Identification of pyrazolo[1,5-a]pyrimidine-3-carboxylates as B-Raf kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.03.129

B-Raf kinase plays a critical role in the Raf-MEK-ERK signaling pathway and inhibitors of B-Raf could be used in the treatment of melanomas, colorectal cancer, and other Ras related human cancers. We have identified novel small molecule pyrazolo[1,5-a]pyr

Full text of DOI:10.1016/j.bmcl.2009.03.129

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2735–2738
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of pyrazolo[1,5-a]pyrimidine-3-carboxylates as B-Raf kinase
inhibitors
a
a
a
b
b
Ariamala Gopalsamy a, , Greg Ciszewski , Yongbo Hu , Frederick Lee , Larry Feldberg , Eileen Frommer ,
*
Steven Kim b, Karen Collins b, Donald Wojciechowicz b, Robert Mallon b
a Chemical and Screening Sciences, Wyeth Research, 401 N. Middletown Road., Pearl River, NY 10965, USA
b Discovery Oncology, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
B-Raf kinase plays a critical role in the Raf-MEK-ERK signaling pathway and inhibitors of B-Raf could be
used in the treatment of melanomas, colorectal cancer, and other Ras related human cancers. We have
identified novel small molecule pyrazolo[1,5-a]pyrimidine derivatives as B-Raf kinase inhibitors. Struc-
ture–activity relationship was generated for various regions of the scaffold to improve the biochemical
profile.
Received 13 February 2009
Revised 20 March 2009
Accepted 25 March 2009
Available online 28 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
B-Raf
B-Raf Inhibitor
Pyrazolo[1,5-a]pyrimidine-3-carboxylate
Raf kinases are serine/threonine protein kinases. They play cen-
tral role in cell growth and survival and are components of Raf-
MEK-ERK signaling pathway.1 This pathway normally mediates
cellular responses to growth signals, however, Ras mutations that
are associated with 30% of all cancers constitutively activate this
pathway. Of the three Raf human isoforms (A-, B- and C-), activat-
ing B-Raf mutations have been found in 66% of malignant melano-
mas2 and in a smaller fraction of other cancers including those of
the colorectum. The most common B-Raf mutation is valine substi-
tution by glutamic acid at amino acid position (V600E). B-Raf
mutations in these cancers were found in a systematic genome-
wide screening effort to detect alterations in genes that control cell
proliferation, differentiation, and death. Inhibitors of B-Raf could
be used in the treatment of melanomas, colorectal cancer, and
other Ras related human cancers thus making B-Raf kinase a com-
pelling drug discovery target.
F
O
N
NH
O
H
N
H
N
O
CF3
Cl
O
Cl
S
N
F
O
H
O
N
N
H
PLX4720
Sorafenib
N
H
N
O
N
N
N
SB-590885
HO
Figure 1. Examples of B-Raf kinase inhibitors.
A number of small molecule inhibitors of B-Raf have emerged in
the recent past3 including the most intensively studied Sorafenib,4
triarylimidazole SB-5908855 and azaindole PLX-47206 (Fig. 1). As
part of our on-going effort to identify B-Raf inhibitors, we estab-
lished an HTS assay to monitor the kinase activity of both wt and
mutant (V600E) forms of this protein. This assay was an adaptation
of our previously described assay system to detect inhibitors of
both C-Raf and MEK1 kinases.7 Briefly, B-Raf was used to phos-
phorylate GST-tagged inactive MEK1. MEK1 phosphorylation was
measured by a phospho-specific antibody that detected B-Raf
phosphorylation of the two serine residues at positions 217 and
221 on MEK1.8 From this effort pyrazolo[1,5-a]pyrimidine-3-car-
boxylate 1 (Fig. 2) was identified as a promising B-Raf kinase
inhibitor.
Compound 1 showed an IC50 of 1.5
lM in the B-Raf kinase as-
say. It inhibited the growth of a variety of tumor cell lines including
BXPC-3 (IC50: 3.25
SW620 (IC50: 8.3
l
l
M), HT29 (IC50: 7.0
M), LOVO (IC50: 3.87
6.2 lM) and CaCo-2 (IC50: 6.6 lM). It is a novel scaffold for B-Raf
l
M), A375 (IC50: 3.8
lM),
l
M), WM266-4 (IC50
:
kinase inhibition. This compound was found to be selective when
* Corresponding author.
tested against a panel of kinases including PDK-1, m-TOR, Tpl2,
E-mail address: gopalsa@wyeth.com (A. Gopalsamy).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.129

2736
A. Gopalsamy et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2735–2738
NO2
NO2
a
H
N
O
N
CF3
b
NH2
N
NH
N
+
O
COOEt
O
N
2
3
4
NO2
N
NH2
N
CO2Et
1
O
c
F3C
+
Cl
N
Figure 2. B-Raf inhibitor hit 1 (pyrazolo[1,5-a]pyrimidine-3-carboxylate) identified
N
N
from high through-put screen.
N
N
COOEt
COOEt
7
6
5
BTK, AKT, CDK-4, LCK, LYN, KDR, IGFR-1, SRC and PI3 K
a
(IC50 > 10 M). The key structural features of the compound had
l
H
N
high degree of resemblance to the reported B-Raf kinase inhibitor
Sorafenib. A docking study was performed with the standard
Glide2.0 SP procedure, using the crystal structure of Sorafenib_B-
Raf complex.9 Illustrated in Figure 3 is a binding model of com-
pound 1 in complex with B-Raf. The model indicated that the
amide of compound 1 makes two hydrogen bonds to the enzyme:
one to the side chain of Glu500 and another to the backbone NH of
Asp593. No specific polar interaction was found between the ester
moiety and B-Raf, but the ester does make hydrophobic interac-
tions with Ile462, Trp530 and Phe582 with the ethyl group point-
ing toward a solvent accessible region. On the other end of the
molecule, the aromatic ring of the amide region sits at a hydropho-
bic pocket which consists of Ile 512, His 573 and Ile 571. The ring is
partially exposed to solvent at the C4 position. Based on this infor-
mation, the initial structure–activity relationship for the ester moi-
ety, amide linker and the aromatic ring of the amide region were
explored.
CF3
O
d
N
N
N
COOEt
1
Scheme 1. Reagents and conditions: (a) DMF–DMA, reflux, 18 h, 79%; (b) acetic
acid, 80 oC, 12 h, 72%; (c) Fe, acetic acid, 80 °C, 3 h, 60%; (d) pyridine, rt, 70%.
H
H
N
N
CF3
CF3
O
O
a
b
1
N
N
N
N
The analogs designed to obtain structure–activity relationship
were synthesized by following the synthetic sequence employed
for compound 1 as shown in Scheme 1.10 Reaction of 3-nitroaceto-
phenone 2 with N,N-dimethylformamidedimethylacetal followed
by condensation with 5-aminopyrazole-4-carboxylate 4 afforded
ester 5. Selective reduction of the nitro group of compound 5 affor-
ded the key intermediate aniline 6 which was further derivatized
to the required amides by reacting with appropriate acid chlorides.
Reacting the common intermediate amine 6 with various isocya-
nates or triphosgene and appropriate amines generated urea ana-
N
N
COOH
CONHR
9
8
Scheme 2. Reagents and conditions: (a) 2 M LiOH, THF–MeOH, 40 °C, 6 h, 90%; (b)
RNH2, PYBOP, DIEA, DMF, rt, 12 h.
Initial understanding of the SAR was directed towards deter-
mining the importance of the trifluoromethyl group and its posi-
tion on the aromatic ring (Table 1) while maintaining the rest of
the molecule intact. Elimination of the group resulted in an unsub-
stituted phenyl analog 10 with significantly reduced activity.
Replacing the CF3 group with other substituents was also at-
tempted. Both electron withdrawing groups and electron releasing
groups were introduced to probe the electronic effects. Most of the
aromatic substituents including halogens were not favorable.
However, introduction of trifluoromethoxy group (compound 16)
maintained the activity indicating that the lipophilicity of the
group is probably more important than the electronics for binding
interactions as indicated by our docking studies. Moving the CF3
group from meta position of the aromatic ring (R2) to the para
position (R3) reduced the activity as shown by analog 17.
logs.
Pyrazolo[1,5-a]pyrimidine-3-carboxylic
acid
8
and
carboxamides 9 were prepared starting from the ester 1, which
was hydrolyzed to the acid and coupled with different amines
(Scheme 2).
Having established the requirement of a lipophilic group like
CF3 in the R2 position of the aromatic ring, we explored the toler-
ance of an additional substituent in that ring as shown by analogs
18–22 in Table 1. Introduction of a small group like methyl, meth-
oxy and chloro in the R3 position along with CF3 group in the R2
position of the aromatic ring was very well tolerated (compounds
18–20). However, moving these substituents to the R5 position of
the aromatic ring in the presence of the CF3 group in the R2 posi-
tion (compound 21 and 22) was not favorable. This is probably
due to the steric hindrance introduced by these groups around
the amide linker which forms two key hydrogen bond interactions
Figure 3. Docked conformation of compound 1 in the active site of B-Raf.

A. Gopalsamy et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2735–2738
2737
Table 1
Table 3
B-Raf kinase activity of pyrazolopyrimidine analogs
SAR for ester modification
R4
L
CF3
R3
R5
R3
R2
NH
O
N
N
N
W
N
N
R3
W
B-Raf kinase
N
Compd
LINKER (L)
a
O
O
IC50
(l
M)
31
32
8
–NH–CO–
–NH–CO–
–NH–CO–
–NH–CO–
–NH–CO–
–NH–CO–
–NH–CO–
–NH–CO–
–NH–CO–
–NH–CO–
–NH–CO–NH–
–NH–CO–NH–
–NH–CO–NH–
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
H
CN
COOH
COOMe
COOCH2CF3
COOCH2CH2N(CH3)2
CONH2
CONHCH3
CONHCH2CH2N(CH3)2
CONHCH2CH2–N–morpholinyl
CONHCH2CH2N(Et)2
CONH(CH2)3OCH3
CONHCH2CH2-N-morpholinyl
>10
>10
>10
0.41
1.45
7.07
>10
>10
5.88
6.34
0.17
0.27
0.16
a
Compd
R2
R3
R4
R5
B-Raf kinase IC50 (lM)
1
CF3
H
CH3
OCH3
Cl
H
H
H
H
H
H
H
H
CF3
CH3
OCH3
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
Cl
1.54
>10
>10
>10
>10
>10
>10
1.57
>10
0.5
33
34
35
36
37
38
39
40
41
42
10
11
12
13
14
15
16
17
18
19
20
21
22
Br
CN
OCF3
H
CF3
CF3
CF3
CF3
CF3
1.03
4.27
>10
>10
a
Values are means of two or more experiments.
pound 26). Additional substituents in the R3 position provided
slight increase in activity in the urea analogs as well, as shown
by compounds 27-29 (Table 2). From our docking studies, this ob-
served conservation of SAR for the hydrophobic region is explained
by the movement of the protein residues in the binding pocket to
accommodate the longer urea linker.
a
Values are means of two or more experiments.
with the enzyme. Replacing the entire aromatic group with
aliphatic groups like methyl or neopentyl was not encouraging.
Similarly replacing the m-trifluoromethylphenyl ring with m-tri-
fluoromethylbenzyl ring was not accommodated. Replacing with
hetroaromatics like thiophene or pyridyl rings resulted in loss of
activity.
As predicted by our proposed binding of compound 1 to the en-
zyme, SAR for the amide linker variation indicated that the reverse
amide and sulfonamides are not ideal. However, the analogs with
urea linker which maintains the required hydrogen bond interac-
tions, showed slight increase in potency as shown in Table 2. As ob-
served in the case of amide linkers, moving the CF3 group to ortho
or para position was not beneficial. A similar effect was observed
with CF3 replacement with other substituents like chloro (com-
In order to determine the importance of the ethyl ester group in
the hit, modifications were attempted at this region (Table 3).
Substituting the ethyl ester with other esters (compounds 33–35)
maintained the activity. However removal of the ester
(analog 31) or replacing the ethyl ester with acid 8 or nitrile 32
or unsubstituted amide 36 rendered the compounds less active.
Since in our proposed binding pose the ethyl group of the ester is
solvent exposed, we explored this region further to introduce
water solubilizing groups through the carboxamide. A number of
analogs were generated (compounds 37–42) using either amide
linker or urea linker. This modification was tolerated and some in-
crease in the enzyme activity was observed for the urea analogs.
However this modification did not lead to significant boost in the
cell activity.11 The next phase of this hit to lead optimization pro-
cess will utilize the observed SAR to improve the pharmaceutical
properties to realize significant improvement in cell activity.
In summary, as detailed above pyrazolopyrimidine scaffold
identified as B-Raf inhibitor from HTS has been optimized to infer
SAR for various regions of the molecule. A lipophilic group in the
meta position of the aromatic group in the amide region was pre-
ferred with tolerance for additional substituents. Of the linkers at-
tempted the urea linker was favorable with slight increase in
potency. Replacement of the ester with amides was tolerated with
slight increase in enzyme potency and provides a handle for intro-
ducing water solubilizing groups to improve the pharmaceutical
properties of the scaffold. Our continued efforts to elaborate this
scaffold to further improve the overall biological and pharmaceuti-
cal profile will be reported in future.
Table 2
SAR for amide linker modification
R5
H
N
H
N
R4
R3
O
R2
N
N
N
O
O
R2
R3
R4
R5
B-Raf kinase IC50 (lM)
a
Compd
23
24
25
26
27
28
29
30
CF3
H
H
H
CF3
H
H
H
H
H
H
H
H
H
H
H
CF3
H
H
H
H
Cl
0.54
3.6
>10
>10
0.36
0.36
0.24
>10
Cl
H
CF3
CF3
CF3
CF3
CH3
Cl
F
Acknowledgments
H
The authors thank Dr. John Ellingboe for his support of this
work. We also thank discovery analytical chemistry group for ana-
lytical data.
a
Values are means of two or more experiments.

2738
A. Gopalsamy et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2735–2738
8. B-Raf kinase assay: Flag/6his-tagged recombinant human B-Raf (from Sf9
References and notes
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containing 20 mM MOPS, pH 7.2, 25 mM b-glycerol phosphate, 5 mM EGTA,
1 mM sodium orthovanadate, 1 mM dithiothreitol; Magnesium/ATP Cocktail
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(0.05%). Kinase assays were carried out in a shaking incubator. The protocol
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l
l
l