Discovery of highly potent and selective type I B-Raf kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.10.030

A series of pyrazolo[1,5-α]pyrimidine analogs has been prepared and found to be potent and selective B-Raf inhibitors. Molecular modeling suggests they bind to the active conformation of the enzyme.

Full text of DOI:10.1016/j.bmcl.2009.10.030

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6571–6574
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of highly potent and selective type I B-Raf kinase inhibitors
a
a
a
a
a
Xiaolun Wang a, , Dan M. Berger , Edward J. Salaski , Nancy Torres , Yongbo Hu , Jeremy I. Levin ,
*
Dennis Powell a, Donald Wojciechowicz b, Karen Collins b, Eileen Frommer b
a Chemical 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:
A series of pyrazolo[1,5-a]pyrimidine analogs has been prepared and found to be potent and selective
B-Raf inhibitors. Molecular modeling suggests they bind to the active conformation of the enzyme.
Received 15 September 2009
Revised 6 October 2009
Accepted 7 October 2009
Available online 13 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
B-Raf
Kinase
Inhibitors
RAS
Raf
The extracellular signal-regulated Mitogen-Activated Protein
(MAP) kinase pathway plays an important role in cell survival,
growth, and proliferation.1 B-Raf, a key component in this cascade,
is mutationally activated in approximately 8% of all human cancers
with the highest incidence (66%) in malignant melanomas.2 It has
thus received considerable attention as the target of small mole-
cule inhibitors as potential cancer therapeutics.3
It was found that aryl or heteroaryl groups on C-7 position of
the pyrazolopyrimidine core improved activity, particularly basic
amines appended to these aryl or heteroaryl further enhanced en-
zyme and cell potency.7 Our exploration of the SAR resulted in the
potent new lead 3-(3-hydroxyphenyl)-2-(pyridin-4-yl)pyrazol-
o[1,5-a]pyrimidine 4, (Fig. 3, B-Raf kinase8 IC50 = 0.9 nM). It also
showed good activity against proliferation of tumor cell lines with
In our ongoing efforts to develop B-Raf inhibitors we identified
B-RafV600E including A375 (IC50 = 0.300
0.270 M) while it is much less potent against a wild-type B-Raf
cell line CaCo-2 (IC50 = 1.3 M). The synthesis of 4 (Scheme 1)
lM) and HT29 (IC50 =
the lead pyrazolo[1,5-a]pyrimidine-3-carboxylate 14 (B-Raf kinase
l
IC50 = 1.5
l
M) (Fig. 1), which binds in the same manner as sorafe-
l
nib,3a,5 a type II kinase inhibitor. Subsequently, a Letter3b appeared
describing a series of novel B-Raf inhibitory tri-aryl imidazoles
lacking a large hydrophobic group which occupies the DFG-out
pocket.5 We envisioned these tri-aryl imidazoles binding to the ac-
tive conformation of B-Raf as type I kinase inhibitors. This was con-
firmed by a recent report from the same group.3c This led us to
investigate the design of a type I B-Raf inhibitor based on the pyr-
started from 2-(3-methoxyphenyl)acetonitrile 8. Condensation of
8 and methyl isonicotinate (9) followed by reaction with phospho-
rus oxychloride afforded chloroacrylonitrile 11, which then reacted
with hydrazine to give aminopyrazole 12. The annulation of 12 and
enaminone 6 followed by a Buchwald coupling with diazabicy-
clo[3.2.1]octanyl (A1) afforded compound 14. Finally, demethyla-
tion with boron tribromide provided compound 4.
azolo[1,5-a]pyrimidine core.
Analogous to the triaryl imidazoles, our initial work involved
putting pyridyl and phenolic moieties onto the 2 and 3 positions
NH
of the pyrazolo[1,5-a]pyrimidine (Fig. 2). The analogs of 2 and 3
N
overlay well in CHEM3D, which suggests 3 may be able to bind
to B-Raf in the same manner as do tri-aryl imidazoles. Molecular
modeling6 also suggested a good fit of 3 into the ATP binding pock-
et of the active conformation of B-Raf.
O
O
Sorafenib
N
H
N
N
O
Cl
N
O
CF3
1
EtOOC
N
N
CF3
H
H
* Corresponding author.
E-mail address: xxw104@gmail.com (X. Wang).
Figure 1. B-Raf inhibitor 1 and sorafenib.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.030

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X. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6571–6574
Br
Br
a
N
Me
N
H
O
5 O
7
6
Me2N
O
CN
COOMe
N
NC
c
b
+
N
MeO
8
9
10
MeO
Cl
N
HN
H2N
N
N
NC
e
d
MeO
Br
MeO
12
11
N
Me
N
g
f
4
N
N
N
N
Figure 2. Design of a B-Raf inhibitor with pyrazolopyrimidine core.
N
N
N
N
To further optimize 4, we prepared a number of analogs
(Table 1) by following the same synthetic sequence. It was found
that the phenolic hydrogen is crucial for the activity as compounds
with an anisole moiety are much less potent (14, 16). An increase
in potency was observed when a halogen atom was introduced
next to the hydroxyl groups (15, 16). Analogs (17–19) with a short
linker between the phenyl and bicycles were also prepared and
found to be inferior to analogs without the linker. The replacement
of the A1 moiety with a diazabicyclo[2.2.1]heptanyl group (Table
2) resulted in the improvement of both enzyme and cell activities
(4 vs 25). Anisole derivatives (33–38) were much less potent than
corresponding phenolic compounds (20, 22, 24, 29, 30), which fur-
ther confirmed the importance of the phenolic hydrogen. Tertiary
amines (R4 = alkyl) showed better cell potency than secondary
amines (R4 = H) while being almost equipotent in the enzyme as-
say. Ethyl and acetyl groups (31, 32) were deleterious to cell po-
tency and a methyl group was optimal for the tertiary amines.
When a small ortho-group (R5) was introduced into the phenyl lin-
ker, a set of extremely potent compounds (21–23 and 26–28) re-
sulted. The B-Raf IC50 of these analogs were below the test limit
(<0.32 nM) and compound 28 had an A375 cell IC50 less than
MeO
13
14
MeO
Scheme 1. Reagents and conditions: (a) DMF-DMA, reflux, 4 d, 70%; (b) NaOEt,
EtOH, reflux, 3 h, 34%; (c) POCl3, 80 °C, 18 h, 57%; (d) N2H4, EtOH, reflux, 6.5 h, 94%;
(e) 6, HOAc, heat, 41%; (f) 7, cat. BINAP and Pd2(dba)3, NaOtBu, 100 °C, 20 h, 49%; (g)
BBr3, rt, 1 h, 83%.
Table 1
Activities of pyrazolopyrimidine analogs
4, 14-16 X = CH
17-19
X = N
R1
HN
N
Me
N
X
N
A2
N
A1
N
N
N
N
N
0.010 lM.
HN
HN
R2
O
R3
A3
A4
N
Me
N
Compd
R1
R2
R3
B-Raf IC50 (nM)
A375 Cell IC50 (lM)
4
14
15
16
17
18
19
A1
A1
A1
A1
A2
A3
A4
H
Me
H
Me
H
H
H
H
Cl
Cl
H
H
H
0.9
17
0.300
1.900
0.170
0.630
0.930
0.370
0.385
<0.32
9.7
2.0
1.5
1.0
7
N
N
N
3
N
H
HO
Illustrated in Figure 4 is a binding model of compound 28 in
complex with B-Raf.6 In the model, the pyridyl nitrogen forms a
hydrogen bond with the hinge region residue Cys532.3c The
4
Figure 3. Lead compound 4.

X. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6571–6574
6573
Table 2
Table 3
Activities of pyrazolopyrimidines with diazabicyclo[2.2.1]heptanyl
The selectivity profile of compound 25
R4
N
a
Kinase
IC50
ABL1
0.563
25.5
28.4
26.4
18.6
9.51
>50
Aurora B
CDK1
CDK2
CHK
CK1c1
ERK2
N
R5
N
N
FYN
GCK
HCK
1.94
4.10
1.24
>50
N
N
IKK
a
IKKb
LYN
MET
MK2
>50
1.76
3.10
17.1
0.069
6.56
1.79
2.46
0.119
17.6
9.71
0.675
0.222
R6
O
R7
p38a
Compd
R4
R5
R6
R7
B-Raf IC50 (nM)
A375 Cell IC50 (lM)
PDGFR
PKA
a
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
H
H
H
H
H
Me
F
Cl
H
H
Me
F
Cl
H
Me
H
H
H
F
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
F
F
F
F
H
H
F
H
F
0.49
<0.32
<0.32
<0.32
<0.32
0.38
<0.32
<0.32
<0.32
<0.32
<0.32
0.51
0.50
12
0.300
0.065
0.023
0.016
0.150
0.077
0.031
0.012
<0.010
0.076
0.015
0.200
0.210
1.100
0.504
1.200
0.760
6.000
0.933
PKCa
PKCb
ROCK1
RSK1
SRC
VEGFR2
H
Me
Me
Me
Me
Me
Me
Et
Ac
H
H
a
lM, ATP concentration = Km,
of the tested kinase.
ATP
selectivity for all kinases tested. The most potent off target activity
was seen for p38 , PKCb, and VEGFR2 with IC50s less than 0.3 M.
H
a
l
Me
Me
Me
Me
Me
Me
While some analogs such as 25–28 showed excellent cell activ-
ity, it was found that these compounds were labile in microsomes.
Metabolite identification studies indicated that the phenol was
glucuronidated in liver microsomes. Therefore, a search for a phe-
nol replacement was undertaken,9 which led to the discovery of
compound 39 containing an indazole group. Compound 39 was
prepared from 3-(pyridin-4-yl)-1H-pyrazol-5-amine 40 in five
steps (Scheme 2). Although compound 39 is slightly less potent
0.44
8.7
H
Me
Me
Me
8.2
21
1.0
F
(B-Raf kinase IC50 = 1.2 nM; A375 IC50 = 0.202 lM) than the phenol
H
N
Br
N
H
N
H2N
N
NH
N
N
H
a
N
b
N
N
N
N
N
N
N
40
41
42
N
N
N
O
O
Figure 4. Docked model of compound 28 in the active site of B-Raf.
B
N
N
c,d
hydroxyl group makes two hydrogen bonds to the side chain of
Glu501 and the backbone NH of Asp 594, which may make an
important contribution to the affinity. The model also indicated
that the pyrazolo[1,5-a]pyrimidine core sits in a hydrophobic
N
N
H
N
N
e
N
N
N
N
N
pocket formed by Phe583 and Val471 with a possible p–p stacking
N
I
interaction with Phe583. The chlorine atom occupies another small
hydrophobic pocket defined by Ile463, Gly464, and Val471, which
may explain the potency enhancement brought by the substituents
ortho to the pyrazolopyrimidine core.
The selectivity profile of compound 25 was assessed against a
panel of 24 protein kinases (Table 3). It shows at least 182-fold
N
H
43
39
Scheme 2. Reagents and conditions: (a) 6, HOAc, 100 °C, 15 h, 57%; (b) cat. BINAP
and Pd2(dba)3, NaOBut, 100 °C, 2 h, 57%; (c) HCHO (aq), NaBH(OAc)3, DMF, rt, 1 h,
97%; (d) NIS, HOAc/CH2Cl2, rt, 30 min, 69%; (e) cat. Pd(PPh3)4, Na2CO3, 130 °C (lW),
1 h, 43%.

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X. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6571–6574
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analog 25, it shows excellent microsomal stability (t1/2 >30 min,
nude mice). Moreover, it also has a better selectivity profile
(>245-fold for the 24-kinase panel, data not shown) compared to
analog 25.
In summary, a series of pyrazolopyrimidine B-Raf inhibitors has
been designed and evaluated. SAR studies resulted in an extremely
potent analog, compound 28, possessing an optimal ortho substitu-
ent on the phenyl linker and
a novel N-methyl-diazabicy-
clo[2.2.1]heptanyl headpiece. Modeling studies suggest that it
binds to the active conformation of B-Raf as type I kinase inhibi-
tors. These compounds also show good kinase selectivity profiles.
An indazole was introduced to replace the metabolically labile
phenol group and further optimization of the indazole analogs is
ongoing and will be reported in the future.
Acknowledgments
We thank Drs. Tarek Mansour, Robert Abraham, and John
Ellingboe for their support of this work. We also thank the mem-
bers of the Wyeth Chemical Technologies group for analytical
and spectral determination.
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References and notes
6. A model built from 2FB8(PDB)3c was used.
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