Pyrazolo[1,5-a]pyrimidines as orally available inhibitors of cyclin-dependent kinase 2

Article abstract of DOI:10.1016/j.bmcl.2007.09.017

Properly substituted pyrazolo[1,5-a]pyrimidines are potent and selective CDK2 inhibitors. Compound 15j is orally available and showed efficacy in a mouse A2780 xenograft model.

Full text of DOI:10.1016/j.bmcl.2007.09.017

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6220–6223
Pyrazolo[1,5-a]pyrimidines as orally available inhibitors
of cyclin-dependent kinase 2q
a,
a
a
Kamil Paruch,a,* Michael P. Dwyer, Carmen Alvarez, Courtney Brown,
*
Tin-Yau Chan,a Ronald J. Doll,a Kerry Keertikar,a Chad Knutson,a Brian McKittrick,a
Jocelyn Rivera,a Randall Rossman,a Greg Tucker,a Thierry O. Fischmann,a Alan Hruza,a
Vincent Madison,a Amin A. Nomeir,a Yaolin Wang,a Emma Lees,b David Parry,b
Nicole Sgambellone,b Wolfgang Seghezzi,b Lesley Schultz,b Fran Shanahan,b
Derek Wiswell,b Xiaoying Xu,a Quiao Zhou,a Ray A. James,c Vidyadhar M. Paradkar,c
Haengsoon Park,c Laura R. Rokosz,c Tara M. Staufferc and Timothy J. Guzia
aSchering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
bSchering-Plough Biopharma, Palo Alto, CA 94304, USA
cPharmacopeia, 3000 East Park Boulevard, Cranbury, NJ 08512, USA
Received 17 July 2007; revised 31 August 2007; accepted 5 September 2007
Available online 8 September 2007
Abstract—Properly substituted pyrazolo[1,5-a]pyrimidines are potent and selective CDK2 inhibitors. Compound 15j is orally avail-
able and showed efficacy in a mouse A2780 xenograft model.
Ó 2007 Elsevier Ltd. All rights reserved.
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 execu-
tion 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 colorec-
tal, ovarian, breast, and prostate cancers.3 CDK inhibitors
havebeenshown toinduceapoptosis in different tumor cell
lines.4 Therefore, CDK inhibitors have the potential to en-
large the group of anticancer agents.
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
N
Ph
NH
N
OH
O
HO
N
N
N
HO
Cl
N
H
OH
O
A number of more or less selective CDK inhibitors have
been described in the literature5; those undergoing clin-
ical trials are flavopiridol (1),6 roscovitine (2),7 and BMS
387032 (3).8 Recently, an article on a series of pyrazol-
o[1,5-a]pyrimidines (4) (with amines linked through
NH or O at the 5-position and arylsulfones at the
2
1
R3
NH
N
R5
N
H
S
S
N
O
N
N
N
O
NHArSO2R
3
Cl
4
Cl
Keywords: CDK2; Kinase; Inhibitors; Pyrazolo[1,5-a]pyrimidine.
q
N
The coordinates of compound 13 bound to CDK2 have been
deposited in the Protein Databank pdb ID 2R3Q.
N
N
N
*
Corresponding authors. Tel.: +1 908 740 4378; fax: +1 908 740
7305; e-mail: kamil.paruch@spcorp.com
NH
5
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.017

K. Paruch et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6220–6223
6221
R3
containing pyridines and pyridine-N-oxides. In addition,
unlike the aniline series,9 those compounds exhibited
good oral PK profile.
R3
R5
N
H2N
HN
c,d
a,b
R3CH2CN
N
N
N
6
Cl
8
7
e
R3
Br
R5
N
R5
N
Table 1. CDK2 inhibitory activity of pyrazolo[1,5-a]pyrimidines 12a–
12y
R5
N
h,i
f,g
N
N
N
N
N
N
CH3
HN
R3
BocN
HN
Br
R7
R7
R7
N
N
11
10
9
N
N
N
N
N+
NH
13
Scheme 1. Reagents: (a) HCO2Et, t-BuOK, THF; (b) N2H4, AcOH,
EtOH; (c) R5COCH2CO2Me, PhCH3; (d) POCl3, N,N-dimethylaniline;
(e) R7NH2, DIPEA, dioxane; (f) Boc2O, DMAP, CH2Cl2; (g) NBS,
CH3CN; (h) R3B(OH)2, Pd[PPh3]4, Na2CO3, DME, H2O or R3SnBu3,
Pd[PPh3]4, dioxane; (i) TFA, CH2Cl2.
N
NH
12a-y
-O
Compound R3
CDK2/cyclin GSK3b
A IC50 (lM) IC50 (lM) IC50 (lM)
MAPK
12a
12b
12c
12d
12e
12f
12g
12h
12i
H
Br
0.25
—
—
0.011
0.072
0.008
0.890
1.20
0.57
—
0.37
1.40
2.00
—
Me
Et
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 b-keto-
esters as shown in Scheme 1. Desired substitution at the
3-position was achieved by choosing properly substi-
tuted acetonitriles 6 or via Pd-catalyzed coupling of
intermediate 10.
2.80
—
Pr
Bu
—
—
Ethynyl
Vinyl
0.048
0.090
0.84
9.82
—
7.48
—
Propynyl
c-Pr
CF3
—
—
—
—
12j
0.071
0.095
0.71
12k
12l
—
—
—
—
CH2CF3
SCH3
12m
12n
12o
12p
12q
12r
12s
12t
12u
12v
12w
12x
12y
0.007
1.10
2.17
1.80
—
0.90
—
Incorporation of halogens and other small substituents
at the 3-position resulted in significant improvement of
potency (Table 1). Most active compounds were selec-
tive against GSK3b and MAPK kinases. 12b exhibited
activity in cells (measured by incorporation of radiola-
beled thymidine) with IC50 = 350 nM. The X-ray crys-
tal 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.
OCH3
CH2OH
—
—
—
—
CH(OH)CF3 3.40
i-Pr
Ph
0.37
>50
16.0
>50
34.0
>50
—
—
—
—
CH2Ph
NO2
—
—
—
—
CH2N(Me)2
COCH3
—
—
—
—
S(CH2)2NHAc 0.002
S(CH2)2OH
CH2CN
0.034
—
1.10
12.00
—
1.33
0.030
0.049
Exploration of the 5-position led to a variety of inhibi-
tors whose IC50s were below 50 nM (Table 2). A some-
what 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[1,5-
a]pyrimidine by sequential displacements at the 7-posi-
tion with 3-(aminomethyl)pyridine and at the 5-position
with the corresponding nucleophiles followed by bro-
mination with NBS.
A variety of substituents were tolerated at the 7-position
(Table 3), which is close to the solvent-exposed part of
the enzyme. The best cell activity (measured by radiola-
beled thymidine uptake) was noted for the subclass
Figure 1. X-ray of crystal structure of 13 in CDK2.

6222
K. Paruch et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6220–6223
Table 4. Pharmacokinetic parameters of 15j
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 summa-
rized in Table 4.
Species
Rat
Dose, mpk vehicle AUC (lM h) cmax (lM) tmax (h)
10
0.4% MC
40
16.4
2.29
6.81
2.58
3.2
2.0
Mouse
Dog
17.9
2.0
20% HPBCD
5
20% HPBCD
2.44
43.0
Monkey 10
0.4% HPMC
3.3
Table 2. CDK2 inhibitory activity of pyrazolo[1,5-a]pyrimidines 14a–
14p
550
500
450
400
350
300
250
200
150
100
50
Control (no dosing)
Vehicle
Br
20 mpk
R5
N
40 mpk
40 mpk(2-5)
N
N
N
NH
14a-p
Compound R5
CDK2/cyclin GSK3b
Thym
A IC50 (lM) IC50 (lM) IC50 (lM)
0
14a
14b
14c
14d
14e
14f
14g
14h
14i
Me
Et
0.060
0.018
0.017
0.045
3.53
0.92
0.42
1.13
0.50
0.79
4.6
1.80
0.52
0.95
1.50
1.40
2.90
1.20
2.00
0.99
0.50
0.20
0.06
0.16
8
11
14
18
Days (post inoculation)
i-Pr
CycloPr
Figure 2. Efficacy of 15j in A2780 xenograft model (mouse).
CH(CH3)OH 0.038
CO2Et
0.089
0.043
0.035
0.038
0.003
0.004
NHCH3
OCH3
SCH3
1.41
1.18
0.054
0.027
0.18
0.41
—
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.
14j
Ph-2-Cl
Cyclohexyl
14k
14l
3-Piperidinyl 0.008
4-Piperidinyl 0.016
14m
14n
14o
14p
Piperazine
2-Furyl
2-Thienyl
0.210
0.008
0.013
—
15.19
0.75
The compound was well tolerated and only a moderate
and reversible decrease of white blood cells was
observed.
0.31
0.48
In conclusion, we demonstrated that properly substi-
tuted pyrazolo[1,5-a]pyrimidines can serve as potent,
selective, and efficacious orally available CDK2
inhibitors.
Table 3. CDK2 inhibitory activity of pyrazolo[1,5-a]pyrimidines 15a–
15l
Br
N
X
N
N
References and notes
HN
R7
15a-l
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15c
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15e
15f
15g
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13.31
0.45
15.2
0.054
0.035
0.13
0.60
—
Cl Ph-4-SO2CH3 0.35
0.300
0.004
0.90
—
F Bn
Cl CH2-3Pyr
0.50
0.17
0.21
0.14
—
Cl CH2-3Pyr-O 0.011
F CH2-3Pyr-O 0.013
H CH2-3Pyr-O 0.034
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