Pyrazolo[1,5-a]pyrimidines as orally available inhibitors
of cyclin-dependent kinase 2q
Kamil Paruch,a,* Michael P. Dwyer,a,* Carmen Alvarez,a
Courtney Brown,a
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
a
Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
b
Schering-Plough Biopharma, Palo Alto, CA 94304, USA
c
Pharmacopeia, 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 available
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 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
havebeenshown toinduceapoptosis 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
O
Cl
OOH
HO
N
OH
N
N N
N
NH
Ph
N
H
HO
1 2
N
S
H
N
O
NH
S
N
O
3
N
N N
R3
R5
NHArSO2R
4
N
N N
NH 5
Cl
Cl
N
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.017
Keywords: CDK2; Kinase; Inhibitors; Pyrazolo[1,5-a]pyrimidine.
q
The coordinates of compound 13 bound to CDK2 have been
deposited in the Protein Databank pdb ID 2R3Q.
* Corresponding authors. Tel.: +1 908 740 4378; fax: +1 908 740
7305; e-mail: kamil.paruch@spcorp.com
Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6220­6223
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-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.
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 GSK3b 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.
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.
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 radiolabeled
thymidine uptake) was noted for the subclass
containing pyridines and pyridine-N-oxides. In addition,
unlike the aniline series,9
those compounds exhibited
good oral PK profile.
N
N N
R5
Cl
N
N N
R5
HN
R3
R7
7
c,d
8
H2N
HN N
R3
R3CH2CN
R3
a,b
e
N
N N
R5
HN
R7
N
N N
R5
BocN
R7
Br
f,g h,i
6
9 10 11
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.
Table 1. CDK2 inhibitory activity of pyrazolo[1,5-a]pyrimidines 12a­
12y
N
N N
NH
13
Br
N+
CH3
-O
N
N N
NH
12a-y
R3
N
Compound R3
CDK2/cyclin
A IC50 (lM)
GSK3b
IC50 (lM)
MAPK
IC50 (lM)
12a H 0.25 -- --
12b Br 0.011 0.57 0.37
12c Me 0.072 -- 1.40
12d Et 0.008 2.80 2.00
12e Pr 0.890 -- --
12f Bu 1.20 -- --
12g Ethynyl 0.048 9.82 7.48
12h Vinyl 0.090 -- --
12i Propynyl 0.84 -- --
12j c-Pr 0.071 -- --
12k CF3 0.095 -- --
12l CH2CF3 0.71 -- --
12m SCH3 0.007 1.80 0.90
12n OCH3 1.10 -- --
12o CH2OH 2.17 -- --
12p CH(OH)CF3 3.40 -- --
12q i-Pr 0.37 -- --
12r Ph >50 -- --
12s CH2Ph 16.0 -- --
12t NO2 >50 -- --
12u CH2N(Me)2 34.0 -- --
12v COCH3 >50 -- --
12w S(CH2)2NHAc 0.002 0.034 12.00
12x S(CH2)2OH 0.030 -- --
12y CH2CN 0.049 1.10 1.33
Figure 1. X-ray of crystal structure of 13 in CDK2.
K. Paruch et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6220­6223 6221
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 nonCDK
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 120­390 nM. The compound
is orally available and its PK parameters are summarized
in Table 4.
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.
In conclusion, we demonstrated that properly substituted
pyrazolo[1,5-a]pyrimidines can serve as potent,
selective, and efficacious orally available CDK2
inhibitors.
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.
Table 2. CDK2 inhibitory activity of pyrazolo[1,5-a]pyrimidines 14a­
14p
N
N N
R5
NH
14a-p
Br
N
Compound R5
CDK2/cyclin
A IC50 (lM)
GSK3b
IC50 (lM)
Thym
IC50 (lM)
14a Me 0.060 3.53 1.80
14b Et 0.018 0.92 0.52
14c i-Pr 0.017 0.42 0.95
14d CycloPr 0.045 1.13 1.50
14e CH(CH3)OH 0.038 0.50 1.40
14f CO2Et 0.089 0.79 2.90
14g NHCH3 0.043 4.6 1.20
14h OCH3 0.035 1.41 2.00
14i SCH3 0.038 1.18 0.99
14j Ph-2-Cl 0.003 0.054 0.50
14k Cyclohexyl 0.004 0.027 0.20
14l 3-Piperidinyl 0.008 0.18 0.06
14m 4-Piperidinyl 0.016 0.41 0.16
14n Piperazine 0.210 -- --
14o 2-Furyl 0.008 0.31 15.19
14p 2-Thienyl 0.013 0.48 0.75
Table 3. CDK2 inhibitory activity of pyrazolo[1,5-a]pyrimidines 15a­
15l
N
N N
HN
15a-l
Br
R7
X
Compound X R7
CDK2/cyclin
A IC50 (lM)
GSK3b
IC50 (lM)
Thym
IC50 (lM)
15a H H 0.25 -- --
15b Cl Me 0.020 0.247 1.80
15c Cl Pr 0.163 -- --
15d Cl c-Pr 0.825 -- --
15e F Ph 0.408 13.31
15f Cl Ph-4-SO2CH3 0.35 0.45 0.90
15g F Bn 0.300 15.2 --
15h Cl CH2-3Pyr 0.004 0.054 0.50
15i Cl CH2-3Pyr-O 0.011 0.035 0.17
15j F CH2-3Pyr-O 0.013 0.13 0.21
15k H CH2-3Pyr-O 0.034 0.60 0.14
15l H CH(Me)-3Pyr 3.000 -- --
Table 4. Pharmacokinetic parameters of 15j
Species Dose, mpk vehicle AUC (lM h) cmax (lM) tmax (h)
Rat 10 16.4 2.29 2.0
0.4% MC
Mouse 40 17.9 6.81 2.0
20% HPBCD
Dog 5 2.44 2.58
20% HPBCD
Monkey 10 43.0 3.2 3.3
0.4% HPMC
0
50
100
150
200
250
300
350
400
450
500
550
8 11 14 18
Days (post inoculation)
TumorSize(mm3
)
Control (no dosing)
Vehicle
20 mpk
40 mpk
40 mpk(2-5)
Figure 2. Efficacy of 15j in A2780 xenograft model (mouse).
6222 K. Paruch et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6220­6223
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.
K. Paruch et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6220­6223 6223