1H-pyrazolo[3,4-b]pyridine inhibitors of cyclin-dependent kinases: Highly potent 2,6-difluorophenacyl analogues

Article abstract of DOI:10.1016/S0960-894X(03)00381-0

Structure-activity studies of 1H-pyrazolo[3,4-b]pyridine 1 have resulted in the discovery of potent CDK1/CDK2 selective inhibitor 21h, BMS-265246 (CDK1/cycB IC₅₀=6 nM, CDK2/cycE IC₅₀=9 nM). The 2,6-difluorophenyl substitution was cri

Full text of DOI:10.1016/S0960-894X(03)00381-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2405–2408
1H-Pyrazolo[3,4-b]pyridine Inhibitors of Cyclin-Dependent
Kinases: Highly Potent 2,6-Difluorophenacyl Analogues
Raj N. Misra,* Hai-yun Xiao, David B. Rawlins, Weifang Shan,
Kristen A. Kellar, Janet G. Mulheron,y John S. Sack, John S. Tokarski,
S. David Kimbally and Kevin R. Webster{
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA
Received 30 December 2002; accepted 13 March 2003
Abstract—Structure–activity studies of 1H-pyrazolo[3,4-b]pyridine 1 have resulted in the discovery of potent CDK1/CDK2 selec-
tive inhibitor 21h, BMS-265246 (CDK1/cycB IC50=6 nM, CDK2/cycE IC50=9 nM). The 2,6-difluorophenyl substitution was cri-
tical for potent inhibitory activity. A solid state structure of 21j, a close di-fluoro analogue, bound to CDK2 shows the inhibitor
resides coincident with the ATP purine binding site and forms important H-bonds with Leu83 on the protein backbone.
# 2003 Elsevier Science Ltd. All rights reserved.
Cyclin-dependentkinases (CDKs) are a family of pro-
tein kinases which along with their regulatory subunit
cyclins play a key role in the growth, development,
proliferation and death of eukaryotic cells and are
responsible for insuring integrity in the coordination of
events through the cell cycle.1 Due to both their central
role in the cell cycle and their misregulation in a number
of cancers, CDKs have been implicated as contributing
factors to the development of cancer. Consequently,
oncology drug discovery programs have directed major
efforts towards the identification of small molecule inhi-
bitors of CDKs as potential therapeutic agents.2 Our
screening efforts recently revealed that pyrazolo[3,4-
b]pyridines SQ-67563 (1) and SQ-67454 (2) as CDK
inhibitors with relatively potent enzyme inhibitory
activity and selectivity for the CDK family (Fig. 1).3ꢀ5
Described herein are the synthesis and SAR of the
5-phenacyl substituent and the solid-state structure of an
optimized inhibitor of this chemotype bound to CDK2.
of PMB-protected aminopyrazole 35 and condensation
with commercial acrylate 4 followed by pyrolysis in
diphenyl ether solution (240 ꢁC) afforded annulated
pyrazole 5. Treatment of 5 with neat phosphorous oxy-
chloride gave 4-chloropyrazolopyridine 6. Introduction
of the 4-butyloxy substituent via the alkoxide followed
by ester hydrolysis afforded acid 7. Acid chloride cou-
pling of 7 with N,O-dimethylhydroxylamine followed by
removal of the PMB group with TFA yielded key
intermediate 9. In general, treatment of 9 with excess
(5–10 equiv) of the appropriate organolithium afforded
the desired 5-keto substituted analogues 10 and 21
(Tables 1 and 2).6,7
In contrast to organolithium reagents, addition of
Grignard reagents to 9 gave competing substitution for
the 4-butoxy group. Attempts to add aryllithiums to
PMB-protected intermediate 8 also resulted in pre-
dominately substitution for the 4-butyloxy group.
Amido analogues 10h and 10i were available from acid
7 as shown in Scheme 2 by generation of the acid chlo-
ride, addition of appropriate amine followed by removal
of the PMB group with hot TFA.
Acyl analogues (Tables 1 and 2) were prepared via
addition to key intermediate amide 9. The overall syn-
thetic route is shown in Scheme 1. Thus, neutralization
The 5-thio analogues4d were prepared as shown in
Scheme 3 from bicyclic pyridinol ester 5. Hydrolysis of
the ester gave acid 12 which upon heating smoothly
decarboxylated to afford pyridinone 13. Treatment of
13 with bromine gave aryl bromide 14. The 4-butoxy
group group was introduced by chlorination of 14 with
*Corresponding author. E-mail: raj_n_misra@hotmail.com
yCurrent address: Lexicon Pharmaceuticals, 350 Carter Road, Prince-
ton, NJ 08540, USA.
{Current address: Astra-Zeneca R&D Boston, 35 Gatehouse Dr.,
Waltham, MA 02451, USA.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00381-0

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R. N. Misra et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2405–2408
Figure 1. Structure of pyrazolo[3,4-b]pyridine screening hits SQ-67563
(1) and SQ-67454 (2).
Table 1. SAR of pyrazolopyridine acyl substituent 10
Compd R Group CDK1/cycB IC50, mMa CDK2/cycE IC50, mMa
10a
10b
10c
1
10d
10e
10f
10g
10h
10i
10j
Me
Cyclopropyl
Benzyl
—
—
—
0.15
—
0.28
0.23
0.45
—
>1.0
2.7
>1.0
0.11
2.4
0.18
0.20
0.21
19
8.1
>1.0
Scheme 1. Synthesis of 5-keto analogues of pyrazolopyridine 1: (a)
desalt; (b) 130 ꢁC, 2.5 h; (c) Ph2O, 240 ꢁC, 1–2 h, 59% from 3; (d)
POCl3, 120 ꢁC 1 h, 66%; (e) nBuONa (3 equiv)/nBuOH, 65 ꢁC, 3 h
then H2O added, 65 ꢁC, 3 h, 100%; (f) (COCl)2/catDMF/CH 2Cl2,
25 ꢁC, then (MeO)NH(Me)HCl, Et3N, 0–25 ꢁC, 93%; (g) TFA, 65 ꢁC,
2.5 h, 89%; (h) ArLi (5–10 equiv)/THF, ꢀ78 to 0 ꢁC, 10–90%.
Ph–
3-Pyridyl
2-Furanyl
2-Thienyl
2-Thiazolyl
PhNH–
EtNH–
—
—
MeO–
aSee ref 8 for description of biological assays.
Table 2. SAR of pyrazolopyridine phenyl substitution
Scheme 2. Synthesis of 5-amido analogues: (a) (COCl)2/catDMF/
CH2Cl2, 25 ꢁC then EtNH2/Et3N; (b) TFA, 65 ꢁC, 2.5 h, 58% from 7.
Table 1 shows the effect of replacement of the phenyl
group in the 5-position on in vitro CDK inhibitory
activity in a cell-free enzyme assay.8 Thus, small alkyl
groups (10a,b) or benzyl (10c) resulted in >10-fold loss
of CDK2 potency. Replacement of the phenyl group
with a pyridyl ring (10d), a small basic heterocycle, also
resulted in a ꢂ200-fold decrease in potency. In contrast,
small neutral heterocycles, such as furan, thiophene and
thiazole (10e–g), afforded analogues with CDK1 and
CDK2 inhibitory potency comparable to phenyl analo-
gue 1. An amide or methyl ester functionality (10h–j) at
the 5-position resulted in a >10 to 200-fold loss in
CDK2 inhibitory potency. Interestingly, both racemic
sulfoxide 19 and sulfone 20 were comparatively potent
inhibitors of CDK2 with IC50=0.34 and 0.52 mM,
respectively, indicating that a sulfone or sulfoxide may
serve as a surrogate for the carbonyl group.
Compd
R Group(s)
CDK1/
cycB
CDK2/
cycE
CDK4/
cycD
IC50, mMa IC50, mMa IC50, mMa
21a (1)
21b
21c
21d
21e
21f
21g
21h
21i
H
2-Me
3-Me
0.15
0.41
—
0.11
0.73
0.66
0.21
0.036
0.064
0.036
0.009
0.027
0.020
0.022
>1.00
>25
—
—
—
24
21
0.69
0.23
0.31
0.37
0.23
4-Me
2-F
2,6-Difluoro
—
0.36
0.032
0.092
0.006
0.013
0.017
0.007
—
2,4,6-Trifluoro
2,6-Difluoro-4-methyl
2,6-Difluoro-4-chloro
2,6-Difluoro-4-bromo
2,6-Difluoro-4-methoxy
2,4,6-Trimethyl
21j
21k
21l
—
aSee ref 8 for description of biological assays.
POCl3 followed by stirring the intermediate 4-chloro
analogue 15 with sodium n-butoxide to give 16. The
5-thio substituent was introduced by low temperature
metal–halogen exchange of 16 and quenching the
resulting anion with diphenyl disulfide to provide 17.
TFA removal of the PMB group afforded 5-thiophenyl
analogue 18. Oxidation of 18 with 1 equiv of mCPBA
provided the racemic 5-sulfoxide 19 while oxidation
with excess mCPBA gave and 5-sulfone analogue 20.
Shown in Table 2 is an examination of phenyl ring sub-
stituent effects on CDK inhibitory potency. Thus, sub-
stitution of a methyl group in the ortho or meta position
of 1 (21b,c) gave a 6- to 7-fold loss in CDK2 inhibitory
potency, while para methyl substitution (21d) resulted in
only a modest 2-fold loss in CDK2 inhibitory potency
relative to 1. In contrast, mono- and di-ortho fluoro
substitution (21e and 21f) both resulted in 2- to 3-fold
increases in CDK2 inhibitory potency relative to 1. Para

R. N. Misra et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2405–2408
2407
Scheme 3. Synthesis of 5-thio analogues 18–20: (a) aq NaOH/EtOH,
95 ꢁC, 94%; (b) 230 ꢁC, 15 min, 100%; (c) Br2, EtOH, 0 ꢁC, 97%; (d)
POCl3, 110 ꢁC, 1 h, 95%; (e) nBuONa/nBuOH, 60 ꢁC, 2 h, 86%; (f)
nBuLi/THF, ꢀ78 ꢁC, 30 min then PhSSPh, 29%; (g) TFA, 65 ꢁC, 2.5
h, 62%; (h) mCPBA (1 equiv)/CH2Cl2, 0 ꢁC, 74% for 19 or mCPBA(3
equiv)/CH2Cl2, 25 ꢁC, 78% for 20.
substitution coupled with 2,6-difluoro substitution
(21g–k) afforded analogues that were ꢂ3- to 12-fold
more potent CDK2 inhibitors than 1. In particular, the
2,6-difluoro-4-methylphenyl analogue 21h exhibited
CDK1 and CDK2 potency that was 25- and 11-fold
more potent versus CDK1 and CDK2, respectively,
than phenyl analogue 1 and represented the most potent
CDK/CDK2 selective analogue from this chemotype. In
contrast to 21h, the 2,4,6-trimethylphenyl analogue 21l
was a very poor inhibitor of CDK2 indicating that the
ortho fluoro substituents were a key for potent inhibi-
tory activity. In an ovarian cancer cell line (A2780)
inhibitor 21h produced a cytotoxic effect with an
IC50=0.76 mM.8
Figure 2. Top: Solid-state structure of pyrazolopyridine 21j bound in
the ATP-pocket of CDK2 (no cyclin). The inhibitor carbon atoms are
colored green, the nitrogen atoms are colored blue and oxygen atoms
are colored red. The protein carbons are colored gray. Hydrogen
bonds are shown by the magenta dotted lines. Bottom: Overlay of
compound 1 (magenta) and 21j (green) bound to CDK2 showing
location of 5-phenacyl substituent. The nitrogen atoms are colored
blue, the oxygen atoms are colored red, the fluorine atoms are colored
slate and the bromine atom is colored in brown.
The three-dimensional structure of 2,6-difluoro analo-
gue 21j in complex with CDK2 was determined by
X-ray crystallography. Crystals were obtained by incu-
bating inhibitor 21j (72 h) with crystalline protein in the
absence of cyclin.8 The crystal structure (Fig. 2)
revealed that 21j binds in the ATP-binding site as seen
previously with 1 forming important hydrogen bonds
between the pyrazolopyridine ring and Leu83.
In this arrangement small, flat pendent aryl groups
would be expected to bind optimally to the protein
while larger groups would create unfavorable steric
interactions with Phe80. This is consistent with the SAR
shown in Table 1 in which benzyl (10c) and amide sub-
stitution (10h and 10i) showed diminished activity.
Interestingly, although the pendent phenyl rings of 1
and 21j occupy nearly the same region in space the car-
bonyl groups appear to be directed orthogonally. The
contrasting orientation of the carbonyl group indicates
that it is unlikely that the carbonyl oxygen is involved in
a H-bonding interaction. The 2,6-difluorophenyl ring
lies nearly coplanar with the carbonyl group (torsional
angle=7.4ꢁ) which may provide an explanation for the
The 4-butoxy substituent extends into the space occu-
pied by the ribose of ATP and does not appear to form
any specific contacts with the protein while the pendent
difluorophenyl ring lies buried within the protein,
stacking with Phe80. An overlay of inhibitors 1 and 21j
clearly show similar binding modes (see Fig. 2, bottom).

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R. N. Misra et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2405–2408
reduced activity of the 2,4,6-trimethylphenyl analogue
21l which requires significantskewing of the phenyl and
carbonyl groups.
2. (a) Webster, K. R. Exp. Opin. Invest. Drugs 1998, 7, 1. (b)
Webster, K. R.; Kimball, S. D. Emerg. Drugs 2000, 5, 45. (c)
Sielecki, T. M.; Boylan, J. F.; Trainor, G. L. J. Med. Chem.
2000, 43, 1. (d) Kimball, S. D.; Webster, K. R. Annu. Rpt.
Med. Chem. 2001, 36, 139.
3. (a) Hoehn, H.; Denzel, T.; Janssen, W. J. Heterocycl.
Chem. 1972, 9, 235. (b) Denzel, T.; Hoehn, H. Arch. Pharm
1976, 309, 486.
In summary, SAR studies of pyrazolopyridine 1 have
resulted in the discovery of potent, CDK1/CDK2
4. (a) Presented in part: Misra, R. N.; Rawlins, D. B.; Bursu-
ker, I.; Kellar, K. A.; Kimball, S. D.; Mulheron, J. G.; Sack, J.
S.; Shan, W.; Xiao, H. Y.; Webster, K. R. Abstracts of Papers,
219th National Meeting of the American Chemical Society,
San Francisco, CA, Mar 26–30, 2000; MEDI038. (b) Misra,
R. N.; Rawlins, D. B.; Batorsky, R.; Bursuker, I.; Kellar, K.
A.; Kimball, S. D.; Mulheron, J. G.; Sack, J. S.; Shan, W.;
Xiao, H. Y.; Webster, K. R. Abstracts of Papers, 91stAmer-
ican Association of Cancer Research National Meeting, San
Francisco, CA, April 1–5, 2000; Abstract No. 3002. (c) Misra,
R. N.; Kimball, S. D.; Rawlins, D. B.; Webster, K. R.; Bur-
suker, I. US Patent 6,107,305, 2000. (d) Misra, R. N. US
Patent 6,448,264 B2, 2002.
selective inhibitor 21h (BMS-265246). The 2,6-difluoro-
phenyl substitution pattern was critical for potent inhi-
bitory activity.
5. Misra, R. N.; Rawlins, D. B.; Xiao, H. Y.; Shan, W.; Bur-
suker, I.; Kellar, K. A.; Mulheron, J. G.; Sack, J. S.; Tokarski,
J. S.; Kimball, S. D.; Webster, K. R. Bioorg. Med. Chem. Lett.
2003, 13, 1133.
6. Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 22, 3815.
7. Ketones 10c and 21l were prepared by addition of benzyl
Grignard and 2,4,6-trimethylphenyllithium, respectively, to
the acid chloride derived from 7, followed by TFA deprotec-
tion.
8. For a description of biological assays and crystallographic
procedures see: Kim, K. S.; Kimball, S. D.; Misra, R. N.;
Rawlins, D. B.; Hunt, J. T.; Xiao, H.-Y.; Lu, S.; Qian, L.;
Han, W.-C.; Shan, W.; Mitt, T.; Cai, Z.-W.; Poss, M. A.; Zhu,
H.; Sack, J. S.; Tokarski, J. S.; Chang, C.-Y.; Pavletich, N.;
Kamath, A.; Humphreys, W. G.; Marathe, P.; Bursuker, I.;
Kellar, K. A.; Roongta, U.; Batorsky, R.; Mulheron, J. G.;
Bol, D.; Fairchild, C. R.; Lee, F. Y.; Webster, K. R. J. Med.
Chem. 2002, 45, 3905.
Acknowledgements
We thank Dr. Bang-Chi Chen and Mr. Mark S. Bed-
narz for preparation of chemical intermediates and Dr.
John T. Huntfor scientific guidance.
References and Notes
1. (a) Hunter, T.; Pines, J. Cell 1994, 79, 573. (b) Sherr, C.
Science 1996, 274, 1672. (c) Review: Pines, J. Semin. Cancer
Biol. 1994, 5, 305.