Discovery of pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors: A template-based approach - Part 2

Article abstract of DOI:10.1016/j.bmcl.2010.10.113

Previous efforts by our group have established pyrazolo[1,5-a]pyrimidine as a viable core for the development of potent and selective CDK inhibitors. As part of an effort to utilize the pyrazolo[1,5-a]pyrimidine core as a template for the design and synth

Full text of DOI:10.1016/j.bmcl.2010.10.113

Bioorganic & Medicinal Chemistry Letters 21 (2011) 467–470
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 1
Michael P. Dwyer a, , Kamil Paruch a, , Marc Labroli a, Carmen Alvarez a, Kerry M. Keertikar a, Cory Poker a,
⇑
Randall Rossman a, Thierry O. Fischmann a, Jose S. Duca a,à, 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 Rd., Kenilworth, NJ 07033, USA
b Merck Research Laboratories, 901 California Avenue, Palo Alto, CA 94304, USA
c Merck Research Laboratories, 320 Bent St., Cambridge, MA 02141, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
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 for the development of potent, selective kinase inhibitors is
detailed.
Received 14 September 2010
Revised 21 October 2010
Accepted 22 October 2010
Available online 27 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Kinase
CHK1
Pyrazolo[1,5-a]pyrimidine
Template
Checkpoint kinase 1 (CHK1) is a serine/threonine kinase that
controls the cellular response to DNA damage. In response to a
DNA-damaging agent, CHK1 is activated by phosphorylation via
ATR and ATM to arrest cells at various cell-cycle checkpoints (G1,
S and G2) in order to initiate the DNA repair process.1 Inhibition
of CHK1 abrogates cell-cycle arrest resulting in genomic instability
and ultimately progression into mitosis and cell death.2 The
inhibition of CHK1 creates a ‘synthetic lethal’ response by which
aberrant cells can not replicate which should impede the progres-
sion of cancer. In contrast, normal cells still arrest at the G1 check-
point, via p53, to repair the DNA damage caused by these agents.
Due to the fact that inhibition of CHK1 represents a targeted
approach to enhance the cytotoxicity of DNA-damaging agents
toward tumor cells while having a lesser effect on normal cells, it
has been an attractive target in the oncology field.3
PF-00477736 (1)5 and AZD7762 (2)6 (Fig. 1), have recently entered
the clinic in combination with various DNA-damaging agents. Due
to the therapeutic value of a CHK1 inhibitor as a chemopotentiator,7
efforts were directed towards the identification of additional, novel
CHK1 inhibitors.
Screening of an internal compound library identified com-
pounds 3 and 4, as early CHK1 program hits (Fig. 2). While these
initial hits possessed better in vitro potency versus CDK2 than
CHK1, it was rationalized that proper substitution around the
pyrazolo[1,5-a]pyrimidine core might improve the potency and
selectivity of this series for CHK1. Previously, the pyrazolo-
[1,5-a]pyrimidine core was shown to be a viable template for the
preparation of CDK2 inhibitors such as compound 5 which was or-
ally bioavailable and found to be efficacious in a mouse tumor
xenograft model (Fig. 2).8 With compound 4 as a starting point,
A
number of small-molecule CHK1 inhibitors have been
described recently and several comprehensive reviews have pro-
vided overviews of the emerging CHK1 small-molecule chemo-
types.4 In addition, several checkpoint kinase inhibitors, such as
NH2
H
O
N
N
O
NH
O
O
N
N
NH
⇑
S
Corresponding author. Tel.: +1 908 740 4478; fax: +1 908 740 7152.
HN
N
N
H
H
E-mail address: michael.dwyer@merck.com (M.P. Dwyer).
Department of Chemistry, Masaryk University, Kamenice 5, 625 00 Brno, Czech
Republic.
NH2
1
2
F
à
Novartis Institutes for BioMedical Research, Inc., 100 Technology Square,
Cambridge, MA 02139, USA.
Figure 1. CHK1 inhibitors currently under clinical evaluation.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.113

468
M. P. Dwyer et al. / Bioorg. Med. Chem. Lett. 21 (2011) 467–470
H
N
treatment or C3 bromide via NBS treatment afforded intermediates
F
8a or 8b. Treatment of 8a, b under either Suzuki or Stille coupling
conditions followed by global deprotection with 3 N HCl in EtOH
afforded the C3 analogs 9a–b, e–r which are summarized in
Table 1. Alternatively in Scheme 2, cyclization of the Cbz-protected
b-keto ester with either the cyano or ethyl ester substituted
Br
Br
Br
3
5
N
N
N
N
N
N
N
N
N
7
NH2
NH2
N+
NH
-O
3
4
5
Table 1
CHK1 IC50 = 2.90 µM
CDK2 IC50 = 0.25 µM
CHK1 IC50 = 1.08 µM
CDK2 IC50 = 0.086 µM
CHK1 IC50 > 50 µM
CDK2 IC50 = 0.013 µM
CHK1 and CDK2 inhibitory activity of C3 substituted pyrazolo[1,5-a]pyrimidines 9a–r
H
N
Figure 2. Initial pyrazolo[1,5-a]pyrimidine CHK1 Hits.
R
N
N
N
Boc
N
NH2
9a-r
Boc
N
N
d, e
a, b, c
N
OMe
N
a
b
Compd
R
CHK1 IC50
36
(l
M)
CDK2/cyclin A IC50
7.8
(lM)
6
NH2
O
O
9a
H
7
9b
9c
18
0.08
nt
Boc
N
H
N
O
R
R
>50
N
N
OEt
CN
f, g
N
N
9d
9e
2.1
9.4
0.81
0.27
N
N
N(SEM)2
NH2
8a: R = I
9a-b, e-r
9f
5.1
0.71
8b: R = Br
Scheme 1. Reagents and conditions: (a) 3-aminopyrazole, PhCH3, 73%; (b) POCl3,
N,N-dimethylaniline, 71%; (c) NH3, 2-propanol, H2O, 98%; (d) SEMCl, DIPEA,
(CH2Cl)2, 76%; (e) NBS, CH3CN or NIS, CH3CN, 92%; (f) RB(OH)2, PdCl2dppf, K3PO4,
DME/H2O or Bu3SnR, Pd[PPh3]4, dioxane, 29–89%; (g) 3 N HCl/EtOH, 23–78%.
9g
4.5
4.6
9h
9i
1.7
0.64
5.4
OH
N
we focused upon making systematic modifications around the
pyrazolo[1,5-a]pyrimidine core in order to enhance the in vitro
potency for CHK1 while monitoring the selectivity versus CDK2.
It was rationalized that selectivity for CHK1 over CDK2 was re-
quired since the inhibition of the CDK function may antagonize
CHK1 ablation/inhibition phenotypes.9
Since a limited set of substituents had been tolerated at the C3
position in the pyrazolo[1,5-a]pyrimidine core in our previous
CDK2 program,8 initial synthetic efforts focused upon modifica-
tions of the 3-position of compound 4 to further explore potential
differences in this region for CHK1. The preparation of C3 analogs
of compound 4 is illustrated in Schemes 1 and 2.10 Cyclization of
b-keto ester 6 with 3-aminopyrazole followed by chlorination
and amination afforded 7. Diprotection of the resultant C7 amino
group11 followed by introduction of either the C3 iodide via NIS
0.28
N
N
9j
0.29
0.54
1.2
nt
N
N
9k
S
9l
1.1
0.59
0.69
nt
S
9m
9n
1.3
O
0.82
Cbz
N
S
N
Cbz
9o
9p
2.0
2.2
10
R
N
H
N
N
a, b
OMe
N
N
N
>50
O
O
Cl
10
11
H
N
N
S
9q
9r
0.060
0.070
6.1
N
N
R
N
9c: R = CO2Et
9d: R = CN
c,d
N
N
0.79
NH2
nt = not tested; values are means of two experiments.
Scheme 2. Reagents and conditions: (a) 3-aminopyrazole-4-carbonitrile or 3-
amino-4-carboethoxypyrazole, PhCH3, 41–61%; (b) POCl3, N,N-dimethylaniline, 91–
98%; (c) NH3, 2-propanol, H2O, 82–90%; (d) TMSI, MeOH, 31–45%.
a
Assay conditions can be found in Ref. 12.
b
Assay conditions can be found in Ref. 8a.

M. P. Dwyer et al. / Bioorg. Med. Chem. Lett. 21 (2011) 467–470
469
Table 2
Boc
N
Boc
N
CHK1 and CDK2 inhibitory activity of C7 substituted amino pyrazolo[1,5-a]pyrimi-
dines 9q, 17a–r
I
N
X
N
c
a,b
N
N
N
N
H
N
N
N
Cl
12
N
13: X = OMe
14: X = SMe
N
R
N
HN
N
N
Boc
N
H
N
N
N
9q, 17a-r
N
X
N
d,e
a
b
Compd
R
CHK1 IC50
(l
M)
CDK2/cyclin A IC50 (lM)
N
N
N
N
9q
17a
17b
H
Me
Et
0.06
0.14
0.48
6.06
na
na
HN
R
15: X = OMe
16: X = SMe
17a-r
17c
17d
17e
1.5
5.0
6.8
na
na
na
f, g, e
Scheme 3. Reagents and conditions: (a) NIS, CH3CN, 97%; (b) NaOMe or NaSMe,
THF, 85–97%; (c) 1-methylpyrazole-4-boronic acid pinacol ester, PdCl2dppf, K3PO4,
DME/H2O, 52–75%; (d) H2NR, NaH, DMF, 38–98%; (e) TFA, CH2Cl2, 13–98%; (f)
MCPBA, CH2Cl2, 85%; (g) H2NR, n-BuOH, 100 °C, 21–85%.
NH
17f
1.0
31
na
pyrazole followed by subsequent chlorination under standard con-
ditions afforded 11. Treatment with NH3 in isopropanol followed
by Cbz deprotection with TMSI afforded the final adducts 9c and
9d.
17g
0.86
OH
N
17h
17i
0.41
na
na
As shown in Table 1, small linear substituents (9b–f) at the C3
position improved the CHK1 potency versus the 3-H adduct (9a)
with the exception of ester 9c. Despite the potency improvements
for CHK1, these analogs also retained good potency for CDK2.
Incorporation of aryl (9g,h) and heteroaryl substituents into the
C3 position substituents (9i–n) resulted in marked improvement
in CHK1 potency and selectivity versus CDK2 in the biochemical
assay. Additionally, several heteroaryl derivatives in Table 1
(9o,p) showed weaker CHK1 activity which suggested that the
proper placement of heteroatoms in the heteroaryl ring at C3 is
critical to retain CHK1 potency. From this SAR survey of the C3 po-
sition, compound 9q emerged as a key compound which demon-
strated good CHK1 potency (IC50 = 60 nM) and nearly 100-fold
selectivity for CHK1 over CDK2. Compound 9q was selected as a
lead structure for further SAR optimization efforts.
With compound 9q in hand, attention turned toward the explo-
ration of additional substitution of the primary amine located at
the C7 position of this compound. The preparation of these analogs
is outlined in Scheme 3.10 Iodination of 12 followed by treatment
with either sodium methoxide or sodium thiomethoxide in THF
afforded compounds 13 or 14, respectively. Treatment of 13 or
14 with 1-methylpyrazole-4-boronic acid pinacol ester under
Suzuki coupling conditions afforded the corresponding coupled
products 15 and 16, respectively. For the preparation of C7 amino
derivatives bearing either aryl or heteroaryl functionality, com-
pound 15 was treated with the anion of the anilinic/heteroaryl
coupling partners using NaH followed by TFA treatment to afford
the title compounds 17i–r (Table 2). Surprisingly, treatment of 15
with simple alkyl and benzyl amines with heating yielded none of
the desired addition product but only the demethylated (7-OH) ad-
duct. This issue was circumvented by utilization of the correspond-
ing thiomethyl adduct 16. Oxidation of the thiomethyl group with
mCPBA afforded a mixture of sulfoxide/sulfone which was treated
with either alkyl or branched alkyl amine derivatives in hot n-BuOH
in the presence of Et3N to afford the desired addition products.
Deprotection of the intermediate Boc adducts with TFA afforded
the C7 substituted amino derivatives 17a–h listed in Table 2.
0.087
17j
0.051
na
CO2Et
O
17k
17l
0.39
0.61
na
na
O
S
N
17m
34
nt
N
S
N
N
N
17n
17o
17p
17q
17r
2.2
na
na
na
na
40
N
N
0.028
0.028
0.021
0.009
S
N
S
O
N
na = not active up to >50
l
M; nt = not tested.
a
Assay conditions can be found in Ref. 12.
Assay conditions can be found in Ref. 8a.
b
As summarized in Table 2, incorporation of simple alkyl,
branched alkyl, and cycloalkyl groups at the C7 amino group
(17a–e) resulted in poorer in vitro potency for CHK1 versus the
parent NH2 derivative (9q). Simple alkyl modifications with pen-

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M. P. Dwyer et al. / Bioorg. Med. Chem. Lett. 21 (2011) 467–470
References and notes
1. (a) Sancar, A.; Lindsey-Boltz, L. A.; Unsal-Kacmaz, K.; Linn, S. Annu. Rev.
Biochem. 2004, 73, 39; (b) Kastan, M. B.; Bartek, J. Nature 2004, 432, 316.
2. (a) Bucher, N.; Britten, C. D. Br. J. Cancer 2008, 98, 523; (b) Luo, Y.; Leverson, J. D.
Expert Rev. Anticancer Ther. 2005, 5, 333.
3. (a) Powell, S. N.; DeFrank, J. S.; Connell, P.; Eogan, M.; Preffer, F.; Dombkowski,
D.; Tang, W.; Friend, S. Cancer Res. 1995, 55, 1643; (b) Zhou, B.-B. S.; Elledge, S. J.
Nature 2000, 408, 433; (c) Li, Q.; Zhu, G.-D. Curr. Top. Med. Chem. 2002, 2, 939.
4. (a) 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; (b) Janetka, J. W.; Ashwell, S.
Expert Opin. Ther. Patents 2009, 19, 165; (c) Janetka, J. W.; Ashwell, S.; Zabludoff,
S.; Lyne, P. Curr. Opin. Drug Discov. Dev. 2007, 10, 473; (d) Arrington, K. L.;
Dudkin, V. Y. ChemMedChem 2007, 2, 1571.
5. 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.
6. 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.
Figure 3. X-ray of crystal structure of 17r in CHK1.13
7. (a) Tao, Z.-F.; Lin, N.-H. Anti-Cancer Agents Med. Chem. 2006, 6, 377; (b)
Prudhomme, M. Recent Patents Anti-Cancer Drug Discov. 2006, 11, 55.
8. (a) 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.
2007, 17, 6216; (b) Paruch, K.; Dwyer, M. P.; Alvarez, C.; Brown, C.; Chan, T.-Y.;
Doll, R. J.; Keertikar, K.; Knutson, C.; McKittrick, B.; Rivera, J.; Rossman, R.;
Tucker, G.; Fischmann, T.; Hruza, A.; Madison, V.; Nomeir, A. A.; Wang, Y.; Lees,
E.; Parry, D.; Sgambellone, N.; Seghezzi, W.; Schultz, L.; Shanahan, F.; Wiswell,
D.; Xu, X.; Zhou, Q.; James, R. A.; Paradkar, V. M.; Park, H.; Rokosz, L. R.; Stauffer,
T. M.; Guzi, T. J. Bioorg. Med. Chem. Lett. 2007, 17, 6220; (c) Paruch, K.; Dwyer,
M. P.; Alvarez, C.; Brown, C.; Chan, T.-Y.; Doll, R. J.; Keertikar, K.; Knutson, C.;
McKittrick, B.; Rivera, J.; Rossman, R.; Tucker, G.; Fischmann, T.; Hruza, A.;
Madison, V.; Nomeir, A. A.; Wang, Y.; Kirschmeier, P.; Lees, E.; Parry, D.;
Sgambellone, N.; Seghezzi, W.; Schultz, L.; Shanahan, F.; Wiswell, D.; Xu, X.;
Zhou, Q.; James, R. A.; Paradkar, V. M.; Park, H.; Rokosz, L. R.; Stauffer, T. M.;
Guzi, T. J. ACS Med. Chem. Lett. 2010, 1, 204.
9. Walton, M. I.; Eve, P. D.; Hayes, A.; Valenti, M.; De Haven Brandon, A.; Box, G.;
Boxall, K. J.; Aherne, G. W.; Eccles, S. A.; Raynaud, F. I.; Williams, D. H.; Reader, J.
C.; Collins, I.; Garrett, M. M. D. Mol. Cancer Ther. 2010, 9, 89.
10. Full experimental details have appeared elsewhere: Guzi, T. J.; Paruch, K.;
Dwyer, M. P.; Parry, D. A. US 2007/0082900.
11. Protection of the C7 amino group as the di-SEM analog proved to be optimal for
efficient coupling reactions using either the Suzuki or Stille coupling protocols.
12. CHK1 SPA assay. An in vitro assay utilizing recombinant His-CHK1 expressed in
the baculovirus expression system as an enzyme source and biotinylated
peptide based upon CDC25C as substrate. His-CHK1 was diluted to 32 nM in
kinase buffer containing 50 mM Tris pH 8.0, 10 mM MgCl2, and 1 mM DTT.
CDC25C (CDC25 Ser216 C-term biotinylated peptide, Research Genetics)
dant functionality (17f–h) resulted in derivatives with modest
CHK1 activity. All of the C7 substituted amino derivatives in Table
2 demonstrated improved selectivity versus CDK2 (IC50 >30 lM).
While simple aryl derivatives or heteroaryl derivatives demon-
strated good CHK1 activity (17i–j, o–p), the proper placement of
heteroatom functionality in these motifs was important for retain-
ing CHK1 potency as illustrated by 17k–n. Aminoisothiazole 17r
emerged from the SAR work at the C7 position with excellent po-
tency for CHK1 (<10 nM) and very good selectivity over CDK2.
In order to better understand the SAR trends observed in Tables 1
and 2, a single crystal X-ray structure of 17r bound to CHK1 (shown
in Fig. 3) was obtained.13 In the X-ray structure, three key interac-
tions were observed between 17r and the CHK1 protein. First, the
N1 moiety and C7 NH of the pyrazolo[1,5-a]pyrimidine core bind
to the peptide backbone in the hinge area. Secondly, the 1-methyl-
pyrazole moiety at the C3 position interacts with an array of ordered
water molecules in the kinase specificity domain of CHK1. The SAR
observed for the C3 heterocyclic derivatives shown in Table 1 may
be explained in part by the propensity of these motifs to interact
favorably with these water molecules which may mediate interac-
tions with other amino acids. Lastly, the C5 piperidine nitrogen
interacts with the carboxylate of Glu 91 as well as the amide
carbonyl of Glu 134. Interestingly, the SAR observed at the C7 amino
group (Table 2) is difficult to rationalize since this substitution
projects into the solvent-exposed region based upon the X-ray
structure depicted in Figure 3. Additional SAR efforts directed
toward trying to elucidate the structural/electronic requirements
in this region of this class of CHK1 inhibitors will be discussed in
the accompanying manuscript.14
peptide was diluted to 1.93
20 of 32 nM CHK1 enzyme solution and 20
solution were mixed and combined with 10 L of compound diluted in 10%
l
M in kinase buffer. For each kinase reaction,
lL
lL
of 1.926 substrate
lM
l
DMSO, making final reaction concentrations of 6.2 nM CHK1, 385 nM CDC25C
and 1% DMSO after addition of start solution. The reaction was started by
addition of 50
ATP (Amersham, UK), making a final reaction concentration of 1
0.2 Ci of 33P-ATP per reaction. Kinase reactions ran for 2 h at room
temperature and were stopped by the addition of 100 L of stop solution
lL of start solution consisting of 2
l
M ATP and 0.2
lCi of 33P-
lM ATP, with
l
l
consisting of 2 M NaCl, 1% H3PO4, and 5 mg/mL Streptavidin-coated SPA beads
(Amersham, UK). SPA beads were captured using a 96-well GF/B filter plate
(Packard/Perkin Elmer Life Sciences) and
a Filtermate universal harvester
(Packard/Perkin Elmer Life Sciences). Beads were washed twice with 2 M NaCl
and twice with 2 M NaCl with 1% phosphoric acid. Signal was then assayed
using a TopCount 96-well liquid scintillation counter (Packard/Perkin Elmer
Life Sciences). Dose–response curves were generated from duplicate 8 point
serial dilutions of inhibitory compounds. IC50 values were derived by nonlinear
regression analysis.
In summary, systematic optimization of both the C3 and C7
positions of pyrazolo[1,5-a]pyrimidine CHK1 hit 4 led to the
discovery of potent, selective CHK1 inhibitors represented by
17r. Single X-ray crystallography of 17r in CHK1 elucidated several
key interactions with the protein that appear to be critical to the
improvement of CHK1 potency and selectivity versus CDK2 for this
class of compounds. Additional SAR development and further
analysis of this novel class of CHK1 inhibitors is found in the
accompanying paper.14
13. The coordinates of compound 17r bound to CHK1 have been deposited in the
Protein Databank: pdb ID 3OT8.
14. Labroli, M.; Paruch, K.; Dwyer, M. P.; Alvarez, C.; Keertikar, K.; Poker, C.;
Rossman, R.; Fischmann, T. O.; Duca, J. A.; Madison, V.; Parry, D.; Davis, N.;
Seghezzi, W.; Wiswell, D.; Guzi, T. J. Bioorg. Med. Chem. Lett. 2010, 21, 471.