Potent and selective pyrazole-based inhibitors of B-Raf kinase

Article abstract of DOI:10.1016/j.bmcl.2008.07.002

Herein we describe a novel pyrazole-based class of ATP competitive B-Raf inhibitors. These inhibitors exhibit both excellent cellular potency and striking B-Raf selectivity. A subset of these inhibitors has demonstrated the ability to inhibit downstream ERK phosphorylation in LOX tumors from mouse xenograft studies.

Full text of DOI:10.1016/j.bmcl.2008.07.002

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4692–4695
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent and selective pyrazole-based inhibitors of B-Raf kinase
a
a
a
a
a
Joshua D. Hansen a, , Jonas Grina , Brad Newhouse , Mike Welch , George Topalov , Nicole Littman ,
Michele Callejo a, Susan Gloor a, Matthew Martinson a, Ellen Laird a, Barbara J. Brandhuber a, Guy Vigers a,
Tony Morales a, Rich Woessner a, Nikole Randolph a, Joseph Lyssikatos b, Alan Olivero b
*
a Lead Optimization, Array BioPharma, 3200 Walnut Street, Boulder, CO 80301, USA
b Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080-4990, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we describe a novel pyrazole-based class of ATP competitive B-Raf inhibitors. These inhibitors
exhibit both excellent cellular potency and striking B-Raf selectivity. A subset of these inhibitors has
demonstrated the ability to inhibit downstream ERK phosphorylation in LOX tumors from mouse xeno-
graft studies.
Received 12 May 2008
Revised 26 June 2008
Accepted 1 July 2008
Available online 5 July 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
B-Raf
Raf
RAF
RAS
MEK
ERK
V600E
Inhibitors
Cancer
The Ras/Raf/MEK/ERK signaling pathway is critical for cell sur-
vival, growth, and proliferation. This intensely studied signal trans-
duction pathway has been implicated in a number of human
cancers. Examinations of cell lines and primary tumor samples
have shown constitutive activation of the ERK pathway in cancers
of the lung, colon, pancreas, kidney, and ovary.1
The serine/threonine kinase B-Raf, when mutated plays an
important role in the development of cancer. B-Raf is mutated
in approximately 7% of human cancers, and a majority of malig-
nant melanomas (50–70%) and papillary thyroid cancers (40–
70%) harbor B-Raf mutations.2 The substitution of valine for glu-
tamic acid at position 600 (V600E) accounts for around 90% of
identified mutations.3 The basal rate of MEK phosphorylation
from V600EB-Raf is 500-fold greater than wild type B-Raf, provid-
ing a strong signal for growth and proliferation to cancer cells.4
The high prevalence of B-Raf mutations in human cancer and
their prominent role in the Ras/Raf/MEK/ERK signaling pathway
in tumorigenesis and progression suggests that disruption of this
signaling cascade could offer a beneficial approach for cancer
chemotherapy.
Several research programs which are currently targeting inhibi-
tion of B-Raf have been cited in recent reviews.5 Figure 1 shows
two distinct structural classes of B-Raf inhibitors, SB-5908856
and RAF-265, of which the later has advanced into phase I clinical
trials.7 We recently became interested in pursuing a research pro-
gram on B-Raf inhibition, and discovered a substituted pyrazole as
a potentially novel lead structure (Fig. 2).
Our studies began with the synthesis of unsubstituted pyra-
zole 1 which defined our minimum pharmacophore. Methylation
of the pyrazole led to a mixture of N1 and N2 methyl isomers.
The N2 methylated pyrazole 2b showed a threefold loss in cellu-
lar potency (pERK), whereas the N1 methyl pyrazole 2a dis-
played a modest gain in potency relative to 1 (Table 1).8 This
trend was seen for a number of analogs (data not shown), which
focused our efforts toward further exploration of N1-modified
pyrazoles. Extension of the alkyl chain by one carbon further in-
creased cellular potency (3) and in general, increased bulk at this
position, such as with the cyclopentyl analog 4, was well toler-
ated. Further substitution on the cycloalkyl ring showed
a
dependence on stereochemistry. While the cis hydroxy analog
5 was less potent, the trans isomer 6 was nearly sevenfold more
active. A further increase in potency was observed in analog 7,
which was slightly more active than its corresponding trans iso-
mer 8. Although ethers (e.g., 9) were in general less active, incor-
* Corresponding author. Tel.: +1 303 386 1312.
E-mail address: josh.hansen@arraybiopharma.com (J.D. Hansen).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.002

J. D. Hansen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4692–4695
4693
Figure 1. Selected B-Raf inhibitors.
Table 2
R3
C4
N1R1
B-Raf activity of N1-modified pyrazoles: amino substituents
N
N
C3
R2
HO
N
N2
Figure 2. Substituted pyrazoles as B-Raf inhibitors.
N
R
N
N
a
Compound
R
B-Raf IC50 (nM)
2.4
pERK IC50 (nM)
119
Table 1
B-Raf activity of N1-modified pyrazoles: alkyl and hydroxylalkyl substituents
NH2
14
HO HO
N
N
NMe2
15
16
17
18
19
20
21
4.02
0.39
0.47
0.03
0.34
0.22
1.2
607
20
NH2
N
N
R
N
N
122
9
N
N
NMe2
2b
a
Compound
R
B-Raf IC50 (nM)
pERK IC50 (nM)
NH
1
H
CH3
—
CH2CH3
Cyclopentyl
OH
0.15
0.02
0.48
0.57
3.46
53
33
165
18
2a
2b
3
10
N
O
48
4
57
5
6
3.91
245
37
332
NH
NH
OH
0.33
22
0.85
0.23
154
18
HN
OH
OH
23
7
8
0.04
0.09
7
a
IC50 values reflect the average from at least three separate experiments.
17
9
CH2CH2OCH3
CH2CH2OH
CH2CH2CH2OH
CH2CH(OH)CH2OH
CH(CH2OH)2
1.16
0.13
0.05
0.87
0.56
569
63
26
22
171
10
11
12
13
minal nitrogen caused a decrease in potency (14 vs 15 and
16 vs 17). Cyclic amines, such as piperidine 18, led to in-
creased potency, and further substitution (19) maintained com-
parable cellular activity. Extending the cyclic amines with a
one-carbon spacer caused a reduction in potency (21 and 22)
while extension of amines directly from the carbocycle dis-
played good activity in both enzymatic and cellular assays
(e.g., 23).
Although modifications at N1 simultaneously increased solu-
bility and potency, some compounds in this series displayed an
undesirable CYP inhibition profile. To explore this property, sev-
eral 4-pyridyl replacements were made (Table 3). Under the as-
say conditions, both indazole 24 and the 3-pyridyl 25 analogs
showed no detectable cellular activity, while the oxazole 26
showed a 70-fold loss in activity relative to 2. This seemed con-
sistent with a theory that improved binding occurs when the
a
IC50 values reflect the average from at least three separate experiments.
poration of alcohols was well tolerated (e.g., 10–13). Such deriv-
atives led to increased aqueous solubility and provided a conve-
nient handle for further substitution and modulation of
physiochemical properties.
In addition to solubilizing tails such as hydroxyl alkyls, a
number of tethered amino substituents were examined (Table
2). The ethyl-linked amines were tolerated, but less active in
the cellular assay than their propyl-linked counterparts ( 14,
15 vs 16, 17). In both chain lengths, methylation of the ter-

4694
J. D. Hansen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4692–4695
Table 3
B-Raf activity of C3-modified pyrazoles
HO
N
N
CH3
N
R
a
a
Compound
R
B-Raf IC50 (nM)
pERK IC50 (nM)
N
N
24
969.6
—
25
26
27
433.8
4.1
—
2400
1000
N
O
N
NH2
2.1
N
28
2.9
3.1
800
N
HO
29
1500
N
N
a
IC50 values reflect the average from at least three separate experiments.
C3 substituent is aligned to accept an H-bond from the hinge
region of the protein (Fig. 3). Attempts to retain potency by
keeping an H-bond acceptor in this region (e.g., 27–29) were
ineffective.
H-bond interactions made by the oxime moiety were re-
vealed in the X-ray structure of the B-Raf complex co-crystal-
lized with inhibitor 18 (Fig. 3a).9 Our SAR evaluation at the C4
position of the pyrazole ring was aimed toward retaining these
contacts and simultaneously improving the poor aqueous sta-
bility at low pH that characterized the oxime series.10 While
the hydroxyamidine analogs 30–32 had the potential H-bond-
ing network intact, only analog 31 displayed good potency.
This suggests that, in certain derivatives, potency increases
Figure 3. (a) X-ray crystal structure of 18 bound to B-Raf, at 2.8 Å resolution.
The electron density shows an Fo À Fc omit map, contoured at 2.5sigma. The
structure is viewed from the N-terminal domain of B-Raf, looking into the ATP-
binding site. The ‘‘hinge” region of B-Raf is on the left of the figure. (b) Cut
away view of 18 on surface of B-Raf. Hydrogen bonds are depicted as dashed
red lines.
with both
a proper display and appropriate filling of the
hydrophobic pocket with which the indane moiety makes con-
tact (Fig. 1b). A similar SAR trend was also observed for pyra-
zole analogs 33–35 in which conformational restraint and
additional hydrophobic contact serve to increase potency. Both
the lactam 36 and phenol isostere11 37 lost potency relative to
2 (Table 4).
The synthesis used to access analogs such as 10 is outlined in
Scheme 1. Pyrazole 3912 was prepared from 4-acetylpyridine via
cyclization of the intermediate enaminoketone with hydrazine
and subsequent bromination. Alkylation of the pyrazole at N1 with
halides or tosylates was substrate-dependant, but generally affor-
ded selectivity better than 6:1. Other electrophiles such as cyclic
epoxides and enones, (e.g., 5–8) reacted with exclusive N1 selectiv-
ity. Palladium-mediated coupling of 40 and commercially available
41 followed by treatment with hydroxylamine provided the E-
oxime analogs.
ma level of the compound. Tumors were harvested, then pro-
cessed for Western blot quantitation of phosphorylated ERK
(p44/42, thr202/tyr204) and total ERK (Fig. 4). Compound 18
had no detectable plasma levels and correspondingly, no effect
on pERK levels. Compounds 2, 7, and 10 all showed plasma lev-
els at the 1 hour time-point, which correlated well with pERK
inhibition.
The selectivity of a subset of these inhibitors was examined.
Compounds 2, 7, and 10 were screened against a panel of over
70 protein kinases at 1 lM. None of the compounds tested
showed greater than 50% inhibition against any of the enzymes
with the exception of C-Raf. Furthermore, when compound 10
was screened in an expanded panel of 212 protein kinases, it
was again selective, only showing expected activity against C-
Raf.
In summary, we have utilized a pyrazole core to produce B-
Raf inhibitors with excellent potency, physiochemical proper-
Four compounds were selected for further evaluation based
upon physiochemical (Table 5)13 and in vivo properties (data
not shown). Female nude mice bearing established subcutane-
ous LOX tumor xenografts were given a single 25 mg/kg po
dose of compound in 50% PEG400/0.05 N HCl vehicle. At 1
and 4 h post dose, blood was collected for analysis of the plas-
ties, and selectivity profiles.
A subset of these compounds
showed significant in vivo inhibition of pERK in xenograft
studies. Further progress on these inhibitors will be reported
in due course.

J. D. Hansen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4692–4695
4695
Table 4
Table 5
B-Raf activity of C4-modified pyrazoles
In vitro stability, permeability, and solubility
R
2
10
7
18
31
N
CH3
Hepat. Cla
Caco-2
7
6
8.5
High
3c
7.8
Med
52
8
High
590
N
High
High
N
Sol. @pH6.5b
4c
76c
a
Compound
R
B-Raf IC50 (nM)
pERK IC50 (nM)
20,000
a
Human hepatocyte clearance (mL/min/kg).
b
c
Aqueous thermodynamic solubility (
lg/mL).
Crystalline solid.
NH2
OH
30
18.4
N
NH
5000
ng/ml
180
160
140
120
100
80
31
32
33
34
35
36
<2.0
16.0
68.4
4.4
56
4500
N
OH
% control
4000
3500
3000
2500
2000
1500
1000
500
NH
10,000
—
N
OH
60
N
N
NH
40
18,000
1900
—
20
NH
0
0
1.5
NH
N
Figure 4. PK/PD evaluation of substituted pyrazoles after 25 mg/kg po dose in LOX
tumor-bearing mice. Four per group, error bars are SEM.
NH
68.4
70.0
O
References and notes
H
N
37
—
N
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Weber, B. L.; Seigler, H. F.; Darrow, T. L.; Paterson, H.; Marais, R.; Marshall, C. J.;
Wooster, R.; Stratton, M. R.; Futreal, P. A. Nature 2002, 417, 949; (b) Cohen, Y.;
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A. W.; King, F. D.; Lovell, P. J.; Naylor, A.; Reith, A. D.; Steadman, J. G.; Wilson, D.
M. Bioorg. Med. Chem. Lett. 2006, 16, 378 .
a
IC50 values reflect the average from at least three separate experiments.
Br
Br
a,b,c
NH
OH
O
d,e
N
N
N
N
N
38
39
40
N
HO
N
O
f,g
+ 40
O
B
N
N
OH
O
41
10
N
(GDC-0879)
(AR00341677)
7. ClinicalTrials.gov.; Identifier: NCT00304525.
Scheme 1. Reagents and conditions: (a) DMF–DMA, tol,
EtOH,
potassium carbonate, DMF; (e) water, MeOH, potassium carbonate,
tetrakis(triphenylphosphine)palladium, potassium carbonate, ACN–water,
D
, (78%); (b) hydrazine,
8. For assay description see: Laird, E.; Lyssikatos, J.; Welch, M.; Grina, J.; Hansen,
J.; Newhouse, B.; Olivero, A.; Topolav, G. WO 2006/084015 A2, 2006.
9. Coordinates for the B-Raf crystal structure have been deposited in PDB: 3d4q.
10. Compound 2 has an aqueous stability half life of 1.2 h at pH 1.2.
11. Bamborough, P.; Angell, R. M.; Bhamra, I.; Brown, D.; Bull, J.; Christopher, J. A.;
Cooper, A. W. J.; Fazal, L. H.; Giordano, I.; Hind, L.; Patel, V. K.; Ranshaw, L. E.;
Sims, M. J.; Skone, P. A.; Smith, K. J.; Vickerstaff, E.; Washington, M. Bioorg. Med.
Chem. Lett. 2007, 17, 4363.
D
,
(96%); (c) bromine, NaOAc, AcOH, (70%); (d) 2-bromoethyl acetate,
, (76%); (f)
, (74%);
D
D
(g) hydroxylamine hydrochloride, EtOH,
D, (78%).
Acknowledgement
12. Compound 39 has been described: Desbordes, P.; Guigues, F. WO 94/29300,
1994.
13. Detailed description of these assays can be found in the supporting information
from: Wallace, E. M.; Lyssikatos, J.; Blake, J. F.; Seo, J.; Yang, H. W.; Yeh, T. C.;
Perrier, M.; Jarski, H.; Marsh, V.; Poch, G.; Livingston, M. G.; Otten, J.; Hingorani,
G.; Woessner, R.; Lee, P.; Winkler, J.; Koch, K. J. Med. Chem. 2006, 49, 441.
The authors thank Andrew Allen for NMR analysis and Susan
Rhodes, Jennifer Otten, and Michelle Livingston for Caco, P450,
and solubility determinations.