Design and synthesis of 5,6-fused heterocyclic amides as Raf kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.04.023

Two scaffolds based on 5,6-fused heterocyclic backbones were designed and synthesized as Raf kinase inhibitors. The scaffolds were assessed for in vitro pan-Raf inhibition, activity in cell proliferation and target modulation assays, and pharmacokinetic p

Full text of DOI:10.1016/j.bmcl.2011.04.023

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3286–3289
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of 5,6-fused heterocyclic amides as Raf kinase inhibitors
Savithri Ramurthy a, , Mina Aikawa b, Payman Amiri b, Abran Costales a, Ahmad Hashash c,
⇑
Johanna M. Jansen a, Song Lin a, Sylvia Ma b, Paul A. Renhowe a, Cynthia M. Shafer a,
Sharadha Subramanian a, Leonard Sung, Joelle Verhagen
a Global Discovery Chemistry/Oncology and Exploratory Chemistry, Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, CA 94608, USA
b Oncolog Research, Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, CA 94608, USA
c Gilead, 333 Lakeside Drive, Foster City, CA 94404, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 February 2011
Revised 2 April 2011
Accepted 7 April 2011
Available online 14 April 2011
Two scaffolds based on 5,6-fused heterocyclic backbones were designed and synthesized as Raf kinase
inhibitors. The scaffolds were assessed for in vitro pan–Raf inhibition, activity in cell proliferation and tar-
get modulation assays, and pharmacokinetic parameters.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Raf kinase inhibitors
5,6-Fused heterocycles
Benzoxazoles
Benzothiazoles
Pharmacokinetic profile
O
The Ras-mitogen activated protein kinase (MAPK) signaling
pathway was the first signaling pathway elucidated from the cell
membrane to the nucleus.1 The MAPK signaling pathway consists
of the Ras/Raf/MEK/ERK signal transduction cascade which is a vi-
tal mediator of a number of cellular activities including growth,
proliferation, survival and other aspects of cellular behavior that
can contribute to the transformed phenotype, making it an attrac-
tive pathway to target in several cancer types. The three Raf iso-
forms (Raf-1 or c-Raf, A-Raf and B-Raf) are all able to interact
with Ras and activate the MAP kinase pathway.2–5
O
N
NHMe
HN
N
X
1
X= NMe
2 X= O
3
X= S
Figure 1. 5,6-Fused heterocyclic cores explored for Raf inhibition.
Inhibition of the Raf/MEK/ERK pathway at the level of Raf ki-
nases is expected to be effective against tumors driven by this
pathway. It has been shown that B-Raf mutation V600E in skin nevi
is a critical step in the initiation of melanocytic neoplasia.6 Further-
more, activating mutations in the kinase domain of B-Raf occur in
roughly 66% of malignant melanomas, 40–70% of papillary thyroid
carcinomas, 12% of colon carcinomas and 14% of liver cancers.5,7–9
The many effects of Raf kinases on cancer cell growth and survival,
together with the high prevalence of mutation in melanoma, for
which there is no good treatment, make Raf a very attractive target
for anticancer therapy.
The first reported Raf inhibitor, BAY43-9006 (Sorafenib,)10–14
,
while effective in RCC, has shown a lack of efficacy in patients
expressing the BrafV600E mutation suggesting its mechanism of ac-
tion is through inhibition of VEGFR rather than Raf.15,16 However,
several other Raf inhibitors (PLX-403217 GSK211843618) are now
in clinical trials and have shown indications of clinical benefit.
Our group has previously disclosed the benzimidazole amide series
as orally available potent Raf inhibitors.19 In particular, the 3-t-
butylphenylbenzimidazole amide 1 ( Fig. 1) potently inhibited
BrafV600E and the phosphorylation of the downstream target ERK
Abbreviations: MAPK, ras-mitogen activated protein kinase; VEGFR, vascular
endothelial growth factor receptor; PDGFR, platelet-derived growth factor receptor;
CSF-1, colony stimulating factor-1 receptor; RTK’s, receptor tyrosine kinases; STK’s,
serine threonine kinases; Lck, Leukocyte specific protein tyrosine kinase.
in the SKMEL-28 cell line with an EC50 of 0.3 lM. The benzoxazole
and benzothiazole amide series (2 and 3, respectively) were syn-
thesized and compared to the lead benzimidazole scaffold to
⇑
Corresponding author. Tel.: +1 510 923 7767; fax: +1 510 923 3360.
E-mail address: savithri.ramurthy@novartis.com (S. Ramurthy).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.023

S. Ramurthy et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3286–3289
3287
cokinetic profiles for representative examples will also be pre-
sented in this communication.
O2N
HO
OMe H2N
a
OMe
b,c
OMe
N
O
Cl
The synthetic22 routes to key intermediates (6 and 8) for the
benzoxazole and benzothiazole scaffolds are depicted in Scheme
1. The synthesis of 6 began with the commercially available nitro-
phenol 4 which was reduced by hydrogenation to give aminophe-
nol (5). Compound 5 was treated with ethyl potassium xanthate to
afford the sulfuryl benzoxazole, which was then chlorinated to af-
ford 6. Bromination of commercially available mercapto benzothi-
azole 7 yielded the desired intermediate 8 required for the
synthesis of the benzothiazole core.
HO
4
5
6
OMe
OMe
N
S
N
d
HS
Br
S
7
8
With the key intermediates for both scaffolds in hand, introduc-
tion of an aniline was effected by SNAr conditions using microwave
to afford 9 and 10 (Scheme 2). Demethylation with 48% HBr fol-
lowed by O-arylation using KHMDS and 4-chloro-pyridylaceta-
mide (11)23 in DMF in the microwave gave the desired
compounds 2 and 3.24
An evaluation of the structure activity relationship of the ani-
line for the benzoxazole series was undertaken (Table 1). As ob-
served in the benzimidazole series, the 3-tert-butyl group was
important for in vitro potency and inhibition of phosphorylation
of ERK.19 It was also determined that compounds with ortho sub-
stituents (12–14) were less potent than compounds with substitu-
ents at the meta or para positions. Compounds substituted at the
meta position (2, 15 and 16) potently inhibited both c-Raf and mu-
tant-B-Raf. In addition, two analogs (2 and 16) were tested in a tar-
get modulation assay and inhibited ERK phosphorylation in the
Scheme 1. Synthesis of benzoxazole intermediate 6 and benzothiazole intermedi-
ate 8. Reagents and conditions: (a) H2, 10% Pd/C, MeOH; (b) potassium ethylxant-
hate, KOH, EtOH, reflux, 18h; (c) SOCl2, DMF, 5 min; (d) bromine, CH2Cl2, rt.
OMe
N
X
OMe
a
HN
N
X
Y
6
9
Y = Cl, X = O
X = O
Y = Br, X = S 8
X = S 10
O
O
N
X
b-c
NHMe
HN
N
O
Cl
1.0 lM range which is three-fold less potent than the lead benz-
NHMe
imidazole (1). Finally, the para position of the aniline tolerated a
large range of substituents ranging from halogens to aliphatics to
oxygen and nitrogen linked moieties (17–24). Only the appendage
of a piperazine to the ring (25) led to degradation of in vitro
potency.
In the exploration of the aniline SAR for the benzothiazole ser-
ies, we focused on the meta and para positions (Table 2, 3, 28–33),
to obtain the best combination of in vitro and cellular potency,
consistent with what we learned from the benzoxazole series.
We found that the SAR tracked well between the two series. The
N
11
2
3
X = O
X = S
Scheme 2. Reagents and conditions: (a) 3-t-butylaniline, NMP, 200 °C, microwave,
7 min; (b) 48% HBr, 140 °C, microwave, 6 min; (c) KHMDS, K2CO3, DMF, 170 °C
microwave 7 min.
further explore the structure activity relationship of additional het-
erocycle ring systems against Raf. Benzoxazoles were reported first
by our group20 and they were also developed by Potashman et al.
as VEGFR inhibitors.21 In addition to the in vitro Raf SAR, pharma-
Table 1
Structure–activity relationship of the benzoxazole scaffold
O
O
N
HN
NHR'
N
O
R
Compd
R
R0
c-Raf IC50
(
lM)
BrafV600E IC50
(l
M)
p-ERK SKMEL28 EC50 (lM)25
1
2
—
0.001
0.20
0.12
0.11
0.027
0.003
0.019
0.003
0.001
0.001
0.002
0.002
0.053
0.012
7.3
0.12
0.006
0.58
0.26
0.34
0.11
—
0.021
0.025
0.005
0.006
0.011
0.012
0.68
0.13
17
0.28
1.0
—
—
—
—
1.0
—
10
7.0
—
—
—
—
—
—
3.7
—
3-t-Bu
2-F
2-Cl
2-Et
3-CF3
3-i-Pr
4-Cl
4-Br
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
4-Et
4-i-Pr
4-n-Bu
4-OCF3
4-OPh
4-NMe2
4-N-Et-piperazine
3-i-Pr
3-i-Pr
0.004
0.14
0.004
0.2
Morpholinoethyl

3288
S. Ramurthy et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3286–3289
Table 2
Structure–activity relationship of the benzothiazole scaffold
O
O
N
S
NHR'
HN
N
R
Compd
R
R0
c-Raf IC50
(lM)
BrafV600E IC50
(l
M)
p-ERK SKMEL28 EC50 (lM)
BAY43-9006
1
3
—
0.12
0.28
2.1
—
>10
—
—
3.2
—
3-t-Bu
4-Cl
4-Br
4-Me
4-n-Bu
4-i-Pr
4-OCF3
3-t-Bu
4-Br
Me
Me
Me
Me
Me
Me
Me
H
0.001
0.004
0.002
0.007
0.009
0.003
0.004
0.001
0.041
0.004
0.015
0.007
0.026
0.065
0.019
0.018
0.002
0.24
28
29
30
31
32
33
34
35
2.9
>10
Morpholinoethyl
Figure 2. Binding site models for compounds 3 (left, in cyan) and 2 (right, in cyan) derived by docking into the crystal structure of B-Raf (PDB accession code 1UWH). The left
and right pictures show cartoon representations of the kinase with selected residues in stick model (Glu501, Cys532, Phe593, Asp594) and the co-crystallized BAY43-9006 in
green. The middle picture shows a surface representation, of the docking model for 3 with the surface colored by atom type (red = oxygen, blue = nitrogen, yellow = sulfur,
grey = carbon/hydrogen).
effect of altering the amide substituent was investigated for both
benzoxazole and benzothiazole series. The primary carboxamides
(26, 34) were found to be equipotent to the methyl amide analogs
(2, 3) and showed a similar effect in the cellular target modulation
structure published for B-Raf (PDB accession code 1UWH) Figure
2 shows the overlap of the docking models with the co-crystallized
conformation of BAY43-9006. The model suggests a very similar
binding mode when comparing 2 or 3 with BAY43-9006.27 Specific
interactions of the benzothiazole and benzoxazole compounds in
the B-Raf model include hydrogen bonds to 1) the backbone NH
and C@O of Cys532 in the hinge region through the pyridyl-amide
moiety, 2) the backbone NH of Asp594 through the benzothiazole
or benzoxazole nitrogen, and 3) the sidechain COO– of Glu501
through the aniline NH.
(3.7 lM for 26 and 2.9 lM for 34). Incorporation of the solubilizing
2-morpholinoethyl group (27, 35) gave a >30-fold loss in BrafV600E
potency in both series, indicating that bulkier groups are not toler-
ated at this position.
While both series yielded extremely potent Raf inhibitors, nei-
ther series inhibited phosphorylation of ERK (SKMEL-28, RafV600E
)
as well as our original benzimidazole series. The discrepancy be-
tween biochemical and cellular potency does not appear to be
due to permeability limitations. Compounds from both series were
tested in the Caco2 assay and shown to have good permeability
(Papp A and B in the range of 10–20 ꢀ 10ꢁ6 cm/s). A working
hypothesis could be that in the biochemical assay, purified kinase
domain of Raf is not representative of the full length protein in
cells where Raf exists as a complex with chaperons, cytoskeleton,
phosphatases and kinases.25
The kinase profiles of two representative compounds, 3 and 16,
were tested against 50 kinases including receptor tyrosine kinases
(RTKS), serine/threonine kinases (STKs), tyrosine kinases (TKs) and
AGC kinases. The compounds exhibited a fairly narrow kinase pro-
file, inhibiting five RTKs (CSFR1, Flt3, KDR, cABL and Ret) out of 50
These models use the ‘DFG-out’ conformation of the protein
where the substituted anilines take the place of Phe593, which
swings out and interacts with the aromatic systems in the hinge
region and the selectivity pocket. The surface model in Figure 2
shows that the space available at the meta and para positions of
the aniline is larger than the space around the ortho position which
could be the reason the ortho-substituted analogues show a reduc-
tion in potency.
Addition of a 2-morpholinoethyl group on the pyridyl-amide
side of the molecules (27, 35) leads to a reduction in potency. Eval-
uation of these compounds in the docking model shows that there
isn’t enough space to accommodate the morpholinoethyl group in
a low-energy conformation.
The pharmacokinetic properties for the two series were exam-
ined. Following a single 20 mg/kg oral administration to female
mice in 15% captisol, benzothiazole 30 exhibited a clearance of
14.1 mL/min/kg, a low volume of distribution (1042 mL/kg), a short
kinases with an IC50 <1 lM.
In order to understand the binding mode for these series, 2 and
3 were docked26 in the active site of the public domain crystal

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half-life (80 min), and an oral bioavailability of 90%. On the other
hand, benzoxazole 19 in the same formulation exhibited a low
clearance (0.93 mL/min/kg), very low volume of distribution
(82 mL/kg), a short half-life (32 min) and an oral bioavailability
of 33%.
In conclusion, we developed two novel series of potent Raf
inhibitors with acceptable pharmacokinetic properties. The cellular
potency for these series was lower than for our previously dis-
closed benzimidazole series and therefore further work on the
benzoxazoles and benzothiazoles was discontinued.
Acknowledgments
The authors wish to acknowledge Keshi Wang for formulation
and bioanalytics done on these compounds and Darrin Stuart for
expert opinion on pharmacology.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.04.023.
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