Discovery of pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors: A template-based approach - Part 1

Article abstract of DOI:10.1016/j.bmcl.2010.10.114

The synthesis and hit-to-lead SAR development of a pyrazolo[1,5-a] pyrimidine hit 4 is described leading to a series of potent, selective CHK1 inhibitors such as compound 17r. In the Letter, the further utility of the pyrazolo[1,5-a]pyrimidine template fo

Full text of DOI:10.1016/j.bmcl.2010.10.114

Bioorganic & Medicinal Chemistry Letters 21 (2011) 471–474
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors:
A template-based approach—Part 2
Marc Labroli a, , Kamil Paruch a, , Michael P. Dwyer a, Carmen Alvarez a, Kartik Keertikar a, Cory Poker a,
⇑
Randall Rossman a, Jose S. Duca a,à, Thierry O. Fischmann a, Vincent Madison a, David Parry b, Nicole Davis b,
Wolfgang Seghezzi b, Derek Wiswell b, Timothy J. Guzi c
a Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, United States
b Merck Research Laboratories, 901 California Avenue, Palo Alto, CA 94304, United States
c Merck Research Laboratories, 320 Bent Street, Cambridge, MA 02141, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Previous efforts by our group have established pyrazolo[1,5-a]pyrimidine as a viable core for the devel-
opment of potent and selective CDK inhibitors. As part of an effort to utilize the pyrazolo[1,5-a]pyrimi-
dine core as a template for the design and synthesis of potent and selective kinase inhibitors, we focused
on a key regulator in the cell cycle progression, CHK1. Continued SAR development of the pyrazolo
[1,5-a]pyrimidine core at the C5 and C6 positions, in conjunction with previously disclosed SAR at the
C3 and C7 positions, led to the discovery of potent and selective CHK1 inhibitors.
Received 14 September 2010
Revised 21 October 2010
Accepted 22 October 2010
Available online 28 October 2010
Keywords:
Kinase
Ó 2010 Elsevier Ltd. All rights reserved.
CHK1
Pyrazolo[1,5-a]pyrimidine
CDK
Cell cycle
Inhibitors
As one of the key regulators of the cell cycle progression, CHK1 is
a serine/threonine kinase which has been an attractive target in
oncology.1 A number of small-molecule ATP-competitive CHK1
inhibitors have been described2 and several compounds have re-
cently entered the clinic including PF-00477736 (1)3 and AZD7762
(2)4 (Fig. 1). Herein, we describe our continued SAR development
Initial screening of our internal compound library identified
compound 3 as a CHK1 program hit which was derived from an
earlier CDK program (Fig. 2). Initial SAR optimization work on
the pyrazolo[1,5-a]pyrimidine scaffold at both the C3 and
C7–NH2 positions led to several promising targets, including 4
and 5, which displayed improved in vitro potency for CHK1 and in-
creased selectivity against CDK2 versus the early hit 3 (Fig. 2).5
As discussed in the preceding Letter,5 the SAR study of the C3
position demonstrated that the 4-N-methylpyrazole moiety con-
ferred optimal CHK1 potency while maintaining selectivity against
CDK2. Additionally, structural modifications of the C7–NH2 posi-
tion were extremely challenging as a majority of substituents
NH2
H
O
N
N
O
NH
O
O
N
N
NH
S
HN
N
N
H
H
NH2
1
2
F
N
N
H
N
H
N
H
N
Figure 1. CHK1 inhibitors currently under clinical evaluation.
N
N
Br
of the pyrazolo[1,5-a]pyrimidine core to develop potent and selec-
tive CHK1 inhibitors.
3
N
N
N
5
6
N
N
N
N
N
N
7
NH
1
NH2
NH2
S
⇑
Corresponding author. Tel.: +1 908 740 5573; fax: +1 908 740 7441.
N
5
3
4
E-mail address: marc.labroli@merck.com (M. Labroli).
Current address: Department of Chemistry, Masaryk University, Kamenice 5, 625
00 Brno, Czech Republic.
CHK1 = 0.009 µM
CDK2 = 40 µM
CHK1 = 1.08 µM
CDK2 = 0.086 µM
CHK1 = 0.06 µM
CDK2 = 6.10 µM
à
Current address: Novartis Institutes for BioMedical Research, Inc., 100 Technology
Square, Cambridge, MA 02139, United States.
Figure 2. Pyrazolo[1,5-a]pyrimidine hits for CHK1 program.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.114

472
M. Labroli et al. / Bioorg. Med. Chem. Lett. 21 (2011) 471–474
incorporated in this region displayed a loss in CHK1 potency versus
the C7–NH2 analog with the exception of compound 5. These
observations could, in part, be attributed to the deleterious effect
of the C7–NH2 substituents on the crucial H-bonding motif in the
hinge region.5 In light of these observations, we felt there might
be additional opportunities to explore the solvent exposed region
using C6 substitution. Initial synthetic efforts focused upon the
preparation of the 6-halo compounds 9a–c which maintained key
functionality at C3 (N-methyl pyrazole) and C5 (3-piperdine) that
was previously noted for CHK1 potency.5 It was envisioned that
the C6 halo functionality could serve as handles for further elabo-
ration at the C6 position. The preparation of these analogs was dis-
cussed previously and shown in Scheme 1.5,6 Interestingly, the
6-halo compounds 9a–c demonstrated a 20-fold improvement in
CHK1 activity relative to the parent compound 4 (Table 1).
play a role in modulating the acidity of the adjacent C7–NH2
group.
Based upon the SAR observed in Table 1, we decided to briefly
investigate a series of C3 heteroaromatic groups bearing the
C6–Br to determine if we were maintaining optimal potency and
selectivity among the C3, C6, and C7 substituents. Representative
examples are depicted in Table 2. Although compound 9b re-
mained the most potent analog, other C3 heteroaromatic groups,
for example, 12c, demonstrated comparable CHK1 potency with
a comparable selectivity profile against CDK2.
With the initial SAR investigations at the C3, C6, and C7–NH2
positions of the pyrazolo[1,5-a]pyrimidine core complete, atten-
tion was turned toward exploration of the C5 position. From the
X-ray structure of compound 5 bound to CHK1,5 the NH of the
C5 3-piperidinyl group was observed to form several key hydrogen
bond interactions with several acidic residues as well as a con-
served water molecule in this region. Initial SAR efforts at the C5
position focused upon further optimization of this key H-bonding
interaction. In order to more rapidly examine the SAR in this re-
gion, an alternative synthesis was developed taking into account
the optimal substituents at the C3, C6, and C7 positions of the pyr-
azolo[1,5-a]pyrimidine core. Retrosynthetically, we envisioned a
more convergent assembly of the pyrazolo[1,5-a]pyrimidine core
(e.g., 4) via the cyclocondensation of b-keto nitrile 13 and 3-ami-
no-1-methyl-1H-10H-4,40-bispyrazole 14 (Fig. 3).
Boc
N
Boc
N
a, b, c
d, e
OMe
N
O
O
N
N
NH2
6
7
N
Boc
N
H
N
N
The preparation of aminopyrazole 1411 began with Vilsmeier-
Haack formylation of N-methyl-1H-pyrazole 15 to afford aldehyde
16 (Scheme 3). Treatment of 16 with tosyl methyl isocyanide12
(TosMIC) in the presence of potassium t-butoxide in DME resulted
in the one-step homologation and cyanation process to yield the
I
N
N
N
f, g, h
N
R1
N
N
N(SEM)2
NH2
8
9a-c
acetonitrile 17. a-Formylation, followed by cyclization with hydra-
zine monohydrochloride in ethanol yielded the final bispyrazole
14. It should be noted that this preparation of bispyrazole 14 was
amenable to large-scale synthesis and the use of TosMIC avoids
the need for toxic cyanide reagents commonly employed in other
cyanation protocols.
Scheme 1. Reagents: (a) 3-aminopyrazole, PhCH3, 75%; (b) POCl3, N,N-dimethyl-
aniline, 71%; (c) NH3, 2-propanol, H2O, 98%; (d) SEMCl, DIPEA (CH2Cl)2, 76%; (e) NIS,
CH3CN, 92%; (f) 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyr-
azole, PdCl2dppf, K3PO4, DME, 81%; (g) HCl, EtOH, 90%; (h) NCS, CH3CN, 72% or NBS
or Br2, t-BuNH2, CH2Cl2, 73% or NIS, CH3CN, 83%.
With the bispyrazole 14 in hand, the syntheses of the C5 targets
22a–k were accomplished utilizing the route displayed in Scheme
4. Treatment of acids 19 with 1,1-carbonyldiimidazole followed by
addition of the anion of acetonitrile provided the substituted
b-keto nitriles 20 in good yield. Cyclocondensation of 20 with
bispyrazole 14 from Scheme 4 in EtOH or toluene provided the core
21. Regioselective bromination with NBS followed by acid depro-
Unfortunately, all attempts to install additional functionality at
the C6 position via the 6-halo precursors using either Suzuki or
Stille coupling protocols were unsuccessful (not shown). While
the limitations of using these coupling protocols in sterically con-
gested systems has been documented,7 the initial synthetic route
had to be modified to allow for more facile incorporation of sub-
stituents at the C6 position which is shown in Scheme 2. Treatment
of 7 with bromine in t-butylamine8 yielded the corresponding
6-bromo derivative 10 for further elaboration (Scheme 2). Bis-
protection of 10 with SEMCl9 followed by Pd-mediated couplings
introduced desired substitution at the C6 position to yield com-
pound 11. Functionalization of the C3 position was achieved by
bromination, Suzuki or Stille coupling with the appropriate hetero-
aromatic group, and global deprotection to afford targets 9d–q.
The biochemical assay results for both CHK1 and CDK2 are summa-
rized in Table 1 for compounds 4 and 9a–q.
Boc
N
Boc
N
N
N
a
b, c
N
N
N
N
Br
NH2
10
NH2
7
As evident from Table 1, small alkyl or cycloalkyl substituents
at the C6 position retained reasonable CHK1 potency with varying
levels of CDK2 selectivity (9d–i) similar to the 6-halo compounds
9a–c. Incorporation of either aryl or heteroaryl derivatives at the
C6 position generally led to a loss of CHK1 potency versus the
smaller substituents while maintaining some selectivity over
CDK2 (9j–p). Although it appears that the solvent exposed region
would be more accommodating to a variety of substituents, the
SAR observed at the C6 position may be a combination of both
size and polarity requirements of the substituents. Additionally,
the electron withdrawing substituents such as the 6-halo deriva-
tives 9a–c seem to be preferred at this position and may in fact
Boc
N
H
N
R2
N
N
N
d, e, f
N
R1
N
R1
N
N(SEM)2
NH2
11
9d-q
Scheme 2. Reagents: (a) Br2, t-BuNH2, CH2Cl2, 79%; (b) SEMCl, DIPEA (CH2Cl)2, 39%;
(c) R1B(OH)2, PdCl2dppf, K3PO4, DME, H2O or Bu3SnR1, Pd(PPh3)4, dioxane, 27–83%;
(d) NBS, CH3CN, 66–92%; (e) R2B(OH)2, PdCl2dppf, K3PO4, DME, H2O or Bu3SnR2,
Pd[PPh3]4, dioxane, 41–90%; (f) HCl, EtOH, 17–80%.

M. Labroli et al. / Bioorg. Med. Chem. Lett. 21 (2011) 471–474
473
Table 1
N
Boc
N
H
CHK1 and CDK2 inhibitory activity of pyrazolo[1,5-a]pyrimidines 4 and 9a–p
N
N
N
N
O
N
H2N
NH
N
+
H
N
N
N
N
CN
N
N
NH2
4
13
14
N
R1
N
NH2
Figure 3. Retrosynthetic analysis of 4.
R1
CHK1 IC50
CDK2/cyclin
A IC50 (lM)
a
Compds
b
(lM)
4
H
Cl
Br
I
0.060
0.003
0.003
0.003
0.031
0.045
0.017
0.026
0.052
0.10
6.1
0.31
0.16
0.33
0.33
0.52
0.17
0.048
2.2
well as different positional isomers (22b,c) led to a loss of CHK1
potency. Additional heteroatoms are tolerated with varying
degrees of CHK1 potency (e.g., 22d,e) while ring-contracted (22f)
or the acyclic amine derivative (22g) generally showed reduced
CHK1 potency. Several homologated analogs (22i,j) as well as exo-
cyclic amine analog 22k demonstrated comparable CHK1 potency
to 9b with a comparable or improved selectivity profile against
CDK2 (Table 3).
9a
9b
9c
9d
9e
9f
9g
9h
9i
Et
Propynyl
Cypr
(CH2)2OH
CH@CH–CH2OMe
CN
1.7
A single X-ray crystal structure of 22k bound to CHK1 (Fig. 4)
was obtained which confirmed a similar binding mode to that ob-
served for compound 5.5 From Figure 4, the key H-bond network
from the C3 pyrazole in 22k with ordered waters is maintained
while the C7–NH2 maintains a key hinge contact with the C6–Br
group projecting into the solvent exposed region as expected.
Interestingly, the exocyclic NH2 of 22k occupies a similar position
as the piperidine NH of 5 to maintain the key interactions with an
acidic group and a conserved water molecule which is imperative
for CHK1 potency as demonstrated in Table 3.
9j
9k
9l
9m
9n
9o
9p
Ph
0.56
0.086
0.16
0.34
0.51
8.3
0.99
2.2
2.3
1.5
2-Thienyl
3-Thienyl
3-Furyl
3-Pyridyl
4-Pyridyl
0.23
0.10
11
22
4-N-Methylpyrazolyl
Values reported are means of two experiments.
a
Assay conditions can be found in Ref. 5.
Assay conditions can be found in Ref. 10.
b
Starting with a CDK2 selective pyrazolo[1,5-a]pyrimidine CHK1
lead 3, systematic SAR studies of the C3, C5, C6, and C7–NH2 posi-
tions of the core led to the identification of potent and selective
CHK1 inhibitors, for example, 5, 9a–c, 12c, and 22i–k.5 Interest-
ingly, potent CHK1 inhibition can be retained through the appro-
priate combination of C6 substitution with a primary amine at
tection of Boc intermediates, when necessary, provided the desired
targets 22a–k listed in Table 2.
As summarized in Table 3, the SAR at C5 clearly demonstrates
the necessity of both the proper positioning and basicity of the pip-
erdine NH for optimal CHK1 potency and selectivity against other
kinases, specifically CDK2. Loss of the piperdine NH (e.g., 22a) as
H
O
NC
N
a
b
N
N
N
N
N
Table 2
CHK1 and CDK2 inhibitory activity of C3-substituted pyrazolo[1,5-a]pyrimidines 9b
and 12a–c
16
17
15
NH2
NC
c
N
N
d
NH
N
N
N
H
N
H
R2
O
14
18
N
N
N
Scheme 3. Reagents: (a) POCl3, DMF, 50%; (b) KO-t-Bu, TosMIC, DME, 65%; (c) ethyl
formate, KO-t-Bu, DME, 82%; (d) N2H4–HCl, EtOH, 90%.
Br
NH2
R2
CHK1 IC50
0.003
(
lM)
CDK2/cyclin A IC50
0.16
(lM)
a
b
Compds
N
N
R3
OH
R3
O
a, b
c
9b
O
CN
20
19
N
12a
12b
0.017
0.025
0.17
0.10
N
N
N
N
O
R3
N
R3
Br
N
d, e
N
N
N
N
S
N
12c
0.008
0.20
NH2
21
NH2
22a-k
Values reported are means of two experiments.
Scheme 4. Reagents and conditions: (a) CDI, THF; (b) LiHMDS, CH3CN, THF, ꢀ78 °C,
22–78% overall for two steps; (c) 14, EtOH, 45 °C, or toluene, 115 °C, 37–87%; (d)
NBS, DCM, 71–89%; (e) TFA, DCM, 39–93%.
a
Assay conditions can be found in Ref. 5.
b
Assay conditions can be found in Ref. 10.

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M. Labroli et al. / Bioorg. Med. Chem. Lett. 21 (2011) 471–474
Table 3
CHK1 and CDK2 inhibitory activity of C5-substituted pyrazolo[1,5-a]pyrimidines 9b
and 22a–k
N
N
R3
Br
N
N
N
NH2
a
b
Compds
R3
CHK1 IC50
0.003
(
lM)
CDK2/cyclin A IC50
0.16
(lM)
H
N
9b
N
22a
4.0
1.3
Figure 4. X-ray of crystal structure of 22k in CHK1.13
NH
22b
22c
2.9
28.7
0.49
HN
0.035
as well as to calibrate the extent of CDK2 selectivity required in a
CHK1 inhibitor utilizing a mechanistically based in-cell evaluation
assay will be the subject of a future Letter.
H
N
22d
22e
0.13
1.2
O
References and notes
H
N
0.006
0.15
1. Zhou, B.-B. S.; Elledge, S. J. Nature 2000, 408, 433.
S
2. (a) Janetka, J. W.; Ashwell, S.; Zabludoff, S.; Lyne, P. Curr. Opin. Drug Discov.
Devel. 2007, 10, 473; (b) Matthews, T. P.; Klair, S.; Burns, S.; Boxall, K.; Cherry,
M.; Fisher, M.; Westwood, I. M.; Walton, M. I.; McHardy, T.; Cheung, K.-M. J.;
Van Montfort, R.; Williams, D.; Aherne, G. W.; Garrett, M. D.; Reader, J.; Collins,
I. J. Med. Chem. 2009, 52, 4810. and references cited therein.
3. Blasina, A.; Hallin, J.; Chen, E.; Arango, M. E.; Kraynov, E.; Register, J.; Gant, S.;
Ninkovic, S.; Chen, P.; Nichols, T.; O’Connor, P.; Anderas, K. Mol. Cancer Ther.
2008, 7, 2394.
H
N
22f
0.031
0.034
0.51
0.92
H2N
22g
4. Zabludoff, S. D.; Deng, C.; Grondine, M. R.; Sheehy, A. M.; Ashwell, S.; Caleb, B.
L.; Green, S.; Haye, H. R.; Horn, C. L.; Janetka, J. W.; Liu, D.; Mouchet, E.; Ready,
S.; Rosenthal, J. L.; Queva, C.; Schwartz, G. K.; Taylor, K. J.; Tse, A. N.; Walker, G.
E.; White, A. M. Mol. Cancer Ther. 2008, 7, 2955.
H
N
22h
0.087
2.8
5. Dwyer, M. P.; Paruch, K.; Labroli, M. A.; Alvarez, C.; Keertikar, K.; Poker, C.;
Rossman, R.; Duca, J. A.; Madison, V.; Parry, D.; Davis, N.; Seghezzi, W.; Wiswell,
D.; Guzi, T. J. Bioorg. Med. Chem. Lett. 2010, 21, 467.
NH
6. Full experimental details have appeared elsewhere: Guzi, T. J.; Paruch, K.;
Dwyer, M. P.; Parry, D. A. US 2007/0082900.
7. (a) Mei, X.; Martin, R. M.; Wolf, C. J. Org. Chem. 2006, 71, 2854; (b) Johnson, M.
G.; Foglesong, R. J. Tetrahedron Lett. 1997, 38, 7001.
8. (a) Pearson, D. E.; Wysong, R. D.; Breder, C. V. J. Org. Chem. 1967, 32, 2358; (b)
Ishizaki, M.; Ozaki, K.; Kanematsu, A.; Isoda, T.; Hoshino, O. J. Org. Chem. 1993,
58, 3877.
9. Protection of the 7-amino group as the di-SEM analog proved to be optimal as it
was shown that this protection was imperative for efficient coupling reactions
via either the Suzuki or Stille couplings.
10. Dwyer, M. P.; Paruch, K.; Alvarez, C.; Doll, R. J.; Keertikar, K.; Duca, J.;
Fischmann, T. O.; Hruza, A.; Madison, V.; Lees, E.; Parry, D.; Seghezzi, W.;
Sgambellone, N.; Shanahan, F.; Wiswell, D.; Guzi, T. J. Bioorg. Med. Chem. Lett.
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11. Labroli, M. A.; Poker, C.; Guzi, T. J.; Paruch, K.; Dwyer, M. P.; Keertikar, K. M.;
Alvarez, C. S. Process for preparation of substituted 3-aminopyrazoles via
formylation, cyanation, and cyclocondensation reactions. PCT Int. Appl. WO
2008153873.
22i
0.007
0.007
0.005
0.84
2.4
NH
22j
NH2
22kc
0.44
Values reported are means of two experiments.
a
Assay conditions can be found in Ref. 5.
Assay conditions can be found in Ref. 10.
10,30-Syn.
b
c
12. van Leusen, A. M.; Oomkes, P. G. Synth. Commun. 1980, 10, 399.
13. The co-ordinates of 22k bound to CHK1 have been deposited in the Protein
Databank pdb 3OT3.
C7. Approaches to differentiate between this novel class of pyraz-
olo[1,5-a]pyrimidines and those identified in the preceding Letter5