Synthesis and evaluation of pyrazolo[3,4-b]pyridine CDK1 inhibitors as anti-tumor agents

Article abstract of DOI:10.1016/j.bmcl.2007.05.029

A series of 3,5-disubstituted pyrazolo[3,4-b]pyridine cyclin-dependent kinase (CDK) inhibitors was synthesized. These compounds showed potent and selective CDK inhibitory activities and inhibited in vitro cellular proliferation in cultured human tumor cel

Full text of DOI:10.1016/j.bmcl.2007.05.029

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4297–4302
Synthesis and evaluation of pyrazolo[3,4-b]pyridine
CDK1 inhibitors as anti-tumor agents
Ronghui Lin,* Peter J. Connolly, Yanhua Lu, George Chiu, Shengjian Li, Yang Yu,
Shenlin Huang, Xun Li, Stuart L. Emanuel, Steven A. Middleton, Robert H. Gruninger,
Mary Adams, Angel R. Fuentes-Pesquera and Lee M. Greenberger
Johnson & Johnson Pharmaceutical Research & Development L.L.C., 1000 Route 202, PO Box 300, Raritan, NJ 08869, USA
Received 7 April 2007; revised 7 May 2007; accepted 9 May 2007
Available online 16 May 2007
Abstract—A series of 3,5-disubstituted pyrazolo[3,4-b]pyridine cyclin-dependent kinase (CDK) inhibitors was synthesized. These
compounds showed potent and selective CDK inhibitory activities and inhibited in vitro cellular proliferation in cultured human
tumor cells. Selected compounds were evaluated in an in vivo tumor xenograft model. The synthesis and biological evaluation of
these pyrazolo[3,4-b]pyridines and related compounds are reported.
Ó 2007 Elsevier Ltd. All rights reserved.
Cyclin-dependent kinases (CDKs), such as CDK1,
CDK2, and CDK4, constitute a class of serine–threo-
nine protein kinases that plays an important role in reg-
ulation of the cell cycle.1 Abnormal CDK control of the
cell cycle has been strongly linked to the molecular
pathology of cancer. CDKs have thus become attractive
therapeutic targets for cancer therapy.2 The CDKs reg-
ulate cell cycle progression through complexes with their
corresponding cyclin partners such as cyclin A, B, D,
and E. For example, CDK1 associated with cyclin B reg-
ulates the cell cycle at the G2 and mitosis (cell division)
phases. CDK1 inhibitors could block mitosis entry and
arrest cell growth, and therefore may be useful therapeu-
tic agents with potentially fewer side effects than conven-
tional cytotoxic drugs targeting DNA synthesis.
structurally different CDK1 inhibitors with improved
pharmacokinetic and solubility properties, we have de-
signed, synthesized, and evaluated several new series of
compounds.9 Herein we report the synthesis of 3,5-
disubstituted pyrazolopyridine analogs (1) and their bio-
logical evaluation as inhibitors of CDK activity and tu-
mor cell proliferation.
R3
R4
R2
X
HN
R1
O
Br
N
Ar
N
N
R
N
H
N
N
N
1
2a X = H
1a, R,R1-4 = H
Several CDK inhibitors have entered clinical evaluation
for the treatment of cancer.3 These include flavopiridol,
7-hydroxystaurosporine (UCN-01), roscovitine (CYC202),
BMS-387032 (SNS-032),4 PD0332991,5 and R5476. In
our program to develop CDK inhibitors as anti-cancer
agents, we recently reported that 1-acyl-1H-[1,2,4]tria-
zole-3,5-diamine analogs7 and 2-amino-3-benzoyl-6-ani-
linopyridine analogs8 are novel anti-cancer CDK
inhibitors and anti-proliferative agents. To discover
2b X = OMe
2c X = OH
Ar = isoquinolin-4-yl
1b, R,R2-4 = H, R1= Me
Ar = isoquinolin-4-yl
A convergent approach to synthesize the designed com-
pounds (1) was envisioned to employ core intermediates
such as 5-bromo-pyrazolo[3,4-b]pyridine-3-carboxalde-
hyde (2a) or 5-bromo-pyrazolo[3,4-b]pyridine-3-carbox-
ylic acid or its ester (2b or 2c). Key steps for the
synthesis would involve (1) a benzimidazole ring
formation by cyclization of the 3-carboxaldehyde or 3-
carboxylic acid group of 2a and 2c with a substituted
Keywords: Pyrazolo[3,4-b]pyridine; Cyclin dependent kinase inhibitor.
*
Corresponding author. Tel.: +1 908 704 5268; fax: +1 908 526
6465; e-mail: Ronghui.Lin@yahoo.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.029

4298
R. Lin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4297–4302
1,2-benzene diamine, and (2) arylation at the 5-position
of the resultant 5-bromo-3-benzimidazolyl-pyrazolo-
pyridine, via Suzuki or Stille coupling.
pling offered better commercial availability and low tox-
icity of the boron reagents compared to the organotin
reagents. The Boc protection protocol for the Suzuki
coupling was convenient for its readily useable crude
Boc protected intermediate and simultaneous in situ
Boc deprotection under Suzuki reaction conditions. In
cases where the Boc group of some substrates was too
labile and fell off, thus leading to poor Suzuki reaction
yield, SEM protection would be an alternative choice.
A series of target compounds 1c–i was prepared by this
general approach.
A multi-step approach to prepare 4-[3-(1H-benzimi-
dazol-2-yl)-1H-pyrazolo[3,4-b]pyridine-5-yl]-isoquinoline
(1a) through 5-bromo-pyrazolo[3,4-b]pyridine-3-carbox-
aldehyde (2a) has been recently reported.9a This
approach employed benzimidazole ring formation by
treating 3-carboxaldehyde compound 2a and a 1,2-benz-
ene diamine with sulfur(0), followed by Stille coupling
with 4-trimethylstannylisoquinoline. 4-[3-(4-Methyl-
1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridine-5-yl]-
isoquinoline (1b) was similarly prepared according to
these procedures. However, poor reproducibility of this
approach prompted us to explore an alternative way
through 5-bromo-pyrazolo[3,4-b]pyridine-3-carboxylic
acid (2b) or its ester (2c).
In order to diversify physicochemical properties and im-
prove ADME properties of target compounds, the benz-
imidazole ring was further derivatized with suitable
functional groups such as hydroxy and carboxylic acid.
Thus, following the general synthetic approach as
shown in Scheme 2, compounds 9a–d were first prepared
from 3-hydroxymethyl-benzene-1,2-diamine (4a) and 2c.
The hydroxyl group of 9a was treated with mesyl chlo-
ride to give corresponding mesylate, which was then dis-
placed with isopropanol to give 9b in 17% yield and with
isopropyl amine and diethyl amine to give 9c and 9d cor-
respondingly. Similarly, compound 10a was prepared
from 2,3-diamino-benzoic acid methyl ester and com-
pound 2c via the similar multi-step protocol. Subsequent
hydrolysis of 10a gave acid 10b. The carboxylic acid
group of compound 10b was converted to amide analogs
10c and 10d in a straightforward manner by coupling
with isopropyl amine and diethyl amine in 32% and
46% yield, respectively (Scheme 2).
As outlined in Scheme 1, bromination of commercially
available pyrazolo[3,4-b]pyridine-3-carboxylic acid
methyl ester (3) afforded intermediate 2b in 43% yield;
2b was then hydrolyzed to 5-bromo-pyrazolo[3,4-b]pyr-
idine-3-carboxylic acid 2c quantitatively. Heterocycliza-
tion of 3-carboxylic acid group of 2c with a substituted
1,2-benzene diamine (4) gave the desired 5-bromo-3-
benzimidazolyl-pyrazolopyridine (6) in good yield, via
a two-step sequence: amide formation to 5, followed
by treatment with glacial acetic acid. Next, arylation
at the 5-position of the resultant 6 could be achieved
either via Stille coupling with an organotin reagent, or
more preferably via Suzuki coupling with a heteroaryl
boronic acid or boronate. Efficient Suzuki or Stille cou-
pling on the pyrazol[3,4-b]pyridine substrates usually re-
quired protection on pyrazole ring of 6 by a Boc or SEM
group giving 7a or 7b correspondingly. For unsymmet-
ric substitution patterns on the benzimidazole ring, 7a
or 7b could be a mixture of isomers. The Suzuki cou-
To evaluate the effect of the pyrazole NH on activity, a
methyl group was introduced. As shown in Scheme 3,
1H-1-methyl-pyrazolo[3,4-b]pyridine analog 12 was pre-
pared from 3-methoxymethyl-benzene-1,2-diamine (4c)
and 2e, which itself was prepared by methylation of 2b
to 2d and then saponification.
R3
R4
H2N
R3
R4
R2
R1
R2
CO2Me
CO2Me
CO2H
N
Br
Br
H2N
H2N
O
R1
N
NH
N
4
c
Br
N
H
N
N
H
N
2c
N
N
a
b
H
N
2b
3
N
H
N
5
d
R3
R3
R4
R4
R3
R4
R2
R2
R1
HN
R2
R1
PGN
R1
HN
N
Ar
N
Br
g for 7a
g, h for 7b
N
e to 7a
f to 7b
N
Br
N
N
N
H
N
N
N
N
PG
N
1c-i
H
7a, PG = Boc
7b, PG = SEM
see Table 1 for structure details
6
Scheme 1. General synthetic approach to 3-(1H-benzimidazol-2-yl)-5-heteroaryl-pyrazolo[3,4-b]pyridine analogs. Reagents and conditions: (a) Br2,
NaOAc, HOAc, 115 °C, sealed flask, 43%; (b) NaOH, H2O, MeOH, reflux, 4 h, 100%; (c) HATU, DIPEA, DMF, 57–90%; (d) glacial HOAc, 80–
120 °C, 3–4 h, 55–87%; (e) (Boc)2O, DMAP, Et3N, THF, 91–100%; (f) SEM-Cl, NaH, THF, 56–96%; (g) heteroaryl boronic acid or boronate,
Pd(PPh3)4, Na2CO3, dioxane, H2O, 90 °C, overnight, 11–91%; (h) 1:1 4 M HCl/EtOH, 38–97%.

R. Lin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4297–4302
4299
in Scheme 4, the 5-bromo-3-benzimidazolyl-pyrazolo-
pyridine, protected with a Boc (7a) or SEM (7b) group,
was coupled with commercially available 5-(4,4,5,5-tetra-
methyl-[1,3,2]dioxaborolan-2-yl)-pyridine-3-carboxalde-
hyde (13). Usually, Boc groups were simultaneously
removed under Suzuki reaction conditions. The carboxal-
dehyde group of compounds 14a and 14b was efficiently
converted to a series of target compounds with various
amino side chains (15a–h) by reductive amination with
primary or secondary amines, followed by subsequent
deprotection in cases of SEM protection.
NH2
NH2
b,c,d,e
a
NH2
CH2OTBDMS
NH2
CH2OH
4b
4a
HN
HN
N
N
N
N
N
HO
f
N
N
N
N
N
N
8
H
H
9a
g
h
Alternatively, SEM-protected 5-bromo-3-benzimidazol-
yl-pyrazolopyridine 7b was converted to 3-benzimidazol-
yl-pyrazolopyridine 5-boronate 16, by treating with
bis(pinacolato)diboron by the catalysis of 1,10-bis(diph-
HN
HN
CH2
N
N
N
NR5R6
i-PrO
N
N
N
enylphosphino)ferrocenedichloro
palladium(II)
as
N
H
N
N
H
9b
depicted in Scheme 5. Coupling of intermediate 3-benzim-
idazolyl-pyrazolopyridine-5-boronate 16 with aryl
halides is complementary to Suzuki coupling at the 5-po-
sition of 5-bromo-3-benzimidazolyl-pyrazolopyridines
7a and 17b. For example, 3,5-dibromo-4-methyl-pyridine
(17) was formylated upon lithiation with n-BuLi and
treatment with DMF to give compound 18. Suzuki
coupling of 16 with 3-bromo-pyridine- 18 gave pyridine-
3-carboxaldehyde 19 in 69% yield. As before, reductive
amination of the carboxaldehyde group of compound
18 with ethylamine gave the target compound having an
amino side chain (20).
9c R5R6 = H, iPr
9d R5R6 = Et2
HN
HN
N
O
O
N
N
N
j from 10b
N
N
N
H
N
N
N
H
10c R5R6 = H, iPr
10a R7 = Me
i
10d R5R6 = Et2
10b R7 = OH
Scheme 2. Derivatization at 3-benzimidazol-2-yl group of pyrazol-
o[3,4-b]pyridine analogs. Reagents and conditions: (a) TBDMS-Cl,
95%; (b) 2c, HATU, DIPEA, DMF, 61%; (c) glacial HOAc, 80 °C,
3.5 h, 88%; (d) (Boc)2O, DMAP, Et3N, THF, 100%; (e) 4-isoquinoline
boronic acid, Pd(PPh3)4, Na2CO3, dioxane, H2O, 90 °C, overnight,
63%; (f) TBAF, THF, 72%; (g) 1—MsCl, DIPEA, THF, 0 °C, 3 h; then
2—i-PrOH, NaH, rt, 2 h, 17%; (h) 1—MsCl, DIPEA, THF, 0 °C, 3 h;
then 2—i-PrNH2, or Et2NH, rt, 2 h, 11–48%; (i) NaOH, H2O, MeOH,
80 °C, 5 h, 83%; (j) i-PrNH2, or Et2NH, HATU, DIPEA, DMSO, 33–
46%.
Table 1 lists the structures and inhibitory activities of
the 3,5-disubstituted pyrazolo[3,4-b]pyridine analogs
against CDK1 and four other protein kinase (VEGF-
R2, HER2, Aurora-A, and RET kinase). The in vitro
anti-proliferative activities in three human tumor cell
On the other hand, derivatization of the left side of the
scaffold was directed toward introducing solubilizing
groupsonto the 5-pyridin-3-yl substituent. As exemplified
R3
R3
R4
PGN
R4
PGN
CHO
R2
CHO
R2
R1
R1
N
O
B
O
N
N
Br
N
CO2Me
CO2H
N
Br
N
N
Br
13
a
b
N
N
N
N
N
2b
PG
PG
a
N
Me
N
N
N
14a, PG = H, from 7a
7a, PG = Boc
7b, PG = SEM
Me
2e
14b, PG = SEM, from 7b
2d
b for 14a
b,c for 14b
c
H2N
H2N
R3
4a
R4
CH2OMe
Br
d, e
CH2NR8R9
R2
R1
4c
HN
HN
N
HN
N
CH2OMe
CH2OMe
f, g
N
N
N
N
N
N
N
N
Me
H
N
N
15a-h
N
N
R8, R9 = H, Me, Et, or n-Pr
Me
12
R1-4 = H, F, CH2OMe, OMe, OCF3,
or 1H-4-Me-piperizin-1-yl
11
Scheme 3. Synthesis of 1H-1-methylated pyrazolo[3,4-b]pyridine ana-
log 12. Reagents and conditions: (a) MeI, NaH, THF, 97%; (b) NaOH,
H2O, MeOH, 90%; (c) MeI, NaH, THF, 34%; (d) HATU, DIPEA,
DMF, 48%; (e) glacial HOAc, 80 °C, 3.5 h, 89%; (f) (Boc)2O, DMAP,
Et3N, THF, 100%; (g) 4-isoquinoline boronic acid, Pd(PPh3)4,
Na2CO3, dioxane, H2O, 90 °C, overnight, 48%.
Scheme 4. Derivatization of the 5-pyridin-3-yl group of pyrazolo[3,4-
b]pyridine analogs. Reagents and conditions: (a) Pd(PPh3)4, Na2CO3,
dioxane, H2O, 90 °C, overnight, 43–62%; (b) MeNH2, EtNH2, n-
PrNH2, or Et2NH, NaBH4 or NaBH3CN, 28–99%; (c) 1:1 4 M HCl/
EtOH, 38–86%.

4300
R. Lin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4297–4302
Replacing the isoquinolin-3-yl group with the pyridin-3-
yl group at the left side of the scaffold usually gave bet-
ter solubility properties while retaining favorable CDK1
potency. Thus derivatization of the left side of the scaf-
fold was focused to introduce solubilizing groups onto
the pyridin-3-yl substituent. The most favorable groups
among those studied were 5-(alkylaminomethyl)-pyri-
din-3-yls such as those in compounds 15a–h, which all
have single digit or sub-nM IC50 values against
CDK1. Various substituents such as alkyl, halogen, alk-
oxyl, and dialkylamino groups on the benzimidazole
ring on the right side were well tolerated. Interestingly,
the 5-dialkylaminomethyl substituted pyridin-3-yl in
compound 15h (IC50 = 120 nM), instead of 5-mono-
alkylamino methyl groups such as those in compounds
15a–h, was detrimental to CDK1 potency. Introduction
of one extra methyl group at 4-position of the pyridine
substituent also drastically reduced CDK1 potency
(20, IC50 = 110 nM).
SEMN
SEMN
O
B
N
N
Br
O
a
MeO
Br
MeO
N
N
N
N
N
N
SEM
SEM
7c
16
CHO
Me
Br
Me
Br
b
c
N
N
18
17
CHO
CH2NHEt
Me
Me
SEMN
HN
N
N
N
MeO
N
d,e
MeO
N
N
N
N
N
H
N
20
SEM
19
Scheme 5. Synthesis and derivatization of 5-(2-methyl-pyridin-3-yl)-
pyrazolo[3,4-b]pyridine analogs. Reagents and conditions: (a) bis(pina-
colato)diboron, 1,10-bis(diphenylphosphino)ferrocenedichloro palladi-
um(II), 80 °C, overnight, 58%; (b) n-BuLi, ꢁ78 °C, then DMF; (c)
Pd(PPh3)4, 90 °C, overnight, 69%; (d) EtNH2, NaBH4 or NaBH3CN,
99%; (e) 1:1 4 M HCl/EtOH, 38%.
Comparison of CDK1 activity with the other four pro-
tein kinases tested, VEGF-R2, HER2, Aurora-A, and
RET kinase, revealed that these pyrazolopyridine ana-
logs are generally quite selective toward CDK1. Selectiv-
ity in favor of CDK1 inhibition versus the other kinase
inhibitory activities was commonly seen in the range of
10–100-fold. In fact, many compounds showed IC50
values >10 lM or >100 lM for VEGF-R2, HER2, Aur-
ora-A, and RET kinases, indicating no observed 50%
inhibition at the highest dose tested.
lines, HeLa (cervical carcinoma), HCT116 (colon carci-
noma), and A375 (melanoma), are also shown.
Examination of substitution on the benzimidazole group
reveals that a small methoxymethyl group as R1 at the 4-
position (1c) gave very favorable CDK1 potency (IC50
5.6 nM), compared to the unsubstituted, methyl,
hydroxymethyl, or larger isopropyloxymethyl substi-
tuted compounds (1a, 1b and 9a, 9b). Small alkylami-
nomethyl or dialkylaminomethyl groups as R1 (9c and
9d) gave roughly comparable CDK1 potencies
(IC50 = 13 and 1.8 nM, respectively). However, replace-
ment of the methoxymethyl group as R1 at the 4-posi-
tion (1c) with methoxycarbonyl or carboxyl (10a and
10b, IC50 ꢀ 10 and 0.54 lM correspondingly) reduced
the CDK1 potency significantly. Still, converting the
carboxylic acid to a primary or secondary amide (10c
and 10d) regained modest CDK1 potency (IC50 = 39
and 53 nM, respectively).
These pyrazolopyridine analogs also proved to be active
in vitro as anti-proliferatives in three cultured human
tumor cell lines, such as HeLa, HCT116, and A375.
The lead CDK1 inhibitors showed potent inhibition of
cell proliferation with IC50 values ranging from 10 to
500 nM among the tumor cell lines tested.
Preliminary pharmacokinetic studies were conducted in
rats with selected compounds. Table 2 lists the pharma-
cokinetic data for compounds 1h and 15e. Pharmacoki-
netic parameters were determined after single oral (po)
and intravenous (iv) doses in 10% SolutolÒ D5W and
20% HPbCD in water vehicle, respectively. Both com-
pounds demonstrated poor oral availability (F%), low
exposure (AUC), and high clearance in rats.
On the other hand, the effect of various heteroaryl
groups on CDK1 potency seems drastic. For example,
compound 1c with the isoquinolin-3-yl group is much
more potent than 1d with the quinolin-3-yl group
(IC50 = 5.6 nM vs 1.36 lM). Similarly, the pyridin-3-yl
group in 1e (IC50 = 5.6 nM) is much more favorable
than the pyridin-4-yl group in 1f (>1 lM) and pyrimi-
din-5-yl group in 1g (1.28 lM). Large-sized substituents
such as 4-methyl-piperazin-1-yl and morpholin-1-yl as
R2 at the 5-position of the benzimidazole group (com-
pounds 1h–i) are generally tolerant to CDK1 potency
(IC50 14 and 8.5 nM correspondingly).
Selected compounds were also evaluated for their
in vitro safety and ADME properties. They were tested
for stability in human liver microsomes, inhibition of
cloned cytochrome P450 enzymes, and inhibition of
astemizole binding to hERG channel. Most of the lead
compounds had human liver microsome stability with
t1/2 > 30 min and had IC50 values for cloned P450s and
hERG generally >10 lM. Solubility was found to be
around 12.5 mg/mL in 20% HPbCD for compounds
15b–g.
The in vivo efficacy of selected compounds was exam-
ined in the A375 human melanoma cell xenograft model
in nude mice, administered intraperitoneally (ip) at
25 mg/kg in 20% HPbCD, which is close to the maximal
tolerated dose. Limited animal tolerance and poor anti-
The importance of the pyrazole NH group in the bicycle
core was also remarkable as its methylation led to a sub-
stantial reduction on CDK1 activity (compound 12 vs
compound 1c, IC50 = 5.6 vs 120 nM).

Table 1. Structures, CDK1 and other protein kinase inhibitory activity, and cellular anti-proliferative activity on various tumor cells of the 3,5-disubstituted pyrazolo[3,4-b]pyridine analogs
R3
R2
R1
R4
HN
N
Ar
N
R
N
N
a
Compound
Ar
R, R1, R2, R3, R4
IC50 (lM)
CDK1/ cyclin B
VEGF-R2
HER2
Aurora-A
RET
HeLa
HCT-116
A375
1a
1b
Isoquinolin-4-yl
Isoquinolin-4-yl
H, H, H, H, H
H, Me, H, H, H
0.023
0.097
0.041
0.0056
0.099
1.36
1.46
10
0.14
1
ND
ND
>10
0.32
>1
ND
ND
>100
>10
>100
>100
1.1
1.7
0.55
1.64
0.87
1.72
3.09
1.76
0.015
2.14
1.46
0.65
3.09
9.91
0.13
0.029
0.45
0.40
10.5
>10
9a
1c
Isoquinolin-4-yl
Isoquinolin-4-yl
H, CH2OH, H, H, H
H, CH2OMe, H, H, H
H, CH2O(i-Pr), H, H, H
H, CH2OMe, H, H, H
H, CH2OMe, H, H, H
H, CH2OMe, H, H, H
H, CH2OMe, H, H, H
H, CH2NH(i-Pr), H, H, H
H, CH2NEt2, H, H, H
>100
0.46
ꢀ0.1
>100
0.16
0.11
0.11
ꢀ1
0.24
0.085
>100
>100
>100
>100
>100
>100
0.089
6.51
>100
>100
>100
>10
0.89
0.010
2.88
1.47
0.010
0.91
9b
1d
Isoquinolin-4-yl
Quinolin-3-yl
>100
>1
>1
0.0032
0.019
0.062
0.33
0.011
0.033
0.023
0.16
1e
Pyridin-3-yl
Pyridin-4-yl
0.029
>1.0
1.28
1f
1g
>100
>100
>100
1
Pyrimidin-5-yl
Isoquinolin-4-yl
ꢀ10
3.62
1
9c
9d
0.013
0.0018
0.014
0.0085
ꢀ10
0.11
0.14
Isoquinolin-4-yl
Isoquinolin-4-yl
1.55
1.00
1.07
ꢀ100
>10
0.031
0.36
0.37
0.047
0.16
0.30
1h
1i
H, H, (1H-4-Me-piperizin-1-yl), H, H
H, H, (1H-morpholin-1-yl), H, H
H, CO2Me, H, H, H
ꢀ1
ꢀ1
Isoquinolin-4-yl
Isoquinolin-4-yl
0.041
ꢀ100
>10
>0.1
ꢀ1
ꢀ1
10a
10b
10c
10d
12
>100
>100
0.41
1.23
>100
ꢀ1
8.01
>10
7.38
>10
Isoquinolin-4-yl
Isoquinolin-4-yl
H, CO2H, H, H, H
0.54
H, CONH(i-Pr), H, H, H
H, CONEt2, H, H, H
Me, CH2OMe, H, H, H
H, CH2OMe, H, H, H
H, CH2OMe, H, H, H
H, H, (1H-4-Me-piperizin-1-yl), H, H
H, H, F, F, H
0.039
0.053
0.12
0.38
ꢀ10
>100
0.64
ND
0.035
0.17
1.17
0.46
0.26
0.47
0.14
0.059
0.093
0.032
1.23
2.07
0.042
0.055
0.20
0.065
0.071
0.26
Isoquinolin-4-yl
Isoquinolin-4-yl
>10
>100
>100
>10
ꢀ100
>1
15a
15b
15c
15d
15e
15f
15g
15h
20
5-(MeNHCH2)-pyridin-3-yl
5-(n-PrNHCH2)-pyridin-3-yl
5-(EtNHCH2)-pyridin-3-yl
5-(EtNHCH2)-pyridin-3-yl
5-(EtNHCH2)-pyridin-3-yl
5-(MeNHCH2)-pyridin-3-yl
5-(EtNHCH2)-pyridin-3-yl
5-(Me2NCH2)-pyridin-3-yl
5-(EtNHCH2)-4-Me-pyridin-3-yl
0.0025
0.0074
0.0030
0.0011
0.0007
0.009
0.0008
0.12
0.31
0.27
0.27
0.25
3.99
0.28
>1
>10
0.14
0.039
0.11
>100
ꢀ1
0.025
ꢀ0.1
0.15
>0.1
>0.1
1.60
4.67
1.07
3.60
5.12
3.82
4.17
>100
>100
0.54
0.63
0.095
0.094
0.055
0.049
0.19
0.046
0.051
0.047
0.10
H, H, F, H, H
H, H, OCF3, H, H
0.80
ꢀ1
H, H, OCH3, H, H
H, CH2OMe, H, H, H
H, CH2OMe, H, H, H
0.36
3.75
8.02
3.23
>10
0.33
0.12
0.11
0.44
a See Ref. 9b for descriptions of the kinase and cellular anti-proliferation assays. IC50 data are the average of at least two separate experiments and are rounded up to two significant figures. IC50 values
listed as >1, or >10, or >100 indicate no observed 50% inhibition at the highest dose tested, nor was an inhibition maximum observed.

4302
R. Lin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4297–4302
Table 2. Pharmacokinetics of the selected compounds in rats
4. Misra, R. N.; Xiao, H.; Kim, K. S.; Lu, S.; Han, W.;
Barbosa, S. A.; Hunt, J. T.; Rawlins, D. B.; Shan, W.;
Ahmed, S. J.; Qian, L.; Chen, B.; 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.
5. (a) Toogood, P. L.; Harvey, P. J.; Repine, J. T.; Sheehan,
D. J.; VanderWel, S. N.; Zhou, H.; Keller, P. R.;
McNamara, D. J.; Sherry, D.; Zhu, T.; Brodfuehrer, J.;
Choi, C.; Barvian, M. R.; Fry, D. W. J. Med. Chem. 2005,
48, 2388; (b) Fry, D. W.; Harvey, P. J.; Keller, P. R.; Elliott,
W. L.; Meade, M.; Trachet, E.; Albassam, M.; Zheng, X.;
Leopold, W. R.; Pryer, N. K.; Toogood, P. L. Mol. Cancer
Ther. 2004, 3, 1427.
Compound
1h
15e
% Oral bioavailability
Vehicle
4
8
10% SolutolÒ
20% HPbCD
in D5W
3.3
65
in water
31.3
3.8
t1/2 (h), po
Cmax (ng/mL), po
AUC (ng h/mL), po
Dose (mg/kg), po
t1/2 (h), iv
173
10
150
10
0.73
1000
959
34.9
2
16.1
217
372
93.6
2
Cmax (ng/mL), iv
AUC (ng h/mL), iv
Clearance (mL/min kg)
Dose (mg/kg), iv
6. Chu, X.; DePinto, W.; Bartkovitz, D.; So, S.; Vu, B. T.;
Packman, K.; Lukacs, 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.
tumor activity were found in this preliminary efficacy
model.
7. (a) Lin, R.; Connolly, P. J.; Wetter, S.; Huang, S.; Emanuel,
S.; Gruninger, R.; Middleton, S. PCT Int. Appl. 2002, WO
2002057240; . Chem. Abstr. 2002, 137, 125160; (b) Emanuel,
S. L.; Rugg, C.; Lin, R.; Connolly, P. J.; Napier, C.;
Hollister, B.; Hall, C.; Middleton, S. Proc. Am. Assoc. Can.
Res. 45, 191. 95th Annual Meeting of the American
Association for Cancer Research, Orlando, Florida, March
27–31, 2004; Abstract #833; (c) Emanuel, S.; Gruninger,
R.; Lin, R.; Rugg, C.; Fuentes-Pesquera, A. R.; Connolly,
P.; Wetter, S.; Hollister, B.; Kruger, W. W.; Napier, C.;
Johnson, D.; Jolliffe, L.; Middleton, S. Proc. Am. Assoc.
Can. Res. 44, 162. 94th Annual Meeting of the American
Association for Cancer Research, Washington, DC, July
11–14, 2003; Abstract #707.
8. (a) Lin, R.; Lu, Y.; Wetter, S. K.; Connolly, P. J.; Turchi, I. J.;
Murray, W. V.; Emanuel, S. L.; Gruninger, R. H.; Fuentes-
Pesquera, A.; Middleton, S. A.; Jolliffe, L. K. Bioorg. Med.
Chem. Lett. 2005, 15, 2221; (b) Lin, R.; Wetter, S. K.; Lu, Y.;
Connolly, P. J.; Emanuel, S.; Gruninger, R. H.; Middleton, S.
A. PCT Int. Appl. 2005, WO2005051387A1.
9. (a) Huang, S.; Lin, R.; Yu, Y.; Lu, Y.; Connolly, P. J.;
Chiu, G.; Li, S.; Emanuel, S. L.; Middleton, S. A. Bioorg.
Med. Chem. Lett. 2007, 17, 1243; (b) Chiu, G.; Li, S.;
Connolly, P. J.; Middleton, S. A.; Emanuel, S. L.; Huang,
S.; Lin, R.; Lu, Y. PCT Int. Appl. 2006, WO2006130673A1;
(c) Huang, S.; Connolly, P. J.; Lin, R.; Emanuel, S. L.;
Middleton, S. A. Bioorg. Med. Chem. Lett. 2006, 16(14),
3639; (d) Huang, S.; Li, R.; Connolly, P. J.; Emanuel, S.;
Middleton, S. A. Bioorg. Med. Chem. Lett. 2006, 16, 4818.
In summary, we have discovered a novel series of 3,5-
disubstituted pyrazolo[3,4-b]pyridine analogs that are
potent and selective cyclin-dependent kinase and cellular
anti-proliferative inhibitors. The key steps for the syn-
thesis employed benzimidazole ring formation by
cyclization of the 3-carboxyl group of 5-bromo-pyrazolo-
[3,4-b]pyridine-3-carboxylic acid with substituted 1,2-
benzene diamines and arylation at the 5-position via
Suzuki coupling. Representative compounds were po-
tent inhibitors of in vitro cellular proliferation in HeLa,
HCT116, and A375 human tumor cell lines. Selected
compounds were evaluated for other biological proper-
ties including an in vivo tumor xenograft efficacy model.
References and notes
1. Harper, J. W.; Adams, P. D. Chem. Rev. 2001, 101, 2511.
2. (a) Sielecki, T. M.; Boylan, J. F.; Benfiled, P. A.; Trainor,
G. T. J. Med. Chem. 2000, 43, 1; (b) Toogood, P. I. Med.
Res. Rev. 2001, 21, 487; (c) Hirai, H.; Kawanishi, N.;
Iwasawa, Y. Curr. Top. Med. Chem. 2005, 5, 167.
3. (a) Fischer, P. M.; Gianella-Borradori, A. Expert Opin.
Invest. Drugs 2003, 12, 955; (b) Sausville, E. A. Curr. Med.
Chem. – Anti-cancer Agents 2003, 3, 47; (c) Benson, C.;
Kaye, S.; Workman, P.; Garrett, M.; Walton, M.; de Bono,
J. Br. J. Cancer 2005, 92, 7.