.94 (s, 1H), 8.32 – 8.20 (m, 3H), 8.11 (d, J = 8.6 Hz, 1H), 8.00 – 7.91
(m, 2H), 7.87 (d, J = 7.8 Hz, 1H), 7.81 (d, J = 8.6 Hz, 1H), 7.56 – 7.47 (m, 3H).
1
H NMR (300 MHz, DMSO-d6) δ 13.04 (b, 1H), 9.12 (s, 1H), 8.95 (s, 1H), 8.52 – 8.20 (m, 3H), 8.14
– 7.99 (m, 3H), 7.99 – 7.90 (m, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.63 – 7.49 (m, 2H).
13
C NMR (126 MHz, CDCl3) δ 148.6, 147.8, 146.0, 145.5, 133.8, 133.0, 128.5, 128.4, 128.2, 127.9,
126.4, 126.1, 124.9, 121.5, 119.2, 116.1.
HRMS (APCI): calcd. for C20H13N3O [M+H]+
= 312.1131; found [M+H]+
= 312.1129.
Preparative example 7C
169
1-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (41 mg, 0,17 mmol), the
product from Preparative Example 5A (40 mg, 0.14 mmol), K3PO4 (91 mg, 0.43 mmol), 1,2dimethoxyethane
(2 mL), H2O (0.5 mL) and PdCl2(dppf) (3.1 mg, 4.3 µmol) were added into a 25 mL
round bottom flask and the mixture was refluxed under N2 for 2 h. The solvent was evaporated and
the residue was loaded on silica gel and purified by column chromatography (EtOAc/hexane; 1:1) to
yield the product as a light yellow wax (40 mg, 79 %).
1
H NMR (500 MHz, CDCl3) δ 8.91 (s, 1H), 8.23 (s, 1H), 8.13 – 8.07 (m, 3H), 7.98 – 7.92 (m, 2H),
7.87 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.6 Hz, 1H), 7.55 – 7.47 (m, 3H), 4.60 (sep, J = 13.4, 6.7 Hz,
1H), 1.62 (s, 3H), 1.60 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 149.1, 147.6, 145.9, 145.3, 137.3, 133.8, 133.0, 128.4, 128.4, 128.3,
127.8, 126.3, 126.0, 125.4, 125.0, 123.6, 121.5, 119.2, 115.8, 54.2, 23.1.
HRMS (APCI): calcd. for C23H19N3O [M+H]+
= 354.1601; found [M+H]+
= 354.1596.
Preparative example 7D
Tert-butyl 3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate
(55 mg, 0.17 mmol), the product from Preparative Example 5A (40 mg, 0.14 mmol), K3PO4 (91 mg,
0.43 mmol), 1,2-dimethoxyethane (2 mL), H2O (0.5 mL) and PdCl2(dppf) (3.1 mg, 4.3 µmol) were
added into a 25 mL round bottom flask and the mixture was refluxed under N2 for 24 h. Then,
additional tert-butyl 3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1carboxylate
(30 mg, 0.09 mmol) and PdCl2(dppf) (4 mg, 5.4 µmol) were added and the mixture was
refluxed under N2 for additional 24 h. The solvent was evaporated and the residue was loaded on
silica gel and purified by column chromatography (EtOAc/MeOH; 30:1) and then rechromatographed
(EtOAc/hexane; 1:1). So obtained material was purified by preparative TLC
(EtOAc/hexane; 1:1) and then by another preparative TLC (CH2Cl2/MeOH; 15:1). The product was
obtained as a colorless wax (7.2 mg, 29 % yield).
170
1
H NMR (300 MHz, CDCl3) δ 8.99 (s, 1H), 8.28 (s, 1H), 8.06 (dd, J = 8.6, 1.6 Hz, 1H), 7.99 – 7.79
(m, 4H), 7.57 – 7.43 (m, 2H), 7.39 (d, J = 8.6 Hz, 1H), 2.62 (s, 6H).
13
C NMR (126 MHz, CDCl3) δ 150.4, 147.0, 145.8, 145.3, 144.7, 143.4, 133.8, 132.9, 128.4, 128.3,
128.2, 127.8, 126.4, 126.3, 126.0, 124.7, 121.6, 118.9, 118.8, 118.1, 12.9, 12.3.
HRMS (APCI): calcd. for C22H17N3O [M+H]+
= 340.1444; found [M+H]+
= 340.1441.
Preparative example 7E
(1,3,5-trimethyl-1H-pyrazol-4-yl)boronic acid (33 mg, 0.21 mmol), the product from Preparative
Example 5A (50 mg, 0.18 mmol), K3PO4 (133 mg, 0.63 mmol), 1,2-dimethoxyethane (2.4 mL), H2O
(0.6 mL) and PdCl2(dppf) (6.5 mg, 8.9 µmol) were added into a 10 mL round bottom flask and the
mixture was refluxed under N2 for 18 h. Additional PdCl2(dppf) (4 mg, 5.4 µmol) and K3PO4 (118
mg, 0.56 mmol) were added and the mixture was refluxed for additional 12 h. Then, another portion
of PdCl2(dppf) (4 mg, 5.4 µmol) was added and the mixture was refluxed for additional 10 h. The
solvent was evaporated and the residue was loaded on silica gel and purified by column
chromatography (EtOAc/hexane; from 1:1 to 2:1) and then by preparative TLC (CH2Cl2/MeOH;
15:1) to yield the product as a colorless wax (5 mg, 8 %).
1
H NMR (500 MHz, CDCl3) δ 8.99 (s, 1H), 8.28 (s, 1H), 8.06 (dd, J = 8.5, 1.7 Hz, 1H), 7.94 – 7.88
(m, 2H), 7.88 – 7.82 (m, 2H), 7.54 – 7.44 (m, 2H), 7.35 (d, J = 8.6 Hz, 1H), 3.85 (s, 3H), 2.59 (s, 3H),
2.51 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 150.7, 147.0, 145.9, 145.8, 145.3, 138.1, 133.9, 132.9, 128.4, 128.3,
128.2, 127.8, 126.4, 126.3, 126.0, 124.7, 121.6, 119.2, 118.9, 118.8, 36.1, 13.7, 11.2.
HRMS (APCI): calcd. for C23H19N3O [M+H]+
= 354.1601; found M+H]+
= 354.1599.
171
Preparative example 7F
A mixture of 5-thiazole boronic acid MIDA ester (69 mg, 0.29 mmol), the product from Preparative
Example 5A (62 mg, 0.22 mmol), K3PO4 (165 mg, 0.78 mmol), 1,2-dimethoxyethane (2 mL), H2O
(0.5 mL) and PdCl2(dppf) (8.1 mg, 11 µmol) was stirred at 60 °C under N2 for 2 h and then at 80 °C
for 7 h. Additional PdCl2(dppf) (4 mg, 5.4 µmol), the mixture was refluxed for 27 h, then additional
PdCl2(PPh3)2 (4 mg, 5.7 µmol) was added and the mixture was refluxed for additional 24 h. Then,
additional Pd(PPh3)4 (5 mg, 4.3 µmol) and 5-thiazole boronic acid MIDA ester (20 mg, 0.083 mmol)
were added and the mixture was refluxed for additional 24 h. The solvent was evaporated and the
residue was loaded on silica gel and purified by column chromatography (EtOAc/hexane; from 1:2 to
1:1) and then by preparative TLC (EtOAc/hexane; 1:1). The product was obtained as a light yellow
solid (7 mg, 10%).
1
H NMR (300 MHz, CDCl3) δ 8.98 (s, 1H), 8.88 (s, 1H), 8.41 (s, 1H), 8.30 (s, 1H), 8.07 (dd, J = 8.6,
1.7 Hz, 1H), 8.03 – 7.84 (m, 4H), 7.74 (d, J = 8.6 Hz, 1H), 7.58 – 7.48 (m, 2H).
13
C NMR (126 MHz, CDCl3) δ 154.4, 148.4, 147.0, 146.3, 146.1, 141.4, 139.7, 133.8, 133.0, 128.6,
128.5, 127.8, 127.7, 126.5, 126.5, 126.3, 124.6, 121.4, 119.5, 116.0.
HRMS (APCI): calcd. for C20H12N2OS [M+H]+
= 329.0743; found [M+H]+
= 329.0747.
Preparative Example 8A
172
To a freshly prepared solution of (S)-1-[(RP)-2-(dicyclohexylphosphino)ferrocenyl]ethyldi-tertbutylphosphine
(0.013 g; 0.023 mmol) and Pd(OAc)2 (0.007 g; 0.030 mmol) in anhydrous
1,2-dimethoxyethane (2 mL) were added the product from Preparative Example 5B (0.053 g; 0.23
mmol), t-BuOK (0.036 g; 0.32 mmol) and 3-methoxy-4-(4-methylpiperazin-1-yl)aniline (0.064 g;
0.29 mmol) and the resulting mixture was stirred under N2 at 100 °C for 16 h. Then it was cooled to
25 °C, diluted with EtOAc (10 mL), poured into brine (25 mL) and extracted with EtOAc (3×10 mL).
The organic extracts were dried over MgSO4, filtered and the solvent was evaporated. The residue
was purified by column chromatography on silica gel (eluent: CH2Cl2/7N NH3 in MeOH – 30:1) to
yield the product as a dark orange semi – solid (0.079 g; 83 %).
1
H NMR (500 MHz, CDCl3) δ 8.05-8.02 (m, 2H); 7.99 (s, 1H); 7.59 (d, J = 8.93 Hz, 1H); 7.44-7.39
(m, 2H); 7.33-7.27 (m, 2H); 6.91-6.81 (m, 2H); 6.70 (d, J = 8.94 Hz, 1H); 6.49 (brs, 1H); 3.80 (s,
3H); 3.10 (s, 4H); 2.68 (s, 4H); 2.38 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 154.0, 153.1, 144.6, 143.7, 137.3, 136.6, 131.3, 128.9, 127.6, 127.2,
121.5, 121.0, 118.9, 112.3, 106.5, 104.8, 55.8, 55.6, 50.9, 46.2.
HRMS (APCI): calcd. for C25H27N4O2 [M+H]+
= 415.2129; found [M+H]+
= 415.2129.
Preparative Example 8B
To a freshly prepared solution of (S)-1-[(RP)-2-(dicyclohexylphosphino)ferrocenyl]ethyldi-tertbutylphosphine
(0.007 g; 0.012 mmol) and Pd(OAc)2 (0.004 g; 0.018 mmol) in anhydrous
1,2-dimethoxyethane (2 mL) were added the product from Preparative Example 5B (0.067 g; 0.29
mmol), t-BuOK (0.041 g; 0.43 mmol) and 4-morpholinoaniline (0.062 g; 0.35 mmol) and the
resulting mixture was stirred under N2 at 100 °C for 14 h. Then it was cooled to 25 °C, diluted with
EtOAc (10 mL), poured into brine (25 mL) and extracted with EtOAc (3×10 mL). The organic
extracts were dried over MgSO4, filtered, and the solvent was evaporated. The residue was purified
173
by column chromatography on silica gel (eluent: CH2Cl2/MeOH – 50:1) to yield the product as an
orange solid (0.062 g; 58 %).
1
H NMR (500 MHz, DMSO-d6) δ 8.92 (s, 1H); 8.59 (s, 1H); 8.25-8.20 (m, 2H); 7.84 (d, J = 9.01 Hz,
1H); 7.72-7.66 (m, 2H); 7.54-7.47 (m, 2H); 7.38-7.31 (m, 1H); 6.98-6.91 (m, 2H); 6.79 (d, J = 9.04
Hz, 1H); 3.78-3.72 (m, 4H); 3.08-3.03 (m, 4H).
13
C NMR (126 MHz, DMSO-d6) δ 153.6, 145.3, 145.0, 143.0, 141.6, 134.6, 131.0, 128.5, 127.0,
126.2, 121.0, 119.7, 118.9, 115.8, 107.7, 66.1, 49.4.
HRMS (APCI): calcd. for C23H22N3O2 [M+H]+
= 372.1707; found [M+H]+
= 372.1704.
Preparative Example 8C
To a freshly prepared solution of (S)-1-[(RP)-2-(dicyclohexylphosphino)ferrocenyl]ethyldi-tertbutylphosphine
(0.008 g; 0.014 mmol) and Pd(OAc)2 (0.010 g; 0.042 mmol) in anhydrous
1,2-dimethoxyethane (2 mL) were added the product from Preparative Example 5B (0.061 g; 0.26
mmol), t-BuOK (0.038 g; 0.40 mmol) and 4-(4-methyl-1-piperazinyl)aniline (0.057 g; 0.30 mmol)
and the resulting mixture was stirred under N2 at 100 °C for 19 h. Then it was cooled to 25 °C, diluted
with EtOAc (15 mL), poured into brine (25 mL) and extracted with EtOAc (3×15 mL). The organic
extracts were dried over MgSO4, filtered and the solvent was evaporated. The resulting residue was
purified by column chromatography on silica gel (eluent: CH2Cl2/7N NH3 in MeOH – 15:1) to yield
the product as a pale brown solid (0.069 g; 68 %).
1
H NMR (500 MHz, CDCl3) δ 8.08-8.04 (m, 2H); 7.99 (s, 1H); 7.56 (d, J = 8.96 Hz, 1H); 7.46-7.41
(m, 2H); 7.37-7.28 (m, 3H); 6.93 (d, J = 5.55 Hz, 2H); 6.66 (d, J = 8.61 Hz, 1H); 6.41 (brs, 1H); 3.18
(brs, 4H); 2.63-2.57 (m, 4H); 2.36 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 147.8, 144.5, 143.8, 134.0, 131.3, 128.9, 127.5, 127.2, 122.8, 122.7,
121.3, 120.9, 117.5, 105.4, 55.4, 50.0, 46.3.
174
HRMS (APCI): calcd. for C24H25N4O [M+H]+
= 385.2023; found [M+H]+
= 385.2030.
Preparative Example 8D
To a freshly prepared solution of (S)-1-[(RP)-2-(dicyclohexylphosphino)ferrocenyl]ethyldi-tertbutylphosphine
(0.009 g; 0.016 mmol) and Pd(OAc)2 (0.015 g; 0.068 mmol) in anhydrous
1,2-dimethoxyethane (2 mL) were added the product from Preparative Example 5B (0.054 g; 0.23
mmol), t-BuOK (0.033 g; 0.34 mmol) and 4-aminopyridine (0.024 g; 0.26 mmol) and the resulting
mixture was stirred under N2 at 100 °C for 14 h. Then it was cooled to 25 °C, diluted with EtOAc (10
mL), poured into brine (25 mL) and extracted with EtOAc (3×10 mL). The organic extracts were
dried over MgSO4, filtered and the solvent was evaporated. The residue was purified by column
chromatography on silica gel (eluent: CH2Cl2/7N NH3 in MeOH – 10:1) to yield the product as a pale
yellow solid (0.034 g; 50 %).
1
H NMR (500 MHz, DMSO-d6) δ 9.71 (s, 1H); 8.71 (s, 1H); 8.35 (d, J = 5.44 Hz, 2H); 8.19 (d, J =
7.31, 2H); 8.02 (d, J = 8.93 Hz, 1H); 7.77 (d, J = 5.66 Hz, 2H); 7.58-7.50 (m, 3H); 7.41-7.35 (m, 1H);
6.98 (d, J = 8.94 Hz, 1H).
13
C NMR (126 MHz, DMSO-d6) δ 152.0, 149.7, 147.8, 145.9, 143.8, 141.8, 130.5, 128.6, 127.3,
126.3, 121.5, 120.0, 111.3, 109.2.
HRMS (APCI): calcd. for C18H14N3O [M+H]+
= 288.1131; found [M+H]+
= 288.1131.
Preparative Example 8E
175
To a freshly prepared solution of (S)-1-[(RP)-2-(dicyclohexylphosphino)ferrocenyl]ethyldi-tertbutylphosphine
(0.011 g; 0.020 mmol) and palladium catalyst Pd(OAc)2 (0.008 g; 0.033 mmol) in
anhydrous 1,2-dimethoxyethane (2 mL) were added the product from Preparative Example 5B (0.053
g; 0.23 mmol), t-BuOK (0.032 g; 0.34 mmol) and aniline (0.025 mL; 0.27 mmol) and the resulting
mixture was stirred under N2 at 100 °C for 15 h. Then it was cooled to 25 °C, diluted with EtOAc (10
mL), poured into brine (25 mL) and extracted with EtOAc (3×10 mL). The organic extracts were
dried over MgSO4, filtered and the solvent was evaporated. The residue was purified by column
chromatography on silica gel (eluent: hexane/EtOAc – 5:1) to yield the product as a brownish semi –
solid (0.050 g; 76 %).
1
H NMR (500 MHz, CDCl3) δ 8.08-8.04 (m, 2H); 8.02 (s, 1H); 7.64 (d, J = 8.96 Hz, 1H); 7.49-7.43
(m, 4H); 7.37-7.29 (m, 3H); 7.07-7.00 (m, 1H); 6.81 (d, J = 8.96 Hz, 1H).
13
C NMR (126 MHz, CDCl3) δ 153.4, 144.8, 144.7, 141.2, 130.9, 129.4, 129.0, 127.8, 127.2, 122.7,
121.3, 119.9, 106.5.
HRMS (APCI): calcd. for C19H15N2O [M+H]+
= 287.1179; found [M+H]+
= 287.1180.
Preparative Example 8F
To a solution of the product from Preparative Example 5B (0.045 g; 0.20 mmol), (R)-BINAP (0.017
g; 0.027 mmol), Pd2(dba)3 (0.014 g; 0.015 mmol) and t-BuOK (0.033 g; 0.29 mmol) in anhydrous
toluene (2 mL) was added N,N-dimethylethylenediamine (0.022 mL; 0.20 mmol) and the resulting
mixture was stirred under N2 at 80 °C for 20 h. Then it was cooled to 25 °C, diluted with EtOAc
(10mL), poured into water (25 mL) and extracted with EtOAc (3×10 mL). The organic extracts were
washed with brine (15 mL), dried over MgSO4, filtered and the solvent was evaporated. The residue
was purified by preparative TLC (eluent: CH2Cl2/7N NH3 in MeOH – 17:1) to yield the product as an
orange wax (0.024 g; 43 %).
1
H NMR (500 MHz, CDCl3) δ 8.13-8.08 (m, 2H); 7.96 (s, 1H); 7.51 (d, J = 8.93 Hz, 1H); 7.45-7.39
(m, 2H); 7.32-7.26 (m, 1H); 6.39 (d, J = 8.93 Hz, 1H); 5.04-4.95 (m, 1H); 3.50 (dd, J = 5.71 Hz,
11.41 Hz, 2H); 2.60 (t, J = 6.06 Hz, 2H); 2.29 (s, 3H).
176
13
C NMR (126 MHz, CDCl3) δ 156.9, 143.9, 143.8, 143.5, 131.7, 128.8, 127.3, 127.0, 121.1, 120.7,
105.4, 58.6, 45.6, 40.1.
HRMS (APCI): calcd. for C17H20N3O [M+H]+
= 282.1601; found [M+H]+
= 282.1600.
Preparative Example 8G
To a freshly prepared solution of (S)-1-[(RP)-2-(dicyclohexylphosphino)ferrocenyl]ethyldi-tertbutylphosphine
(0.011 g; 0.023 mmol) and Pd(OAc)2 (0.007 g; 0.030 mmol) in anhydrous
1,2-dimethoxyethane (2 mL) were added the product from Preparative Example 5C (0.054 g; 0.22
mmol), t-BuONa (0.030 g; 0.31 mmol) and 3-methoxy-4-(4-methylpiperazin-1-yl)aniline (0.056 g;
0.25 mmol) and the resulting mixture was stirred under N2 at 100 °C for 18 h. Then it was cooled to
25 °C, diluted with EtOAc (10 mL), poured into brine (25 mL) and extracted with EtOAc (3×10 mL).
The organic extracts were dried over MgSO4, filtered, and the solvent was evaporated. The residue
was purified by column chromatography on silica gel (eluent: CH2Cl2/7N NH3 in MeOH – 17:1) to
yield the product as a pale orange foam (0.041 g; 42 %).
1
H NMR (500 MHz, CDCl3) δ 7.99-7.95 (m, 2H); 7.91 (s, 1H); 7.56 (d, J = 8.92 Hz, 1H); 7.27-7.22
(m, 1H); 6.91-6.81 (m, 2H); 6.68 (d, J = 8.93 Hz, 1H); 6.50 (brs, 1H); 3.83 (s, 3H); 3.80 (s, 3H); 3.08
(brs, 4H); 2.64 (brs, 4H); 2.36 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 159.2, 153.9, 153.1, 144.4, 143.7, 137.3, 136.6, 128.4, 123.8, 121.1,
120.9, 118.8, 114.3, 112.3, 106.3, 104.8, 55.7, 55.6, 55.5, 51.1, 46.3.
HRMS (APCI): calcd. for C26H29N4O3 [M+H]+
= 445.2234; found [M+H]+
= 445.2235.
177
Preparative Example 9
To a stirred solution of the product from Preparative Example 6C (0.049 g; 0.14 mmol) in ethanol (2
mL) was added aqueous solution of HCl (3M; 0.9 mL; 2.7 mmol) and the resulting mixture was
stirred under N2 at 60 °C for 8 h. Then, the ethanol and HCl were evaporated and the oily residue was
mixed with CH2Cl2 (2 mL), MeOH (1mL) and Na2CO3 (200 mg) and the mixture was stirred at 25 °C.
After 20 min., the solvents were evaporated and the solid residue was purified by column
chromatography on silica gel (eluent: CH2Cl2/7N NH3 in MeOH – 10:1) to yield the product as a pale
yellow solid (0.026 g; 75 %).
1
H NMR (500 MHz, CD3OD) δ 8.36 (s, 1H); 8.31-8.08 (m, 4H); 7.85 (d, J = 8.64 Hz, 1H); 7.63 (d, J
= 8.64 Hz, 1H); 7.49-7.44 (m, 2H); 7.36-7.31 (m, 1H).
13
C NMR (126 MHz, CD3OD) δ 150.2, 149.0, 147.1, 146.8, 138.6, 132.2, 129.7, 128.6, 128.1, 122.5,
120.3, 117.2.
HRMS (APCI): calcd. for C16H12N3O [M+H]+
= 262.0975; found [M+H]+
= 262.0976.
Preparative Example 10
The product from Preparative Example 3 (4.5 g; 17.62 mmol) was placed into a 250 mL round bottom
flask. TEA (32 mL) and 1,4-dioxane (32 mL) were added and the mixture was purged with N2.
Ethynyltrimethylsilane (2.25 g; 22.9 mmol), CuI (0,168 g; 0,881 mmol) and PdCl2(PPh3)2 (0.247 mg,
0.352 mmol) were added and the mixture was stirred at 45 °C under N2 for 2.5 h. The solvent was
evaporated and the residue was purified by column chromatography (hexane/EtOAc; from 15:1 to
10:1) to yield the product as an orange solid (2.90 g; 73 % yield).
178
1
H NMR (500 MHz, CDCl3) δ 7.67 (dd, J = 0.85 Hz, 8.56 Hz, 1H); 7.17 (d, J = 8.56 Hz, 1H); 7.03 (d,
J = 0.84 Hz, 1H); 0.35 (m, 9H).
13
C NMR (126 MHz, CDCl3) δ 170.8, 149.9, 148.5, 146.7, 120.7, 119.1, 116.8, -1.9.
HRMS (APCI): calcd. for C10H13ClNOSi [M+H]+
= 226.0449; found [M+H]+
= 226.0446.
Preparative Example 11
To a solution of the product from Preparative Example 10 (1.013 g; 4.49 mmol) in methanol (20 mL)
was added KF (0.803 g; 13.82 mmol) and the resulting mixture was stirred under N2 at 60 °C for 15 h.
Then it was cooled to 25 °C, poured into aqueous solution of HCl (0.1M; 100 mL) and extracted with
EtOAc (3×60 mL). The organic extracts were dried over Na2SO4, filtered, and the solvent was
evaporated. The residue was purified by column chromatography on silica gel (eluent: hexane/ EtOAc
– 10:1) to yield the product as a pale yellow solid (0.579 g; 84 %).
1
H NMR (500 MHz, CDCl3) δ 7.84 (d, J = 2.28 Hz, 1H); 7.70 (dd, J = 0.84 Hz, 8.60 Hz, 1H); 7.21 (d,
J = 8.60 Hz, 1H); 6.90 (dd, J = 0.84 Hz, 2.26 Hz, 1H).
13
C NMR (126 MHz, CDCl3) δ 150.3, 147.6, 147.2, 146.9, 121.1, 119.5, 108.1.
HRMS (APCI): calcd. for C7H5ClNO [M+H]+
= 154.0054; found [M+H]+
= 154.0055.
Preparative Example 12A
To a mixture of the product from Preparative Example 11 (0.201 g; 0.89 mmol), 1-Boc-pyrazole-4boronic
acid pinacol ester (0.0.317 g; 1.07 mmol), K3PO4 (0.781 g; 3.68 mmol) and PdCl2.dppf
(0.039 g; 0.053 mmol) were added under N2 1,2-dimethoxyethane (4 mL) and water (1 mL). The
reaction mixture was refluxed for 19 h. Then it was cooled to 25 °C, diluted with EtOAc (20 mL),
179
poured into brine (30 mL) and extracted with EtOAc (3×20 mL). The organic extracts were dried
over Na2SO4, filtered and the solvent was evaporated. The residue was purified by column
chromatography on silica gel (eluent: CH2Cl2/MeOH – 15:1). So obtained solid was further purified
by preparative TLC (eluent: CH2Cl2/MeOH – 15:1) to yield the pale yellow crystalline product (0.080
g; 49 %).
1
H NMR (500 MHz, CD3OD) δ 8.17 (s, 2H); 8.04 (d, J = 2.28 Hz, 1H); 7.89 (dd, J = 0.83 Hz, 8.64
Hz, 1H); 7.62 (d, J = 8.65 Hz, 1H); 6.96 (dd, J = 0.85 Hz, 2.26 Hz, 1H); 4.86 (brs, 1H).
13
C NMR (126 MHz, CD3OD) δ 151.4, 150.1, 148.2, 148.1, 133.4, 123.8, 120.9, 117.7, 108.2.
HRMS (APCI): calcd. for C10H8N3O [M+H]+
= 186.0662; found [M+H]+
= 186.0659.
Preparative Example 12B
To a mixture of the product from Preparative Example 11 (0.052 g; 0.34 mmol), 1-methylpyrazole-4boronic
acid pinacol ester (0.092 g; 0.44 mmol), K3PO4 (0.308 g; 1.45 mmol) and PdCl2.dppf (0.017
g; 0.023 mmol) were added under N2 1,2-dimethoxyethane (2 mL) and water (0.5 mL). The reaction
mixture was refluxed for 14 h. Then it was cooled to 25 °C, diluted with EtOAc (15 mL), poured into
brine (25 mL) and extracted with EtOAc (3×15 mL). The organic extracts were dried over Na2SO4,
filtered and the solvent was evaporated. The residue was purified by column chromatography on
silica gel (eluent: CH2Cl2/MeOH – 10:1) to yield the product as a brown solid (0.045 g; 66 %).
1
H NMR (500 MHz, CDCl3) δ 7.94 (s, 2H); 7.80 (d, J = 2.21 Hz, 1H); 7.72 (d, J = 8.53 Hz, 1H); 7.39
(d, J = 8.59 Hz, 1H); 6.95 (d, J = 1.64 Hz, 1H); 3.94 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 149.3, 149.0, 147.5, 146.7, 137.7, 128.9, 124.1, 119.2, 116.0, 108.3,
39.4.
HRMS (APCI): calcd. for C11H10N3O [M+H]+
= 200.0818; found [M+H]+
= 200.0817.
180
Preparative Example 13
To a mixture of the product from Preparative Example 10 (1.62 g; 7.16 mmol), phenylboronic acid
(1.13 g; 9.26 mmol), triethylamine (10 mL; 71.8 mmol) and PdCl2.dppf (0.160 g; 0.22 mmol) were
added under N2 1,2-dimethoxyethane (12 mL) and water (3 mL). The reaction mixture was refluxed
under N2 for 20 h. Then it was cooled to 25 °C, diluted with EtOAc (70 mL), poured into brine (90
mL) and extracted with EtOAc (3×70 mL). The organic extracts were dried over Na2SO4, filtered and
the solvent was evaporated. The residue was purified by column chromatography on silica gel (eluent:
hexane/EtOAc – 15:1) to yield the product as a pale yellow solid (1.61 g; 84 %).
1
H NMR (500 MHz, CDCl3) δ 8.01-7.96 (m, 2H); 7.78 (dd, J = 0.93 Hz, 8.61 Hz, 1H); 7.61 (d, J =
8.62 Hz, 1H); 7.49-7.43 (m, 2H); 7.41-7.35 (m, 1H); 7.20 (d, J = 0.81 Hz, 1H); 0.39-0.39 (m, 9H).
13
C NMR (126 MHz, CDCl3) δ 169.5, 154.3, 150.3, 148.6, 140.3, 128.9, 128.6, 127.5, 118.7, 117.6,
116.9, -1.8.
HRMS (APCI): calcd. for C16H18NOSi [M+H]+
= 268.1152; found [M+H]+
= 268.1153.
Preparative Example 14
To a stirred solution of the product from Preparative Example 13 (1.61 g, 6.02 mmol) in anhydrous
CH2Cl2 (20 ml) was added MCPBA (1.88 g, 10.9 mmol) and the resulting mixture was stirred under
N2 at 25 °C for 72 h. Then it was poured into saturated aqueous solution of NaHCO3 (110 mL) and
extracted with CH2Cl2 (3×60 mL). The organic extracts were washed with brine (50 mL), dried over
Na2SO4, filtered and the solvent was evaporated. The residue was purified by column
chromatography on silica gel (eluent: CH2Cl2/MeOH – 12:1) to yield the product as a pale yellow
solid (1.60 g; 94 %).
181
1
H NMR (500 MHz, CDCl3) δ 7.86-7.80 (m, 2H); 7.50-7.39 (m, 5H); 7.30 (d, J = 8.58 Hz, 1H); 0.38-
0.36 (m, 9H).
13
C NMR (126 MHz, CDCl3) δ 169.0, 153.2, 144.7, 139.6, 133.1, 129.8, 129.4, 128.5, 122.2, 112.0,
110.4, -1.9.
HRMS (APCI): calcd. for C16H17NO2Si [2M+H]+
= 567.213; found [2M+H]+
= 567.2134.
Preparative Example 15
To a stirred solution of the product from Preparative Example 14 (0.575 g, 2.03 mmol) in CHCl3 (10
ml) was added POCl3 (3.4 mL; 36.5 mmol) and the resulting mixture was refluxed under N2 for 1 h.
Then, the CHCl3 and POCl3 were evaporated under reduced pressure. The dark oily residue was
diluted with CH2Cl2 (50 mL), poured into saturated aqueous solution of NaHCO3 (200 mL) and
extracted with CH2Cl2 (3×70 mL). The organic extracts were washed with water (50 mL), brine (80
mL), dried over Na2SO4, filtered, and the solvent was evaporated. The residue was purified by
column chromatography on silica gel (eluent: hexane/CH2Cl2 – 1:1) to yield the product as a colorless
wax (0.339 g; 55 %).
1
H NMR (500 MHz, CDCl3) δ 7.99-7.94 (m, 2H); 7.63 (s, 1H); 7.49-7.44 (m, 2H); 7.42-7.37 (m, 1H);
7.20 (s, 1H); 0.41-0.38 (m, 9H).
13
C NMR (126 MHz, CDCl3) δ 170.6, 155.4, 149.8, 146.8, 139.3, 129.1, 129.0, 127.4, 126.5, 118.1,
117.3, -1.8.
HRMS (APCI): calcd. for C16H16ClNOSi [M+H]+
= 302.0762; found [M+H]+
= 302.0764.
182
Preparative Example 16
To a solution of the product from Preparative Example 15 (1.11 g; 3.68 mmol) in methanol (10 mL)
was added KF (0.646 g; 11.1 mmol) and the resulting mixture was stirred under N2 at 60 °C for 20 h.
Then it was cooled to 25 °C, poured into aqueous solution of HCl (0.1M; 40 mL) and extracted with
EtOAc (3×30 mL). The organic extracts were washed with brine (30 mL), dried over Na2SO4, filtered
and the solvent was evaporated. The residue was purified by column chromatography on silica gel
(eluent: CH2Cl2/ MeOH – 20:1) to yield the product as a white solid (0.796 g; 94 %).
1
H NMR (500 MHz, CDCl3) δ 7.99-7.95 (m, 2H); 7.90 (d, J = 2.18 Hz, 1H); 7.68 (s, 1H); 7.51-7.44
(m, 2H); 7.43-7.39 (m, 1H); 7.09 (d, J = 2.15 Hz, 1H).
13
C NMR (126 MHz, CDCl3) δ 155.8, 150.2, 148.8, 144.0, 138.8, 129.4, 129.1, 127.5, 127.0, 117.6,
109.2.
HRMS (APCI): calcd. for C13H9ClNO [M+H]+
= 230.0367; found [M+H]+
= 230.0365.
Preparative Example 17A
To a solution of the product from Preparative Example 16 (0.103 g; 0.45 mmol), (R)-BINAP (0.016
g; 0.026 mmol), Pd(dba)2 (0.017 g; 0.030 mmol) and t-BuOK (0.078 g; 0.69 mmol) in anhydrous
toluene (3 mL) was added 3-picolylamine (0.050 mL; 0.49 mmol) and the resulting mixture was
stirred under N2 at 80 °C for 17 h. Then it was cooled to 25 °C, diluted with EtOAc (10 mL), poured
into water (25 mL) and extracted with EtOAc (3×10 mL). The organic extracts were washed with
183
brine (15 mL), dried over MgSO4, filtered, and the solvent was evaporated. The residue was purified
by column chromatography on silica gel (eluent: CH2Cl2/ MeOH – 10:1) to yield the product as an
orange foam (0.056 g; 42 %).
1
H NMR (500 MHz, CDCl3) δ 8.62 (d, J = 59.05 Hz, 2H); 7.84 (d, J = 7.29 Hz, 2H); 7.75-7.66 (m,
2H); 7.44-7.25 (m, 4H); 6.96 (d, J = 2.02 Hz, 1H); 6.81 (s, 1H); 5.09 (brs, 1H); 4.63 (d, J = 5.71 Hz,
2H).
13
C NMR (126 MHz, CDCl3) δ 156.5, 149.5, 149.3, 147.5, 146.7, 140.8, 139.4, 136.8, 135.2, 133.7,
128.8, 128.6, 127.5, 124.0, 109.1, 99.6, 45.0.
HRMS (APCI): calcd. for C19H16N3O [M+H]+
= 302.1288; found [M+H]+
= 302.1285.
Preparative Example 17B
To a solution of the product from Preparative Example 16 (0.207 g; 0.90 mmol), (R)-BINAP (0.035
g; 0.056 mmol), Pd(dba)2 (0.050 g; 0.087 mmol) and t-BuOK (0.146 g; 1.30 mmol) in anhydrous
toluene (3 mL) was added benzylamine (0.120 mL; 1.10 mmol) and the resulting mixture was stirred
under N2 at 80 °C for 17 h. Then it was cooled to 25 °C, diluted with EtOAc (10 mL), poured into
water (25 mL) and extracted with EtOAc (3×10 mL). The organic extracts were washed with brine
(15 mL), dried over MgSO4, filtered and the solvent was evaporated. The residue was purified by
column chromatography on silica gel (eluent: CH2Cl2/ MeOH – 15:1). So obtained solid was further
purified by preparative TLC (eluent: CH2Cl2/MeOH – 20:1) to yield the product as a brownish foam
(0.233 g; 86 %).
1
H NMR (500 MHz, CDCl3) δ 7.90-7.85 (m, 2H); 7.69 (d, J = 2.19 Hz, 1H); 7.44-7.28 (m, 8H); 6.95
(d, J = 2.19 Hz, 1H); 6.85 (s, 1H); 4.96 (t, J = 5.13 Hz, 1H); 4.59 (d, J = 5.63 Hz, 2H).
184
13
C NMR (126 MHz, CDCl3) δ 156.6, 147.2, 146.7, 141.2, 139.7, 138.1, 136.9, 129.1, 128.7, 128.4,
128.0, 127.7, 127.6, 109.2, 99.5, 47.4.
HRMS (APCI): calcd. for C20H17N2O [M+H]+
= 301.1335; found [M+H]+
= 301.1335.
Preparative Example 17C
To a freshly prepared solution of xantphos (0.012 g; 0.021 mmol) and Pd3(dba)2 (0.022 g; 0.024
mmol) in anhydrous 1,2-dimethoxyethane (2 mL) were added the product from Preparative Example
16 (0.049 g; 0.22 mmol), t-BuOK (0.052 g; 0.46 mmol) and 4-morpholinoaniline (0.048 g; 0.27
mmol) and the resulting mixture was stirred under N2 at 100 °C for 16 h. Then it was cooled to 25 °C,
diluted with EtOAc (15 mL), poured into brine (25 mL) and extracted with EtOAc (3×15 mL). The
organic extracts were dried over MgSO4, filtered, and the solvent was evaporated. The residue was
purified by column chromatography on silica gel (eluent: CH2Cl2/MeOH – 15:1). So obtained oil was
further purified by preparative TLC (eluent: CH2Cl2/MeOH – 20:1) to yield the product as a brownish
solid (0.018 g; 22 %).
1
H NMR (500 MHz, CDCl3) δ 7.86-7.83 (m, 2H); 7.74 (d, J = 2.20 Hz, 1H); 7.42-7.37 (m, 2H); 7.36-
7.31 (m, 1H); 7.26-7.21 (m, 2H); 7.15 (s, 1H); 6.99 (d, J = 2.20 Hz, 1H); 6.97-6.93 (m, 2H); 6.34
(brs, 1H); 3.90-3.85 (m, 4H); 3.20-3.14 (m, 4H).
13
C NMR (126 MHz, CDCl3) δ 156.3, 149.1, 147.5, 147.2, 140.9, 137.9, 137.0 131.4, 128.7, 128.5,
127.6, 124.7, 116.9, 109.2, 100.3, 67.1, 49.8.
HRMS (APCI): calcd. for C23H22N3O2 [M+H]+
= 372.1707; found [M+H]+
= 372.1707.
185
Preparative Examples 17D-17I
By essentially same procedure set forth in Preparative Example 17C, using proper amines instead of
4-morpholinoaniline, the compounds given below were prepared.
Preparative Example 17D
Brown semi – solid.
1
H NMR (500 MHz, CDCl3) δ 7.87-7.83 (m, 2H); 7.74 (d, J = 2.21 Hz,; 1H); 7.42-7.37 (m, 2H);
7.35-7.31 (m, 1H); 7.25 (s, 1H); 6.98 (d, J = 2.20 Hz, 1H); 6.95 (d, J = 8.36 Hz, 1H); 6.87 (dd, J =
2.33 Hz, 8.36 Hz, 1H); 6.83 (d, J = 2.33 Hz, 1H); 6.38 (brs, 1H); 3.84 (s, 3H); 3.20-3.02 (m, 4H);
2.69-2.58 (m, 4H); 2.36 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 156.4, 153.3, 147.6, 147.4, 140.9, 138.8, 137.4, 137.0, 134.4, 128.7,
128.5, 127.5, 119.2, 115.3, 109.3, 107.1, 100.6, 55.9, 55.6, 50.9, 46.3.
HRMS (APCI): calcd. for C25H27N4O2 [M+H]+
= 415.2129; found [M+H]+
= 415.2130.
Preparative Example 17E
Orange solid.
186
1
H NMR (500 MHz, DMSO-d6) δ 8.36 (d, J = 2.26 Hz, 1H); 8.01-7.97 (m, 2H); 7.93 (s, 1H); 7.61-
7.50 (m, 3H); 7.48-7.43 (m, 1H); 7.38 (s, 1H); 7.21 (d, J = 2.25 Hz, 1H); 2.33 (s, 3H).
13
C NMR (126 MHz, DMSO-d6) δ 168.5, 153.3, 150.6, 146.5, 143.5, 138.8, 130.8, 128.7, 128.7,
126.6, 121.5, 110.7, 108.0, 53.7, 20.0.
HRMS (APCI): calcd. for C17H14N3OS [M+H]+
= 308.0852; found [M+H]+
= 308.0850.
Preparative Example 17F
Pale yellow solid.
1
H NMR (500 MHz, CDCl3) δ 7.89-7.86 (m, 2H); 7.76 (d, J = 2.21 Hz, 1H); 7.44-7.29 (m, 8H); 7.18-
7.14 (m, 1H); 7.00 (d, J = 2.22 Hz, 1H); 6.48 (brs, 1H).
13
C NMR (126 MHz, CDCl3) δ 156.4, 147.7, 147.6, 140.9, 139.5, 137.2, 136.4, 129.9, 128.8, 128.5,
127.5, 124.5, 121.8, 109.3, 101.0.
HRMS (APCI): calcd. for C19H15N2O [M+H]+
= 287.1179; found [M+H]+
= 287.1178.
Preparative Example 17G
Orange semi – solid.
187
1
H NMR (500 MHz, CDCl3) δ 7.95-7.90 (m, 2H); 7.69 (d, J = 2.17 Hz, 1H); 7.46-7.40 (m, 2H); 7.38-
7.33 (m, 1H); 6.94 (d, J = 2.17 Hz, 1H); 6.81 (s, 1H); 5.36-5.23 (m, 1H); 3.43 (dd, J = 5.19 Hz, 11.56
Hz, 2H); 2.69-2.62 (m, 2H); 2.30 (s, 6H).
13
C NMR (126 MHz, CDCl3) δ 156.4, 147.1, 146.6, 141.3, 140.1, 137.0, 128.7, 128.4, 127.6, 109.0,
99.5, 57.8, 45.3, 40.2.
HRMS (APCI): calcd. for C17H20N3O [M+H]+
= 282.1601; found [M+H]+
= 282.1602.
Preparative Example 17H
Pale yellow solid foam.
1
H NMR (500 MHz, CDCl3) δ 8.41 (s, 1H); 8.03-7.98 (m, 2H); 7.76 (d, J = 2.21 Hz, 1H); 7.46-7.42
(m, 2H); 7.39-7.35 (m, 1H); 7.15 (brs, 1H); 7.02 (d, J = 2.21 Hz, 1H); 1.57 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 156.5, 152.0, 148.1, 147.3, 140.3, 136.9, 131.4, 128.8, 128.8, 127.7,
109.4, 105.5, 82.4, 28.5.
HRMS (APCI): calcd. for C18H19N2O3 [M+H]+
= 311.1390; found [M+H]+
= 311.1394.
Preparative Example 17I
Orange wax.
188
1
H NMR (500 MHz, CDCl3) δ 7.93-7.89 (m, 2H); 7.73 (d, J = 2.22 Hz, 1H); 7.47-7.41 (m, 2H), 7.40-
7.35 (m, 1H); 6.99 (d, J = 1.60 Hz, 1H); 6.92 (s, 1H); 3.67-3.60 (m, 8H); 1.48 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 156.3, 154.9, 147.2, 142.3, 138.0, 128.8, 128.7, 127.6, 108.9, 102.9,
80.4, 48.1, 28.7.
HRMS (APCI): calcd. for C22H26N3O3 [M+H]+
= 380.1969; found [M+H]+
= 380.1970.
Preparative Example 18A
To a stirred solution of the product from Preparative Example 17H (0.031 g; 0.10 mmol) in ethanol
(2 mL) was added aqueous solution of HCl (3M; 0.7 mL; 2.1 mmol) and the resulting mixture was
stirred under N2 at 60 °C for 18 h. Then the ethanol and HCl were evaporated and the oily residue was
treated with CH2Cl2 (2 mL), MeOH (1mL) and Na2CO3 (200 mg) and the mixture was stirred at 25
°C. After 20 min., the solvents were evaporated and the solid residue was purified by preparative
TLC (eluent: CH2Cl2/7N NH3 in MeOH – 50:1) to yield the product as a white solid (0.019 g; 88 %).
1
H NMR (500 MHz, CDCl3) δ 7.92-7.87 (m, 2H); 7.72 (d, J = 2.20 Hz, 1H); 7.45-7.40 (m, 2H); 7.38-
7.33 (m, 1H); 6.95 (d, J = 2.20 Hz, 1H); 6.92 (s, 1H); 4.49 (brs, 2H).
13
C NMR (126 MHz, CDCl3) δ 156.1, 147.8, 147.5, 140.6, 138.5, 137.0, 128.8, 128.5, 127.5, 109.0,
103.2.
HRMS (APCI): calcd. for C13H11N2O [M+H]+
= 211.0866; found [M+H]+
= 211.0866.
Preparative Example 18B
By essentially same procedure set forth in Preparative Example 18A, using the product from
Preparative Example 17I, the compound given below was prepared.
189
Pale yellow semi – solid.
1
H NMR (500 MHz, CDCl3) δ 7.93-7.89 (m, 2H); 7.71 (d, J = 2.21 Hz, 1H); 7.46-7.40 (m, 2H); 7.38-
7.34 (m, 1H); 6.96 (d, J = 2.22 Hz, 1H); 6.92 (s, 1H); 3.65-3.60 (m, 4H); 3.13-3.04 (m, 4H).
13
C NMR (126 MHz, CDCl3) δ 156.4, 148.4, 146.9, 142.7, 141.0, 138.2, 128.8, 128.5, 127.6, 108.9,
102.8, 49.2, 46.0.
HRMS (APCI): calcd. for C17H18N3O [M+H]+
= 280.1444; found [M+H]+
= 280.1443.
Preparative Example 19A
To a freshly prepared solution of (S)-1-[(RP)-2-(dicyclohexylphosphino)ferrocenyl]ethyldi-tertbutylphosphine
(0.012 g; 0.022 mmol) and Pd(OAc)2 (0.008 g; 0.037 mmol) in anhydrous
1,2-dimethoxyethane (2 mL) were added the product from Preparative Example 16 (0.049 g; 0.21
mmol), t-BuOK (0.038 g; 0.34 mmol) and 4-amino-N,N-dimethyl-benzenesulfonamide (0.051 g; 0.25
mmol) and the resulting mixture was stirred under N2 at 100 °C for 16 h. Then it was diluted with
EtOAc (15 mL), poured into brine (25 mL) and extracted with EtOAc (3×15 mL). The organic
extracts were dried over MgSO4, filtered and the solvent was evaporated. The residue was purified by
column chromatography on silica gel (eluent: CH2Cl2/MeOH – 15:1). So obtained oil was further
purified by preparative TLC (eluent: CH2Cl2/MeOH – 20:1) to yield the yellow semi – solid product
(0.036 g; 43 %).
190
1
H NMR (500 MHz, CDCl3) δ 7.90-7.86 (m, 2H); 7.78-7.71 (m, 3H); 7.49 (s, 1H); 7.45-7.32 (m, 5H);
7.03 (brs, 1H); 7.00 (d, J = 2.15 Hz, 1H); 2.72 (s, 6H).
13
C NMR (126 MHz, CDCl3) δ 156.4, 148.3, 148.3, 144.5, 140.3, 137.6, 134.1, 129.8, 129.2, 128.9,
128.9, 127.5, 118.8, 109.2, 103.2, 38.2.
HRMS (APCI): calcd. for C21H20N3O3S [M+H]+
= 394.1220; found [M+H]+
= 394.1217.
Preparative Example 19B
By essentially same procedure set forth in Preparative Example 19A, using cyclohexylamine instead
of 4-amino-N,N-dimethyl-benzenesulfonamide, the compound given below was prepared.
Yellow wax.
1
H NMR (500 MHz, CDCl3) δ 7.92-7.87 (m, 2H); 7.66 (d, J = 2.17 Hz, 1H); 7.45-7.39 (m, 2H); 7.38-
7.32 (m, 1H); 6.96 (d, J = 2.17 Hz, 1H); 6.81 (s, 1H); 4.70 (brs, 1H); 3.65-3.52 (m, 1H); 2.16-2.05
(m, 2H); 1.84-1.75 (m, 2H); 1.71-1.62 (m, 1H); 1.48-1.36 (m, 2H); 1.35-1.19 (m, 4H).
13
C NMR (126 MHz, CDCl3) δ 156.0, 147.1, 146.0, 140.8, 139.4, 136.7, 128.7, 128.5, 127.6, 108.8,
99.5, 51.5, 33.4, 25.8, 25.0.
HRMS (APCI): calcd. for C19H21N2O [M+H]+
= 293.1648; found [M+H]+
= 293.1647.
191
Preparative Example 19C
Degassed 1,2-dimethoxyethane (2.5 mL) was added under N2 into a 10 mL round bottom flask
containing Pd2(dba)3 (15.9 mg, 0.017 mmol) and SPhos (7.1 mg, 0.017 mmol). After 5 min, the
product from Preparative Example 16 (40 mg, 0.17 mmol), 5-methylisoxazol-3-amine (20 mg, 0.212
mmol) and Cs2CO3 (125 mg, 0.383 mmol) were added. The mixture was stirred at 80 °C under N2 for
24 h, then the temperature was elevated to 120 °C and the mixture was stirred for additional 24 h.
H2O (15 mL) was added and the mixture was extracted with EtOAc (3×25 mL). The organic phase
was dried over Na2SO4, filtered, and then the solvent was evaporated. The residue was purified by
column chromatography (CH2Cl2/EtOAc; 2:1) and then by preparative TLC (CH2Cl2/EtOAc; 2:1).
The product was obtained as a colorless wax (14 mg, 30 % yield).
1
H NMR (300 MHz, CDCl3) δ 8.21 (s, 1H), 8.03 (d, J = 7.9 Hz, 2H), 7.77 (d, J = 1.7 Hz, 1H), 7.54 –
7.34 (m, 3H), 7.09 – 6.96 (m, 2H), 5.92 (s, 1H), 2.42 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 169.4, 159.5, 156.4, 147.6, 146.7, 140.0, 136.4, 132.8, 128.5, 128.4,
127.3, 109.0, 104.3, 94.8, 12.4.
HRMS (APCI): calcd. for C17H13N3O2 [M+H]+
= 292.1081; found [M+H]+
= 292.1082.
Preparative Example 19D
By essentially same procedure set forth in Preparative Example 19C, using isoxazol-3-amine instead
of 5-methylisoxazol-3-amine, the compound given below was prepared.
192
Colorless wax.
1
H NMR (500 MHz, CDCl3) δ 8.30 (d, J = 1.7 Hz, 1H), 8.28 (s, 1H), 8.07 – 8.01 (m, 2H), 7.78 (d, J =
2.2 Hz, 1H), 7.52 – 7.44 (m, 2H), 7.44 – 7.37 (m, 1H), 7.14 (s, 1H), 7.04 (d, J = 2.2 Hz, 1H), 6.28 (d,
J = 1.7 Hz, 1H).
13
C NMR (126 MHz, CDCl3) δ 158.2, 157.6, 155.8, 146.8, 146.1, 139.3, 135.6, 131.8, 127.7, 127.7,
126.6, 108.3, 103.7, 97.0.
HRMS (APCI): calcd. for C16H11N3O2 [M+H]+
= 278.0924; found [M+H]+
= 278.0926.
Preparative Example 19E
By essentially same procedure set forth in Preparative Example 19C, using pyridin-3-amine instead of
5-methylisoxazol-3-amine, the compound given below was prepared.
Colorless wax.
1
H NMR (300 MHz, CDCl3) δ 8.65 (s, 1H), 8.42 (d, J = 4.2 Hz, 1H), 7.93 – 7.84 (m, 2H), 7.78 (d, J =
2.2 Hz, 1H), 7.71 – 7.62 (m, 1H), 7.46 – 7.31 (m, 5H), 7.02 (d, J = 2.2 Hz, 1H), 6.66 (s, 1H).
13
C NMR (126 MHz, CDCl3) δ 156.4, 148.0, 147.9, 145.3, 143.5, 140.4, 137.1, 136.4, 135.5, 128.7,
128.6, 128.2, 127.4, 124.1, 109.2, 101.1.
193
HRMS (APCI): calcd. for C18H13N3O [M+H]+
= 288.1131; found [M+H]+
= 288.1132.
Preparative Example 19E
By essentially same procedure set forth in Preparative Example 19C, using 1-methyl-1H-pyrazol-4amine
instead of 5-methylisoxazol-3-amine, the compound given below was prepared.
Yellow wax.
1
H NMR (500 MHz, CDCl3) δ 7.89 – 7.84 (m, 2H), 7.75 (d, J = 2.2 Hz, 1H), 7.55 (s, 1H), 7.46 – 7.39
(m, 3H), 7.38 – 7.34 (m, 1H), 6.99 (d, J = 2.2 Hz, 1H), 6.98 (s, 1H), 6.00 (s, 1H), 3.94 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 156.4, 147.5, 146.9, 140.8, 138.8, 136.6, 136.1, 128.6, 128.4, 127.4,
125.9, 121.4, 109.0, 100.0, 39.7.
HRMS (APCI): calcd. for C17H14N4O [M+H]+
= 291.1240; found [M+H]+
= 291.1237.
Preparative Example 20
To a mixture of the product from Preparative Example 3 (4.34 g; 17.0 mmol) and K2CO3 (7.07 g;
51.1 mmol) in N,N-dimethylformamide (30 mL) was added under N2 crotyl bromide (2.6 mL; 25.3
mmol). The resulting reaction mixture was stirred under N2 at 60 °C for 2 h. Then the solvent was
evaporated and the residue was suspended between H2O (120 mL) and CH2Cl2 (90 mL). The water
phase was extracted with CH2Cl2 (3×100 mL). The organic extracts were washed with brine (100
mL), dried over Na2SO4, filtered and the solvent was evaporated. The residue was purified by column
194
chromatography on silica gel (eluent: hexane/EtOAc – 1:1). So obtained pale yellow solid was
washed with cold pentane (3 × 25 mL) to yield the white crystalline product (4.24 g; 81 %).
1
H NMR (500 MHz, CDCl3) δ 7.19-7.11 (m, 1H); 6.93 (d, J = 8.46 Hz, 1H); 5.93-5.83 (m, 1H); 5.71-
5.61 (m, 1H); 4.52 (d, J = 5.49 Hz, 2H); 1.75 (d, J = 6.42 Hz, 3H).
13
C NMR (126 MHz, CDCl3) δ 154.4, 141.6, 131.7, 124.7, 123.7, 121.3, 110.0, 70.8, 18.1.
HRMS (APCI): calcd. for C9H10ClINO [M+H]+
= 309.9490; found [M+H]+
= 309.9488.
Preparative Example 21
The mixture of the product from Preparative Example 20 (4.24 g; 13.7 mmol), K2CO3 (4.75 g;
34.4 mmol), HCOONa (0.934 g; 13.7 mmol); tetrabutylammonium chloride (4.21 g; 15.1 mmol) and
Pd(OAc)2 (0.185 g; 0.82 mmol) in N,N-dimethylformamide (30 mL) was stirred under N2 at 80 °C for
3 h. Then the solvent was evaporated and the residue was suspended between H2O (180 mL) and
CH2Cl2 (100 mL). The aqueous phase was extracted with CH2Cl2 (3×100 mL). The organic extracts
were washed with brine (100 mL), dried over Na2SO4, filtered and the solvent was evaporated. The
residue was purified by column chromatography on silica gel (eluent: hexane/EtOAc – 30:1) to yield
the product as a pale yellow solid (0.774 g; 31 %).
1
H NMR (500 MHz, CDCl3) δ 7.66-7.62 (m, 2H); 7.19 (d, J = 8.55 Hz, 1H); 2.76 (dq, J = 1.23 Hz,
7.51 Hz, 2H); 1.33 (t, J = 7.52 Hz, 3H).
13
C NMR (126 MHz, CDCl3) δ 147.8, 147.4, 146.6, 146.2, 123.6, 120.9, 119.2, 16.0, 13.5.
HRMS (APCI): calcd. for C9H9ClNO [M+H]+
= 182.0367; found [M+H]+
= 182.0365.
Preparative Example 22
195
To a freshly prepared solution of (S)-1-[(RP)-2-(dicyclohexylphosphino)ferrocenyl]ethyldi-tertbutylphosphine
(0.022 g; 0.039 mmol) and Pd(OAc)2 (0.009 g; 0.039 mmol) in anhydrous
1,2-dimethoxyethane (2 mL) were added the product from Preparative Example 21 (0.087 g; 0.48
mmol), t-BuONa (0.064 g; 0.66 mmol) and N,N-dimethylethylenediamine (0.063 mL; 0.57 mmol)
and the resulting mixture was stirred under N2 at 100 °C for 15 h. Then it was cooled to 25 °C, diluted
with EtOAc (15 mL), poured into brine (25 mL) and extracted with EtOAc (3×15 mL). The organic
extracts were dried over MgSO4, filtered and the solvent was evaporated. The residue was purified by
column chromatography on silica gel (eluent: CH2Cl2/7N NH3 in MeOH – 15:1) to yield the product
as an orange oil (0.081 g; 73 %).
1
H NMR (500 MHz, CDCl3) δ 7.46-7.41 (m, 2H); 6.32 (d, J = 8.87 Hz, 1H); 5.00-4.89 (m, 1H); 3.42
(dd, J = 5.69 Hz, 11.45 Hz, 2H); 2.68 (dq, J = 1.14 Hz, 7.51 Hz, 2H ); 2.58 (t, J = 6.06 Hz, 2H); 2.28
(s, 6H); 1.31 (t, J = 7.51 Hz, 3H).
13
C NMR (126 MHz, CDCl3) δ 156.7, 145.3, 143.7, 143.2, 123.0, 120.4, 104.5, 58.6, 45.5, 40.2, 16.2,
13.5.
HRMS (APCI): calcd. for C13H20N3O [M+H]+
= 234.1601; found [M+H]+
= 234.1601.
Preparative Example 23
To a mixture of the product from Preparative Example 21 (0.053 g; 0.29 mmol), 1-methylpyrazole-4boronic
acid pinacol ester (0.074 g; 0.36 mmol), K3PO4 (0.262 g; 1.23 mmol) and PdCl2.dppf (0.013
g; 0.017 mmol) were added under N2 1,2-dimethoxyethane (2 mL) and water (0.5 mL). The reaction
mixture was refluxed for 19 h. Then it was cooled to 25 °C, diluted with EtOAc (15 mL), poured into
brine (25 mL) and extracted with EtOAc (3×15 mL). The organic extracts were dried over Na2SO4,
filtered and the solvent was evaporated. The residue was purified by column chromatography on
silica gel (eluent: CH2Cl2/MeOH – 10:1) to yield the product as a brown semi – solid (0.055 g; 83 %).
1
H NMR (500 MHz, CDCl3) δ 8.04-7.91 (m, 2H); 7.64 (d, J = 8.49 Hz, 1H); 7.58 (s, 1H); 7.36 (d, J =
8.51 Hz, 1H); 3.94 (s, 3H); 2.81 (q, J = 7.35 Hz, 2H); 1.37 (t, J = 7.49 Hz, 3H).
196
13
C NMR (126 MHz, CDCl3) δ 148.1, 147.5, 147.1, 145.1, 137.7, 129.0, 124.3, 123.7, 118.9, 115.7,
39.3, 16.2, 13.5.
HRMS (APCI): calcd. for C13H14N3O [M+H]+
= 228.1131; found [M+H]+
= 228.1133.
Preparative Example 24
To a mixture of the product from Preparative Example 21 (1.43 g; 7.85 mmol), phenylboronic acid
(1.24 g; 10.2 mmol), K3PO4 (6.86 g; 32.3 mmol) and PdCl2.dppf (0.530 g; 0.72 mmol) were added
under N2 1,2-dimethoxyethane (50 mL) and water (10 mL). The reaction mixture was refluxed under
N2 for 18 h. Then it was cooled to 25 °C, diluted with EtOAc (70 mL), poured into brine (100 mL)
and extracted with EtOAc (3×70 mL). The organic extracts were dried over Na2SO4, filtered and the
solvent was evaporated. The residue was purified by column chromatography on silica gel (eluent:
hexane/EtOAc – 15:1) to yield the product as a pale yellow solid (1.59 g; 91 %).
1
H NMR (500 MHz, CDCl3) δ 8.04 (d, J = 7.51 Hz, 2H); 7.73 (d, J = 8.54 Hz, 1H); 7.63 (d, J = 8.51
Hz, 2H); 7.51-7.43 (m, 2H); 7.42-7.35 (m, 1H); 2.86 (q, J = 7.48 Hz, 2H); 1.41 (t, J = 7.37 Hz, 3H).
13
C NMR (126 MHz, CDCl3) δ 153.8, 147.9, 147.7, 145.2, 140.4, 128.9, 128.6, 127.4, 124.1, 118.7,
116.6, 16.2, 13.6.
HRMS (APCI): calcd. for C15H14NO [M+H]+
= 224.107; found [M+H]+
= 224.1068.
Preparative Example 25
To a stirred solution of the product from Preparative Example 24 (1.59 g, 7.12 mmol) in anhydrous
CH2Cl2 (20 ml) was added MCPBA (2.22 g, 12.9 mmol) and the resulting mixture was stirred under
197
N2 at 25 °C for 72 h. Then it was poured into saturated aqueous solution of NaHCO3 (150 mL) and
extracted with CH2Cl2 (3×80 mL). The organic extracts were washed with brine (80 mL), dried over
Na2SO4, filtered, and the solvent was evaporated. The residue was purified by column
chromatography on silica gel (eluent: CH2Cl2/EtOAc – 5:1) to yield the product as a pale yellow solid
(0.545 g; 32 %).
1
H NMR (500 MHz, CDCl3) δ 7.83-7.76 (m, 2H); 7.50-7.35 (m, 5H); 7.27 (d, J = 8.60 Hz, 1H); 3.07
(q, J = 7.37 Hz, 2H); 1.34 (t, J = 7.41, 3H).
13
C NMR (126 MHz, CDCl3) δ 151.0, 145.4, 144.0, 137.7, 133.1, 129.9, 129.3, 128.4, 122.2, 110.4,
18.1, 14.9.
HRMS (APCI): calcd. for C15H14NO2 [M+H]+
= 240.1019; found [M+H]+
= 240.1017.
Preparative Example 26
To a stirred solution of the product from Preparative Example 25 (0.713 g, 2.98 mmol) in CHCl3 (15
ml) was added POCl3 (6 mL, 64.4 mmol) and the resulting mixture was refluxed under N2 for 1 hr.
Then, the CHCl3 and POCl3 were evaporated under reduced pressure. The dark oily residue was
diluted with CH2Cl2 (50 mL), poured into saturated aqueous solution of NaHCO3 (200 mL) and
extracted with CH2Cl2 (3×50 mL). The organic extracts were washed with water (50 mL), with brine
(80 mL), dried over Na2SO4, filtered and the solvent was evaporated. The residue was purified by
column chromatography on silica gel (eluent: hexane/CH2Cl2 – 2:1) to yield the product as a white
solid (0.282 g; 37 %).
1
H NMR (500 MHz, CDCl3) δ 8.07-7.98 (m, 2H); 7.69-7.64 (m, 2H); 7.52-7.36 (m, 3H); 2.84 (dd, J =
6.89 Hz, 14.36 Hz, 2H); 1.45-1.39 (m, 3H).
13
C NMR (126 MHz, CDCl3) δ 155.0, 149.1, 145.8, 144.2, 139.3, 129.1, 129.0, 127.4, 126.3, 124.8,
117.1, 16.3, 13.5.
198
HRMS (APCI): calcd. for C15H13ClNO [M+H]+
= 258.0680; found [M+H]+
= 258.0678.
Preparative Example 27A
To a solution of the product from Preparative Example 26 (0.202 g; 0.78 mmol), (R)-BINAP (0.032
g; 0.052 mmol), Pd2(dba)3 (0.044 g; 0.048 mmol) and t-BuOK (0.146 g; 1.30 mmol) in anhydrous
toluene (4 mL) was added benzylamine (0.100 mL; 0.92 mmol) and the resulting mixture was stirred
under N2 at 80 °C for 17 h. Then it was cooled to 25 °C, diluted with EtOAc (10 mL), poured into
water (25 mL) and extracted with EtOAc (3×10 mL). The organic extracts were washed with brine
(15 mL), dried over MgSO4, filtered and the solvent was evaporated. The residue was purified by
column chromatography on silica gel (eluent: CH2Cl2/ MeOH – 20:1) to yield a brownish solid (0.168
g; 65 %).
1
H NMR (500 MHz, CDCl3) δ 7.94-7.89 (m, 2H); 7.49-7.46 (m, 1H); 7.43-7.27 (m, 8H); 6.85 (s, 1H);
4.89 (brs, 1H); 4.58 (d, J = 5.67 Hz, 2H); 2.83 (dd, J = 1.08 Hz, 7.49 Hz, 2H); 1.38 (t, J = 7.51 Hz,
3H).
13
C NMR (126 MHz, CDCl3) δ 155.7, 146.6, 143.1, 141.4, 139.6, 138.3, 137.2, 129.1, 128.7, 128.3,
128.0, 127.7, 127.6, 124.6, 99.3, 47.4, 16.4, 13.6.
HRMS (APCI): calcd. for C22H21N2O [M+H]+
= 329.1648; found [M+H]+
= 329.1650.
Preparative Example 27B
By essentially same procedure set forth in Preparative Example 27A, using N,Ndimethylethylenediamine
instead of benzylamine, the compound given below was prepared.
199
Yellow wax.
1
H NMR (500 MHz, CDCl3) δ 7.99-7.95 (m, 2H); 7.48-7.46 (m, 1H); 7.45-7.40 (m, 2H); 7.37-7.32
(m, 1H); 6.81 (s, 1H); 5.19-5.11 (m, 1H); 3.41 (dd, J = 5.16 Hz, 11.70 Hz, 2H); 2.82 (qd, J = 1.20 Hz,
7.50 Hz, 2H); 2.66-2.59 (m, 2H); 2.28 (s, 6H); 1.38 (t, J = 7.51 Hz, 3H).
13
C NMR (126 MHz, CDCl3) δ 155.6, 146.7, 142.9, 141.6, 139.9, 137.3, 128.7, 128.2, 127.6, 124.5,
99.3, 57.9, 45.4, 40.3, 16.4, 13.6.
HRMS (APCI): calcd. for C19H24N3O [M+H]+
= 310.1914; found [M+H]+
= 310.1915.
Preparative Example 28
To a stirred solution of the product from Preparative Example 27A (0.052 g, 0.16 mmol) in hot EtOH
(2 ml) were added Pd(OH)2 (37 mg) and ammonium formate (0.059 g; 0.94 mmol) and the resulting
mixture was refluxed under N2 for 42 h. Then it was cooled to 25 °C, filtereded, and the solvent was
evaporated. The residue was purified by preparative TLC (eluent: CH2Cl2/NH3 in MeOH – 50:1) to
yield the product as a white solid (0.012 g; 32 %).
1
H NMR (500 MHz, CDCl3) δ 7.96 (d, J = 7.43 Hz, 2H); 7.50 (s, 1H); 7.46-7.39 (m, 2H); 7.38-7.31
(m, 1H); 6.92 (s, 1H); 4.36 (brs, 2H); 2.82 (q, J = 7.26 Hz, 2H); 1.38 (t, J = 7.45 Hz, 3H).
13
C NMR (126 MHz, CDCl3) δ 155.3, 147.7, 143.6, 140.9, 138.1, 137.4, 128.7, 128.3, 127.4, 124.5,
103.0, 16.4, 13.6.
200
HRMS (APCI): calcd. for C15H15N2O [M+H]+
= 239.1179; found [M+H]+
= 239.1179.
Preparative Example 29
To a freshly prepared solution of xantphos (0.022 g; 0.038 mmol) and Pd3(dba)2 (0.042 g; 0.046
mmol) in anhydrous 1,2-dimethoxyethane (2 mL) were added the product from Preparative Example
26 (0.104 g; 0.40 mmol), t-BuOK (0.097 g; 0.86 mmol) and aniline (0.048 mL; 0.53 mmol) and the
resulting mixture was stirred under N2 at 100 °C for 16 h. Then it was cooled to 25 °C, diluted with
EtOAc (15 mL), poured into brine (25 mL) and extracted with EtOAc (3 × 15 mL). The organic
extracts were dried over MgSO4, filtered and the solvent was evaporated. The residue was purified by
column chromatography on silica gel (eluent: hexane/EtOAc – 15:1) to yield the product as a pale
solid (0.064 g; 51 %).
1
H NMR (500 MHz, CDCl3) δ 7.96-7.91 (m, 2H); 7.56-7.53 (m, 1H); 7.45-7.27 (m, 8H); 7.17-7.12
(m, 1H); 6.40 (brs, 1H); 2.86 (qd, J = 1.15 Hz, 7.50 Hz, 2H); 1.41 (t, J = 7.51 Hz, 3H).
13
C NMR (126 MHz, CDCl3) δ 155.5, 147.7, 143.5, 141.1, 139.8, 137.6, 136.1, 129.9, 128.7, 128.4,
127.6, 124.8, 124.2, 121.5, 100.9, 16.4, 13.6.
HRMS (APCI): calcd. for C21H19N2O [M+H]+
= 315.1492; found [M+H]+
= 315.1492.
Preparative Example 30
201
To a freshly prepared solution of xantphos (0.023 g; 0.039 mmol) and Pd3(dba)2 (0.036 g; 0.039
mmol) in anhydrous 1,2-dimethoxyethane (2 mL) were added the product from Preparative Example
26 (0.104 g; 0.40 mmol), t-BuOK (0.158 g; 1.40 mmol) and 5-amino-3-methyl-isothiazole
hydrochloride (0.115 g; 0.76 mmol) and the resulting mixture was stirred under N2 at 100 °C for 22 h.
Then it was cooled to 25 °C, diluted with EtOAc (15 mL), poured into brine (25 mL) and extracted
with EtOAc (3×15 mL). The organic extracts were dried over MgSO4, filtered, and the solvent was
evaporated. The residue was purified by column chromatography on silica gel (eluent: CH2Cl2/MeOH
– 20:1). So obtained oil was further purified by preparative TLC (eluent: CH2Cl2/MeOH – 30:1) to
yield the product as a pale orange solid (0.053 g; 39 %).
1
H NMR (500 MHz, DMSO-d6) δ 8.15-8.12 (m, 1H); 8.03-7.98 (m, 2H); 7.94-7.88 (m, 1H); 7.56-
7.50 (m, 3H); 7.47-7.43 (m, 1H); 7.37 (s, 1H); 2.77 (qd, J = 1.05 Hz, 7.48 Hz, 2H); 2.32 (s, 3H); 1.35
(t, J = 7.51 Hz, 3H).
13
C NMR (126 MHz, DMSO-d6) δ 168.4, 152.7, 146.4, 146.0, 143.9, 138.9, 130.6, 128.7, 128.6,
126.6, 122.8, 121.5, 110.6, 53.9, 20.0, 15.5, 13.3.
HRMS (APCI): calcd. for C19H18N3OS [M+H]+
= 336.1165; found [M+H]+
= 336.1164.
Preparative Example 31A
To a freshly prepared solution (S)-1-[(RP)-2-(dicyclohexylphosphino)ferrocenyl]ethyldi-tertbutylphosphine
(0.011 g; 0.021 mmol) and Pd(OAc)2(0.006 g; 0.028 mmol) in anhydrous
1,2-dimethoxyethane (2 mL) were added the product from Preparative Example 26 (0.109 g; 0.42
mmol), t-BuONa (0.060 g; 0.62 mmol) and 3-picolylamine (0.045 mL; 0.44 mmol) and the resulting
mixture was stirred under N2 at 100 °C for 17 h. Then it was cooled to 25 °C, diluted with EtOAc (15
mL), poured into brine (25 mL) and extracted with EtOAc (3×15 mL). The organic extracts were
dried over MgSO4, filtered and the solvent was evaporated. The residue was purified by column
202
chromatography on silica gel (eluent: CH2Cl2/MeOH – 10:1). So obtained oil was further purified by
preparative TLC (eluent: CH2Cl2/NH3 in MeOH – 30:1) to yield the product as a pale yellow solid
(0.081 g; 58 %).
1
H NMR (500 MHz, CDCl3) δ 8.67 (s, 1H); 8.55 (d, J = 4.20 Hz, 1H); 7.92-7.86 (m, 2H); 7.73-7.68
(m, 1H); 7.48 (s, 1H); 7.43-7.37 (m, 2H); 7.36-7.31 (m, 1H); 7.29-7.25 (m, 1H); 6.81 (s, 1H); 5.01-
4.90 (m, 1H); 4.62 (d, J = 5.83 Hz, 2H); 2.82 (qd, J = 0.84 Hz, 7.45, 2H); 1.38 (t, J = 7.51 Hz, 3H).
13
C NMR (126 MHz, CDCl3) δ 155.7, 149.5, 149.3, 146.9, 143.3, 141.1, 139.2, 137.1, 135.2, 133.9,
128.7, 128.4, 127.5, 124.6, 123.9, 99.4, 45.0, 16.4, 13.6.
HRMS (APCI): calcd. for C21H20N3O [M+H]+
= 330.1601; found [M+H]+
= 330.1598.
Preparative Examples 31B-31D
By essentially same procedure set forth in Preparative Example 31A, using proper amines instead of
3-picolylamine, the compounds given below were prepared.
Preparative Example 31B
Pale yellow solid.
1
H NMR (500 MHz, CDCl3) δ 7.97-7.91 (m, 2H); 7.78-7.73 (m, 2H); 7.59-7.55 (m, 1H); 7.50 (s, 1H);
7.46-7.32 (m, 5H); 6.90 (brs, 1H); 2.86 (qd, J = 0.86 Hz, 7.44 Hz, 2H); 2.76-2.70 (m, 6H); 1.40 (t, J =
7.51 Hz, 3H).
13
C NMR (126 MHz, CDCl3) δ 155.3, 148.1, 144.6, 144.3, 140.1, 138.0, 133.9, 129.9, 129.3, 128.9,
127.6, 126.6, 124.7, 118.7, 103.1, 38.2, 16.4, 13.6.
203
HRMS (APCI): calcd. for C23H24N3O3S [M+H]+
= 422.1533; found [M+H]+
= 422.1534.
Preparative Example 31C
Brown solid.
1
H NMR (500 MHz, CDCl3) δ 7.92-7.86 (m, 2H); 7.55-7.52 (m, 1H); 7.42-7.30 (m, 4H); 7.23-7.20
(m, 1H); 7.17-7.13 (m, 1H); 6.98-6.92 (m, 2H); 6.31 (brs, 1H); 3.90-3.84 (m, 4H); 3.20-3.14 (m, 4H);
2.87 (q, J = 7.42 Hz, 2H); 1.39 (t, J = 7.50 Hz, 3H).
13
C NMR (126 MHz, CDCl3) δ 155.3, 149.0, 143.5, 137.3, 131.6, 128.7, 128.5, 127.7, 124.6, 124.6,
124.5, 123.8, 117.0, 116.4, 100.3, 67.1, 49.8, 16.5, 13.6.
HRMS (APCI): calcd. for C25H26N3O2 [M+H]+
= 400.2020; found [M+H]+
= 400.2019.
Preparative Example 31D
Brown solid foam.
1
H NMR (500 MHz, CDCl3) δ 7.98-7.93 (m, 2H); 7.53-7.50 (m, 1H); 7.46-7.40 (m, 2H); 7.39-7.33
(m, 1H); 6.92 (s, 1H); 3.66-3.56 (m, 8H); 2.84 (q, J = 7.39 Hz, 2H); 1.48 (s, 9H); 1.38 (t, J = 7.51 Hz,
3H).
204
13
C NMR (126 MHz, CDCl3) δ 155.4, 154.9, 143.0, 142.2, 138.4, 128.8, 128.5, 127.6, 124.2, 102.8,
80.4, 48.1, 28.6, 16.3, 13.6.
HRMS (APCI): calcd. for C24H30N3O3 [M+H]+
= 408.2282; found [M+H]+
= 408.2281.
Preparative Example 32
To a stirred solution of the product from Preparative Example 31D (0.088 g; 0.22 mmol) in ethanol (2
mL) was added aqueous HCl (3 M; 1.4 mL; 4.2 mmol) and the resulting mixture was stirred under N2
at 60 °C for 17 h. Then, the ethanol and HCl were evaporated and the oily residue was mixed with
CH2Cl2 (2 mL), MeOH (1mL) and Na2CO3 (200 mg) and the mixture was stirred at 25 °C. After 20
min., the solvents were evaporated and the solid residue was purified by column chromatography on
silica gel (eluent: CH2Cl2/7N NH3 in MeOH – 15:1) to yield the product as a pale solid (0.048 g; 72
%).
1
H NMR (500 MHz, CDCl3) δ 7.99-7.94 (m, 2H); 7.51-7.48 (m, 1H); 7.46-7.40 (m, 2H); 7.38-7.32
(m, 1H); 6.93 (s, 1H); 3.63-3.56 (m, 4H); 3.11-3.04 (m, 4H); 2.82 (qd, J = 1.22 Hz, 7.50 Hz, 2H);
2.04 (brs, 1H); 1.37 (t, J = 7.51 Hz, 3H).
13
C NMR (126 MHz, CDCl3) δ 155.4, 148.4, 142.7, 142.6, 141.3, 138.5, 128.7, 128.3, 127.5, 124.2,
102.5, 49.3, 46.1, 16.3, 13.6.
HRMS (APCI): calcd. for C19H22N3O [M+H]+
= 308.1757; found [M+H]+
= 308.1755.
205
Preparative Example 33
To a mixture of the product from Preparative Example 26 (0.047 g; 0.18 mmol), 1-methylpyrazole-4boronic
acid pinacol ester (0.045 g; 0.22 mmol), K3PO4 (0.163 g; 0.77 mmol) and PdCl2(dppf) (0.009
g; 0.013 mmol) were added under N2 1,2-dimethoxyethane (2 mL) and water (0.5 mL). The reaction
mixture was refluxed for 15 hrs. Then it was cooled to 25 °C, diluted with EtOAc (15 mL), poured
into brine (20 mL) and extracted with EtOAc (3×15 mL). The organic extracts were dried over
Na2SO4, filtered and the solvent was evaporated. The residue was purified by column
chromatography on silica gel (eluent: CH2Cl2/EtOAc – 10:1). So obtained oil was further purified by
preparative TLC (eluent: CH2Cl2) to yield the product as a white solid (0.024 g; 43 %).
1
H NMR (500 MHz, CDCl3) δ 8.14 (d, J = 3.08 Hz, 2H); 8.07-8.02 (m, 2H); 7.73 (s, 1H); 7.67-7.64
(m, 1H); 7.50.7.44 (m, 2H); 7.41-7.36 (m, 1H); 3.99 (s, 3H); 2.87 (qd, J = 1.10 Hz, 7.49 Hz, 2H);
1.41 (t, J = 7.52 Hz, 3H).
13
C NMR (126 MHz, CDCl3) δ 154.4, 148.2, 144.7, 144.2, 140.4, 138.4, 130.5, 128.9, 128.6, 127.5,
124.3, 124.2, 116.1, 112.5, 39.5, 16.3, 13.6.
HRMS (APCI): calcd. for C19H18N3O [M+H]+
= 304.1444; found [M+H]+
= 304.1444.
Preparative Example 34A
Into a 10 mL flask were placed 1,2-dimethoxyethane (3 mL), Pd(OAc)2 (1.8 mg, 0.008 mmol) and
CyPF(t-Bu) (4.4 mg, 0.008 mmol) and the mixture was stirred at 25 °C under N2 for 5 min. Then, the
206
product from Preparative Example 5A (55 mg, 0.20 mmol), N1
,N1
-dimethylpropane-1,3-diamine (24
mg, 0.24 mmol) and t-BuONa (28 mg, 0.30 mmol) were added and the mixture was refluxed for 22 h.
Brine (30 mL) was added and the mixture was extracted with EtOAc (30+20+20 mL). The organic
phase was dried over Na2SO4, filtered, and the solvent was evaporated. The residue was purified by
preparative TLC on silica gel (CH2Cl2/7 M solution of NH3 in MeOH; 30:1). The product was
obtained as a green solid (24 mg, 35 %).
1
H NMR (500 MHz, CDCl3) δ 8.09 (s, 1H), 8.04 (dd, J = 8.5, 1.7 Hz, 1H), 7.94 – 7.86 (m, 2H), 7.84
(d, J = 7.9 Hz, 1H), 7.56 (d, J = 8.9 Hz, 1H), 7.52 – 7.43 (m, 2H), 6.42 (d, J = 8.9 Hz, 1H), 3.56 (t, J
= 6.6 Hz, 2H), 2.53 (t, J = 6.9 Hz, 2H), 2.31 (s, 6H), 1.94 (p, J = 6.8 Hz, 2H).
13
C NMR (126 MHz, CDCl3) δ 156.9, 144.02, 143.7, 143.4, 133.8, 132.6, 128.9, 128.3, 128.1, 127.7,
126.1, 125.8, 125.7, 124.8, 120.7, 120.6, 105.0, 58.0, 45.4, 41.5, 27.0.
HRMS (ESI): calcd. for C22H23N3O [M+H]+
= 346.1914; found [M+H]+
= 346.1912.
Preparative Example 34B
By essentially same procedure set forth in Preparative Example 34A, using 2-methoxyethanamine
instead of N1
,N1
-dimethylpropane-1,3-diamine, the compound given below was prepared.
Dark red solid.
1
H NMR (500 MHz, CDCl3) δ 8.84 (s, 1H), 8.10 (s, 1H), 8.04 (dd, J = 8.5, 1.7 Hz, 1H), 7.96 – 7.87
(m, 2H), 7.87 – 7.81 (m, 1H), 7.59 (d, J = 8.9 Hz, 1H), 7.53 – 7.44 (m, 2H), 6.46 (d, J = 8.9 Hz, 1H),
3.72 (s, 4H), 3.43 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 156.5, 144.3, 143.9, 133.9, 132.8, 128.8, 128.4, 128.2, 127.8, 126.2,
125.9, 125.8, 124.9, 121.0, 120.8, 105.6, 71.6, 58.9, 42.4.
207
HRMS (APCI): calcd. for C20H18N2O2 [M+H]+
= 319.1441; found [M+H]+
= 319.1437.
Preparative Example 35
5-bromopyridin-3-ol (1.1 g, 6.3 mmol), iodine (1.6 g, 6.3 mmol), Na2CO3 (1.4 g, 13.2 g) and H2O (21
mL) were placed into a 100 mL round bottom flask. The mixture was stirred under N2 at 25 °C for 3
h. The mixture was neutralized with 1 M aqueous solution of HCl and extracted with EtOAc
(60+40+40 mL). The organic phase was washed with brine (50 mL), dried over MgSO4 and filtered.
The product was obtained as a brown solid (1.89 g; 100 %).
1
H NMR (300 MHz, CDCl3) δ 8.08 (d, J = 2.1 Hz, 1H), 7.38 (d, J = 2.1 Hz, 1H), 5.39 (s, 1H).
Preparative Example 36
The product from Preparative Example 35 (1.88 g, 6.27 mmol), 1,4-dioxane (12 mL) and TEA (12
mL) were placed into a 100 mL round bottom flask. The mixture was purged with N2, then
ethynyltrimethylsilane (1.15 mL, 8.15 mmol), PdCl2(PPh3)2 (132 mg, 0.188 mmol) and CuI (71 mg,
0.376 mmol) were added. The mixture was stirred under N2 at 45 °C for 3 h. The solvent was
evaporated and the residue was purified by column chromatography on silica gel (EtOAc/hexane;
1:15). The product was obtained as an orange solid (1.04 g, 61 %).
1
H NMR (500 MHz, CDCl3) δ 8.58 (d, J = 1.9 Hz, 1H), 7.91 (dd, J = 1.9, 1.0 Hz, 1H), 7.10 (d, J = 1.0
Hz, 1H), 0.37 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 170.2, 150.9, 147.1, 146.8, 121.1, 117.1, 115.2, -1.9.
HRMS (APCI): calcd. for C10H12BrNOSi [M+H]+
= 269.9944; found [M+H]+
= 269.9954.
208
Preparative Example 37
The product from Preparative Example 36 (47 mg, 0.174 mmol), phenylboronic acid (28 mg, 0.226
mmol), 1,2-dimethoxyethane (8 mL), TEA (1 mL) and H2O (2 mL) were placed into a 25 mL round
bottom flask and the mixture was purged with N2. Then, PdCl2(dppf) (3.8 mg, 5.2 µmol) was added
and the mixture was refluxed under N2 for 75 min. After addition of brine (25 mL), the mixture was
extracted with EtOAc (3×20 mL). The organic phase was dried over MgSO4, filtered, and the solvent
was evaporated. The residue was purified by column chromatography on silica gel (EtOAc/hexane;
1:10). The product was obtained as a white solid (39 mg, 84 %).
1
H NMR (300 MHz, CDCl3) δ 8.78 (d, J = 1.8 Hz, 1H), 7.93 (d, J = 0.7 Hz, 1H), 7.69 – 7.59 (m, 2H),
7.55 – 7.35 (m, 3H), 7.17 (d, J = 0.8 Hz, 1H), 0.40 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 169.6, 151.2, 147.5, 145.1, 138.5, 132.9, 129.7, 129.2, 127.9, 127.6,
117.1, 116.4, 115.5, -1.8.
HRMS (APCI): calcd. for C16H17NOSi [M+H]+
= 268.1152; found [M+H]+
= 268.1160.
Preparative Example 38
The product from Preparative Example 36 (47 mg, 0.174 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-1H-pyrazole (47 mg, 0.226 mmol), 1,2-dimethoxyethane (8 mL), TEA (1
mL) and H2O (2 mL) were placed into a 25 mL round bottom flask and the mixture was purged with
N2. Then, PdCl2(dppf) (3.8 mg, 5.2 µmol) was added and the mixture was refluxed under N2 for 70
min. After addition of brine (25 mL), the mixture was extracted with EtOAc (3×20 mL). The organic
phase was dried over MgSO4, filtered, and the solvent was evaporated. The residue was purified by
column chromatography on silica gel (CH2Cl2/MeOH; 20:1). The product was obtained as a yellow
wax (51 mg, 99 %).
209
1
H NMR (500 MHz, CDCl3) δ 8.67 (d, J = 1.7 Hz, 1H), 7.79 (m, 2H), 7.68 (s, 1H), 7.12 (d, J = 1.0
Hz, 1H), 3.97 (s, 3H), 0.37 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 168.9, 151.3, 146.7, 143.8, 137.1, 127.3, 124.6, 120.5, 117.2, 114.6,
39.3, -1.9.
HRMS (APCI): calcd. for C14H17N3OSi [M+H]+
= 272.1214; found [M+H]+
= 272.1219.
Preparative Example 39
5-chloropyridin-3-ol (5.12 g, 39.7 mmol), iodine (10.1 g, 39.7 mmol), Na2CO3 (8.83 g, 83.3 mmol)
and H2O (80 mL) were placed into a 500 mL round bottom flask and the mixture was stirred under N2
at 25 °C for 3.5 h. The mixture was neutralized with 1 M aqueous solution of HCl (ca. 120 mL) and
extracted with EtOAc (120+70+70 mL). The organic phase was washed with brine (80 mL), dried
over MgSO4 and filtered. The product was obtained as a brown solid (10.13 g; 100 %).
1
H NMR (300 MHz, DMSO) δ 11.38 (s, 1H), 7.95 (d, J = 2.3 Hz, 1H), 7.17 (d, J = 2.3 Hz, 1H).
Preparative Example 40
The product from Preparative Example 39 (8.34 g, 32.7 mmol), ethynyltrimethylsilane (6.0 mL, 42.5
mmol), PdCl2(PPh3)2 (688 mg, 0.98 mmol), CuI (373 mg, 1.96 mmol), 1,4-dioxane (25 mL) and TEA
(25 mL) were placed into a 250 mL round bottom flask. The mixture was stirred under N2 at 45 °C for
2.5 h. The solvent was evaporated and the residue was purified by column chromatography on silica
gel (EtOAc/hexane; 1:15). The product was obtained as an orange solid (4.37 g, 59 %).
1
H NMR (500 MHz, CDCl3) δ 8.52 (dd, J = 1.9, 1.4 Hz, 1H), 7.81 – 7.76 (m, 1H), 7.16 – 7.12 (m,
1H), 0.40 (d, J = 1.0 Hz, 9H).
13
C NMR (126 MHz, CDCl3) δ 170.3, 150.5, 146.9, 144.9, 127.2, 118.3, 117.1, -1.9.
210
HRMS (APCI): calcd. for C10H12ClNOSi [M+H]+
= 226.0449; found [M+H]+
= 226.0458.
Preparative Example 41
The product from Preparative Example 37 (1.60 g, 5.98 mmol), CH2Cl2 (12 mL) and mCPBA (1.86 g,
10.8 mmol) were placed into a 100 mL round bottom flask and the mixture was stirred under N2 at 25
°C for 3 h. The mixture was neutralized with saturated aqueous solution of NaHCO3 (30 mL) and
extracted with CH2Cl2 (3×25 mL). The organic phase was dried over MgSO4 and filtered. The solvent
was evaporated and the residue was purified by column chromatography on silica gel
(EtOAc/acetone; 9:1). The product was obtained as a white solid (1.38 g, 82 %).
1
H NMR (500 MHz, CDCl3) δ 8.46 (d, J = 1.1 Hz, 1H), 7.62 (m, 1H), 7.57 (m, 2H), 7.49 (m, 2H),
7.45 (m, 1H), 7.41 (d, J = 0.9 Hz, 1H), 0.39 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 169.6, 154.5, 137.7, 136.3, 135.3, 133.4, 129.5, 129.0, 127.4, 111.1,
109.3, -2.0.
HRMS (APCI): calcd. for C16H17NO2Si [M+H]+
= 284.1101; found [M+H]+
= 284.1099.
Preparative Example 42
The product from Preparative Example 41 (1.35 g, 4.75 mmol), chloroform (10 mL) and POCl3 (7.96
mL, 8.54 mmol) were placed into a 100 mL round bottom flask and the mixture was refluxed under
N2 for 1 h. The solvent and POCl3 were evaporated and the residue was mixed with saturated aqueous
solution of NaHCO3 (50 mL) and extracted with CH2Cl2 (50+25+25 mL). The organic phase was
dried over MgSO4 and filtered. The solvent was evaporated and the residue was purified by column
211
chromatography on silica gel (EtOAc/CH2Cl2; 1:20). The product was obtained as a white solid
(0.824 g, 57 %).
1
H NMR (500 MHz, CDCl3) δ 8.49 (s, 1H), 7.55 – 7.42 (m, 5H), 7.20 (s, 1H), 0.42 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 170.4, 148.3, 147.9, 147.6, 135.6, 132.0, 130.1, 128.5, 128.3, 124.3,
117.7, -1.8.
HRMS (APCI): calcd for C16H16ClNOSi [M+H]+
= 302.0762; found [M+H]+
= 302.0765.
The structural integrity of this compound was also confirmed by X-ray crystallography.
Preparative Example 43
Pd(OAc)2 (16.7 mg, 74 µmol), SPhos (40 mg, 99 µmol) and 1-butanol (5 mL) were placed into a 25
mL flask and stirred for 5 min. The product from Preparative Example 42 (750 mg, 2.48 mmol), 1methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole
(724 mg, 3.48 mmol), TEA
(10.4 mL, 74 mmol) and H2O (1 mL) were added and the mixture was refluxed for 15 h. The solvent
was evaporated and the residue was purified by column chromatography on silica gel
(EtOAc/CH2Cl2; 1:10). The product was obtained as a pale yellow solid (427 mg, 50 %).
1
H NMR (500 MHz, CDCl3) δ 8.41 (s, 1H), 7.58 (s, 1H), 7.47 – 7.41 (m, 3H), 7.38 – 7.32 (m, 2H),
7.27 (s, 1H), 7.20 (s, 1H), 3.84 (s, 3H), 0.42 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 168.5, 148.0, 147.8, 147.6, 140.9, 139.1, 131.4, 130.3, 130.1, 128.9,
128.0, 122.5, 117.5, 114.3, 39.2, -1.7.
HRMS (APCI): calcd. for C20H21N3OSi [M+H]+
= 348.1527; found [M+H]+
= 348.1529.
212
Preparative Example 44
The product from Preparative Example 43 (420 mg, 1.21 mmol), CH2Cl2 (5 mL) and mCPBA (375
mg, 2.18 mmol) were placed into a 25 mL round bottom flask and the mixture was stirred under N2 at
25 °C for 1 h. The mixture was mixed with saturated aqueous solution of NaHCO3 (40 mL), brine (30
mL) was added and the mixture was extracted with CH2Cl2 (50+25+25 mL). The organic phase was
dried over MgSO4 and filtered. The solvent was evaporated and the residue was purified by column
chromatography on silica gel (EtOAc/acetone; 2:1). The product was obtained as a light yellow semisolid
(357 mg, 81 %).
1
H NMR (500 MHz, CDCl3) δ 8.13 (s, 1H), 7.49 (s, 1H), 7.47 – 7.42 (m, 4H), 7.34 – 7.28 (m, 2H),
7.15 (s, 1H), 3.82 (s, 3H), 0.42 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 168.8, 151.0, 140.1, 137.5, 136.8, 135.5, 132.2, 130.6, 129.7, 129.1,
128.8, 116.2, 113.5, 111.4, 39.2, -1.9.
HRMS (APCI): calcd. for C20H21N3O2Si [M+H]+
= 364.1476; found [M+H]+
= 364.1478.
Preparative Example 45
The product from Preparative Example 44 (351 mg, 0.966 mmol) and POCl3 (4 mL) were placed into
a 25 mL round bottom flask and the mixture was stirred under N2 at 100 °C for 25 min. The POCl3
was evaporated, the residue was mixed with saturated aqueous solution of NaHCO3 (25 mL) and
extracted with CH2Cl2 (20+15+15 mL). The organic phase was dried over MgSO4 and filtered. The
213
solvent was evaporated and the residue was purified by column chromatography on silica gel
(EtOAc/hexane; 1:1). The product was obtained as a white solid (316 mg, 86 %).
1
H NMR (500 MHz, CDCl3) δ 7.53 – 7.47 (m, 4H), 7.28 – 7.26 (m, 1H), 7.26 – 7.25 (m, 1H), 7.13 (s,
1H), 6.96 (s, 1H), 3.78 (s, 3H), 0.42 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 169.6, 147.4, 147.3, 146.9, 140.9, 138.5, 131.6, 130.3, 129.2, 128.5,
127.8, 126.0, 116.9, 114.5, 39.2, -1.8.
HRMS (APCI): calcd. for C20H20ClN3OSi [M+H]+
= 382.1137; found [M+H]+
= 382.1141.
Preparative Example 46
The product from Preparative Example 45 (285 mg, 0.746 mmol), KF (130 mg, 2.24 mmol) and
MeOH (16 mL) were placed into a 50 mL round bottom flask and the mixture was stirred under N2 at
62 °C for 43 h.. The solvent was evaporated and the residue was purified by column chromatography
on silica gel (EtOAc/hexane; 1:1). The product was obtained as a white solid (213 mg, 92 %).
1
H NMR (500 MHz, CDCl3) δ 7.93 (d, J = 2.3 Hz, 1H), 7.52 – 7.47 (m, 3H), 7.31 – 7.26 (m, 3H),
7.21 (s, 1H), 7.00 (d, J = 2.3 Hz, 1H), 3.80 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 149.4, 148.0, 146.0, 144.4, 140.7, 138.2, 131.9, 130.3, 129.2, 128.6,
128.3, 126.6, 114.0, 108.3, 39.2.
HRMS (APCI): calcd. for C18H12ClN3O [M+H]+
= 310.0742; found [M+H]+
= 310.0750.
214
Preparative Example 47
The product from Preparative Example 38 (2.36 g, 8.7 mmol), CH2Cl2 (15 mL) and mCPBA (2.1 g,
15.7 mmol) were placed into a 100 mL round bottom flask and the mixture was stirred under N2 at 25
°C for 45 h. Then, additional mCBPA (0.62 g, 3.6 mmol) was added and the mixture was stirred for
additional 4 h. The mixture was mixed with saturated aqueous solution of NaHCO3 (20 mL), brine
(30 mL) was added, and the mixture was extracted with CH2Cl2 (3×70 mL). The organic phase was
dried over MgSO4 and filtered. The solvent was evaporated and the residue was purified by column
chromatography on silica gel (EtOAc/MeOH; from 20:1 to 5:1). The product was obtained as a light
yellow solid (1.67 g, 66 %).
1
H NMR (300 MHz, CDCl3) δ 8.36 (d, J = 0.9 Hz, 1H), 7.76 (s, 1H), 7.63 (s, 1H), 7.49 (s, 1H), 7.36
(d, 1H), 3.98 (s, 3H), 0.38 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 168.9, 154.6, 137.1, 131.9, 127.6, 126.8, 118.6, 111.1, 107.4, 39.5, -
2.0.
HRMS (APCI): calcd. for C14H17N3O2Si [2M+H]+
= 575.2253; found [2M+H]+
= 575.2255.
Preparative Example 48
The product from Preparative Example 47 (1.67 g, 5,81 mmol), chloroform (12 mL) and POCl3 (9.75
mL, 105 mmol) were placed into a 100 mL round bottom flask and the mixture was refluxed under N2
for 45 min. The solvent and POCl3 were evaporated and the residue was mixed with saturated
aqueous solution of NaHCO3 (40 mL) and extracted with CH2Cl2 (50+30+30 mL). The organic phase
215
was dried over MgSO4 and filtered. The solvent was evaporated and the residue was purified by
column chromatography on silica gel (CH2Cl2/MeOH; 14:1). The product was obtained as a white
solid (1.15 g, 65 %).
1
H NMR (500 MHz, CDCl3) δ 8.58 (s, 1H), 7.87 (s, J = 0.6 Hz, 1H), 7.83 (s, 1H), 7.15 (s, 1H), 4.00
(s, 3H), 0.40 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 169.9, 148.2, 147.3, 146.4, 139.0, 129.8, 123.5, 123.2, 117.7, 116.5,
39.3, -1.9.
HRMS (APCI): calcd. for C14H16N3OSi [M+H]+
= 306.0824; found [M+H]+
= 306.0825.
Preparative Example 49
Pd(OAc)2 (25 mg, 113 µmol), SPhos (62 mg, 150 µmol) and 1-butanol (8 mL) were placed into a 50
mL flask and stirred for 5 min. The product from Preparative Example 48 (1.15 g, 3.76 mmol),
phenylboronic acid (688 mg, 5.64 mmol), TEA (15.7 mL, 113 mmol) and H2O (1.6 mL) were added
and the mixture was refluxed under N2 for 90 min. The solvent was evaporated and the residue was
purified by column chromatography on silica gel (EtOAc/MeOH; from 20:1 to 15:1). The product
was obtained as a light grey solid (1.32 g, 100 %).
1
H NMR (500 MHz, CDCl3) δ 8.64 (s, 1H), 7.45 – 7.39 (m, 5H), 7.25 (d, J = 0.6 Hz, 1H), 7.17 (s,
1H), 7.04 (s, 1H), 3.81 (s, 3H), 0.32 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 169.0, 149.3, 147.1, 146.8, 139.1, 133.5, 130.1, 130.1, 129.2, 128.5,
128.5, 122.8, 118.7, 117.3, 39.0, -1.8.
HRMS (APCI): calcd. for C20H21N3OSi [M+H]+
= 348.1527; found [M+H]+
= 348.1530.
216
Preparative Example 50
The product from Preparative Example 49 (1.3 g, 3.74 mmol), CH2Cl2 (10 mL) and mCPBA (1.16 g,
6.73 mmol) were placed into a 50 mL round bottom flask and the mixture was stirred under N2 at 25
°C for 135 min. The mixture was mixed with saturated aqueous solution of NaHCO3 (40 mL) and
then extracted with CH2Cl2 (50+40+40 mL). The organic phase was dried over MgSO4 and filtered.
The solvent was evaporated and the residue was purified by column chromatography on silica gel
(EtOAc/MeOH; from 10:1 to 7:1). The product was obtained as a white semi-solid (1.13 g, 83 %).
1
H NMR (500 MHz, CDCl3) δ 8.33 (s, 1H), 7.47 – 7.40 (m, 4H), 7.40 – 7.33 (m, 2H), 7.21 (s, 1H),
7.00 (s, 1H), 3.81 (s, 3H), 0.31 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 169.2, 152.6, 138.9, 136.9, 134.2, 132.4, 130.2, 129.5, 128.8, 128.7,
125.3, 123.3, 116.9, 111.2, 39.2, -2.0.
HRMS (APCI): calcd. for C20H21N3O2Si [M+H]+
= 364.1476; found [M+H]+
= 364.1479.
Preparative Example 51
The product from Preparative Example 50 (1.13 g, 3.11 mmol) and POCl3 (6 mL) were placed into a
50 mL round bottom flask and the mixture was stirred under N2 at 100 °C for 20 min. The POCl3 was
evaporated, the residue was mixed with saturated aqueous solution of NaHCO3 (100 mL) and
extracted with CH2Cl2 (60+40+40 mL). The organic phase was dried over MgSO4 and filtered. The
solvent was evaporated and the residue was purified by column chromatography on silica gel
(EtOAc/hexane; 1:2). The product was obtained as a white solid (1.02 g, 86 %).
217
1
H NMR (300 MHz, CDCl3) δ 7.38 – 7.32 (m, 3H), 7.31 – 7.26 (m, 3H), 7.14 (s, 1H), 7.10 (s, 1H),
3.83 (s, 3H), 0.32 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 170.8, 148.5, 147.6, 146.6, 141.0, 134.6, 133.4, 131.2, 130.2, 128.5,
128.2, 121.5, 116.7, 116.5, 39.0, -1.9.
HRMS (APCI): calcd. for C20H20ClN3OSi [M+H]+
= 382.1137; found [M+H]+
= 382.1141.
Preparative Example 52
By essentially same procedure set forth in Preparative Example 46, using the product from
Preparative Example 51, the compound given below was prepared.
White solid.
1
H NMR (500 MHz, CDCl3) δ 7.83 (d, J = 2.3 Hz, 1H), 7.38 – 7.35 (m, 3H), 7.29 – 7.26 (m, 3H),
7.17 (s, 1H), 6.98 (d, J = 2.3 Hz, 1H), 3.83 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 150.3, 148.0, 145.6, 140.9, 135.2, 133.0, 131.1, 130.0, 128.8, 128.4,
122.2, 116.2, 108.1, 39.1.
HRMS (APCI): calcd. for C17H12ClN3O [M+H]+
= 310.0742; found [M+H]+
= 310.0746.
Preparative Example 53
218
The product from Preparative Example 52 (30 mg, 0.969 mmol), 1,2-dimethoxyethane (2 mL), K3PO4
(61.7 mg, 0.291 mmol), (4-(methoxycarbonyl)phenyl)boronic acid (26.1 mg, 0.145 mmol) and
PdCl2(dppf) (4.3 mg, 5.8 µmol) were placed into a 25 mL round bottom flask. The mixture was
refluxed under N2 for 25 h, then additional PdCl2 2O (0.4 mL) were
added and the mixture was refluxed for additional 14 h. The solvent was evaporated and the residue
was purified by column chromatography on silica gel (EtOAc/hexane; 1:1). The product was obtained
as a colorless wax (17 mg, 43 %).
1
H NMR (500 MHz, CDCl3) δ 8.00 – 7.94 (m, 2H), 7.85 (d, J = 1.7 Hz, 1H), 7.48 – 7.43 (m, 2H),
7.39 – 7.35 (m, 3H), 7.32 – 7.27 (m, 2H), 7.08 (s, 1H), 6.80 (s, 1H), 6.63 (s, 1H), 3.91 (s, 3H), 3.65
(s, 3H).
13
C NMR (126 MHz, CDCl3) δ 171.2, 167.1, 155.4, 149.9, 146.5, 146.2, 145.8, 140.7, 133.3, 133.1,
130.9, 130.1, 129.9, 129.3, 129.1, 128.5, 128.4, 121.9, 117.5, 108.6, 52.2, 38.9.
HRMS (APCI): calcd. for C25H19N3O3 [M+H]+
= 410.1499; found [M+H]+
= 410.1492.
Preparative Example 54
The product from Preparative Example 5D (40 mg, 0.14 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate (49 mg, 0.168 mmol), K3PO4 (104 mg, 0.49
mmol), 1,2-dimethoxyethane (2.4 mL), H2O (0.6 mL) and PdCl2(dppf) (6.2 mg, 8.4 µmol) were
placed into a 10 mL round bottom flask and the mixture was refluxed under N2 for 22 h. Then,
additional PdCl2(dppf) (8 mg, 10.9 µmol) was added and the mixture was refluxed for additional 4 h.
The solvent was evaporated and the residue was purified by column chromatography on silica gel
(EtOAc/hexane; 1:1). The product was obtained as a white solid (18.5 mg, 42 %).
219
1
H NMR (500 MHz, DMSO-d6) δ 13.04 (s, 1H), 8.74 (s, 1H), 8.52 – 8.10 (m, 4H), 8.05 (d, J = 8.6
Hz, 1H), 7.73 (d, J = 8.7 Hz, 1H), 7.54 (d, J = 8.2 Hz, 2H), 1.35 (s, 9H).
13
C NMR (126 MHz, DMSO-d6) δ 149.8, 148.8, 146.7, 146.1, 144.8, 128.7, 127.7, 126.3, 125.4,
122.4, 119.9, 119.5, 115.9, 34.3, 31.1.
HRMS (APCI): calcd. for C20H19N3O [M+H]+
= 318.1601; found [M+H]+
= 318.1599.
Preparative Example 55
The product from Preparative Example 5E (11 mg, 52.5 µmol), degassed 1,2-dimethoxyethane (2
mL) and H2O (0,5 mL) were placed into a 5 mL round bottom flask. Then, K3PO4 (39 mg, 0.184
mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate (18.5
mg, 63 µmol) and PdCl2(dppf) (2 mg, 2.6 µmol) were added and the mixture was stirred under N2 at
60 °C for 16 h. Then, additional PdCl2(dppf) (2 mg, 2.6 µmol), the mixture was refluxed for
additional 5 h, another portion of PdCl2(dppf) (2 mg, 2.6 µmol) and the mixture was refluxed for
additional 4 h. The solvent was evaporated and the residue was purified by column chromatography
on silica gel (EtOAc/hexane; 1:1). The product was obtained as a yellow wax (2 mg, 15 %).
1
H NMR (500 MHz, CDCl3) δ 8.24 (b, J = 47.6 Hz, 2H), 7.66 (d, J = 8.2 Hz, 1H), 7.52 (s, 1H), 7.37
(d, J = 8.1 Hz, 1H), 1.52 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 147.7, 143.5, 131.1, 118.7, 115.2, 31.2, 29.8.
HRMS (APCI): calcd. for C14H15N3O [M+H]+
= 242.1288; found [M+H]+
= 242.1292.
220
Preparative Example 56
The product from Preparative Example 11 (1.78 g, 11.6 mmol) and degassed 1,4-dioxane (62 mL)
were placed into a 250 mL round bottom flask, then AcCl (0.825 mL, 11.6 mmol) was added and the
mixture was stirred under N2 at 25 °C. After 3 min, NaI (17.4 g, 116 mmol) was added, the flask was
wrapped with aluminum foil and the mixture was stirred at 106 °C for 70 h. Additional portions of
AcCl were added at these times: 15 h, (0.70 mL, 9.8 mmol); 24 h, (0.80 mL, 11.2 mmol); 40 h (0.80
mL, 11.2 mmol); 48 h (0.825 mL, 11.6 mmol; 64 h (0.825 mL, 11.6 mmol). The solvent was
evaporated, the residue was mixed with saturated solution of NaHCO3 (50 mL) and with Na2S2O3 (5
g), and extracted with CH2Cl2 (3x90 mL). The organic phase was dried over MgSO4 and filtered. The
mixture was concentrated to the volume of 75 mL and hexane (25 mL) was added. To the solution,
upon cooling in ice bath, HCl (15 mL, 1 M solution in Et2O) was added. The precipitate was collected
by filtration. To the solid, TEA (1.9 mL, 14 mmol) and CH2Cl2 (20 mL) were added, the mixture was
cooled in ice bath, H2O (60 mL) was added, and the mixture was extracted with CH2Cl2 (3×60 mL).
The organic phase was dried over MgSO4, filtered, and the solvent was evaporated. The product was
obtained as a white solid (1.90 g, 67 %).
1
H NMR (500 MHz, CDCl3) δ 7.81 (d, J = 2.3 Hz, 1H), 7.60 (d, J = 8.5 Hz, 1H), 7.47 (dd, J = 8.5, 0.9
Hz, 1H), 6.96 (dd, J = 2.3, 0.9 Hz, 1H).
13
C NMR (126 MHz, CDCl3) δ 149.7, 149.5, 147.5, 129.5, 120.4, 112.0, 108.0.
HRMS (APCI): calcd. for C7H4INO [M+H]+
= 245.9410; found [M+H]+
= 245.9407.
Preparative Example 57
The product from Preparative Example 56 (1.86 g, 7.60 mmol) and CCl4 (20 mL) were placed into a
100 mL round bottom flask and the mixture was cooled to -18 °C. Then, bromine (6.49 mL, 114
mmol) was added slowly. The mixture was stirred under N2 while allowed to warm up to 25 °C for 90
221
min. The mixture was poured into a mixture of water (100 mL), ice (50 mL) and Na2S2O5 (30 g). The
resulting mixture was extracted with CH2Cl2 (2x100 mL) and EtOAc (100 mL). The organic phase
was washed with brine (100 mL), dried over MgSO4, filtered, and the solvent was evaporated.
Toluene (24 mL) and DBU (3.4 mL, 22.8 mmol) were added to the residue and the mixture was
stirred under N2 at 80 °C for 45 min. The solvent was evaporated and the residue was and purified by
column chromatography on silica gel (EtOAc/hexane; from 1:7 to 1:5). The product was obtained as a
white solid (1.77 g, 72 %).
1
H NMR (500 MHz, CDCl3) δ 7.85 (s, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.47 (d, J = 8.5 Hz, 1H).
13
C NMR (126 MHz, CDCl3) δ 147.4, 147.2, 146.8, 131.1, 120.9, 113.0, 98.9.
HRMS (APCI): calcd. for C7H3BrINO [M+H]+
= 323.8515; found [M+H]+
= 323.8512.
Preparative Example 58
A mixture of the product from Preparative Example 57 (167 mg, 0.515 mmol), 1-methyl-4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole
(113 mg, 0.54 mmol), K3PO4 (383 mg, 1.80
mmol), and PdCl2(dppf) (18.8 mg, 26 µmol) in 1,2-dimethoxyethane (2 mL) and H2O (0.5 mL) was
stirred under N2 at 25 °C for 2.5 h. The solvent was evaporated and the residue was purified by
column chromatography on silica gel (hexane/EtOAc; 2:3). The product was obtained as an orange
solid (80 mg; 56 %).
1
H NMR (500 MHz, CDCl3) δ 8.04 (s, 1H), 7.97 (s, 1H), 7.85 (s, 1H), 7.73 (d, J = 8.6 Hz, 1H), 7.46
(d, J = 8.6 Hz, 1H), 3.96 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 149.9, 146.6, 146.3, 144.6, 137.6, 129.3, 123.6, 119.6, 116.9, 99.6,
39.3.
HRMS (APCI): calcd. for C11H8BrN3O [M+H]+
= 277.9924; found [M+H]+
= 277.9920.
222
Preparative Example 59
By essentially same procedure set forth in Preparative Example 58, using tert-butyl 4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate
instead of 1-methyl-4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole,
the compound given below was prepared.
White solid.
1
H NMR (500 MHz, CDCl3) δ 8.65 (s, 1H), 8.27 (s, 1H), 7.90 (s, 1H), 7.79 (d, J = 8.6 Hz, 1H), 7.54
(d, J = 8.6 Hz, 1H), 1.69 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 148.2, 147.6, 147.1, 146.7, 145.0, 142.5, 128.7, 125.7, 119.8, 117.5,
99.7, 86.0, 28.1.
HRMS (APCI): calcd. for C15H14BrN3O3 [M+H]+
= 364.0291; found [M+H]+
= 364.0294.
The structural integrity of this compound was also confirmed by X-ray crystallography.
Preparative Example 60
A mixture of the product from Preparative Example 57 (127 mg, 0.393 mmol), K3 PO4 (292 mg, 1.38
mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole (80.5 mg, 0.413 mmol), and
PdCl2(dppf) (14.4 mg, 19.7 µmol) in 1,2-dimethoxyethane (2.8 mL) and H2O (0.7 mL) was stirred
under N2 at 40 °C for 25 h. The solvent was evaporated and the residue was purified by column
chromatography on silica gel (CH2Cl2/EtOAc; 20:1). The product was obtained as a white solid (51
mg; 49 %).
1
H NMR (500 MHz, CDCl3) δ 9.00 (s, 1H), 8.88 (s, 1H), 7.93 (s, 1H), 7.82 (d, J = 8.6 Hz, 1H), 7.50
(d, J = 8.6 Hz, 1H).
223
13
C NMR (126 MHz, CDCl3) δ 155.7, 148.4, 147.5, 146.8, 146.1, 145.3, 122.2, 119.9, 117.8, 99.7.
HRMS (APCI): calcd. for C10H5BrN2O2 [M+H]+
= 264.9607; found [M+H]+
= 264.9603.
Preparative Example 61
Degassed 1-butanol (2.0 mL) and H2O (0.4 mL) were placed into a 10 mL round bottom flask,
Pd(OAc)2 (1 mg, 4 µmol) and SPhos (2.2 mg, 5.4 µmol) were added and the mixture was stirred at 25
°C for 3 min. Then, the product from Preparative Example 58 (25 mg, 90 µmol), [1,1'-biphenyl]-3ylboronic
acid (25 mg, 0.126 mmol) and TEA (1.0 mL, 7.2 mmol) were added. The mixture was
stirred under N2 at 40 °C for 1 h, then at 50 °C for 2 h. The solvent was evaporated and the residue
purified by column chromatography on silica gel (hexane/EtOAc; 1:1) and then by preparative TLC
(hexane/acetone; 10:7). The product was obtained as a white solid (7 mg; 22 %).
1
H NMR (500 MHz, CDCl3) δ 8.57 – 8.53 (m, 1H), 8.18 (s, 1H), 8.14 – 8.10 (m, 1H), 8.03 (s, 1H),
8.00 (s, 1H), 7.77 (d, J = 8.6 Hz, 1H), 7.75 – 7.71 (m, 2H), 7.63 – 7.54 (m, 2H), 7.53 – 7.44 (m, 3H),
7.42 – 7.37 (m, 1H), 3.99 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 148.8, 147.6, 145.8, 145.2, 141.7, 141.4, 137.7, 131.3, 129.2, 128.9,
128.9, 127.5, 127.3, 126.5, 126.2, 125.9, 124.3, 121.4, 119.2, 115.7, 39.3.
HRMS (APCI): calcd. for C23H17N3O [M+H]+
= 352.1444; found [M+H]+
= 352.1449.
224
Preparative Example 62
The product from Preparative Example 60 (21 mg, 79.2 µmol), Pd(OAc)2 (1 mg, 4 µmol), SPhos (2
mg, 4.8 µmol), naphthalen-2-ylboronic acid (17.7 mg, 103 µmol), 1-butanol (2 mL), H2O (0.4 mL)
and TEA (1.0 mL, 7.17 mmol) were placed into a 10 mL round bottom flask. The mixture was stirred
under N2 at 45 °C for 3 h. The solvent was evaporated, the residue was purified by column
chromatography (hexane/EtOAc; 3:1) and then by preparative TLC (CH2Cl2/EtOAc; 30:1). The
product was obtained as a colorless wax (2.5 mg; 10 %).
1
H NMR (500 MHz, CDCl3) δ 9.02 (s, 1H), 8.94 (s, 1H), 8.87 (s, 1H), 8.30 (s, 1H), 8.09 (dd, J = 8.5,
1.6 Hz, 1H), 8.00 – 7.93 (m, 2H), 7.91 – 7.85 (m, 2H), 7.57 – 7.50 (m, 3H).
13
C NMR (126 MHz, CDCl3) δ 155.2, 148.5, 146.5, 146.1, 145.1, 133.8, 133.1, 128.5, 128.5, 127.9,
127.7, 126.6, 126.4, 126.3, 124.8, 122.8, 121.6, 119.5, 116.9.
HRMS (APCI): calcd. for C20H12N2O2 [M+H]+
= 313.0972; found [M+H]+
= 313.0975.
Preparative Example 63
By essentially same procedure set forth in Preparative Example 61, using [1,1'-biphenyl]-4-ylboronic
acid instead of[1,1'-biphenyl]-3-ylboronic acid, the compound given below was prepared.
White solid.
225
1
H NMR (500 MHz, CDCl3) δ 8.28 – 8.24 (m, 2H), 8.16 (s, 1H), 8.04 (d, J = 2.9 Hz, 2H), 7.77 (d, J =
8.6 Hz, 1H), 7.76 – 7.72 (m, 2H), 7.70 – 7.66 (m, 2H), 7.50 – 7.45 (m, 3H), 7.40 – 7.35 (m, 1H), 4.00
(s, 3H).
13
C NMR (126 MHz, CDCl3) δ 148.8, 147.6, 145.8, 145.1, 141.0, 140.5, 137.7, 129.9, 129.0, 128.9,
127.6, 127.5, 127.4, 127.1, 124.3, 121.3, 119.2, 115.8, 39.3.
HRMS (APCI): calcd. for C23H17N3O [M+H]+
= 352.1441; found [M+H]+
= 352.1442.
Preparative Example 64
By essentially same procedure set forth in Preparative Example 61, using [1,1'-biphenyl]-2-ylboronic
acid instead of[1,1'-biphenyl]-3-ylboronic acid, the compound given below was prepared.
Colorless wax.
1
H NMR (500 MHz, CDCl3) δ 8.18 (d, J = 7.6 Hz, 1H), 7.98 – 7.91 (m, 2H), 7.67 (d, J = 8.6 Hz, 1H),
7.54 – 7.48 (m, 1H), 7.47 – 7.42 (m, 2H), 7.38 (d, J = 8.6 Hz, 1H), 7.35 – 7.30 (m, 2H), 7.30 – 7.22
(m, 3H), 7.17 (s, 1H), 3.97 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 148.5, 147.5, 146.6, 146.1, 142.2, 141.6, 137.7, 130.9, 130.6, 129.5,
129.1, 128.6, 128.3, 127.8, 127.6, 127.1, 124.1, 120.8, 119.0, 115.5, 39.3.
HRMS (APCI): calcd. for C23H17N3O [M+H]+
= 352.1444; found [M+H]+
= 352.1448.
Preparative Example 65
226
Degassed 1-BuOH (2.5 mL) and H2O (0.5 mL) were placed into a 10 mL round bottom flask, then the
product from Preparative Example 58 (40 mg, 144 µmol), 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)-1H-indazole (49.2 mg, 0.201 mmol), Pd(PPh3)4 (8.3 mg, 7.2 µmol) and K3PO4 (92 mg, 0.432
mmol) were added. The mixture was stirred under N2 at 90 °C for 18 h. The solvent was evaporated
and the residue was purified by column chromatography on silica gel (MeOH/EtOAc; 1:10) and then
by preparative TLC (CH2Cl2/acetone; 3:2). The product was obtained as a white solid (10 mg; 22 %).
1
H NMR (500 MHz, acetone-d6) δ 12.35 (b, 1H), 9.06 (d, J = 1.0 Hz, 1H), 8.66 (s, 1H), 8.27 (s, 1H),
8.14 (s, 1H), 8.06 (d, J = 0.9 Hz, 1H), 7.95 (d, J = 8.7 Hz, 1H), 7.89 – 7.84 (m, 2H), 7.68 (d, J = 8.6
Hz, 1H), 3.98 (s, 3H).
13
C NMR (126 MHz, acetone-d6) δ 150.2, 148.5, 147.3, 146.5, 142.0, 138.4, 134.8, 129.9, 129.9,
124.9, 123.6, 122.0, 121.6, 120.5, 120.3, 116.8, 109.7, 39.4.
HRMS (APCI): calcd. for C18H12N5O [M+H]+
= 316.1193; found [M+H]+
= 316.1197.
Preparative Example 66
Degased 1-butanol (2.0 mL) and H2O (0.4 mL) were placed into a 5 mL round bottom flask. Then,
the product from Preparative Example 58 (30 mg, 108 µmol), (4-carbamoylphenyl)boronic acid (26.7
mg, 0.162 mmol), Pd(PPh3)4 (6.2 mg, 5.4 µmol) and K3PO4 (68.6 mg, 0.323 mmol) were added. The
mixture was stirred under N2 at 80 °C for 45 min. The solvent was evaporated and the residue was
purified by column chromatography on silica gel (MeOH/EtOAc; 1:10) and then by preparative TLC
(MeOH/EtOAc; 1:10). The product was obtained as a white solid (12 mg; 35 %).
1
H NMR (500 MHz, acetone-d6) δ 8.68 (s, 1H), 8.50 – 8.45 (m, 2H), 8.28 (s, 1H), 8.10 – 8.05 (m,
3H), 7.95 (d, J = 8.7 Hz, 1H), 7.69 (d, J = 8.7 Hz, 1H), 3.97 (s, 3H).
13
C NMR (126 MHz, acetone-d6) δ 168.8, 150.5, 148.5, 147.8, 146.2, 138.2, 135.1, 134.3, 130.0,
128.9, 127.6, 124.8, 121.3, 120.4, 116.9, 39.4.
227
HRMS (APCI): calcd. for C18H14N4O2 [M+H]+
= 319.1190; found [M+H]+
= 319.1187.
Preparative Example 67
By essentially same procedure set forth in Preparative Example 66, using (3-chloro-4methoxyphenyl)boronic
acid instead of (4-carbamoylphenyl)boronic acid, the compound given below
was prepared.
White solid.
1
H NMR (500 MHz, acetone-d6) δ 8.56 (s, 1H), 8.43 (d, J = 2.1 Hz, 1H), 8.34 (dd, J = 8.6, 2.2 Hz,
1H), 8.22 (s, 1H), 8.07 (d, J = 0.6 Hz, 1H), 7.92 (d, J = 8.7 Hz, 1H), 7.66 (d, J = 8.7 Hz, 1H), 7.25 (d,
J = 8.6 Hz, 1H), 3.97 (d, J = 2.0 Hz, 6H).
13
C NMR (126 MHz, acetone-d6) δ 155.5, 150.2, 148.3, 146.5, 146.2, 138.2, 129.8, 129.3, 127.6,
125.6, 124.8, 123.1, 120.4, 120.3, 116.8, 113.7, 56.7, 39.4.
HRMS (APCI): calcd. for C18H14ClN3O2 [M+H]+
= 340.0847; found [M+H]+
= 340.0842.
Preparative Example 68
228
Degassed 1-butanol (2.0 mL) and H2O (0.4 mL) were placed into a 10 mL round bottom flask. Then,
the product from Preparative Example 67 (25 mg, 0.074 mmol), phenylboronic acid (11.7 mg, 95.7
µmol), Pd(OAc)2 (1.0 mg, 3.7 µmol), SPhos (1.8 mg, 44 µmol) and K3PO4 (46.9 mg, 0.220 mmol)
were added. The mixture was stirred under N2 at 80 °C for 4 h. The solvent was evaporated and the
residue was purified by column chromatography on silica gel (EtOAc) and then by preparative TLC
(CH2Cl2/EtOAc; 2:1; 3 runs). The product was obtained as a colorless wax (9 mg; 32 %).
1
H NMR (300 MHz, acetone-d6) δ 8.56 (s, 1H), 8.43 – 8.33 (m, 2H), 8.19 (s, 1H), 8.05 (d, J = 0.6 Hz,
1H), 7.90 (d, J = 8.7 Hz, 1H), 7.70 – 7.61 (m, 3H), 7.50 – 7.33 (m, 3H), 7.24 (d, J = 8.5 Hz, 1H), 3.96
(s, 3H), 3.89 (s, 3H).
13
C NMR (75 MHz, acetone-d6) δ 157.2, 150.0, 148.4, 146.6, 146.2, 139.9, 138.2, 131.7, 130.6,
130.4, 129.8, 129.0, 128.3, 127.9, 125.0, 124.8, 121.5, 120.1, 116.6, 112.9, 56.2, 39.4.
HRMS (APCI): calcd. for C24H19N3O2 [M+H]+
= 382.1550; found [M+H]+
= 382.1547.
Preparative Example 69
By essentially same procedure set forth in Preparative Example 66, using (4-formylphenyl)boronic
acid MIDA ester instead of (4-carbamoylphenyl)boronic acid, the compound given below was
prepared.
White solid.
1
H NMR (500 MHz, acetone-d6) δ 10.11 (s, 1H), 8.78 (s, 1H), 8.65 (d, J = 8.3 Hz, 2H), 8.32 (s, 1H),
8.11 (s, 1H), 8.09 – 8.04 (m, 2H), 7.99 (d, J = 8.7 Hz, 1H), 7.73 (d, J = 8.7 Hz, 1H), 3.99 (s, 3H).
13
C NMR (126 MHz, acetone-d6) δ 192.6, 192.5, 150.7, 148.6, 148.6, 146.0, 138.3, 136.7, 130.9,
130.1, 128.2, 124.7, 121.0, 120.5, 117.1, 39.4.
HRMS (APCI): calcd. for C18H13N3O2 [M+H]+
= 304.1081; found [M+H]+
= 304.1079.
229
Preparative Example 70
The product from Preparative Example 69 (31 mg, 0.102 mmol), NaBH4 (8 mg, 0.204 mmol) and
MeOH (7 mL) were placed into a 10 mL round bottom flask. The mixture was stirred under N2 at 25
°C for 90 min. Aqueous saturated solution of NH4Cl (10 mL) was added and the mixture was
extracted with EtOAc (2x20 mL). The organic phase was washed with brine (10 mL), dried over
MgSO4 and filtered. The solvent was evaporated and the residue was purified by column
chromatography on silica gel (MeOH/EtOAc; 1:12). The product was obtained as a white solid (26
mg; 84 %).
1
H NMR (500 MHz, acetone-d6) δ 8.54 (s, 1H), 8.33 – 8.29 (m, 2H), 8.25 (s, 1H), 8.06 (d, J = 0.6 Hz,
1H), 7.92 (d, J = 8.7 Hz, 1H), 7.66 (d, J = 8.7 Hz, 1H), 7.52 – 7.47 (m, 2H), 4.72 – 4.68 (m, 2H), 3.96
(s, 3H).
13
C NMR (126 MHz, acetone-d6) δ 150.2, 148.4, 146.7, 146.5, 142.9, 138.2, 130.6, 129.9, 127.8,
127.8, 124.9, 122.0, 120.2, 116.7, 64.7, 39.4.
HRMS (APCI): calcd. for C18H15N3O2 [M+H]+
= 306.1237; found [M+H]+
= 306.1242.
Preparative Example 71
By essentially same procedure set forth in Preparative Example 66, using (4(methylsulfonyl)phenyl)boronic
acid instead of (4-carbamoylphenyl)boronic acid, the compound
given below was prepared.
230
White solid.
1
H NMR (300 MHz, acetone-d6) δ 8.78 (s, 1H), 8.70 – 8.63 (m, 2H), 8.31 (s, 1H), 8.12 – 8.02 (m,
3H), 7.98 (d, J = 8.7 Hz, 1H), 7.71 (d, J = 8.7 Hz, 1H), 3.97 (s, 3H), 3.17 (s, 3H).
13
C NMR (75 MHz, acetone-d6) δ 150.7, 148.7, 148.5, 145.9, 141.0, 138.2, 137.4, 130.1, 128.7,
128.3, 124.6, 120.6, 120.4, 117.1, 44.6, 39.4.
HRMS (APCI): calcd. for C18H15N3O3S [M+H]+
= 354.0907; found [M+H]+
= 354.0901.
Preparative Example 72
By essentially same procedure set forth in Preparative Example 66, using (4(methylthio)phenyl)boronic
acid instead of (4-carbamoylphenyl)boronic acid, the compound given
below was prepared.
White solid.
1
H NMR (500 MHz, acetone-d6) δ 8.55 (s, 1H), 8.35 – 8.29 (m, 2H), 8.24 (s, 1H), 8.06 (d, J = 0.5 Hz,
1H), 7.91 (d, J = 8.7 Hz, 1H), 7.65 (d, J = 8.6 Hz, 1H), 7.43 – 7.38 (m, 2H), 3.96 (s, 3H), 2.55 (s, 3H).
13
C NMR (126 MHz, acetone-d6) δ 150.2, 148.4, 146.7, 146.4, 139.0, 138.2, 129.9, 128.8, 128.3,
127.5, 124.9, 121.5, 120.2, 116.7, 39.4, 15.7.
HRMS (ACPI): calcd. for C18H25N3OS [M+H]+
= 322.1009; found [M+H]+
= 322.1003.
231
Preparative Example 73
The product from Preparative Example 72 (13 mg, 40.4 µmol) and CH2Cl2 (2 mL) were placed into a
5 mL round bottom flask. The mixture was cooled to 0 °C, then mCPBA (7.0 mg, 40.4 mmol) was
added and the mixture was stirred under N2 at 0 °C for 45 min. Aqueous saturated solution of
NaHCO3 (5 mL) and H2O (5 mL) were added and the mixture was extracted with CH2Cl2 (2×10 mL).
The organic phase was dried over MgSO4 and filtered. The solvent was evaporated and the residue
was purified by preparative TLC on silica gel (EtOAc/MeOH; 20:1). The product was obtained as a
yellow wax (5 mg; 38 %).
1
H NMR (300 MHz, acetone-d6) δ 8.70 (s, 1H), 8.63 – 8.55 (m, 2H), 8.29 (s, 1H), 8.09 (s, 1H), 7.96
(d, J = 8.7 Hz, 1H), 7.81 (d, J = 8.6 Hz, 2H), 7.69 (d, J = 8.7 Hz, 1H), 3.97 (s, 3H), 2.76 (s, 3H).
13
C NMR (75 MHz, acetone-d6) δ 150.4, 148.4, 147.8, 147.1, 146.1, 138.2, 134.6, 130.0, 128.4,
124.8, 124.7, 120.9, 120.4, 116.9, 44.4, 39.3.
HRMS (APCI): calcd. for C18H15N3O3S [M+H]+
= 338.0958; found [M+H]+
= 338.0955.
Preparative Example 74
By essentially same procedure set forth in Preparative Example 66, using (3-(tertbutyl)phenyl)boronic
acid instead of (4-carbamoylphenyl)boronic acid, the compound given below
was prepared.
Colorless wax.
232
1
H NMR (500 MHz, CDCl3) δ 8.41 (d, J = 0.9 Hz, 1H), 8.12 (s, 1H), 8.04 (s, 1H), 7.97 (s, 1H), 7.84
(m, 1H), 7.74 (d, J = 8.6 Hz, 1H), 7.42 (m, 3H), 3.97 (s, 3H), 1.44 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 151.7, 148.6, 147.6, 145.9, 145.0, 137.7, 130.4, 128.8, 128.5, 124.8,
124.7, 124.4, 124.1, 122.0, 119.1, 115.6, 35.0, 31.6, 31.0.
HRMS (APCI): calcd. for C21H21N3O [M+H]+
= 332.1757; found [M+H]+
= 332.1754.
Preparative Example 75
6-chloro-5-methylpyridin-3-ol (2.51 g, 17.5 mmol), iodine (4.44 g, 17.5 mmol), H2O (35 mL), THF
(30 mL) and Na2CO3 (3.90 g, 36.8 mmol) were placed into a 100 mL round bottom flask. The mixture
was stirred under N2 at 25 °C for 18 h. The solvent was evaporated and the solution was neutralized
with 1 M aqueous solution of HCl (38 mL). Then, saturated aqueous solution of NH4Cl (30 mL) and
H2O (100 mL) were added and the mixture was extracted with CH2Cl2 (80 mL) and EtOAc (2×80
mL). The organic phase was washed with brine (30 mL), dried over MgSO4, filtered, and the solvent
was evaporated. The product was obtained as a white solid (4.24 g; 90 %).
1
H NMR (500 MHz, CDCl3) δ 7.12 (d, J = 0.7 Hz, 1H), 2.31 (d, J = 0.7 Hz, 3H).
13
C NMR (126 MHz, CDCl3) δ 124.8, 19.3.
HRMS (APCI): calcd. for C6H5ClINO [M+H]+
= 269.9178; found [M+H]+
= 269.9179.
Preparative Example 76
The product from Preparative Example 75 (2.2 g, 8.16 mmol), degassed 1,4-dioxane (17 mL) and
TEA (17 mL) were placed into a 100 mL round bottom flask. Then, ethynyltrimethylsilane (1.49 mL,
10.6 mmol), CuI (78 mg, 0.408 mmol) and PdCl2(PPh3)2 (114 mg, 0.163 mmol) were added. The
mixture was stirred under N2 at 45 °C for 3 h. The solvent was evaporated and the residue was
233
purified by column chromatography on silica gel (hexane/EtOAc; 10:1). The product was obtained as
an orange solid (930 mg, 47 %).
1
H NMR (500 MHz, CDCl3) δ 7.62 (s, 1H), 7.01 (d, J = 0.8 Hz, 1H), 2.48 (s, 3H), 0.36 (s, 9H).
13
C NMR (126 MHz, CDCl3) δ 169.5, 150.4, 147.1, 146.1, 127.3, 121.1, 116.5, 20.6, -1.9.
HRMS (APCI): calcd. for C11H14ClNOSi [M+H]+
= 240.0606; found [M+H]+
= 240.0604.
Preparative Example 77
The product from Preparative Example 76 (0.90 g, 3.75 mmol), MeOH (28 mL) and KF (654 mg,
11.3 mmol) were placed into a 100 mL round bottom flask. The mixture was stirred under N2 at 25 °C
for 14 h, then at 60 °C for additional 8 h. The solvent was evaporated and the residue was purified by
column chromatography on silica gel (hexane/EtOAc; from 10:1 to 5:1). The product was obtained as
a white solid (560 mg, 88 %).
1
H NMR (500 MHz, CDCl3) δ 7.79 (d, J = 2.0 Hz, 1H), 7.65 (s, 1H), 6.89 (m, 1H), 2.49 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 149.3, 147.6, 147.4, 145.3, 127.8, 121.4, 107.7, 20.6.
HRMS (APCI): calcd. for C8H6ClNO [M+H]+
= 168.0211; found [M+H]+
= 168.0209.
Preparative Example 78
Degassed 1,2-dimethoxyethane (4.0 mL) and H2O (1.0 mL) were placed into a 25 mL round bottom
flask. Then, the product from Preparative Example 77 (160 mg, 0.94 mmol), 1-methyl-4-(4,4,5,5-
234
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (294 mg, 1.42 mmol), PdCl2(dppf) (34 mg, 47
µmol) and K3PO4 (599 mg, 2.82 mmol) were added. The mixture was stirred under N2 at 80 °C for 5
h. The solvent was evaporated and the residue was purified by column chromatography on silica gel
(EtOAc/MeOH; from 1:0 to 10:1) and then by another column chromatography (EtOAc). The product
was obtained as a white solid (120 mg; 60 %).
1
H NMR (500 MHz, CDCl3) δ 7.90 (s, 1H), 7.82 (s, 1H), 7.76 (d, J = 2.3 Hz, 1H), 7.60 (s, 1H), 6.92
(dd, J = 2.2, 0.9 Hz, 1H), 3.97 (s, 3H), 2.56 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 148.6, 146.9, 145.3, 139.3, 137.0, 130.3, 126.6, 123.2, 120.4, 108.0,
39.1, 21.6.
HRMS (APCI): calcd. for C12H11N3O [M+H]+
= 214.0975; found [M+H]+
= 214.0972.
Preparative Example 79
The product from Preparative Example 78 (115 mg, 0.54 mmol) and CCl4 (5 mL) were placed into a
100 mL round bottom flask. The mixture was cooled to -18 °C, then bromine (0.56 mL, 10.8 mmol)
was added slowly. The mixture was allowed to warm to 12 °C and stirred under N2 for 60 min. The
mixture was poured into a mixture of water (30 mL), ice (20 mL) and Na2S2O5 (2 g). The resulting
mixture was extracted with CH2Cl2 (20 mL) and EtOAc (2×15 mL). The organic phase was dried over
MgSO4, filtered, and the solvent was evaporated. Toluene (12 mL) and DBU (0.241 mL, 1.62 mmol)
were added to the residue and the mixture was stirred under N2 at 80 °C for 45 min. The solvent was
evaporated and the residue was purified by column chromatography on silica gel (EtOAc/hexane; 1:1)
and then by preparative TLC (EtOAc/hexane; 2:1). The product was obtained as a white solid (19 mg,
12 %).
1
H NMR (500 MHz, CDCl3) δ 7.93 (d, J = 2.3 Hz, 2H), 7.80 (s, 1H), 7.61 (s, 1H), 3.98 (s, 3H), 2.60
(s, 3H).
13
C NMR (126 MHz, CDCl3) δ 149.2, 146.5, 146.1, 142.5, 139.3, 130.9, 127.9, 122.98 (s), 121.0,
99.5, 39.2, 21.7.
235
HRMS (APCI): calcd. for C12H10BrN3O [M+H]+
= 292.0080; found [M+H]+
= 292.0075.
Preparative Example 80
Degassed 1-butanol (2.0 mL) and H2O (0.4 mL) were placed into a 10 mL round bottom flask. Then,
the product from Preparative Example 79 (15 mg, 51.3 µmol), naphthalen-2-ylboronic acid (13.3 mg,
77 µmol), Pd(PPh3)4 (3.0 mg, 2.6 µmol) and K3PO4 (32.7 mg, 0.154 mmol) were added. The mixture
was stirred under N2 at 80 °C for 70 min. The solvent was evaporated and the residue was purified by
column chromatography on silica gel (EtOAc/CH2Cl2; 1:2) and then by preparative TLC
(EtOAc/CH2Cl2; 2:3). The product was obtained as a colorless wax (8.5 mg; 49 % yield).
1
H NMR (300 MHz, CDCl3) δ 8.88 (s, 1H), 8.18 (s, 1H), 8.14 – 8.07 (m, 2H), 7.97 – 7.84 (m, 4H),
7.64 (d, J = 0.5 Hz, 1H), 7.55 – 7.44 (m, 2H), 4.03 (s, 3H), 2.64 (s, 3H).
13
C NMR (75 MHz, CDCl3) δ 148.1, 148.0, 144.9, 143.9, 139.8, 133.9, 133.0, 130.5, 128.6, 128.5,
128.4, 127.9, 126.5, 126.4, 126.3, 126.1, 125.0, 123.9, 121.4, 120.7, 39.3, 21.9.
HRMS (APCI): calcd. for C22H17N3O [M+H]+
= 340.1444; found [M+H]+
= 340.1440.
Preparative Example 81
Degassed 1-butanol (2.0 mL) and H2O (0.4 mL) were placed into a 10 mL round bottom flask. Then,
the product from Preparative Example 58 (23.8 mg, 85.6 µmol), (4-(1H-tetrazol-5-yl)phenyl)boronic
236
acid (19.5 mg, 0.103 mmol), Pd(PPh3)4 (5.0 mg, 4.3 µmol) and K3PO4 (54.5 mg, 0.257 mmol) were
added. The mixture was refluxed under N2 for 150 min. The solvent was evaporated and the residue
purified by column chromatography on silica gel (EtOAc/MeOH; from 3:1 to 2:1) and then by
preparative TLC (THF/MeOH; 2:1). The product was obtained as a colorless semi-solid (12 mg; 41
%).
1
H NMR (300 MHz, CD3OD) δ 8.44 (s, 1H), 8.37 – 8.30 (m, 2H), 8.20 – 8.13 (m, 3H), 8.05 (d, J =
0.7 Hz, 1H), 7.83 (d, J = 8.7 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H), 3.96 (s, 3H).
13
C NMR (75 MHz, CD3OD) δ 149.9, 148.9, 147.3, 146.6, 138.4, 132.7, 130.7, 130.0, 128.2, 127.9,
126.5, 125.4, 122.0, 120.3, 116.9, 39.1.
HRMS (APCI): calcd. for C18H13N7O [M+H]+
= 344.1254; found [M+H]+
= 344.1252.
ASSAYS:
In vitro essays were performed by the company Merck Millipore in their KinaseProfiler radiometric
protein kinase assay as paid commercial service. The compounds’ IC50 values for inhibition of
individual protein kinases were determined. Dose-response curves were plotted from inhibition data
generated, each in duplicate, from 10 point serial dilutions of inhibitory compounds. Concentration of
compound was plotted against % kinase activity. To generate IC50 values, the dose-response curves
were fitted to a standard sigmoidal curve and IC50 values were derived by standard nonlinear
regression analysis.
All tested compounds were prepared in 100% DMSO to final assay concentrations either 0.5 mM (for
the concentration row A, see below) or 0.05 mM (for the concentration row B). This working stock of
the compound was added to the assay well as the first component in the reaction, followed by the
remaining components as detailed below. The stock solution was added to the individual assay wells
in such amounts that the concentrations of the compound were either in the row A (0.001 μM, 0.003
μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1.0 μM, 3.0 μM, and 10.0 μM) or in the row B (0.0001 μM,
0.0003 μM, 0.001 μM, 0.003 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, and 1.0 μM). There was no
pre-incubation step between the compound and the kinase prior to initiation of the reaction.
237
The positive control wells contained all components of the reaction, except the compound of interest;
however, DMSO (at a final concentration of 2%) was included in these wells to control for solvent
effects. The blank wells contained all components of the reaction, with staurosporine as a reference
inhibitor replacing the compound of interest. This abolished kinase activity and established the baseline
(0% kinase activity remaining).
CLK2 assay
CLK2 (h) was diluted in the buffer (20 mM MOPS (3-(N-morpholino)propanesulfonic acid), 1 mM
EDTA (ethylendiaminotetraacetic acid), 0.01% Brij-35 (detergent), 5% Glycerol, 0.1% βmercaptoethanol,
1 mg/mL BSAs) to the concentration of 1.01 mg/mL prior to addition to the
reaction mix.
The above stock solution of CLK2(h) was added to a mixture containing 8 mM MOPS pH 7.0, 0.2
mM EDTA, and 20 μM YRRAAVPPSPSLSRHSSPHQS(p) EDEEE in such amount that the resulting
concentration of CLK2(h) was 2.1 nM. This mixture was added to the stock solution of the tested
compound. The reaction was initiated by the addition of the MgATP mix in such amount that the
resulting concentration of Mg acetate in the reaction mixture was 10 mM and [γ-33
P-ATP] (specific
reaction was stopped by the addition of 3% phosphoric acid solution. 10 μL of the reaction was then
spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and
once in methanol prior to drying and scintillation counting.
CLK4 assay
CLK4 (h) was diluted in the buffer (20 mM MOPS, 1 mM EDTA, 0.01% Brij-35, 5% Glycerol, 0.1%
β-mercaptoethanol, 1 mg/mL BSAs) to the concentration of 1.01 mg/mL prior to addition to the
reaction mix.
The above stock solution of CLK4(h) was added to a mixture containing 8 mM MOPS pH 7.0, 0.2
mM EDTA, and 200 μM YRRAAVPPSPSLSRHSSPHQS(p) EDEEE in such amount that the
resulting concentration of CLK4(h) was 140.8 nM. This mixture was added to the stock solution of
the tested compound. The reaction was initiated by the addition of the MgATP mix in such amount
that the resulting concentration of Mg acetate in the reaction mixture was 10 mM and [γ-33
P-ATP]
238
(specific activity approx. 500 cpm/pmol) was 15 μM. After incubation for 40 minutes at room
temperature, the reaction was stopped by the addition of 3% phosphoric acid solution. 10 μL of the
reaction was then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM
phosphoric acid and once in methanol prior to drying and scintillation counting.
HIPK1 assay
HIPK1(h) was diluted in the buffer (20 mM MOPS, 1 mM EDTA, 0.01% Brij-35, 5% Glycerol, 0.1%
β-mercaptoethanol, 1 mg/mL BSAs) to the concentration of 1.01 mg/mL prior to addition to the
reaction mix.
The above stock solution of HIPK1(h) was added to a mixture containing 8 mM MOPS pH 7.0, 0.2
mM EDTA, and 0.33 mg/mL myelin basic protein in such amount that the resulting concentration of
HIPK1(h) was 4.7 nM. This mixture was added to the stock solution of the tested compound. The
reaction was initiated by the addition of the MgATP mix in such amount that the resulting
concentration of Mg acetate in the reaction mixture was 10 mM and [γ-33
P-ATP] (specific activity
approx. 500 cpm/pmol) was 45 μM. After incubation for 40 minutes at room temperature, the reaction
was stopped by the addition of 3% phosphoric acid solution. 10 μL of the reaction was then spotted
onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in
methanol prior to drying and scintillation counting.
HIPK2 assay
HIPK2(h) was diluted in the buffer (20 mM MOPS, 1 mM EDTA, 0.01% Brij-35, 5% Glycerol, 0.1%
β-mercaptoethanol, 1 mg/mL BSAs) to the concentration of 1.01 mg/mL prior to addition to the
reaction mix.
The above stock solution of HIPK2(h) was added to a mixture containing 8 mM MOPS pH 7.0, 0.2
mM EDTA, and 0.33 mg/mL myelin basic protein in such amount that the resulting concentration of
HIPK2(h) was 1.4 nM. This mixture was added to the stock solution of the tested compound. The
reaction was initiated by the addition of the MgATP mix in such amount that the resulting
concentration of Mg acetate in the reaction mixture was 10 mM and [γ-33
P-ATP] (specific activity
approx. 500 cpm/pmol) was 10 μM. After incubation for 40 minutes at room temperature, the reaction
was stopped by the addition of 3% phosphoric acid solution. 10 μL of the reaction was then spotted
239
onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in
methanol prior to drying and scintillation counting.
HIPK3 assay
HIPK3(h) was diluted in the buffer (20 mM MOPS, 1 mM EDTA, 0.01% Brij-35, 5% Glycerol, 0.1%
β-mercaptoethanol, 1 mg/mL BSAs) to the concentration of 1.01 mg/mL prior to addition to the
reaction mix.
The above stock solution of HIPK3(h) was added to a mixture containing 8 mM MOPS pH 7.0, 0.2
mM EDTA, and 1.0 mg/mL myelin basic protein in such amount that the resulting concentration of
HIPK3(h) was 6.4 nM. This mixture was added to the stock solution of the tested compound. The
reaction was initiated by the addition of the MgATP mix in such amount that the resulting
concentration of Mg acetate in the reaction mixture was 10 mM and [γ-33
P-ATP] (specific activity
approx. 500 cpm/pmol) was 15 μM. After incubation for 40 minutes at room temperature, the reaction
was stopped by the addition of 3% phosphoric acid solution. 10 μL of the reaction was then spotted
onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in
methanol prior to drying and scintillation counting.
FLT3 assay
FLT3(h) was diluted in the buffer (20 mM MOPS, 1 mM EDTA, 0.01% Brij-35, 5% Glycerol, 0.1%
β-mercaptoethanol, 1 mg/mL BSAs) to the concentration of 1.01 mg/mL prior to addition to the
reaction mix.
The above stock solution of Flt3(h) was added to a mixture containing 8 mM MOPS pH 7.0, 0.2 mM
EDTA, and 50 μM EAIYAAPFAKKK, in such amount that the resulting concentration of FLT3(h)
was 28.3 nM. This mixture was added to the stock solution of the tested compound. The reaction was
initiated by the addition of the MgATP mix in such amount that the resulting concentration of Mg
acetate in the reaction mixture was 10 mM and [γ-33
P-ATP] (specific activity approx. 500 cpm/pmol)
was 200 μM. After incubation for 40 minutes at room temperature, the reaction was stopped by the
addition of 3% phosphoric acid solution. 10 μL of the reaction was then spotted onto a P30 filtermat
and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying
and scintillation counting.
240
TRKA assay
TRKA(h) was diluted in the buffer (20 mM MOPS, 1 mM EDTA, 0.01% Brij-35, 5% Glycerol, 0.1%
β-mercaptoethanol, 1 mg/mL BSAs) to the concentration of 1.01 mg/mL prior to addition to the
reaction mix.
The above stock solution of TRKA(h) was added to a mixture containing 8 mM MOPS pH 7.0, 0.2
mM EDTA, and 250 μM KKKSPGEYVNIEFG, in such amount that the resulting concentration of
TRKA(h) was 28.2 nM. This mixture was added to the stock solution of the tested compound. The
reaction was initiated by the addition of the MgATP mix in such amount that the resulting
concentration of Mg acetate in the reaction mixture was 10 mM and [γ-33
P-ATP] (specific activity
approx. 500 cpm/pmol) was 120 μM. After incubation for 40 minutes at room temperature, the
reaction was stopped by the addition of 3% phosphoric acid solution. 10 μL of the reaction was then
spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and
once in methanol prior to drying and scintillation counting.
241
Results
A: IC50 < 0.100 M
B: IC50 < 1.00 M
C: IC50 < 5.00 M
compound CLK2 CLK4 FLT3 HIPK1 HIPK2 HIPK3 DYRK2 TRKA
6A B A B B C C
6D C B C C
7A A A B B B
7B A A A A A C
7C C C C
7D C C C B C
7F B B B A
8B B B C
8C C C C
8D C
8E C C C
9 A A A A B C
242
compound CLK2 CLK4 FLT3 HIPK1 HIPK2 HIPK3 DYRK2 TRKA
12B C B
17E C C
23 B B C
54 C C B B
55 B B
61 A A B A C
62 C C B B
65 A B B
66 B B C
70 B B C
71 B C C
73 C C
74 B C
80 B
81 C C C
243
Part 4a
Syntheses of 5´-amino-2´,5´-dideoxy-2´, 2´-difluorocytidine derivatives
as novel anticancer nucleoside analogs*
*published as:
Labroli, M.; Dwyer, M. P.;*
Shen, R.; Popovici-Muller, J.; Pu, Q.; Richard, J.; Rosner, K.; Paruch, K.; Guzi, T.
J. Syntheses of 5´-amino-2´,5´-dideoxy-2´,2´-difluorocytidine Derivatives as Novel Anticancer Nucleoside
Analogs. Tetrahedron Lett. 2014, 55, 598.
2´-Deoxy-2´, 2´-difluorocytidine, the antimetabolite nucleoside known as gemcitabine (1), is
approved for the treatment of pancreatic, breast, and non-small cell lung cancers either as a single
agent or in combination with other chemotherapeutic agents.1
Gemcitabine acts as a prodrug which is
intracellularly phosphorylated to its active diphosphate and triphosphate intermediates.2
Gemcitabine
triphosphate competes with deoxycytidine triphosphate for incorporation into DNA which results in
termination of DNA polymerization.3
The diphosphate intermediate effectively inhibits
ribonucleotide reductase (RRM1) which leads to the depletion of the deoxynucleotide pool and halt of
DNA synthesis.4
Since the discovery of gemcitabine by Hertel et al.,5
numerous gemcitabine
derivatives have been synthesized in search for new anticancer or antiviral agents. For example, the
base-modified gemcitabine derivatives include adenosine, guanine, and uracil analogs.6
The ribosemodified
derivatives include 4´-azido analogs,7
4´-allene substituted analogs,8
3´-deoxy analogs,9
and
thio/aza/carbocyclic analogs.10
However, to our knowledge, the 5´-amino derivatives of gemcitabine,
a novel and potentially biologically interesting class of compounds, have not been actively
investigated. As part of our program to identify novel and selective anticancer compounds, we
initiated efforts to develop efficient syntheses of 5´- amino-2´,5´-dideoxy-2´,2´-difluorocytidine
derivatives in order to profile this novel series. Herein, we describe our chemistry efforts to prepare
5´-amino derivatives of gemcitabine.11
Our initial route to the 5´-amino-2´,5´-dideoxy-20,20-difluorocytidine analogs is outlined in Scheme
1. The synthesis began with protection of the 5´-hydroxy group of gemcitabine hydrochloride (1) as
the tert-butyldimethylsilyl (TBDMS) ether followed by global benzoyl protection to afford compound
2. Desilylation of the silyl ether was achieved with TBAF at 0 °C to provide alcohol 312
which was
converted to tosylate 4 under standard conditions. Treatment of intermediate 4 with excess amine
244
coupling partner at 100 °C promoted both tosylate displacement and benzoyl group cleavage to afford
products 6a-d,i (Table 1).
Scheme 1. Reagents and conditions: (i) TBSCl, imidazole, DMF, rt, 12 h, 92%; (ii) BzCl, DMAP, pyridine, rt,
12 h, 90%; (iii) TBAF, THF, 0 °C, 3.5 h then AcOH, 85%; (iv) TsCl, pyridine, Et3N, 85%; (v) HNRR1
, DMF,
90–100 °C; (vi) NH3, MeOH, 58%; HNRR1
, DMF; (vii) MeP(OPh)3I, DMF, 60 °C, 84%; (viii) NH3, MeOH,
92%.
Alternatively, this protocol could be carried out in a step-wise fashion by first removing the benzoyl
groups of 4 by treatment with 7 N ammonia in methanol to provide the penultimate product.
Treatment of the resulting tosylate with excess amine provided the desired compounds 6e,f (Table 1).
While the displacement reaction with the amine coupling afforded modest yields of the desired
products (Table 1), multiple purifications were required to completely remove the resultant TsOH
from desired product. In order to circumvent this issue, alcohol 3 was converted to the iodide
intermediate 5 using methyltriphenoxyphosphonium iodide followed by benzoyl deprotection.
Treatment of iodide 5 with 3 equiv of amines at 90 °C provided 5´-amino-2´,5´-dideoxy-2´,2´difluorocytidine
6g,h in respectable yields (Table 1).
245
Table 1. Preparation of 6a–i by SN2 substitution.
a Yield for one step.
b Combined yield for two steps.
c Rxn heated at 40 °C.
246
The use of iodide 5 allowed us to scale back the number of equivalents of amine for the displacement
reaction while making the purification of the final analogs much easier.
While the SN2 strategy depicted in Scheme 1 represented a concise preparation of 5´-amino-2´,5´dideoxy-2´,2´-difluorocytidine
derivatives, we decided to pursue other alternatives due to the poor to
modest yields for the final compounds. Toward this end, a reductive amination approach was
explored not only to improve the yield of the final 5´-amino-2´,5´-dideoxy-2´,2´-difluorocytidine
analogs but to broaden the scope of coupling partners. As shown in Scheme 2, alcohol 3 was
converted to the mesylate followed by treatment with NaN3 to afford the corresponding azide 7.
Treatment of azide 7 with trimethylphosphine provided amine intermediate 8 which was used directly
without silica gel purification to prevent benzoyl transfer to the 5´-amino group.
Scheme 2. Reagents and conditions: (i) MsCl, Et3N, pyridine, 89%; (ii) NaN3, DMF, 70 °C, 98%; (iii) Me3P,
MeCN/H2O, 85%; (iv) RCHO, NaBH3CN, MeOH, AcOH, 35–75%; (v) 7 M NH3/MeOH, 45–85%.
Treatment of 8 with various aldehydes under reductive amination conditions (NaBH3CN) afforded the
N-alkylated products which were treated with 7 M NH3 in MeOH to afford 5´-substituted amino
analogs 9a-f shown in Table 2. The reductive amination/deprotection protocol worked well for both
aromatic and aliphatic aldehydes (entries 9a-d) with respectable yields observed for both steps (Table
2). Additionally, this protocol was adapted to a stepwise format by employing two distinct aldehydes
to afford 9e or by employing a dicarbonyl substrate 10 to afford bicyclic derivative 9f.
247
Table 2. Reductive amination protocol/deprotection from 8 to afford 9a–f (Scheme 2).
a
Amine 8 was used as the crude product of reduction of azide 7.
b
Prepared by one-pot stepwise reductive amination.
While the reductive amination approach utilizing amine 8 proceeded well with aldehydes, this
protocol yielded very low yields of -substituted amine products when ketones were used as coupling
partners. Owing to the reduced electrophilicity and increased steric hindrance of the ketone coupling
partners, it was rationalized that a strong Lewis acid may be required to promote the initial imine
formation for these substrates. It was rationalized that one might need an alternative 5´-amino
intermediate with a suitable protecting group for harsher imine formation conditions due to
potentially labile 3´-benzoate of 8. Additionally, it was desirable to develop a flexible route which
could allow for rapid base modification (other than cytosine) while at the same time circumventing
the need to use expensive gemcitabine as a starting material. Based on the above considerations, a
248
second reductive amination synthetic route was developed to prepare 5´-amino-2´,5´-dideoxy-2´,2´difluorocytidine
derivatives derived from ketone coupling partners.
The synthesis started with commercially available 2-deoxy-2,2-difluoro-D-erythro-pentafuranous-1ulose-3,5-dibenzoate
(11) (Scheme 3). Following the protocol described by Chou et al.,13
lactone 11
was converted to nucleoside 12 in three steps as a mixture of both anomers (: = 1:1.5). The 4amino
group of the cytosine was protected as the Boc derivative followed by hydrolysis of the 3´,5´dibenzoate
under mild basic conditions to provide diol 13. Based on the selective iodination protocol
of thymidine reported by Verheyden and Moffatt,14
treatment of diol 13 with
methyltriphenoxyphosphonium iodide afforded only the 5´-iodo nucleoside anomers 14 and 15.
Scheme 3. Reagents and conditions: (i) Boc2O, DMAP, THF, 75%; (ii) Et3N/MeOH/H2O, rt, 12 h, 92%; (iii)
MeP(OPh)3I, DMF, rt, 1 h, 31% (14), 45% (15); (iv) NaN3, DMF, 40 °C, quant.; (v) Me3P, MeCN/H2O, 73%;
(vi) RCOMe, Ti(i-OPr)4, then NaBH3CN, 51–68%; (vii) TFA, CH2Cl2, 71–98%.
Presumably, the selectivity of this transformation is driven by the reduced nucleophilicity of the 3´hydroxyl
caused by the geminal di-fluorine atoms which yields only the 5´-iodo products.
Fortunately, the 5´-iodo anomers 14 and 15 could be separated by regular silica gel chromatography.
Treatment of -anomer 14 with sodium azide followed by reduction furnished the 5´-amino
intermediate 16. Treatment of amine 16 with aromatic ketones in the presence of Ti(i-OPr)4
15
249
followed by treatment with NaBH3CN and Boc deprotection leads to a-methylated 5´-amino
compounds 17a–d as a mixture of diastereomers in moderate to good yields (Scheme 3). The specific
yields for the reductive amination step as well as deprotection are detailed in Table 3.
Table 3. Reductive amination/deprotection protocol from 16 to afford 17a–d (Scheme 3).
In summary, we have described three unique synthetic routes for the syntheses of a novel class of 5´amino-2´,5´-dideoxy-2´,2´-
difluorocytidine derivatives. The first route relied upon a SN2
displacement of either a 5´-tosylate or 5´-iodide intermediate using excess amine as coupling partners.
To circumvent the modest yields and challenging purifications of final products using the first route, a
second route was developed which relied upon a reductive amination of a 5´-amino derivative with
aldehydes to afford a wide variety of 5´-N-alkylated derivatives. Finally, the final route relies upon a
reductive amination step with ketones but also offers the ability to change the base with fewer
manipulation of protecting groups than the previous routes. While the 5´-amino intermediates 8 and
16 were critical components for the reductive amination protocols described, one could envision the
utility of these materials for preparation of such non-basic analogs such as amides, sulfonamides, and
ureas. The biological activity associated with this novel class of 5´-amino-2´,5´-dideoxy-2´,2´difluorocytidine
derivatives will be reported in due course.16
250
References and notes
1. (a) Burris, H. A., III; Moore, M. J.; Andersen, J.; Green, M. R.; Rothenberg, M. L.; Modiano, M.
R.; Cripps, M. C.; Portenoy, R. K.; Storniolo, A. M.; Tarassoff, P.; Nelson, R.; Dorr, F. A.; Stephens,
C. D.; Von Hoff, D. D. J. Clin. Oncol. 1997, 15, 2403; (b) Manegold, C. Expert Rev. Anticancer
Ther. 2004, 4, 345; (c) Heinemann, V. Expert Rev. Anticancer Ther. 2005, 5, 429.
2. Heinemann, V.; Hertel, L. W.; Grindey, G. B.; Plunkett, W. Cancer Res. 1988, 48, 4024.
3. Huang, P.; Chubb, S.; Hertel, L. W.; Grindey, G. B.; Plunkett, W. Cancer Res. 1991, 51, 6110.
4. (a) van der Donk, W. A.; Yu, G.; Pérez, L.; Sanchez, R. J.; Stubbe, J.; Samano, V.; Robins, M. J.
Biochemistry 1998, 37, 6419; (b) Artin, E.; Wang, J.; Lohman, G. J. S.; Yokoyama, K.; Yu, G.;
Griffin, R. G.; Bar, G.; Stubbe, J. Biochemistry 2009, 48, 11622.
5. Hertel, L. W.; Kroin, J. S.; Misner, J. W.; Tustin, J. M. J. Org. Chem. 1988, 53, 2406.
6. (a) Hertel, L. W.; Grossman, C. S.; Kroin, J. S.; Mineishib, S.; Chubb, S.; Nowak, B.; Plunkett, W.
Nucleos. Nucleot. 1989, 8, 951; (b) Fahrig, R.; Lohmann, D.; Rolfs, A.; Dieks, H.; Teubner, J.;
Heinrich, J.-C. WO 2008017515; Chem. Abstr. 2008, 148, 239457.
7. Smith, D. B.; Kalayanov, G.; Sund, C.; Winqvist, A.; Maltseva, T.; Leveque, V. J.-P., et al J. Med.
Chem. 2009, 52, 2971.
8. Qiu, Y.-L.; Wang, C.; Peng, X.; Ying, L.; Or, Y. S. WO 2010030858; Chem. Abstr. 2010, 152,
335423.
9. Hertel, L. W.; Grossman, C. S.; Kroin, J. S. EP 329348, 1989; Chem. Abstr. 1989, 112, 56592.
10. (a) Qiu, X.-L.; Xu, X.-H.; Qing, F.-L. Tetrahedron 2010, 66, 789; (b) Devos, R.; Dymock, B. W.;
Hobbs, C. J.; Jiang, W.-R.; Martin, J. A.; Merrett, J. H.; Najera, I.; Shimma, N.; Tsukuda, T. WO
2002018404; Chem. Abstr. 2002, 136, 217007.
11. Guzi, T. J.; Parry, D. A.; Labroli, M. A.; Dwyer, M. P.; Paruch, K.; Rosner, K. E.; Shen, R.;
Popovici-Muller, J. WO 2009061781; Chem. Abstr. 2009, 150, 515402.
12. It was necessary to maintain the desilylation reaction of 2 at low temperature and higher dilution
while quenching the reaction with acetic acid to suppress gemcitabine 3´,5´,4-tribenzoate formation
as a side product. This side product
could be formed by intermolecular benzoyl transfer from the less stable 3´-benzoate to 5´-hydroxyl
group.
13. Chou, T. S.; Heath, P. C.; Patterson, L. E.; Poteet, L. M.; Lakin, R. E.; Hunt, A. H. Synthesis
1992, 565.
14. Verheyden, J. P. H.; Moffatt, J. G. J. Org. Chem. 1970, 35, 2319.
15. Mattson, R. J.; Pham, K. M.; Leuck, D. J.; Cowen, K. A. J. Org. Chem. 1990, 55, 2552.
251
16. Labroli, M. A.; Dwyer, M. P.; Shen, R.; Popovici-Muller, J.; Pu, Q.; Wyss, D.; McCoy, M.;
Barrett, D.; Davis, N.; Seghezzi, W.; Shanahan, F.; Taricani, L.; Parry, D.; Guzi, T. J. Bioorg. Med.
Chem. submitted for publication.
Note: Experimental details can be found in our publicly available patent WO 2009/061781 A1.
252
253
Part 4b
New carbocyclic nucleosides: synthesis of carbocyclic pseudoisocytidine
and its analogs*
*published as:
Maier, L.; Hylse, O.; Nečas, M.; Trbušek, M.; Ytre-Arne, M.; Dalhus, B.; Bjorås, M.; Paruch, K.*
New
Carbocyclic Nucleosides: Synthesis of Carbocyclic Pseudoisocytidine and its Analogs. Tetrahedron Lett. 2014,
55, 3713.
Nucleoside analogs represent a diverse group of organic compounds. Appropriate modifications of
the nucleoside scaffold can result in significantly altered biological activity.1
As natural nucleosides
contain the relatively labile aminal motif (structure A in Figure 1), significant effort has been invested
in order to identify more stable analogs.
Figure 1. The common scaffold of natural nucleosides (A) and the structures of their analogs B, C, D.
One strategy consists of replacing the tetrahydrofuran ring with an appropriate carbocyclic isostere.
The resulting carbanucleosides have been synthesized by diverse synthetic methods,2
and in many
cases, it has been observed that the tetrahydrofuran ring can be replaced with cyclopentane (structure
B in Figure 1) without significant loss of biological activity.3
Naturally occurring representatives of
this series iclude aristeromycin and its unsaturated analog (-)-neplanocin A.4
Another strategy is
based on attachment of the heterocyclic base to the tetrahydrofuran via a C-C bond linkage (structure
C in Figure 1). While the resulting C-nucleosides are in general more stable than natural nucleosides,
the synthesis of even relatively simple systems (e.g., tiazofurin and its analogs) in this series is often
not trivial.5
In addition, some C-nucleosides can still undergo ring-opening of the furan (see below).
Structure D in Figure 1 combines elements of structures B and C: carbocyclic C-nucleosides with a
254
C-C connection between the (heterocyclic) base and the carbocyclic scaffold. It is conceivable that, at
least in some cases, these compounds might be more robust versions of nucleoside analogs B and C.
Furthermore, the installation of certain substituents (e.g., R = OH) is meaningful only in this series, as
this would lead to chemically unstable ketals and aminals in the other series. Interestingly,
compounds with general structure D (where R = H) are quite rare6
and we have been unable to find
any analogs of type D (containing R = OH) with nucleoside-like substitution patterns.
One attractive biologically active candidate for the tetrahydrofuran-cyclopentane replacement is
pseudoisocytidine (1), which has been shown to be active against cytarabine-resistant leukemias,7
but
hepatotoxic in vivo,8
which may be the result of opening of the tetrahydrofuran core (Scheme 1).9
Scheme 1. Opening of pseudoisocytidine 1 and the structures of its direct carbocyclic analog 2a and related
compounds 2b and 2c.
The direct carbocyclic analog 2a cannot undergo such a ring-opening process and its toxicological
profile might therefore be superior to that of pseudoisocytidine, while its biological activity could be
retained (Scheme 1). Herein, we report the first synthesis of the previously unknown carbocyclic
pseudoisocytidine analog 2a and its derivatives 2b and 2c.
255
Scheme 2. Retrosynthetic analysis of pseudoisocytidine analogs (PG = protecting group).
The overall retrosynthetic strategy is depicted in Scheme 2. It includes the previously described
substituted norbornene intermediate 5,10
which could yield ketoester 4 after oxidative cleavage. The
desired stereochemistry of 4 is dictated by the geometry of the bicyclo[2.2.1]heptene scaffold
produced in a Diels-Alder reaction between cyclopentadiene and appropriately substituted dienophile
6, possessing a leaving group that is utilized in a subsequent elimination-diastereoselective cisdihydroxylation
sequence. Compound 4 was envisioned as a precursor to the novel aldehyde-ester 3,
which upon reaction with urea, thiourea or guanidine, and global deprotection would provide the
target compounds 2a-c.
The synthesis started from commercially available methyl propiolate, which was converted into
methyl (Z)-3-bromo-2-propenoate (6a) (Scheme 3).11
256
Scheme 3. Reagents and conditions: a) EtAlCl2, cyclopentadiene, CH2Cl2, 0 °C; 80% for 7a, 70-95% for 7b. b)
OsO4, NMO, acetone:H2O (4:1), 40 °C; 80% for 8a, 90% for 8b. c) Me2CH(OMe)2, cat. TsOH, acetone, r. t.;
99% for 9a and 9b. d) DBU, Et2O, 0 °C to r. t.; 95%, for 9a, DBU, CH3CN, 90 °C; 86% for 9b. e) O3, CH2Cl2, -
78 °C then Me2S -78 °C to room temp. f) Li(Al-O-tBu)3H, THF, 0 °C to r. t.; 50-80% from 10. g) TBDPSCl,
imidazole, CH2Cl2, r. t.; 70-92%.
Since we have found that the bromo compound 6a irritates skin (especially upon repeated exposure),
we have utilized an alternative starting material for the Diels-Alder reaction, sulfone 6b.12
Thermal
Diels-Alder reaction between cyclopentadiene and 6a was very sluggish - the conversion after 24
hours in refluxing benzene or toluene was negligible and significant dimerization of cyclopentadiene
occurred. On the other hand, the reaction proceeded smoothly at low temperature (0 °C), catalysed by
257
EtAlCl2, with very good diastereoselectivity (9:1 endo:exo). Sulfone 6b underwent an uncatalyzed
Diels-Alder reaction quite efficiently (60% yield, r. t., 14 h), although a higher conversion and yield
were obtained in the presence of the catalyst (EtAlCl2). The structure of the major endo diastereomer
7b was confirmed by X-ray crystallography (see Supporting Information). Diastereoselective cisdihydroxylation
of both adducts 7a and 7b provided the corresponding diols 8a and 8b, which were
subsequently protected as acetonides. Elimination of the bromide or phenylsulfonyl group under basic
conditions afforded the key intermediate 10, which underwent ozonolytic cleavage to produce the
rather unstable aldehyde 11 that was immediately used in the next step without purification. It should
be noted that, in principle, compound 10 could be prepared more directly by dihydroxylation and
protection of the Diels-Alder adduct of methyl propiolate and cyclopentadiene, which we prepared in
60-80% yield (cat. AlCl3, PhH, 0 °C). Attempted dihydroxylations of the adduct, however, yielded
complex hydroxylation mixtures that contained the desired product together with the unwanted
diastereomer, the regioisomer with a dihydroxylated double bond in the vicinity of the ester group as
well as a tetrahydroxylated product. In accordance with the published results,10
our attempts to
selectively reduce the aldehyde in the presence of an α-ketoester were not successful. On the other
hand, reduction with excess Li(Al-O-t-Bu)3H yielded an inseparable mixture of epimeric diols in
which the primary hydroxyl group could be selectively silylated to provide α-hydroxyester 13
(Scheme 3).
Oxidation of the hydroxyl group in 13 proved challenging. Many standard methods (e.g., MnO2, PDC,
KMnO4, Swern oxidation) including RuO2 plus NaIO4 conditions that were previously used for a
structurally similar intermediate,10
failed to give the desired product 14 in acceptable yield.
Fortunately, oxidation with Dess-Martin periodinane yielded pure α-ketoester 14 in very good yield
and purity (Scheme 4).
258
Scheme 4. Reagents and conditions: a) Dess-Martin periodinane, CH2Cl2, 0 °C to r. t.; 80-90%. b)
Ph3P+
CH2OMeCl,
LDA, THF, 0 °C to r. t.; 37-65% (Z:E 7:5). c) guanidinium.HCl for compound 16a (20-
35%), urea for 16b (30-45%) and thiourea for 16c (40-50%), t-BuOK, t-BuOH, reflux. d) TBAF, wet THF, r. t.;
90% for 17a, 96% for 17b, 90% for 17c. e) HCl:H2O:MeOH 1:1:1, r. t.; 69% for 17a, 76% for 17b, 71% for
17c.
One-carbon homologation was accomplished via the Wittig reaction with
methoxymethylenetriphenylphosphorane. The reaction produced enol ether 15 in 37-65% yield as a
separable mixture of Z and E isomers (Z:E ~7:5). It should be noted that the Wittig olefination was
rather sensitive to the type and quality of base. Generation of the phosphonium ylide with LDA gave
the most consistent and reproducible results, while reactions with LiHMDS, KHMDS, or t-BuOK
afforded olefination products in substantially lower yields. Attempts to hydrolyze selectively enol
ether 15 into the desired aldehyde 3 in the presence of the acetonide and TBPDS groups met with
259
only limited success. With PPTS or acetic acid, partial cleavage of the acetonide and/or TBDPS
groups was observed, while the enol ether moiety remained intact. We thus attempted direct
transformation of 15 into pyrimidine 16b by reaction with urea. With sodium ethoxide in ethanol or
NaH in THF, we observed mainly cleavage of the TBDPS group and the desired product 16b was
formed in very low yield. However, using t-BuOK in t-BuOH as the base, the desired product was
formed in good yield. Reactions of 15 with thiourea and guanidine under similar conditions yielded
compounds 16c and 16a, respectively. Selective deprotection of the TBDPS group in pyrimidines
16a-c with TBAF revealed the primary hydroxyl group, which could be utilized for further selective
derivatization, e.g., the preparation of phosphates and its isosteres. Final hydrolysis of the acetonide
under acidic conditions provided the target compounds 2a, 2b and 2c in good overall yields. The
relative configurations of 2a-c were confirmed by 2D NMR experiments (shown in Supporting
Information). We were able to separate the enantiomers of intermediates 17b and 17c as well as 16a
by HPLC on a chiral stationary phase (see Supporting Information).
Clearly, the strategy described above can only be applied to construct the (hetero)cyclic bases by
elaboration of the ketoester 14. In order to target compounds that are unaccessible by the
methodology described above, we envisioned a different and perhaps more general route that utilizes
a versatile cyclopentanone intermediate (19, Scheme 5).
Scheme 5. Reagents and conditions: a) LiHMDS, TBSOTf, THF, -78 °C. b) O3, CH2Cl2, -78 °C, then Me2S -78
°C to r. t.; 52 % from 14.
In order to access quickly and evaluate the potential of compound 19, we converted one of the
synthetic intermediates, ketoester 14, into the corresponding silyl enol ether 18. Subsequent
260
ozonolysis of this, rather unusual,13
substrate afforded 19 in an acceptable yield (Scheme 5). The twostep
sequence provided sufficient amounts of material for preliminary studies. We initially studied the
introduction of a phenyl group using PhMgBr or PhLi under a variety of conditions (e.g., variable
temperature and solvent, presence or absence of CeCl3) and found that the best results were obtained
when PhLi was added to a solution of 19 in THF at 0 °C. Under these conditions, a single
diastereomer of addition product 20, resulting from attack of the reagent from the less sterically
hindered side of 19, was obtained in 75% yield (Scheme 6). We were unable to detect the other
diastereomer by NMR spectroscopy.
Scheme 6. Reagents and conditions: a) PhLi, THF, 0 °C; 75% b) TBAF, wet THF, r. t.; then PPTS, MeOH r. t.;
42% from 20. c) TBAF, wet THF, r. t., then TsCl, Et3N, DMAP, CH2Cl2, 0 °C; then NaH, BrPhCOCl, THF, 0
°C to r. t., 42% from 20.
The relative stereochemistry of the addition product 20 (supported by 2D NMR; see Supporting
Information) was unambiguously assigned by X-ray crystallography of its p-bromobenzoyl derivative
24 (Scheme 6 and Figure 3).
We tested the effect of compounds 2a, 2b and 2c on the viability of leukemia cell lines that were
available to us: SU-DHL-4 (diffuse large B-cell lymphoma, del/mut TP53), JEKO-1 (mantle cell
lymphoma, del/mut TP53), and JVM-3 (mantle cell lymphoma, wt-TP53). The viability of SU-DHL-
4 and JEKO-1 was not affected by 10 µM nor 100 µM concentrations of the compounds. However,
261
JVM-3 was more sensitive: 90% viability was observed upon treatment with 100 µM 2c; with 10 µM
and 100 µM 2a we observed 90% and 83% viability, respectively.
Figure 3. X-ray crystal structure of compound 24 (CCDC ref. No. 937690)
Since the arrangement around the tertiary alcohol carbon of compound 22 mimics that of the acetal
product of glycosylase-mediated cleavage,14
this compound was tested against glycosylases NEIL1,
NEIL2, NTH1, and hOGG1, and was found to inhibit selectively NEIL1 in a dose-dependent manner:
at 1 mM we observed 46% inhibition, and at 0.5 mM and 0.125 mM concentrations 22% and 2%
inhibition, respectively.
In summary, we have completed the first syntheses of three new racemic carbocyclic nucleoside
analogs (2a-c) of pseudoisocytidine, each in 13 steps. The synthetic approach builds on a userfriendly
preparation of sulfone 7b, which can be diastereoselectively dihydroxylated and ultimately
elaborated into tetrasubstituted chiral cyclopentanes 11 with good diastereoselectivity. While DielsAlder
reactions between cyclopentadiene with a beta-sulfonyl enoate are known,15
the strategic
elaboration of readily accessed sulfones 7b into cyclopentanes seems largely undeveloped, and can be
used for the construction of nucleoside analogs and for target directed syntheses. Analogs of αketoester
intermediate 14 have been previously used for the construction of the heterocyclic ring
directly,6d
or after a two carbon homologation;6f
our one-carbon homologation enables the synthesis
of additional (hetero)cycles. Furthermore, we have performed preliminary studies towards a
potentially more versatile strategy for the preparation of carbocyclic nucleoside analogs that utilizes
highly diastereoselective additions of organometallic reagents onto cyclopentanone 19, which itself is
available in two-steps from α-ketoester intermediate 14. While analogs of 19 are known and have
been used in synthesis of carbocyclic nucleosides,16
tetraol 22, to our knowledge, is the only
carbocyclic C-nucleoside represented by generic structure D, where R is an oxygenated substituent.
262
Unlike the analogs in the series A, B, and C (Figure 1), compounds such as 22 are stable and their
biological evaluation should enable mapping of part of the chemical space that is currently
unaccessible. Along this line, we have tested the prepared carbocyclic analogs in three leukemia cell
lines, and compounds 2a and 2c were found to be moderately active against wt-TP53 - mantle cell
lymphoma cell line JVM-3, which is generally the most sensitive to chemotherapeutic treatment. The
ability of compound 22 to inhibit glycosylase NEIL1 has served as the starting point for a more
thorough exploration of the biological activity of this series of novel carbocyclic nucleoside analogs.
Further studies are currently in progress and the results will be published elsewhere.
References and notes
1. Modified Nucleosides in Biochemistry, Biotechnology and Medicine; Herdewijn, P., Ed.; WileyVCH,
2008.
2. a) Agrofoglio, L.; Suhas, E.; Farese, A.; Condom, R.; Challand, R. S.; Earl, R. A; Guedj, R.
Tetrahedron 1994, 50, 10611; b) Hildbrand, S.; Troxler, T.; Scheffold, R. Helv. Chim. Acta
1994, 77, 1236; c) Altmann, K. H.; Kesselring, R. Synlett 1994, 853; d) Crimmins, M. T.
Tetrahedron 1998, 54, 9229; e) Zhu, X.-F. Nucleosides, Nucleotides, Nucleic Acids 2000, 19,
651; f) Agrofoglio, L. A Curr. Org. Chem. 2006, 10, 333.
3. a) Borchardt, R. T.; Wu, Y. S.; Huber, J. A.; Wycpalek, A. F. J. Med. Chem. 1976, 19, 1104; b)
Secrist, J. A.; Clayton, S. J.; Montgomery, J. A. J. Med. Chem. 1984, 27, 534; c) Neres, J.;
Labello, N. P.; Somu, R. V.; Boshoff, H. I.; Wilson, D. J.; Vannada, J.; Chen, L.; Barry, C. E.;
Bennett, E. M.; Aldrich, C. A. J. Med. Chem. 2008, 51, 5349; d) Choi, W. J.; Chung, H.;
Chandra, G.; Alexander, V.; Zhao, L. X.; Lee, H. W.; Nayak, A.; Majik, M. S.; Kim, H. O.; Kim,
J.-H.; Lee, Y. B.; Ahn, C. H.; Lee, S. K.; Jeong, L. S. J. Med. Chem. 2012, 55, 4521.
4. a) Arita, M.; Adachi, K.; Ito, Y.; Sawai, H.; Ohno, M. J. Am. Chem. Soc. 1983, 105, 4049; b)
Arai, Y.; Hayashi, Y.; Yamamoto, M.; Takayema, H.; Koizumi, T. J. Chem. Soc., Perkin Trans.
1 1988, 3133.
5. a) Popsavin, M.; Spaic, S.; Svirčev, M.; Kojic, V.; Bogdanovic, G.; Popsavin, V. Bioorg. Med.
Chem. Lett. 2006, 16, 5317; b) Štambaský, J. ; Hocek, M. ; Kočovský, P. Chem. Rev. 2009, 109,
6729; c) Bárta, J.; Slavětínská, L.; Klepetářová, B.; Hocek, M. Eur. J. Org. Chem. 2010, 5432.
6. a) Playtis, A. J.; Fissekis, J. D. J. Org. Chem. 1975, 40, 2488; b) Chu, C. K. I.; Wempen, I.;
Watanabe, K. A.; Fox, J. J. J. Org. Chem. 1976, 41, 2793; c) Just, G.; Kim, S. Tetrahedron Lett.
1976, 14, 1063; d) Just, G.; Kim, S. Can. J. Chem. 1977, 55, 427; e) Saksena, A. K.; Ganguly, A.
K. Tetrahedron Lett. 1981, 22, 5227; f) Takahashi, T.; Kotsubo, H.; Koizumi, T. Tetrahedron:
263
Asymmetry 1991, 2, 1035; g) Dishington, A. P.; Humber, D. C.; Stoodley, R. J. J. Chem., Soc.
Perkin Trans. 1 1993, 57; h) Díaz, M.; Ortuño, R. M. Tetrahedron: Asymmetry 1997, 8, 3421.
7. Burchenal, J. H.; Ciovacco, K.; Kalaher, K.; O´Toole, T.; Kiefner, R.; Dowling, M. D.; Chu, C.
K.; Watanabe, K. A.; Wempen, I.; Fox, J. J. Cancer Res. 1976, 36, 1520.
8. Woodcock, T. M.; Chou, T. C.; Tan, C. T. C.; Sternberg, S. S.; Philips, F. S.; Young, C. W.;
Burchenal, J. H. Cancer Res. 1980, 40, 4243.
9. Grierson, J. R.; Shields, A. F.; Zheng, M.; Kozawa, S. M.; Courter, J. H. Nucl. Med. Biol. 1995,
22, 671.
10. a) Just, G.; Reader, G. Tetrahedron Lett. 1973, 17, 1521; b) Just, G.; Reader, G.; Faure, B. C.
Can. J. Chem. 1975, 54, 849.
11. a) Ma, S.; Lu, X. Org. Synth. 1999, 9, 415; b) Rossi, R.; Bellina, F.; Catanese, A.; Mannina, L.;
Valensin, D. Tetrahedron 2000, 56, 479.
12. Hirst, G. C.; Parsons, P. J. Org. Synth. 1990, 69, 169.
13. We have been unable to find any examples of the ozonolysis of such tetrasubstituted alkenes.
14. a) Slupphaug, G.; Mol, C. D.; Kavli, B.; Arvai, A. S.; Krokan, H. E.; Tainer, J. A. Nature
1996, 384, 87; b) Parikh, S. S.; Mol, C. D.; Slupphaug, G.; Bharati, S.; Krokan, H. E.; Tainer,
J. A. EMBO J. 1998, 17, 5214.
15. Downey, C. W. .; Craciun, S.; Vivelo, C. A.; Neferu, A. M.; Mueller, C. J.; Corsi, S. Tetrahedron
Lett. 2012, 53, 5766.
16. Tanaka, M.; Yoshioka, M.; Sakai, K. Tetrahedron: Asymmetry 1993, 4, 981.
Experimental Section
General
All reagents and solvents were of reagent grade and were used without further purification.
Anhydrous solvents (THF, dichloromethane, CH3CN) were used from commercial suppliers (Aldrich,
Acros) or distilled and stored over 4Ǻ molecular sieves. All reactions were carried out in oven-dried
glasware and under N2 atmosphere. Column chromatography was carried on silica gel (230-400
mesh). TLC plates were visualized under UV and with phosphomolybdic acid or KMnO4 solution.
NMR spectra were recorded on Bruker Avance 300 and 500 MHz instruments, with operating
frequencies 300.13, 500.13 MHz for 1
H a 75.48, 125.77 MHz for 13
C. The 1
H and 13
C NMR chemical
shifts (δ in ppm) were referenced to the residual signals of solvents: CDCl3 [7.24 (1
H) and 77.23
(13
C)] and DMSO-d6 [2.50 (1
H) and 39.51 (13
C)]. Structural assignments of resonances has been
264
performed with the help of 2D NMR gradients experiments (COSY, multiplicity edited 1
H-13
C
HSQC, 1
H-13
C HMBC, NOESY, 1
H-15
N HSQC and 1
H-15
N HMBC).
Diffraction data were collected on KM4CCD four-circle area diffractometer (Oxford Diffraction,
Abingdon, UK) equipped with an Oxford Cryosystems, Oxford, UK. The crystallographic
packageShelXTL was used to solve and refine the structures and to prepare the figures.
High resolution mass spectra have been measured on Agilent 6224 Accurate-Mass TOF LC-MS with
dual electrospray/chemical ionization mode with mass accuracy greater than 2 ppm, applied mass
range from 25 to 20,000 Da.
IR spectra (4000-400 cm-1
) were collected on an EQUINOX 55/S/NIR FTIR spectrometer. Samples
were prepared as KBr pellets.
(Z)-methyl 3-bromoacrylate (6a)11
:
The compound was prepared according to the literature procedure11
for synthesis of ethyl-(Z)-3bromo-2-propenoate.
LiBr (1.303 g, 15.0 mmol) was dissolved in anhydrous CH3CN (11 mL).
Methyl propiolate (1.074 mL, 12.0 mmol) and AcOH (0.839 mL, 15 mmol) were added under N2 and
the resulting mixture was refluxed under N2 for 24 h. After cooling down water (10 mL) was added
and the mixture was carefully neutralized by 200 mg of solid K2CO3. The aqueous phase was
subsequently extracted with Et2O (3x 15 mL). Organic extracts were dried with MgSO4, filtered and
the solvents were evaporated under reduced pressure to yield a colourless oil (1.58 g, 80%). The
crude product was used without further purification. GC-MS and 1
H, 13
C NMR analysis indicated the
presence of (Z)-isomer only. NMR data were consistent with the literature.11
GC-MS: m/z = 164
[M+
], 166 [M+
], 133 [M+
-OMe], 85 [M+
-Br]. 1
H NMR (300 MHz, CDCl3): δ = 6.97 (d, J = 8.3 Hz,
1H), 6.60 (d, J = 8.3 Hz, 1H), 3.74 (s, 3H) ppm. 13
C NMR (75 MHz, CDCl3): δ = 164.47 (-COOMe),
124.35, 121.63, 51.79 (-OMe) ppm.
(1R*,2R*,3R*,4S*)-methyl 3-bromobicyclo[2.2.1]hept-5-ene-2-carboxylate (7a):
265
Methyl-(Z)-3-bromo-2-propenoate 5 (1.789 g, 10.84 mmol) was dissolved in anhydrous CH2Cl2 (15
mL). The resulting mixture was cooled with ice bath to 0°C and EtAlCl2 (1.8M solution in toluene, 3
mL, 5.42 mmol, 0.5 equiv) was added dropwise over period of 5 minutes. The reaction mixture was
stirred for 30 min at 0°C. Freshly distilled cyclopentadiene (4.5 mL, 54.2 mmol) was then added in
one portion. The resulting mixture was stirred for an additional 1 hour while maintaining reaction
temperature between 0-5°C. The ratio of endo:exo isomers of the product in an aliquot was
determined to be 9:1 by GC-MS analysis. The reaction mixture was catiously poured into a mixture of
15 mL of 10% HCl, 50 g of ice and 50 mL of Et2O and stirred at 0°C till a white solid precipitated
from the mixture. The precipitate was removed by filtration and the resulting filtrate was extracted
with Et2O (5x 30 mL). Combined organic extracts were dried over MgSO4, filtered and the solvent
was removed under vacuum to produce yellow viscous oil. The residue was purified by flash column
chromatography (hexane/EtOAc 10:1) to yield pure endo diastereomer as colourless oil which
solidified upon freezing to white solid (1.88 g, 75%); m.p.: 31-32°C. GC-MS: m/z = 232 [M+
], 230
[M+
], 199 [M+
-OMe], 133 [M+
-OMe], 165, 151, 66 [cyclopentadiene]. 1
H NMR (300 MHz, CDCl3): δ
= 6.53 (dd, J = 5.4, 3.0 Hz, 1H), 6.10 (dd, J = 5.4, 3.0 Hz, 1H), 4.61 (dd, J = 9.2, 3.0 Hz, 1H), 3.66 (s,
3H), 3.20 (dd, J = 9.2, 3.0 Hz, 1H,), 3.20 (br s, 1H), 3.09 (br s, 1H), 1.64 (d, J = 9.2 Hz, 1H), 1.37 (d,
J = 9.2 Hz, 1H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 171.75, 136.94, 134.32, 51.71, 51.32, 50.15,
50.01, 47.70, 40.05 ppm. IR (KBr): ν˜max = 3452, 2987, 1733, 1430, 1189, 1039, 835, 746 cm–1
. HRMS
(ESI): calcd for C9H11BrO2 [M+H]+
: 231.0014. Found 231.0018.
(1R*,2S*,3S*,4R*,5S*,6R*)-methyl 3-bromo-5,6-dihydroxybicyclo[2.2.1]heptane-2-carboxylate
(8a):
Into a solution of 7a (1.056 g, 4.57 mmol) in acetone/water (12 mL, 4:1) was added NMO (4.57
mmol, 0.5354 g) and 4% solution of OsO4 in water (0.036 mmol, 0.227 mL). The reaction mixture
was stirred at 40°C, after 14 h solid Na2S2O5 (0.5 g) was added and the resulting black solution was
stirred for additional 30 min at room temperature. Volatiles were removed under reduced presssure
and the black residue was preadsorbed on silica gel and then purified by flash column
chromatography (petrolether/EtOAc 2:1) to yield a white crystalline solid (969 mg, 80%). m.p. = 144-
146°C. 1
H NMR (300 MHz, CDCl3): δ = 4.90-4.87 (m, 1H), 4.49-4.44 (m, 2H), 3.68 (s, 3H), 3.09
(dd, J = 11.2, 3.2 Hz, 1H); 2.87 (d, J = 5.2Hz, -OH, 1H,), 2.63 (d, J = 5.5 Hz, -OH, 1H), 2.51 (d, 1H,
J = 3.5 Hz), 2.37 (br s, 1H), 2.07 (dm, J = 11.1 Hz, 1H), 1.27 (dm, J = 11.1 Hz, 1H) ppm. 13
C NMR
266
(75 MHz, CDCl3): δ = 170.93, 71.62, 68.53, 51.79, 50.82, 48.39, 48.02, 46.52, 33.13 ppm. IR (KBr):
ν˜max = 3382, 3263, 2981, 1738, 1430, 1367, 1170, 1047, 860 cm–1
. HR-MS (APCI): calcd for
C9H13BrO4 [M-H2O+H]+
= 246.9964. Found 246.9964.
(3aR*,4R*,5S*,6S*,7R*,7aS*)-methyl 6-bromo-2,2-dimethylhexahydro-4,7methanobenzo[d][1,3]dioxole-5-carboxylate
(9a):
Diol 8a (1.1302 g; 4.26 mmol) was dissolved in acetone (12 mL), 2,2-dimethoxypropane (1.776 g, 4
eq., 17.05 mmol) and 3 mg of TsOH were added and the resulting mixture was stirred at room temp.
till the TLC indicated disappearance of the starting material (30-60 min). The remaining 2,2dimethoxypropane
and acetone were evaporated under reduced pressure yield crude acetonide 9a,
whose purity (analyzed by GC-MS, 1
H NMR and 13
C NMR) was satisfactory and it was therefore
used without further purification directly in the next step. GC-MS: m/z = 289 [M+
-Me], 275 [M+
OMe],
229 [M+
-OOCMe2], 135, 79. 1
H NMR (300 MHz, CDCl3): δ = 4.01-3.94 (m, 3H); 3.69 (s,
3H); 2.61-2.54 (m, 3H), 2.08-2.04 (dm, 1H); 1.89-1.85 (dm, 1H); 1.43 (s, 3H), 1.25 (s, 3H) ppm. 13
C
NMR (75 MHz, CDCl3): δ = 171.02, 110.72, 80.79, 79.98, 51.96, 51.11, 48.80, 47.25, 43.54, 29.44,
25.56, 24.36 ppm.
(Z)-methyl 3-(phenylsulfonyl)acrylate (6b):
Compound 6b was prepared by a slightly modified literature procedure.12
Into a solution of sodium
benzensulfinate (3.671g, 22.36 mmol) and Bu4NHSO4 (1.138 g 3.354 mmol) in H2O/THF 1:1 (80
mL), were added methyl propiolate (1.88 g, 22.36 mmol) followed by H3BO3 (2.074 g, 33.54 mmol).
The mixture was vigorously stirred at room temp. for 48 h. After that pH was adjusted by 1M HCl to
pH = 4 and the mixture was extracted with CH2Cl2 (4x 50 mL). The organic phase was washed with
brine (1x 30 mL), dried with MgSO4, filtered and the solvents were removed under reduced pressure.
The resulting yellow oil was purified by flash column chromatography (petrolether/EtOAc 3:2) to
267
afford both isomers as white crystals (101 mg, 2% of E isomer and 2.93 g, 58% of Z isomer 6b). E
isomer: m.p. = 99-100°C. 1
H NMR (300 MHz, CDCl3): δ = 7.91-7.89 (m, 2H), 7.69-7.54 (m, 3H),
7.32 (d, J = 15.2 Hz, 1H), 6.82 (d, J = 15.2 Hz, 1H), 3.78 (s, 3H) ppm. 13
C NMR (75 MHz, CDCl3): δ
= 164.11, 143.71, 138.75, 134.60, 130.73, 129.86, 128.56, 52.98 ppm. Z isomer: m.p. = 64-65°C. 1
H
NMR (300 MHz, CDCl3): δ = 7.99-7.97 (m, 2H), 7.67-7.53 (m, 3H), 6.54 (d, J = 11.5 Hz, 1H), 6.48
(d, 1H, J = 11.5 Hz), 3.88 (s, 3H) ppm. 13
C NMR (75 MHz, CDCl3): δ = 164.53, 139.40, 135.60,
134.22, 131.73, 129.46, 128.24, 52.83 ppm. IR (KBr): ν˜max = 3456, 3037, 2954, 1734, 1633, 1340,
1311, 1238, 1153, 763, 731 cm–1
.
(1R*,2R*,3R*,4S*)-methyl 3-(phenylsulfonyl)bicyclo[2.2.1]hept-5-ene-2-carboxylate (7b):
EtAlCl2 (15.92 mmol, 8.85 ml of 1.8M solution in toluene) was added dropwise over the period of 5
min to a cooled (ice bath, 0°C) solution of compound 6b (7.206 g, 31.85 mmol) in anhydrous CH2Cl2
(30 mL). The reaction mixture was stirred for 30 min, then freshly distilled cyclopentadiene (1.32
mL, 159.25 mmol) was added. The mixture was stirred for additional 1 h at 0°C, then it was poured
onto 10% HCl (80 mL) with ice (200 g). The mixture was filtered to remove white polymeric
byproducts, to the filtrate was added brine (200 mL) and it was extracted with CH2Cl2 (4x 100 ml).
The organic extracts were dried with MgSO4, filtered and concentrated in a vacuum to afford a yellow
oil. The product was precipitated by addition of Et2O (20 mL); the resulting white crystalline solid
was filtered and washed with Et2O (3x 15 mL). The product (pure endo diastereomer by NMR) was
dried in a vacuum and used directly without further purification. Analytically pure material was
isolated by flash column chromatography (petrolether/EtOAc 3:1) as white crystals (7.72 g, 83 %).
m.p. = 150-151°C. 1
H NMR (300 MHz, CDCl3): δ = 7.93-7.84 (m, 2H), 7.67-7.51 (m, 3H), 6.59 (dd,
J = 5.3, 3.0 Hz. 1H), 6.26 (dd, J = 5.3, 3.0 Hz, 1H), 4.12 (dd, J = 10.0, 3.1Hz, 1H), 3.42 (dd, J = 10.0,
3.1 Hz, 1H), 3.21 (br s, 1H), 3.02 (br s, 1H), 1.47 (dm, J = 8.9 Hz, 1 H), 1.25 (dm, J = 8.9 Hz, 1 H)
ppm. 13
C NMR (75 MHz, CDCl3): δ = 170.87, 141.40, 137.70, 133.40, 132.13, 129.07, 127.86, 69.17,
51.87, 49.16, 48.12, 47.07, 46.64 ppm. HR-MS (APCI): calcd for C15H16O4S [M+H]+
: 293.0842.
Found 293.0841.
Crystal data for 7b: CCDC ref. No. 929386. Crystallized from CH2Cl2, C15H16O4S, Mrel = 292.35, T =
120 K, space group P-1, a = 8.2134(4) Å, b = 8.3868(4) Å, c = 10.7465(6) Å, α = 108.251(5), β =
94.702(4), γ = 99.040(4), V = 687.466 Å3
, R = 0.031.
268
X-ray crystal structure of compound 7b
(1R*,2S*,3S*,4R*,5S*,6R*)-methyl 5,6-dihydroxy-3-(phenylsulfonyl)bicyclo[2.2.1]heptane-2carboxylate
(8b):
To a stirred solution of alkene 7b (4.511 g, 15.4 mmol) in acetone/H2O (45 mL, 4:1) were added
NMO (1.8 g, 15.4 mmol) followed by OsO4 (700 μL, 4% wt. solution in H2O, 0.12 mmol). The
yellow solution was stirred for 14 h at 40°C. Solid Na2S2O5 (0.5 g) was then added and the resulting
black mixture was stirred for 30 min. Volatiles were removed under reduced pressure and the black
residue was preadsorbed on silica gel. Flash column chromatography (EtOAc) yielded a white
crystalline compound (4.52 g, 90 %). m.p.: 144-145°C. 1
H NMR (500 MHz, CDCl3): δ = 7.93-7.85
(m, 2H), 7.66-7.51 (m, 3H), 4.96-4.89 (m, 1H), 4.78-4.70 (m,1H), 3.76 (dd, J =11.9, 4.1Hz, 1H), 3.18
(d, J = 4.9Hz, 1H), 3.11 (dd, J = 11.9, 4.1Hz, 1H), 3.06 (d, J = 4.9 Hz, 1H), 2.56 (br s, 1H), 2.43 (br s,
1H), 2.09 (d, J = 10.9 Hz, 1H), 1.14 (d, J = 10.9 Hz, 1H) ppm. 13
C NMR (126 MHz, CDCl3): δ =
170.13, 141.25, 133.85, 129.39, 128.00, 77.48, 77.23, 76.98, 69.32, 68.39, 64.65, 52.31, 48.00, 47.90,
44.73, 33.24 ppm. IR (KBr): ν˜max = 3456, 1747, 1637, 1385, 1144, 752, 721 cm–1
. HR-MS (ESI):
calcd for C15H18O6S [M+H]+
: 327.0897. Found 327.0892.
Crystal data for 8b: CCDC ref. No. 929387. Crystallized from CH2Cl2, C15H18O6S, Mrel = 326.36, T =
120 K, space group P-1, a = 10.4471(4) Å, b = 11.4450(6) Å, c = 13.4617(6) Å, α = 65.794(4), β =
87.155(3), γ = 82.891(4), V = 1456.77 Å3
, R = 0.030.
269
X-ray crystal structure of compound 8b
(3aR*,4R*,5S*,6S*,7R*,7aS*)-methyl 2,2-dimethyl-6-(phenylsulfonyl)hexahydro-4,7methanobenzo[d][1,3]dioxole-5-carboxylate
(9b):
To a solution of diol 8b (2.00 g, 6.13 mmol) in acetone (24 mL) were added 2,2-dimethoxypropane
(3.1 mL, 24.5 mmol) and TsOH (2 mg). The reaction mixture was stirred at room temp. till the
starting material was not detected by TLC (petrolether/EtOAc 1:1); within ca. 30 min. The reaction
mixture was evaporated to dryness (orange oil) and the crude product was used without further
purification directly in the next step. Pure product could be obtained by flash column chromatography
(petrolether/EtOAc 1:1) as colourless crystals (2.22 g, 99%). m.p.: 149-150°C. 1
H NMR (500 MHz,
CDCl3): δ = 7.90 (m, 2H), 7.63 (m, 1H), 7.55 (m, 1H), 5.18 (d, J = 5.4 Hz, 1H), 4.97 (d, J = 5.4 Hz,
1H), 3.75 (dd, J = 11.7, 4.1 Hz, 1H), 3.71 s (3H), 3.17 (dd, J = 11.7, 4.1 Hz, 1H), 2.69 (m, 1H), 2.53
(m, 1H), 1.99 (m, 1H), 1.41 (s, 3H), 1.33 (s, 3H), 1.04 (m, 1H) ppm. 13
C NMR (126 MHz, CDCl3): δ
= 169.69, 141.35, 133.83, 129.41, 128.05, 108.88, 77.29, 76.06, 64.27, 52.35, 45.11, 45.07, 44.01,
32.86, 25.46, 24.52 ppm. IR (KBr): ν˜max = 3456, 1747, 1637, 1385, 1144, 752, 721 cm–1
. HR-MS
(ESI): calcd for C18H22O6S [M+H]+
: 367.1210. Found 367.1214.
(3aR*,4R*,7S*,7aS*)-methyl 2,2-dimethyl-3a,4,7,7a-tetrahydro-4,7methanobenzo[d][1,3]dioxole-5-carboxylate
(10):
270
Method a) Into a solution of acetonide 9a (1.299 g; 4.26 mmol) in anhydrous Et2O (10 mL) was
added DBU (1.6 mL, 10.65 mmol) at 0°C. The resulting mixture was allowed to warm to room temp.
and then it was stirred for 16 h. The resulting suspension was filtered to remove the solid residue. The
glass filter was rinsed by Et2O (3x 15 mL). The filtrates were collected, the solvents were evaporated
under reduced pressure and the resulting yellow viscous liquid was purified by flash column
chromatography (hexane/EtOAc 3:1) to yield a white solid (908 mg, 95% from 8a).
Method b) Into a stirred solution of crude acetonide 9b (1.37 g, 6.13 mmol) in dry MeCN (10 mL)
was added dropwise DBU (2.8 mL, 18.4 mmol). The reaction mixture was then stirred at 90°C for 1 h
(monitored by TLC with hexane/EtOAc 3:1). The solvent was then evaporated and the orange residue
was purified by flash column chromatography (hexane/EtOAc 3:1) to afford a colourless oil which
upon freezing crystallized to white crystals (1.18 g, 86% from 8b). m.p.: 61-62°C. 1
H NMR (300
MHz, CDCl3): δ = 6.91 (d, J = 3.1 Hz, 1H), 4.27 (d, J = 5.3 Hz, 1H), 4.21 (d, J = 5.3 Hz, 1H), 3.70 (s,
3H), 3.14 (br s, 1H), 2.93 (br s, 1H), 2.05 (m, 1H), 1.79 (m, 1H), 1.46 (s, 3H), 1.31 (s, 3H) ppm. 13
C
NMR (75 MHz, CDCl3): δ = 164.65, 147.55, 142.37, 114.40, 80.23, 80.15, 51.76, 47.40, 45.81,
42.99, 26.19, 24.59 ppm. IR (KBr): ν˜max = 3452, 3000, 2931, 1712, 1643, 1597, 1267, 1062, 856,
756 cm–1
. HR-MS (ESI): calcd for C12H16O4 [M+H]+
: 225.1121. Found 225.1124.
(R*)-methyl 2-hydroxy-2-((3aR*,4R*,6S*,6aS*)-6-(hydroxymethyl)-2,2-dimethyltetrahydro-
3aH-cyclopenta[d][1,3]dioxol-4-yl)acetate (12):
O3/O2 mixture (5mL/min oxygen flow, ozonolysis rate ~ 12 mmol/5min) was bubbled through a
cooled solution (-78°C) of compound 10 (2.7447 g, 12.24 mmol) in CH2Cl2 (35 mL) till the TLC
(hexane/EtOAc 3:1) indicated disappearance of the starting material and blue colour of the reaction
mixture persisted. After that N2 was bubbled through the reaction mixture to remove residual ozone
and oxygen. Me2S (4.5 mL, 61.2 mmol) was added in one portion and the reaction mixture was stirred
for 4 h while allowed to warm to room temperature. Brine (20 mL) was then added and the mixture
was extracted with CH2Cl2 (1x 30 mL). Organic phase was washed with brine (2x 10 mL), dried with
MgSO4, filtered and the solvent was evaporated. The resulting colourless oil was immediately used in
the next step without additional purification. The purity of crude aldehyde 11 was satisfactory by 1
H
271
NMR (the main impurity is DMSO as a product of Me2S oxidation, see below). Attempts to purify the
compound by standard flash column chromatography failed due to the compound‘s instability.
Into a cooled (ice bath, 0°C) solution of crude aldehyde 11 (1.742 g, 6.8 mmol) in anhydrous THF (15
mL) was added Li(AlO-tBu)3H (3.98g, 15.64 mmol) portionwise. The reaction mixture was then
stirred overnight while allowed to warm to room temp. It was then poured into 5% aqueous solution
of NaHSO4 (50 mL) with crushed ice (50 g). The resulting white liquid was then diluted with brine
(30 mL) and extracted with EtOAc (4x 50 mL). The organic phase was dried with MgSO4, filtered
and concentrated in a vacuum to give a yellow oil. Flash column chromatography (CH2Cl2/MeOH
15:1) yielded a colourless oil (620 mg, 65 % from compound 10) as a mixture of both epimers with
the ratio of 5:2 based on 1
H NMR. 1
H NMR (500 MHz, CDCl3): δ = 4.62 (dd, 1H major epimer), 4.43
dd, 0.4 H minor epimer), 4.39 (d), 4.34 (m), 4.15 (d), 3.78 (s), 3.77 (s), 3.65 (m), 3.01 (br s), 2.46 (m),
2.23 (m), 2.02 (m), 1.76 (m), 1.48 (s), 1.44 (s), 1.29 (s), 1.26 (s) ppm. 13
C NMR (126 MHz, CDCl3): δ
= 174.98, 112.91, 83.53, 83.49, 82.24, 80.95, 70.97, 70.19, 64.85, 64.73, 53.61, 52.92, 52.80, 49.04,
48.50, 47.48, 30.66, 27.89, 27.86, 27.46, 25.56, 25.46 ppm. HR-MS (ESI): calcd for C12H20O6
[M+H]+
: 261.1333. Found 261.1330, calcd for C12H20O6 [M1+Na]+
: 283.1152. Found 283.1156.
methyl 2-((3aR*,4R*,6S*,6aS*)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-2-hydroxyacetate
(13):
Into a solution of starting material 12 (692 mg, 2.66 mmol) in anhydrous CH2Cl2 (8 mL) was added in
one portion TBDPSCl (691 μL, 2.79 mmol) followed by imidazole (453 mg, 6.65 mmol). The
reaction mixture was then stirred overnight at room temp. The solvent was evaporated and the viscous
residue was purified by flash column chromatography (hexane/EtOAc 3:1) to yield a colourless oil
(1.07 g, 81%) as a mixture of epimers with the ratio of 5:2 based on 1
H NMR. 1
H NMR (500 MHz,
CDCl3): δ = 7.66-7.60 (m), 7.43-7.32 (m), 4.57-4.51 (m), 4.43-4.30 (m), 4.18-4.14 (m), 3.76 (s), 3.73-
3.64 (m), 2.84 d (0.4 H, minor epimer), 2.74 (d, 1H, major epimer), 2.45-2.35 (m), 2.29-2.19 (m),
2.01-1.94 (m), 1.71-1.64 (m), 1.61-1.53 (m), 1.47 ((-C(CH3)2), major epimer), 1.43 ((-C(CH3)2),
minor epimer), 1.27 ((-C(CH3)2), major epimer), 1.24 ((-C(CH3)2), minor epimer), 1.04 ppm. 13
C
NMR (126 MHz, CDCl3): δ = 175.23, 175.15, 135.87, 133.92, 133.89, 129.86, 127.88, 112.67, 82.59,
272
82.48, 81.86, 80.76, 70.83, 70.71, 70.11, 69.99, 65.02, 64.94, 52.87, 52.71, 49.36, 48.87, 47.10,
46.62, 30.70, 28.00, 27.97, 27.67, 27.12, 25.66, 25.54, 19.55 ppm. IR (KBr): ν˜max = 3452, 2933,
2858, 1740, 1639, 1429, 1211, 1112, 704 cm–1
. HR-MS (ESI): calcd C28H38O6Si [M+Na]+
: 521.2330.
Found 521.2334.
methyl 2-((3aR*,4S*,6S*,6aS*)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-2-oxoacetate
(14):
Into a cooled (0°C, ice bath) solution of starting material 13 (487 mg, 0.977 mmol) in anhydrous
CH2Cl2 (8 mL) was added Dess-Martin periodinane (581 mg, 1.37 mmol). The reaction mixture was
then allowed to warm to room temp. and then stirred for 14 h. The solvent was evaporated, the
residue was suspended in cold Et2O (50 mL) and the solid was removed by filtration. The filtrate was
washed with saturated aqueous NaHCO3 solution (2x 10 mL), dried with MgSO4, filtered and
concentrated in a vacuum to provide a colourless oil (412 mg, 85%) which solidified upon freezing.
Attempts to purify the compound by column chromatography failed due to partial epimerization and
decomposition on silica gel. m.p.: 76-78°C. 1
H NMR (500 MHz, CDCl3): δ = 7.65-7.56 (m, 4H),
7.43-7.31 (m, 6H), 4.82-4.76 (m, 1H), 4.41 (dd, J = 6.0, 3.6Hz, 1H), 3.85 (s, 3H), 3.69-3.53 (m, 3H),
2.41-2.29 (m, 2H), 1.82-1.75 (m, 1H), 1.47 (s, 3H), 1.26 (s, 3H), 1.02 (s, 9H) ppm. 13
C NMR (126
MHz, CDCl3): δ = 194.60, 161.73, 135.81, 133.68, 129.94, 127.94, 112.66, 82.70, 81.74, 64.18,
54.18, 53.24, 47.69, 30.98, 27.68, 27.09, 25.27, 19.50 ppm. IR (KBr): ν˜max = 3448, 3415, 2956,
2933, 1738, 1714, 1259, 1114, 1032, 708 cm–1
. HR-MS (ESI): calcd for C28H36O6Si [M+Na]+
:
519.2173. Found 519.2176.
Methyl 2-((3aR*,4R*,6S*,6aS*)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyltetra
hydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-3-methoxyacrylate (15):
273
Into a cooled (0°C, ice bath) suspension of (methoxymethyl)triphenylphosphonium chloride (1.929g,
5.63 mmol) in anhydrous THF (15 mL) was added under N2 atmosphere LDA (2.63 mL of 2M
solution in THF, 5.26 mmol) dropwise over the period of 10 min. The resulting orange mixture was
stirred at 0°C for 30 min. and then a solution of ketone 14 (932 mg, 1.88 mmol) in anhydrous THF
(20 mL) was added in one portion. The yellow mixture was stirred for additional 3 h while allowed to
warm to room temp., then it was quenched with saturated aqueous solution of NH4Cl (15 mL). The
aqueous phase was extracted with EtOAc (3x 20 mL), the combined organic parts were dried with
MgSO4, filtered and volatiles were evaporated. The dark brown residue was purified by flash column
chromatography (CH2Cl2/EtOAc 20:1) to afford two isomeric enol ethers in the overall yield of 503
mg (51%) with ratio of Z:E ~7:5. Z isomer (293 mg, 30%, yellow oil): 1
H NMR (500 MHz, CDCl3): δ
= 7.63 (m, 4H), 7.36 (m, 6H), 6.51 (s, 1H), 4.59 (m, 1H), 4.41 (m, 1H), 3.79 (s, 3H), 3.77 (m, 1H),
3.70 (s, 3H), 3.68 (m, 1H), 2.61 (m, 1H), 2.20 (m, 1H), 1.97 (m, 1H), 1.83 (dd, J = 12.24, 11.98 Hz,
1H), 1.47 (s, 3H), 1.27 (s, 3H), 1.04 (s, 9H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 166.60, 157.05,
135.85, 135.84, 134.01, 129.80, 127.83, 112.60, 108.56, 84.56, 82.37, 65.02, 62.21, 51.27, 47.78,
47.34, 34.85, 28.14, 27.11, 25.61, 19.56 ppm. HR-MS (ESI): calcd for C30H40O6Si [M+H]+
:
525.2667. Found 525.2671.
E isomer (210 mg, 21%, yellow oil): 1
H NMR (500 MHz, CDCl3): δ = 7.64 (m, 4H), 7.36 (m, 6H),
7.33 (s, 1H), 4.74 (m, 1H), 4.44 (m, 1H), 3.79 (s, 3H), 3.79 (m, 1H), 3.69 (s, 1H), 3.66 (s, 3H), 3.27
(m, 1H), 2.24 (m, 1H), 1.94 (m, 2H), 1.48 (s, 3H), 1.26 (s, 3H), 1.04 (s, 9H) ppm.
13
C NMR (126 MHz, CDCl3): δ = 168.30, 160.08, 135.87, 134.13, 134.11, 129.76, 127.82, 112.47,
110.71, 84.37, 82.52, 65.13, 61.76, 51.30, 48.20, 40.88, 33.65, 28.23, 27.11, 25.78, 19.59 ppm. IR
both isomers (KBr): ν˜max = 3450, 2935, 2858, 1693, 1639, 1429, 1378, 1211, 1113, 704, 505 cm–1
.
HR-MS (ESI): calcd for C30H40O6Si [M+Na]+
: 547.2486. Found 547.2485.
General procedure for synthesis of compounds 16a-16c
To a 0.05 M solution of starting material 15 (mixture of Z+E enol ethers) in anhydrous t-BuOH was
added urea (or thiourea or guanidinium.HCl) (3 equiv.) and t-BuOK (6 equiv. for 16a and 4 equiv. for
16b and 16c ). The resulting solution was refluxed for 14 h. The reaction mixture was cooled to room
274
temp., diluted with H2O (15 mL) and pH was adjusted to 6-7 with 1M HCl. The resulting solution
was extracted with EtOAc (4x 20 mL). Combined organic extracts were dried with Na2SO4, filtered
and concentrated in a vacuum. The resulting brown oil was purified by flash column chromatography
(CH2Cl2/MeOH 15:1 for 16a and 20:1 for 16b and 16c).
2-amino-5-((3aR*,4R*,6S*,6aS*)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-4(1H)-one
(16a):
Colourless amorphous solid (166 mg, 43%). 1
H NMR (500 MHz, DMSO-d6): δ = 10.83 (br s, 1H),
7.61 (m, 4H), 7.44 (m, 6H), 6.34 (br s, 2H), 4.64 (m, 1H), 4.38 (m, 1H), 3.68 (m, 2H), 2.83 (m, 1H),
2.18 (m, 1H), 1.95 (m, 1H), 1.80 (m, 1H), 1.39 (s, 3H), 1.18 (s, 3H), 1.00 (s, 9H) ppm. 13
C NMR (126
MHz, DMSO-d6): δ = 161.80*, 155.08, 134.99, 133.15, 129.73, 127.79, 114.27*, 111.38, 83.20,
81.83, 64.89, 47.26, 44.07, 33.58, 27.72, 26.61, 25.21, 18.84 ppm.* - these resonances were indirectly
detected by 1
H-13
C HMBC experiment. HR-MS (ESI): calcd for C29H37N3O4Si [M-H]:
518.2481.
Found 518.2481.
Separation of enantiomers of 16a:
Chiralpak AD 4.6x250 mm column; hexane/2-propanol 90:10; 1mL/min; UV detection (254 nm)
5-((3aR*,4R*,6S*,6aS*)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyltetrahydro-3aHcyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione
(16b):
Slightly yellow amorphous solid (85 mg, 38%). 1
H NMR (500 MHz, CDCl3): δ = 9.23 (br s, 1H),
8.82 (s, 1H), 7.63 (m, 4H), 7.37 (m, 6H), 7.08 (d, J = 6.49 Hz, 1H), 4.63 (m, 1H), 4.46 (dd, J = 6.8,
275
5.2 Hz, 1H), 3.73 (ddd, J = 16.4, 10.2, 5.7 Hz, 2H), 2.87 (m, 1H), 2.31 (m, 1H), 2.13 (dd, J = 12.9,
6.8 Hz, 1H), 1.89 (dd, J = 24.3, 12.9 Hz, 1H), 1.48 (s, 3H), 1.26 (s, 3H), 1.05 (s, 9H) ppm. 13
C NMR
(126 MHz, CDCl3): δ = 163.50, 152.09, 136.83, 135.86, 133.93, 129.89, 127.90, 114.64, 113.09,
83.33, 82.37, 65.01, 47.45, 45.49, 33.68, 28.00, 27.14, 25.48, 19.57 ppm. HR-MS (ESI): calcd for
C29H36N2O5Si [M-H]:
519.2321. Found 519.2303.
5-((3aR*,4R*,6S*,6aS*)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyltetrahydro-3aHcyclopenta[d][1,3]dioxol-4-yl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one
(16c):
Yellow ammorphous solid (198 mg , 45%). 1
H NMR (300 MHz, CDCl3): δ = 10.66 (br s, 1H), 10.21
(s, 1H), 7.63 (m, 4H), 7.38 (m, 6H), 7.07 (d, J = 5.2 Hz, 1H), 4.70 (dd, J = 6.60, 13.61 Hz, 1H), 4.48
(dd, J = 6.60, 11.77 Hz, 1H), 3.74 (m, 2H), 2.90 (m, 1H), 2.34 (m, 1H), 2.10 (m, 1H), 1.90 (m, 1H),
1.51 (s, 3H), 1.29 (s, 3H), 1.05 (s, 9H) ppm. 13
C NMR (75 MHz, CDCl3): δ = 174.93, 160.80, 137.14,
135.84, 133.86, 129.92, 127.92, 119.13, 113.38, 83.01, 82.43, 64.97, 47.33, 45.72, 33.41, 28.02,
27.14, 25.54, 19.56 ppm. HR-MS (ESI): calcd for C29H36N2O4SSi [M+Na]+
: 559.2057. Found
559.2021.
General procedure for synthesis of compounds 17a-c
To a 0.1 M solution of starting material 16b-c in wet THF was added TBAF (1.1 equiv., 1 M solution
in THF) and the reaction mixture was stirred at room temp for 14h. THF was evaporated and the
brown oily residue was purified on a short silica gel column (CH2Cl2/MeOH 2:1 for 17a and 10:1 for
17b and 17c).
2-amino-5-((3aR*,4R*,6S*,6aS*)-6-(hydroxymethyl)-2,2-dimethyltetrahydro-3aHcyclopenta[d][1,3]dioxol-4-yl)pyrimidin-4(1H)-one
(17a):
276
White crystalline solid (80 mg, 90%). m.p.: > 250°C, decomp. 1
H NMR (500 MHz, DMSO-d6): δ =
10.80 (s, 1H), 7.43 (s, 1H), 6.33 (s, 2H), 4.63 (m, 1H), 4.54 (m, 1H), 4.32 (m, 1H), 3.40 (m, 2H), 2.79
(m, 1H), 2.04 (m, 1H), 1.89 (m, 1H), 1.65 (m, 1H), 1.40 (s, 3H), 1.19 (s, 3H) ppm.13
C NMR (126
MHz, DMSO-d6): δ = 162.04*, 155.03, 153.10*, 114.52*, 111.28, 83.10, 82.21, 62.57, 47.41, 44.31,
33.84, 27.72, 25.19 ppm. * - resonances were indirectly detected through 1
H-13
C HSQC or 1
H-13
C
HMBC experiments. IR (KBr): ν˜max = 3434, 3097, 2983, 2736, 1670, 1614, 1562, 1207, 1051, 867
cm–1
. HR-MS (ESI): calcd for C13H19N3O4 [M+H]+
: 282.1448. Found 282.1447
5-((3aR*,4R*,6S*,6aS*)-6-(hydroxymethyl)-2,2-dimethyltetrahydro-3aHcyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione
(17b):
White crystalline solid (89 mg, 96%). m.p.: 246-247°C, decomp. 1
H NMR (500 MHz, DMSO-d6): δ =
10.99 (s, 1H), 10.70 (br s, 1H), 7.30 (s, 1H), 4.58 (m, 2H), 4.33 (dd, J = 5.3, 12 Hz, 1H), 3.40 (m,
2H), 2.83 (m, 1H), 2.04 (m, 1H), 1.94 (m, 1H), 1.56 (m, 1H), 1.40 (s, 3H), 1.20 (s, 3H) ppm. 13
C
NMR (126 MHz, DMSO-d6): δ = 164.00, 151.06, 137.93, 111.99, 111.43, 82.94, 82.05, 62.40, 47.24,
43.43, 33.83, 27.66, 25.18 ppm. IR (KBr): ν˜max = 3490, 3228, 2933, 1720, 1666, 1384, 1377, 1065,
1040, 860, 791 cm–1
. HR-MS (ESI): calcd for C13H18N2O5 [M-H]:
calcd for C13H18N2O5 [M-H]-
:
283.1288. Found 283.1287.
Separation of enantiomers of 17b:
Chiralpak AD 4.6x250 mm column; hexane/2-propanol 80:20; 1mL/min; UV detection (254 nm)
5-((3aR*,4R*,6S*,6aS*)-6-(hydroxymethyl)-2,2-dimethyltetrahydro-3aHcyclopenta[d][1,3]dioxol-4-yl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one
(17c):
277
White crystalline solid (99 mg, 90%). m.p. 199-201°C. 1
H NMR (500 MHz, DMSO-d6): δ = 12.41 (s,
1H), 12.24 (br s, 1H), 7.35 (s, 1H), 4.59 (m, 1H), 4.58 (m, 1H), 4.34 (dd, J = 5.20, 12 Hz, 1H), 3.40
(m, 2H), 2.88 (m, 1H), 2.05 (m, 1H), 1.96 (m, 1H), 1.56 (dd, 1H, J = 12, 23.7 Hz), 1.40 (s, 3H), 1.20
(s, 3H) ppm. 13
C NMR (126 MHz, DMSO-d6): δ = 174.59, 160.97, 137.86, 117.78, 111.53, 82.78,
82.06, 62.31, 47.18, 43.36, 33.44, 27.63, 25.19 ppm. IR (KBr): ν˜max = 3450, 3263, 2927, 1658,
1541, 1456, 1383, 1371, 1213, 1155, 866 cm–1
. HR-MS (ESI): calcd for C13H18N2O4S [M-H]:297.0915.
Found: 297.0904.
Separation of enantiomers of 17c:
Chiralpak AD 4.6x250 mm column; hexane/2-propanol 75:25; 1mL/min; UV detection (254 nm)
General procedure for synthesis of compounds 2a-c
To a 0.2 M solution of starting material 17a-c in MeOH were added 35% HCl (1 mL) and H2O (1
mL) and the reaction mixture was stirred at room temp for 30 min. The solvents were evaporated in a
vacuum and the resulting brown solid was purified by flash column chromatography (CH2Cl2/7 N
NH3 in MeOH 5:1 for compound 2a and CH2Cl2/MeOH 5:1 to 1:1 for compounds 2b and 2c.
2-amino-5-((1R*,2R*,3S*,4S*)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)pyrimidin-4(1H)one
(2a):
278
White crystalline solid (46 mg, 69 %). m.p.: > 250°C, decomp. 1
H NMR (500 MHz, DMSO-d6): δ =
10.94 (br s, 1H), 7.37 (s, 1H), 6.33 (br s 2H), 4.73 (br s –OH), 4.45 (m, 1H), 4.09 (br s – OH), 3.81
(m, 1H), 3.67 (m, 1H), 3.36 (m, 2H), 2.76 (m, 1H), 1.91 (m, 1H), 1.84 (m, 1H), 1.25 (m, 1H).13
C
NMR (126 MHz, DMSO-d6): δ = 164.10*, 155.40, 151.76*, 115.31, 75.14, 73.33, 63.39, 46.66,
42.42, 29.12.*- these resonances were indirectly detected through 1
H-13
C HSQC or 1
H-13
C HMBC
experiments. IR (KBr): ν˜max = 3421, 2921, 1652, 1606, 1486, 1118, 1043, 777 cm–1
. HR-MS (ESI):
calcd for C10H15N3O4[M-H]:
240.0990. Found: 240.0969.
5-((1R*,2R*,3S*,4S*)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)pyrimidine-2,4(1H,3H)dione
(2b):
White solid (60 mg, 76 %). m.p. > 300°C, decomp. 1
H NMR (500 MHz, DMSO-d6): δ = 10.94 (br s,
1H), 10.62 (br s, 1H), 7.15 (s, 1H), 4.46 (m, 1H), 4.37 (d, J = 6.40 Hz, 1H), 4.22 (d, J = 4.19 Hz,
1H), 3.79 (m, 1H), 3.68 (m, 1H), 3.34 (m, 2H), 2.76 (m, 1H), 1.91 (m, 2H), 1.11 (m, 1H) ppm. 13
C
NMR (126 MHz, DMSO-d6): δ = 164.52, 151.07, 137.32, 113.18, 74.41, 73.03, 63.13, 46.36, 41.35,
29.50. IR (KBr): ν˜max = 3448, 2926, 1709, 1659, 1446, 1225, 1113, 766, cm–1
. HR-MS (ESI): calcd
for C10H14N2O5 [M-H]:
241.0830. Found: 241.0833.
5-((1R*,2R*,3S*,4S*)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)-2-thioxo-2,3dihydropyrimidin-4(1H)-one
(2b):
White solid (60 mg, 71 %). m.p: 172-175°C, decomp.1
H NMR (500 MHz, DMSO-d6): δ = 12.34 (br
s, 1H), 12.18 (br s, 1H), 7.20 (s, 1H), 4.49 (m, 1H), 4.43 (d, J = 6.44 Hz, 1H), 4.25 (d, J = 4.47 Hz,
1H), 3.81 (m, 1H), 3.69 (m, 1H), 3.34 (m, 2H), 2.81 (m, 1H), 1.93 (m, 2H), 1.12 (m, 1H) ppm. 13
C
NMR (126 MHz, DMSO-d6): δ = 174.30, 161.40, 137.33, 119.02, 74.24, 73.03, 63.09, 46.37, 41.40,
29.12 ppm. IR (KBr): ν˜max = 3437, 3162, 3070, 2931, 1654, 1587, 1463, 1232, 1033, 766 cm–1
. HRMS
(ESI): calcd for C10H14N2O4S [M-H]:
257.0602 Found:257.0608.
279
(3aR*,6R*,6aR*)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyldihydro-3aHcyclopenta[d][1,3]dioxol-4(5H)-one
(19):
LiHMDS (280 μL of 1 M solution in THF, 0.28 mmol) followed by TBDMSOTf (0.28 mmol, 64 μL)
were added dropwise to a cooled (-78°C) solution of starting material 14 (140 mg, 0.28 mmol) in
THF (2 mL) and the resulting mixture was stirred for additional 2 h at -78°C. All volatiles were
evaporated and the crude mixture was immediately dissolved in anhydrous CH2Cl2 (6 mL) and cooled
to -78°C. O3/O2 mixture (5 mL/min oxygen flow) was bubbled through the solution until TLC
(hexane/EtOAc 10:1) indicated disappearance of starting material and blue colour of the reaction
mixture persisted. Then N2 was bubbled through the reaction mixture to remove residual ozone and
oxygen. Me2S (120 μL, 1.4 mmol) was added in one portion and the reaction mixture was stirred for 4
h while allowed to warm to room temperature. Brine (10 mL) was then added and the mixture was
extracted with CH2Cl2 (2x 15 mL). The organic phase was washed with brine (2x 10 mL), dried with
MgSO4, filtered and the solvents were evaporated. The resulting yellow oil was purified by flash
column chromatography (hexane/EtOAc 5:1) to yield a white crystalline solid (62 mg, 52 %). m.p:
96-97°C. 1
H NMR (500 MHz, CDCl3): δ = 7.60-7.57 (m, 4H), 7.45-7.36 (m, 6H), 4.61 (d, J = 5.4 Hz,
1H), 4.33 (d, J = 5.4 Hz, 1H), 3.78 (dd, J = 10.2, 2.8 Hz, 1H), 3.59 (dd, J = 10.2, 2.8 Hz, 1H), 2.73
(dd, J = 18.4, 9.0 Hz, 1H), 2.47 (m, 1H), 2.17 (m, 1H), 1.41 (s, 3H), 1.32 (s, 3H), 1.00 (s, 9H) ppm.
13
C NMR (126 MHz, CDCl3): δ = 213.41, 135.94, 135.82, 132.97, 132.61, 130.24, 130.19, 128.09,
111.48, 81.79, 79.32, 77.48, 77.23, 76.98, 66.13, 39.31, 37.49, 27.05, 27.02, 24.89, 19.35. IR (KBr):
ν˜max = 3486, 2933, 1755, 1589, 1429, 1108, 705.85 cm–1
. HR-MS (ESI): calcd for C25H32O4Si
[M+Na]+
: 447.1962 Found:447.1962.
(3aR*,4R*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-dimethyl-4-phenyltetrahydro-
3aH-cyclopenta[d][1,3]dioxol-4-ol (20):
280
PhLi (138 μL of 1.8 M solution in Bu2O, 0.25 mmol) was added dropwise to a cooled (0°C, ice bath)
solution of ketone 19 (70 mg, 0.17 mmol) in THF (4 mL) and the resulting mixture was stirred for 1h
at 0°C. Then it was quenched with saturated aqueous solution of NH4Cl (15 mL). The aqueous phase
was extracted with EtOAc (3x 15 mL), the combined organic parts were dried with MgSO4, filtered
and volatiles were evaporated. The dark brown residue was purified by flash column chromatography
(hexane/EtOAc 10:1) to yield a colourless oil (125 mg, 75%). 1
H NMR (500 MHz, CDCl3): δ = 7.69-
7.62 (m, 4H), 7.47-7.33 (m, 10H), 7.28-7.23 (m, 1H), 4.70-4.63 (m, 2H), 3.87 (dd, J = 10.3, 5.0 Hz,
1H), 3.79 (dd, J = 10.3, 4.4 Hz, 1H), 3.32 (d, J = 1.5 Hz, 1H), 2.73-2.66 (m, 1H), 2.31 (dd, J = 13.6,
6.8 Hz, 1H), 2.18 (dd, 13.6, 1.5 Hz, 1H), 1.59 (s, 3H), 1.35 (s, 3H), 1.08 (s, 9H) ppm. 13
C NMR (126
MHz, CDCl3): δ = 145.25, 135.86, 135.84, 133.85, 133.81, 129.92, 128.53, 127.93, 127.92, 127.26,
125.32, 114.74, 87.11, 82.23, 78.57, 64.46, 46.14, 42.73, 27.16, 26.61, 24.89, 19.60.
(3aR*,4R*,6R*,6aR*)-6-(hydroxymethyl)-2,2-dimethyl-4-phenyltetrahydro-3aHcyclopenta[d][1,3]dioxol-4-ol
(21)
To a solution of compound 20 (72 mg, 0.14 mmol) in wet THF (3 mL) was added TBAF (154 μL of 1
M solution in THF, 154 mmol) and the reaction mixture was stirred at 25°C for 14h. The solvent was
evaporated and the brown oily residue was purified on a short silica gel column (CH2Cl2/MeOH 20:1)
to yield a white crystalline solid (33 mg, 87%). 1
H NMR (500 MHz, CDCl3): δ = 7.480-7.42 (m, 2H),
7.36-7.31 (m, 2H), 7.26-7.22 (m, 1H), 4.70-4.63 (m, 1H), 3.83 (dd, J = 10.7, 5.0 Hz, 1H), 3.67 (dd, J
= 10.7, 6.7 Hz, 1H), 3.38 (d, J = 1.5 Hz, 1H), 2.73-2.65 (m, 1H), 2.32 (dd, J = 13.5, 6.6 Hz, 1H),
1.97 (dd, J = 13.5, 1.5 Hz, 1H), 1.60 (s, 3H), 1.37 (s, 3H) ppm. 13
C NMR (126 MHz, CDCl3): δ =
144.87, 128.52, 127.36, 125.32, 115.08, 86.83, 83.11, 78.29, 64.06, 46.33, 42.28, 26.57, 24.93.
(1R*,2R*,3R*,4R*)-4-(hydroxymethyl)-1-phenylcyclopentane-1,2,3-triol (22)
To a solution of compound 21 (33 mg, 0.124 mmol) in MeOH (4 mL) was added PPTS (156 mg,
0.620 mmol) and the mixture was stirred at 25°C for 24h. The solvent was evaporated and the residue
281
was purified by flash column chromatography (CH2Cl2/MeOH 20:1 to 2:1) to yield a white crystalline
solid (14 mg, 48 %). m.p: 141.3-143°C. 1
H NMR (500 MHz, DMSO-d6): δ = 7.49-7.44 (m, 2H), 7.33-
7.27 (m, 2H), 7.22-7.17 (m, 1H), 4.66 (s, 1H), 4.58 (d, J = 6.09 Hz, 1H), 4.55 (m, 1H), 4.44 (d, J =
7.68 Hz, 1H), 3.82 (dd, J = 14.14, 6.83 Hz, 1H), 3.79 (m, 1H), 2.81 (m, 1H), 2.22 (m, 1H), 1.95 (dd, J
= 13.5, 8.4 Hz, 1H), 1.66 (dd, J = 13.5, 10.07 Hz, 1H) ppm. 13
C NMR (126 MHz, DMSO-d6): δ =
146.27, 127.55, 126.03, 125.33, 80.73, 77.89, 72.91, 62.70, 46.85 ppm. IR (KBr): ν˜max = 3465,
3272, 2960, 2933, 1645, 1417, 1130, 1022, 700 cm–1
. HR-MS (ESI): calcd for C12H6O4 [M+H]+
:
224.0976 Found:224.0970.
((3aR*,4R*,6R*,6aR*)-6-hydroxy-2,2-dimethyl-6-phenyltetrahydro-3aHcyclopenta[d][1,3]dioxol-4-yl)methyl
4-methylbenzenesulfonate (23)
To a cooled (0°C, ice bath) solution of compound 21 (73 mg, 0.276 mmol) in CH2Cl2 (5 mL) were
added Et3N (154 μL, 1.1), DMAP (3 mg, 0.028 mmol) and TsCl (58 mg, 0.304 mmol). The mixture
was stirred at 25°C for 3h. The solvent was evaporated and the residue was purified by flash column
chromatography (CH2Cl2/EtOAc 20:1) to yield a colourless solid (107 mg, 93 %). m.p: 141.3-143°C.
1
H NMR (500 MHz, CDCl3): δ = 7.81-7.76 (m, 2H), 7.44-7.27 (m, 6H), 7.26-7.21 (m, 1H), 4.64 (d, J
= 7.90 Hz, 1H), 4.53 (m, 1H), 4.22 (dd, J = 10.10, 4.74 Hz 1H), 4.10 (dd, J = 10.10, 4.84 Hz, 1H),
3.28 (brs, 1H), 2.79-2.72 (m, 1H), 2.43 (s, 3H), 2.24 (dd, J = 13.6, 6.6 Hz, 1H), 1.97 (m, J = 13.6 Hz,
1H), 1.55 (s, 3H), 1.31 (s, 3H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 145.19, 144.32, 130.16,
128.61, 128.20, 125.29, 115.39, 86.43, 81.60, 78.08, 70.71, 43.46, 42.08, 26.51, 24.92, 21.85 ppm.
(3aR*,4R*,6R*,6aR*)-2,2-dimethyl-4-phenyl-6-(tosyloxymethyl)tetrahydro-3aHcyclopenta[d][1,3]dioxol-4-yl
4-bromobenzoate (24)
To a cooled (0°C, ice bath) stirred suspension of NaH (60%, 5 mg, 0.135 mmol) in anhydrous THF (2
mL) was added a solution of compound 23 (47 mg, 0.113 mmol) in anhydrous THF (1 mL) dropwise.
282
The resulting mixture was vigorously stirred at 25°C for 2 h. Then, 4-bromobenzoyl chloride (27 mg,
0.124 mmol) was added in one portion and the mixture was stirred at 25°C for additional 24 h. The
reaction was quenched with saturated aqueous NH4Cl (0.2 mL) and the solvent was evaporated. The
residue was dissolved in CH2Cl2 (1 mL) and purified using preparative TLC (silica gel, CH2Cl2) to
give benzoate 24 as a white solid (35 mg, 51 % yield). 1
H NMR (500 MHz, CDCl3): δ = 7.85 (m,
2H), 7.73 (m, 2H), 7.52 (m, 2H), 7.40 (m, 2H), 7.34-7.23 (m, 5H), 4.82 (d, J =7.00 Hz, 1H), 4.51 (dd,
J = 7.1, 4.4 Hz, 1H), 3.96 (m, 2H), 3.04 (dd, J = 13.9, 7.1 Hz 1H), 2.56 (m, 1H), 2.43 (s, 3H), 2.19
(dd, J = 13.9, 10.2 Hz, 1H), 1.56 (s, 3H), 1.33 (s, 3H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 164.63,
145.24, 131.85, 131.42, 130.29, 130.16, 128.73, 128.36, 128.19, 125.96, 114.30, 86.83, 85.57, 81.11,
70.34, 42.33, 37.35, 26.67, 25.09, 21.85 ppm. Crystal data for 24: CCDC ref. No. 937690.
Crystallized from toluene, C29H29BrO7S, Mrel = 601.51, T = 120 K, space group P-1, a = 8.0723(4) Å,
b = 12.4224(5) Å, c = 13.9348(6) Å, α = 92.267(4), β = 96.617(4), γ = 106.814(4), V = 1324.79 Å3
, R
= 0.026.
WST-1 assay of compounds 2a, 2b, and 2c
The following B-lymphoid cell lines were used: SU-DHL-4 (diffuse large B-cell lymphoma, del/mut
TP53), JEKO-1 (mantle cell lymphoma, del/mut TP53), and JVM-3 (mantle cell lymphoma, wtTP53).
Cells were seeded in 96-well plates in duplicates (1 × 10(5) cells per well, volume 200 µl) and
subjected to 72 h exposure of the studied compounds at 10 µM and 100 µM concentrations (the
concentrations used were obtained by dilution of 50 mM stock solutions of the compounds in
DMSO). DMSO was added to mock control. The cell viability was assessed by the metabolic WST-1
assay (Roche) using spectrophotometer 1420 Multilabel Counter Victor (PerkinElmer).
Glycosylase activity assays
Samples Positive control
(Enzyme + DMSO)
Enzyme + compound Negative Control
Protein extract 1 l 1 l milliQ-filtered
H2O 3 l 3 l 4 l
Compound - 1 l DMSO
1 l - 1 l
DMSO was added to the compounds to give 10 mM stock solutions. 1 l of the stock solution was
added to each sample for initial testing (final concentration 1 mM). Compounds that decreased
enzyme activity were further diluted with DMSO to 5 mM, 2,5 mM and 1 mM (which gave
compound concentrations of 0,5 mM, 0,25 mM and 0,1 mM, respectively, after adding 1 l to the
samples).
283
Protein extracts were diluted to the desired concentration using protein dilution buffer.
A master mix containing reaction buffer and substrate (labelled oligonucleotide) was made for the
appropriate number of reactions.
Neil1 (bifunctional glycosylase)
Enzyme concentration: 7-10 nM
Substrate: 5ohU/G
Samples were prepared according to the table above.
5 l mastermix was added to each sample. Incubated at 37°C for 10 minutes.
10 l glycosylase stop solution was added to each sample followed by incubation at 95°C for 5
minutes. Samples were analyzed on 20% denaturing urea gels. Gels were run at 200 volt for 50
minutes. The gels were transferred to 3M paper and dried at 80°C for 45 minutes. The dry gels were
placed in a storage phosphor screen over night, and subsequently scanned on the Typhoon 9410
Variable Mode Image. ImageQuant TL Version 2003.02 (Amersham Biosciences) was used to
analyze the results.
284
Part 4c
Highly diastereoselective flexible synthesis of new carbocyclic C-nucleosides*
*manuscript submitted as:
Maier, L.; Khirsariya, P.; Hylse, O.; Adla, S. K.; Černová, L.; Poljak, M.; Krajčovičová, S.; Weis, E.; Drápela,
S.; Souček, K.; Paruch, K.*
Highly Diastereoselective Flexible Synthesis of New Carbocyclic C-nucleosides J.
Org. Chem. 2016.
Introduction
For several decades, nucleoside analogs have been of high interest to medicinal chemists. Numerous
biologically active nucleosides have been identified and more than 30 of them are clinically used.1
Classical nucleosides (structure A in Figure 1) possess the hemiaminal motif; their chemical and
metabolic stability is therefore often limited and the resulting metabolites can be a source of
undesired side effects.1a,2
Significant effort has thus been invested into identification of more stable
analogs while preserving the biological activity. Two main strategies involve replacement of the C-N
bond between sugar and base by the more stable C-C bond (C-nucleosides, structure B in Figure 1)3
and replacement of the tetrahydrofuran motif by a carbocyclic ring (e. g., cyclopentane), which leads
to carbocyclic N-nucleosides (structure C in Figure 1).4
Figure 1. Generic structures of natural nucleosides (A) and their analogs B, C and D.
Structure D in Figure 1 combines the stabilizing elements of structures B and C (i. e. C-C connection
between the (heterocyclic) base and the carbocyclic scaffold) and represents carbocyclic Cnucleosides,
which are only sporadically documented in the literature. It is conceivable that, at least in
some cases, those compounds might be more robust versions of nucleoside analogs B and C.
Furthemore, installation of certain substituents (e. g. R1´
= OH) is meaningful only in this series, as
this would lead to chemically unstable ketals and aminals in the other analog series. Compounds with
general structure D where R1´
= H are quite rare and we are aware of only one analog of type D
285
containing R1´
= OH with ribose-like substitution pattern – moderately active inhibitor of human
glycosylase Neil1 (compound 1 in Figure 1) which we reported recently.5
This scarcity could be
caused by the lack of sufficiently efficient and versatile synthetic routes to these compounds that
would allow flexible variation of the substituents on the cyclopentane core. To our best knowledge,
the reported syntheses are focused on the production of single target carbocylic C-nucleosides6
and do
not allow easy manipulation of the substituents, which would enable the SAR mapping and facile
identification of direct analogs of nucleosides A-C with attractive biological activity.
We envisioned that a properly protected cyclopentanone 2 could be a suitable flexible precursor for
three sub-series of target carbocyclic C-nucleosides (Scheme 1). Specifically, stereoselective addition
of organometallic reagents5
was to produce series 3 and transition metals catalyzed coupling of
(heretofore unknown) enol triflate of 2 was to afford unsaturated analogs 4. Stereoselective
hydrogenation of 4 was envisioned to yield sub-series 5. Similarly to 3, unsaturated compounds 4 are
also meaningful in the class of carbocyclic C-nucleosides, but not for A, B or C, where the presence
of double bond would lead to unstable oxonium salts and/or enamines. Precursor 2 with the desired
stereochemistry was to be prepared from inexpensive norbornadiene.7
Scheme 1. Retrosynthetic Analysis of Carbocyclic C-nucleosides 3, 4 and 5
Results and discussion
In order to prepare analogs represented by generic structure 3 (Scheme 1), we first focused on
improvement of preparation of the acetonide-protected cyclopentanone 2a, which we had previously
used to prepare compound 1.5
We modified the route reported for a closely related analog of 2a (with
R5
= Bn).7
Briefly, diastereoselective cis dihydroxylation of norbornadiene followed by reaction with
2,2-dimethoxypropane provided acetonide-protected diol 6a, subsequent ozonolytical cleavage,
reduction, and monosilylation afforded intermediate 7a, which was then converted into iodide 8a
(Scheme 2). Finally, elimination followed by oxidative cleavage yielded the desired cyclopentanone
2a.
286
Scheme 2. Preparation of Key Cyclopentanone Intermediatesa
a
Reagents and conditions: i) a) OsO4 or K2Os2O8.2H2O, NMO acetone:H2O 4:1, 40 °C then Na2S2O5, (40-
55%); b) 2,2-DMP, TsOH, acetone, rt, (95% of 6a); Ph2C(OCH3)2, TsOH, CH2Cl2, rt, (90 % of 6b); CDI,
PhCH3, 55 °C, (80% of 6c); DTBSOTf, imidazole, DMAP, DMF, 0 °C to rt, (78% of 6d); NaH, BnBr, TBAI,
DMF, rt, (75-90% of 6e); NaH, PMBCl, TBAI, DMF, rt, (93% of 6f); TBSCl, imidazole, CH2Cl2, rt, (76% of
6g); TBDPSCl, imidazole, CH2Cl2, rt, (43% of 6h), TIPSOTf, imidazole, DMAP, DMF, 65 C, (88% of 6i) ii)
a) O3, CH2Cl2/MeOH, -78 °C then NaBH4, -78 °C to rt, (50-65% from 6a, 70-80% of 7b) b) NaH, TBDPSiCl,
THF, rt, (76 % of 7a); iii) Piv-Cl, DIPEA, DMAP, CH2Cl2, rt (70% of 7b); iv) NaH, BnBr, THF, rt, (65-75% of
7d); v) Ph3P, I2, imidazole, CH2Cl2, 0 °C to rt, (85% of 8a); vi) a) DBU, PhCH3, 110 °C, (75% from 8a); b) O3,
CH2Cl2/MeOH, -78 °C then thiourea, -78 °C to rt, (92% of 2a) or OsO4, NaIO4, THF:H2O 1:1, rt, (65-85% of
2a); vii) Bu3P, 3-NO2PhSeCN, THF, rt, then H2O2, 0 °C to rt (80%, over 2 steps of 9b, 75% over 2 steps of 10b)
viii) O3, CH2Cl2, -78 °C then thiourea, rt, (90% of 2b), (86% for 2c).
Unfortunately, we soon realized that the utility of acetonide-protected cyclopentanone 2a was
somewhat limited. While it did undergo highly diastereoselective additions5
with a variety of
nucleophiles (the other diastereomers could not be detected by TLC or NMR), the final deprotection
287
of acetonide in many cases proved to be extremely difficult. For instance, we were not able to utilize
this route to prepare uracil-containing target compound 15a (Scheme 3).
Scheme 3. Nucleophilic Addition to Cyclopentanone 2ab
b
Reagents and conditions: i) 2,4-bis(benzyloxy)-5-bromopyrimidine, n-BuLi, THF, -78 °C to rt, (65-80%); ii)
Pd/C, H2, EtOH, 80 C, (85-95%); iii) TBAF, THF, rt, (90-98%); iv) CSA, CH2Cl2: MeOH 3:1, rt, (46%).
We tried to deprotect advanced intermediate 14 under a variety of standard conditions8
(e. g., aqueous
HCl in MeOH, CH3COOH, CF3COOH, CSA, PPTS, I2 in MeOH, FeCl3.6H2O, BCl3, Dowex®
50WX8 100-200 mesh, In(OTf)3
9
) at different temperatures as well as in the presence of additives
(e.g. ethylene glycol or propan-1,3-dithiol in order to promote transketalization). Typically, we
observed either low conversion or decomposition. Similarly, when we tried to cleave the acetonide in
an earlier intermediate (13), only desilylation and decomposition were observed. This general failure
can be rationalized by facile carbocation formation at position 1´ under acidic conditions. Indeed,
when intermediate 14 was treated with CSA in a mixture of dichloromethane and MeOH, elimination
occured and alkene 16 was isolated in 46% yield.
We thus needed to identify suitable alternative protecting group(s) for the hydroxyls at 2´ and 3´
positions, which i) would be compatible with the conditions of the synthetic sequence in Scheme 2, ii)
their cleavage would be facile (preferably under non-acidic conditions) and iii) ideally, they would be
orthogonal to the group protecting the hydroxyl at position 5´.
First, we utilized intermediate 6b with diphenyl ketal in place of acetonide hoping to remove it
hydrogenolytically10
and prepared analogs of 13 and 14. Unfortunately, the deprotection failed under
288
different conditions (H2, HCOONH4, different metal catalysts, e. g., PtO2, Pd/C, Pd(OH)2/C), in
different solvents at various temperatures with or without additives (AcOH, TFA). We mainly
observed only the starting material and no traces of the desired product. Then, we tried a variety of
several cyclic and non-cyclic protecting groups; e. g., carbonate, bis(t-butyl)silyl, Bn, PMB, TBS, and
TBDPS. Rather surprisingly, even very early intermediates, i. e. protected diols 6b-6i (Scheme 2)
were not known, except for recently reported but poorly described carbonate 6c.11
TBDPS-protected
intermediate 6h was obtained in low yield and thus was not elaborated further. Carbonate 6c and
TBS-protected intermediate 6g underwent undesired transformations during the ozonolytical cleavage
followed by reduction with NaBH4. The stability of the intermediates derived from 6d bearing cyclic
silicon-based protecting group was limited and purification by flash column chromatography of the
corresponding alkene and ketone provided only low yields of the compounds. On the other hand, we
were able to convert intermediates protected with Bn, PMB and TIPS groups (compounds 6e, 6f and
6i, respectively) into the desired cyclopentanones efficiently on multigram scale. However, the
stability of some of the cyclopentanones proved to be limited. For instance, the analogs of compound
2a with benzyls or PMB in place of acetonide underwent elimination even during the purification on
neutral alumina and epimerization at position 2´ in the presence of triethylamine in dichloromethane.
Gratifyingly, we realized that the stability of TIPS-protected cyclopentanones 2b and 2c was much
better – we could purify the compounds by column chromatography and store them at 25 °C for
months without noticeable decomposition. However, we had to modify the route used for 2a as the
elimination of HI from intermediate 8b was extremely sluggish. Fortunately, one-pot selenation of 7c
and 7d and subsequent oxidation followed by intramolecular elimination12
proceeded smoothly and
provided the desired exocyclic alkenes 9b and 10b, respectively, which enabled preparation of
cyclopentanones 2b and 2c on relatively large (> 5 g) scale (Scheme 2). It is likely that while the
steric hindrance caused by the TIPS group at 2´-hydroxyl makes abstraction of proton at position 1´ in
8b difficult during the elimination, analogous shielding provided by the TIPS group in
cyclopentanones 2b and 2c positively contributes to their stability by protecting the otherwise easily
enolizable position 2´.13
The TIPS-protected cyclopentanone 2c underwent highly diastereoselective addition with lithiated
bis(benzyloxy)pyrimidine (Scheme 4) and the resulting adduct was successfully converted into the
desired target compound 15a. By the same methodology, we prepared additional target compounds
(15b-e) bearing alkyl and (hetero)aryl moieties (Scheme 4).
289
Scheme 4. Synthesis of Pseudouridine Analog 15ac
c
Reagents and conditions: i) 11, n-BuLi, THF, -78 °C then 2c, -78 °C to rt, (45%); ii) Pd/C, H2, EtOH, 80 C,
(93%); iii) TBAF, THF, rt, (85%); iv) a) 2c, n-BuMgCl, THF, 0 °C to rt (38% of 17b); PhLi, THF, 0°C (65% of
17c); BnMgCl, THF, 0 °C to rt, (79% of 17d); 4-bromothiazole, (CH3)2MgCl·LiCl (90 % of 17e); b) TBAF,
THF, rt, (82% of 19a, 96% of 19b, 90% of 19c); Li, naphthalene, THF, rt, then TBAF, THF, rt (53% of 15e
over 2 steps) c) Pd(OH)2/C, H2 (50 bar), THF, 70 C, (93% 15b, for 15c 92%, for 15d 75%); v) a) Pd/C, H2,
EtOH, 80 °C, (75%); b) NaH, CH3I, THF, 0 °C to rt, (67%); c) TBAF, THF, rt, (42%).
In addition, orthogonal deprotection of the benzyl group allowed us to selectively modify the
hydroxyl at position 5´: debenzylation of compound 20 followed by methylation and cleavage of the
TIPS groups provided target compound 21 (Scheme 4).
Next, we addressed the sub-series 4 depicted in Scheme 1. Treatment of acetonide-protected
cyclopentanone 2a with LDA at -78 C followed by addition of N-phenylbis(trifluoromethansulfonimide)
provided stable enol triflate 22a in good yield (Scheme 5). In
contrast, in order to get a clean and complete conversion of TIPS-protected 2b into enol triflate 22b,
we had to optimize the conditions (KHMDS added to a mixture of 2b and Commins’ reagent). The
Suzuki coupling of both 22a and 22b with phenylboronic acid proceeded smoothly and afforded
compounds 23a and 25a, respectively, in good yields (Scheme 5). In addition, we were able to
convert enol triflate 22a into the corresponding boronate 24a by Pd-catalyzed borylation;14
but, in
contrast, we were not able to perform analogous transformation on enol triflate 22b under a variety of
conditions.
290
Scheme 5. Formation of Enol Triflates and Their Transformationsd
d
Reagents and conditions: i) LDA, THF, -78 °C then PhNTf2, -78 °C to rt, (60-80% for 22a); KHMDS,
Commins reagent, THF, -78 C to rt, (97% for 22b ii) PhB(OH)2, Pd(dppf)Cl2, K3PO4, DME, H2O, 80 C, (70-
80%); iii) pin2B2, Pd(Ph3P)2Cl2, Ph3P, KBr, KOPh, PhCH3, 50 C (70 %) iv) PhB(OH)2, Pd(dppf)Cl2, K3PO4,
DME, H2O, 80 C, (92% for 25a), 2,4-difluorophenylboronic acid, Pd(PPh3)4, LiCl, Na2CO3, DME, H2O, 80
°C, (76% for 25b), 1-methylpyrazole-4-boronic acid pinacol ester, Pd(dppf)Cl2, K3PO4, DME, H2O, 80 C, (87
% for 25c), 4-(methoxycarbonyl)furan-2-boronic acid pinacol ester, Pd(dppf)Cl2, K3PO4, DME, H2O, 80 C,
(81% for 25d), 3-benzothienylboronic acid, Pd(dppf)Cl2, K3PO4, DME, H2O, 80C, (75% for 25e), boronic acid
29, Pd(dppf)Cl2, K3PO4, DME, H2O, 80 C, (87 % for 25f); v) a) TBAF, THF, rt, (89% for 26a, 90% for 26b,
91% for 26c, 86% for 26d, 83% for 26e, 88% for 26f); vi) MeONa, MeOH, 65 °C, (89% for 27a, 87% for 27b,
91% for 27c, 63% for 27d, 91 % for 27e, 88 % for 27f); vii) PPPTS, MeOH, H2O, 55 C, (75 %).
Once again, acetonide cleavage turned out to be problematic. We obtained best results (38% yield)
when we used PPTS to deprotect intermediate 23a. More forcing conditions (aq. HCl, CH3COOH,
CF3COOH) were incompatible with the presence of the double bond and (partial) cleavage of the
TBDPS group was also observed. On the other hand, selective TIPS deprotection on 25a was
successful and yielded the desired intermediate 26a in high yield (89%). Final cleavage of the
pivaloate with DIBAL or sodium methoxide proceeded uneventfully. Using different boronic
291
acids/boronates, we prepared additional target compounds 27b-f with diverse substituents at position
1´. In order to prepare the new unsaturated carbocyclic analog of tubercidine 28, we utilized
heretofore unknown boronic acid 29, whose preparation (described in SI) from commercially
available 7-bromopyrrolo[1,2-f][1,2,4]triazin-4-amine as well as the coupling were in our hands more
reliable than with the (known) unprotected analog.15
The SEM groups were removed from
intermediate 27f under mild conditions (Scheme 6).
Scheme 6. Hydrogenation of Cyclopentene Intermediatese
e
Reagents and conditions: i) Pd/C, H2, EtOH, 80 C, (85%); ii) a) PPTS, MeOH, rt, (38% for 31, 41% of 23a
recovered); b) Pd/C, H2, EtOH, 80 C, (60% of 31a and 27% of 31b); iii) Crabtree’s catalyst, H2, CH2Cl2, rt,
(94%); iv) MeONa, MeOH, 65 °C, (89%); v) a) Crabtree’s catalyst, H2, CH2Cl2, rt, (92% for 32b, 95% for 32d,
86% for 32e); Pd(OH)2/C, H2, THF, rt, (44% of 32c, 49% of 32f); vi) MeONa, MeOH, 65 °C, (90% for 33b,
53% for 33c, 74% for 33d, 88% for 33e, 79% of 33f). vii) PPPTS, MeOH, H2O, 55 C, (80 %).
X-ray crystal structure
of compound 33a
X-ray crystal structure
of compound 33c
292
Finally, we focused on preparation of saturated analogs represented by generic structure 5 in Scheme
1.
Hydrogenation of the acetonide-protected intermediate 23a proceeded exclusively from the less
hindered top face of the cyclopentene scaffold and yielded only the undesired diastereomer 30. The
diastereoselectivity was more promising when the acetonide in intermediate 23a was cleaved prior to
the hydrogenation, which then afforded a separable mixture of the desired intermediate 31a (60%)
and its epimer 31b (27 %). Gratifyingly, we realized that diol 26a can be hydrogenated with
practically perfect diastereoselectivity in the presence of Crabtree’s catalyst16
to yield pivaloate 32a.
Final deprotection afforded triol 33a in high overall yield (66 % over 5 steps from 2b). The relative
configuration of triol 33a was unambiguously confirmed by X-ray crystallography. We then
investigated the scope of the highly diastereoselective hydrogenation in preparation of target
compounds 33b-f.
The hydrogenation of intermediates which afforded compounds possessing aromatic and O and S
heteroaromatic moieties (33b, 33d and 33e) proceeded with excellent diastereoselectivity. On the
other hand, the rate and diastereoselectivity of the hydrogenation of the substrates with N-containing
substituents (target compounds 33c and 33f) were significantly worse, which is likely caused by
preferential coordination of the catalyst to the nitrogen atoms. We tried a variety of conditions,
varying pressure (up to 150 bar), temperature, solvents (THF, CH2Cl2, PhCH3) and the catalyst (Ir, Rh
or Ru based catalysts); however, we did not observe any significant improvement. For the Ncontaining
substrates we eventually used Pd(OH)2. Although these hydrogenations gave
diastereomeric mixtures with the ratio of ca. 2:1, in favor of the desired epimers, the diastereomers
were in all cases separable by column chromatography. This route also provided another heretofore
unknown carbocyclic C-analog of tubercidine (compound 34 in Scheme 6), which we briefly tested
for cytotoxicity. Compared to tubercidine, 34 was found to be less active against tumorigenic MCF7
cells (IC50 values 110 nM for tubercidine and 30 M for 34), but comparatively less toxic to human
foreskin fibroblast HFF1 cells (56 nM for tubercidine and >100 M for 34). On the other hand, the
1´-epimer of 34 and unsaturated analog 28 were inactive.
Next, we utilized the orthogonality of TIPS and pivaloate protecting groups to further selectively
manipulate the position 5´. Cleavage of the pivaloate followed by tosylation provided tosylate 35,
which proved to be a versatile intermediate for manipulation of position 5´ via nucleophilic
substitution: it underwent substitutions with ammonia or primary amines; amine 37 was used for
293
subsequent reactions with different electrophiles, which ultimately provided target compounds 38a-c
(Scheme 7).
Scheme 7. Modifications of Position 5´ f
f
Reagents and conditions: i) a) DIBAl-H, CH2Cl2, -78 °C to rt, (90%); b) TsCl, TEA, DMAP, CH2Cl2 rt,
(98%), ii) a) 2-methoxyethylamine, DIPEA, DMF 100 °C, (69 %) b) TBAF, THF, rt, (90%); iii) NH3 in IPA,
aq. NH3, 75 °C, (87 %), iv) a) AcCl, DIPEA, CH2Cl2, rt, (73%); b) TBAF, THF, rt, (89% of 38a); v) a)
phenylisocyanate, TEA, CH2Cl2, rt, (93%); b) TBAF, THF, rt, (67% of 38b); vi) a) N,N-dimethylsulfamoyl
chloride, TEA, DMF, rt, (86%); b) TBAF, THF, rt, (89% of 38c).
We also attempted to manipulate position 2´ utilizing compound 33a, which we were able to produce
in gram quantities by the routes depicted in Schemes 5 and 6 above. Standard di-protection of 5´- and
3´- hydroxyls by TIPDS17
provided cyclic siloxane 39 (Scheme 8). Alkylation of the remaining free
hydroxyl at position 2´ followed by final deprotection yielded target compound 40. Our attempts to
invert the stereochemistry at position 2´ by Mitsunobu reaction18
failed and we recovered only
unreacted 39. However, we were able to perform the inversion via diastereoselective reduction of
ketone 41 (Scheme 8). While with typically used NaBH4
19
the reduction proceeded with no
diastereoselectivity, the results were more satisfactory with sterically demanding agents: for
LiAlH(O-tBu)3, the ratio of 42 to its epimer was 90:10 and for LiAlH[O-C(CH2CH3)3]3 the formation
of 42 was exclusive. Subsequent removal of the protecting group produced triol 43 in high overall
yield.
294
Scheme 8. Manipulation of Position 2´ g
g
Reagents and conditions: i) TIPDS-Cl, Pyr, 0 °C, (88%); ii) a) n-BuLi, MeOTf, -78 °C, (49%), b) TBAF,
THF, rt, 64 %; iii) IBX, CH3CN, 80 °C, (81%), iv) LiAlH[O-C(CH2CH3)3]3, THF, 0 °C, (92%); v) TBAF, THF,
rt, (73%).
Ultimately, we attempted to develop a route that would allow enantioselective selective synthesis of
all three sub-series 3, 4, and 5. The utility of known chiral precursors proved to be limited:
monoacetate 4420
was not optimal due to the presence of difficult to remove acetonide, while the
synthesis of compound 4521
(especially the alkylation of cyclopentadienide22
) was in our hands
extremely irreproducible. Similarly, attempted desymmetrization of diol 7b by enantioselective
silylation23
or by pivaloylation with chiral variant of DMAP24
also failed. Fortunately, upon extensive
experimentation we found that racemic 7d can be converted into diastereomeric camphanates (-)-46a
and (+)-46b, which we were able to separate by standard column chromatography and re-benzylate25
without the loss of stereochemical integrity (Scheme 9). The absolute configuration of the crystalline
camphanate (-)-46a was confirmed by X-ray crystallography. Interestingly, attempts to prepare
camphanates 46a and 46b directly from diol 7b failed and provided mixtures containing substantial
amounts of poorly separable dicamphanate. Subsequent removal of camphanate from (-)-46a
provided (-)-7d, which was converted into optically pure (-)-2c and (-)-2b. (-)-2b was then elaborated
into (+)-26b, whose enantiomeric purity was confirmed by HPLC on chiral stationary phase, and
ultimately into (+)-27b (Scheme 9).
X-ray crystal structure
of compound 40
X-ray crystal structure
of compound 42
295
Scheme 9. Enantioselective routeh
h
Reagents and conditions: i) a) (1S)-(-)-camphanic chloride, DIPEA, DMAP, CH2Cl2, rt (97%, 1:1
diastereomeric mixture); b) Pd(OH)2/C, H2, THF, 65 C, (67% of (-)46a and 76% of (+)-46b); ii) a) TriBOT,
TfOH, 5Å MS, 1,4-dioxane, rt, (75-90%), b) MeONa, MeOH, rt, (90%); iii) Pd(OH)2/C, H2, THF, 65 °C then
PivCl, DMAP, DIPEA, CH2Cl2, rt (80% over two steps).
In summary, our route enables flexible and diastereoselective synthesis of new carbocyclic Cnucleosides
with a variety of substituents at position 1´ and selective manipulations of positions 3´
and 5´ of the cyclopentane core. Identification of suitable versatile intermediates (i. e. TIPS-protected
cyclopentanones 2b and 2c) required extensive optimization of the protecting groups. By the
methodology presented above, we have prepared a series of more than 60 new carbocyclic Cnucleosides.
Compared to tubercidine, analog 34 was found to be less potent, but more selectively
toxic to MCF7 cells (breast cancer) than to HFF1 (human foreskin fibroblasts). A detailed account of
the compounds‘ biological activity will be reported elsewhere in the near future.
X-ray crystal structure
of compound (-)46a
296
Experimental procedures
General
All reagents and solvents were of reagent grade and were used without further purification.
Anhydrous solvents (THF, dichloromethane, CH3CN, toluene, DMF) were used and stored over 4Å
molecular sieves as received from commercial suppliers (Aldrich, Acros). All reactions were carried
out in oven-dried glasware and under nitrogen atmosphere unless stated otherwise. Flash column
chromatography was carried on silica gel (230-400 mesh). TLC plates were visualized under UV
and/or with phosphomolybdic acid, KMnO4, CAM or H2SO4 in MeOH.
NMR spectra were recorded on spectrometers Bruker Ascend 500 MHz (with operating frequencies,
500.13 MHz for 1
H, 125.77 MHz for 13
C, 470.53 MHz for 19
F and 160.46 MHz for 11
B) and Bruker
Avance 300 MHz (with operating frequencies 300.13 MHz for 1
H, 75.48 MHz for 13
C). The 1
H, and
13
C NMR chemical shifts (δ in ppm) were referenced to the residual signals of solvents: CDCl3 [7.26
(1
H) and 77.23 (13
C)], CD2Cl2 [5.32 (1
H) and 53.50 (13
C)], CD3OD [3.35 (1
H) and 49.30 (13
C)],
acetone-d6 [2.09 (1
H) and 29.90, 206.7 (13
C)], and DMSO-d6 [2.50 (1
H) and 39.51 (13
C)]. 19
F NMR
chemical shifts (δ in ppm) were referenced to the signal of trifluorotoluene (-63.72). Structural
assignment of resonances have been performed with the help of 2D NMR gradients experiments
(COSY, multiplicity edited 1
H-13
C HSQC, 1
H-13
C HMBC, NOESY, 1
H-15
N HSQC and 1
H-15
N
HMBC).
HPLC analysis was performed on Agilent 1260 InfinityTM
HPLC/MS system or UltimateTM
3000
Thermo Scifentific UHPLC system.
The diffraction data were collected with a Rigaku partial χ geometry diffractometer equipped with a
Saturn 944+ HG CCD detector and a Cryostream Cooler (Oxford Cryosystems, UK). Cu Kα radiation
(λ= 1.54184 Å, MicroMax-007HF rotating anode source, multilayer optic VariMax) was used. Data
reduction and final cell refinement were carried out using the CrysAlisPro software (CrysAlisPRO,
Agilent Technologies UK Ltd).
High resolution mass spectra have been measured on Agilent 6224 Accurate-Mass TOF LC-MS with
dual electrospray/chemical ionization mode with mass accuracy greater than 2 ppm, applied mass
range from 25 to 20,000 Da.
IR spectra (4000-400 cm–1
) were collected on an EQUINOX 55/S/NIR FTIR spectrometer and on
Bruker Platinum ATR (4000-400 cm–1
). Solid samples were measured neat or as KBr pellets and oily
samples as a film evaporated from CH2Cl2 or CH3OH solutions.
297
Melting points were measured on SMP 40 Stuart®
apparatus in capillary and are uncorrected. Optical
rotations were measured on polarimeter AUTOPOL III (Rudolph Research Analytical) in cuvettes
with the path length of 10 cm.
CD spectra were recorded at room temperature using Chirascan spectrometer (Applied Photophysics,
UK). Data were collected from 195 nm to 280 nm in a 1 cm quartz cuvette.
Experimental procedures for cyclopentanone preparation
(1R*,4S*,5S*,6R*)-5,6-bis(triisopropylsilyloxy)bicyclo[2.2.1]hept-2-ene (6i).
Imidazole (14.88 g, 218.73 mmol) and DMAP (0.87 g, 7.14 mmol) were added to a solution of
starting material S-1 (6.00 g, 47.55 mmol) in anhydrous DMF (60 mL), followed by dropwise
addition of TIPSOTf (29.4 mL, 109.38 mmol) at 25 °C. The reaction mixture was stirred at 65 °C for
5 days. The reaction mixture was cooled to 25 °C, quenched with H2O (60 mL), and extracted with
Et2O (3 × 70 mL). The organic extracts were dried over Na2SO4, filtered, and the solvent was
evaporated. The residual yellow oil was purified by flash column chromatography (hexane) to afford
6i as a colorless oil (20.1 g, 88 %). 1
H NMR (500 MHz, CDCl3): δ = 6.00 (m, 2H), 3.84 (d, J = 1.7
Hz, 2H), 2.62 (m, 2H), 2.20 (dm, J = 8.4 Hz, 1H), 1.60 (dm, J = 8.4 Hz, 1H), 1.12-1.07 (m, 42H)
ppm. 13
C NMR (126 MHz, CDCl3): δ = 136.91, 71.40, 49.84, 43.45, 18.51, 18.44, 13.07 ppm. IR
(ν˜max) = 2927 (w), 2800 (w), 1403 (m), 1049 (s), 1111 (s), 702 (m), 646 (m) cm–1
. HR-MS (APCI)
calculated for C25H50O2Si2 [M+H]+
: 439.3422. Found: 439.3427.
General procedure A: ozonolytical cleavage of protected norbornenes
298
Mixture of O3/O2 (O2 flow = 5L/min, ozonolysis rate ~ 12 mmol/5 min) was bubbled into a cooled (–
78 °C) solution of the starting material in CH2Cl2:MeOH (1:3, ~ 15 mmol of the starting material/10
mL). After completion of ozonolysis (indicated by persistent blue color of the reaction mixture),
excess of O3 was removed by bubbling N2 into the reaction mixture. Thiourea (6 eq.) was added in
five portions and the reaction mixture was stirred at –78 °C for 1 h. NaBH4 (0.25 eq.) was added in
one portion and the reaction mixture was stirred at –78 °C for 1 h. Another portion of NaBH4 (1.025
eq.) was added and the reaction mixture was stirred for additional 3-14 h while allowed to warm to 25
°C. The reaction mixture was concentrated under reduced pressure and the residual viscous oil was
partitioned between dichloromethane (100 mL/15 mmol) and brine (50 mL). The aqueous phase was
reextracted with CH2Cl2. The organic extracts were dried over Na2SO4, filtered, and the solvent was
evaporated. The residue was purified by flash column chromatography to afford the corresponding
diol.
(1R*,3S*,4S*,5R*)-4,5-bis(triisopropylsilyloxy)cyclopentane-1,3-diyl)dimethanol (7b).
Prepared by general procedure A using 10.09 g (22.8 mmol) of 6i; flash column chromatography
(CH2Cl2/MeOH = 20:1) afforded 7b as a white solid (10.03 g, 80%), m.p. = 107 – 109 °C. 1
H NMR
(500 MHz, CDCl3): δ = 4.05 (dd, J = 2.7, 7.4 Hz, 2H), 3.63 (ddd, J = 27.0, 10,5, 5.7 Hz, 4H), 2.28
(m, 2H), 2.07 (m, 1H), 1.69 (br s,-OH, 2H), 1.16 (m, 1H), 1.09 (m, 42H) ppm. 13
C NMR (126 MHz,
CDCl3): δ = 77.73, 65.14, 45.02, 25.75, 18.52 ppm. IR (ν˜max) = 3301 (br), 2890 (m), 2859 (m), 1432
(m), 1039 (s), 825(m), 631 (m) cm–1
. HR-MS (ESI) calculated for C25H54O4Si [M+Na]+
: 497.34528.
Found: 497.34518.
((3aS*,4S*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-dimethyltetrahydro-3aHcyclopenta[d][1,3]dioxol-4-yl)methanol
(7a).
A solution of starting material S-2 in 15 mL of anhydrous THF (1.95 g, 9.64 mmol) was added to a
suspension of NaH (0.464 g, 11.6 mmol, 60 % dispersion in mineral oil) in THF (2 mL). The reaction
299
mixture was stirred at 25 °C for 20 min, then TBDPSCl (2.63 mL, 10.12 mmol) was added and the
reaction mixture was stirred for 14 h. The reaction mixture was quenched by addition of silica gel (0.5
g) and adsorbed on silica gel (10 g). Flash column chromatography (hexane/EtOAc = 3:1) afforded 7a
as a colorless oil (3.24 g, 76 %). 1
H NMR (500 MHz, CDCl3): δ = 7.66-7.64 (m, 4H), 7.44-7.36 (m,
6H), 4.41-4.38 (m, 1H), 4.33-4.31 (m, 1H), 3.75-3.60 (m, 4H), 2.31-2.22 (m, 4H), 2.08-2.03 (m, 1H),
1.49 (s, 3H), 1.39 (dd, J = 23.7, 10.9 Hz, 1H), 1.29 (s, 3H), 1.06 (s, 9H) ppm. 13
C NMR (126 MHz,
CDCl3): δ = 135.86, 135.85, 133.89, 129.88, 127.89, 112.62, 83.90, 82.91, 65.25, 65.14, 48.12, 47.36,
30.87, 27.91, 27.13, 25.43, 19.55. ppm. IR (ν˜max) = 3456 (br), 2930 (w), 2857 (w), 1471 (w), 1427
(w), 1110 (s), 1060 (s), 701 (s), 504 (s) cm–1
. HR-MS (ESI) calculated for: C26H36O4Si [M+Na]+
:
463.22571. Found: 463.22752.
(1R*, 2R*, 3S*, 4S*)-4-(hydroxymethyl)-2, 3-bis((triisopropylsilyl)oxy)cyclopentyl)methyl
pivalate (7c).
DMAP (0.153 g, 1.26 mmol) and DIPEA (2.13 mL, 25.26 mmol) were added into a solution of 7b
(6.00 g, 12.63 mmol) in CH2Cl2 (30 mL). Pivaloyl chloride (1.55 mL, 12.63 mmol) was added
dropwise and the reaction mixture was stirred at 25 °C for 14 h. The reaction mixture was quenched
with water (50 mL) and extracted with CH2Cl2 (3 × 50 mL). The organic phase was dried over
MgSO4, filtered and concentrated under reduced pressure to yield the crude product, which was
purified by flash column chromatography (hexane/EtOAc = 10:1) to afford 7c as white crystals (3.87
g, 71 %), m.p. = 63-65 °C. NMR (500 MHz, CDCl3): δ = 4.10 (dd, J = 11.2, 6.0 Hz, 1H), 4.05 (app d,
overlapped, 1H), 3.95 (dd, J = 11.2, 6.0 Hz, 1H), 3.64 (dd, J = 10.5, 6.2, Hz, 1H), 3.56 (dd, J = 10.5,
6.2 Hz, 1H), 2.40 (m, 1H), 2.29 (m, 1H), 2.07 (m, 1H), 1.20 (s, 9H), 1.09 (m, 1H, overlapped,
resolved by 1
H-13
C HSQC experiment), 1.09 (m, 42H) ppm. 13
C NMR (126 MHz, CDCl3): δ =
178.78, 77.24 (overlapped with CDCl3 signal, resolved by 1
H-13
C HSQC experiment), 76.65, 65.79,
65.14, 44.96, 42.10, 39.06, 27.44, 25.95, 18.53, 18.50, 18.48, 18.44, 13.2 ppm. IR (ν˜max) = 2942 (m),
2865 (m), 1731 (m), 1713 (m), 1463 (m), 1143 (s), 881 (s), 677 (s) cm–1
. HR-MS (ESI) calculated for
C30H62O5Si2 [M+Na]+
: 581.4033. Found: 581.4033.
((1S*,2S*,3R*,4R*)-4-((benzyloxy)methyl)-2,3-bis((triisopropylsilyl)oxy)cyclopentyl) methanol
(7d).
300
To a stirred suspension of NaH (0.590 g, 14.76 mmol, 60% dispersion in mineral oil) in THF (10 mL)
was added a solution of compound 7b (5.01 g, 10.54 mmol) in THF (15 mL). After stirring for 30
min and cooling to 0 °C, benzyl bromide (1.28 mL, 10.54 mmol) was added dropwise over the period
of 2 h and the resulting mixture was stirred for 24 h at 25 °C. The reaction was quenched by addition
of silica gel (10 g) and the solvent was evaporated. The pre-adsorbed crude mixture was then purified
by flash column chromatography (hexane/EtOAc = 10:1) to afford 7d as a pale yellow oil (4.55 g, 77
% yield). 1
H NMR (500 MHz, CDCl3): δ = 7.37-7.27 (m, 5H), 4.51 (AB d, J = 12.1 Hz, 1H), 4.44
(AB d, J = 12.1 Hz, 1H), 4.11 (m, 1H), 4.00 (dd, J = 6.6, 3.5 Hz, 1H), 3.67 (dd, J = 10.6, 5.8 Hz, 1H),
3.58 (dd, J = 10.6, 5.8 Hz, 1H), 3.41 (m, 2H), 2.36-2.24 (m, 2H), 2.12-2.04 (m, 1H), 1.19 (m, 1H),
1.13-0.97 (m, 42H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 138.58, 128.51, 127.86, 127.74, 77.88,
77.54, 73.44, 72.43, 65.36, 44.90, 43.46, 43.64, 26.39, 18.52, 18.49, 18.44, 13.37, 13.19 ppm. IR
(ν˜max) = 3438 (br w), 2941 (m), 2864 (m), 1496 (m), 1067 (m), 882 (s), 677 (s) cm–1
. HR-MS (APCI)
calculated for C32H60O4Si2 [M+H]+
: 565.4103. Found: 565.4099.
tert-butyl(((3aR*,4R*,6R*,6aS*)-6-(iodomethyl)-2,2-dimethyltetrahydro-3aHcyclopenta[d][1,3]dioxol-4-yl)methoxy)diphenylsilane
(8a).
Iodine (1.96 g, 7.74 mmol) was added to a cooled (0 °C, ice bath) solution of PPh3 (2.33 g, 8.88
mmol) and imidazole (1.44 g, 21.12 mmol) in CH2Cl2 (5 mL) and the reaction mixture was stirred at
0 °C for 20 min. A solution of starting material 7a (3.1 g, 7.04 mmol) in CH2Cl2 (15 mL) was added
and the reaction mixture was stirred while allowed to warm to 25 °C for 14 h. The solvent was
removed under reduced pressure and the residue was purified by flash column chromatography
(hexane/EtOAc = 20:1 to 15:1) to afford 8a as a colorless oil (3.29 g, 85%). 1
H NMR (500 MHz,
CDCl3): δ = 7.67-7.64 (m, 4H), 7.45-7.37 (m, 6H), 4.45 (dd, J = 6.9, 4.9 Hz, 1H), 4.17 (dd, J = 6.9
Hz, 1H), 3.70 (d, J = 5.6 Hz, 2H), 3.34 (dd, J = 9.9, 5.3 Hz, 1H), 3.24 (dd, J = 9.9, 6.9 Hz, 1H), 2.30
(m, 1H), 2.18 (m, 1H), 2.10 (m, 1H), 1.48 (s, 3H), 1.48 (m, 1H), 1.29 (s, 3H), 1.07 (s, 9H) ppm. 13
C
301
NMR (126 MHz, CDCl3): δ = 135.88, 133.83, 133.81, 129.92, 127.92, 112.79, 85.46, 83.11, 64.81,
47.54, 46.92, 35.54, 27.89, 27.17, 25.49, 19.56, 10.37 ppm. IR (ν˜max) = 2930 (m), 2857 (m), 1471
(w), 1210 (w), 1111 (m), 1069 (m), 702 (s) cm–1
.
(1R*,2R*,3S*)-4-methylene-2,3-bis(triisopropylsilyloxy)cyclopentyl)methyl pivalate (9b).
2-Nitrophenyl selenocyanate (1.88 g, 8.28 mmol) and Bu3P (3.22 mL, 12.88 mmol) were added to a
solution of 7c (2.58 g, 4.6 mmol) in THF (15 mL) and the reaction mixture was stirred at 25 °C for 14
h. Aqeous H2O2 (30% solution, 15.5 mL) was added at 0 °C (ice bath) and the reaction mixture was
stirred at 25 °C for 2 h. The crude mixture was concentrated, diluted by saturated aqueous NaHCO3
(20 mL) and extracted with EtOAc (3 × 30 mL). The organic extracts were dried over Na2SO4 and the
brown residue was purified by flash column chromatography (hexane/EtOAc = 50:1) to afford 9b as a
pale yellow oil (1.99 g, 80 % over two steps). 1
H NMR (500 MHz, CDCl3): δ = 5.12 (m, 1H), 4.92
(m, 1H), 4.40 (d, J = 2.7 Hz, 1H), 4.17 (dd, J = 11.2, 5.9 Hz, 1H), 4.00 (dd, J = 5.0, 2.7 Hz, 1H), 3.97
(dd, J = 11.2, 5.9 Hz, 1H), 2.63 (app dd, J = 16.7, 10.2 Hz, 1H), 2.49 (m, 1H), 2.00 (dm, J = 16.7 Hz,
1H), 1.19 (s, 9H), 1.11-1.06 (m, 42 H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 178.75, 149.52,
109.37, 77.94, 76.41, 65.57, 42.19, 39.10, 29.94, 27.42, 18.50, 18.47, 18.45, 18.40, 13.07 ppm. IR
(ν˜max) = 2942 (m), 2865 (m), 1732 (m), 1463 (w), 1282 (w), 1139 (s), 881 (s), 678 (s) cm–1
. HR-MS
(ESI) calculated for C30H60O4Si [M+H]+
: 563.3930. Found: 563.3929.
((1S*,2R*,3R*)-3-((benzyloxy)methyl)-5-methylenecyclopentane-1,2-diyl)bis(oxy))bis
(triisopropylsilane) (10b).
By the essentially same procedure used for compound 9b, 4.52 g (8.01 mmol) of 7d afforded
(purification by flash column chromatography (hexane/EtOAc = 50:1)) 10b as a pale yellow oil (3.28
g, 75% over the two steps). 1
H NMR (500 MHz, CDCl3): δ = 7.35-7.26 (m, 5H), 5.08 (m, 1H), 4.89
(m, 1H), 4.52 (AB d, J = 12.0 Hz, 1H), 4.45 (AB d, J = 12.0 Hz, 1H), 4.41 (d, J = 2.8 Hz, 1H), 4.00
(dd, J = 3.7 Hz, 1H), 3.47 (dd, J = 9.1, 5.4 Hz, 1H), 3.41 (dd, J = 9.1, 5.4 Hz, 1H), 2.61 (m, 1H), 2.39
302
(m, 1H), 2.11 (dm, J = 17.2 Hz, 1H), 1.08-0.91 (m, 42H) ppm. 13
C NMR (126 MHz, CDCl3): δ =
150.55, 138.75, 128.49, 127.81, 127.68, 108.23, 77.92, 76.88, 73.38, 72.41, 43.36, 30.40, 18.49,
18.48, 18.45, 18.41, 13.11, 13.04 ppm. IR (ν˜max) = 2942 (m), 2865 (m), 1463 (m), 1110 (s), 882 (s),
679 (s) cm–1
. HR-MS (APCI) calculated for C32H58O3Si2 [M+H]+
: 547.3997. Found: 373.2551, Δ
mass: 174.1446 (-OTIPS).
General procedure B: ozonolysis of exocyclic alkenes
A mixture of O3/O2 (O2 flow = 5L/min, ozonolysis rate ~ 12 mmol/5 min) was bubbled into a cooled
(–78 °C) solution of the starting material in CH2Cl2:MeOH (1:3, 3 mmol of starting material/25 mL).
After full conversion of ozonolysis (indicated by persistent blue color of the reaction mixture), excess
of O3 was removed by bubbling N2 into the reaction mixture. Thiourea (2 eq.) was added and the
reaction mixture was stirred for 3 h at 25 °C. The solvents were removed and the solid residue was
purified by flash column chromatography to afford the desired ketone.
(3aR*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-dimethyldihydro-3aHcyclopenta[d][1,3]dioxol-4(5H)-one
(2a).
Prepared by general procedure B using alkene S-7 (3.25 g, 7.69 mmol); flash column chromatography
(hexane/EtOAc = 5:1) afforded 2a as a white crystalline solid (3.0 g, 92%), m.p. = 96-97 °C. 1
H
NMR (500 MHz, CDCl3): δ = 7.62-7.59 (m, 4H), 7.47-7.38 (m, 6H), 4.63 (d, J = 5.4 Hz, 1H), 4.35 (d,
J = 5.4 Hz, 1H), 3.80 (dd, J = 10.2, 2.8 Hz, 1H), 3.61 (dd, J = 10.2, 2.8 Hz, 1H), 2.75 (dd, J = 18.4,
9.0 Hz, 1H), 2.49 (m, 1H), 2.19 (m, 1H), 1.43 (s, 3H), 1.34 (s, 3H), 1.02 (s, 9H) ppm. 13
C NMR (126
MHz, CDCl3): δ = 213.41, 135.94, 135.82, 132.97, 132.61, 130.24, 130.19, 128.09, 111.48, 81.79,
79.32, 77.48, 77.23, 76.98, 66.13, 39.31, 37.49, 27.05, 27.02, 24.89, 19.35. IR (KBr, ν˜max) = 3486,
2933, 1755, 1589, 1429, 1108, 705.85 cm–1
. HR-MS (ESI) calculated for C25H32O4Si [M+Na]+
:
447.1962. Found: 447.1962. The spectral data were consistent with those reported.5
303
(1R*,2R*,3R*)-4-oxo-2,3-bis(triisopropylsilyloxy)cyclopentyl)methyl pivalate (2b).
Prepared by general procedure B using 9b (5.74 g, 10.57 mmol); flash column chromatography
(hexane/EtOAc = 30:1) afforded 2b as a colorless oil (5.46 g, 95 %). 1
H NMR (500 MHz, CDCl3): δ
= 4.41 (dd, J = 3.9, 1.5 Hz, 1H), 4.37 (m, J = 3.9 Hz, 1H), 4.13 (dd, J = 11.6, 8.6 Hz, 1H), 4.05 (dd, J
= 11.6, 6.2 Hz, 1H), 2.62 (m, 1H), 2.51 (dd, J = 18.9, 10.1 Hz, 1H), 1.96 (dm, J = 18.9, 1H), 1.20 (s,
1H), 1.10-1.05 (m, 42H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 212.01, 178.52, 78.76, 74.25, 65.40,
39.64, 39.05, 34.29, 27.36, 18.33, 18.31, 18.29, 18.17, 12.81, 12.78 ppm. IR (ν˜max) = 2942 (m), 2866,
1763 (m), 173 (s), 1463 (m), 1132 (s), 881 (s), 676 (s) cm–1
. HR-MS (APCI) calculated for:
C29H58O5Si2 [M+H]+
543.3896. Found: 543.3892.
(2R*,3R*,4R*)-4-((benzyloxy)methyl)-2,3-bis((triisopropylsilyl)oxy)cyclopentanone (2c).
Prepared by general procedure B using 10b (4.00 g, 7.31 mmol); flash column chromatography
(hexane/EtOAc = 25:1) afforded 2c as a yellow oil (3.41 g, 85 % yield). 1
H NMR (500 MHz, CDCl3):
δ = 7.35-7.25 (m, 5H), 4.61 (dd, J = 4.1, 1.6 Hz, 1H), 4.52 (AB d, J = 11.8 Hz, 1H), 4.46 (AB d, J =
11.8 Hz, 1H), 4.36 (d, J = 4.1 Hz, 1H), 3.61 (dd, J = 9.4, 4.0 Hz, 1H), 3.49 (dd, J = 9.4, 5.1 Hz, 1H),
2.49-2.41 (m, 2H), 2.14-2.06 (m, 1H), 1.09-1.00 (m, 42H). 13
C NMR (126 MHz, CDCl3): δ = 213.87,
138.02, 128.61, 127.98, 127.89, 79.52, 75.73, 73.76, 71.79, 40.92, 34.54, 18.33, 18.31, 18.27, 18.18,
12.82, 12.78. IR (ν˜max) = 2942 (m), 2865 (m), 1760 (m), 1463 (m), 1064 (s), 882 (s), 676 (s) cm–1
.
HR-MS (APCI) calculated for C31H56O4Si2 [M+H]+
: 549.3790. Found: 549.3783.
(3aR*,4R*,6R*,6aR*)-4-(2,4-bis(benzyloxy)pyrimidin-5-yl)-6-((tertbutyldiphenylsilyloxy)methyl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-ol
(12).
304
n-BuLi (1.6 M in hexanes, 0.646 mL, 1.035 mmol) was added dropwise to a cooled (–78 C) solution
of bromide 11 (0.384 g, 1.035 mmol,) in THF (3 mL) and the reaction mixture was stirred at –78 °C
for 1 h. A solution of ketone 2a (0.256 g, 0.69 mmol) in THF (2 mL) was added dropwise and the
reaction mixture was stirred at –78 °C for 1 h. The mixture was allowed to warm to 25 °C, quenched
with saturated aqueous NH4Cl (10 mL) and extracted with EtOAc (3 × 15 mL). The combined
organic extracts were dried over Na2SO4, filtered and concentrated in a vacuum. The residue was
purified by flash column chromatography ( hexane/EtOAc = 5:1) to afford 12 as a white wax (0.250
g, 76 %). 1
H NMR (500 MHz, CDCl3): δ = 8.50 (s, 1H), 7.65-7.63 (m, 4H), 7.48-7.25 (m, 16H), 5.48
(AB d, J = 12.2 Hz, 1H), 5.42 (m, 2H), 5.34 (d, J = 12.2 Hz, 1H), 4.79 (d, J = 7.7 Hz, 1H), 4.37 (dd,
J = 7.7, 5.9 Hz, 1H), 3.73 (dd, J = 10.2, 5.3 Hz, 1H), 3.63 (dd, J = 10.2, 6.4 Hz, 1H), 3.49 (d, J = 1.4
Hz, 1H, -OH), 2.63 (m, 1H), 2.28 (m, 1H), 2.16 (m, 1H), 1.51 (s, 3H), 1.31 (s, 3H), 1.03 (s, 9H) ppm.
13
C NMR (126 MHz, CDCl3): δ = 167.35, 164.37, 157.13, 136.96, 136.10, 135.84, 133.93, 133.89,
129.87, 128.77, 128.65, 128.50, 128.32, 128.26, 128.18, 127.88, 117.89, 114.69, 83.67, 82.62, 75.87,
69.31, 68.78, 65.17, 46.28, 41.21, 27.10, 26.69, 25.07, 19.54 ppm. IR (ν˜max) = 3515 (w), 1600 (w),
1473 (m), 1124 (m), 859 (s) 694 (s) cm–1
. HR-MS (APCI) calculated for C43H48N2O6Si [M+H]+
:
717.3359. Found: 717.3358.
(1R*,2R*,3R*,4R*)-4-((benzyloxy)methyl)-1-(2,4-bis(benzyloxy)pyrimidin-5-yl)-2,3bis((triisopropylsilyl)oxy)cyclopentanol
(17a).
305
Compound 17a was prepared by essentially same procedure used for compound 12 from bromide 11
(1.09 g, 2.95 mmol) and ketone 2c (1.08 g, 1.97 mmol); flash column chromatography
(hexane/EtOAc = 10:1) afforded 17a as a yellow crystalline solid (0.741 g, 45 % yield), m.p. = 75-77
°C. 1
H NMR (500 MHz, CDCl3): δ = 8.62 (s, 1H), 7.42-7.38 (m, 2H), 7.29- 7.17 (m, 11H), 7.14-7.11
(m, 2H), 5.37 (s, 2H), 5.34 (d, J = 11.7 Hz, 1H), 5.10 (d, J = 11.7 Hz, 1H), 4.72 (s, 1H,-OH), 4.45 (d,
J = 4.0 Hz, 1H), 4.19 (d, J = 4.0 Hz, 1H), 4.11 (d, J = 12.0 Hz, 1H), 3.97 (d, J = 12.0 Hz, 1H), 2.95-
2.91 (m, 1H), 2.84-2.79 (m, 1H), 2.41-2.34 (m, 1H), 2.10-1.99 (m, 2H), 1.01-0.95 (m, 21H), 0.87-
0.72 (m, 18H), 0.68-0.60 (m, 3H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 166.51, 164.08, 159.69,
138.69, 137.19, 135.84, 129.75, 128.90, 128.57, 128.43, 128.20, 128.03, 127.62, 127.52, 116.95,
79.89, 79.09, 75.05, 73.07, 72.91, 69.14, 69.08, 43.42, 39.21, 18.43, 18.42, 18.27, 17.98, 13.09, 12.93
ppm. IR (ν˜max) = 3457 (br w), 2942 (m), 2865 (m), 1559 (m), 1422 (s), 825 (s), 685 (s) cm–1
. HR-MS
(APCI) calculated for C49H72N2O6Si2 [M+H]+
: 841.5002, found: 841.4999.
(1S*,2R*,3R*,4R*)-4-(benzyloxymethyl)-1-butyl-2,3-bis(triisopropylsilyloxy)cyclopentanol
(17b).
Butylmagnesium chloride (2M in THF, 0.20 mL, 0.41 mmol) was added slowly into a solution of 2c
(0.150 g, 0.27 mmol) in THF (2 mL) at 0 °C. The reaction mixture was allowed to warm to 25 °C,
stirred for 14 h, then quenched with saturated aqueous NH4Cl (15 mL), and extracted with EtOAc (3
× 15 mL). The organic extracts were dried over MgSO4, filtered, and solvent was evaporated. The
residual oil was purified by flash column chromatography (hexane/ EtOAc = 25:1) to afford 17b as a
colorless oil (0.062 g, 38 %). 1
H NMR (500 MHz, CDCl3): δ = 7.35-7.27 (m, 5H), 4.47 (d, AB, J =
12.1 Hz, 1H), 4.43 (d, AB, J = 12.1 Hz, 1H), 4.24 (d, J = 3.9 Hz, 1H), 3.90 (s, 1H, -OH), 3.77 (d, J =
3.9 Hz, 1H), 3.38 (dd, J = 8.7, 5.1 Hz, 1H), 3.23 (dd, J = 9.0, 6.6 Hz, 1H), 2.36 (m, 1H), 2.14 (dd, J =
14.2, 10.5 Hz, 1H), 1.77 (m, 1H), 1.49 (dd, J = 14.3, 5.2 Hz, 1H), 1.36-1.25 (m, 4H), 1.24-1.16 (m,
2H), 1.12-1.05 (m, 42H), 0.9 (t, J = 6.7 Hz, 3H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 138.48,
128.51, 127.86, 127.79, 80.55, 79.74, 78.63, 73.48, 72.73, 43.05, 39.48, 38.30, 26.43, 23.71, 18.57,
18.49, 14.29, 13.44, 13.13 ppm. IR (ν˜max) = 2942 (m), 2865 (s), 1463 (m), 1154 (m), 825 (s), 679 (s)
cm–1
. HR-MS (ESI): calculated for C35H66O4Si2 [M+Na]+
: 629.43918. Found: 629.43918.
306
(1R*,2R*,3R*,4R*)-4-((benzyloxy)methyl)-1-phenyl-2,3-bis((triisopropylsilyl)oxy) cyclopentanol
(17c).
Phenyllithium (1.54 mL, 2.77 mmol, 1.8 M in dibutyl ether) was added to a cooled (0 °C, ice bath)
solution of 2c (1.01 g, 1.84 mmol) in THF (10 mL). The reaction mixture was stirred for 6 h while
allowed to warm to 25 C. The reaction was quenched with aqueous saturated NH4Cl (5 mL) and
extracted with EtOAc (4 × 20 mL). The combined organic extracts were washed with brine (20 mL),
dried over MgSO4 and concentrated in vacuo. The residue was purified by flash column
chromatography (hexane/EtOAc = 50:1) to afford 17c as a viscous brown oil (0.628 g, 55 %) and
recovered starting material (0.147 g, 15 %). 1
H NMR (500 MHz, CDCl3): δ = 7.51 (m, 2H), 7.39-7.34
(m, 4H), 7.34-7.29 (m, 1H), 7.25-7.20 (m, 2H), 7.18-7.13 (m, 1H), 4.56 (d, AB, J = 11.7 Hz, 1H),
4.53 (d, AB, J = 11.7 Hz, 1H), 4.44 (s, 1H), 4.38 (d, J = 4.3 Hz, 1H), 4.34 (d, J = 4.3 Hz, 1H), 3.62
(ddm, J = 9.3, 4.1 Hz, 1H), 3.46 (ddm, J = 9.3, 4.1 Hz, 1H), 2.52-2.44 (m, 2H), 2.11 (dd, AB, J =
20.0, 10.0 Hz, 1H), 1.19-1.09 (m, 21H), 0.89-0.79 (m, 18H), 0.62-0.54 (m, 3H).13
C NMR (126 MHz,
CDCl3): δ = 145.69, 138.28, 128.61, 128.24, 127.98, 127.73, 126.50, 126.19, 82.74, 81.45, 80.67,
73.89, 72.93, 44.15, 43.51, 18.51, 18.48, 18.43, 18.20, 13.11, 13.08, 1.24 ppm. IR (ν˜max) = 3511 (br
w), 2915 (s), 2866 (s), 1730 (m), 1495 (m), 1047 (m), 883 (s), 681 (s) cm–1
. HR-MS (APCI):
calculated for C37H62O4Si2 [M+H]+
: 627.4259. Found: 609.4152, Δ mass: 18.0107 (H2O).
(1R*,2R*,3R*,4R*)-1-benzyl-4-(benzyloxymethyl)-2,3-bis(triisopropylsilyloxy)cyclopentanol
(17d).
The compound was prepared by essentially same procedure used for 17c, using benzylmagnesium
chloride (2M in THF, 0.21 ml, 0.41 mmol) and ketone 2c (0.150 g, 0.27 mmol); flash column
chromatography (hexane/ EtOAc = 25:1) afforded 17d as a colorless oil (0.063 g, 38 %). 1
H NMR
307
(500 MHz, CDCl3): δ = 7.35-7.22 (m, 9H), 7.18 (m, 1H), 4.47 (d, AB, J = 12.1 Hz, 1H), 4.43 (d, AB,
J = 12.1 Hz, 1H), 4.26 (dd, J = 4.0, 1.4 Hz, 1H), 3.91 (d, J = 4.0 Hz, 1H), 3.75 (d, J = 1.0 Hz, 1H, OH),
3.40 (dd, J = 9.1, 4.8 Hz, 1H), 3.23 (dd, J = 9.1, 5.8 Hz, 1H), 3.13 (d, J = 13.1 Hz, 1H), 2.44 (d,
J = 13.1 Hz, 1H), 2.29 (m, 1H), 1.87 (dd, J = 14.3, 10.5 Hz, 1H), 1.63 (dd, J = 14.3, 5.5 Hz, 1H),
1.20-0-96 (m, 42H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 138.98, 138.48, 130.56, 128.55, 128.04,
127.93, 127.84, 126.17, 80.53, 79.47, 79.28, 73.53, 72.43, 45.98, 43.18, 38.28, 18.59, 18.53, 18.50,
13.61, 13.11 ppm. IR (ν˜max) = 2942 (m), 2865 (m), 1463 (m), 1153 (m), 881 (s), 678 (s) cm–1
. HRMS
(ESI) calculated for C38H64O4Si2 [M+H]+
: 641.44159. Found: 641.44170.
(1R*,2R*,3R*,4R*)-4-((benzyloxy)methyl)-1-(thiazol-4-yl)-2,3bis((triisopropylsilyl)oxy)cyclopentanol
(17e).
Isopropylmagnesium chloride - lithium chloride complex (1.3 M THF, 0.34 mL, 0.44 mmol) was
added dropwise into a solution of 4-bromothiazole (0.04 mL, 0.41 mmol) in THF (1.5 mL). The
reaction mixture was stirred at 25 °C for 30 min, then a solution of 2c (0.150 g, 0.27 mmol) in THF
(1.5 mL) was added and the reaction mixture was stirred at 25 °C for 14 h. The solvent was
evaporated, aqueous saturated NH4Cl (15 mL) was added to the residue and the mixture was extracted
with EtOAc (3 × 15 mL). The organic extracts were dried over MgSO4, filtered, and the solvent was
evaporated. The residual yellow oil was purified by flash column chromatography (hexane/EtOAc =
25:1) to afford 17e as a yellow oil (0.156 g, 90 %). 1
H NMR (500 MHz, CDCl3): δ = 7.40-7.33 (m,
4H), 7.28 (m, 1H), 7.13 (s, 1H), 4.86 (s, 1H), 4.43 (d, J = 4.0 Hz, 1H), 4.55 (d, AB, J = 11.4 Hz, 1H),
4.50 (d, AB, J = 11.4 Hz, 1H), 3.95 (d, J = 3.8 Hz, 1H), 2.53-2.48 (m, 1H, overlapped), 2.48 (d, AB, J
= 10.7 Hz, 1H, partially overlapped), 2.43 (d, AB, J = 10.7 Hz, 1H), 2.24 (dd, J = 13.5, 4.1 Hz, 1H),
1.14-1.08 (m, 21H), 0.97-0.93 (m, 9H), 0.89-0.86 (m, 21H), 0.82-0.73 (m, 3H) ppm. 13
C NMR (126
MHz, CDCl3): δ = 178.39, 138.46, 128.54, 127.91, 127.71, 124.52, 117.63, 83.90, 79.57, 79.49,
73.57, 72.40, 43.48, 41.95, 18.48, 18.45, 18.30, 18.09, 13.04 ppm. IR (ν˜max) = 2942 (s), 2865 (s),
1463 (m), 1256 (m), 1089 (s), 882 (s), 680 (s) cm–1
.
308
General procedure C: hydrogenolysis of the benzyl protecting group or hydrogenation
Pd/C (10%, 10 mol %) or Pd(OH)2 (10%-20%, 10 mol%) was added to a solution of the starting
material in EtOH (0.1 mmol/5 mL) or THF (0.1 mmol/ 5 mL). The reaction mixture was thoroughly
purged with H2 and heated to 80 C under H2 atmosphere (1-50 bar, depending on the substrate) for 2-
14 h (monitored by TLC and/or by 1
H NMR). The reaction mixture was cooled to 25 C, filtered
through Celite and concentrated under reduced pressure. The residue was purified by flash column
chromatography to afford the desired product.
5-((3aR*,4R*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-4-hydroxy-2,2dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione
(13).
Prepared by general procedure C using compound 12 (0.195 g, 0.272 mmol), Pd/C (0.003 g, 0.027
mmol), H2 (1 bar) in EtOH flash column chromatography (CH2Cl2/MeOH = 10:1) afforded 13 as a
pale yellow solid (0.140 g, 96 %). 1
H NMR (500 MHz, CDCl3): δ = 10.22 (d, J = 4.9 Hz, 1H, -NH),
9.79 (s, 1H, -NH), 7.68-7.65 (m, 4H), 7.42-7.34 (m, 6H), 5.05 (d, J = 7.7 Hz, 1H), 4.64 (dd, J = 7.6,
5.4 Hz, 1H), 3.78 (m, 2H), 3.42 (d, J = 0.7 Hz, 1H, -OH), 2.61 (m, 1H), 2.51 (m, 1H), 1.92 (dd, J =
13.0, 6.05 Hz, 1H), 1.54 (s, 3H), 1.32 (s, 3H), 1.07 (s, 9H) ppm. 13
C NMR (126 MHz, CDCl3): δ =
163.31, 153.09, 138.99, 135.85, 133.96, 133.89, 129.81, 127.85, 116.46, 114.66, 82.60, 82.03, 76.05,
64.83, 46.21, 40.67, 27.08, 26.62, 24.96, 19.53 ppm. IR (ν˜max) = 1710 (s), 1682 (s), 1502 (w), 1245
(m), 1049 (m), 719, 657 (s) cm–1
. HR-MS (ESI) calculated for C29H36O6N2Si [M+Na]+
: 559.22348.
Found: 559.22340.
5-((1R*,2R*,3R*,4R*)-1-hydroxy-4-(hydroxymethyl)-2,3bis(triisopropylsilyloxy)cyclopentyl)pyrimidine-2,4(1H,3H)-dione
(18a).
309
Prepared by general procedure C using compound 17a (0.376 g, 0.45 mmol), Pd/C (0.005 g, 0.045
mmol), H2 (1 bar) in EtOH. The solid residues were removed by filtration through a pad of Celite and
the resulting crude product was used in the next step without further purification. Analytical sample
of 18a was obtained by flash column chromatography (hexane/EtOAc = 1:1) to afford 18a as a white
crystalline solid, m.p. > 200 °C (dec). 1
H NMR (300 MHz, DMSO-d6): δ = 11.05 (s, 1H), 10.80 (d, J
= 5.9 Hz, 1H), 7.31 (d, J = 6.0 Hz, 1H), 4.68-4.66 (m, 1H), 4.47 (s, 1H), 4.32 (d, J = 3.9 Hz, 1H),
3.46-3.39 (m, 2H, partially overlapped with residual H2O), 2.30-2.16 (m, 1H), 2.03 (dd, J = 14.3, 3.8
Hz, 1H), 1.87 (dd, J = 14.3, 10.3 Hz, 1H), 1.14-1.04 (m, 21H), 1.02-0.88 (m, 21H) ppm. 13
C NMR
(126 MHz, DMSO-d6): δ = 163.31, 151.09, 139.34, 113.13, 78.79, 73.77, 63.27, 45.02, 30.61, 17.97,
17.95, 17.87, 17.69, 12.32, 12.11 ppm. IR (ν˜max) = 3507 (br w), 3234 (br w), 3159 (br w), 2943 (m),
2866 (m), 1714 (s), 1652 (s), 1147 (m), 881 (s), 679 (s) cm–1
. HR-MS (APCI) calculated for
C28H54N2O6Si2 [M+Na]+
: 593.3413, found: 593.3413.
(1R*,2R*,3R*,4R*)-4-(hydroxymethyl)-1-phenyl-2,3-bis((triisopropylsilyl)oxy)cyclopentanol.
Prepared by general procedure C using compound 20 0.100 g (0.16 mmol), Pd/C (0.002 g, 0.016
mmol) and H2 (1 bar) in EtOH flash column chromatography (hexane/EtOAc = 20:1) afforded
compound the title compound as a white crystalline solid (0.065 g, 75 %), m.p. = 87-89 °C. 1
H NMR
(300 MHz, CDCl3): δ = 7.58-7.52 (m, 2H), 7.32-7.24 (m, 2H), 7.22-7.15 (m, 1H), 4.48 (s, 1H), 4.41
(d, AB, J = 4.1 Hz, 1H), 4.33 (d, AB, J = 4.1 Hz, 1H) 3.78 (dd, J = 10.1, 4.7 Hz, 1H), 3.68 (dd, J =
10.1, 4.7 Hz, 1H), 2.55-2.38 (m, 2H), 2.06 (ddm, J = 13.4, 3.2 Hz, 1H), 1.18-1.10 (m, 21H), 0.94-0.81
(m, 18H), 0.72-0.54 (m, 3H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 138.58, 128.52, 127.87, 127.75,
77.89, 77.55, 73.45, 72.44, 65.39, 44.91, 43.47, 26.39, 18.53, 18.49, 18.45, 13.38, 13.20 ppm. IR
310
(ν˜max) = 3435 (br w), 3251 (br w), 2942 (s), 2865 (s), 1498 (m), 1151 (s), 867 (s), 680 (s) cm–1
. HRMS
(APCI) calculated for C30H56O4Si2 [M+H]+
: 537.3790. Found: 519.3682, Δ mass: 18.0108 (H2O).
(1S*,2R*,3R*,4R*)-1-butyl-4-(hydroxymethyl)cyclopentane-1,2,3-triol (15b).
Prepared by general procedure C using compound 19a (0.039 g, 0.13 mmol) and Pd(OH)2/C (0.002 g,
0.013 mmol), H2 (50 bar) in THF flash column chromatography (CH2Cl2 to CH2Cl2/CH3OH = 5:1)
afforded 15b as a white wax (0.025 g, 93 %). 1
H NMR (500 MHz, DMSO-d6): δ= 4.42 (app t, J = 5.1
Hz, 1H), 4.34 (dd, J = 15.4, 6.3 Hz, 2H), 3.91 (s, 1H), 3.61 (dd, J = 5.4, 11.3 Hz, 1H), 3.39-3.32 (m,
3H), 2.02 (m, 1H), 1.69 (dd, J = 8.8, 13.3 Hz, 1H), 1.49-1.41 (m, 1H), 1.40-1.32 (m, 1H), 1.32-1.22
(m, 4H), 1.19 (dd, J = 9.0, 13.3 Hz, 1H), 0.86 (t, J = 7.1 Hz, 3H) ppm. 13
C NMR (126 MHz, DMSOd6)
δ: 78.73, 75.94, 73.30, 62.76, 45.92, 36.79, 25.70, 22.81, 14.00 ppm. IR (ν˜max) = 3424 (br w),
3235 (br w), 2955 (m), 2854 (m), 1376 (m), 1131 (s), 1020 (s), 867 (s), 529 (m) cm–1
. HR-MS (ESI)
calculated for C20H20O4N2[M+Cl]–
: 239.1056. Found: 239.1056.
(1R*,2R*,3R*,4R*)-4-(hydroxymethyl)-1-phenylcyclopentane-1,2,3-triol (15c).
Prepared by general procedure C using compound 19b (0.061 g, 0.194 mmol), Pd(OH)2/C (0.003 g,
0.019 mmol), H2 (50 bar) in THF flash column chromatography (CH2Cl2 to CH2Cl2/CH3OH = 3:1)
afforded compound 15c as a white solid (0.040 g, 92 %). 1
H NMR (500 MHz, DMSO-d6): δ = 7.49-
7.44 (m, 2H), 7.33-7.27 (m, 2H), 7.22-7.17 (m, 1H), 4.66 (s, 1H), 4.58 (d, J = 6.09 Hz, 1H), 4.55 (m,
1H), 4.44 (d, J = 7.68 Hz, 1H), 3.82 (dd, J = 14.14, 6.83 Hz, 1H), 3.79 (m, 1H), 2.22 (m, 1H), 1.95
(dd, J = 13.5, 8.4 Hz, 1H), 1.66 (dd, J = 13.5, 10.07 Hz, 1H) ppm. 13
C NMR (126 MHz, DMSO-d6): δ
= 146.27, 127.55, 126.03, 125.33, 80.73, 77.89, 72.91, 62.70, 46.85 ppm. The spectral data were
consistent with those reported.5
(1R*,2R*,3R*,4R*)-1-benzyl-4-(hydroxymethyl)cyclopentane-1,2,3-triol (15d).
311
Prepared by general procedure C using compound 19c (0.030 g, 0.09 mmol), Pd(OH)2/C (0.001 g,
0.009 mmol), H2 (50 bar) in THF flash column chromatography (CH2Cl2/CH3OH = 20:1 to 1:1)
afforded 15d as a white semi-solid (0.020 g, 92 %). 1
H NMR (500 MHz, DMSO-d6): δ = 7.27-7.21
(m, 4H), 7.19-7.15 (m, 1H), 4.43 (d, J = 6.7 Hz, 1H, -OH), 4.40 (app t, J = 5.1 Hz, 1H, -OH), 4.35 (d,
J = 7.1 Hz, 1H), 4.10 (s, 1H, -OH), 3.64 (dd, J = 11.8, 5.8 Hz, 1H), 3.38 (app t, J = 6.4 Hz, 1H), 3.32-
3.26 (m, 2H, overlapped with H2O), 2.77 (d, J = 13.1 Hz, 1H), 2.61 (d, J = 13.1 Hz, 1H), 2.03-1.97
(m, 1H), 1.49 (dd, J = 13.5, 8.9 Hz, 1H), 1.25 (dd, 13.5, 8.9 Hz, 1H) ppm. 13
C NMR (126 MHz,
DMSO-d6): δ = 138.43, 130.24, 127.51, 125.65, 79.33, 75.30, 72.79, 62.66, 45.95, 44.46, 35.96 ppm.
IR (ν˜max) = 3528 (m), 2912 (w), 2862 (w), 1366 (w), 1018 (s), 697 (s) cm–1
. HR-MS (ESI) calculated
for C13H18O4 [M-H]–
: 237.1132. Found: 237.1132.
5-((3aS*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-dimethyl-6,6a-dihydro-3aHcyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione
(16).
CSA (11 mg, 0.047 mmol) was added to a stirred solution of 14 (0.025 g, 0.047 mmol) in CH2Cl2 and
MeOH (3+1 mL). The resulting mixture was stirred at 25 °C for 16 h. The reaction was quenched
with saturated aqueous NaHCO3 (15 mL) and extracted with CH2Cl2 (3 × 10 mL). The organic
extracts were dried over MgSO4, filtered and concentrated in a vacuum. The crude product was
purified by flash column chromatography (CH2Cl2/MeOH = 20:1) to afford 16 (0.011 g, 46 %) as a
white solid, m.p. = 223-228 C. 1
H NMR (500 MHz, CDCl3): δ = 9.63 (d, J = 5.3 Hz, 1H, N-H), 8.88
(br s, 1H, N-H), 7.65 (d, J = 5.8 Hz, 1H), 7.63-7.58 (m, 4H), 7.42-7.32 (m, 6H), 6.76 (d, J = 2.6 Hz,
1H), 5.22 (d, J = 5.8 Hz, 1H), 4.63 (app d, J = 5.7 Hz, 1H), 3.82 (dd, J = 10.2, 4.4 Hz, 1H), 3.59 (dd,
J = 10.2, 5.0 Hz), 3.07 (m, 1H), 1.36 (s, 3H), 1.35 (s, 3H), 0.99 (s, 9H) ppm. 13
C NMR (126 MHz,
CDCl3): δ = 162.61, 151.54, 137.63, 135.89, 135.81, 134.32, 133.71, 133.47, 132.25, 129.94, 127.94,
312
127.92, 110.85, 109.82, 85.73, 81.05, 64.89, 54.33, 27.71, 27.03, 26.16, 19.44 ppm. IR (ν˜max) = 1707
(s), 1679 (s), 1447 (w), 1109 (m), 1047 (m), 698 (s), 503 (s) cm–1
. HR-MS (ESI) calculated for
C29H34O5N2Si [M+Na]+
: 541.21292. Found: 541.21291.
(1R*,2R*,3R*,4R*)-4-(methoxymethyl)-1-phenyl-2,3-bis((triisopropylsilyl)oxy)cyclopentanol.
To a stirred suspension of NaH (0.016 g, 0.24 mmol, 60% dispersion in mineral oil) in THF (1.5 mL)
was added a solution of (1R*,2R*,3R*,4R*)-4-(hydroxymethyl)-1-phenyl-2,3bis((triisopropylsilyl)oxy)cyclopentanol
(0.065 g, 0.12 mmol) in THF (1 mL) at 25 °C. After stirring
for 30 min and cooling to 0 °C (ice bath), methyl iodide (53 µL, 0.85 mmol) was added dropwise and
the resulting mixture was stirred at 25 °C for 24 h. The reaction was quenched by addition of silica
gel (0.150 g) and the solvent was evaporated under reduced pressure. The residue was then purified
by flash column chromatography (hexane/EtOAc = 75:1 to 10:1) to afford the title compound as a
colorless crystalline solid (0.044 g, 67 %), m.p. = 61-69 °C. 1
H NMR (500 MHz, CDCl3): δ = 7.57-
7.54 (m, 2H), 7.30-7.26 (m, 2H), 7.20-7.15 (m, 1H), 4.36 (d, AB, J = 4.0 Hz, 1H), 4.34 (d, AB, J =
4.0 Hz, 1H), 3.48 (dd, J = 9.1, 4.5 Hz, 1H), 3.39 (s, 3H), 3.36 (dd, J = 9.1, 5.2 Hz, 1H), 2.52-2.41 (m,
2H, overlapped), 2.05 (dd, J = 13.7, 3.6 Hz, 1H), 1.16-1.11 (m, 21H), 0.95-0.81 (m, 18H), 0.67-0.57
(m, 3H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 145.73, 127.79, 126.55, 126.15, 82.61, 81.36, 80.61,
77.44, 59.34, 44.23, 43.37, 18.50, 18.48, 18.42, 13.13 ppm. IR (ν˜max) = 3511 (m), 2939 (s), 2865 (s),
1461 (m), 1148 (m), 1067 (m), 882 (s), 681 (s) cm–1
. HR-MS (ESI) calculated for C31H58O4Si2
[M+Na]+
: 573.37658 Found: 573.37635.
General procedure D: removal of the TBDPS and TIPS protecting groups
TBAF (1M in THF, 1.1-1.3 eq.) was added to a stirred solution of starting material in THF (0.3
mmol/mL). The reaction mixture was stirred at 25 C for 14-24 h. The solvent was evaporated and
the residue was purified by flash column chromatography to afford the product.
313
5-((1R*,2R*,3R*,4R*)-1,2,3-trihydroxy-4-(hydroxymethyl)cyclopentyl)pyrimidine-2,4(1H,3H)dione
(15a).
Prepared by general procedure D using compound 18a (0.235 g, 0.41 mmol) flash column
chromatography (CH2Cl2 to CH2Cl2/MeOH = 10:1 to 1:1) afforded compound 15a (0.090 g, 85 %)
slightly contaminated by residual TBAF. Analytically pure sample of 15a was obtained by RP-HPLC
(Nucleodur®
C18 HTec, details given in Supporting Information) as a white solid, m.p. > 250 °C. 1
H
NMR (500 MHz, DMSO-d6): δ = 10.96 (bs, 1H, -NH), 10.71 (bs, 1H, -NH), 7.29 (s, 1H), 4.54 (s, 1H,
-OH), 4.42-4.50 (m, 3H, -3 x OH), 4.11 (m, 1H), 3.74 (dd, J = 10.5, 6.0 Hz, 1H), 3.43 (m, 1H), 3.34
(m, 1H), 2.13 (m, 1H), 1.89 (dd, J = 13.3, 9.7 Hz, 1H), 1.63 (dd, J = 13.3, 8.4 Hz, 1H) ppm. 13
C NMR
(126 MHz, DMSO-d6): δ = 163.32, 151.29, 138.64, 114.46, 78.29, 72.91, 72.60, 63.17, 46.74, 36.25
ppm. IR (ν˜max) = 3088 (w), 3077 (w), 1704 (s), 1654 (s), 1015 (m), 849 (m), 542 (m) cm–1
. HR-MS
(ESI) calculated for C10H14N2O6 [M+H]+
: 257.0779. Found: 257.0775.
(1S*,2R*,3R*,4R*)-4-(benzyloxymethyl)-1-butylcyclopentane-1,2,3-triol (19a).
Prepared by general procedure D using compound 17b (0.198 g, 0.33 mmol) flash column
chromatography (CH2Cl2/MeOH = 20:1) afforded 19a as a slightly yellow wax (0.078 g, 82%). 1
H
NMR (500 MHz, CDCl3): δ = 7.37-7.26 (m, 5H), 4.53 (d, AB, J = 12 Hz, 1H), 4.50 (d, AB, J = 12
Hz, 1H), 3.90 (m, 1H), 3.64 (d, J = 6.4 Hz, 1H), 3.53 (dd, J = 9.1, 5.2 Hz, 1H), 3.38 (dd, J = 9.1, 6.7
Hz, 1H), 3.16 (br s, 1H, -OH), 2.93 (br s, 1H, -OH), 2.70 (br s, 1H, -OH), 2.44-2.35 (m, 1H), 1.95
(dd, J = 14.0, 8.8 Hz, 1H), 1.62-1.53 (m, 1H), 1.49-1.28 (m, 6H), 0.90 (t, J = 7.23, 3H) ppm. 13
C
NMR (126 MHz, CDCl3): δ = 138.39, 128.63, 127.89, 127.77, 80.84, 76.27, 76.15, 73.43, 72.45,
44.29, 39.14, 36.79, 26.23, 23.40, 14.23 ppm. IR (ν˜max) = 3260 (m), 2937 (m), 1409 (w), 1096 (s),
802 (s), 698 (s) cm–1
. HR-MS (ESI) calculated for C17H26O4 [M-H]–
: 293.1758. Found: 293.1758.
314
(1R*,2R*,3R*,4R*)-4-(benzyloxymethyl)-1-phenylcyclopentane-1,2,3-triol (19b).
Prepared by general procedure D using compound 17c (0.256 g, 0.408 mmol) flash column
chromatography (CH2Cl2/MeOH = 20:1) afforded compound 19b as a colorless oil (0.123 g, 96%).
1
H NMR (500 MHz, CDCl3): δ = 7.48 (m, 2H), 7.38-7.27 (m, 7H), 7.26 (m, 1H, overlapped with
CHCl3), 4.56 (d, AB, J = 12.0 Hz, 1H), 4.53 (d, AB, J = 12.0 Hz, 1H), 4.19 (d, J = 6.4 Hz, 1H), 4.06
(dd, J = 6.3, 3.7 Hz, 1H), 3.63 (dd, J = 9.1, 4.9 Hz, 1H), 3.47 (dd, J = 9.1, 6.1 Hz, 1H), 3.35-2.77 (br
s, 1H, -OH), 2.58-2.50 (m, 1H), 2.28 (dd, J = 14.1, 8.7 Hz, 1H), 1.81 (dd, J = 14.1, 9.7 Hz, 1H) ppm.
13
C NMR (126 MHz, CDCl3): δ = 144.13, 138.36, 128.67, 128.56, 127.94, 127.85, 127.49, 125.45,
82.29, 77.46, 75.86, 73.53, 72.16, 44.95, 39.78 ppm. IR (ν˜max) = 2955 (s), 2849 (s), 1729 (s), 1254
(m), 1065 (m), 702 (s) cm–1
. HR-MS (ESI) calculated for C19H22O4 [M-H]–
: 313.14453. Found:
313.14421.
(1R*,2R*,3R*,4R*)-1-benzyl-4-(benzyloxymethyl)cyclopentane-1,2,3-triol (19c).
Prepared by general procedure D using compound 17d (0.218 g, 0.33 mmol) flash column
chromatography (hexane/EtOAc = 20:1) afforded 19c as a white semi-solid (0.098 g, 90 %). 1
H NMR
(500 MHz, DMSO-d6): δ = 7.36-7.22 (m, 10H), 4.49 (d, AB, J = 12.3 Hz, 1H), 4.46 (d, AB, J = 12.3
Hz, 1H), 3.90 (dd, J = 6.0, 4.0 Hz, 1H), 3.72 (d, J = 6.0 Hz, 1H), 3.49 (dd, J = 9.1, 5.1 Hz, 1H), 3.33
(dd, J = 9.1, 6.4 Hz, 1H), 3.03 (br s, 1H, -OH), 2.92 (d, J = 13.5 Hz, 1H), 2.80 (d, J = 13.5 Hz, 1H),
2.67 (br s, 1H, -OH), 2.36 (m, 1H), 1.83 (dd, J = 14.0, 8.8 Hz, 1H), 1.47 (dd, J = 14.0, 9.7 Hz, 1H)
ppm. 13
C NMR (126 MHz, DMSO-d6): δ = 138.40, 137.23, 130.45, 128.62, 128.48, 127.85, 127.74,
126.82, 81.06, 77.23, 76.98, 75.74, 75.57, 73.37, 72.13, 44.82, 44.41, 36.43 ppm. IR (ν˜max) = 3432
(br w), 2844 (br w), 2955 (m), 1354 (m), 1183 (m), 1090 (s), 755 (m), 695 (s) cm–1
. HR-MS (ESI)
calculated for C20H24O4[M-H]:
327.1602. Found: 327.1604.
315
(1R*,2R*,3R*,4R*)-4-(methoxymethyl)-1-phenylcyclopentane-1,2,3-triol (21).
Prepared by general procedure D using (1R*,2R*,3R*,4R*)-4-(methoxymethyl)-1-phenyl-2,3bis((triisopropylsilyl)oxy)cyclopentanol
(0.068 g, 0.12 mmol) flash column chromatography
(hexane/EtOAc = 1:5) afforded 21 as a pale yellow crystalline solid (0.012 g, 42 %), m.p. = 59-60 °C.
1
H NMR (500 MHz, CDCl3): δ = 7.52-7.49 (m, 2H), 7.38-7.34 (m, 2H), 7.29-7.26 (m, 1H), 4.19 (d, J
= 6.5 Hz, 1H), 4.06 (dd, J = 6.4, 3.9 Hz, 1H), 3.55 (dd, J = 9.1, 4.9 Hz, 1H), 3.39 (dd, J = 9.1, 6.4 Hz,
1H), 3.38 (s, 3H), 2.56-2.49 (m, 1H), 2.28 (dd, J = 14.1, 8.5 Hz, 1H), 1.78 (dd, J = 14.1, 10.0 Hz, 1H)
ppm. 13
C NMR (126 MHz, CDCl3): δ = 144.13, 128.61, 127.57, 125.46, 82.20, 77.36, 75.82, 74.83,
59.34, 45.02, 39.53 ppm. IR (ν˜max) = 3427 (br w), 3333 (br w), 3209 (br m), 2932 (w), 2857 (w),
1443 (m), 1066 (s), 1031 (s), 699 (s) cm–1
. HR-MS (ESI) calculated for C13H17O4 [M-H]:
237.11323.
Found: 237.11285.
5-((3aR*,4R*,6R*,6aR*)-4-hydroxy-6-(hydroxymethyl)-2,2-dimethyltetrahydro-3aHcyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione
(14).
Prepared by general procedure D using compound 13 (0.060 g, 0.111 mmol) flash column
chromatography (CH2Cl2/MeOH = 15:1 to 10:1) afforded 14 as a colorless solid (0.031 g, 94%). m.p.
> 250 C (dec.) 1
H NMR (500 MHz, DMSO-d6): δ = 11.03 (br s, 1H, -NH), 10.79 (br s, 1H, -NH),
7.36 (s, 1H), 4.79 (d, J = 7.7 Hz, 1H), 4.57 (m, 1H, -OH), 4.41 (dd, J = 7.6, 5.1 Hz, 1H), 4.10 (d, J =
1.6 Hz), 3.40 (m, 2H), 2.36 (m, 1H), 2.19 (m, 1H), 1.66 (dd, J = 12.9, 6.9 Hz, 1H), 1.45 (s, 3H), 1.23
(s, 3H) ppm. 13
C NMR (126 MHz, DMSO-d6): δ = 163.30, 151.12, 138.59, 114.29, 113.17, 82.17,
316
82.03, 75.72, 62.53, 45.32, 40.04 (CH2-6´ - overlapped with DMSO, detected through 1
H-13
C HSQC),
26.19, 25.11 ppm. IR (ν˜max) = 1711 (s), 1681 (s), 1452 (m), 1033 (m), 719 (s) cm–1
. HR-MS (APCI)
calculated for C13H18N2O6 [M+Na]+
: 321.1062, found: 321.1063.
(1R*,2R*,3R*,4R*)-4-(hydroxymethyl)-1-(thiazol-4-yl)cyclopentane-1,2,3-triol (15e).
Lithium (0.010 g, 1.43 mmol) in several pieces was added into a solution of naphthalene (0.244 g,
1.91 mmol) in THF (9 mL). The reaction mixture was stirred at 25 °C under until the lithium was
completely dissolved (2-3 h). The resulting dark green solution of lithium naphthalenide was slowly
added into a solution of 17e (0.1500 g, 0.24 mmol) in THF (2 mL). The reaction mixture was stirred
at 25 °C for 1 h. The reaction mixture was quenched with saturated aqueous NH4Cl (10 mL) and
extracted with EtOAc (3 × 15 mL). The organic extracts were dried over MgSO4, filtered, and the
solvent was evaporated. The crude product was dissolved in THF (3 mL) and TBAF (1 M in THF,
0.42 mL, 0.42 mmol) was added. The reaction mixture was stirred for 14 h at 25 °C. The solvent was
evaporated and the crude product was purified by flash column chromatography (CH2Cl2/MeOH =
30:1 to 1:1) to afford 15e (0.029 g, 53 %) slightly contaminated by residual TBAF. Analytically pure
sample of 15e was obtained by RP-HPLC (Nucleodur®
C18 HTec, details given in Supporting
Information) as a colorless glassy solid. 1
H NMR (500 MHz, CD3OD): δ = 7.77 (d, J = 3.4 Hz, 1H),
7.50 (d, J = 3.4 Hz, 1H), 4.19 (d, J = 6.5 Hz, 1H), 4.06 (dd, J = 6.5, 4.7 Hz, 1H), 3.71 (ddm, J =
10.6, 6.1 Hz, 2H), 2.45 (m, 1H), 2.23 (dd, J = 13.9, 8.5 Hz, 1H), 2.08 (dd, J = 13.9, 10.2 Hz, 1H)
ppm. 13
C NMR (126 MHz, CD3OD): δ = 180.11, 143.70, 120.90, 83.58, 79.64, 74.73, 64.91, 40.67
ppm. IR (ν˜max) = 3345 (w), 2932 (w), 2873 (w), 1499 (w), 1063 (s), 1024 (s), 727 (m) cm–1
. HR-MS
calculated for C9H13NO4S [M+Cl]–
: 266.0259. Found: 266.0259.
(3aR*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-dimethyl-6,6a-dihydro-3aHcyclopenta[d][1,3]dioxol-4-yl
trifluoromethanesulfonate (22a).
317
LDA (2M solution in THF, 1.21 mL, 2.42 mmol) was added dropwise to a cooled solution (–78 °C)
of compound 2a (0.79 g, 1.86 mmol) in THF (6 mL). The reaction mixture was stirred at –78 °C for 2
h. A solution of N-phenyl-bis(trifluoromethansulfonimide (0.79 g, 2.22 mmol) in THF (5 mL) was
added and the reaction mixture was stirred for 14 h while allowed to warm to 25 °C. The reaction
mixture was quenched with saturated aqueous NH4Cl (20 mL), diluted with water (30 mL), and
extracted with EtOAc (3 × 50 mL). The organic extracts were dried over Na2SO4, filtered, and the
solvent was evaporated. The dark brown residue was purified by flash column chromatography
(hexane/EtOAc = 20:1) to afford 22a as a colorless oil (0.770 g, 74%). 1
H NMR (500 MHz, CDCl3):
δ = 7.64-7.60 (m, 4H), 7.45-7.38 (m, 6H), 5.64 (d, J = 2.5 Hz, 1H), 5.05 (dd, J = 5.8, 1.8 Hz, 1H),
4.56 (d, J =1.8 Hz, 1H), 3.77 (dd, J = 10.3, 4.9 Hz, 1H), 3.66 (dd, J = 10.3, 4.4 Hz, 1H), 2.94 (m,
1H), 1.45 (s, 3H), 1.35 (s, 3H), 1.05 (s, 9H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 149.15, 135.78,
133.22, 133.07, 130.16, 128.05, 128.04, 118.92, 118.90 (q, C-F
J = 320.7 Hz), 111.91, 80.99, 79.68,
64.22, 49.57, 27.27, 27.00, 25.87, 19.35. 19
F (470 MHz, CDCl3): δ = –73.30 ppm. IR (ν˜max) = 2933
(w), 2860 (w), 1443 (m), 1424 (s), 1209 (s), 1129 (m), 1111 (m), 702 (m), 601 (m), 504 (m) cm–1
.
HR-MS (APCI) calculated for C26H31F3O6SSi [M+H]+
: 557.1635. Found: 557.1636.
(1R*,4R*,5R*)-3-(trifluoromethylsulfonyloxy)-4,5-bis(triisopropylsilyloxy)cyclopent-2enyl)methyl
pivalate (22b).
KHMDS (1.21 mL, 1 M THF solution) was added at –78 °C to a solution of compound 2b (0.55 g,
1.01 mmol) and Commins’ reagent (0.476 g, 1.21 mmol) in THF (4 mL) and the mixture was stirred
at –78 °C for 1 h. The reaction mixture was then allowed to warm to 25 °C, stirred for 1 h, then
quenched with saturated aqueous solution of NH4Cl (10 mL), and extracted with EtOAc (3 × 20 mL).
The combined extracts were dried over MgSO4, filtered and concentrated under reduced pressure. The
residue was purified by flash column chromatography (hexane/EtOAc = 50:1) to afford 22b as a
colorless wax (0.540 g, 79 %). 1
H NMR (500 MHz, CDCl3): δ = 5.71 (d, J = 1.7 Hz), 4.61 (app d, J =
4.8 Hz, 1H), 4.47 (dd, J = 11.6, 3.4 Hz, 1H), 4.18 (dd, J = 6.3, 4.9 Hz, 1H), 4.06 (dd, J = 11.6, 4.1
Hz, 1H), 3.08 (m, 1H), 1.17 (s, 9H), 1.12-1.04 (m, 42H) ppm. 13
C NMR (126 MHz, CDCl3): δ =
178.50, 150.91, 120.91, 118.79 (q, C-F
J = 319.8 Hz), 75.51, 74.24, 61.76, 47.29, 39.09, 27.27, 18.39,
18.36, 18.33, 18.30, 13.27, 13.08 ppm. 19
F (470 MHz, CDCl3): δ = –73.35 ppm. IR (ν˜max): 2944 (m),
318
2867 (m), 1735 (m), 1427 (m), 1211 (s), 1138 (s), 882 (m), 825 (m), 682 (m), 609 (m) cm–1
. HR-MS
(ESI) calculated for C30H57F3O7SSi2 [M+H]+
: 675.33884. Found: 675.33895.
General procedure E: Suzuki coupling of enol triflates and (hetero)aryl boronic acids
Hetero(aryl) boronic acid or boronate (1.5 eq.) and K3PO4 (3 eq.) were added to a solution of triflate
in DME/H2O 4:1 (0.1 mmol/mL). The reaction mixture was thoroughly flushed with N2 and
Pd(dppf)Cl2.CH3CN (10 mol %) was added to the reaction mixture. The reaction mixture was then
stirred at 80 °C for 2-14 h. After cooling to 25 C, the reaction mixture was partitioned between H2O
(10 mL/0.5 mmol) and EtOAc (20 mL/0.5 mmol). The organic extracts were dried over Na2SO4,
evaporated, and the residue was purified by flash column chromatography to afford the product.
tert-butyl(((3aR*,4R*,6aS*)-2,2-dimethyl-6-phenyl-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-
4-yl)methoxy)diphenylsilane (23a).
Prepared by general procedure E using triflate 22a (0.44 g, 0.79 mmol) and PhB(OH)2 (0.145 g, 1.18
mmol) flash column chromatography (hexane/EtOAc = 20:1) afforded 23a as a colorless oil (0.326
g, 85%). 1
H NMR (500 MHz, CDCl3): δ = 7.65-7.60 (m, 6H), 7.42-7.29 (m, 9H), 6.05 (d, J = 2.6 Hz,
1H), 5.50 (dd, J = 5.8, 1.8 Hz, 1H), 4.71 (dm, J = 5.8 Hz, 1H), 3.84 (dd, J = 10.3, 4.9 Hz, 1H), 3.69
(dd, J = 10.3, 4.4 Hz, 1H), 3.09 (m, 1H), 1.42 (s, 3H), 1.39 (s, 3H), 1.02 (s, 9H) ppm. 13
C NMR (125
MHz, CDCl3): δ = 143.82, 135.86, 135.81, 134.81, 133.78, 133.62, 129.92, 128.65, 128.11, 127.91,
127.88, 126.56, 110.57, 85.40, 81.89, 65.23, 53.80, 27.73, 27.06, 26.23, 19.45 ppm. IR (ν˜max) = 1588
(m), 1557 (m), 1401 (s), 1335 (w), 1239 (w), 1212 (s), 1098 (m), 708 (s) cm–1
. HR-MS (ESI)
calculated for C31H36O3Si [M+Na]+
: 507.23259. Found: 507.23253.
tert-butyl(((3aR*,4R*,6aS*)-2,2-dimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4,6adihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)methoxy)diphenylsilane
(24a).
319
Bis(pinacolato)diboron (0.140 g, 0.549 mmol), KBr (0.090 g, 0.749 mmol), and KOPh (0.099 g,
0.749 mmol) were added to a solution of 22a (0.278 g, 0.499 mmol) in toluene (5 mL). The solution
was evacuated and backfilled with argon (3 cycles). Pd(Ph3P)2Cl2 (0.011 mg, 0.0149 mmol, 3 mol %)
and Ph3P (0.008 g, 0.0298 mmol, 6 mol %) were added and the reaction mixture was stirred at 60 C
for 3 h. The reaction mixture was cooled to 25 C, diluted with saturated aqueous NaHCO3 (30 mL)
and extracted with EtOAc (4 × 10 mL). The organic extracts were dried over Na2SO4, the solvent was
evaporated and the brown residue was purified by flash column chromatography (hexane/EtOAc =
20:1 to 10:1) to afford 24a as a colorless oil (0.187 g, 70%). 1
H NMR (500 MHz, CDCl3): δ = 7.65-
7.59 (m, 4H), 7.45-7.35 (m, 6H), 6.44 (d, J = 2.8 Hz, 1H), 5.29 (d, J = 5.8 Hz, 1H), 4.59 (d, J = 6.2
Hz, 1H), 3.77 (dd, J = 10.1, 4.6 Hz, 1H), 3.59 (dd, J = 10.1, 4.6 Hz, 1H), 3.02 (m, 1H), 1.37 (s, 3H),
1.35 (s, 3H), 1.30 (s, 6H), 1.28 (s, 6H), 1.02 (s, 9H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 149.07,
137.72 (C-B observed indirectly by 1
H-13
C HMBC), 135.93, 135.80, 133.79, 133.51, 129.91, 129.90,
127.90, 127.88, 109.97, 87.64, 83.66, 82.37, 64.46, 56.16, 27.76, 27.03, 26.09, 24.95, 19.47 ppm. 11
B
NMR (160.5 MHz, CDCl3): δ = 29.92 ppm. IR (ν˜max) = 1592 (m), 1542 (m), 1328 (m), 1248 (m), 828
(s), 745 (m) cm–1
. HR-MS (APCI) calculated for C31H43O5BSi [M+Na]+
: 557.28650. Found:
557.28666.
((1R*,4S*,5R*)-3-phenyl-4,5-bis(triisopropylsilyloxy)cyclopent-2-enyl)methyl pivalate (25a).
Prepared by general procedure E using triflate 22b (0.540 g, 0.8 mmol) and PhB(OH)2 (0.126 g, 1.04
mmol) flash column chromatography (hexane/EtOAc = 25:1) afforded 25a as a colorless oil (0.455
g, 94%). 1
H NMR (500 MHz, CDCl3): δ = 7.42-7.39 (m, 2H), 7.31 (m, 2H), 7.25 (m, 1H), 6.02 (app
s, 1H), 5.10 (d, J = 4.2 Hz, 1H), 4.63 (dd, J = 11.2, 3.3 Hz, 1H), 4.13 (dd, J = 6.7, 4.6 Hz, 1H), 4.02
(dd, J = 11.2, 5.6 Hz, 1H), 3.24 (m, 1H), 1.15-1.13 (m, 30H), 0.98-0.89 (m, 18), 0.86-0.77 (m, 3H)
320
ppm. 13
C NMR (126 MHz, CDCl3): δ = 178.72, 145.73, 135.85, 130.31, 128.49, 127.89, 126.28,
77.36, 77.04, 63.20, 49.46, 39.10, 27.41, 18.60, 18.53, 18.46, 13.75, 13.21 ppm. IR (ν˜max) = 2943
(m), 2866 (m), 1733 (m), 1141 (s), 882 (m), 682 (m) cm–1
. HR-MS (ESI) calculated for C35H62O4Si2
[M+Na]+
: 625.40788. Found: 625.40787.
((1R*,4S*,5R*)-3-(2,4-difluorophenyl)-4,5-bis(triisopropylsilyloxy)cyclopent-2-enyl)methyl
pivalate (25b).
Degassed dimethoxyethane and water (10+5 mL) were added under argon to a mixture of 22b (2.7 g;
3.99 mmol), 2,4-difluorophenylboronic acid (1.3 g; 7.98 mmol), LiCl (0.012 g; 0.28 mmol), Na2CO3
(1.7 g; 16.15 mmol) and Pd(PPh3)4 (322 mg; 0.28 mmol) and the reaction mixture was stirred at 85 °C
for 18 h. The reaction mixture was cooled to 25 C, mixed with brine (80 mL) and extracted with
EtOAc (3 × 80 mL). The combined organic layers were dried over MgSO4, filtered and concentrated
under reduced pressure. The residue was purified by column chromatography (hexane/EtOAc = 15:1)
to afford 25b as a pale yellow oil (2.3 g; 89 %). 1
H NMR (500 MHz, CDCl3): δ = 7.31 (td, J = 8.6,
6.5 Hz, 1H), 6.84 (ddd, J = 8.6, 3.4, 1.7 Hz, 1H), 6.79 (ddd, J = 11.2, 8.6, 2.5 Hz, 1H), 6.02 (app s,
1H), 5.08 (d, J = 4.4 Hz, 1H), 4.59 (dd, J = 11.3, 3.8 Hz, 1H), 4.17 (dd, J = 6.8, 4.4 Hz, 1H), 4.03 (dd,
J = 11.3, 5.5 Hz, 1H), 3.21 (m, 1H), 1.18-1.06 (m, 32H), 0.97-0.91 (m, 16H), 0.86-0.77 (m, 3H) ppm.
13
C NMR (126 MHz, CDCl3): δ = 178.68, 163.53 (m, C-F
J = 12.0 Hz), 161.62 (ddm, C-F
J = 12.0, 17.5
Hz), 159.69 (m, C-F
J = 11.1 Hz), 140.34 (d, C-F
J = 1.6 Hz), 134.45 (d, C-F
J = 4.8 Hz), 130.54 (dd, C-F
J =
5.6, 9.6 Hz), 120.67 (dd, C-F
J = 4.1, 13.1 Hz), 111.24 (dd, C-F
J = 3.1, 21.4 Hz), 104.37 (dd, C-F
J =
26.22 Hz), 78.07 (d, C-F
J = 3.4 Hz), 77.47 (detected by 1
H-13
C HSQC, overlapped with CDCl3), 62.97,
49.36, 39.09, 27.38, 18.51, 18.44, 18.41, 17.92, 13.70, 13.29, 12.53 ppm. 19
F{1
H} NMR (471MHz,
CDCl3): δ = –109.22 (d, J = 7.8 Hz), –110.87 (d, J = 7.9 Hz) ppm. IR (ν˜max) = 2943 (m), 2886 (m),
1732 (s), 1501 (s), 1463 (w), 1137 (s), 881 (m), 679 (s) cm–1
. HR-MS (ESI) calculated for
C35H60F2O4Si2 [M+Na+
]: 661.3890. Found: 661.3886.
321
((1R*,4S*,5R*)-3-(1-methyl-1H-pyrazol-4-yl)-4,5-bis(triisopropylsilyloxy)cyclopent-2enyl)methyl
pivalate (25c).
Prepared by general procedure E using triflate 22b (0.575 g, 0.851 mmol) and 1-methylpyrazole-4boronic
acid pinacol ester (0.194 g, 0.936 mmol) flash column chromatography (hexane/EtOAc =
20:1) afforded 25c as a colorless wax (0.45 g, 87 %). 1
H NMR (500 MHz, CDCl3): δ = 7.51 (s, 1H),
7.38 (s, 1H), 5.75 (s, 1H), 4.81 (d, J = 4.2 Hz, 1H), 4.56 (dd, J = 11.2, 3.8 Hz, 1H), 4.09 (dd, J = 6.1,
4.6 Hz, 1H), 3.98 (dd, J = 11.2, 5.5 Hz, 1H), 3.88 (s, 3H), 3.15 (m, 1H), 1.16 (s, 9H), 1.11 (s, 22H),
1.03-0.89 (m, 22H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 178.71, 137.61, 137.42, 127.66, 127.00,
118.75, 78.52, 77.23 (overlapped with CDCl3), 63.25, 49.23, 39.13, 39.08, 27.41, 18.64, 18.55, 18.50,
18.44, 13.75, 13.23 ppm. IR (ν˜max) = 2941 (w), 2893 (w), 1721 (s), 1462 (m), 1256 (s), 1239 (s), 679
(s) cm–1
. HR-MS (APCI) calculated for C33H62N2O4Si2 [M+H]+
: 607.4321. Found: 607.4322.
Methyl 5-((3R*,4R*,5S*)-3-(pivaloyloxymethyl)-4,5-bis(triisopropylsilyloxy)cyclopent-1enyl)furan-3-carboxylate
(25d).
Prepared by general procedure E using 22b (0.700 g, 1.037 mmol) and methyl 5-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)furan-3-carboxylate (0.287 g, 1.14 mmol). Flash column chromatography
(hexane/EtOAc = 25:1) afforded 25d as a colorless oil (0.55 g, 81%). 1
H NMR (500 MHz, CDCl3): δ
= 7.92 (s, 1H), 6.67 (s, 1H), 6.10 (d, J = 1.4 Hz, 1H), 4.92 (d, J = 4.1 Hz, 1H), 4.62 (dd, J = 11.3, 3.6
Hz, 1H), 4.06 (dd, J = 7.4, 4.2 Hz, 1H), 4.01 (dd, J = 11.3, 5.6 Hz, 1H), 3.84 (s, 3H), 3.23 (m, 1H),
1.16 (s, 9H), 1.13-0.98 (m, 42H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 178.63, 163.60, 152.43,
146.85, 135.61, 130.84, 120.79, 106.32, 77.12, 76.43, 62.77, 51.86, 49.11, 39.11, 27.40, 18.60, 18.46,
18.41, 13.66, 13.14 ppm. IR (ν˜max) = 1730 (s), 1580 (w), 1281 (s), 881 (m), 679 (s) cm–1
. HR-MS
(ESI) calculated for C35H62O7Si2 [M+Na]+
: 673.39263. Found: 673.39266.
322
((1R*,4S*,5R*)-3-(3a,7a-dihydrobenzo[b]thiophen-3-yl)-4,5-bis(triisopropylsilyloxy)cyclopent-2enyl)methyl
pivalate (25e).
Prepared by general procedure E using using 22b (1.2 g, 1.83 mmol) and benzothiophen-2-ylboronic
acid (0.490 g, 2.75 mmol) flash column chromatography (hexane/EtOAc = 15:1) afforded compound
25e as a pale yellow oil (0.913 g; 75 %). 1
H NMR (500 MHz, CDCl3): δ = 7.89 (dd, J = 6.8, 1.4 Hz,
1H), 7.85 (dd, J = 6.8, 1.4 Hz, 1H), 7.39-7.32 (m, 2H), 7.36 (s, 1H), 6.09 (d, J = 2.0 Hz, 1H), 5.13 (d,
J = 4.3 Hz, 1H), 4.57 (dd, J = 11.3, 4.3 Hz, 1H), 4.30 (dd, J = 5.7, 4.5 Hz, 1H), 4.10 (dd, J = 11.3, 5.4
Hz, 1H), 1.16 (m, 30H), 0.92-0.87 (m, 21 H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 178.73, 140.99,
140.52, 138.28, 132.57, 130.67, 124.56, 124.41, 123.99, 123.54, 122.99, 79.40, 77.12, 63.29, 49.93,
39.10, 27.42, 18.57, 18.50, 18.45, 13.56, 13.37 ppm. IR (ν˜max) = 2942 (m), 2865 (m), 1730 (m), 1460
(m), 1139 (s), 881 (s), 679 (s) cm–1
. HR-MS (APCI) calculated for C37H62O4SSi2 [M+Na]+
: 681.3800.
Found: 681.3803.
((1R*,4S*,5R*)-3-(4-(bis((2-(trimethylsilyl)ethoxy)methyl)amino)pyrrolo[1,2-f][1,2,4]triazin-7yl)-4,5-bis(triisopropylsilyloxy)cyclopent-2-enyl)methyl
pivalate (25f).
Prepared by general procedure E using 22b (0.162 g, 0.240 mmol) and boronic acid 29 (0.137 g,
0.312 mmol) flash column chromatography (hexane/EtOAc = 20:1) afforded 25f as a yellow semisolid
(0.164 g, 75%). 1
H NMR (500 MHz, CDCl3): δ = 7.98 (s, 1H), 6.98 (d, J = 4.8 Hz, 1H), 6.76
(app s, 1H), 6.74 (d, J = 4.8 Hz, 1H), 5.24-5.17 (m, 5H), 4.64 (dd, J = 11.3, 3.4 Hz, 1H), 4.12 (dd, J
= 7.5, 4.3 Hz, 1H), 4.05 (dd, J = 11.3, 5.4 Hz, 1H), 3.67 (m, 4H), 1.12 (m, 30H), 1.07-0.79 (m, 29H),
323
-0.01 (s, 18H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 178.67, 156.18, 146.49, 135.40, 133.27,
127.22, 115.79, 111.69, 105.51, 78.01, 77.85, 76.80, 66.17, 63.07, 49.09, 39.09, 27.41, 18.56, 18.49,
18.43, 18.41, 13.71, 13.12, -1.17 ppm. IR (ν˜max) = 2945 (w), 2858 (w), 1732 (s), 1582 (s), 1513 (s),
1248 (w), 1142 (s), 1079 (s), 855 (s), 681 (m) cm–1
. HR-MS (ESI) calculated for C47H91O6Si4
[M+H]+
: 919.6012. Found: 919.6012.
((1R*,4S*,5R*)-4,5-dihydroxy-3-phenylcyclopent-2-enyl)methyl pivalate (26a).
Prepared by general procedure D using 25a (0.120 g, 0.198 mmol) flash column chromatography
(CH2Cl2/EtOAc = 10:3) afforded 26a as a colorless wax (0.055 g, 95%). 1
H NMR (500 MHz, DMSOd6):
δ = 7.56-7.53 (m, 2H), 7.35-7.31 (m, 2H), 7.24 (dt, J = 9.1, 4.3 Hz, 1H), 6.26 (d, J = 2.2 Hz, 1H),
4.67 (m, 2H), 4.57 (dd, J = 9.2, 5.2 Hz, 2H, overlapped), 3.82 (dm, J = 4.9, 1H), 3.66 (m, 1H), 3.38
(m, 1H), 2.73 (dm, J = 5.9 Hz, 1H), 1.17 (s, 9H) ppm. 13
C NMR (126 MHz, DMSO-d6): δ = 142.48,
135.29, 129.61, 128.19, 127.01, 125.71, 73.94, 72.73, 61.80, 53.56 ppm. IR (ν˜max) = 2985 (w), 1729
(s), 1569 (m), 1507 (s), 1282 (s), 1107 (s), 857 (s) cm–1
. HR-MS (ESI) calculated for C17H22O4
[M+Na]+
: 313.1411. Found: 313.1410.
((1R*,4S*,5R*)-3-(2,4-difluorophenyl)-4,5-dihydroxycyclopent-2-enyl)methyl pivalate (26b.)
Prepared by general procedure D using 25b (0.178 g, 0.28 mmol) flash column chromatography
(CH2Cl2/EtOAc = 3:1 to 1:1) afforded 26b as a colorless oil (0.088 g, 97%). 1
H NMR (500 MHz,
CDCl3) δ = 7.55-7.50 (ddm, J = 6.6, 2.2 Hz, 1H), 6.91-6.87 (dm, J = 2.6, 1.04 Hz, 1H), 6.87-6.81 (m,
J = 9.9, 2.6 Hz, 1H), 6.24 (app t, J = 1.8 Hz, 1H), 4.96 (dm, J = 5.9 Hz, 1H), 4.35 (dd, J = 11.1, 5.6
Hz, 1H), 4.19 (dd, J = 7.0, 4.1 Hz, 1H), 4.17 (m, 1H), 3.11 (m, 1H), 1.19 (s, 9H) ppm. 13
C NMR (126
MHz, CDCl3): δ = 178.72, 163.50 (dd, C-F
J = 250.2, 10.6 Hz), 162.18 (dd, C-F
J = 254.2, 11.8 Hz),
161.54 (dd, C-F
J = 250.2, 10.6 Hz), 160.16 (dd, C-F
J = 254.2, 11.8 Hz), 138.26 (d, C-F
J = 2.4 Hz),
132.68 (dd, J = 8.4, 1.7 Hz), 130.27 (dd, C-F
J = 9.9, 5.9 Hz), 118.9 (dd, C-F
J = 12.8, 4.8 Hz), 111.73
324
(dd, C-F
J = 21.1, 3.5 Hz), 104.70 (app t, C-F
J = 25.8 Hz), 76.31 (d, C-F
J = 1.6 Hz), 73.93, 64.40, 51.69,
39.07, 27.40, 17.92, 12.53 ppm. 19
F{1
H} NMR (471MHz, CDCl3): δ= –109.48 (d, J = 7.8 Hz), –
110.33 (d, J = 7.9 Hz) ppm. IR (ν˜max) = 2973 (w), 1727 (s), 1591 (m), 1504 (s), 1282 (s), 1104 (s),
848 (s) cm–1
. HR-MS (ESI) calculated for C17H20F2O4 [M+Na]+
: 349.1222. Found: 349.1219.
((1R*,4S*,5R*)-4,5-dihydroxy-3-(1-methyl-1H-pyrazol-4-yl)cyclopent-2-enyl)methyl pivalate
(26c).
Prepared by general procedure D using 25c (0.378 g, 0.62 mmol) flash column chromatography
(CH2Cl2/EtOAc = 1:1) afforded 26c as a yellow wax (0.167 g, 91%). 1
H NMR (500 MHz, CDCl3) δ =
7.62 (s, 1H), 7.54 (s, 1H), 5.78 (d, J = 2.2 Hz, 1H), 4.76 (d, J = 5.8 Hz, 1H), 4.27 (dd, J = 11.0, 5.8
Hz, 1H), 4.13 (dd, J = 5.8, 4.3 Hz, 1H), 4.09 (dd, J = 11.0, 5.5 Hz, 1H), 3.87 (s, 2H), 3.04 – 2.99 (m,
1H), 1.17 (s, 9H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 178.74, 137.65, 136.24, 128.21, 124.08,
117.28, 77.23, 74.01, 64.81, 51.76, 39.11, 39.03, 27.39 ppm. IR (ν˜max) = 3416 (w), 2952 (w), 1717
(s), 1284 (s), 1167 (s), 1151 (s), 622 (m) cm–1
. HR-MS (ESI) calculated for C15H22N2O4 [M+Cl]–
:
329.1274. Found: 329.1273.
methyl 5-((3R*,4R*,5S*)-4,5-dihydroxy-3-(pivaloyloxymethyl)cyclopent-1-enyl)furan-3carboxylate
(26d).
Prepared by general procedure D using 25d (0.415 g, 0.64 mmol) flash column chromatography
(CH2Cl2/EtOAc = 3:1) afforded 26d as a yellow wax (0.185 g, 86%). 1
H NMR (500 MHz, CDCl3) δ
7.96 (s, 1H), 6.81 (s, 1H), 6.12 (d, J = 2.3 Hz, 1H), 4.86 (m, 1H), 4.33 (dd, J = 11.1, 5.6 Hz, 1H), 4.13
(m, 2H, overlapped), 3.84 (s, 3H), 3.10 (dm, J = 4.7 Hz, 1H), 1.18 (s, 9H) ppm. 13
C NMR (126 MHz,
CDCl3): δ = 178.70, 163.61, 151.45, 147.63, 134.04, 128.02, 120.82, 107.48, 74.96, 74.15, 64.40,
325
51.91, 51.71, 39.06, 27.39. IR (ν˜max) = 3427 (m), 2958 (m), 1719 (m), 1235 (s), 760 (s) cm–1
. HR-MS
(ESI) calculated for C17H22O7 [M+Cl]–
: 373.1060. Found: 373.1058.
((1R*,4S*,5R*)-3-(3a,7a-dihydrobenzo[b]thiophen-3-yl)-4,5-dihydroxycyclopent-2-enyl)methyl
pivalate (26e).
Prepared by general procedure D using 25e (0.873 g, 0.64 mmol) flash column chromatography
(CH2Cl2/EtOAc = 3:1) afforded 26e as a yellow oil (0.378 g, 83%). 1
H NMR (500 MHz, CDCl3) δ =
7.99 (dd, J = 7.3, 0.6 Hz, 1H), 7.88 (dd, J = 7.3, 0.6 Hz, 1H), 7.43-7.36 (m, 2H), 6.24 (d, J = 2.4 Hz,
1H), 4.97 (dd, J = 5.7, 1.0 Hz, 1H), 4.43 (dd, J = 11.1, 5.4 Hz, 1H), 4.25 (m, 2H, overlapped), 3.18
(m, 1H), 1.20 (s, 9H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 178.79, 140.76, 139.09, 137.75, 131.19,
129.22, 125.23, 124.80, 124.77, 123.23, 77.65, 73.77, 64.42, 51.91, 39.10, 27.44, 17.92, 12.52 ppm.
IR (ν˜max) = 2958 (m), 2939 (m), 2865 (m), 1726 (s), 1282 (s), 1150 (s), 758 (s), 731 (s) cm–1
. HR-MS
(ESI) calculated for C19H22O4S [M+Na]+
= 369.1131. Found: 369.1128.
((1R,4S,5R)-3-(4-(bis((2-(trimethylsilyl)ethoxy)methyl)amino)pyrrolo[1,2-f][1,2,4]triazin-7-yl)-
4,5-dihydroxycyclopent-2-enyl)methyl pivalate (26f).
Prepared by general procedure D using 25f (0.140 g, 0.153 mmol) flash column chromatography
(CH2Cl2 to CH2Cl2/MeOH = 20:1) afforded 26f as a yellow semi-solid (0.075 g, 81 %). 1
H NMR (500
MHz, CD2Cl2): δ = 7.99 (s, 1H), 7.08 (d, J = 4.8 Hz, 1H), 6.86 (d, J = 4.8 Hz, 1H), 6.44 (d, J = 2.2
Hz, 1H), 5.22 (m, 4H), 4.97 (dd, J = 6.0, 0.9 Hz, 1H), 4.40 (dd, J = 11.0, 5.2 Hz), 4.15 (m, 2H), 3.69
(m, 4H), 3.13 (m, 1H), 1.19 (s, 9H), 0.99 (m, 4H), -0.02 (s, 18H) ppm. 13
C NMR (126 MHz, CD2Cl2):
δ = 178.32, 156.40, 146.88, 135.21, 131.32, 127.61, 115.21, 111.54, 106.78, 77.79, 74.46, 74.09,
326
66.08, 64.28, 51.49, 38.82, 29.78, 27.07, 18.22, –1.41, –1.62 ppm. IR (ν˜max) = 3278 (w), 2954 (w),
2918 (w), 1725 (m), 1578 (m), 1160 (w), 1084 (s), 1008 (m), 858 (s), 833 (s) cm–1
. HR-MS (ESI)
calculated for C29H50N4O6Si2 [M+Na]+
: 607.3352. Found: 607.3352.
General procedure F: cleavage of pivaloate
Sodium methoxide (5 eq.) was added into a solution of the starting material in MeOH (0.1 mmol/mL)
and the reaction mixture was stirred at 65 °C for 14 h. The reaction mixture was cooled to 25 °C, the
solvent was evaporated and the residue was purified by flash column chromatography.
(1R*,2S*,5R*)-5-(hydroxymethyl)-3-phenylcyclopent-3-ene-1,2-diol (27a).
Prepared by general procedure F using 26a (0.334 g, 1.15 mmol) flash column chromatography
(CH2Cl2/MeOH = 20:1 to 10:1) afforded 27a as a white solid (0.211 g, 89 %), m.p. = 99-101 °C. 1
H
NMR (500 MHz, DMSO-d6): δ = 7.56-7.53 (m, 2H), 7.33 (tm, J = 7.2 Hz, 2H), 7.23 (tt, J = 7.2, 1.0
Hz, 1H), 6.26 (d, J = 2.2 Hz), 4.67 (m, 2H), 4.57 (m, 2H), 3.82 (app d, J = 4.5 Hz, 1H, -OH), 3.66 (m,
1H), 3.38 (m, 1H), 2.73 (m, 1H) ppm. 13
C NMR (126 MHz, DMSO-d6): δ = 142.48, 135.29, 129.61,
128.19, 127.01, 125.71, 73.94, 72.73, 61.80, 53.56 ppm. IR (ν˜max) = 2927 (m), 2855 (m), 1471 (m),
1427 (m), 1104 (s), 699 (s) cm–1
. HR-MS (ESI) calculated for C12H14O3[M+Cl]–
: 241.0637. Found:
241.0637.
(1R*,2S*,5R*)-3-(2,4-difluorophenyl)-5-(hydroxymethyl)cyclopent-3-ene-1,2-diol (27b)
Prepared by general procedure F using 26b (0.126 g, 0.39 mmol) flash column chromatography
(CH2Cl2/MeOH = 10:1 to 5:1) afforded 27b as a white solid (0.083 g, 89 %), m.p. = 122-124 C
327
(racemic), 107-108 °C as (+)-enantiomer. NMR (500 MHz, DMSO-d6) δ = 7.61 (ddm, J = 6.8, 2.1
Hz, 1H), 7.22 (ddd, J = 11.9, 9.3, 2.6 Hz, 1H), 7.09 (ddm, J = 8.6, 2.8 Hz, 1H), 6.29 (app t, J = 2.1
Hz, 1H), 4.73 (m, 1H, -OH), 4.68 (app t, J = 5.9, 1H,-OH), 4.63-4.59 (m, 2H, overlapped), 3.81 (dd, J
= 12.0, 5.8 Hz, 1H), 3.64 (m, 1H), 3.40-3.35 (m, 1H), 2.76 (m, 1H) ppm. 13
C NMR (126 MHz,
DMSO-d6): δ = 161.87 (dd, C-F
J = 246.0, 13.5 Hz), 161.40 (dd, C-F
J = 252.6, 13.2 Hz), 159.90 (dd, C-F
J
= 246.0, 13.5 Hz), 159.40 (dd, C-F
J = 252.6, 13.2 Hz), 136.54 (d, C-F
J = 3.3 Hz), 134.20 (dd, J = 10.1,
1.9 Hz), 130.27 (dd, C-F
J = 9.8, 6.0 Hz), 119.82 (dd, C-F
J = 13.0, 3.5 Hz), 111.26 (dd, C-F
J = 20.9, 3.5
Hz), 104.17 (app t, C-F
J = 26.1 Hz), 74.92, 71.98, 61.63, 54.07 ppm. 19
F{1
H} NMR (471MHz, DMSOd6):
δ= –108.75 (d, J = 8.3 Hz), –111.87 (d, J = 7.8 Hz) ppm. IR (ν˜max) = 3072 (w), 3051 (w), 2931
(w), 2858 (w), 1613 (m), 1254 (m), 849 (m), 821 (m), 739 (s) cm–1
. HR-MS (ESI) calculated for
C12H12O3F2 [M+Na]+
: 265.0652. Found: 265.0657. Crystal data for 27b: Crystallized from MeOH,
C12H12F2O3, Mrel = 242.22, T = 120 K, space group P-1, a = 5.2120(4) Å, b = 7.4380(5) Å, c =
14.3549(9) Å, α = 80.834(6), β = 85.227(6), γ = 74.865(6), V = 529.826 Å3
. CCDC ref. No. 1452238.
(1R*,2S*,5R*)-5-(hydroxymethyl)-3-(1-methyl-1H-pyrazol-4-yl)cyclopent-3-ene-1,2-diol (27c).
Prepared by general procedure F using 26c (0.096 g, 0.33 mmol) flash column chromatography
(CH2Cl2/MeOH = 10:1 to 4:1) afforded 27c as a white wax (0.063 g, 91 %). 1
H NMR (500 MHz,
DMSO-d6): δ = 7.73 (s, 1H), 7.56 (s, 1H), 5.82 (d, J = 2.1 Hz, 1H), 4.60 (d, J = 6.0 Hz, 1H, -OH),
4.54 (dd, J = 4.9 Hz, 1H), 4.49-4.44 (m, 2H, overlapped), 3.80 (s, 3H), 3.78 (dd, J = 11.5, 5.5 Hz,
1H), 3.55 (m, 1H), 2.65 (m, 1H) ppm. 13
C NMR (126 MHz, DMSO-d6): δ = 136.58, 135.40, 127.94,
124.76, 117.55, 75.15, 72.58, 62.25, 53.85, 38.33 ppm. IR (ν˜max) = 2956 (m), 2923 (s), 1727 (s),
1461 (m), 1260 (s), 1071 (m), 798 (m) cm–1
. HR-MS (ESI) calculated for C10H14N2O30 [M+Cl]–
:
245.0698. Found: 245.0698.
methyl 5-((3R,4R,5S)-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-1-enyl)furan-3-carboxylate
(27d).
328
Prepared by general procedure F using 26d (0.120 g, 0.35 mmol) flash column chromatography
(CH2Cl2/MeOH = 10:1 to 4:1) afforded 27d as a white wax (0.077 g, 86 %). 1
H NMR (500 MHz,
DMSO-d6): δ = 8.35 (s, 1H), 6.72 (s, 1H), 6.13 (d, J = 2.2 Hz, 1H), 4.92 (d, J = 6.7 Hz, 1H), 4.60 (m,
2H), 4.70 (d, J = 6.7 Hz, 1H), 3.82 (dd, J = 12.2, 5.8 Hz, 1H), 3.78 (s, 3H), 3.60 (ddd, J = 10.3, 5.1
Hz, 1H), 3.40 – 3.34 (m, 1H), 2.72 (m, 1H) ppm. 13
C NMR (126 MHz, DMSO-d6): δ = 162.70,
152.19, 147.70, 133.34, 129.36, 119.68, 105.80, 73.69, 72.50, 61.48, 53.90, 51.46 ppm. IR (ν˜max) =
3203 (m), 1724 (s), 1580 (m), 1515 (m), 1233 (s), 760 (s) cm–1
. HR-MS (ESI) calculated for C12H14O6
[M+Cl]–
: 289.0484. Found: 289.0483.
(1R*,2S*,5R*)-3-(3a,7a-dihydrobenzo[b]thiophen-3-yl)-5-(hydroxymethyl)cyclopent-3-ene-1,2diol
(27e).
Prepared by general procedure F using 26e (0.070 g, 0.20 mmol) flash column chromatography
(CH2Cl2/MeOH = 10:1 to 5:1) afforded 27e as a white solid (0.044 g, 83 %). m.p. = 123-125 °C. 1
H
NMR (500 MHz, DMSO-d6): δ = 7.94 (tm, J = 6.8 Hz, 2H), 7.43 (app s, 1H), 7.40-7.33 (m, 2H), 4.59
(d, J = 6.1 Hz, 1H), 4.55 (dd, J = 5.23 Hz, 1H), 4.43 (d, J = 4.8 Hz, 1H), 3.92 (m, 1H), 3.76 (dd, J =
9.9, 4.9 Hz, 1H), 3.50 (m, 1H), 3.45-3.36 (m, 2H, overlapped), 2.22 (ddm, J = 12.8, 8.22 Hz, 1H),
2.12-2.04 (m, 1H), 1.42 (ddd, J = 12.9, 10.2, 8.9 Hz) ppm. 13
C NMR (126 MHz, DMSO-d6): δ =
139.78, 139.16, 139.10, 124.10, 123.74, 122.70, 122.43, 120.34, 76.99, 73.32, 62.79, 46.32, 42.91,
30.35 ppm. IR (ν˜max) = 3311 (m), 1254 (m), 1126 (m), 1029 (s), 1018 (m), 737 (s) cm–1
. HR-MS
(ESI) calculated for C14H14O3S [M+Na]+
= 285.0556. Found: 285.0554.
329
(1R*,2S*,5R*)-3-(4-(bis((2-(trimethylsilyl)ethoxy)methyl)amino)pyrrolo[1,2-f][1,2,4]triazin-7yl)-5-(hydroxymethyl)cyclopent-3-ene-1,2-diol
(27f).
Prepared by general procedure F using 26f (0.066 g, 0.108 mmol) flash column chromatography
(CH2Cl2 to CH2Cl2/MeOH = 10:1) afforded 27f as a yellow solid (0.055 g, 98 %). m.p. = 250 °C. 1
H
NMR (500 MHz, CD3OD): δ = 8.10 (s, 1H), 7.18 (d, J = 4.8 Hz, 1H), 7.13 (d, J = 2.2 Hz, 1H), 7.03
(d, J = 4.8 Hz, 1H), 5.32 (app s, 4H), 5.01 (dd, J = 5.8, 0.6 Hz, 1H), 4.11 (m, 1H), 3.96 (dd, J = 10.8,
4.8 Hz, 1H), 3.80 (m, 4H), 3.73 (dd, J = 10.8, 6.6 Hz, 1H), 3.09 m (1H), 1.06 (m, 4H), -0.08 (s, 18H)
ppm. 13
C NMR (126 MHz, CD3OD): δ = 157.47, 147.69, 134.83, 133.46, 128.61, 116.93, 113.42,
107.78, 79.63, 77.39, 74.56, 67.21, 63.99, 55.20, 19.29, –0.98 ppm. IR (ν˜max) = 3280 (w), 2920 (m),
1725 (m), 1579 (w), 1087 (s), 833 (s), 759 (s) cm–1
. HR-MS (ESI) calculated for C24H43N4O5Si2
[M+Na]+
: 523.2772. Found: 523.2776.
(1R*,2S*,5R*)-3-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-(hydroxymethyl)cyclopent-3-ene-
1,2-diol (28).
PPTS (0.360 g, 1.44 mmol) was added into a solution of 27f (0.150 g, 0.28 mmol) in MeOH : H2O
(5+1 mL), the reaction mixture was stirred at 55 °C for 12 h, then cooled to 25 °C and the solvents
were evaporated. The residue was purified by preparative TLC (SiO2, CH2Cl2/MeOH 10:1, repeated
elution) to afford 28 (0.115 g) contaminated by TsOH. Analytically pure sample of 28 was obtained
by RP-HPLC (Nucleodur®
C18 HTec, details given in Supporting Information) as a white solid, m.p.
> 250 °C. 1
H NMR (500 MHz, DMSO-d6): δ = 7.92 (s, 1H), 7.64 (br s, 2H, -NH2), 7.13 (d, J = 2.2
Hz, 1H), 6.90 (d, J = 4.5 Hz, 1H), 6.88 (d, J = 2.1 Hz, 1H), 6.76 (d, J = 4.5 Hz, 1H), 4.74 (d, J = 6.5
Hz, 1H), 4.70 (app t, J = 5.8 Hz, 1H), 4.60 (app t, J = 5.3 Hz, 1H), 4.51 (d, J = 7.2 Hz), 3.78 (dd, J =
13.1, 6.5 Hz, 1H), 3.68 (m, 1H), 3.40-3.35 (m, 1H), 2.80 (m, 1H) ppm. 13
C NMR (126 MHz, DMSO-
330
d6): δ = 155.46, 147.98, 133.05, 130.16, 125.88, 115.42, 110.58, 101.38, 75.15, 72.17, 62.01, 53.89
ppm. IR (ν˜max) = 3333 (w), 3218 (w), 2924 (m), 1651 (m), 1602 (m), 1122 (m), 1010 (m), 731 (s)
cm–1
. HR-MS (APCI) calculated for C12H14N4O3 [M+H]+
: 263.1139. Found: 263.1139.
tert-butyl(((3aR*,4R*,6R*,6aS*)-2,2-dimethyl-6-phenyltetrahydro-3aHcyclopenta[d][1,3]dioxol-4-yl)methoxy)diphenylsilane
(30).
Prepared by general procedure C using compound 23a (0.140 g, 0.288 mmol), Pd/C (0.030 g, 0.028
mmol), H2 (1 bar) in EtOH flash column chromatography (hexane/EtOAc = 20:1) afforded 30 as a
colorless oil (0.120 g, 86 %). 1
H NMR (500 MHz, CDCl3): δ = 7.70-7.65 (m, 6H), 7.46-7.38 (m, 6H),
7.34-7.28 (m, 4H), 7.25-7.20 (m, 1H), 4.62 (m, 1H), 4.56 (m, 1H), 3.66 (m, 2H), 3.23 (m, 1H), 2.37
(m, 2H), 1.87 (dd, J = 11.8, 6.3 Hz, 1H), 1.47 (s, 3H), 1.27 (s, 3H), 1.09 (s, 9H) ppm. 13
C NMR (126
MHz, CDCl3): δ = 139.65, 135.87, 135.83, 133.66, 129.96, 129.06, 128.21, 127.97, 126.73, 110.10,
83.93, 82.90, 65.18, 48.00, 47.14, 31.45, 27.14, 26.52, 24.24, 19.47 ppm. IR (ν˜max) = 1525 (m), 1515
(s), 1282 (s), 1107 (s), 848 (s), 719 (m) cm–1
. HR-MS (ESI) calculated for C31H38O3Si [M+Na]+
:
509.2492. Found: 509.2491.
(1S*,2R*,3R*,5S*)-3-((tert-butyldiphenylsilyloxy)methyl)-5-phenylcyclopentane-1,2-diol (31a).
Prepared by general procedure C using S-12 (0.030 g, 0.067 mmol), Pd/C (0.007 g, 0.0067 mmol),
H2 (1 bar) in EtOH preparative TLC (CH2Cl2/EtOAc = 40:1) afforded 31a as a colorless oil (0.018 g,
60 %) and 0.008 g (27%) of the epimer 31b. 1
H NMR (500 MHz, CDCl3): δ = 7.71-7.67 (m, 4H),
7.47-7.38 (m, 6H), 7.34-7.29 (m, 2H), 7.26-7.21 (m, 3H), 4.13 (m, 1H), 4.03 (m, 1H), 3.86 (dd, J =
10.1, 4.8 Hz, 1H), 3.71 (dd, J = 10.1, 6.4 Hz, 1H), 3.17 (m, 1H), 2.72 (br s, 1H, -OH), 2.45 (br s, 1H,
-OH), 2.36-2.29 (m, 1H), 2.20-2.13 (m, 1H), 1.57-1.47 (m, 1H), 1.09 (s, 9H) ppm. 13
C NMR (126
MHz, CDCl3): δ = 142.96, 135.84, 135.83, 133.55, 133.50, 130.04, 130.03, 128.81, 128.01, 127.61,
331
126.75, 79.20, 75.90, 66.07, 50.53, 46.64, 31.53, 27.15, 19.49 ppm. IR (ν˜max) = 3200 (w), 2949 (w),
1215 (s), 760 (s), 697 (s) cm–1
. HR-MS (APCI) calculated for C28H34O3Si [M+H]+
: 437.2355. Found:
437.2355.
General procedure G: directed hydrogenation with Crabtree´s catalyst
H2 was gently bubbled into a solution of Crabtree´s catalyst (1 mol %) and the starting material in
CH2Cl2 (0.2 mmol/mL) at 25 °C till the full conversion was observed by TLC or 1
H NMR (up to 3h).
The solvent was evaporated and the residue was purified by flash column chromatography.
((1R*,2R*,3S*,4S*)-2,3-dihydroxy-4-phenylcyclopentyl)methyl pivalate (32a).
Prepared by general procedure G using 26a (0.466 g, 1.6 mmol) flash column chromatography
(CH2Cl2/EtOAc = 1:1) afforded 32a as a pale yellow oil (0.439 g, 94 %). 1
H NMR (500 MHz, CDCl3)
δ = 7.33 (m, 2H), 7.27-7.22 (m, 3H), 7.43-7.36 (m, 2H), 4.22 (dd, J = 11.1, 5.6 Hz, 1H), 4.16 (dd, J =
11.1, 5.8 Hz, 1H), 4.04-3.97 (m, 2H), 3.16 (m, 1H), 2.55 (brs, 1H), 2.40 (m, 1H), 2.31 (brs, 1H), 2.25
(m, 1H), 1.48 (dd, J = 22.4, 11.5 Hz), 1.23 (s, 9H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 178.86,
142.17, 128.94, 127.52, 127.01, 78.87, 74.65, 65.77, 50.22, 44.25, 39.12, 31.90, 27.48 ppm. IR
(ν˜max) = 3452 (m), 2955 (m), 2892 (m), 1724 (s), 1283 (m), 719 (s) cm–1
. HR-MS (ESI) calculated
for C17H24O4 [M+H]+
= 293.1747. Found: 293.1747.
((1R*,2R*,3S*,4S*)-4-(2,4-difluorophenyl)-2,3-dihydroxycyclopentyl)methyl pivalate (32b).
Prepared by general procedure G using 26b (0.290 g, 0.89 mmol) flash column chromatography
(CH2Cl2/EtOAc = 3:1) afforded 32b as a colorless wax (0.269 g, 92 %). 1
H NMR (500 MHz, CDCl3)
332
δ = 7.19 (ddm, J = 8.5, 6.4 Hz, 1H), 6.87-6.78 (m, 2H), 4.22 (dd, J = 11.5, 5.7 Hz, 1H), 4.15 (dd, J =
11.5, 5.7 Hz, 1H), 4.12 (m, 1H), 3.99 (app t, J = 5.6 Hz, 1H), 3.32 (ddm, J = 11.3, 7.8 Hz, 1H), 2.44-
2.35 (m, 1H), 2.21 (m, 1H), 1.47 (m, 1H), 1.22 (s, 9H) ppm. 13
C NMR (126 MHz, CDCl3): δ =
178.90, 162.97 (dd, C-F
J = 247.1, 12.5 Hz), 162.51 (dd, C-F
J = 249.7, 10.7 Hz), 161.00 (dd, C-F
J =
247.1, 12.5 Hz), 160.52 (dd, C-F
J = 249.7, 10.7 Hz), 129.66 (dd, C-F
J = 9.8, 7.3 Hz), 125.04 (dd, C-F
J =
14.2, 4.0 Hz), 111.53 (dd, C-F
J = 20.9, 3.8 Hz), 104.36 (app t, C-F
J = 26.2 Hz), 77.38, 74.26, 65.42,
44.59, 44.37, 39.12, 30.80, 27.45 ppm. 19
F{1
H} NMR (471MHz, CDCl3): δ= –112.65 (d, J = 6.6 Hz),
–113.20 (d, J = 7.1 Hz). IR (ν˜max) = 3365 (w), 2970 (w), 2931 (w), 1703 (s), 1601 (m), 1505 (s),
1287 (s), 1175 (s), 964 (s), 849 (s) cm–1
. HR-MS (ESI) calculated for C17H22F2O4 [M+Na]+
=
351.1378. Found: 351.1376.
(1R*,2R*,3S*,4S*)-2,3-dihydroxy-4-(1-methyl-1H-pyrazol-4-yl)cyclopentyl)methyl pivalate
(32c).
Prepared by general procedure C using 26c (0.091 g, 0.309 mmol), Pd(OH)2/C (0.0043 g, 0.0309
mmol), H2 (1 bar) in EtOH flash column chromatography (EtOAc/MeOH = 99:1) afforded 32c
(0.040 g, 44 %) as a pale yellow solid. m.p. = 80-83 °C. 1
H NMR (500 MHz, CDCl3): δ = 7.35 (s,
1H), 7.24 (s, 1H), 4.13 (ddd, J = 11.2, 5.8 Hz, 2H), 3.92 (dd, J = 5.8, 4.6 Hz, 1H), 3.85 (s, 3H), 3.80
(dd, J = 8.2, 5.8 Hz, 1H), 3.05 (ddd, J = 11.1, 7.9 Hz, 1H), 2.41 – 2.32 (m, 1H), 2.22 (ddd, J = 13.0,
8.0 Hz, 1H), 1.31 (m, 1H), 1.20 (s, 9H). 13
C NMR (126 MHz, CDCl3): δ = 178.83, 137.60, 128.11,
123.07, 79.14, 74.32, 65.86, 44.31, 40.16, 39.07, 39.00, 31.42, 27.42 ppm. IR (ν˜max) = 3449 (m),
2955 (m), 2889 (m), 1723 (s), 1283 (m), 1158 (s), 711 (s) cm–1
. HR-MS (ESI) calculated for
C15H24N2O4 [M+H]+
: 297.1809. Found: 297.1806.
methyl 5-((1R*,2S*,3R*,4R*)-2,3-dihydroxy-4-(pivaloyloxymethyl)cyclopentyl)furan-3carboxylate
(32d).
333
Prepared by general procedure G using 26d (0.312 g, 0.92 mmol) flash column chromatography
(CH2Cl2/EtOAc = 2:1) afforded 32d as a colorless wax (0.298 g, 95 %). 1
H NMR (500 MHz, CDCl3):
δ = 7.91 (d, J = 0.7 Hz, 1H), 6.45 (s, 1H), 4.19 (dd, J = 11.2, 5.5 Hz, 1H), 4.13 (dd, J = 11.2, 5.7 Hz,
1H), 4.08 (m, 1H), 3.95 (m, 1H), 3.08 (s, 3H), 3.24 (m, 1H), 2.40 (m, 1H), 2.26 (m, 1H), 1.53 (m,
1H), 11.21 (s, 9H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 178.87, 163.83, 157.70, 146.97, 119.97,
105.50, 76.91, 74.62, 65.42, 51.76, 43.66, 43.32, 39.11, 28.99, 27.43 ppm. IR (ν˜max) = 3434 (m),
2956 (m) , 1580 (m), 1515 (m), 1233 (s), 760 (s) cm–1
. HR-MS (ESI) calculated for C17H24O7 [M+Cl]–
: 375.1216. Found: 375.1216.
((1R*,2R*,3S*,4S*)-4-(3a,7a-dihydrobenzo[b]thiophen-3-yl)-2,3-dihydroxycyclopentyl)methyl
pivalate (32e).
Prepared by general procedure G using 26e (0.102 g, 0.29 mmol) flash column chromatography
(CH2Cl2/EtOAc = 2:1) afforded 32e as a pale yellow wax (0.088 g, 86 %). 1
H NMR (500 MHz,
CDCl3): δ = 7.93-7.90 (m, 1H), 7.87-7.85 (m, 1H), 7.42-7.34 (m, 2H), 7.16 (d, J = 0.8 Hz, 1H), 4.27
(dd, J = 11.2, 5.4 Hz, 1H), 4.21-4.16 (m, 2H), 4.00 (dd, J = 5.6 Hz, 1H), 3.61 (m, 1H), 2.54-2.46 (m,
1H), 2.44-2.37 (m, 1H), 1.65-1.57 (m, 1H), 1.23 (s, 9H) ppm. 13
C NMR (126 MHz, CDCl3): δ =
178.69, 140.76, 138.97, 137.42, 124.55, 124.06, 122.91, 122.21, 120.10, 77.61, 74.52, 65.36, 43.62,
43.52, 30.31, 27.26, 17.69 ppm. IR (ν˜max) = 3434 (m), 2956 (m), 1712 (s), 1580 (m), 1515 (m), 1233
(s), 760 (s) cm–1
. HR-MS (ESI) calculatedd for C19H24O4S [M+H]+
: 349.1468. Found: 349.1472.
((1R*,2R*,3S*,4S*)-4-(4-(bis((2-(trimethylsilyl)ethoxy)methyl)amino)pyrrolo[1,2f][1,2,4]triazin-7-yl)-2,3-dihydroxycyclopentyl)methyl
pivalate (32f).
334
Prepared by general procedure C using 26f (0.275 g, 0.45 mmol), Pd(OH)2/C (0.006 g, 0.045 mmol),
H2 (1 bar) in EtOH preparative TLC (CH2Cl2/EtOAc = 1:1) afforded 32f as a yellow wax (0.134 g,
49%). 1
H NMR (500 MHz, CDCl3): δ = 7.94 (s, 1H), 6.98 (d, J = 4.7 Hz, 1H), 6.52 (d, J = 4.7 Hz,
1H), 5.23 (app s, 4H), 4.18 (d, J = 6.1 Hz, 1H), 4.06-3.98 (m, 2H), 3.75 (m, 1H), 3.68 (app t, J = 8.2
Hz, 1H), 2.51 (m, 1H), 2.35 (m, 1H), 1.79 (ddm, J = 11.9, 8.2 Hz, 1H), 1.21 (s, 9H), 0.99 (app t, J =
8.2 Hz), -0.01 (s, 18H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 13
C NMR (126 MHz, CDCl3) δ
178.76, 156.55, 146.66, 133.04, 114.74, 109.55, 105.79, 78.22, 77.76, 74.76, 66.30, 65.81, 44.19,
42.00, 39.09, 28.36, 27.46, 18.44, –1.16 ppm. IR (ν˜max) = 3339 (m), 2964 (m), 1580 (m), 1701 (s),
1226 (s), 749 (s) cm–1
. HR-MS (ESI) calculated for C29H52N4O6Si2 [M+Na]+
: 631.3323. Found:
631.3323.
(1S*,2R*,3R*,5S*)-3-(hydroxymethyl)-5-phenylcyclopentane-1,2-diol (33a).
Prepared by general procedure F using 32a (0.578 g, 1.97 mmol). Flash column chromatography
(CH2Cl2/MeOH = 10:1) afforded 33a as a white solid (0.365 g, 89 %). m.p. = 105-106 °C. 1
H NMR
(500 MHz, DMSO-d6): δ = 7.30-7.24 (m, 4H), 7.16 (m, 1H), 4.53 (app t, J = 5.1 Hz, 1H, -OH), 4.40
(d, J = 6.0 Hz, 1H, -OH), 4.35 (d, J = 2.7 Hz, 1H, -OH), 3.72 (m, 1H), 3.48-3.37 (m, overlapped, 2H),
2.97 (m, 1H), 2.07-1.95 (m, 2H), 1.32-1.22 (m, 2H) ppm. 13
C NMR (126 MHz, DMSO-d6): δ =
143.99, 128.00, 127.41, 125.70, 78.12, 73.47, 63.05, 49.03, 46.73, 31.74 ppm. IR (ν˜max) = 3290 (m),
2938 (w), 1396 (w), 1213 (s), 1111 (s), 1178 (s), 759 (s), 697 (s) cm–1
. HR-MS (APCI) calculated for
C12H16O3 [M+NH4]+
: 226.1438. Found: 226.1436. Crystal data for 33a: Crystallized from MeOH,
C12H16O3, Mrel = 601.51, T = 120 K, space group Pbca, a = 9.8445(4) Å, b = 6.9522(5) Å, c =
30.4659(12) Å, α = 90.00, β = 90.00, γ = 90.00, V = 2085.11 Å3
. CCDC ref. No. 1452773.
335
(1R*,2S*,3S*,5R*)-3-(2,4-difluorophenyl)-5-(hydroxymethyl)cyclopentane-1,2-diol (33b).
Prepared by general procedure F using 32b (0.173 g, 0.53 mmol). Flash column chromatography
(CH2Cl2/MeOH = 10:1 to 5:1) afforded 33b as a white solid (0.115 g, 90 %). m.p. = 101-103 °C. 1
H
NMR (500 MHz, DMSO-d6) δ = 7.41 (ddm, J = 8.6, 6.9 Hz, 1H), 7.12 (dddd, J = 12.0, 10.7, 9.7, 2.7
1H), 7.03 (app td, J = 8.5, 2.7 Hz, 1H), 4.55 (app t, J = 5.2 Hz, 1H), 4.46 (d, J = 6.5 Hz, 1H), 4.40 (d,
J = 4.6 Hz, 1H), 3.74 (dd, J = 7.2, 3.6 Hz, 1H), 3.45-3.36 (m, 2H), 3.23 (m, 1H), 2.07-1.95 (m, 2H,
overlapped), 1.25-1.17 (m, 1H) ppm. 13
C NMR (126 MHz, DMSO-d6): δ = 161.67 (dd, C-F
J = 246.9,
11.6 Hz), 161.42 (dd, C-F
J = 242.2, 11.4 Hz), 159.70 (dd, C-F
J = 246.9, 11.6 Hz), 159.48 (dd, C-F
J =
242.2, 11.4 Hz), 129.63 (dd, C-F
J = 10.0, 7.2 Hz), 126.69 (dd, C-F
J = 14.6, 3.8 Hz), 111.14 (dd, C-F
J =
19.7, 4.5 Hz), 103.35 (dd, C-F
J = 26.8, 25.2 Hz), 76.72, 73.13, 62.96, 46.63, 41.60, 30.96 ppm.
19
F{1
H} NMR (471MHz, CDCl3): δ = –114.08 (AB d, J = 6.05 Hz), –114.13 (AB d, J = 6.7 Hz). IR
(ν˜max) = 3276 (m), 1453 (m), 1272 (m), 1208 (m), 1043 (s), 963 (s), 849 (s), 606 (m) cm–1
. HR-MS
(ESI) calculated for C17H22F2O4 [M+Na]+
: 351.1378. Found: 351.1376.
(1S*,2R*,3R*,5S*)-3-(hydroxymethyl)-5-(1-methyl-1H-pyrazol-4-yl)cyclopentane-1,2-diol (33c).
Prepared by general procedure F using 32c (0.212 g, 0.378 mmol). Flash column chromatography
(CH2Cl2/MeOH = 5:1) afforded 33c as a yellow solid (0.043 g, 53 %). m.p. = 112-114 °C. 1
H NMR
(500 MHz, DMSO-d6 + CDCl3): δ = 7.44 (s, 1H), 7.25 (s, 1H), 4.50 (dd, J = 5.1 Hz, 1H, -OH), 4.41
(d, J = 6.4 Hz, 1H,-OH), 4.28 (d, J = 4.1 Hz, 1H,-OH), 3.76 (s, 3H), 3.65 (dd, J = 9.2, 4.5 Hz, 1H),
3.50 (dd, J = 14.0, 5.8 Hz, 1H), 3.42 – 3.35 (m, 2H), 2.83 (ddd, J = 10.6, 8.1 Hz, 1H), 2.06 – 1.97 (m,
1H), 1.94 (m, 1H), 1.17 (ddd, J = 12.4, 10.8, 8.4 Hz, 1H). 13
C NMR (126 MHz, DMSO-d6 + CDCl3):
δ = 137.44, 128.43, 124.15, 79.11, 73.80, 63.72, 47.20, 38.74, 31.66 ppm. IR (ν˜max) = 3415 (m), 2926
(m), 2870 (m), 1265 (s), 1118 (s), 1015 (s), 839 (m), 670 (w) cm–1
. HR-MS (ESI) calculated for
C10H16N2O3 [M+Cl]–
: 247.0855. Found: 247.0854. Crystal data for 33c: Crystallized from MeOH,
336
C10H16N2O3, Mrel = 212.25, T = 120 K, space group P-1, a = 7.2069(4) Å, b = 7.7420(3), c =
9.5826(4) Å, α = 80.466(4), β = 79.403(4), γ = 77.127(4), V = 507.987 Å3
. CCDC ref. No. 1452237.
methyl 5-((1R,2S,3R,4R)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)furan-3-carboxylate
(33d).
Prepared by general procedure F using 32d (0.334 g, 0.98 mmol) flash column chromatography
(CH2Cl2/MeOH = 5:1) afforded 33d as a yellow wax (0.186 g, 74 %). 1
H NMR (500 MHz, DMSOd6):
δ = 8.23 (d, J = 0.8 Hz, 1H), 6.44 (s, 1H), 4.54 (m, 3H), 3.78 (m, 1H), 3.75 (s, 3H), 3.67 (m, 1H),
3.38 (m, 2H), 3.07 (dd, J = 18.2, 8.2 Hz, 1H), 2.00 (m, overlapped, 2H), 1.34 (ddd, J = 12.5, 10.2, 7.8
Hz, 1H), 1.53 (m, 1H), 11.21 (s, 9H) ppm. 13
C NMR (126 MHz, DMSO-d6): δ = 162.95, 159.47,
146.82, 118.87, 104.24, 76.27, 73.24, 62.74, 51.29, 46.07, 42.28, 28.81 ppm. IR (ν˜max) = 3284 (m),
1707 (s), 1607 (m), 1515 (m), 1438 (m), 1110 (s), 760 (s) cm–1
. HR-MS (ESI) calculated for
C12H6O6Si2 [M+Cl]:
291.0641. Found: 291.0641.
(1R*,2S*,3S*,5R*)-3-(3a,7a-dihydrobenzo[b]thiophen-3-yl)-5-(hydroxymethyl)cyclopentane-1,2diol
(33e).
Prepared by general procedure F using 32e (0.173 g, 0.53 mmol) flash column chromatography
(CH2Cl2/MeOH = 5:1) afforded 33e as a yellow solid (0.123 g, 88 %). m.p. = 102-104 °C. 1
H NMR
(500 MHz, DMSO-d6): δ = 7.98-7.92 (m, 2H), 7.43 (s, 1H), 7.42-7.32 (m, 2H), 4.59 (d, J = 5.8 Hz,
1H, -OH), 4.55 (dd, J = 5.3 Hz, 1H, -OH), 4.43 (d, J = 4.8 Hz, 1H, -OH), 3.92 (dd, J = 12.8, 5.8 Hz,
1H), 3.75 (dd, J = 9.8, 4.8 Hz, 1H), 3.49 (m, 1H), 3.46-3.36 (m, 2H, overlapped), 2.22 (ddd, J = 12.8,
7.5 Hz, 1H), 2.08 (m, 1H) ppm. 13
C NMR (126 MHz, DMSO-d6): δ = 139.78, 139.16, 139.10, 124.10,
123.74, 122.70, 122.43, 120.34, 76.99, 73.32, 62.79, 46.32, 42.91, 30.35 ppm. IR (ν˜max) = 3260 (w),
337
1426 (m), 1318 (m), 1097(m), 1082 (m), 1068 (m), 1022 (m), 960 (m), 906 (m), 708 (m), 634 (m)
cm–1
. HR-MS (ESI) calculated for C19H24O4S [M+Na]+
: 371.1288. Found: 371.1286.
(1R*,2S*,3S*,5R*)-3-(4-(bis((2-(trimethylsilyl)ethoxy)methyl)amino)pyrrolo[1,2-f][1,2,4]triazin-
7-yl)-5-(hydroxymethyl)cyclopentane-1,2-diol (33f).
Prepared by general procedure F using 32f (0.156 g, 0.256 mmol) flash column chromatography
(CH2Cl2/CH3OH = 10:1) afforded 33f as a yellow wax (0.106 g, 79 %). 1
H NMR (500 MHz, CD2Cl2):
δ = 7.94 (s, 1H), 6.99 (d, J = 4.7 Hz, 1H), 6.99 (d, J = 4.7 Hz, 1H), 5.21 (app s, 4H), 4.04 (dd, J = 6.9,
6.4 Hz, 1H), 3.74 (dd, J = 10.8, 4.9 Hz, 1H), 3.68 (app t, J = 8.4 Hz, 6H), 2.30 (m, 2H), 1.66 (m, 1H),
0.98 (app t, J = 8.2 Hz, 4H), –0.02 (s, 18H) ppm. 13
C NMR (126 MHz, CD2Cl2): δ = 156.34, 146.42,
133.23, 114.57, 109.25, 105.54, 77.72, 77.44, 75.28, 66.01, 65.03, 47.18, 42.27, 28.14, 18.21, –1.63
ppm. IR (ν˜max) = 3393 (w), 2951 (w), 2924 (w), 1589 (s), 1521 (s), 1409 (m), 1248 (m), 1074 (s),
832 (s) cm–1
. HR-MS (ESI) calculated for C24H45O5N4Si2 [M+H]+
: 525.29230. Found: 525.29230.
(1R*,2S*,3S*,5R*)-3-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-(hydroxymethyl)cyclopentane-
1,2-diol (34).
The compound was prepared by the essentially same route described for compound 28, using
compound 33f (0.090 g, 0.171 mmol). The crude mixture was purified by preparative TLC
(CH2Cl2/MeOH = 5:1) afforded compound 34 (0.102 g) contaminated by TsOH. Analytically pure
sample of 34 was obtained by RP-HPLC (Nucleodur®
C18 HTec, details given in Supporting
Information) as a white solid, m.p.= > 250 C decomp. 1
H NMR (500 MHz, CD3OD): δ = 7.90 (s,
1H), 7.15 (d, J = 4.6 Hz, 1H), 6.73 (d, J = 4.6 Hz, 1H), 4.23 (dd, J = 7.9, 5.1 Hz, 1H), 4.00 (app t, J =
338
5.1 Hz), 3.79 (m, 1H), 3.70 (dd, J = 10.7, 6.1 Hz), 3.64 (dm, J = 10.7, 6.1 Hz, 1H), 2.40 (m, 1H), 2.27
(m, 1H), 1.55 (dm, J = 10.5, 8.8 Hz, 1H) ppm. 13
C NMR (126 MHz, DMSO-d6): δ = 155.09, 146.72,
133.49, 113.69, 107.48, 101.18, 76.05, 73.19, 62.94, 48.51, 46.37, 29.98 ppm. IR (ν˜max) = 3362 (w),
3225 (w), 1679 (m), 1606 (m), 1108 (m), 1019 (m), 725 (m) cm–1
. HR-MS (ESI) calculated for
C15H21O3N4 [M+Na]+
: 305.16082. Found: 305.16088.
((1R*,4S*,5R*)-3-phenyl-4,5-bis(triisopropylsilyloxy)cyclopent-2-enyl)methyl4-methylbenzene
sulfonate (35).
Dibal-H (1M in hexane, 0.538 mL, 0.538 mmol) was added to a solution of 25a (0.130 g, 0.215
mmol) in CH2Cl2 (4 mL) at –78 °C and the reaction mixture was stirred at –78 °C for 30 min. The
mixture was then allowed to warm to 25 °C, stirred for 1 h, then quenched with saturated aqueous
solution of sodium potassium tartrate (3 mL), and extracted with CH2Cl2 (3 × 15 mL). The organic
extracts were washed with brine (5 mL), dried over MgSO4, filtered, and concentrated under reduced
pressure to yield the crude product, which was purified by flash column chromatography
(hexane/EtOAc 10:1) to afford the de-pivaloylated intermediate (a colorless wax, 0.100 g, 90%),
which was directly used in the next step. Tosyl chloride (0.049 g, 0.258 mmol), triethylamine (0.083
mL, 0.595 mmol) and DMAP (0.002 g, 0.020 mmol) were added to a solution of the intermediate
(0.100 g, 0.198 mmol) in CH2Cl2 (4 mL). The reaction mixture was stirred at 25 °C for 3 h, quenched
with saturated aqueous NaHCO3 (10 mL) and extracted with CH2Cl2 (3 × 20 mL). The organic
extracts were dried over MgSO4 and concentrated under reduced pressure to yield a pale yellow
residue, which was purified by flash column chromatography (hexane/EtOAc = 15 : 1) to afford 35
(0.131 g, 98%) as a colorless wax. 1
H NMR (500 MHz, CDCl3): δ = 7.78 (d, J = 8.3 Hz, 2H), 7.38 –
7.26 (m, 7 H), 5.93 (app s, 1H), 5.04 (d, J = 4.6 Hz, 1H), 4.39 (dd, J = 9.7, 3.7 Hz, 1H), 4.05 (dd, J =
9.7, 7.2 Hz, 1H), 3.99 (dd, J = 7.3, 4.4 Hz, 1H), 3.20 (m, 1H), 2.44 (s, 3H), 1.09-1.01 (m, 21H), 0.95-
0.85 (m, 18H), 0.77 (m, 3H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 146.21, 145.02, 135.42, 133.23,
130.04, 129.19, 128.53, 128.24, 128.16, 126.25, 77.05, 76.61, 70.23, 49.09, 21.85, 18.55, 18.49,
18.44, 18.38, 13.69, 13.11 ppm. IR (ν˜max) = 2943, 2866, 1729, 1495, 1381, 1178 cm–1
. HR-MS (ESI)
calculated for C37H60O5SSi2 [M+Na]+
: 695.3592. Found: [M+Na]+
= 695.3588.
339
(1R*,2S*,5R*)-5-((2-methoxyethylamino)methyl)-3-phenylcyclopent-3-ene-1,2-diol (36).
2-Methoxyethylamine (0.033 mL, 0.386 mmol) and DIPEA (0.100 mL, 0.579 mmol) were added to a
solution of 35 (0.130 g, 0.193 mmol) in DMF (2 mL). The reaction mixture was stirred at 100 °C for
3 h, then quenched with water (10 mL) and extracted with EtOAc (2 × 20 mL). The combined
extracts were dried over MgSO4, filtered and concentrated under reduced pressure to yield crude
product, which was purified by flash column chromatography (hexane/EtOAc 4 : 1) to afford the
substitution intermediate (0.077 g, 69 %) as a pale yellow wax. TBAF (1 M in THF, 0.267 mL, 0.267
mmol) was added to a solution of the intermediate (0.070 g, 0.121 mmol) in THF (5 mL) at 0 °C. The
reaction mixture was stirred at 25 °C for 14 h, then quenched with water (15 mL) and concentrated
under reduced pressure to yield the crude product, which was purified by preparative TLC
(CH2Cl2/7M NH3 in MeOH = 8 : 1) to afford 36 (0.028 g, 90 %) as a yellow wax. 1
H NMR (500
MHz, DMSO-d6): δ = 7.55 (d, J = 7.5 Hz, 2H), 7.33 (t, J = 7.7 Hz, 2H), 7.24 (t, J = 7.3 Hz, 1H), 6.29
(app s, 1H), 4.73 (m, 1H), 4.67 (m, 1H), 3.78 (t, J = 5.4 Hz, 1H), 3.44 (t, J = 5.4 Hz, 2H), 3.26 (s,
3H, partially overlapped with residual H2O), 2.86-2.75 (m, 4H) ppm. 13
C NMR (126 MHz, DMSOd6):
δ = 142.53, 135.13, 129.45, 128.22, 127.13, 125.73, 75.10, 73.79, 70.80, 57.97, 51.39, 50.09,
48.50 ppm. IR (ν˜max) = 3345 (m), 2959 (m), 2875 (m), 1448 (m), 804 (s), 693 (m) cm–1
. HR-MS
(APCI) calculated for C15H21N2O3 [M+H]+
: 264.1594. Found: 264.1593.
(1R*,4S*,5R*)-3-phenyl-4,5-bis(triisopropylsilyloxy)cyclopent-2-enyl)methanamine (37).
A mixture of 35 (0.075 g, 0.11 mmol) and 2 M NH3 in 2-propanol (2.5 mL) was stirred in a pressure
tube at 50 °C for 24 h. Then, aqueous NH3 solution (25–29%, 1 mL) was added and the reaction
mixture was stirred at 75 °C for additional 24 h, after which the TLC showed full consumption of the
starting material. The reaction mixture was concentrated under reduced pressure and the residue was
purified by flash column chromatography (CH2Cl2/MeOH/NH3 in MeOH = 95 : 5: 0.5) to afford 37 as
a yellow wax (0.050 g, 70%). 1
H NMR (500 MHz, DMSO-d6): δ = 7.44 (m, 2H), 7.35 (app t, J = 7.5
340
Hz, 2H), 7.27 (app t, J = 7.1 Hz, 1H), 6.40 (s, 1H), 5.07 (d, J = 4.3 Hz, 1H), 3.97 (dd, J = 7.0, 4.3 Hz,
1H), 3.07 (dd, J = 12.1, 3.4 Hz, 1H), 2.96 (m, 1H), 2.55 (dd, J = 11.5, 8.9 Hz, 1H), 1.16-1.05 (s,
21H), 0.94-0.86 (m, 18H), 0.83-0.75 (m, 3H) ppm. 13
C NMR (126 MHz, DMSO-d6): δ = 143.91,
135.28, 131.85, 128.23, 127.56, 125.68, 77.79, 76.37, 51.14, 41.67, 18.16, 18.11, 18.05, 18.01, 12.91,
12.33 ppm. IR (ν˜max) = 2943, 2866, 1464, 1155, 1118 cm–1
. HR-MS (ESI) calculated for
C30H55NO2Si2 [M+Na]+
: 540.3664. Found: 540.3660.
N-(((1R*,4S*,5R*)-4,5-dihydroxy-3-phenylcyclopent-2-enyl)methyl)acetamide (38a).
DIPEA (30 µL, 0.17 mmol) and AcCl (4.5 µL, 0.07 mmol) were added dropwise into cooled (0 C)
solution of 37 (30 mg, 0.06 mmol) in CH2Cl2 (1.5 mL). The reaction mixture was stirred at 25 C for
2 h, then mixed with saturated aqueous NaHCO3 (5 mL) and extracted with EtOAc (3 × 10 mL). The
combined organic layers were washed with brine (15 mL), dried over MgSO4 and concentrated in
vacuo. The residue was dissolved in THF (2 mL) and TBAF in THF (1 M, 86 µL) was added
dropwise at 0 °C. The reaction mixture was allowed to warm to 25 C and stirred for 14 h. The
solvent was evaporated and the residue was purified by preparative TLC (CH2Cl2/MeOH = 15:1) to
afford 38a (8.2 mg, 89%) as a yellow wax. 1
H NMR (500 MHz, Acetone-d6): δ = 7.63 (dm, J = 8.5
Hz, 2H), 7.36 (m, 2H), 7.30 – 7.26 (m, 1H), 7.21 (br s, 1H, -NH), 6.28 (d, J = 2.2 Hz, 1H), 4.88 (dd, J
= 5.8, 1.1 Hz, 1H), 4.02 (app t, J = 5.7 Hz, 1H), 3.49 – 3.43 (m, 1H), 3.42-3.35 (m, 1H), 2.93 (app dd,
J = 11.8, 5.9 Hz, 1H), 1.91 (s, 3H) ppm. 13
C NMR (126 MHz, Acetone-d6): δ = 170.27, 144.45,
136.53, 130.25, 129.19, 128.24, 127.00, 76.00, 75.70, 52.76, 41.83, 23.06 ppm. IR (ν˜max) = 3308,
2922, 2852, 1655, 1633, 1109 cm–1
. HR-MS (ESI) calculated mass for C14H17NO3 [M+Na]+
=
270.1101. Found: 270.1104.
341
1-(((1R*,4S*,5R*)-4,5-dihydroxy-3-phenylcyclopent-2-enyl)methyl)-3-phenylurea (38b).
TEA (28 µL, 0.20 mmol) and phenyl isocyanate (12 µL, 0.11 mmol) were added dropwise at 0C into
a solution of 37 (52 mg, 0.10 mmol) in CH2Cl2 (2 mL). The reaction mixture was stirred at 25 C for
2.5 h, then mixed with H2O (10 mL) and extracted with CH2Cl2 (3 × 20 mL). The combined extracts
were dried over MgSO4 and concentrated in vacuo. The residue was dissolved in THF (3 mL) and
TBAF in THF (1 M, 0.18 mL) was added. The reaction mixture was stirred at 25 C for 14 h. The
solvent was removed in vacuo and the crude product was purified by flash column chromatography
(CH2Cl2/MeOH = 95:5) to afford 38b (17 mg, 67%) as a white semi-solid.1
H NMR (500 MHz,
DMSO-d6): δ = 8.60 (s, 1H,- NH), 7.56 (app d, J = 7.6 Hz, 2H), 7.39 (app d, J = 7.7 Hz, 2H), 7.34
(app d, J = 7.7 Hz, 2H), 7.27-7.17 (m, 3H) 6.87 (app t, J = 7.3 Hz, 1H), 6.32 (app t, J = 5.7 Hz, 1H, NH),
6.22 (d, J = 2.0 Hz, 1H), 4.75 (br s, 2H, -OH), 4.70 (m, 1H), 3.81 (m, 1H), 3.35-3.23 (m, 2H,
overlapped with residual H2O signal), 2.81 (app dd, J = 11.9, 5.20 Hz) ppm.13
C NMR (126 MHz,
DMSO-d6): δ = 155.34, 143.03, 140.59, 135.10, 129.01, 128.53, 128.23, 127.20, 125.80, 120.80,
117.51, 74.37, 73.93, 51.05, 40.83 ppm. IR (ν˜max) = 3340, 3284, 3058, 3031, 2959, 2923, 2853, 1649,
1555, 1257 cm–1
. HR-MS (ESI): calculated mass for C19H20N2O3 [M+H]+
= 325.1547. Found:
325.1545.
N-(((1R*,4S*,5R*)-4,5-dihydroxy-3-phenylcyclopent-2-en-1-yl)methyl)-N,N-dimethylsulfuric
diamide (38c).
TEA (50 µL, 0.36 mmol) and N,N-dimethylsulfamoyl chloride (20 µL, 0.18 mmol) were added at 0
C to a solution of 37 (0.062 g, 0.12 mmol) in DMF (3 mL). The reaction mixture was stirred for 30
min at 0 C and then at 25 C for 3 h. The reaction mixture was quenched with H2O (10 mL) and
extracted with EtOAc (3 × 20 mL). The combined organic extracts were dried over MgSO4 and
342
concentrated under reduced pressure. The residue was dissolved in THF (2 mL) and TBAF in THF (1
M, 0.19 mL) was added. The reaction mixture was stirred at 25 C for 14 h. The solvent was removed
in vacuo and the crude product was purified by flash column chromatography (CH2Cl2/MeOH 15:1 to
5:1) to afford 38c (0.024 g, 89%) as a colorless wax. 1
H NMR (500 MHz, CDCl3): δ = 7.53 (app d, J
= 8.0 Hz, 2H), 7.36 (m, 2H), 7.30 (m, 1H), 6.12 (d, J = 2.0 Hz, 1H), 4.94 (dd, J = 5.9, 1.3 Hz, 1H),
4.67 (br s, 1H), 4.10 (app t, J = 5.8 Hz, 1H), 3.36 (m, 1H), 3.19 (dd, J = 12.6, 7.9 Hz, 1H), 3.02 (dd, J
= 12.1, 6.0 Hz, 1H), 2.83 (m, 6H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 144.29, 133.99, 128.96,
128.56, 128.25, 126.25, 76.19, 75.34, 51.11, 38.32 ppm. IR (ν˜max) = 3445, 3301, 2924, 1457, 1320,
1145, 1093 cm–1
. HR-MS (ESI) calculated for C14H20N2O4S [M+Na]+
: 335.1036. Found: 335.1032.
(6aR*,8S*,9S*,9aR*)-2,2,4,4-tetraisopropyl-8-phenylhexahydrocyclopenta[f][1,3,5,2,4]
trioxadisilocin-9-ol (39).
1,3-Dichloro-1, 1, 3, 3-tetraisopropyldisiloxane (0.307 mL, 0.960 mmol) was added to a solution of
33a (0.200 g, 0.960 mmol) in pyridine (4 mL) at 0 °C. The reaction mixture was stirred at 25 °C for
16 h, then quenched with 2 M aqueous HCl (15 mL), and extracted with EtOAc (2 × 40 mL). The
combined organic extracts were dried over MgSO4, filtered and concentrated under reduced pressure
to yield the crude product, which was purified by flash column chromatography (hexane/EtOAc =
20:1) to afford 39 (0.380 g, 88 %) as a colorless wax. 1
H NMR (500 MHz, DMSO-d6): δ = 7.31-7.25
(m, 2H), 7.23-7.16 (m, 3H), 4.29 (br s, 1H, -OH), 4.07 (dd, J = 7.6, 5.9 Hz, 1H), 3.90 (dd, J = 11.6,
3.6 Hz, 1H), 3.79 (app t, J = 5.3 Hz, 1H), 3.75 (dd, J = 11.6, 3.6 Hz, 1H), 2.95 (m, 1H), 2.19 (m, 1H),
1.97 (ddm, J = 12.5, 7.70 Hz, 1H), 1.09 – 0.95 (m, 28H) ppm. 13
C NMR (126 MHz, DMSO-d6): δ =
144.07, 128.21, 127.02, 125.90, 77.59, 73.72, 62.14, 49.83, 46.29, 30.26, 17.39, 17.27, 17.24, 17.21,
17.03, 17.00, 16.96, 16.91, 12.93, 12.80, 12.29, 12.08 ppm. IR (ν˜max) = 2943 (m), 2886 (m), 1028
(m), 883 (m), 694 (s) cm–1
. HR-MS (ESI) calculated for C24H42O4Si2 [M+H]+
: 451.26944. Found:
451.26950.
343
(1R*,2S*,3S*,5R*)-5-(hydroxymethyl)-2-methoxy-3-phenylcyclopentanol (40).
n-BuLi (0.070 mL, 0.110 mmol) was added to a solution of 39 (0.050 g, 0.107 mmol) in THF (1.5
mL) at –78 °C and the mixture was stirred for 15 min. Methyl trifluoromethanesulfonate (0.012 mL,
0.110 mmol) was added dropwise at –78 °C and the reaction mixture was stirred for 3 h while
allowed to warm to 25 °C. The mixture was mixed with saturated aqueous solution of NH4Cl (5 mL)
and extracted with EtOAc (2 × 20 mL). The combined extracts were dried over MgSO4, filtered, and
concentrated under reduced pressure. The residue was dissolved in THF (2 mL) and TBAF (1 M in
THF, 0.220 mL) was added. The reaction mixture was stirred at 25 C for 14 h. The solvent was
removed in vacuo and the crude product was purified by flash column chromatography
(CH2Cl2/CH3OH = 15:1 to 5:1) to afford 40 (0.008 g, 32% over the 2 steps) as a colorless solid. m.p.
= 103-105 °C. 1
H NMR (500 MHz, CDCl3): δ = 7.34-7.29 (m, 2H), 7.25-7.20 (m, 3H), 4.08 (app t, J
= 5.8 Hz, 1H), 3.80 (dd, J = 10.6, 4.9 Hz, 1H), 3.71 (d, J = 10.6, 6.7 Hz, 1H), 3.64 (app t, J = 6.2 Hz,
1H), 3.34 (s, 3H), 3.21 (m, 1H), 2.24 (m, 2H), 1.38 (m, 1H) ppm.13
C NMR (126 MHz, CDCl3): δ =
143.61, 128.84, 127.44, 126.68, 88.00, 75.01, 65.39, 58.13, 48.55, 46.99, 32.36 ppm. IR( ν˜max): 3251
(m), 2925 (w) 2907 (w), 1350 (w), 1193 (s), 1055 (s), 697 (s), 551 (s) cm–1
HR-MS (APCI) calculated
for C13H18O3 [M+H]+
: 223.1329. Found: 223.1330. Crystal data for 40: Crystallized from MeOH,
C13H18O3, Mrel = 222.28, T = 120 K, space group P21/n, a = 12.5947(2) Å, b = 6.74830(10) Å, c =
14.1169(3) Å, α = 90.00, β = 105.382(2), γ = 90.00, V = 1156.86 Å3
. CCDC ref. No. 1452235.
(6aR*,8S*,9aR*)-2,2,4,4-tetraisopropyl-8-phenyltetrahydrocyclopenta[f][1,3,5,2,4]
trioxadisilocin-9(9aH)-one (41).
IBX (0.102 g, 0.366 mmol) was added to a solution of 39 (0.110 g, 0.244 mmol) in acetonitrile (2.5
mL) and the reaction mixture was stirred at 80 °C for 4 h, then cooled down to 25 C, diluted with
344
Et2O (20 mL) and filtered through a pad of Celite. The filtrate was washed with saturated aqueous
solution of NaHCO3 (15 mL), dried over MgSO4, filtered, and concentrated under reduced pressure.
The residue was purified by flash column chromatography (hexane/EtOAc = 20 : 1) to afford 41
(0.088 g, 81 %) as a colorless wax. 1
H NMR (500 MHz, CDCl3): δ = 7.32 (m, 2H), 7.23 (m, 3H), 4.32
(d, J = 11.6 Hz, 1H), 4.12 (dd, J = 11.6, 2.6 Hz, 1H), 3.93 (d, J = 11.6 Hz, 1H), 3.41 (m, 1H), 2.34-
2.28 (m, 1H), 2.19-2.05 (m, 2H), 1.14-0.96 (m, 28H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 212.57,
138.54, 128.92, 127.82, 127.19, 76.92, 60.22, 51.85, 43.66, 27.12, 17.67, 17.62, 17.58, 17.33, 17.22,
17.15, 17.13, 13.88, 13.50, 12.96, 12.71 ppm. IR (ν˜max) = 2944 (m), 2866 (m), 1726 (m), 1449 (m),
1025 (m), 856 (m), 688 (s) cm–1
. HR-MS (ESI) calculated for C24H42O4Si2 [M-H]:
447.2387. Found:
447.2386.
(6aR*,8S*,9R*,9aR*)-2,2,4,4-tetraisopropyl-8-phenylhexahydrocyclopenta[f][1,3,5,2,4]
trioxadisilocin-9-ol (42).
LiAlH[OC(C2H5)3]3 (0.5 M in THF, 0.459 mL, 0.229 mmol) was added dropwise to a solution of 41
(0.103 g, 0.229 mmol) in THF (4 mL) at 0 °C. The reaction mixture was stirred at 25 °C for 3 h, then
quenched with saturated aqueous NH4Cl (6 mL), and extracted with EtOAc (3 × 30 mL). The
combined extracts were dried over MgSO4, filtered, and concentrated under reduced pressure. The
residue was purified by preparative TLC (hexane/EtOAc = 10 : 1) to afford 42 (0.095 g, 92 %) as a
white solid, m.p. = 61-63 °C. 1
H NMR (500 MHz, CDCl3): δ = 7.36-7.31 (m, 2H), 7.31-7.28 (m, 2H),
7.25 (m, J = 1H), 4.20 (dd, J = 5.5, 2.7 Hz, 1H), 4.16 (m, 1H), 4.04 (dd, J = 11.3, 3.9 Hz, 1H), 3.77
(dd, J = 11.3, 8.6 Hz, 1H), 3.44 (m, 1H), 2.19 (m, 1H), 2.02 (m, 1H), 1.85 (dd, J = 24.2, 12,1 Hz),
1.15-1.04 (m, 28H) ppm.13
C NMR (126 MHz, CDCl3): δ = 139.35, 128.88, 128.80, 127.05, 81.37,
81.32, 65.58, 49.68, 47.46, 30.20, 17.92, 17.78, 17.75, 17.70, 17.49, 17.42, 17.41, 17.36, 13.85,
13.79, 13.29, 12.81 ppm. IR (ν˜max) = 2941 (w), 2864 (m), 1060 (m), 551 (s) cm–1
. HR-MS (ESI)
calculated for C24H42O4Si2: [M+H]+
: 451.2694. Found: 451.2694. Crystal data for 42: Crystallized
from MeOH, C24H42O4Si2, Mrel = 450.76, T = 120 K, space group P21/n, a = 9.6577(2) Å, b =
345
26.6774(5) Å, c = 10.5938(2) Å, α = 90.00, β = 109.914(2), γ = 90.00, V = 2566.21 Å3
. CCDC ref.
No. 1452236.
(1R*,2R*,3R*,5S*)-3-(hydroxymethyl)-5-phenylcyclopentane-1,2-diol (43).
Prepared by general procedure D using compound 42 (0.070 g, 0.155 mmol) flash column
chromatography (CH2Cl2/MeOH = 10:1) afforded 43 as a white wax (0.024 g, 73 %). 1
H NMR (500
MHz, Acetone-d6): δ = 7.38 (dm, J = 7.3 Hz, 2H), 7.29 (app t, J = 7.0 Hz, 2H), 7.19 (tm, J = 7.40
Hz), 4.01 (m, 2H), 3.94 (br s, 1H), 3.74 (m, 2H), 3.61 (d, J = 5.6 Hz, 1H), 3.44 (m, 1H), 2.24-2.17 (m,
1H), 2.16-2.11 (m, 1H), 2.04-1.95 (m, 1H) ppm. 13
C NMR (126 MHz, Acetone-d6): δ = 142.10,
129.79, 128.68, 126.71, 82.35, 81.58, 65.20, 50.25, 49.09, 31.40 ppm. IR (ν˜max): 3310 (w), 2964 (m),
1154 (s), 798 (s) cm–1
. HR-MS (APCI) calculated for C12H16O3: [M+H]+
: 209.1172. Found:
209.1167.
(1S,4S)-((1S,2S,3R,4R)-4-(benzyloxymethyl)-2,3-bis(triisopropylsilyloxy)cyclopentyl)methyl
4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylate.
(1S,4S)-((1R,2R,3S,4S)-4-(benzyloxymethyl)-2,3-bis(triisopropylsilyloxy)cyclopentyl)methyl
4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylate.
346
DMAP (0.214 g, 1.75 mmol) and DIPEA (1.22 mL, 7.02 mmol) were added to a cooled (0 °C, ice
bath) solution of 7d (1.98 g, 3.51 mmol) in CH2Cl2 (6 mL) followed by dropwise addition of (1S)-(-)camphanic
chloride solution in CH2Cl2 (6 mL). The reaction mixture was stirred at 25 °C for 14 h.
The solvent was evaporated and the resulting yellow oil was purified by flash column
chromatography (SiO2, hexane/EtOAc = 10:1) to afford unseparable mixture of the diastereomeric
camphanates as a colorless oil (2.53 g, 97 %), which was used in the next step.
(1S,4S)-((1R,2R,3S,4S)-4-(hydroxymethyl)-2,3-bis(triisopropylsilyloxy)cyclopentyl)methyl
4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylate ((+)-46b)
(1S,4S)-((1S,2S,3R,4R)-4-(hydroxymethyl)-2,3-bis(triisopropylsilyloxy)cyclopentyl)methyl
4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylate ((−)-46a).
Pd(OH)2/C (0.150 g, 1.07 mmol) was added to a degassed solution of the mixture of the
diastereomeric camphanates (2.53 g 3.4 mmol) in THF (35 mL). The reaction mixture was stirred in a
hydrogenation apparatus at 65 °C under H2 atmosphere (50 bar) for 24 h. The reaction mixture was
cooled to 25 C and filtered through pad of Celite, which was washed with additional THF (3 × 15
mL). The filtrate was concentrated in a vacuum and the resulting colorless oil was purified by flash
column chromatography (hexane/EtOAc = 10:1 to 4:1). The less polar diastereomer (+)-46b was
obtained as a colorless oil (0.853 g, 76 %). 𝛼 𝐷
𝑅𝑇
= + 7.5 (c = 1, CHCl3). IR (ν˜max) = 2942 (m), 2865
(m), 1791 (s), 1735 (m), 1464 (m), 1102 (s), 1061 (s), 882 (s) cm–1
. 1
H NMR (500 MHz, CDCl3): δ =
4.30 (dd, J = 4.9, 11.3 Hz, 1H), 4.14 (d, J = 6.0, 11.3 Hz, 1H), 4.06 (ddm, J = 13.1, 6.1 Hz, 2H),
2.50-2.39 (m, 2H), 2.24 (m, 1H), 2.09 (m, 1H), 2.02-1.88 (m, 2H), 1.69 (m, 1H), 1.20 (m, 2H), 1.08
(s, 40H), 0.95 (s, 3H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 178.33, 167.91, 91.35, 77.36, 76.52,
347
67.04, 64.67, 54.97, 54.43, 45.22, 41.86, 30.85, 29.19, 26.20, 18.50, 18.46, 16.97, 16.90, 13.30,
13.21, 9.91 ppm. HR-MS (APCI) calculated for C35H66O7Si2[M+Na]+
: 677.42393. Found: 677.42385.
The more polar diastereomer (−)-46a (0.746 g, 67%) was obtained as a white crystalline compound.
m.p. = 91-93 °C. 𝛼 𝐷
𝑅𝑇
= –7.6 (c = 1, CHCl3). IR (ν˜max) = 2941 (m), 2867 (m), 1790 (s), 1735 (m),
1465 (m), 1102 (s), 1058 (s), 882 (s) cm–1
. 1
H NMR (500 MHz, CDCl3): δ = 4.36 (dd, J = 5.2, 11.0
Hz, 1H), 4.11 (d, J = 6.7, 11.0 Hz, 1H), 4.07 (m, 1H), 4.03 (m, 1H), 3.59 (ddm, 6.0, 15.5 Hz, 2H),
2.51-2.38 (m, 1H), 2.26 (m, 1H), 2.11 (m, 1H), 2.02 (m, 1H), 1.91 (m, 1H), 1.69 (m, 1H), 1.09 (m,
42H), 0.96 (s, 3H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 178.21, 167.86, 91.35, 77.27, 76.66, 67.33,
64.70, 55.00, 54.29, 45.20, 41.99, 30.97, 29.18, 26.29, 18.51, 18.46, 16.99, 16.97, 13.29, 13.23, 9.92
ppm. HR-MS (APCI: calculated for C35H66O7Si2[M+Na]+
: 677.42393. Found: 677.42385. Crystal
data for (-)-46a: Crystallized from EtOAc, C35H66O7Si2, Mrel = 655.07, T = 120 K, space group P21212
a = 11.0527(3) Å, b = 44.5049(19) Å, c = 7.8273(3) Å, α = 90.00, β = 90 γ = 90.00, V = 3850.24 Å3
.
CCDC ref. No. 1452234.
General procedure H: benzylation of camphanates (−)-46a and (+)-46b
TriBOT (0.8 eq.) and dried MS 5Å (30 mg/mL of 1,4-dioxane) were added into a solution of
camphanate (0.6 mmol) in 1,4-dioxane (3 mL). TfOH (0.4 eq.) was added dropwise and the reaction
mixture was stirred at 25 °C for 2 h. The reaction was quenched by addition of DIPEA (50 μL),
diluted with brine (15 mL) and extracted with EtOAc (3 × 20 mL). The organic extracts were dried
over MgSO4, filtered and concentrated. The residue was purified by flash column chromatography.
(1S,4S)-((1R,2R,3S,4S)-4-(benzyloxymethyl)-2,3-bis(triisopropylsilyloxy)cyclopentyl)methyl
4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylate ((+)-S-14).
Prepared by general procedure H using compound (+)-46b (0.302 g, 0.461 mmol); flash column
chromatography (hexane/EtOAc = 7: 1) afforded (+)-S14 as a colorless oil (0.274 g, 80 %). 1
H NMR
(500 MHz, CDCl3): δ = 7.34-7.26 (m, 5H), 4.48 (d, AB, J = 12.5 Hz, 2H), 4.35 (dd, J = 11.0, 5.1 Hz,
1H), 4.14-4.07 (m, 2H), 4.01 (d, J = 7.0, 3.6 Hz, 1H), 3.37 (d, J = 6.3 Hz, 2H), 2.47 (m, 1H), 2.40
348
(ddd, J = 13.4, 10.7, 4.2 Hz, 1H), 2.28 (m, 1H), 2.01-1.86 (m, 2H), 2.11 (m, 1H), 1.67 (ddd, J = 13.4,
9.3, 4.2 Hz, 1H), 1.19 (m, 2H), 1.06 (m, 42H), 0.94 (s, 3H) ppm. 13
C NMR (126 MHz, CDCl3): δ =
178.29, 167.85, 138.73, 128.47, 127.80, 127.63, 91.35, 76.38, 73.37, 72.22, 67.45, 54.99, 54.31,
43.51, 41.84, 30.86, 29.21, 27.10, 18.51, 18.49, 18.47, 18.44, 16.99, 16.92, 13.34, 13.13, 9.95 ppm.
IR (ν˜max) = 2942 (m), 2867 (m), 1794 (s), 1734 (m), 1463 (m), 1098 (s), 1060 (s), 882 (s) cm–1
. HRMS
(APCI) calculated for C42H72O7Si2 [M+H]+
: 745.4889. Found: 745.4892.
(1S,4S)-((1S,2S,3R,4R)-4-(benzyloxymethyl)-2,3-bis(triisopropylsilyloxy)cyclopentyl)methyl
4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylate ((−)-S-14).
Prepared by general procedure H using compound (−)-46a (0.321 g, 0.490 mmol); flash column
chromatography (hexane/EtOAc = 7: 1) afforded (−)-S14 as a colorless oil (0.339 g, 93 %). 1
H NMR
(500 MHz, CDCl3): δ = 7.35-7.25 (m, 5H), 4.47 (d, AB, J = 11.7 Hz, 2H), 4.37 (dd, J = 10.9, 5.0 Hz,
1H), 4.14-4.06 (m, 2H), 3.99 (d, J = 7.2, 3.4 Hz, 1H), 3.37 (m, 2H), 2.48 (m, 1H), 2.38 (m, 1H), 2.28
(m, 1H), 2.12 (m, 1H), 1.97 (m, 1H), 1.88 (m, 1H), 1.66 (m, 1H), 1.19 (m, 2H), 1.06 (m, 40H), 0.94
(s, 3H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 178.25, 167.78, 138.67, 128.49, 127.76, 127.68, 91.32,
77.18, 76.52, 73.37, 72.12, 67.61, 54.97, 54.22, 43.49, 41.92, 30.90, 29.21, 27.07, 18.51, 18.49,
18.47, 18.44, 16.99, 16.94, 13.32, 13.14, 9.93 ppm. IR (ν˜max): 2944 (m), 2862 (m), 1794 (s), 1734
(m), 1463 (m), 1098 (s), 1060 (s), 882 (s) cm–1
. HR-MS (APCI) calculated for C42H72O7Si2 [M+H]+
:
745.4889. Found: 745.4892.
(1R,2R,3S,4S)-4-(benzyloxymethyl)-2,3-bis(triisopropylsilyloxy)cyclopentyl)methanol
((+)-7d).
Sodium methoxide (0.088 g, 1.69 mmol) was added to a solution of (+)-S-14 (0.243 g, 0.326 mmol)
in MeOH (3 mL) and the mixture was stirred at 25 °C for 14 h. The solvent was removed in a
vacuum, the residue was diluted with saturated aqueous NH4Cl (10 mL) and the mixture was
349
extracted with EtOAc (4 × 10 mL). The organic extracts were dried over MgSO4, filtered, and the
solvent was evaporated. The crude mixture was purified by flash column chromatography
(hexane/EtOAc = 15:1) to afford product (+)-7 as colorless oil (0.165 g, 90%). The spectral data were
identical to those of the racemic compound 7d. 𝛼 𝐷
𝑅𝑇
= +3.3 (c = 1, CHCl3).
(1S,2S,3R,4R)-4-(benzyloxymethyl)-2,3-bis(triisopropylsilyloxy)cyclopentyl)methanol
((−)-7d).
0.270 g (0.362 mmol) of (−)-S-14, using the procedure for compound (+)-7d, afforded (−)-7d as a
colorless oil (0.184 g, 90%). The spectral data were identical to those of the racemic compound 7d.
𝛼 𝐷
𝑅𝑇
= −3.4 (c = 1, CHCl3).
(2R,3R,4R)-4-((benzyloxy)methyl)-2,3-bis((triisopropylsilyl)oxy)cyclopentanone ((–)-2c).
Prepared from (−)-7d by the procedure used for racemic 2c. 𝛼 𝐷
𝑅𝑇
= −21.5 (c = 1, CHCl3).
(2S,3S,4S)-4-((benzyloxy)methyl)-2,3-bis((triisopropylsilyl)oxy)cyclopentanone ((+)-2c).
Prepared from (+)-7d by the procedure used for racemic 2c. 𝛼 𝐷
𝑅𝑇
= +21.2 (c = 1, CHCl3).
(1R,2R,3R)-4-oxo-2,3-bis(triisopropylsilyloxy)cyclopentyl)methyl pivalate ((−)-2b).
Pd(OH)2/C (0.017 g, 0.12 mmol) was added to a degassed solution of (−)-2c (0.220 g, 0.4 mmol) in
THF (8 mL). The reaction mixture was stirred in a hydrogenation apparatus at 65 °C under H2
atmosphere (50 bar) for 24 h. The reaction mixture was cooled to 25 C and filtered through pad of
350
Celite, which was washed with additional THF (3 × 10 mL). The filtrate was concentrated in a
vacuum and the residue was dissolved in CH2Cl2 (8 mL). DIPEA (167 μL, 0.96 mmol) and Piv-Cl (98
μL, 0.8 mmol) were added into the reaction mixture and the mixture was stirred at 25 °C for 14 h. The
solvent was removed in a vacuum and the residue was purified by flash column chromatography
(hexane/EtOAc = 30:1) to afford (−)-2b as a colorless oil (0.173 g, 80%). The spectral data were
identical to those of racemic 2b. 𝛼 𝐷
𝑅𝑇
= −16 (c = 1, CHCl3).
(1S,2S,3S)-4-oxo-2,3-bis(triisopropylsilyloxy)cyclopentyl)methyl pivalate ((+)-2b).
Prepared from (+)-2c by the procedure used for (–)2b. 𝛼 𝐷
𝑅𝑇
= +18 (c = 1, CHCl3).
((1R,4S,5R)-3-(2,4-difluorophenyl)-4,5-dihydroxycyclopent-2-enyl)methyl pivalate ((+)-26b.)
Prepared from (−)-2b by the procedure used for racemic 26b. 𝛼 𝐷
𝑅𝑇
= +63.5 (c = 0.5, CHCl3).
((1S,4R,5S)-3-(2,4-difluorophenyl)-4,5-dihydroxycyclopent-2-enyl)methyl pivalate ((−)-26b.)
Prepared from (+)-2b by the procedure used for racemic 26b. 𝛼 𝐷
𝑅𝑇
= −63.0 (c = 0.5, CHCl3).
(1R,2S,5R)-3-(2,4-difluorophenyl)-5-(hydroxymethyl)cyclopent-3-ene-1,2-diol ((+)-27b).
Prepared from (+)-26b by the procedure used for racemic 27b. 𝛼 𝐷
𝑅𝑇
= + 62.5 (c = 1, MeOH).
351
References and notes
1. a) Modified Nucleosides in Biochemistry, Biotechnology and Medicine; Ed.: P. Herdewijn, WileyVCH,
2008. b) Jordheim, L. P., Durantel, D., Zoulim, F., Dumontet, Ch. Nat. Rev. Drug. Discov.
2013, 447. (c) Chemical Synthesis of Nucleoside Analogues; Ed.: P. Merino, Wiley, 2013.
2. a) Kawaguchi, T.; Fukushima, S.; Ohmura, M.; Mishima, M.; Nakano, M. Chem. Pharm. Bull.
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353
Part 5
Synthesis of carbocyclic analogs of dehydroaltenusin: identification of a stable
inhibitor of calf DNA polymerase *
*published as:
Kováčová, S.; Adla, S. K.; Maier, L.; Babiak, M.; Mizushina, Y.; Paruch, K.*
Synthesis of Carbocyclic Analogs
of Dehydroaltenusin: Identification of a Stable Inhibitor of Calf DNA Polymerase . Tetrahedron 2015, 71,
7575.
1. Introduction
Proteins and pathways involved in DNA damage/repair continue to be intensely studied, especially in
the context of development of new cancer therapeutic agents.1
The inhibition of DNA polymerases
(pols) seems to be a proper component of synthetic lethal combination therapy.2
Specifically, deletion
of pol  was found to be synthetic lethal with elimination or inhibition of CHK1 kinase, but
elimination of additional pols (delta and epsilon) resulted in significantly weaker synthetic lethal
response.3
Selective inhibitors of pol  could therefore be of considerable therapeutic potential. Of
the compounds that are known to inhibit this enzyme,1
only two seem to be sufficiently selective:
nucleoside analog BuPdGTP and its derivatives,4
and dehydroaltenusin.5
Both substances possess
suboptimal pharmacological properties: BuPdGTP exhibits only weak activity in the cell,6
while
chemical stability of dehydroaltenusin is limited, as it undergoes a rearrangement in aqueous
solutions to give a mixture of the spirocyclic and non-spirocyclic forms (structures DHAs and DHA
in Scheme 1; absolute configurations are not known).7
It is not clear which of these forms is active
and it has been suggested that the rearrangement o-quinone intermediate might be also responsible for
the inhibitory activity.8
In order to help elucidate this issue, we decided to carry out bioisosteric
replacement of the lactone ring oxygen by methylene group and prepare the carbocyclic analogs of
the spirocyclic and non-spirocyclic forms of dehydroaltenusin - cDHAs and cDHA, respectively
(Scheme 1). These compounds were envisioned to be significantly more stable and resistant to the
rearrangement.
354
Scheme 1. Spirocyclic and non-spirocyclic isomers of dehydroaltenusin and their carbocyclic analogs.
Of note, the central scaffolds of both cDHA and cDHAs are only sporadically documented in the
literature.
2. Results and Discussion
The retrosynthetic analysis of cDHA, depicted in Scheme 2, relies on benzylic oxidation and
alkylation of protected tetralone 3 to provide intermediate 2 possessing the required quaternary
center, aldol cyclization forming the tricyclic scaffold 1, and final oxidation and deprotection to yield
the target compound (cDHA).
Scheme 2. Retrosynthetic analysis of cDHA. Construction of the quaternary center and the tricyclic scaffold.
355
Starting from 3,5-dimethoxybenzaldehyde, Wittig reaction followed by hydrogenation afforded acid
4, whose intramolecular Friedel-Crafts cyclization gave the required tetralone (not shown).9
Attempted protections of the tetralone using ethanediol, 2-methoxy-1,3-dioxolane, ethanediol plus
CH(OCH3)3, or 2-methoxy-1,3-dioxolane in the presence of TsOH did not provide the desired product
3 in acceptable yields, likely due to the sensitivity of the substance to strong acids. However, reaction
with ethanediol in the presence of PPTS in refluxing benzene afforded 3 in satisfactory yield (Scheme
3).
Benzylic oxidation of 3 using Cr(CO)6 or PDC with tert-butylhydroperoxide10
afforded the desired
ketone 5 in modest yields (10% and 35%, respectively). Best result (55%) was obtained with
dirhodium caprolactamate plus tert-butylhydroperoxide in acetonitrile, buffered with NaHCO3.11
Methylation (t-BuOK, MeI) of 5 led to a mixture of mono- and di-methylated ketones, along with the
unreacted starting material. Reasonable yield (55%) of the desired mono-methylated product was
obtained when the alkylation was carried out in the presence of DMPU. Subsequent reaction with
methyl vinyl ketone (MVK) proved difficult and out of many conditions tried, only the method by
von Doering12
afforded the desired diketone 2 in acceptable (61%) yield.
Intramolecular aldol condensation of 2 using t-BuOH/t-BuOK provided tricyclic ketone 1.
Subsequent oxidation with oxygen under basic conditions, followed by acidic cleavage of the ketal
provided compound 6 in good overall yield (Scheme 3).
Finally, selective demethylation of 6 using BBr3 gave the desired target compound cDHA, whose
structure was confirmed by X-ray crystallography (Scheme 3).
356
Scheme 3. Synthesis and structural assignment of cDHA.
The retrosynthetic analysis of cDHAs (depicted in Scheme 4) relies on double alkylation of ketone 9,
regioselective ring closure of 8 leading to the spirocyclic intermediate 7, and final oxidation and
selective demethylation to afford cDHAs.
357
Scheme 4. Retrosynthetic analysis of cDHAs. Construction of the spirocyclic core.
Ketone 9 was prepared in two steps by condensation of 3,5-dimethoxybenzaldehyde with nitroethane,
followed by reduction with iron in AcOH.13
Because our subsequent attempts to add MVK to 9 failed
under a variety of conditions and in most cases lead only to the recovery of the starting material, we
attempted alkylations with MVK equivalents. Best results were obtained with iodide 11, which we
prepared in 80% yield by modification of the procedure published for the analogous bromide.14
This
way we obtained intermediate 10 that underwent second alkylation with t-butyl-2-iodoacetate.15
The
resulting ester 12 was then deprotected to give diketone 8, which was used for the subsequent
intramolecular aldol closure (Scheme 5).
358
Scheme 5. Construction of the spirocyclic scaffold.
The regioselectivity of the aldol closure proved very dependent on the reaction conditions.
Condensation using piperidine/AcOH in toluene,16
which may proceed via enamine formation at the
less sterically hindered carbonyl, afforded the desired enone 13a (81%). In contrast, when carried out
under basic conditions (t-BuOK/THF), the condensation yielded exclusively the isomer 13b (Scheme
5). Construction of the spirocyclic scaffold was completed by intramolecular Friedel-Crafts reaction
of 13a in the presence of MsOH, which provided the spirocyclic enone 7 in good yield (Scheme 5).
In contrast to the non-spirocyclic series, we were not able to oxidize enone 7 directly. We thus
attempted to convert it into a silyl enol ether that could be used for subsequent Rubottom oxidation.
In accordance with the literature,17
regioselective formation of the desired silyl enol ether proved non-
359
trivial: numerous attempts to carry out mono-silylation of 7 yielded only mixtures of the desired
product, often along with its exocyclic isomer, bis-silyl enol ether 14 and unreacted 7. However, with
excess of TBSOTf the bis-silylation of 7 proceeded cleanly and the subsequent epoxidation occurred
predominantly at the six membered ring (Scheme 6). The resulting hydroxyketone 15 (as ca. 5:1
mixture of diastereomers) was oxidized to 16. However, all our attempts to cleave the phenolic
methyl ether in the vicinity of indanone carbonyl (e.g., using BBr3 or BCl3) failed and yielded
complex mixtures that contained only traces of the desired product cDHAs.
Scheme 6. Attempted conversion of enone 7 into cDHAs.
Fortunately, selective demethylation was possible at the stage of spirocyclic enone 7, which enabled
us to introduce an alternative protecting group that could be cleaved more easily at the end of the
synthesis (Scheme 7). Along this line we decided to prepare pivaloyl ester 17. Interestingly (and in
contrast to 7), ester 17 underwent clean mono-silylation to afford enol ether 18, which was then
oxidized to hydroxyketone 19. Exposure of methanolic solution of 19 to air and K2CO3, which
effected the desired oxidation and cleavage of the pivaloate, afforded the target compound cDHAs,
whose structure was again unambiguously confirmed by X-ray crystallography (Scheme 7).
360
Scheme 7. Completion of synthesis of cDHAs.
In the biochemical assay we developed previously,18
we tested both cDHA and cDHAs against the
following mammalian pols: calf pol  and human pols , , and , which belong to the B-, A-, Y-,
and X-family of pols, respectively. As shown in Table 1, cDHA specifically inhibits pol . In
contrast, cDHAs inhibits none of the tested pols.
Table 1. IC50 values of cDHA and cDHAs for mammalian DNA polymerases.a
polymerase IC50 value (M)
cDHA cDHAs
calf pol  5.5 ± 0.29 > 100
human pol  > 100 > 100
human pol  > 100 > 100
human pol  > 100 > 100
a
Data are shown as the means +/- SE of three independent experiments. Description of the assay is given in
Experimental Section.
361
3. Conclusions
In conclusion, we prepared racemic novel carbocyclic analogs of dehydroaltenusin cDHA and
cDHAs in 11 and 13 steps, respectively, starting from 3,5-dimethoxybenzyldehyde. While cDHA is
approximately eight times weaker inhibitor than dehydroaltenusin itself (previously reported IC50
value for dehydroaltenusin against calf pol  is 0.68 M),5
it is stable and can be stored in the solid
state as well as in aqueous DMSO solution for at least four weeks without noticeable decomposition.
In addition, the enantiomers of cDHA can be efficiently separated by HPLC on chiral stationary
phase (details given in Supporting Information). Bioisosteric replacement of oxygen atom by the
methylene group is commonly used in medicinal chemistry;19
however, in the context of this project,
it enabled not only identification of more stable analogs, but also provided significant (albeit indirect)
information on which of the dehydroaltenusin isomers is responsible for its biological activity. Our
observation of the dramatic difference in activities of cDHA and cDHAs strongly suggests that
dehydroaltenusin’s inhibitory activity toward pols is due to its non-spirocyclic form DHA and while
the presence reactive o-quinone intermediate may contribute to dehydroaltenusin’s inhibitory
activity, it may not be absolutely required.
We believe the methodology presented above will enable preparation of additional analogs possessing
the rare and non-trivial tricyclic central motifs. In addition, our results could be used to guide further
development of potent and selective inhibitors of mammalian polymerases, namely pol , exploiting
the recently published crystal structure.20
4. Experimental Section
General
All reagents and solvents were of reagent grade and used without further purification. Anhydrous
solvents (THF, dichloromethane, CH3CN) were purchased from commercial suppliers (Aldrich,
Acros) and stored over 4Å molecular sieves. All reactions were carried out in oven-dried glassware
and under N2 atmosphere unless stated otherwise. Column chromatography was carried on silica gel
(230-400 mesh). TLC plates were visualized under UV and stained with phosphomolybdic acid or
KMnO4 solution or Vaughn's reagent ((NH4)6Mo7O24•4H2O/Ce(SO4)2•4H2O/conc. H2SO4/H2O).
NMR spectra were recorded on Bruker Avance 300 and 500 MHz spectrometers, with operating
frequencies 300.13, 500.13 MHz for 1
H and 75.48, 125.77 MHz for 13
C. The 1
H and 13
C NMR
chemical shifts (δ in ppm) were referenced to the residual signals of solvents: CDCl3 [7.24 (1
H) and
362
77.23 (13
C)]. Structural assignments of resonances have been performed with the help of 2D NMR
gradients experiments (HSQC, HMBC, NOESY).
High resolution mass spectra were measured on Agilent 6224 Accurate-Mass TOF LC-MS with dual
electrospray/chemical ionization mode with mass accuracy greater than 2 ppm, applied mass range
was from 25 to 20,000 Da.
IR spectra (4000-400 cm-1
) were collected on an EQUINOX 55/S/NIR FTIR spectrometer. Samples
were prepared as KBr pellets.
The diffraction data for sample cDHA (CCDC number 1012667) were collected with a KUMA KM-4
κ-axis diffractometer equipped with a Sapphire2 CCD detector and a Cryostream Cooler (Oxford
Cryosystems, UK). Mo Kα radiation (λ= 0.71073 Å, fine-focus sealed tube, graphite monochromator)
was used.
The diffraction data for sample cDHAs (CCDC number 1012668) were collected with a Rigaku
partial χ geometry diffractometer equipped with a Saturn 944+ HG CCD detector and a Cryostream
Cooler (Oxford Cryosystems, UK). Cu Kα radiation (λ= 1.54184 Å, MicroMax-007HF rotating anode
source, multilayer optic VariMax) was used. Data reduction and final cell refinement were carried out
using the CrysAlisPro software ([
CrysAlisPro] CrysAlisPRO, Agilent Technologies UK Ltd).
4.1. 4-(3,5-dimethoxyphenyl)but-3-enoic acid
To NaH (60% suspension in mineral oil, 1.90 g, 49.7 mmol) was added dry DMSO (36 mL) and the
mixture was stirred and heated at 70 °C. After the evolution of bubbles ceased, the mixture was
cooled to 10 °C. To this mixture, a solution of (2-carboethoxy)triphenylphosphonium bromide (11.00
g, 26.5 mmol) in DMSO (26 mL) was added dropwise. To the resulting dark red reaction mixture, a
solution of 3,5-dimethoxybenzaldehyde (2.0 g, 12.0 mmol) in DMSO (5 mL) was added. The reaction
mixture was stirred at 50 °C for 16 h, then cooled, poured into ice–water (200 mL), acidified with
conc. HCl to pH<4, and extracted with EtOAc (4 x 50 mL). The combined organic layers were
washed with H2O (2 x 50 mL), dried over Na2SO4 and concentrated. The product was purified by
column chromatography (silica gel, EtOAc/hexane (1:1)) to obtain the title 4-(3,5dimethoxyphenyl)but-3-enoic
acid as a yellow solid (1.80 g, 66%). mp 58-60 °C; 1
H NMR (300
MHz, CDCl3) δ = 6.55 (d, J = 2.3 Hz, 2H), 6.47 (d, J = 15.9 Hz, 1H), 6.39 (t, J =2.3 Hz, 1H), 6.28 (dt,
J = 15.9 Hz, J = 7.2 Hz, 1H), 3.80 (s, 6H), 3.30 (d, J = 7.2 Hz, 2H).1
H NMR spectrum was identical
with the published literature.21
363
4.2. 4-(3,5-dimethoxyphenyl)butanoic acid (4)
A solution of 4-(3,5-dimethoxyphenyl)but-3-enoic acid (2.00 g, 9.0 mmol) in EtOH (24 mL) was
added to Pd/C (10% Pd, 478 mg) and ammonium formate (1.70 g, 27.0 mmol). The reaction mixture
was stirred at 60 °C for 14 h, then cooled, filtered through a pad of Celite, and concentrated under
reduced pressure. The residue was diluted with H2O (100 mL), acidified with conc. HCl to pH=4, and
extracted with EtOAc (3 x 50 mL). The combined organic extracts were dried over Na2SO4 and
concentrated. The residue was purified by column chromatography (silica gel, EtOAc/hexane (1:1))
to afford 4 as a white solid (1.44 g, 71%). mp 63-65 °C; 1
H NMR (300 MHz, CDCl3) δ = 6.35 (d, J =
2.0 Hz, 2H), 6.32 (dd, J = 2.0 Hz, 1H), 3.78 (s, 6H), 2.62 (t, J = 7.6 Hz, 2H), 2.38 (t, J = 7.4 Hz, 2H),
2.02 – 1.90 (m, 2H).
4.3. 6,8-dimethoxy-3,4-dihydronaphthalen-1(2H)-one
A mixture of 4 (1.44 g, 6.42 mmol) and polyphosphoric acid (14 mL) was stirred at 90 °C for 4 h,
then it was poured into ice–water (250 mL) and extracted with EtOAc (3 x 100 mL). The combined
organic extracts were washed with saturated aqueous solution of NaHCO3 (2 x 50 mL) and dried over
Na2SO4. Removal of the solvent under reduced pressure afforded the product as a yellow solid (1.24
g, 93%). mp 62-63 °C; 1
H NMR (300 MHz, CDCl3) δ = 6.35 – 6.30 (m, 3H), 3.87 (s, 2H), 3.84 (s,
3H), 2.87 (t, J = 6.0 Hz, 2H), 2.57 (t, J = 6.5 Hz, 2H), 2.07 – 1.96 (m, 2H). 1
H NMR spectrum was
identical with the published literature.22
4.4. 6',8'-dimethoxy-3',4'-dihydro-2'H-spiro[[1,3]dioxolane-2,1'-naphthalene] (3)
A mixture of 6,8-dimethoxy-3,4-dihydronaphthalen-1(2H)-one (206 mg, 1.00 mmol), pyridinium ptoluenesulfonate
(25 mg, 0.01 mmol), and ethylene glycol (170 μl, 3.00 mmol) in benzene (25 mL)
was heated at reflux in a flask equipped with a Dean-Stark apparatus for 14 h. The mixture was
cooled to 25 °C, K2CO3 (100 mg) was added, and the solvent was evaporated. The residue was
purified by column chromatography (silica gel, EtOAc/hexane/Et3N (100:50:1)) to afford 3 as a white
solid (200 mg, 80%), which was used immediately in the next step. mp 85 °C (dec.); 1
H NMR (500
MHz, CDCl3) δ = 6.33 (d, J = 2.4 Hz, 1H), 6.21 (d, J = 2.4 Hz, 1H), 4.25 – 4.18 (m, 2H), 4.08 – 4.01
(m, 2H), 3.80 (s, 3H), 3.76 (s, 3H), 2.75 – 2.70 (m, 2H), 1.96 – 1.91 (m, 2H) 1.86 – 1.79 (m, 2H); 13
C
NMR (126 MHz, CDCl3) δ = 160.2, 160.0, 142.3, 117.8, 108.1, 104.3, 98.0, 65.3 (2C), 55.8, 55.1,
36.8, 31.1, 21.1; HRMS (APCI): calcd. for C14H19O4 [M+H]+
: 251.1283, found: 251.1348.
4.5. 6',8'-dimethoxy-2'H-spiro[[1,3]dioxolane-2,1'-naphthalen]-4'(3'H)-one (5)
364
Rh2(cap)4 (9 mg, 0.01 mol) and t-BuOOH (5.5 M in decane, 900 μL, 4.95 mmol) were added to a
mixture of 3 (250 mg, 1.00 mmol) and NaHCO3 (42 mg, 0.50 mmol) in CH3CN (2 mL). The reaction
mixture was stirred at 25 °C for 16 h, then concentrated, and purified by column chromatography
(silica gel, EtOAc/hexane (1:2)) to yield 5 as a white solid (145 mg, 55%). mp 81 °C (dec.); 1
H NMR
(300 MHz, CDCl3) δ = 7.11 (d, J = 2.5 Hz, 1H), 6.71 (d, J = 2.5 Hz, 1H), 4.31 – 4.23 (m, 2H), 4.13 –
4.05 (m, 2H), 3.86 (s, 3H), 3.84 (s, 3H), 2.79 (m, 2H), 2.28 (m, 2H); 13
C NMR (75 MHz, CDCl3) δ =
197.3, 161.0, 159.1, 135.2, 123.4, 106.8, 106.0, 100.9, 65.8 (2C), 56.3, 55.6, 36.2, 34.4; IR (KBr): ~
= 2962 cm-1
(m), 2939 (w), 2887 (w), 1685 (s), 1603 (s), 1466 (m), 1430 (w), 1354 (s), 1321 (s), 1300
(s), 1217 (s), 1163 (s), 1114 (s), 1040 (s), 980 (w), 916 (m), 842 (m); HRMS (APCI): calcd. for
C14H17O5 [M+H]+
: 265.1071, found: 265.1068.
4.6. 6',8'-dimethoxy-3'-methyl-2'H-spiro[[1,3]dioxolane-2,1'-naphthalen]-4'(3'H)-one:
A solution of compound 5 (100 mg, 0.38 mmol) and 1,3-dimethyltetrahydropyrimidin-2(1H)-one
(330 μL) in THF (1 mL) was cooled to -78 °C, t-BuOK (46.8 mg, 0.42 mmol) was added, and the
mixture was stirred at -78 °C for 45 min. Iodomethane (30 μL, 0.48 mmol) was added, the mixture
was allowed to warm to 25 °C and stirred for 16 h, then diluted with H2O (50 mL), and extracted with
CH2Cl2 (3 x 20 mL). The combined organic extracts were dried over Na2SO4 and concentrated. The
residue was purified by column chromatography (silica gel, EtOAc/hexane (1:2)) to yield 6',8'dimethoxy-3'-methyl-2'H-spiro[[1,3]dioxolane-2,1'-naphthalen]-4'(3'H)-one
as a white solid (58 mg,
55%). mp 99 °C (dec.); 1
H NMR (500 MHz, CDCl3) δ = 7.11 (d, J = 2.6 Hz, 1H), 6.70 (d, J = 2.6 Hz,
1H), 4.33 – 4.24 (m, 2H), 4.17 – 4.13 (m, 1H), 4.11 – 4.06 (m, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.00 –
2.92 (m, 1H) 2.25 (m, 1H), 2.12 (m, 1H), 1.22 (d, J = 6.7 Hz, 3H); 13
C NMR (126 MHz, CDCl3) δ =
199.6, 161.0, 159.1, 135.4, 123.0, 106.7, 105.5, 100.9, 65.9, 65.8, 56.1, 55.5, 42.7, 39.9, 14.6; IR
(KBr): ~ = 2987 cm-1
(w), 2968 (m), 2893 (m), 2860 (m), 2839 (w), 1680 (s), 1603 (s), 1470 (m) ,
1456 (s), 1365 (m), 1323 (s), 1300 (s), 1229 (s) , 1207 (s), 1157 (m), 1115 (s), 1082 (m), 1041 (m),
964 (m), 949 (w), 941 (w), 839 (s); HRMS (APCI): calcd. for C15H19O5 [M+H]+
: 279.1227, found:
279.1227.
4.7. 6',8'-dimethoxy-3'-methyl-3'-(3-oxobutyl)-2'H-spiro[[1,3]dioxolane-2,1'-naphthalen]-
4'(3'H)-one (2)
A stream of nitrogen saturated by MVK was bubbled through a solution of 6',8'-dimethoxy-3'-methyl-
2'H-spiro[[1,3]dioxolane-2,1'-naphthalen]-4'(3'H)-one (40 mg, 0.14 mmol) and DBU (22 μL, 0.14
mmol) in toluene (1 mL). Upon completion, indicated by TLC (silica gel, EtOAc/hexane (1:2)), the
solvent was evaporated. The crude residue was purified by column chromatography (silica gel,
365
EtOAc/hexane (1:2)) to afford 2 as a colorless wax (29.7 mg, 61%). 1
H NMR (300 MHz, CDCl3) δ =
7.10 (d, J = 2.5 Hz, 1H), 6.71 (d, J = 2.5 Hz, 1H), 4.33 – 4.20 (m, 2H), 4.16 – 4.05 (m, 2H), 3.84 (s,
6H), 2.57 – 2.44 (m, 1H), 2.39 – 2.27 (m, 1H), 2.22 (dAB, J = 14.4 Hz, 1H), 2.12 (s, 3H), 2.10 (dAB J =
14.4 Hz, 1H) 2.06 – 1.85 (m, 2H), 1.20 (s, 3H); 13
C NMR (75 MHz, CDCl3) δ = 208.1, 201.5, 161.2,
159.1, 133.9, 122.4, 105.8, 105.6, 101.3, 65.64, 65.56, 56.1, 55.5, 44.9, 44.5, 38.6, 31.3, 29.8, 23.1;
IR (KBr): ~ = 2964 cm-1
(w), 2937 (w), 2899 (w), 1713 (s), 1687 (s), 1601 (s), 1464 (m), 1356 (m),
1323 (s), 1296 (s), 1221 (m), 1159 (s), 1115 (m), 1090 (m), 1045 (m), 1011 (w), 948 (w), 848 (w);
HRMS (APCI): calcd. for C19H25O6 [M+H]+
: 349.1646, found: 349.1647.
4.8. 6',8'-dimethoxy-10a'-methyl-10',10a'-dihydro-1'H-spiro[[1,3]dioxolane-2,9'-phenanthren]-
3'(2'H)-one (1)
t-BuOK (57.9 mg, 0.52 mmol) was added to a solution of 2 (150 mg, 0.43 mmol) in t-BuOH (2 mL),
the reaction mixture was stirred at 25 °C for 3 h. H2O (5 mL) was added and the mixture was
extracted with EtOAc (3 x 10 mL). The combined organic extracts were dried over Na2SO4, filtered,
and concentrated in a vacuum to afford 1 as a white solid (110 mg, 77%), which was used in the next
step without further purification. mp 126 °C (dec.); 1
H NMR (300 MHz, CDCl3) δ = 6.70 (d, J = 2.3
Hz, 1H), 6.60 (d, J = 2.3 Hz, 1H), 6.47 (br. s, 1H), 4.37 – 4.27 (m, 1H), 4.23 – 4.04 (m, 3H), 3.86 (s,
3H), 3.82 (s, 3H), 2.70 – 2.54 (m, 1H), 2.48 – 1.39 (m, 1H), 2.14 – 1.85 (m, 4H), 1.30 (s, 3H); 13
C
NMR (75 MHz, CDCl3) δ = 199.6, 162.2, 160.9, 160.0, 135.6, 121.9, 118.7, 106.3, 102.9, 101.3,
65.8, 65.2, 56.0, 55.4, 49.3, 36.8, 35.6, 33.2, 21.7; IR (KBr): ~ = 2950 cm-1
(m), 2922 (w), 2890 (w),
1660 (s), 1603 (s), 1579 (s), 1466 (s), 1425 (m), 1356 (s), 1327 (s), 1296 (s), 1275 (s), 1207 (s), 1157
(s), 1134 (s), 1101 (s), 1147 (s), 1020 (m), 989 (m), 950 (m), 849 (w), 837 (m), 821 (w); HRMS
(APCI): calcd. for C19H23O5 [M+H]+
: 331.1540, found: 331.1543.
4.9. 2-hydroxy-6,8-dimethoxy-10a-methyl-10,10a-dihydrophenanthrene-3,9-dione (6)
t-BuOK (110 mg, 0.30 mmol) was added to a solution of 1 (186 mg, 0.33 mmol) in t-BuOH (2 mL)
and THF (2 mL) and the mixture was stirred under O2 atmosphere at 25 °C for 4 h. The reaction
mixture was diluted with saturated aqueous solution of NH4Cl (30 mL) and extracted with CH2Cl2 (3
x 20 mL). The combined organic phases were dried over Na2SO4, filtered, and the solvent was
evaporated. The residue was dissolved in acetone (5 mL), TsOH (10 mg, 0.05 mmol) was added, and
the mixture was stirred at 25 °C for 16 h. The solvent was removed under reduced pressure and the
residue was purified by column chromatography (silica gel, EtOAc/CH2Cl2 (1:1)) to afford 6 (71 mg,
72%) as a white solid. mp 218-220 °C; 1
H NMR (300 MHz, CDCl3) δ = 6.84 (s, 1H), 6.73 (d, J = 2.3
Hz, 1H), 6.61 (d, J = 2.3 Hz, 1H), 6.37 (br. s, 1H), 6.13 (s, 1H), 3.95 (s, 3H), 3.94 (s, 3H), 2.83 (dAB, J
366
= 16.1 Hz, 1H), 2.57 (dAB, J = 16.1 Hz, 1H), 1.34 (s, 3H); 13
C NMR (126 MHz, CDCl3) δ = 192.0,
181.7, 164.8, 162.6, 162.3, 146.5, 141.8, 123.8, 122.3, 114.0, 102.5, 101.1, 56.3, 55.7, 51.0, 42.0,
25.8; IR (KBr): ~ = 3234 cm-1
(w), 3018 (w), 2972 (w), 2931 (w), 1657 (s), 1606 (s), 1589 (s), 1558
(s), 1477 (m), 1454 (m), 1407 (m), 1377 (w), 1342 (s), 1306 (w), 1265 (s), 1245 (s), 1215 (s), 1207
(s), 1165 (s), 1115 (s), 1026 (w), 999 (m), 957 (w), 895 (w), 850 (w), 839 (w); HRMS (APCI): calcd.
for C17H17O5 [M+H]+
: 301.1071, found: 301.1067.
4.10. 2,8-dihydroxy-6-methoxy-10a-methyl-10,10a-dihydrophenanthrene-3,9-dione (cDHA)
BBr3 (1 M in CH2Cl2, 0.2 mL, 0.2 mmol) was added dropwise to a solution of 6 (30 mg, 0.1 mmol) in
anhydrous CH2Cl2 (3 mL) at -78 °C. The reaction mixture was stirred at -78 °C for 1 h, then allowed
to warm to 0 °C, and quenched with aqueous 10% NaOH (1 mL). The mixture was acidified with 1 M
HCl to pH=6 and extracted with CH2Cl2 (2 x 10 mL). The combined organic extracts were dried over
Na2SO4, filtered, and concentrated. The residue was purified by preparative TLC (CH2Cl2/EtOAc
(1:1)) to afford cDHA (15 mg, 52%) as a pale yellow solid. mp 199 °C (dec.); 1
H NMR (300 MHz,
CDCl3) δ = 12.61 (s, 1H), 6.84 (s, 1H), 6.72 (d, J = 2.3 Hz, 1H), 6.54 (d, J = 2.3 Hz, 1H), 6.38 (s,
1H), 6.13 (s, 1H), 3.90 (s, 3H), 2.85 (dAB, J = 17.0 Hz, 1H), 2.67 (dAB, J = 17.0 Hz, 1H), 1.35 (s, 3H);
13
C NMR (126 MHz, CDCl3) δ = 199.1, 181.5, 166.5, 165.5, 160.4, 146.6, 139.6, 123.0, 122.3, 109.1,
105.5, 102.8, 55.9, 48.8, 41.6, 26.3; IR (KBr): ~ = 3375 cm-1
(m), 2964 (w), 2926 (w), 1632 (s), 1564
(w), 1479 (w), 1454 (w), 1429 (m), 1389 (m), 1365 (m), 1343 (w), 1292 (s), 1238 (m), 1203 (s), 1159
(s), 1126 (w), 1101 (w), 1067 (w), 978 (w), 903 (w), 879 (w); HRMS (APCI): calcd. for C16H15O5
[M+H]+
: 287.0914, found: 287.0912.
Crystal data for cDHA: CCDC ref. No. 1012667. Crystallized from CHCl3.
4.11. 1,3-dimethoxy-5-(2-nitroprop-1-en-1-yl)benzene
A mixture of 3,5-dimethoxybenzaldehyde (10.0 g, 60.18 mmol), nitroethane (45 mL, 630.68 mmol)
and NH4OAc (3.36 g, 43.57 mmol) was stirred and heated at reflux for 4 h (oil bath 130 °C). The
reaction mixture was cooled to 25 °C, diluted with Et2O (200 mL), and washed with brine (4 x 150
mL). The organic layer was dried over Na2SO4, filtered, and the solvent was evaporated. 1,3Dimethoxy-5-(2-nitroprop-1-en-1-yl)benzene
(13.04 g, 97%) was obtained as a yellow solid. mp 86-
87 °C; 1
H NMR (300 MHz, CDCl3) δ = 7.99 (br. s, 1H), 6.54 (d, J = 2.2 Hz, 2H), 6.51 (d, J = 2.2 Hz,
1H), 3.81 (s, 6H), 2.43 (d, J = 0.9 Hz, 3H).
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4.12. 1-(3,5-dimethoxyphenyl)propan-2-one (9)
Fe powder (1.60 g, 28.7 mmol) was added to a solution of 1,3-dimethoxy-5-(2-nitroprop-1-en-1yl)benzene
(493 mg, 2.21 mmol) in AcOH (13.9 mL, 221.0 mmol) and the mixture was heated at
reflux for 3 h. The mixture was cooled to 25 °C, filtered, diluted with water (70 mL), and extracted
with CH2Cl2 (3 x 30 mL). Combined organic extracts were washed with aqueous 5% NaOH (2 x 30
mL), brine (2 x 30 mL), dried over Na2SO4, and concentrated. The crude residue was purified by
column chromatography (silica gel, EtOAc/hexane (2:1)) to provide 9 (303 mg, 71%) as a colorless
oil. 1
H NMR (300 MHz, CDCl3) δ = 6.37 – 6.31 (m, 3H), 3.74 (s, 6H), 3.57 (s, 2H), 2.11 (s, 3H).
4.13. 2-(2-iodoethyl)-2-methyl-1,3-dioxolane (11)
Iodotrimethysilane (1.7 mL, 12 mmol) was added dropwise at 0 °C to a mixture of methyl vinyl
ketone (90%, 833 μL, 10 mmol) and ethylene glycol (1.24 mL, 22 mmol). The reaction mixture was
stirred for 2 h while allowed to warm to 25 °C, then it was diluted with pentane (20 mL) and washed
successively with 5% aqueous Na2CO3 (10 mL) and 5% aqueous Na2S2O3 (10 mL). The organic layer
was dried over K2CO3 and concentrated under reduced pressure to provide 11 (1.93 g, 80%) as a pale
yellow oil, which was used directly in the next step without further purification. 1
H NMR (300 MHz,
CDCl3) δ = 4.01 – 3.87 (m, 4H), 3.21 – 3.12 (m, 2H), 2.35 – 2.24 (m, 2H), 1.30 (s, 3H).
4.14. 3-(3,5-dimethoxyphenyl)-5-(2-methyl-1,3-dioxolan-2-yl)pentan-2-one (10)
t-BuOK (677 mg, 6.04 mmol) was added to a solution of 9 (1.02 g, 5.24 mmol) in anhydrous THF (12
mL) and the reaction mixture was stirred at 25 °C for 1 h. 11 (1.7 g, 7.02 mmol) was added dropwise
over the period of 5 min and the reaction mixture was stirred at 25 °C for 16 h. Saturated aqueous
solution of NaHCO3 (1 mL) was added, the solvents were evaporated and the residue was directly
loaded onto a column and purified by column chromatography (silica gel, EtOAc/hexane (1:2)). 10
(1.37 g, 85%) was obtained as a pale yellow oil. 1
H NMR (300 MHz, CDCl3) δ = 6.35 (s, 3H), 3.94 –
3.84 (m, 4H), 3.77 (s, 6H), 3.54 (t, J = 7.4 Hz, 1H), 2.16 – 2.02 (m, 1H), 2.05 (s, 3H), 1.86 – 1.71 (m,
1H), 1.64 – 1.43 (m, 2H), 1.29 (s, 3H); 13
C NMR (CDCl3, 75 MHz) δ = 207.9, 161.2 (2C), 141.2,
109.9, 106.4 (2C), 99.2, 64.61, 64.58, 59.7, 55.3 (2C), 36.6, 28.8, 26.0, 23.8; IR (KBr): ~ = 2958 cm-1
(w), 2941 (w), 1712 (s), 1670 (w), 1605 (s), 1595 (s), 1460 (m), 1431 (m), 1352 (w), 1205 (s), 1157
(s), 1065 (s), 858 (w), 837 (w); HRMS (ESI): calcd. for C17H24O5Na [M+Na]+
: 331.1516, found:
331.1517.
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4.15. tert-butyl 3-acetyl-3-(3,5-dimethoxyphenyl)-5-(2-methyl-1,3-dioxolan-2-yl)pentanoate (12)
t-BuOK (580 mg, 5.17 mmol) was added to a solution of 10 (1.39 g, 4.50 mmol) in THF (16 mL) at 0
°C and the resulting yellow mixture was stirred at 0 °C for 1 h. Then, t-butyl-2-iodoacetate (2.0 g,
5.85 mmol) was added, the mixture was allowed to warm to 25 °C and stirred for 20 h. Reaction was
quenched by saturated solution of NaHCO3 (1 mL). The solvents were evaporated and the residue
was purified by column chromatography (silica gel, EtOAc/hexane (1:4 to 1:1)) to yield 12 (924 mg,
62%) as a colorless oil. 1
H NMR (300 MHz, CDCl3) δ = 6.36 – 6.33 (m, 3H), 3.93 – 3.80 (m, 2H),
3.76 (s, 6H), 2.96 (dAB, J = 14.7 Hz, 1H), 2.81 (dAB, J = 14.7 Hz, 1H), 2.40 – 2.27 (m, 1H), 2.07 –
1.96 (m, 3H), 1.98 (s, 3H), 1.45 – 1.37 (m, 2H), 1.35 (s, 9H), 1.30 (s, 3H); 13
C NMR (75 MHz,
CDCl3) δ = 207.8, 170.2, 161.0 (2C), 142.6, 109.8, 105.2 (2C), 98.9, 81.0, 64.6, 64.5, 57.4, 55.3 (2C),
39.5, 33.2, 27.8 (3C), 27.5, 25.4, 23.7; IR (KBr): ~ = 2978 cm-1
(w), 2937 (w), 2885 (w), 1726 (s),
1713 (s), 1598 (s), 1458 (m), 1425 (m), 1369 (m), 1352 (m), 1296 (w), 1205 (s), 1157 (s), 1064 (m),
858 (w), 845 (w); HRMS (ESI): calcd. for C23H34O7Na [M+Na]+
: 445.2197, found: 445.2198.
4.16. tert-butyl 3-acetyl-3-(3,5-dimethoxyphenyl)-6-oxoheptanoate (8)
TsOH (75 mg, 0.17 mmol) was added to a solution of 12 (721 mg, 1.71 mmol) in acetone (10 mL)
and the mixture was stirred at 25 °C for 8 h. The solvent was evaporated, the residue was dissolved in
Et2O (20 mL) and washed with saturated aqueous solution of NaHCO3 (10 mL). The aqueous phase
was re-extracted with Et2O (2 x 10 mL). The combined organic extracts were washed with H2O (10
mL), dried over Na2SO4 and concentrated in a vacuum to yield 8 (641 mg, 100%) as a white solid. mp
94-97 °C; 1
H NMR (500 MHz, CDCl3) δ = 6.36 (t, J = 2.2 Hz, 1H), 6.30 (d, J = 2.2 Hz, 2H), 3.76 (s,
6H), 2.98 (d, J = 14.3 Hz, 1H), 2.79 (d, J = 14.3 Hz, 1H), 2.48 – 2.42 (m, 1H), 2.29 – 2.15 (m, 3H),
2.07 (s, 3H), 1.97 (s, 3H), 1.35 (s, 9H); 13
C NMR (126 MHz, CDCl3) δ = 207.6, 207.4, 170.0, 161.2
(2C), 142.2, 105.0 (2C), 99.0, 81.4, 57.1, 55.4 (2C), 39.8, 38.5, 29.8, 27.8 (3C), 27.5, 25.6; IR (KBr):
~ = 2975 cm-1
(w), 2939 (w), 1720 (s), 1707 (s), 1593 (s), 1466 (m), 1427 (m), 1365 (m), 1352 (s),
1311 (s), 1292 (m), 1211 (s), 1146 (s), 1068 (m), 1051 (m), 924 (w), 840 (w); HRMS (ESI): calcd. for
C21H30O6Na [M+Na]+
: 401.1935, found: 401.1935.
4.17. tert-butyl 2-(3',5'-dimethoxy-6-methyl-4-oxo-1,2,3,4-tetrahydro-[1,1'-biphenyl]-1-yl)acetate
(13a)
AcOH (0.12 mL, 2.13 mmol) and piperidine (0.19 mL, 1.93 mmol) were added to a solution of 8 (732
mg, 1.93 mmol) in toluene (10 mL) and the mixture was heated at reflux (oil bath 110 °C) for 18 h.
The solvent was evaporated in a vacuum, the residue was dissolved in EtOAc (150 mL) and washed
369
with H2O (100 mL) and then with brine (100 mL). The organic layer was dried over Na2SO4,
concentrated, and the residue was purified by column chromatography (silica gel, EtOAc/hexane
(1:2)) to afford 13a as a white semi-solid (563 mg, 81%). 1
H NMR (300 MHz, CDCl3) δ = 6.40 (d, J
= 2.1 Hz, 2H), 6.36 (t, J = 2.1 Hz, 1H), 6.16 (br. s, 1H), 3.76 (s, 6H), 2.91 (s, 2H), 2.72 – 2.60 (m,
1H), 2.32 – 2.01 (m, 3H), 1.98 (d, J = 1.2 Hz, 3H), 1.43 (s, 9H); 13
C NMR (75 MHz, CDCl3) δ =
198.7, 169.8, 163.5, 161.0 (2C), 144.7, 130.4, 105.7 (2C), 96.1, 81.4, 55.3 (2C), 46.8, 43.8, 35.7,
34.2, 28.0 (3C), 21.7; IR (KBr): ~ = 2976 cm-1
(w), 2937 (w), 1724 (s), 1672 (s), 1601 (s), 1456 (s),
1425 (m), 1369 (m), 1348 (m), 1310 (m), 1207 (s), 1159 (s), 1068 (w), 1043 (w), 841 (w); HRMS
(ESI): calcd. for C21H28O5Na [M+Na]+
: 383.1829, found: 383.1829.
4.18. tert-butyl 2-(3',5'-dimethoxy-4-methyl-6-oxo-1,2,3,6-tetrahydro-[1,1'-biphenyl]-1-yl)acetate
(13b)
t-BuOK (14.6 mg, 0.13 mmol) was added to a solution of 8 (50 mg, 0.13 mmol) in THF (1 mL) at 0
°C. The reaction mixture was stirred at 0 °C for 1 h, then saturated aqueous solution of NaHCO3 (1
mL) was added. The solvent was evaporated and the residue was loaded directly onto a column and
purified by column chromatography (silica gel, EtOAc/hexane (1:2)) to give 13b as a colorless oil
(25.2 mg, 54%). 1
H NMR (500 MHz, CDCl3) δ = 6.40 (d, J = 2.2 Hz, 2H), 6.34 (t, J = 2.1 Hz, 1H),
5.95 (br. s, 1H), 3.75 (s, 6H), 2.93 (dAB, J = 16.2, 1H), 2.73 – 2.66 (m, 1H), 2.56 (dAB, J = 16.2, 1H),
2.51 – 2.45 (m, 1H), 2.34 – 2.24 (m, 1H), 2.17 – 2.10 (m, 1H), 1.84 (s, 3H), 1.36 (s, 9H); 13
C NMR
(126 MHz, CDCl3) δ = 199.2, 170.6, 161.4, 160.8 (2C), 141.8, 126.4, 105.3 (2C), 96.7, 80.4, 55.3
(2C), 50.9, 45.1, 31.3, 28.7 (3C), 28.2, 24.1; IR (KBr): ~ = 2974 cm-1
(w), 2931 (w), 1730 (s), 1668
(s), 1637 (w), 1595 (s), 1458 (s), 1425 (m), 1367 (m), 1348 (m), 1308 (m), 1294 (w), 1205 (s), 1159
(s), 1080 (w), 1063 (w), 1049 (w), 843 (w); HRMS (APCI): calcd. for C21H29O5 [M+H]+
: 361.2010,
found: 361.2004.
4.19. 4',6'-dimethoxy-2-methylspiro[cyclohex[2]ene-1,1'-indene]-3',4(2'H)-dione (7)
A solution of 13a (415 mg, 1.15 mmol) in MsOH (4.2 mL) was heated at 50 °C for 15 h. The mixture
was cooled to 25 °C, poured into cold saturated solution of NaHCO3 (30 mL), and extracted with
EtOAc (5 x 30 mL). The combined organic extracts were dried over Na2SO4, evaporated, and the
residue was purified by gradient column chromatography (silica gel, EtOAc/hexane (1:2 to pure
EtOAc)) to afford 7 (275 mg, 84%) as a pale brown solid. mp 159-164 °C; 1
H NMR (300 MHz,
CDCl3) δ = 6.37 (d, J = 1.9 Hz, 2H), 6.32 (d, J = 1.9 Hz, 1H), 6.00 (br. s, 1H), 3.93 (s, 3H), 2.86 (s,
370
3H), 2.82 (dAB, J = 18.5, 1H), 2.65 (dAB, J = 18.5, 1H), 2.56 – 2.48 (m, 2H), 2.46 – 2.29 (m, 1H), 2.06
– 1.97 (m, 1H), 1.64 (d, J = 1.2 Hz, 3H); 13
C NMR (75 MHz, CDCl3) δ = 199.2, 197.9, 167.5, 163.8,
162.5, 159.5, 128.4, 118.8, 100.4, 98.3, 55.91, 55.89, 47.4, 46.7, 37.2, 34.9, 20.4; IR (KBr): ~ = 2953
cm-1
(w), 2927 (w), 1697 (s), 1664 (s), 1576 (s), 1460 (m), 1439 (w), 1427 (w), 1350 (w), 1315 (s),
1234 (s), 1203 (s), 1159 (s), 1107 (w), 1053 (m), 1026 (w), 860 (m); HRMS (APCI): calcd. for
C17H19O4 [M+H]+
: 287.1278, found: 287.1279.
4.20. 4'-hydroxy-6'-methoxy-2-methylspiro[cyclohex[2]ene-1,1'-indene]-3',4(2'H)-dione
BCl3 (1 M solution in CH2Cl2, 0.96 mL, 0.96 mmol) was added dropwise to a solution of 7 (275 mg,
0.96 mmol) in CH2Cl2 (10 mL) at 0 °C and the mixture was stirred at 0 °C. Additional five 0.96 mL
portions of 1M BCl3 in CH2Cl2 (total volume of 4.8 mL, 4.8 mmol) were added in one hour intervals
and then the mixture was stirred for 16 h while allowed to warm to 25 °C. Then the reaction mixture
was cooled to 0 °C, additional four 2.88 mL portions of 1M BCl3 in CH2Cl2 (total volume of 11.5 mL,
11.5 mmol) were added in three hour intervals and the mixture was stirred for additional 16 h while
allowed to warm to 25 °C. Then it was quenched with cold saturated aqueous solution of NaHCO3 (50
mL), and extracted with CH2Cl2 (3 x 50 mL). The combined organic extracts were dried over Na2SO4,
filtered, and concentrated. The residue was purified by column chromatography (silica gel,
EtOAc/hexane (2:1)) to afford 4'-hydroxy-6'-methoxy-2-methylspiro[cyclohex[2]ene-1,1'-indene]-
3',4(2'H)-dione (170 mg, 65%) as a white solid. mp 128-129 °C; 1
H NMR (300 MHz, CDCl3) δ =
9.07 (s, 1H), 6.34 (d, J = 1.9 Hz 1H), 6.31 (d, J = 1.9 Hz, 1H), 5.98 (br. s, 1H ), 3.82 (s, 3H), 2.85
(dAB, J = 18.9 Hz, 1H), 2.69 (dAB, J = 18.9 Hz, 1H), 2.61 – 2.46 (m, 2H), 2.44 – 2.28 (m, 1H), 2.10 -
2.00 (m, 1H), 1.64 (d, J = 1.2 Hz, 3H); 13
C NMR (75 MHz, CDCl3) δ = 204.0, 197.7, 168.4, 162.0,
160.5, 159.1, 128.3, 116.2, 102.8, 100.1, 55.9, 47.6, 46.9, 36.7, 34.8, 20.3; IR (KBr): ~ = 3433 cm-1
(w), 2976 (w), 2928 (w), 2918 (w), 1672 (s), 1659 (s), 1624 (s), 1597 (s), 1491 (m), 1443 (m), 1367
(s), 1325 (m), 1254 (m), 1205 (m), 1151 (s), 1022 (w), 989 (w), 962 (w), 864 (w), 849 (w); HRMS
(APCI): calcd. for C16H17O4 [M+H]+
: 273.1121, found: 273.1125.
4.21. 6'-methoxy-2-methyl-3',4-dioxo-2',3'-dihydrospiro[cyclohex[2]ene-1,1'-inden]-4'-yl
pivalate (17)
A solution of 4'-hydroxy-6'-methoxy-2-methylspiro[cyclohex[2]ene-1,1'-indene]-3',4(2'H)-dione (295
mg, 1.08 mmol) in dry THF (5 mL) was added to NaH (60% dispersion in mineral oil, 65 mg, 1.63
mmol) and the mixture was stirred at 25 °C for 45 min. Pivaloyl chloride (189 μL, 1.52 mmol) was
added and the mixture was stirred for additional 1 h. Water (50 mL) was added and the mixture was
extracted with EtOAc (3 x 50 mL). The combined organic extracts were dried over Na2SO4, filtered,
371
and concentrated. The residue was purified by column chromatography (silica gel, EtOAc/hexane
(1:1)) to afford 17 (352 mg, 92%) as a white solid. mp 136-138 °C; 1
H NMR (300 MHz, CDCl3) δ =
6.63 (br. s, 1H), 6.59 (br. s, 1H), 6.00 (br. s, 1H), 3.86 (s, 3H), 2.81 (dAB, J = 18.7 Hz, 1H), 2.64 (dAB,
J = 18.9 Hz, 1H), 2.57 – 2.30 (m, 3H), 2.11 - 2.01 (m, 1H), 1.64 (br. s, 3H); 1.40 (s, 9H); 13
C NMR
(75 MHz, CDCl3) δ = 198.2, 197.6, 176.0, 166.5, 162.14, 162.09, 149.5, 128.5, 121.9, 109.1, 106.6,
55.1, 47.4, 46.8, 39.1, 37.1, 34.9, 27.1 (3C), 20.4; IR (KBr): ~ = 2978 cm-1
(w), 2956 (w), 2931 (w),
1759 (s), 1711 (s), 1670 (s), 1591 (m), 1481 (m), 1438 (m), 1340 (w), 1332 (w), 1310 (s), 1261 (m),
1244 (m), 1142 (s), 1109 (s), 1084 (s), 1036 (m), 1009 (m), 897 (w), 856 (w); HRMS (APCI): calcd.
for C21H25O5 [M+H]+
: 357.1697, found: 357.1695.
4.22. 4-((tert-butyldimethylsilyl)oxy)-6'-methoxy-2-methyl-3'-oxo-2',3'-dihydrospiro[cyclohexa[2,4]diene-1,1'-inden]-4'-yl
pivalate (18)
TBSOTf (143 μL, 0.61 mmol) was added to a solution of 17 (135 mg, 0.38 mmol) in CH2Cl2 (1 mL)
at -78 °C, then DBU (92 μL, 0.61 mmol) was added dropwise and the reaction mixture was stirred at -
78 °C for 30 min, filtered through a pad of silica gel (1 cm height, 2 cm diameter), and the pad was
quickly washed with EtOAc (100 mL). After evaporation of the solvents from the filtrate, 18 (152
mg, 85%) was obtained as a colorless semi-solid, which was used in the next step without any further
purification. Analytically pure sample was obtained by preparative TLC (silica gel, EtOAc/hexane
(1:2)); 1
H NMR (300 MHz, CDCl3) = 6.84 (d, J = 2.0 Hz, 1H), 6.54 (d, J = 2.0 Hz, 1H), 5.67 (br. s ,
1H), 4.85 – 4.79 (m, 1H), 3.88 (s, 3H), 2.81 (dAB, J = 18.3 Hz, 1H), 2.74 (dd, J = 16.9 Hz, J = 2.6 Hz,
1H), 2.45 (dAB, J = 18.3 Hz, 1H), 2.26 (dd, J = 16.9 Hz, J = 5.8 Hz, 1H), 1.53 (s, 3H), 1.41 (s, 9H),
0.95 (s, 9H), 0.16 (s, 6H); 13
C NMR (126 MHz, CDCl3) δ = 200.3, 176.2, 166.1, 163.9, 149.1, 148.9,
141.0, 124.2, 122.0, 108.6, 107.3, 99.0, 56.0 (2C), 48.4, 46.4, 39.1, 38.6, 27.2 (3C), 25.7 (3C), 19.5,
18.1, - 4.4; IR (KBr): ~ = 2958 cm-1
(m), 2931 (m), 2856 (w), 1761 (s), 1711 (s), 1674 (s), 1610 (s),
1589 (s), 1479 (m), 1462 (m), 1461 (m), 1441 (m), 1396 (w), 1340 (m), 1310 (s), 1273 (m), 1244 (m),
1194 (w), 1144 (s), 1109 (s), 1186 (s), 1036 (m), 862 (w), 839 (w); HRMS (APCI): calcd. for
C27H39O5Si [M+H]:
471.2561, found: 471.2560.
4.23. 5-hydroxy-6'-methoxy-2-methyl-3',4-dioxo-2',3'-dihydrospiro[cyclohex[2]ene-1,1'-inden]-
4'-yl pivalate (19)
mCPBA (77%, 110 mg, 0.49 mmol) was added to a mixture of 18 (155 mg, 0.33 mmol) and KHCO3
(77 mg, 0.77 mmol) in CH2Cl2, (5 mL) at 0 °C and the reaction mixture was stirred at 25 °C for 16 h.
EtOAc (50 mL) was added and the mixture was washed with saturated aqueous solution of NaHCO3
(20 mL), then with H2O (30 mL), dried over Na2SO4, filtered, and concentrated in a vacuum. The
372
residue was dissolved in CH2Cl2 (10 mL), pyr.HF (1 mL) was added, and the mixture was stirred at 25
°C for 16 h. The mixture was diluted with H2O (10 mL) and extracted with EtOAc (3 x 30 mL). The
combined organic extracts were dried over Na2SO4, filtered, and concentrated under reduced pressure.
The residue was purified by preparative TLC (silica gel, EtOAc/hexane (2:1) to afford 19 (45 mg,
37%) as a colorless semi solid. 1
H NMR (500 MHz, CDCl3) δ = 6.71 (d, J = 2.0 Hz, 1H), 6.63 (d, J =
2.0 Hz, 1H), 6.16 – 6.15 (m, 1H), 4.57 (dd, J = 12.9 Hz, J = 5.8 Hz, 1H), 3.87 (s, 3H), 3.08 (dAB, J =
18.7 Hz, 1H), 2.56 (dAB, J = 18.7 Hz, 1H), 2.50 (dd, J = 13.2 Hz, J = 5.8 Hz, 1H), 2.27 (dd, J = 13.2
Hz, J = 12.9 Hz, 1H), 1.78 (d, J = 1.2 Hz, 3H), 1.43 (s, 9H); 13
C NMR (126 MHz, CDCl3) δ = 198.4,
198.1, 176.1, 166.3, 163.5, 159.0, 150.1, 125.9, 121.3, 109.0, 107.6, 69.0, 56.2, 51.1, 48.2, 44.1, 39.2,
27.2 (3C), 20.1; IR (KBr): ~ = 2976 cm-1
(w), 2933 (w), 1759 (s), 1711 (s), 1684 (s), 1608 (s), 1587
(s), 1479 (w), 1440 (w), 1340 (m), 1308 (m), 1244 (m), 1142 (s), 1109 (s), 1053 (w), 1032 (w), 899
(w), 867 (w); HRMS (APCI): calcd. for C21H25O6 [M+H]+
: 373.1646, found: 373.1641.
4.24. 4',5-dihydroxy-6'-methoxy-2-methylspiro[cyclohexa[2,5]diene-1,1'-indene]-3',4(2'H)-dione
(cDHAs)
K2CO3 (44.5 mg, 0.322 mmol) was added to a solution of 19 (38 mg, 0.107 mmol) in MeOH (4 mL),
and the mixture was stirred in an open flask at 50 °C for 6 h. MeOH was then evaporated and the
residue was dissolved in H2O (10 mL) and the pH was adjusted to 7 with 1 M HCl. The mixture was
extracted with EtOAc (4 x 10 mL), the combined organic extracts were dried over Na2SO4, filtered,
and the solvent was evaporated. The residue was purified by preparative TLC (silica gel,
CH2Cl2/EtOAc (2:1)) to afford cDHAs (14.5 mg, 50%) as a white solid. mp 177 °C (dec.); 1
H NMR
(300 MHz, CDCl3) δ = 9.02 (br. s, 1H), 6.42 – 6.33 (m, 3H), 6.14 (d, J = 1.7 Hz, 1H), 6.03, (s, 1H),
3.83 (s, 3H), 2.92 (s, 2H), 1.75 (s, 3H); 13
C NMR (126 MHz, CDCl3) δ = 203.4, 181.6, 168.8, 162.8,
159.3, 156.8, 146.0, 125.5, 120.0, 116.0, 102.6, 101.0, 56.1, 50.8, 47.3, 19.5; IR (KBr): ~ = 3387 cm-
1
(w), 2955 (w), 2922 (m), 2850 (w), 1680 (s), 1639 (s), 1628 (s), 1433 (w), 1420 (w), 1377 (m), 1311
(m), 1205 (s), 1176 (m), 1148 (s), 1105 (w), 1022 (w), 887 (w); HRMS (APCI): calcd. for C16H15O5
[M+H]+
: 287.0914, found: 287.0912.
Crystal data for cDHAs: CCDC ref. No. 1012668. Crystallized from CHCl3.
373
Measurement of inhibitory activities of mammalian pols
The four mammalian pols α, γ, , and  were prepared, and the reaction mixtures for these pols, as
described previously.23
Briefly, poly(dA)/oligo(dT)18 (A/T, 2:1) and [3
H]-dTTP (100 cpm/pmol) were
used as the DNA template-primer substrate and nucleotide (dNTP; 2'-deoxynucleotide-5'triphosphate)
substrate, respectively. The tested compounds were dissolved in distilled dimethyl
sulfoxide (DMSO) at concentrations 0 – 200 µM. Subsequently, 4-µL aliquots were mixed with 16
µL of each enzyme (0.05 units) in 50 mM Tris-HCl (at pH 7.5) containing 1 mM dithiothreitol, 50%
glycerol (v/v), and 0.1 mM ethylenediaminetetraacetic acid, and were held at 0°C for 10 min.
Subsequently, 8 µL of these inhibitor-enzyme mixtures were added to 16-µL aliquots of enzyme
standard reaction mixture containing 50 mM Tris-HCl (at pH 7.5), 1 mM dithiothreitol, 1 mM MgCl2,
15% glycerol, 5 µM poly(dA)/oligo(dT)18 (A/T, 2:1) and 10 µM [3
H]-dTTP, and were incubated at 37
°C for 60 min. The enzyme activity in the absence of inhibitor was taken as 100% (the activity
without the enzyme was considered 0%,), and the inhibitory activity was determined for each
inhibitor concentration. One unit of pol activity was defined as the amount of each enzyme that
catalyzed incorporation of 1 nmol dTTP into synthetic DNA template-primers in 60 min at 37 °C,
under standard reaction conditions.24
The 50% inhibitory concentration (IC50 value) of the enzyme
inhibitor was determined by constructing a dose-response curve and examining the effect of different
concentrations of inhibitor on reversing enzyme activity (functional antagonist assay).
374
References
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[2] Martin, S. A.; McCabe, N.; Mullarkey, M.; Cummings, R.; Burgess, D. J.; Nakabeppu, Y.; Oka,
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130.
[5] (a) Mizushina, Y.; Kamisuki, S.; Mizuno, T.; Takemure, M.; Asahara, H.; Linn, S.; Yamaguchi,
T.; Matsukage, A.; Hanaoka, F.; Yoshida, S.; Saneyoshi, M.; Sugawara, F.; Sakaguchi, K. J. Biol.
Chem. 2000, 275, 33957-33961; (b) Mizushina, Y. Biosci. Biotechnol. Biochem. 2009, 73, 1239-
1251.
[6] Jackson, D. A. Nucleic Acid Res. 1990, 18, 753-758.
[7] Kamisuki, S.; Takahashi, S.; Mizushina, Y.; Sakaguchi, K.; Nakata, T.; Sugawara, F. Bioorg.
Med. Chem. 2004, 12, 5355-5359.
[8] Kuramochi, K.; Fukudome, K.; Kuryiama, I.; Takeuchi, T.; Sato, Y.; Kamisuki, S.; Tsubaki, K.;
Sugawara, F.; Yoshida, H.; Mizushina, Y. Bioorg. Med. Chem. 2009, 17, 7227–7238.
[9] Beugelmans, R.; Chastanet, J.; Ginsburg, H.; Quintero-Cortes, L.; Roussi, G. J. Org. Chem. 1985,
50, 4933-4938.
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5170.
[12] Doering, W. v. E.; Benkhoff, J.; Shao, L. J. Am. Chem. Soc. 1999, 121, 962-968.
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[15] Jiang, S.; Li, P.; Lai, C. C.; Kelley, J. A.; Roller, P. P. J. Org. Chem. 2006, 71, 7307-7314.
[16] Srikrishna, A.; Ramasastry, S. S. V. Tetrahedron Lett. 2005, 46, 7373-7376.
[17] (a) Krafft, M. E.; Holton, R. A. J. Am. Chem. Soc. 1984, 106, 7619-7621; (b) Ihara, M.; Ishida,
Y.; Fukumoto, K.; Kametani, T. Chem. Phar. Bull. 1985, 33, 4102-4105; (c) Jung, Y. C.; Yoon, C.
H.; Turos, E.; Yoo, K. S.; Jung, K. W. J. Org. Chem. 2007, 72, 10114-10122.
[18] Myobatake, Y.; Takeuchi, T.; Kuramochi, K.; Kuriyama, I.; Ishido, T.; Hirano, K.; Sugawara, F.;
Yoshida, H.; Mizushina, Y. J. Nat. Prod. 2012, 75, 135-141.
[19] Bioisosteres in Medicinal Chemistry; Brown, N., Ed.;Wiley-VCH, 2012.
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[21] Findlay, J. A.; Kwan, D. Can. J. Chem. 1973, 51, 3299-3301.
[22] El-Feraly, F. S.; Cheatham, S. F.; McChesney, J. D. Can. J. Chem. 1985, 63, 2232-2236.
[23] (a) Mizushina, Y.; Tanaka, N.; Yagi, H.; Kurosawa, T.; Onoue, M.; Seto, H.; Horie, T.; Ayoagi,
N.; Yamaoka, M.; Matsukage, A.; Yoshida, S.; Sakaguchi, K. Biochim. Biophys. Acta 1996, 1308,
256-262; (b) Mizushina, Y.; Yoshida, S.; Matsukage, A.; Sakaguchi, K. Biochim. Biophys. Acta 1997,
1336, 509-521; (c) Myobatake, Y.; Takeuchi, T.; Kuramochi, K.; Kuriyama, I.; Ishido, T.; Hirano, K.;
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1
Supporting information
Highly Diastereoselective Flexible Synthesis of New Carbocyclic C-nucleosides
Lukáš Maier,a,b#
Prashant Khirsariya,a,b#
Ondřej Hylse,a,b
Santosh Kumar Adla,a
Lenka Černová,a
Michal Poljak,a
Soňa Krajčovičová,a
Erik Weis,a
Stanislav Drápela,b,c
Karel Souček,b,c
and Kamil Paruch a,b*
Table of contents
1. Experimental procedures.................................................................................................................................................................................2
2. 1
H, 13
C NMR spectra related to key cyclopentanone synthesis...................................................................................................................16
3. 1
H, 13
C NMR spectra related to nucleophilic addition pathway.................................................................................................................77
4. 1
H, 13
C NMR spectra related to enol triflate pathway.................................................................................................................................126
5. 1
H, 13
C NMR spectra related to 5´ and 2´modifications..............................................................................................................................214
6. 1
H, 13
C NMR spectra related to the preparation of optically active intermediates..................................................................................236
7. Selected IR spectra, HPLC chromatograms and CD spectra.......................................................................................................................245
8. Cell-based assays...............................................................................................................................................................................................260
2
(1R*,2R*,3S*,4S*)-bicyclo[2.2.1]hept-5-ene-2,3-diol (S-1).
NMO (19.06 g , 162.8 mmol) and OsO4 (4% wt. in H2O, 2.04 mL, 0.2 mol %) were added to a solution of norbornadiene (15.0 g, 162.8 mmol) in
acetone and H2O (200 + 50 mL) and the reaction mixture was stirred at 40 °C for 14 h. After cooling to 25 °C, Na2S2O5 (1. 00 g) was added and
the reaction mixture was stirred at 25 °C for another 30 min. All volatiles were removed under reduced pressure and the black residue was
purified by flash column chromatography (hexane/EtOAc = 2:1) to afford S-1 as a white crystalline solid (8.42 g, 41%). 1
H NMR (500 MHz,
CDCl3): δ = 6.04 (m, 2H), 3.71 (m, 2H), 2.95 (m, 2H), 2.70 m (2H), 1.89 (dm, J = 9.2 Hz, 1H), 1.63 (dm, J = 9.2 Hz, 1H) ppm. 13
C NMR (126
MHz, CDCl3): δ = 136.56, 69.18, 48.21, 42.37 ppm. The spectral data were consistent with the literature.7
(exo, exo)-5,6-dimethylmethylendioxy-bicyclo[2.2.1]hept-2-ene (6a).
2,2-dimethoxypropane (26.0 mL, 196.6 mmol) and TsOH (5 mg) were added to a solution of diol S-1 (6.2 g, 49.2 mmol) in acetone (75 mL). The
reaction mixture was stirred at 25 °C for 20 min., the solvent was evaporated and the residue was purified on a short pad of silica gel
(hexane/EtOAc = 20:1) to afford 6a as a colorless oil which solidified upon standing at -20 °C (7.86 g, 95 %). 1
H NMR (500 MHz, CDCl3): δ =
6.05 (m, 2H), 4.18 (d, J = 1.6 Hz, 2H), 2.76 (m, 2H), 1.97 (m, 1H), 1.67 (m, 1H), 1.47 (s, 3H), 1.32 (s, 3H) ppm. 13
C NMR (126 MHz, CDCl3): δ
= 136.89, 113.81, 80.63, 45.35, 43.02, 26.32, 24.58 ppm. The spectral data were consistent with the literature.7
3
(3aR*,4R*,7S*,7aS*)-2,2-diphenyl-3a,4,7,7a-tetrahydro-4,7-methanobenzo[d][1,3]dioxole (6b).
Benzophenone dimethyl ketal (2.08 g, 9.15 mmol) was added to a solution of S-1 (0.800 g, 6.34 mmol) in CH2Cl2 (8 mL), followed by addition
of TsOH (1 mg), and the reaction mixture was stirred at 25 C for 18 h. The solvent was evaporated and the residue was purified by flash column
chromatography (hexane/EtOAc = 20:1) to afford 6b (1.60 g) contaminated with residual benzophenone and benzophenone dimethyl ketal as a
white crystalline solid. This material was used for the next step (preparation of compound S-3) without further purification. An analytical sample
could be obtained by repeated flash column chromatography with slow gradient elution (hexane to hexane/EtOAc = 20:1) as a white crystalline
solid. m.p. = 123-127 °C. 1
H NMR (500 MHz, CDCl3): δ = 7.58-7.56 (m, 2H), 7.51-7.49 (m, 2H), 7.37-7.34 (m, 2H), 7.31-7.26 (m, 4H), 6.05
(m, 2H), 4.13 (d, J = 1.6 Hz, 2H), 2.97 (m, 2H), 2.21 (dm, J = 8.9 Hz, 1H), 1.77 (dm, J = 8.9 Hz, 1H). 13
C NMR (126 MHz, CDCl3): δ = 143.03,
141.71, 137.06, 128.46, 128.40, 128.28, 128.07, 126.73, 126.24, 114.41, 81.31, 45.43, 43.78 ppm. IR (ν˜max) = 2979 (w), 2946 (w), 2924 (w),
1489 (m), 1270 (m), 1203 (m), 1019 (s), 747 (s), 703 (s), 694 (s) cm–1
. HR-MS (EI) calculated for C20H18O2: 290.1307. Found: 290.1311.
(3aR*,4R*,7S*,7aS*)-3a,4,7,7a-tetrahydro-4,7-methanobenzo[d][1,3]dioxol-2-one (6c).
1,1´-carbonyldiimidazole (0.250 g, 1.98 mmol) was added to a solution of starting material S-1 (0.200 g, 1.58 mmol) in anhydrous toluene (6
mL) and the reaction mixture was stirred at 55 °C for 16 h. The reaction mixture was then cooled to 25 °C and the solvent was evaporated. The
residue was purified by flash column chromatography (hexane/EtOAc = 5:1) to afford 6-c as a white crystalline solid (0.191 g, 80%). m.p. = 86-
4
88 °C. 1
H NMR (500 MHz, CDCl3): δ = 6.12 (m, 2H), 4.56 (d, J = 1.4 Hz, 2H), 3.14 (m, 2H), 1.90-1.82 (m, 2H) ppm. 13
C NMR (126 MHz,
CDCl3): δ = 156.53, 136.17, 78.92, 45.79, 41.15 ppm. IR (ν˜max) = 3011 (w), 1777 (s), 1367 (m), 1160 (s), 1055 (s), 1014 (s), 742 (s), 697 (s) cm–
1
. HR-MS (ESI) calculated for C8H8O3 [M+Na]+
: 175.0371. Found: 175.0370.
(1R*,4S*,5S*,6R*)-5,6-bis(triisopropylsilyloxy)bicyclo[2.2.1]hept-2-ene (6d).
Imidazole (1.78 g, 26.16 mmol) was added to a cooled solution (0 °C, ice bath) of S-1 (1 g, 7.93 mmol) in anhydrous DMF (10 mL) followed by
dropwise addition of di-tert-butylsilyl bis(trifluoromethansulfonate) (2.84 mL, 8.72 mmol). The reaction mixture was stirred while allowed to
warm to 25 C for 14 h. H2O (60 mL) was added slowly and the mixture was extracted with Et2O (3 × 50 mL). The organic extracts were dried
over Na2SO4, filtered, and the solvent was evaporated. The residue was purified by flash column chromatography (hexane/EtOAc = 20:1) to
afford 6d as a colorless oil (1.42 g, 78%). 1
H NMR (500 MHz, CDCl3): δ = 6.06 (m, 2H), 4.15 (d, J = 1.5 Hz, 2H), 2.79 (m, 2H), 2.20 (dm, J =
9.2 Hz, 1H), 1.64 (dm, J = 9.2 Hz, 1H), 1.12 (s, 9H), 1.05 (s, 9H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 137.51, 78.53, 47.20, 42.51, 28.30,
27.73, 22.88, 20.62 ppm. IR (ν˜max) = 2933 (w), 2858 (w), 1475 (m), 1171 (w), 1036 (s), 1021 (s), 853 (s), 823 (s), 704 (m), 648 (m) cm–1
. HRMS
(ESI) calculated for C15H27O2Si [M+H]+
: 267.1780. Found: 267.1779.
(1R*,4S*,5S*,6R*)-5,6-bis(benzyloxy)bicyclo[2.2.1]hept-2-ene (6e).
5
A solution of diol S-1 (4.88 g, 38.70 mmol) in DMF (40 mL) was added dropwise to a suspension of NaH (60% dispersion in mineral oil, 4.64 g,
116.11 mmol) in DMF (20 mL). The reaction mixture was stirred at 25 °C for 20 min, benzyl bromide (11.05 mL, 92.9 mmol) was slowly
added, followed by a solution of tetra-N-butyl ammonium iodide (1.43 g, 3.87 mmol) in DMF (10 mL). The reaction mixture was stirred at 25 °C
for additional 14 h and then quenched by dropwise addition of H2O (5 mL). H2O (100 mL) was added and the mixture was extracted with Et2O (4
× 100 mL). The combined organic extracts were dried over Na2SO4, filtered, and the solvent was evaporated. The resulting yellow oil was
purified by flash column chromatography (hexane/EtOAc = 20:1) to afford 6e as a white crystalline solid (9.08 g, 77%), m.p. = 65.9-66.1 °C. 1
H
NMR (500 MHz, CDCl3): δ = 7.37-7.26 (m, 10H), 6.01 (m, 2H), 4.69-4.63 (m, 4H), 3.53 (m, 2H), 2.85 (m, 2H), 2.20 (m, 1H), 1.67 (m, 1H) ppm.
13
C NMR (126 MHz, CDCl3): δ = 139.23, 137.02, 128.48, 128.00, 127.59, 77.19, 72.50, 45.88, 44.18 ppm. IR (ν˜max) = 3063 (w), 3029 (w), 1496
(w), 1453 (m), 1343 (w), 1114 (m), 733 (s), 696 (s) cm–1
. HR-MS (ESI) calculated for C21H22O2 [M+Na]+
: 329.1517. Found: 329.1524.
(1R*,4S*,5S*,6R*)-5,6-bis(4-methoxybenzyloxy)bicyclo[2.2.1]hept-2-ene (6f).
A solution of diol S-1 (1.18 g. 9.37 mmol), in DMF (20 mL), was added dropwise to a suspension of NaH (60% dispersion in mineral oil, 1.12 g,
28.11 mmol) in DMF (20 mL). The reaction mixture was stirred at 25 °C for 20 min, 4-methoxybenzyl chloride (2.8 mL, 20.62 mmol) was
slowly added, followed by a solution of tetra-N-butyl ammonium iodide (0.346 g, 0.94 mmol) in DMF (10 mL). The reaction mixture was stirred
at 25 °C for additional 14 h and then quenched by dropwise addition of H2O (5 mL). H2O (100 mL) was added and the mixture was extracted
with Et2O (4 × 100 mL). The combined organic extracts were dried over Na2SO4, filtered, and the solvent was evaporated. The resulting yellow
oil was purified by flash column chromatography (hexane/EtOAc = 15:1) to afford 6f as a white wax (3.2 g, 93%). 1
H NMR (500 MHz, CDCl3):
δ = 7.27 (d, J = 8.6 Hz, 4H), 6.85 (d, J = 8.6 Hz, 4H), 5.99 (m, 2H), 4.58 (d, J = 11.7 Hz, 2H), 4.53 (d, J = 11.7 Hz, 2H), 3.80 (s, 6H), 3.48 (d, J =
6
1.8 Hz, 2H), 2.80 (m, 2H), 2.16-2.14 (m, 1H), 1.64-1.62 (m, 1H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 159.29, 137.01, 131.35, 129.59, 113.89,
76.98, 76.79, 72.10, 55.50, 45.87, 44.17 ppm. IR (ν˜max) = 2991 (w), 2952 (w), 1402 (m), 1257 (m), 1109 (s), 741 (s), 694, 650 (s) cm–1
.
(1R*,4S*,5S*,6R*)-5,6-bis(tert-butyldimethylsilyloxy)bicyclo[2.2.1]hept-2-ene (6g).
Imidazole (0.558 g, 8.21 mmol) and TBSCl (0.544 g, 3.61 mmol) were added to a solution of S-1 (0.207 g, 1.64 mmol) in CH2Cl2 (15 mL) and
the reaction mixture was stirred at 25 C for 14 h. The solvent was evaporated and the residue was purified by flash column chromatography
(hexane/EtOAc = 10:1) to afford 6g as a white semi-solid (0.44 g, 76%). 1
H NMR (500 MHz, CDCl3): δ = 6.00 (m, 2H), 3.64 (m, 2H), 2.53 (m,
2H), 2.14 (dm, J = 8.2 Hz, 1H), 1.56 (dm, J = 8.2 Hz, 1H) 0.91 (s, 18H), 0.07 (s, 6H), 0.05 (s, 6H) ppm. 13
C NMR (126 MHz, CDCl3): δ =
136.95, 70.98, 49.65, 43.41, 26.30, 18.60, -4.02, -4.65 ppm. IR (ν˜max) = 1481 (w), 1103 (m), 759 (s), 693 (s) cm–1
.
(1R*,4S*,5S*,6R*)-5,6-bis(tert-butyldiphenylsilyloxy)bicyclo[2.2.1]hept-2-ene (6h).
TBDPSCl (1 mL, 3.96 mmol) and imidazole (0.433 g, 6.36 mmol) were added to a solution of S-1 (0.200 g, 1.59 mmol) in CH2Cl2 (10 mL) and
the reaction mixture was stirred at 25 °C for 14 h. The solvent was evaporated and the yellow residue was purified by flash column
chromatography (hexane/EtOAc = 20:1) to afford 6h as a white crystalline solid (0.24 g, 25 %), along with the mono-silylated side-product
(0.417 g, 43%). m.p. = 133-136 °C. 1
H NMR (500 MHz, CDCl3): δ = 7.76-7.72 (m, 8H), 7.41-7.31 (m, 12H), 5.53 (m, 2H), 3.87 (d, J = 1.5 Hz,
2H), 2.32 (dm, J = 8.6 Hz, 1H), 2.24 (m, 2H), 1.38 (dm, J = 8.6 Hz, 1H), 1.10 (s, 18H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 136.63, 136.27,
7
136.15, 135.28, 134.70, 129.71, 129.60, 127.68, 127.67, 72.30, 48.87, 42.96, 27.43, 19.60 ppm. IR (ν˜max) = 1472 (w), 1102 (m), 1086 (m), 697
(s), 499 (s), 481 (s) cm–1
. HR-MS (ESI) calculated for C39H46O2Si2 [M+Na]+
: 625.29285. Found: 625.29285.
((3aR*,4R*,6S*,6aS*)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4,6-diyl)dimethanol (S-2).
Prepared by general procedure A using 6.2 g (37.32 mmol) of 6a; flash column chromatography (CH2Cl2/MeOH = 20:1 to 10:1) afforded S-2 as
a colorless oil (5.11 g, 67%). 1
H NMR (500 MHz, CDCl3): δ = 4.41 (m, 2H), 3.69 (m, 4H), 2.28 (m, 2H), 2.08 (m, 1H), 1.68 (br s, 2H), 1.51 (s,
3H), 1.32 (s, 3H), 1.26 (m, 1H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 112.66, 83.67, 64.51, 47.63, 30.66, 27.75, 25.32 ppm. HR-MS (APCI)
calculated for C10H18O4 [M+H]+
: 203.1278. Found: 203.1276. The spectral data were consistent with the literature.7
((3aR*,4R*,6S*,6aS*)-2,2-diphenyltetrahydro-3aH-cyclopenta[d][1,3]dioxole-4,6-diyl)dimethanol (S-3).
Prepared by general procedure A using 2.59 g (7.92 mmol) of 6b; flash column chromatography (CH2Cl2/MeOH = 20:1) afforded S-3 as a
colorless oil (0.950 g, 37%). 1
H NMR (500 MHz, CDCl3): δ = 7.54-7.52 (m, 2H), 7.47-7.45 (m, 2H), 7.36-7.26 (m, 6H), 4.41-4.38 (m, 2H), 3.73-
3.66 (m, 4H), 2.49-2.45 (m, 2H), 2.17-2.12 (m, 1H), 1.67 (br s, 2H), 1.40-1.33 (m, 1H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 142.60, 142.29,
8
128.50, 128.39, 128.22, 126.70, 126.64, 113.25, 84.42, 64.78, 47.32, 31.01 ppm. IR (ν˜max) = 3307 (m), 2924 (m), 1446 (m), 1261 (m), 1086
(m), 1067 (m), 1052 (m), 973 (m), 695 (s), 635 (m) cm–1
. HR-MS (ESI) calculated for C20H22O4 [M+H]+
: 327.1591. Found: 327.1581.
((3aR*,4R*,6S*,6aS*)-2,2-di-tert-butyltetrahydro-3aH-cyclopenta[d][1,3,2]dioxasilole-4,6-diyl)dimethanol (S-4).
Prepared by general procedure A using 1.19 g (4.49 mmol) of 6c; flash column chromatography (CH2Cl2/MeOH = 20:1) afforded S-4 as a white
solid (0.643 g, 62 %). m.p. = 99-103 °C. 1
H NMR (500 MHz, CDCl3): δ = 4.29-4.25 (m, 2H), 3.76 (d, J = 6.1 Hz, 2H), 2.17 (m, 2H), 1.88 (m,
1H), 1.08 (s, 9H), 1.08 (overlapped, m, 1H), 1.02 (s, 9H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 81.86, 65.15, 49.47, 28.35, 27.70, 27.03, 22.11,
19.66 ppm. IR (ν˜max) = 3298 (br), 2886 (m), 2858 (m), 1472 (m), 1065 (s), 1032 (s), 849 (m), 819 (m), 651 (m) cm–1
. HR-MS (ESI) calculated
for C15H30O4Si [M+Na]+
: 325.18056. Found: 325.18060.
((1R*,3S*,4S*,5R*)-4,5-bis(benzyloxy)cyclopentan-1,3-diyl)dimethanol (S-5).
Prepared by general procedure A using 9.08 g (29.60 mmol) of 6e; flash column chromatography (CH2Cl2/MeOH = 20:1) afforded S-5 as a
colorless oil (8.95 g, 88%). 1
H NMR (500 MHz, CDCl3): δ = 7.36 – 7.28 (m, 2H), 4.59 (d, AB, J = 11.8 Hz, 2H), 4.52 (d, AB, J = 11.8 Hz, 2H),
3.71 (m, 2H), 3.64 (m, 2H), 3.52 (m, 2H), 2.45 (m, 2H), 1.96 (m, 1H), 1.77 (br s, 2H), 0.93 (m, 1H) ppm.13
C NMR (126 MHz, CDCl3): δ =
9
138.51, 128.62, 128.21, 127.93, 81.16, 71.39, 65.42, 43.99, 25.51 ppm. IR (ν˜max) = 3310 (m), 2928 (m), 1431 (m), 1250 (m), 1060 (m), 973
(m), 670 (s) cm–1
. HR-MS (APCI) calculated for C21H26O4 [M+H]+
: 343.1904. Found: 343.1900.
((1R*,3S*,4S*,5R*)-4,5-bis(4-methoxybenzyloxy)cyclopentane-1,3-diyl)dimethanol (S-6).
Prepared by general procedure A using 3.20 g (8.74 mmol) of 6f; flash column chromatography (CH2Cl2/MeOH = 20:1) afforded S-6 as a
colorless oil (1.81 g, 56%). 1
H NMR (500 MHz, CDCl3): δ = 7.28 (m, 4H), 6.87 (m, 4H), 4.52 (d, AB, J = 11.8 Hz, 2H), 4.43 (d, AB, J = 11.8
Hz, 2H), 3.81 (s, 6H), 3.67-3.60 (m, 4H), 3.51-3.47 (m, 2H), 2.41 (m, 2H), 1.94 (m, 1H), 1.85 (br s, 2H), 0.90 (m, 1H) ppm.13
C NMR (126
MHz, CDCl3): δ = 159.49, 130.60, 129.79, 129.61, 114.02, 80.78, 70.93, 65.49, 55.50, 43.97, 25.51. IR (ν˜max) = 3367 (b), 2924 (w), 2858 (w),
1448 (m), 1203 (m), 1084 (s), 735 (s), 696 (s) cm–1
.
((3aS*,4S*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-diphenyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)methanol (S-7):
The compound was prepared by essentially same procedure used for compound 7a from 0.571 g (1.75 mmol) of S-3; flash column
chromatography (hexane/EtOAc = 5:1) afforded S-7 as a colorless oil (0.742 g, 75 %). 1
H NMR (500 MHz, CDCl3): δ = 7.65-7.63 (m, 4H), 7.53-
10
7.51 (m, 2H), 7.48-7.46 (m, 2H), 7.42-7.40 (m, 2H), 7.38-7.27 (m, 10H), 4.40 (dd, J = 7.1, 4.9 Hz, 1H), 4.31 (dd, J = 7.1, 5.6 Hz, 1H), 3.70 (dd, J
=6. 25, 1.92 Hz, 2H), 3.66 (m, 2H), 2.51 (m, 1H), 2.42 (m, 1H), 1.05 (s, 9H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 142.87, 142.48, 135.86,
135.84, 133.89, 129.87, 128.39, 128.35, 128.19, 128.12, 127.89, 126.67, 126.59, 113.14, 84.25, 83.93, 65.26, 65.01, 47.60, 47.00, 31.15, 27.10,
19.54. IR (ν˜max) = 2930 (w), 1449 (w), 1427 (w), 1264 (m), 1105 (m), 1064 (m), 733 (s), 698 (s) cm–1
. HR-MS (ESI) calculated for C36H40O4Si
[M+H]+
: 565.2769. Found: 565.2762.
tert-butyl(((3aR*,4R*,6R*,6aS*)-6-(iodomethyl)-2,2-diphenyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)methoxy)diphenylsilane (S-
8).
The compound was prepared by essentially same procedure used for compound 8a from 0.735 g (1.3 mmol) of S-7; flash column
chromatography (hexane/EtOAc = 40:1)) afforded S-8 as a colorless oil (0.685 g, 78 %). 1
H NMR (500 MHz, CDCl3): δ = 7.65-7.63 (m, 4H),
7.52-7.50 (m, 2H), 7.47-7.41 (m, 4H), 7.38-7.28 (m, 10H), 4.44 (dd, J = 7.2, 5.04 Hz, 1H), 4.18 (dd, J = 7.2, 6.28 Hz, 1H), 3.74-3.65 (m, 2H),
3.35 (dd, J = 9.9, 5.6 Hz 1H), 3.25 (dd, J = 9.9, 7.0 Hz), 2.51 (m, 1H), 2.37 (m, 1H), 2.11 (m, 1H), 1.40 (m, 1H), 1.06 (s, 9H) ppm. 13
C NMR
(126 MHz, CDCl3): δ = 142.78, 135.87, 135.85, 133.80, 129.91, 128.43, 128.41, 128.23, 128.19, 127.91, 126.57, 126.49, 113.34, 86.00, 84.06,
64.95, 46.96, 46.65, 35.88, 27.12, 19.55, 10.06 ppm. IR (ν˜max) = 2929 (w), 2856 (w), 1489 (w), 1471 (w), 1109 (m), 737 (m), 698 (s), 503 (m)
cm-1
. HR-MS (ESI) calculated for C39H39IO3Si[M+Na]+
: 697.1603. Found: 697.1606.
tert-butyl(((3aR*,4R*,6aS*)-2,2-dimethyl-6-methylenetetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)methoxy)diphenylsilane (S-9).
11
DBU (1.04 mL, 7.54 mmol) was added to a solution of 8a (1.54 g, 2.78 mmol) in toluene (10 mL) and the reaction mixture was stirred at 90 C
for 4 h. The solvent was evaporated under reduced pressure and the residue was purified by flash column chromatography (hexane/EtOAc =
20:1) to afford S-9 as a colorless oil (0.855 g, 72 %). 1
H NMR (500 MHz, CDCl3): δ = 7.65-7.63 (m, 4H), 7.45-7.36 (m, 6H), 5.17 (m, 1H), 5.07
(m, 1H), 4.63 (d, J = 5.6 Hz, 1H), 4.50 (d, J = 5.6 Hz, 1H), 3.48 (m, 2H), 2.78-2.73 (m, 1H), 2.38 (m, 1H), 2.15 (dm, J = 15.9, Hz, 1H), 1.47 (s,
3H), 1.32 (s, 3H), 1.05 (s, 9H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 149.83, 135.83, 133.77, 133.75, 129.91, 129.90, 127.90, 112.65, 110.56,
82.74, 81.96, 64.62, 45.85, 32.74, 27.07, 26.99, 24.69, 19.46 ppm. IR (ν˜max) = 2930 (m), 1732 (w), 1478 (w), 1222 (w), 1127 (s), 679 (s) cm–1
.
tert-butyl(((3aR*,4R*,6aS*)-6-methylene-2,2-diphenyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)methoxy)diphenylsilane (S-10).
The compound was prepared by essentially same procedure used for compound S-9 from 0.685 g (1.015 mmol) of S-8; flash column
chromatography (hexane/EtOAc = 20:1) afforded S-10 as a colorless oil (0.433 g, 78 %). 1
H NMR (500 MHz, CDCl3): δ = 7.59-7.55 (m, 6H),
7.42-7.31 (m, 14H), 5.21 (m, 1H), 4.47 (d, J = 5.90 Hz, 1H), 4.41 (d, J = 5.9 Hz, 1H), 3.44 (m, 2H), 2.85 (m, 1H), 2.57 (m, 1H), 2.18 (m, 1H),
1.03 (s, 9H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 147.24, 138.94, 138.82, 135.85, 135.82, 134.02, 129.81, 129.79, 128.51, 128.45, 128.15,
12
128.00, 127.86, 127.65, 112.32, 110.93, 80.39, 79.53, 71.77, 69.65, 64.04, 43.66, 30.15, 27.11, 19.60 ppm. HR-MS (APCI) calculated for
C36H38O3Si [M+H]+
: 547.2663 Found: 547.2664.
(3aR*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-diphenyldihydro-3aH-cyclopenta[d][1,3]dioxol-4(5H)-one (S-11).
Prepared by general procedure B using 0.43 g (0.792 mmol) alkene S-10; flash column chromatography (hexane/EtOAc = 5:1) afforded S-11 as a
white crystalline solid (0.40 g, 92%). m.p. > 250 °C. 1
H NMR (500 MHz, CDCl3): δ = 7.58-7.53 (m, 4H), 7.51 (m, 2H), 7.44-7.26 (m, 14H), 4.57
(d, J = 6.03, 1H), 4.36 (d, J = 5.6, 1H), 3.76 (dd, J = 10.08, 2.77 Hz, 1H), 3.58 (dd, J = 10.08, 3.26 Hz, 1H), 2.76 (dd, J = 18.18, 9.20 Hz, 1H),
2.66 (m, 1H), 2.19 (d, J = 18.18 Hz, 1H), 0.99 (s, 9H) ppm. 13
C NMR (126 MHz): δ = 212.05, 141.74, 141.09, 135.93, 135.73, 132.84, 132.53,
130.21, 130.13, 128.69, 128.57, 128.48, 128.29, 128.06, 128.00, 126.58, 126.45, 112.05, 82.70, 79.51, 66.17, 39.64, 38.13, 27.04, 19.30 ppm. IR
(ν˜max) = 2961 (w), 1756 (s), 1104 (m), 1068 (s), 699 (s), 502 (m) cm-1
. HR-MS (ESI) calculated for: C35H36O4Si[M+Na]+
: 571.2271. Found:
571.2274.
(3aR*,4R*,6R*,6aR*)-4-(2,4-bis(benzyloxy)pyrimidin-5-yl)-6-(hydroxymethyl)-2,2-diphenyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4ol
(S-12).
13
Prepared by essentially same procedure used for compound 17, from bromide 11 (0.152 g, 0.41 mmol) and ketone S-11 (0.150 g, 0.273 mmol);
flash column chromatography (SiO2, hexane/EtOAc 10:1) afforded the adduct (0.170 g, 75 %), which was directly dissolved in THF (4 mL) and
deprotected with TBAF (1.0 M in THF, 338 μL, 0.338 mmol) according to general procedure D. Flash column chromatography (CH2Cl2/EtOAc
= 1:1) afforded compound S-12 as a colorless glassy solid (0.085 g, 73%). 1
H NMR (500 MHz, CDCl3): δ = 8.48 (s, 1H), 7.46-7.42 (m, 6H),
7.36-7.20 (m, 14H), 5.41 (AB dd, J = 12.2, 0.8 Hz, 2H), 5.38 (m, 2H), 4.48 (d, J = 7.8 Hz, 1H), 4.58 (dd, J = 7.8, 5.5 Hz), 3.67 (m, 1H), 3.53 (m,
1H), 3.44 (d, J = 1Hz, 1H, -OH), 2.83 (m, 1H), 2.24 (m, 1H), 2.13 (dd, J = 13.5, 7.2 Hz, 1H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 167.31,
164.47, 157.20, 141.71, 140.90, 136.89, 135.88, 128.96, 128.83, 128.67, 128.60, 128.52, 128.44, 128.36, 128.23, 126.51, 126.49, 117.58, 115.22,
84.50, 83.57, 76.39, 69.38, 68.98, 64.07, 45.60, 40.89 ppm. IR (ν˜max) = 2929 (w), 1592 (m), 1560 (m), 1422 (s), 1066 (s), 695 (s) cm-1
. HR-MS
(ESI) calculated for C37H34O6N2 [M+H]+
: 603.24896. Found: 603.24908.
5-((3aR*,4R*,6R*,6aR*)-4-hydroxy-6-(hydroxymethyl)-2,2-diphenyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-
2,4(1H,3H)-dione (S-13).
14
Prepared by general procedure C using using compound S-12 (67 mg, 0.111 mmol), Pd/C (0.002 g, 0.022 mmol), H2 (1 bar) in EtOH flash
column chromatography (CH2Cl2/MeOH = 5:1) afforded S-13 as a colorless semi-solid (38 mg, 81%). 1
H NMR (500 MHz, DMSO-d6): δ = 11.06
(br s, 1H, N-H), 10.85 (br s, 1H, N-H), 7.47-7.32 (m, 10H), 4.75 (d, J = 8.0 Hz, 1H), 4.64 (m, 1H), 4.32 (d, J = 4.2 Hz, 1H, -OH), 4.28 (dd, J =
7.9, 5.5 Hz, 1H), 3.46 (m, 2H), 2.62 (m, 1H), 2.27 (m, 1H), 1.74 (dd, J = 13.1, 6.8 Hz, 1H), 1.68 (dd, J = 12.2, 6.0 Hz, 1H) ppm. 13
C NMR (126
MHz, DMSO-d6): δ = 163.37, 151.11, 142.06, 141.23, 138.77, 128.25, 128.02, 127.77, 126.53, 125.96, 114.12, 113.52, 82.83, 82.74, 76.04,
62.04, 44.87, 40.64 ppm. IR (ν˜max) = 1708 (s), 1679 (s), 1455 (w), 1212 (m), 1100 (w), 722 (s) cm–1
. HR-MS (ESI) calculated for C23H22N2O6
[M+Na]+
: 445.1359. Found: 445.1436.
7-bromo-N,N-bis((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[2,1-f][1,2,4]triazin-4-amine (S-14).
A solution of 7-bromopyrrolo[1,2-f][1,2,4]triazin-4-amine (0.500 g, 2.34 mmol) in DMF (4 mL) was added to a suspension of NaH (60 % in
mineral oil, 0.234 g, 5.86 mmol) in DMF (1.5 mL) and the mixture was stirred at 25 °C for 30 min. SEM-Cl (0.872 mL, 4.92 mmol) was added
15
dropwise and the mixture was stirred at 25 °C for 5 h. The reaction mixture was quenched with water (10 mL) and extracted with EtOAc (3 × 25
mL). The organic phase was washed with brine (15 mL), dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was
purified by flash column chromatography (hexane/EtOAc 20 : 1) to afford S-14 (0.650 g, 58 %) as a colorless oil. 1
H NMR (500 MHz, CDCl3): δ
= 8.08 (s, 1H), 7.08 (d, AB, J = 4.8 Hz, 1H), 6.76 (d, AB, J = 4.8 Hz, 1H), 5.21 (s, 4H), 3.68 (m, 4H), 0.98 (m, 4H), 0.01 (s, 9H) ppm. 13
C NMR
(126 MHz, CDCl3): δ = 156.09, 147.52, 115.99, 113.96, 106.86, 102.09, 77.82, 66.32, 18.41, -1.18 ppm. IR (ν˜max) = 2954 (w), 1582 (w), 1517
(w), 1264 (m), 1074 (m), 858 (m), 732 (s) cm–1
. HR-MS (ESI) calculated for C18H34N4O2Si2Br [M+H]+
: 473.1403. Found: 473.1402.
(4-(bis((2-(trimethylsilyl)ethoxy)methyl)amino)pyrrolo[2,1-f][1,2,4]triazin-7-yl)boronic acid (29).
n-BuLi (1.6 M in hexanes, 2.4 mL, 3.84 mmol) was added to a solution of S-14 (0.909 g, 1.919 mmol) in THF (6 mL) at -78 °C and the mixture
was stirred at -78 °C for 30 min. 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.515 mL, 2.303 mmol) was added dropwise to the
reaction mixture at -78 °C. The reaction mixture was then allowed to warm to 25 °C and stirred for 1 h. Saturated aqueous solution of NH4Cl (10
mL) was added and the mixture was extracted with EtOAc (3 × 30 mL). The organic phase was washed with brine (15 mL), dried over MgSO4,
filtered, and concentrated under reduced pressure to yield the crude boronate. The boronate hydrolyzed during the purification by flash column
chromatography on silica gel (CH2Cl2/EtOAc = 9:1) to afford 29 (0.418 g, 50 %) as a yellow wax . 1
H NMR (500 MHz, DMSO-d6): δ = 8.35 (s,
2H, -B(OH)2), 8.16 (s, 1H), 7.20 (d, AB, J = 4.5 Hz, 1H), 7.01 (d, AB, J = 4.8 Hz, 1H), 5.22 (s, 4H), 3.65 (app t, J = 8.13 Hz, 4H), 0.91 (app t, J
= 8.13 Hz, 4H), -0.03 (s, 18H) ppm. 13
C NMR (126 MHz, DMSO-d6): δ = 155.74, 146.08, 126.25 (br, C-B(OH)2, detected through 1
H-13
C
16
HMBC), 120.16, 116.92, 106.21, 77.62, 65.07, 17.52, -1.45 ppm. 11
B NMR (160.5 MHz, DMSO-d6): δ = 25.90 (br) ppm. IR (ν˜max) = 2952 (m),
1585 (m), 1517 (w), 1370 (m), 1248 (m), 1075 (s), 856 (s), 832 (s) cm–1
. HR-MS (ESI) calculated for C18H34N4O4BSi2 [M-H]:
437.22171.
Found: 437.22107.
(1R*,2S*,5R*)-5-((tert-butyldiphenylsilyloxy)methyl)-3-phenylcyclopent-3-ene-1,2-diol (S-15).
Freshly prepared PPTS (0.111 g, 0.443 mmol) was added to a solution of 23a (0.043 g, 0.089 mmol) in MeOH (3 mL). The reaction mixture was
stirred at 25 °C for 96 h. The solvent was evaporated and the residue was purified by flash column chromatography (gradient elution from
hexane/EtOAc 10:1 to initially elute starting material - 18 mg, 33% recovered to CH2Cl2/EtOAc = 10:1) to afford product S-15 as a white semisolid
(0.015 g, 38 %). 1
H NMR (500 MHz, CDCl3): δ = 7.67-7.62 (m, 4H), 7.54-7.50 (m, 2H), 7.44-7.31 (m, 8H), 7.26 (m, 1H), 6.09 (d, J = 2.2
Hz, 1H), 4.96 (m, 1H), 4.22 (dd, J = 9.9, 4.9 Hz, 1H), 3.86 (dd, J = 10.0, 5.5 Hz, 1H), 3.79 (dd, J = 10.0, 5.5 Hz, 1H), 2.99 (m, 1H), 2.69 (d, J =
5.1 Hz, 1H, -OH), 2.43 (d, J = 4.7 Hz, 1H, -OH), 1.04 (s, 9H) ppm. 13
C NMR (126 MHz, CDCl3): δ = 143.55, 135.83, 135.79, 134.69, 133.60,
133.54, 129.99, 129.31, 128.78, 128.05, 127.98, 127.96, 126.26, 75.75, 74.77, 65.09, 54.00, 27.09, 19.45 ppm. IR (ν˜max) = 3300 (m), 2940 (w),
1215 (s), 1182 (s), 765 (s) cm-1
.
17
1
H NMR (500MHz) spectrum of S-1 in CDCl3
18
13
C NMR (126 MHz) spectrum of S-1 in CDCl3
19
1
H NMR (500 MHz) spectrum of 6a in CDCl3
*
*
*
*- EtOAc
20
13
C NMR (126 MHz) spectrum of 6a in CDCl3
21
1
H NMR (500 MHz) spectrum of 6b in CDCl3
22
13
C NMR (126 MHz) spectrum of 6b in CDCl3
23
1
H NMR (500 MHz) spectrum of 6c in CDCl3
24
13
C NMR (126 MHz) spectrum of 6c in CDCl3
25
1
H NMR (500 MHz) spectrum of 6d in CDCl3
26
13
C NMR (126 MHz) spectrum of 6d in CDCl3
27
1
H NMR (500MHz) spectrum of 6e in CDCl3
28
13
C NMR (126 MHz) spectrum of 6e in CDCl3
29
1
H NMR (500 MHz) spectrum of 6f in CDCl3
30
13
C NMR (126 MHz) spectrum of 6f in CDCl3
31
1
H NMR (500 MHz) spectrum of 6g in CDCl3
32
13
C NMR (126 MHz) spectrum of 6g in CDCl3
33
1
H NMR (500 MHz) spectrum of 6h in CDCl3
34
13
C NMR (126 MHz) spectrum of 6h in CDCl3
35
1
H NMR (500 MHz) spectrum of 6i in CDCl3
36
13
C NMR (126 Mhz) spectrum of compound 6i in CDCl3
37
1
H NMR (500 MHz) spectrum of S-2 in CDCl3
38
13
C NMR (126 MHz) spectrum of S-2 in CDCl3
39
1
H NMR (500 MHz) spectrum of S-3 in CDCl3
40
13
C NMR (126 MHz) spectrum of S-3 in CDCl3
41
1
H NMR (500 MHz) spectrum of S-4 in CDCl3
42
13
C NMR (126 MHz) spectrum of S-4 in CDCl3
43
1
H NMR (500 MHz) spectrum of S-5 in CDCl3
44
13
C NMR (126 MHz) spectrum of S-5 in CDCl3
45
1
H NMR (500 MHz) spectrum of S-6 in CDCl3
46
13
C NMR (126 MHz) spectrum of S-6 in CDCl3
47
1
H NMR (500 MHz) spectrum of 7b in CDCl3
48
13
C NMR (126 MHz) spectrum of 7b in CDCl3
49
1
H NMR (500MHz) spectrum of 7a in CDCl3
50
13
C NMR (126 MHz) spectrum of 7a in CDCl3
51
1
H NMR (500MHz) spectrum of S-7 in CDCl3
52
13
C NMR (126 MHz) spectrum of S-7 in CDCl3
53
1
H NMR (500MHz) spectrum of 7c in CDCl3
54
13
C NMR (126 MHz) spectrum of 7c in CDCl3
55
1
H NMR (500MHz) spectrum of 7d in CDCl3
56
13
C NMR (126 MHz) spectrum of 7d in CDCl3
57
1
H NMR (500 MHz) spectrum of 8a in CDCl3
58
13
C NMR (126 MHz) spectrum of 8a in CDCl3
59
1
H NMR (500 MHz) spectrum of S-8 in CDCl3
60
13
C NMR (126 MHz) spectrum of S-8 in CDCl3
61
1
H NMR (500 MHz) spectrum of S-9 in CDCl3
62
13
C NMR (126 MHz) spectrum of S-9 in CDCl3
63
1
H NMR (500 MHz) spectrum of S-10 in CDCl3
64
13
C NMR (126 MHz) spectrum of S-10 in CDCl3
65
1
H NMR (500 MHz) spectrum of 9b in CDCl3
66
13
C NMR (126 MHz) spectrum of 9b in CDCl3
67
1
H NMR (500 MHz) spectrum of 10b in CDCl3
68
13
C NMR (126 MHz) spectrum of 10b in CDCl3
69
1
H NMR (500 MHz) spectrum of 2a in CDCl3
70
13
C NMR (126 MHz) spectrum of 2a in CDCl3
71
1
H NMR (500 MHz) spectrum of S-11 in CDCl3
72
13
C NMR (126 MHz) spectrum of S-11 in CDCl3
73
1
H NMR (500 MHz) spectrum of 2b in CDCl3
74
13
C NMR (126 MHz) spectrum of 2b in CDCl3
75
1
H NMR (500 MHz) spectrum of 2c in CDCl3
76
13
C NMR (126 MHz) spectrum of 2c in CDCl3
77
1
H NMR (500 MHz) spectrum of 12 in CDCl3
78
13
C NMR (126 MHz) spectrum of 12 in CDCl3
79
1
H NMR (500 MHz) spectrum of S-12 in CDCl3
80
13
C NMR (126 MHz) spectrum of S-12 in CDCl3
81
1
H NMR (500 MHz) spectrum of 17a in CDCl3
82
13
C NMR (126 MHz) spectrum of 17a in CDCl3
83
1
H NMR (500 MHz) spectrum of 17b in CDCl3
84
13
C NMR (126 MHz) spectrum of 17b in CDCl3
85
1
H NMR (500 MHz) spectrum of 17c in CDCl3
86
13
C NMR (126 MHz) spectrum of 17c in CDCl3
87
1
H NMR (500 MHz) spectrum of 17d in CDCl3
88
13
C NMR (126 MHz) spectrum of 17d in CDCl3
89
1
H NMR (500 MHz) spectrum of 17e in CDCl3
90
13
C NMR (126 MHz) spectrum of 17e in CDCl3
91
1
H NMR (500 MHz) spectrum of 13 in CDCl3
92
13
C NMR (126 MHz) spectrum of 13 in CDCl3
93
1
H NMR (300 MHz) spectrum of 18a in CDCl3
94
13
C NMR (126 MHz) spectrum of 18a in CDCl3
95
1
H NMR (300 MHz) spectrum in CDCl3
96
13
C NMR (126 MHz) spectrum in CDCl3
97
1
H NMR (500 MHz) spectrum of 15b in DMSO-d6
98
13
C NMR (126 MHz) spectrum of 15b in DMSO-d6
99
1
H NMR (500 MHz) spectrum of 15c in DMSO-d6
100
13
C NMR (126 MHz) spectrum of 15c in DMSO-d6
101
1
H NMR (500 MHz) spectrum of 15d in DMSO-d6
102
13
C NMR (126 MHz) spectrum of 15d in DMSO-d6
103
1
H NMR (500 MHz) spectrum of 16 in CDCl3
104
13
C NMR (126 MHz) spectrum of 16 in CDCl3
105
1
H NMR (500 MHz) spectrum in CDCl3
106
13
C NMR (126 MHz) spectrum in CDCl3
107
1
H NMR (500 MHz) spectrum of 15a in DMSO-d6
108
13
C NMR (126 MHz) spectrum of 15a in DMSO-d6
109
Key NOE interactions observed in molecule 15a
110
1
H NMR (500 MHz) spectrum of 19a in CDCl3
111
13
C NMR (126 MHz) spectrum of 19a in CDCl3
112
1
H NMR (500 MHz) spectrum of 19b in CDCl3
113
13
C NMR (126 MHz) spectrum of 19b in CDCl3
114
1
H NMR (126 MHz) spectrum of 19c in CDCl3
115
13
C NMR (126 MHz) spectrum of 19c in CDCl3
116
1
H NMR (126 MHz) spectrum of 21 in CDCl3
117
13
C NMR (126 MHz) spectrum of 21 in CDCl3
118
1
H NMR (500 MHz) spectrum of 14 in DMSO-d6
119
13
C NMR (126 MHz) spectrum of 14 in DMSO-d6
120
1
H NMR (126 MHz) spectrum of 15e in CD3OD
121
13
C NMR (126 MHz) spectrum of 15e in CD3OD
122
1
H NMR (500 MHz) spectrum of S-12 in CDCl3
123
13
C NMR (126 MHz) spectrum of S-12 in CDCl3
124
1
H NMR (500 MHz) spectrum of S-13 in DMSO-d6
125
13
C (126 MHz) NMR spectra of S-13 in DMSO-d6
126
1
H NMR (500 MHz) spectrum of 22a in CDCl3
127
13
C NMR (126 MHz) spectrum of 22a in CDCl3
128
1
H NMR (500 MHz) spectrum of 22b in CDCl3
129
13
C NMR (126 MHz) spectrum of 22b in CDCl3
130
1
H NMR (500 MHz) spectrum of 23a in CDCl3
131
13
C NMR (126 MHz) spectrum of 23a in CDCl3
132
1
H NMR (500 MHz) spectrum of 24a in CDCl3
133
13
C NMR (126 MHz) spectrum of 24a in CDCl3
134
1
H NMR (500 MHz) spectrum of 25a in CDCl3
135
13
C NMR (126 MHz) spectrum of 25a in CDCl3
136
1
H NMR (500 MHz) spectrum of 25b in CDCl3
137
13
C NMR (126 MHz) spectrum of 25b in CDCl3
138
1
H NMR (500 MHz) spectrum of 25c in CDCl3
139
13
C NMR (126 MHz) spectrum of 25c in CDCl3
140
1
H NMR (500 MHz) spectrum of 25d in CDCl3
141
13
C NMR (126 MHz) spectrum of 25d in CDCl3
142
1
H NMR (500 MHz) spectrum of 25e in CDCl3
143
13
C NMR (126 MHz) spectrum of 25e in CDCl3
144
1
H NMR (500 MHz) spectrum of 25f in CDCl3
145
13
C NMR (126 MHz) spectrum of 25f in CDCl3
146
1
H NMR (500 MHz) spectrum of 26a in DMSO-d6
147
13
C NMR (126 MHz) spectrum of 26a in DMSO-d6
148
1
H NMR (500 MHz) spectrum of 26b in CDCl3
149
13
C NMR (126 MHz) spectrum of 26b in CDCl3
150
1
H NMR (500 MHz) spectrum of 26c in CDCl3
151
13
C NMR (126 MHz) spectrum of 26c in CDCl3
152
1
H NMR (500 MHz) spectrum of 26d in CDCl3
153
13
C NMR (126 MHz) spectrum of 26d in CDCl3
154
1
H NMR (500 MHz) spectrum of 26e in CDCl3
155
13
C NMR (126 MHz) spectrum of 26e in CDCl3
156
1
H NMR (500 MHz) spectrum of 26f in CD2Cl2
157
13
C NMR (126 MHz) spectrum of 26f in CD2Cl2
158
1
H NMR (500 MHz) spectrum of 27a in DMSO-d6
159
13
C NMR (126 MHz) spectrum of 27a in DMSO-d6
160
1
H NMR (500 MHz) spectrum of 27b in DMSO-d6
161
13
C NMR (126 MHz) spectrum of 27b in DMSO-d6
162
X-ray structure of 27b
163
1
H NMR (500 MHz) spectrum of 27c in DMSO-d6
164
13
C NMR (126 MHz) spectrum of 27c in DMSO-d6
165
1
H NMR (500 MHz) spectrum of 27d in DMSO-d6
166
13
C NMR (126 MHz) spectrum of 27d in DMSO-d6
167
1
H NMR (500 MHz) spectrum of 27e in DMSO-d6
168
13
C NMR (126 MHz) spectrum of 27e in DMSO-d6
169
1
H NMR (500 MHz) spectrum of 27f in CD3OD
170
13
C NMR (126 MHz) spectrum of 27f in CD3OD
171
1
H NMR (500 MHz) spectrum of 28 in DMSO-d6
172
13
C NMR (500 MHz) spectrum of 28 in DMSO-d6
173
1
H NMR (500 MHz) spectrum of S-14 in CDCl3
174
13
C NMR (126 MHz) spectrum of S-14 in CDCl3
175
1
H NMR (500 MHz) spectrum of 29 in DMSO-d6
*- EtOAc
*
*
*
176
13
C NMR (126 MHz) spectrum of 29 in DMSO-d6
177
1
H NMR (500 MHz) spectrum of 30 in CDCl3
178
13
C NMR (126 MHz) spectrum of 30 in CDCl3
179
1
H NMR (500 MHz) spectrum of S-15 in CDCl3
180
13
C NMR (126 MHz) spectrum of S-15 in CDCl3
181
1
H NMR (500 MHz) spectrum of 31a in CDCl3
182
13
C NMR (126 MHz) spectrum of 31a in CDCl3
183
1
H NMR (500 MHz) spectrum of 32a in CDCl3
184
13
C NMR (126 MHz) spectrum of 32a in CDCl3
185
1
H NMR (500 MHz) spectrum of 32b in CDCl3
186
13
C NMR (126 MHz) spectrum of 32b in CDCl3
187
1
H NMR (500 MHz) spectrum of 32c in CDCl3
188
13
C NMR (126 MHz) spectrum of 32c in CDCl3
189
1
H NMR (500 MHz) spectrum of 32d in CDCl3
190
13
C NMR (126 MHz) spectrum of 32d in CDCl3
191
1
H NMR (500 MHz) spectrum of 32e in CDCl3
192
13
C NMR (126 MHz) spectrum of 32e in CDCl3
193
1
H NMR (500 MHz) spectrum of 32f in CDCl3
194
13
C NMR (500 MHz) spectrum of 32f in CDCl3
195
1
H NMR (500 MHz) spectrum of 33a in DMSO-d6
196
13
C NMR (126 MHz) spectrum of 33a in DMSO-d6
197
1
H NMR (500 MHz) spectrum in DMSO-d6
198
13
C NMR (126 MHz) spectrum in DMSO-d6
199
Comparison of 1
H NMR resonances for 33a and corresponding epimer
200
Comparison of 13
C NMR resonances for 33a and corresponding epimer
201
1
H NMR (500 MHz) spectrum of 33b in DMSO-d6
202
13
C NMR (126 MHz) spectrum of 33b in DMSO-d6
203
1
H NMR (500 MHz) spectrum of 27c in DMSO-d6 + CDCl3
204
13
C NMR (126 MHz) spectrum of 27c in DMSO-d6 + CDCl3
205
1
H NMR (500 MHz) spectrum of 33d in DMSO-d6
206
13
C NMR (126 MHz) spectrum of 33d in DMSO-d6
207
1
H NMR (500 MHz) spectrum of 33e in DMSO-d6
208
13
C NMR (126 MHz) spectrum of 33e in DMSO-d6
209
1
H NMR (500 MHz) spectrum of 33f in CD2Cl2
210
13
C NMR (126 MHz) spectrum of 33f in CD2Cl2
211
1
H NMR (500 MHz) spectrum of 34 in CD3OD
212
13
C NMR (126 MHz) spectrum of 34 in DMSO-d6
213
Important NOE interactions observed in compound 34
214
1
H NMR (500 MHz) spectrum of 35 in CDCl3
215
13
C NMR (126 MHz) spectrum of 35 in CDCl3
216
1
H NMR (500 MHz) spectrum of 36 in DMSO-d6
217
13
C NMR (126 MHz) spectrum of 36 in DMSO-d6
218
¨
1
H NMR (500 MHz) spectrum of 37 in DMSO-d6
219
13
C NMR (126 MHz) spectrum of 37 in DMSO-d6
220
1
H NMR (500 MHz) spectrum of 38a in acetone-d6
221
13
C NMR (126 MHz) spectrum of 38a in acetone-d6
222
1
H NMR (500 MHz) spectrum of 38b in DMSO-d6
223
13
C NMR (126 MHz) spectrum of 38b in DMSO-d6
224
1
H NMR (500 MHz) spectrum of 38c in CDCl3
225
13
C NMR (126 MHz) spectrum of 38c in CDCl3
226
1
H NMR (500 MHz) spectrum of 39 in DMSO-d6
227
13
C NMR (126 MHz) spectrum of 39 in DMSO-d6
228
1
H NMR (500 MHz) spectrum of 40 in CDCl3
229
13
C NMR (126 MHz) spectrum of 40 in CDCl3
230
1
H NMR (500 MHz) spectrum of 41 in CDCl3
231
13
C NMR (126 MHz) spectrum of 41 in CDCl3
232
1
H NMR (500 MHz) spectrum of 42 in CDCl3
233
13
C NMR (126 MHz) spectrum of 42 in CDCl3
234
1
H NMR (500 MHz) spectrum of 43 in acetone-d6
235
13
C NMR (126 MHz) spectrum of 43 in acetone-d6
236
1
H NMR (500 MHz) spectrum of (+)-46b in CDCl3
237
13
C NMR (126 MHz) spectrum of (+)-46b in CDCl3
238
1
H NMR (500 MHz) spectrum of (-)-46b in CDCl3
239
13
C NMR (126 MHz) spectrum of (-)-46b in CDCl3
240
Portion of 1
H NMR spectrum of diastereomers 46b
241
1
H NMR (500 MHz) spectrum in CDCl3
242
13
C NMR (126 MHz) spectrum in CDCl3
243
1
H NMR (500 MHz) spectrum in CDCl3
244
13
C NMR (126 MHz) spectrum in CDCl3
245
IR spectrum (neat) of S-11
246
IR spectrum (neat) of 2b
247
IR spectrum (neat) of 2c
248
IR spectrum (neat) of 15a
249
IR spectrum (neat) of 21
250
IR spectrum (neat) of 27a
251
IR spectrum (neat) of 33a
252
IR spectrum (neat) of 25f
253
IR spectrum (neat) of 26f
254
IR spectrum (neat) of 34
255
Table 1. Gradient elution profile for RP-HPLC (Nucleodur®
C18 HTec, 5μm, 250 mm × 50 mm) purification of 15a and 15e (solvent A –
MeOH, solvent B – H2O).
Time [min] Solvent B [%] Flow [ml/min]
0.00 90.0 10.00
2.00 60.0 10.00
4.00 55.0 10.00
6.00 50.0 10.00
8.00 45.0 10.00
10.00 40.0 10.00
12.00 30.0 10.00
14.00 15.0 10.00
25.00 90.0 10.00
HPLC Chromatogram of compound 15a (UV detection at 210 nm).
TBAF impurity
256
CD spectra of enantiomers of compound 26 (EtOH, 10-3
M)
-20
-16
-12
-8
-4
0
4
8
12
16
20
24
195 210 225 240 255 270
ellipticity(mdeg)
wavelength (nm)
LM1588
LM1589
257
HPLC chromatogram of racemic compound 27 and corresponding HPLC analysis of enantiomers (+)-26b and (-)-27b. (Daicel-CHIRALPAK AS
4.6 mm × 250 mm; hexane/EtOH = 96.5 / 3.5; 2 mL/min; rt; tR = 6.02 min for (+)-26b, 6.60 min for (-)-26b.
258
Table 2. Gradient elution profile for RP-HPLC (Nucleodur®
C18 HTec, 5μm, 250 mm × 50 mm) purification of compounds 28 and 34 (
(solvent A – MeOH, solvent B – H2O).
Time [min] Solvent B [%] Flow [ml/min]
0.00 90.0 10.00
2.00 65.0 10.00
4.00 55.0 10.00
6.00 52.0 10.00
8.00 50.0 10.00
10.00 45.0 10.00
13.00 90.0 10.00
259
HPLC chromatogram of tubercidine analog 34
260
Cell cultures
MCF7 breast cancer cells was cultivated in Minimum Essential Media (MEM) with L-Glutamine, Proline and Pyruvate (Gibco) supplemented
with penicillin (100 U/mL), streptomycin (0.1 mg/mL) and 10% fetal bovine serum (Gibco).
Human foreskin fibroblasts HFF1 were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 15% fetal bovine
serum (Gibco).
All cells were harvested after a brief incubation in 0.05% ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS), followed
by trypsinization (0.25% w/v trypsin/0.53 mM EDTA in PBS). They were then counted by using a CASY TT automatic cell counter (Roche
Diagnostics, Prague, Czech Republic), diluted in the appropriate volume and seeded for experimental procedure.
MCF7 and HFF1 cell lines were obtained from American Type Culture Collection (LGC Standards, Warsaw, Poland).
Cell proliferation assays
For cytotoxicity screening the cells were seeded at the density of 20 000 cells/cm2
on black Corning 96 well plates with clear flat bottom. After
24 hrs, cells were treated with tubercidine or compound 34 (3 wells per concentration, range 0,0015 uM to 100 uM using 9 points). Vehicle
(DMSO) was added at the same time as the control. The cells were grown during next 24 hrs. The medium was then gently removed, the cells
were refurbished with fresh medium and allowed to proliferate for 48 hrs. Finally, the cells were harvested and analysed using the CyQuant
assay. The CyQuant cell proliferation assay (Invitrogen) was performed according to the manufacturer’s recommendations and the results were
analysed using a Fluostar Galaxy reader (BMG Labtech, Ortenberg, Germany).
Data analysis
The data were evaluated as % of viable cell normalized to the control (DMSO). For calculation of IC50 values, a four-parameter logistic doseresponse
model with a sigmoidal shape was used: Y=Bottom + (Top-Bottom)/1+(x/IC50)^HillSlope where IC50 denoted the concentration of the
inhibitor that gave a response that was halfway between Bottom and Top. HillSlope described the steepness of the curve, and the Top and Bottom
denoted plateaus in the units of the Y-axis. Lower and upper bound of a 95% confidence interval for IC50 was calculated.