1H-Pyrazolo[3,4-b]pyridine inhibitors of cyclin-dependent kinases

Article abstract of DOI:10.1016/S0960-894X(03)00034-9

1H-Pyrazolo[3,4-b]pyridine 3 (SQ-67563) has been shown to be a potent, selective inhibitor of CDK1/CDK2 in vitro. In cells 3 acts as a cytotoxic agent with the ability to block cell cycle progression and/or induce apoptosis. The solid state structure of 3 bound to CDK2 shows 3 resides coincident with the ATP purine binding site and forms important H-bonding interactions with Leu83 on the protein backbone.

Full text of DOI:10.1016/S0960-894X(03)00034-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1133–1136
1H-Pyrazolo[3,4-b]pyridine Inhibitors of Cyclin-Dependent
Kinases
Raj N. Misra,* David B. Rawlins, Hai-yun Xiao, Weifang Shan, Isia Bursuker,
Kristin A. Kellar, Janet G. Mulheron,y John S. Sack, John S. Tokarski,
S. David Kimball,y and Kevin R. Webster{
Bristol-Myers Squibb Pharmaceutical Research Institute, PO 4000, Princeton, NJ 08543-4000, USA
Received 18 October 2002; accepted 18 December 2002
Abstract—1H-Pyrazolo[3,4-b]pyridine 3 (SQ-67563) has been shown to be a potent, selective inhibitor of CDK1/CDK2 in vitro. In
cells 3 acts as a cytotoxic agent with the ability to blockcell cycle progression and/or induce apoptosis. The solid state structure of 3
bound to CDK2 shows 3 resides coincident with the ATP purine binding site and forms important H-bonding interactions with
Leu83 on the protein backbone.
# 2003 Elsevier Science Ltd. All rights reserved.
Cyclin-dependent kinases (CDKs) are a family of pro-
tein kinases which play a key role in the growth, devel-
opment, proliferation and death of eukaryotic cells.1 In
particular, CDKs along with their regulatory subunit
cyclins are responsible for coordinating events which
progress cells through the cell cycle and insure the
genetic integrity of daughter cells. Due to their role as
drivers of cell growth and division, combined with their
hyperactivation in a number of cancers, oncology drug
discovery programs have directed a major effort
towards the identification of small molecule inhibitors
of CDKs as potential therapeutic agents.2 Olomoucine
(1)3 is a purine-based ATP-competitive inhibitor of
CDKs with moderate potency which was identified a
number of years ago. More recently, flavopiridol (2), a
synthetic flavone, was described as a broad-spectrum
ATP-competitive CDK inhibitor and has progressed into
Phase 2 clinical trials as an anti-tumor agent.4 (Fig. 1)
representative from this class bound to human CDK2
are described.6
Our initial focus in this series was on examining the effect
on inhibitory potency of substituents at the 4-position of
the pyrazolopyridine ring. The 4-substituted analogues
in Table 1 were prepared from the 4-chloro intermediate
10 shown in Scheme 1. Thus, PMB-protected aminopyr-
azole 7 was prepared by condensation of acrylonitrile
and p-methoxybenzaldehyde followed by based-induced
rearrangement of the crude imine to give the pyrazole
which was isolated as the hydrochloride salt.7,8
Our screening efforts recently revealed pyrazolo[3,4-
b]pyridines SQ-67563 (3) and SQ-67454 (4)5 were CDK
inhibitors with potent in vitro activity (Table 1).
Described herein are the synthesis and preliminary SAR
of CDK inhibitors based on this chemotype. In addi-
tion, cellular effects and an X-ray crystal structure of a
Neutralization of salt 7 and condensation of the free
amine with acrylate 89,10 followed by pyrolysis (240–
260 ꢀC) in diphenyl ether afforded fused bicyclic inter-
mediate 9. Heating of 9 with phosphorous oxychloride
gave 4–chloropyrazolopyridine 10. Treatment of 10
with the appropriate anion followed by removal of the
PMB protecting group in neat, hot TFA afforded the
desired analogues 11.
*Corresponding author. E-mail: raj.misra@bms.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)00034-9

1134
R. N. Misra et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1133–1136
trast with flavopiridol which also shows potent inhibi-
tory activity towards CDK4. Among other
representative kinases pyrazolopyridine 4 did not show
inhibition of PKA, PKC or LCK (IC50>50 mM, data
not shown). The N-ethyl analogue 5 did not inhibit
CDK2 indicating that an N–H at this position was
either critical to binding to the enzyme or an alkyl
group was not sterically tolerated in this position.
Crystallographic studies confirmed that in this class of
compounds the NH formed an important H-bonding
interaction with the enzyme (vide infra). Examination of
the 4-position (Table 2) showed that replacement of the
oxygen with either nitrogen or sulfur (3 vs 11a,b) results
in a significant loss in CDK2 inhibitory activity.
Straight-chain, branched or cyclic alkoxy groups (3, 5,
and 11c–f) were all tolerated in the 4-position. Benzyl-
oxy was also tolerated although unexpectedly phenoxy
shows a 50-fold loss in CDK2 inhibitory potency (11g
vs 11h). Finally, introduction of polar substituents,
hydroxy and dimethylamino, in the 4-position resulted
in a significant loss in potency (11i and 11j). Exami-
nation of the 5-keto group indicated that it was also
essential for potent CDK inhibitory activity. Thus,
reduction to the alcohol 12 or further to a methylene
analogue 13 afforded compounds devoid of CDK2
inhibitory activity (IC50 >25 mM). Removal of the
ketone while retaining an sp2 carbon at the 5-position to
afford exo methylene analogue 14 also resulted in a loss
of CDK2 inhibitory activity (IC50>25 mM).
Figure 1. Structures of ATP-competitive inhibitors olomucine (1) and
flavopiridol (2).
Table 1. Structure of CDK screening hits SQ-67563 (3) and SQ-
67454 (4) and inhibition of CDKsa
Compd CDK1/cycB CDK2/cycE CDK4/cycD CDK7/cycH
IC50, mM
IC50, mM
IC50, mM
IC50, mM
2
3
4
5
0.03
0.15
0.24
—
0.17
0.11
0.18
0.10
>25
>25
—
0.40
—
1.1
—
>25
aSee ref 11 for description of biological assays.
In order to determine the importance of the carbonyl
functionality in the 5-position, we prepared the corre-
sponding alcohol, des-keto and olefin analogues of 3 as
shown in Scheme 2. Thus, carbinol analogue 12 was
prepared by sodium borohydride reduction of 3. Ionic
reduction of 12 provided benzyl analogue 13 while
methylene analogue 14 was obtained by addition of
methyllithium to ketone 3 followed by acid-catalyzed
dehydration of the crude tertiary carbinol intermediate.
In order to understand the molecular basis of the
observed relationship between structure and biological
activity of this series, the three-dimensional structure of
3 in complex with CDK2 was determined by X-ray
crystallography. Crystals were obtained by incubating
inhibitor 3 (72 h) with crystalline protein in the absence
of cyclin.11 The crystal structure (Fig. 2) revealed that 3
binds in the ATP-binding site with the pyrazolopyridine
ring adjacent to Leu83 in the space occupied by the ATP
purine ring. In this configuration the pyridine nitrogen
acts as an acceptor for the amide NH of Leu83. The
hydrogen on N1 of the pyrazolopyridine ring serves as a
H-bond donor to the carbonyl oxygen of Leu83. This is
consistent with the SAR which indicates that the alkyl-
ation of N1 is not tolerated (4 vs 5) and substituents in
Pyrazolopyridines 3–5 and 11–13 were examined for
their ability to inhibit CDKs in vitro.11 The results from
cell-free enzyme assays are summarized in Tables 1 and
2. Thus, 3 and 5 were found to be selective inhibitors of
CDK1/CDK2 within the CDK family. This is in con-
Scheme 1. Synthesis of 4-substituted pyrazolopyridine analogues 11:
(a) NH2NH.2H2O (1 equiv), THF, 0–25 ꢀC, 2 h; then p-methoxy-
benzaldehyde, (1 equiv), 25 ꢀC, 2 h; (b) nBuONa (1 equiv)/nBuOH,
25–120 ꢀC, 3 h then HCl, 42% from acrylonitrile; (c) desalt, 10%
K2CO3; (d) 8 (1 equiv), 120 ꢀC, 1.5 h then Ph2O, 250 ꢀC, 1.5 h, 40%;
(e) POCl3, 110 ꢀC, 60%; (f) NaX–Y, CH2Cl2, 25 ꢀC, 6 h; (g) TFA,
65 ꢀC, 2.5 h, 50–80% from 10.
Scheme 2. Synthesis of 5-keto analogues 12–14: (a) NaBH4/EtOH,
25 ꢀC, 84%; (b) TFA/Et3SiH (1:4), 25 ꢀC, 69%; (c) MeLi (3 equiv)/
ether, THF, 0 ꢀC; (d) TFA/CH2Cl2, 25 ꢀC, 54%.

R. N. Misra et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1133–1136
Table 2. SAR of 4-substituent of pyrazolopyridines 11a
1135
Compd
X
Y
CDK1/cycB IC50, mM
CDK2/cycE IC50, mM
11a
11b
11c
3
11d
11e
11f
11g
11h
11i
11j
S
NMe
O
O
O
O
O
O
O
n-Butyl
n-Butyl
n-Propyl
n-Butyl
n-Pentyl
—
—
4.4
>25
0.18
0.11
0.24
0.52
0.66
0.28
15
0.22
0.15
0.31
0.31
0.60
0.35
—
(1-Methyl)butyl
Cyclohexyl
Benzyl
Phenyl
(2-Hydroxy)ethyl
(2-Dimethylamino)ethyl
O
O
—
—
3.7
17
aSee ref 11 for description of biological assays.
the 4-position which effect the basicity of the pyridine
nitrogen also have a large effect on activity. The 4-
alkoxy substituent extends into the space occupied by
the ribose of ATP and does not appear to form any
specific contacts with the protein. Although this posi-
tion tolerated a wide number of lipophilic substituents it
was surprising that we were unable to successfully
introduce simple polar substituents (e.g., 11i and 11j)
into the ribose binding region. It was also surprising
that the SAR indicates that the oxygen at the 4-position
cannot be replaced by a sulfur or nitrogen since the
structure did not show specific interactions with the
protein. It is possible that the 4-oxo substituent may
simply provide the optimal electronic characteristics to
maximize hydrogen bonding of the pyrazolopyridine to
Leu83 and/or be sufficiently small in comparison to a
alkylthio or dialkylamino group not to interfere with
the orientation of the 5-phenacyl side chain. The pen-
dent phenyl group was found to lie deep within the
ATP-pocket stacking neatly with the side chain of
Phe80. The SAR indicates the importance of the 5-keto
oxygen which may serve as a hydrogen bond acceptor
although we do not consistently see a hydrogen bonding
interaction with the ketone and Lys33. Finally, we were
unable to explain the observed CDK1/CDK2 selectivity
of this series of inhibitors based on the solid-state
structure since there are few interactions with either
Ile10 or Lys89 which are the only residues in the binding
site that differs between CDK1, CDK2, and CDK4.
In an ovarian cancer cell line (A2780) inhibitor 3 was
found to act as a cytotoxic agent with an IC50=5.8 mM.
FACS analysis demonstrated 3 blocked cell cycle pro-
gression and/or induced apoptosis. Specifically, A2780
and HCT116 cells treated with inhibitor 3 exhibited a
significant increase in the G2/M population, while CEM
cells predominantly showed an increase in the apoptotic
cell fraction versus untreated cells. In addition, exami-
nation of A2780 cells treated with inhibitor 3 (IC90
concentration for 4 h) showed inhibition of phospor-
ylation of the CDK substrates Rb protein, histone H1
and DNA pola consistent with a mechanism of action
involving CDK inhibition.11
Figure 2. Top: Solid-state structure of pyrazolopyridine 3 bound in
the ATP-pocket of CDK2 (no cyclin). The inhibitor carbon atoms are
in green, the nitrogen atoms are in blue and oxygen atoms are in red.
The protein carbons are in gray. Hydrogen bonds are shown by the
magenta dotted lines. Bottom: Overlap showing relative binding
modes of compound 3 (magenta) bound to CDK2 with the that of
ATP (green) bound to CDK2.11,12
In summary, pyrazolopyridine 3 has been been shown
to be a potent, selective inhibitor of CDK1/CDK2 in
vitro. In cells 3 acts as a cytotoxic agent with the ability
to blockcell progression and/or induce apoptosis and

1136
R. N. Misra et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1133–1136
has been shown to act by a mechanism of action con-
sistent with CDK inhibition. The solid-state structure of
3 bound to CDK2 shows that 3 resides in the ATP
purine pocket with important hydrogen bonding inter-
actions between the pyridine nitrogen and N1-hydrogen
of the heterocyclic ring and Leu83 of the enzyme.
Chem. 1972, 9, 235. (b) Denzel, T.; Hoehn, H. Arch. Pharm.
1976, 309, 486.
6. Presented in part: (a) 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; American Chemical
Society: Washington, DC, 2000; MEDI038. (b) Misra, R. N.;
Kimball, S. D.; Rawlins, D. B.; Webster, K. R.; Bursuker, I.
US Patent 6,107,305; 2000.
Acknowledgements
7. (a) Synthetic compounds were characterized by HPLC, 400
1
We thankDr. Bang-Chi Chen and Mr. MarkS. Bed-
narz for preparation of chemical intermediates and Dr.
John T. Hunt for scientific guidance.
or 500 MHz H NMR, and LC/MS.
8. (a) Hoehn, H. Z. Chem. 1970, 10, 386. (b) Preparation of 7:
To a solution of 7.50 g (141 mmol) of acrylonitrile in 30 mL of
dry THF cooled in an ice-bath was added dropwise 7.41 g (148
mmol) of hydrazine monohydrate. Warmed to room tem-
perature, and after 2 h added dropwise was 20.1 g (148 mmol)
of p-anisaldehyde. The solution was stirred for 2 h then con-
centrated in vacuo to give 29.1 g of a yellow oil. To a solution
of the crude oil in 30 mL of dry nBuOH was added at 25 ꢀC 70
mL of nBuOH containing 1 equiv of nBuONa. The resulting
darkmixture was heated to 120 ꢀC for 3 h, cooled then added
to 300 mL of water and extracted with 3–100 mL portions of
ether. The combined ether extracts were washed with 2–150
mL portions of 1 M aq HCl solution. The combined acidic
aqueous washes were basified (pH 14) with 50% aq NaOH
solution then extracted with 3–100 mL portions of ether. The
combined ether extracts were dried (Na2SO4) and treated with
excess ethereal HCl, a precipitate formed. The mixture was
concentrated in vacuo then MeOH was added and the product
precipitated by addition of ether and cooling. The solid pre-
cipitate was collected by filtration, washed with ether and
dried in vacuo to give 14.1 g (42% based on acrylonitrile) of
compound 7 as a yellow solid.
References and Notes
1. (a) Hunter, T.; Pines, J. Cell 1994, 79, 573. (b) Sherr, C.
Science 1996, 274, 1672. (c) Review: Pines, J. Seminars Cancer
Biol. 1994, 5, 305.
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. Rep.
Med. Chem. 2001, 36, 139.
3. (a) Vesely, J.; Havlicek, L.; Strnad, M.; Blow, J.; Donella-
Deana, A.; Pinna, L.; Letham, D. S.; Kato, J.; Detivaud, L.;
Leclerc, S.; Meijer, L. Eur. J. Biochem. 1994, 224, 771. (b)
Abraham, R.; Acquarrrrone, M.; Andersen, A.; Asensi, A.;
Belle, R.; Berger, F.; Bergounioux, C.; Brun, G.; Buquet-
Fagot, C.; Fagot, D.; Glab, N.; Goudeau, H.; Goudeau, M.;
Guerrier, P.; Houghton, P.; Hendriks, H.; Kloareg, B.; Lippai,
M.; Marie, D.; Maro, B.; Meijer, L.; Mester, J.; Mulner-Lor-
illon, O.; Poulet, S.; Schierenberg, E.; Schutte, B.; Vaulot, D.;
Verlhac, M. Biol. Cell 1995, 83, 105. (c) Havlicek, L.; Hanus,
J.; Vesely, J.; Leclerc, S.; Meijer, L.; Shaw, G.; Strand, M. J.
Med. Chem. 1997, 40, 408.
4. (a) Kaur, G.; Stetler-Stevenson, M.; Sebers, S.; Worland,
P.; Sedlacek, H.; Myers, C.; Czeck, J.; Naik, R.; Sausville, E.
J. Nat. Cancer Inst. 1992, 84, 1736. (b) Carlson, B.; Dubay,
M. M.; Sausville, E. A.; Brizuela, L.; Worland, P. J. Cancer
Res. 1996, 56, 2973. (c) Sedlacek, H. H.; Czech, J.; Naik, R.;
Kaur, G.; Worland, P.; Losiewicz, M.; Parker, B.; Carlson, B.;
Smith, A.; Senderowicz, A.; Sausville, E. Int. J. Oncol. 1996, 9,
1143. (d) Senderowicz, A. M. Invest. New Drugs 1999, 17, 313.
(e) Stadler, W. M.; Vogelzang, J.; Amato, R.; Sosman, J.;
Taber, D.; Liebowitz, D.; Vokes, E. E. J. Clin. Oncol. 2000,
18, 371. (f) Zhai, S.; Senderowicz, A. M.; Sausville, E. A.;
Figg, W. D. Ann. Pharmacother. 2002, 36, 905.
9. Prepared in 40% yield by heating ethylbenzoyl acetate (1
equiv), triethylorthoformate (1.5 equiv) and acetic anhydride
(2.5 equiv) at 145 ꢀC for 6 h. Compound 8 was isolated by
distillation as a brown oil (bp 165–169 ꢀC, 3 mm).
10. Su, T.; Huang, J.-T.; Chou, T.-C.; Otter, G. M.; Sirotnak,
F. M.; Wantanabe, K. A. J. Med. Chem. 1988, 31, 1209.
11. 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.
12. Structure of ATP-CDK2 used for comparison of binding
modes was from: Schulze-Gahmen, U.; De Bondt, H. L.; Kim,
S. H. J. Med. Chem. 1996, 39, 4540.
5. (a) Hoehn, H.; Denzel, T.; Janssen, W. J. Heterocycl.