Identification of amidoheteroaryls as potent inhibitors of mutant (V600E) B-Raf kinase with in vivo activity

Article abstract of DOI:10.1016/j.bmcl.2008.10.053

A series of amidoheteroaryl compounds were designed and synthesized as inhibitors of B-Raf kinase. Several compounds from the series show excellent potency in biochemical, phenotypic and mode of action cellular assays. Potent examples from the series have also demonstrated good plasma exposure following an oral dose in rodents and activity against the Ras-Raf pathway in tumor bearing mice.

Full text of DOI:10.1016/j.bmcl.2008.10.053

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1026–1029
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of amidoheteroaryls as potent inhibitors of mutant (V600E)
B-Raf kinase with in vivo activity
*
Paul D. Lyne , Brian Aquila, Donald J. Cook, Les A. Dakin, Jay Ezhuthachan, Stephanos Ioannidis,
Timothy Pontz, Mei Su, Qing Ye, Xiaolan Zheng, Michael H. Block, Scott Cowen, Tracy L. Deegan, John W. Lee,
David A. Scott, Dominique Custeau, Lisa Drew, Srinivasu Poondru, Minhui Shen, Allan Wu
Cancer Research, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, 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 amidoheteroaryl compounds were designed and synthesized as inhibitors of B-Raf kinase. Sev-
eral compounds from the series show excellent potency in biochemical, phenotypic and mode of action
cellular assays. Potent examples from the series have also demonstrated good plasma exposure following
an oral dose in rodents and activity against the Ras-Raf pathway in tumor bearing mice.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 3 September 2008
Revised 7 October 2008
Accepted 10 October 2008
Available online 15 October 2008
Keywords:
B-Raf
V600E
Amidoheteroaryl
B-Raf is a member of the MAPK signaling cascade, sitting down-
stream of Ras. During MAPK signaling B-Raf phosphorylates its
substrates Mek-1/2, which in turn phosphorylate Erk-1/2. Once
phosphorylated the Erks translocate to the nucleus where they en-
gage a number of transcription factors that ultimately leads to sev-
eral biological responses including proliferation.1 The Ras-Raf-Erk
pathway has been implicated previously to play a role in oncogen-
esis having been demonstrated to promote proliferation, angiogen-
esis and survival in several cancer models.2 Mutant forms of the
kinase domain of B-Raf have been identified in a number of human
cancer types, with the highest prevalence found in malignant mel-
anoma tumors.3,4 The most common mutation, the V600E mutant
(mB-Raf), has been demonstrated in vitro to be constitutively active
in carcinoma cells and to simulate the growth of cancer cells inde-
pendent of upstream signaling. Based on this epidemiological data,
inhibition of the kinase domain of B-Raf represents a promising
strategy for the clinical treatment of cancers bearing the V600E
mutation.
A ring
B ring
C ring
CF3
N
H
H
N
N
O
O
Linker 1
Linker 2
Figure 1. Bisamidophenyl screening hit 1.
Modeling6 of the initial hit 1 to the X-ray structure of mB-Raf
(pdb code 1uwj7) suggested that this series bound to mB-Raf in a
DFG-out8 manner, with the quinoline located in the region of the
hinge (Fig. 2). Preliminary efforts to explore the SAR of this series
focused on replacement of the quinoline ring. Initial changes to
the quinoline focused on changing the location of the aryl nitrogen
and reduction in ring size to optimize the interaction between the
Cys531 backbone NH and the inhibitor. As shown in Table 1, the
potency of the series is sensitive to the location of the aryl nitro-
gen, and interestingly, monocyclic heteroaryl rings (5) were toler-
ated. This reduction in the molecular weight of the series was seen
as advantageous for improving the physical properties. 3-Pyridyl
analogs were most potent in the enzyme among the monocyclics.
Furthermore, a significant increase in enzymatic and cellular po-
tency was obtained by reversing the amide (in linker 2, 10), which
A subset of the AstraZeneca collection, known or expected to
possess activity against kinases, including compounds screened
previously against C-Raf,5 was screened against mB-Raf and several
series with good activity were identified. One of the promising hit
series in terms of biochemical (enzyme) and cellular potency is
shown in Figure 1 (1: enzyme IC50 0.002
lM; GI50 (Colo205 cells)
5.9 M; P-Erk (Colo205 cells) IC50 0.64 M).
l
l
* Corresponding author.
E-mail address: paul.lyne@astrazeneca.com (P.D. Lyne).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.053

P. D. Lyne et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1026–1029
1027
was attributed to an improvement in the interaction between the
amide and the gatekeeper Thr528, and an improvement in the
hydrogen bond accepting ability of the pyridyl nitrogen.
Compounds 1–9 were prepared via the route shown in Scheme
1. The strategy was to couple 3-(trifluoromethyl)benzoyl chloride
with 4-methyl-3-nitroaniline followed by reduction with SnCl2 to
provide a key intermediate (92% yield for two steps) to examine
multiple C rings.
Subsequent optimization of 10 focused on the generation of an
A-ring library to explore the SAR of the series in the DFG-out pock-
et. A variety of substituents on the 3-position of the A ring were
found to yield compounds with potent inhibition in the enzyme as-
say. Similarly substituents at the 4-, 3,4-, and 3,5-positions also
yielded potent compounds in the enzyme assay. A variety of sub-
stituents yielded excellent enzymatic potency, with substitution
at the 3-position giving good cellular potency (inhibition of P-Erk
levels), and in particular, the dimethylcyano group (19) resulted
in a compound with the best cellular potency. Although compound
19 was found to have reasonable physical and pharmacokinetic
properties, the clearance and bioavailability precluded testing of
this compound in pharmacological assays (Table 3).
Figure 2. Compound 1 docked into the binding site of V600E B-Raf (pdb code
1uwj). The quinoline makes a hydrogen bond to the backbone of Cys531. The amide
between the B and C rings interacts with the gatekeeper Thr528. The amide
between the
A and B rings contributes to the stabilization of the DFG-out
Compounds 10–25 were prepared via the route shown in
Scheme 2. The dimethylcyano A ring was prepared by reacting
methyl-3-(bromomethyl)benzoate with NaCN and then methyl io-
dide. Hydrolysis of the ester with LiOH provided the key A ring in a
43% overall yield. Coupling with methyl 5-amino-2-methylbenzo-
ate followed by hydrolysis (98% yield for two steps) generated
the acid that allowed extensive investigation of the C ring. Com-
pound 19 was completed by coupling the A–B intermediate to pyri-
din-3-amine with HATU in DMF (98%). Ref. 11 provides detailed
experiments for all analogs described in Tables 2 and 3.
conformation of the protein with the trifluoromethylphenyl group buried in the
induced pocket.
Table 1
mB-Raf enzyme and cell P-Erk inhibition SAR for quinoline analogs.
CF3
H
N
X
O
Optimization efforts for compound 19 focused on exploring
substitution on the pyridyl C ring with a view to adding potency,
blocking metabolic sites and adding steric hindrance to mitigate
any potential for Cyp P450 inhibition (10 Cyp2C9 IC50 = 5
lM). In
addition, based on structural information, a focused exploration
Compound
X
IC50
(
l
M)9 mB-Raf
Cell (
l
M)10P-Erk IC50
N
H
N
1
0.002
0.005
0.64
O
O
O
O
of the B ring was examined. The ortho methyl group of the B ring
N
H
N
2
3
0.73
—
N
N
H
N
10.5
N
N
H
N
4
0.001
0.049
0.40
>30
—
Scheme 1. Preparation of examples 1–9. Reagents and conditions: (a) 3-(trifluo-
romethyl)benzoyl chloride, Et3N, DCM; (b) SnCl2ÁH2O, DMF; (c) RCO2H, HATU, i-
Pr2NEt, DMF.
N
H
N
5
O
O
N
N
H
N
N
Br
6
21.1
a, b, c
N
N
+
H
N
O
O
O
7
0.580
1.2
—
H2N
O
O
O
O
O
N
H
N
8
—
N
d, e, f
O
H
N
N
H
N
N
9
0.625
0.007
—
N
N
H
O
O
Scheme 2. Preparation of reversed amide examples. Reagents and conditions: (a)
NaCN, DMF, 75 °C; (b) MeI, NaH, DMSO; (c) LiOH, THF/MeOH/H2O (3:1:1); (d)
HATU, i-Pr2NEt, DMF; (e) LiOH, THF/MeOH/H2O (3:1:1); (f) Pyridin-3-amine, HATU,
i-Pr2NEt, DMF.
O
10
1.4
N
H

1028
P. D. Lyne et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1026–1029
Table 2
of the compounds did not seem to be alleviated by substitution
on the pyridyl ring, with Cyp2C9 being consistently inhibited, al-
beit generally at low M concentrations. Analogs that combined
the best groups at both the 2- and 3-positions yielded compounds
with excellent cellular potency and acceptable pharmacokinetic
profiles.
SAR (biochemical and cellular) of the A ring.
l
N
O
H
R
N
N
H
O
The kinase selectivity profile of this series was assessed, using
39 as a representative. The dose response data are shown in Table
4. Within this panel, which includes a range of tyrosine and serine/
threonine targets of therapeutic relevance, 39 was found to dem-
onstrate good selectivity over the majority of the targets. Notable
Compound
R
IC50
(
l
M) mB-Raf
Cell (lM) P-Erk IC50
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
3-CF3
3-F
0.007
0.322
0.016
0.015
0.016
0.012
0.015
0.028
0.007
0.237
0.011
0.030
0.115
0.009
0.076
1.4
—
>30
5.9
3-Me
3-iPr
exceptions are the potent inhibitions of p38a and CSF-1R. The
3-NMe2
3-Cl
14.6
>30
14.9
10.1
0.33
>30
>30
>30
>30
3.1
p38
a activity is not surprising given a previous report of p38a
activity for related compounds,14 and data on a related series has
been reported recently for CSF-1R.15
3-OiPr
3-OiBu
3-C(Me2)CN
3-CN
3-SO2Me
3-SO2NH2
4-OMe
Based on the known MAPK pathway biology these particular
off-target activities are not expected to contribute to inhibition
3-CF3, 4-Cl
3-C(Me2)CN, 5-CNMe2
Table 4
—
IC50 data for 39 against a panel of kinases screened at KM for ATP in each case.
Kinase
p38
CSF-1R
PDGFRb
PTK2
EphB4
FGFR1
Jak2
IC50 (lM)
a
0.005
0.005
0.031
0.478
0.672
13.0
29.0
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
could be replaced with chloro and that change resulted in reduced
metabolism without impacting cellular potency. The data for se-
lected analogs are summarized in Table 3.
Substitution at the 2- and 3-positions of the pyridyl C ring led to
improvements in cellular potency and in some cases to improved
pharmacokinetic profiles. Addition of a hydrogen bond donor at
the 2-position of the pyridyl ring resulted in good cellular potency
with excellent potency seen for the acetamide analog 31. Unfortu-
nately, the metabolic lability and poor oral exposure of 31 pre-
vented further progression. Attempts to introduce ionizable basic
groups at the 2-position of the pyridyl ring generally increased sol-
ubility but resulted in decreases in cellular potency. A variety of
groups with a range of electronic properties was tolerated at the
3-position. Introduction of a chloro group was found to be partic-
ularly beneficial for cellular potency. The P450 inhibition profile
Kdr
EGFR
IGFR1
Pak1
Plk
Csk
Src
Chk-1
Cdk2
Jnk1
PKA
Table 3
Enzyme and cellular potency, physical property data and rat PK upon iv (3 mpk) and po (10 mpk) dosing.13
X
N
O
H
N
R
N
H
O
Y
12
Compound
R
X
Y
IC50
M)
Cell (
IC50
lM) P-Erk
GI50
M)
Sol (
7.4
lM) pH
Cyp2C9IC50
M)
Cl (mL/min/
kg)
T1/2
(h)
Vdss (L/
kg)
F
(%)
(
l
(
l
(
l
19
26
27
28
29
30
—
2-NH2
2-Me
2-OMe
2-NMe
2-
C(Me2)CN Me 0.007
C(Me2)CN Me 0.014
C(Me2)CN Me 0.048
C(Me2)CN Me 0.116
C(Me2)CN Me 0.038
C(Me2)CN Me 0.079
0.33
0.20
0.25
>30
0.19
6.0
1.2
0.56
1.58
29
42.5
75.5
47.5
1.6
0.6
0.8
2.5
2.3
2.9
17
36
13
9.8
4.9
73
15
2.9
53.5
0.6
1.7
26
Morpholine
2-
31
C(Me2)CN Me 0.048
1.4
NCCN(Me)2
2-NCOMe
2-CN
3-Me
3-Cl
3-CONMe
3-OMe
2-NH2, 3-Cl
2-NH2, 3-Cl
2-NH2, 3-Cl
32
33
34
35
36
37
38
39
40
C(Me2)CN Me 0.016
C(Me2)CN Me 0.205
C(Me2)CN Me 0.016
C(Me2)CN Me 0.035
C(Me2)CN Me 0.017
C(Me2)CN Me 0.014
C(Me2)CN Me 0.016
0.01
>30
0.20
35
2
23
9
21
24
12
<1
<1
64
0.7
2.7
10
0.23
0.04
0.89
0.08
0.04
0.08
0.11
0.83
0.46
2.1
0.81
0.35
0.86
0.38
1.8
0.4
32.5
60
0.8
0.9
1.4
3.1
4
45
56
9.5
11
8
0.5
1.0
1.4
1.6
1.7
0.8
1.2
1.0
4.0
0.7
1.8
30
96
78
CF3
CF3
Cl
0.027
Me 0.012

P. D. Lyne et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1026–1029
1029
Table 5
9. Activity of purified full length His-tagged human mB-Raf was determined
in vitro using an Amplified Luminescent Proximity Homogeneous Assay
(ALPHA) (Perkin-Elmer, MA). mBRaf was expressed in insect cells and affinity
purified by Ni2+ agarose followed by Q-Sepharose chromatography. mB-Raf
Pharmacodynamic data following a single oral dose to A375 tumor bearing Nude
mice.
Compound
30 mpk
60 mpk
100 mpk
enzyme (0.12 nM), 84 nM biotinylated His-MEK1-AVI, and 24
lM ATP in 1.2Â
buffer was preincubated with compound for 20 min at 25 °C. Reactions were
initiated with 24 mM MgCl2 in 1.2Â buffer and incubated at 25 °C for 60 min
and reactions were stopped by addition of detection mix consisting of 20 mM
Hepes, 102 mM ethylenediamine tetraacetic acid, 1.65 mg/mL BSA, 136 mM
NaCl, 3.4 nM Phospho-MEK1/2 (Ser217/221) antibody (Cell Signaling
P-Erk Inhib. (%) 2 h post-dose
38
39
40
0
50
18
40
64
67
83
71
81
Technology, Danvers, MA), 40
lg/mL Streptavidin donor beads (Perkin-Elmer,
Waltham, MA), and 40 g/mL Protein Aacceptor beads (Perkin-Elmer,
l
Waltham, MA). Plates were incubated at 25 °C for 18 h in the dark.
Phosphorylated substrate was detected by an EnVision plate reader (Perkin-
Elmer, Waltham, MA) 680 nm excitation, 520–620 nm emission. Data was
graphed using Excel and IC50s determined.
of mB-Raf mediated signaling. The compound, 39, was found to
be inactive against Kdr and Cdks, which should aid in the inter-
pretation of biological activity in future efficacy studies with this
series.
Based on these optimization efforts compounds 38–40 were
chosen for profiling in pharmacodynamic models. Each compound
was dosed orally to A375 tumor (average tumor volume 200 mm3)
bearing Nude mice. The mice were sacrificed and tumors were col-
lected at specific timepoints post-dose. The tumors were lysed and
analyzed for P-Erk levels. The activities of these compounds rela-
tive to vehicle control are shown in Table 5. Each of these com-
pounds was able to inhibit the Raf-Mek-Erk pathway in tumors
in a dose dependent manner.
In conclusion, amidoheteroaryl compounds presented here are
potent inhibitors of mutant (V600E) B-Raf protein, which results
in blockade of signaling through the Raf-Mek-Erk pathway in
Colo205 cells in vitro, and in A375 tumor cells in vivo. Selected
members of this series have pharmacokinetic properties that ren-
der them suitable for testing in cancer disease models and the re-
sults of such studies will be reported in due course.
10. Cells were seeded into 96-well microplates (Costar, Corning, Lowell, MA) at
2 Â 105 cells/well in phenol red free DMEM (Invitrogen, Carlsbad, CA)
supplemented with 10% FBS. After 48 h, the cells were treated with DMSO or
multiple concentrations of compound and then returned to the incubator for
75 min. Medium was then aspirated and cells fixed with a 6% formaldehyde
solution for 20 min at room temperature. Cells were washed once with PBS
containing 0.5% Triton X-100 (PBST) and 0.6% hydrogen peroxide added for
20 min at room temperature. After washing again in PBST, cells were blocked
with 10% FBS/PBST solution for 1 h at room temperature. After washing,
phospho-p44/42 monoclonal antibody (Cell Signaling Technology, Danvers,
MA) was added and plates placed at 4 oC overnight. Plates were then washed in
PBST, incubated with goat anti-mouse HRP-conjugated secondary antibody
(Cell Signaling Technology, Danvers, MA) for 2 h at room temperature, washed
in PBST, ABTS solution (Sigma, St. Louis, MO) added and plates incubated for
2 h at 30 oC. Quantification of signal was determined at OD405 using
a
SpectraMax plate reader (Molecular Devices, Sunnyvale, CA). Plates were then
washed twice in PBST and twice in water. Crystal violet was then added and
plates incubated for 20 min at room temperature. Washing was then repeated,
1% SDS solution with plates incubated with shaking for 20 min at room
temperature. Quantification of signal was determined at OD595 using
a
SpectraMax plate reader (Molecular Devices, Sunnyvale, CA). Data was graphed
using Excel and EC50s calculated.
11. Pyridine carboxamide derivatives for use as anticancer agents: Almeida, L.; Aquila,
B.; Cook, D.; Cowen, S.; Dakin, L.; Ezhuthachan, J.; Ioannidis, S.; Lee, J. W.; Lee,
S.; Lyne, P. D.; Pontz, T.; Scott, D.; Su, M.; Zheng, X. AstraZeneca AB,
AstraZeneca UK Ltd, WO2006067446 A1, 2006.
References and notes
12. Cells were seeded into 96-well microplates (Costar, Corning, Lowell, MA) in
phenol red-free RPMI DMEM (Invitrogen, Carlsbad, CA) supplemented with
10% FBS. Cell density was predetermined for each cell line based on linear
phase growth over a 96 hour period. After overnight incubation, the cells were
treated with DMSO or multiple concentrations of compound (day 0), and then
returned to the incubator for 72 h (day 3). The number of viable cells on days 0
and 3 was determined using the CellTiter 96 Aqueous One Cell Proliferation
Assay (Promega, Madison, WI). Quantification of signal was determined using a
SpectraMax plate reader (Molecular Devices, Sunnyvale, CA). Percentage of net
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6. Docking was performed using Glide from the Schrodinger software suite
(http://www.schrodinger.com). The ligand was built and refined using ligprep.
The protein was prepared using pprep and the ligand was docked to the kinase
domain using SP mode.
7. Wan, P. T.; Garnett, M. J.; Roe, S. M.; Lee, S.; Niculescu-Duvaz, D.; Good, V. M.;
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growth at day
3 (100%) relative to day 0 (0%) was calculated and the
concentration of compound required to inhibit growth by 50% (GI50)
determined.
13. Compounds were administered to Han Wistar male rats in DMA/PEG/saline
(40/40/20) solutions (iv dose) and (0.1%) HPMC suspensions (po dose).
14. Brown, D. S.; Belfield, A. J.; Brown, G. R.; Campbell, D.; Foubister, A., et al Bioorg.
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