Identification and optimization of new dual inhibitors of B-Raf and epidermal growth factor receptor kinases for overcoming resistance against vemurafenib

Article abstract of DOI:10.1021/jm500007h

Epidermal growth factor receptor (EGFR) amplification has been demonstrated to be critical for the inherent and/or acquired resistance against current B-Raf^V600E inhibitor therapy for melanoma and colorectal cancer patients. We describe the discovery and structure-activity relationship study of a series of 1H-pyrazolo[3,4-b]pyridine-5-carboxamide analogues as novel dual inhibitors of EGFR and B-Raf^V600E mutant. One of the most promising compounds, 6a, potently inhibited both of the kinases with IC₅₀ values of 8.0 and 51 nM, respectively. The compound also strongly suppressed the proliferation of a panel of intrinsic and acquired resistant melanoma and/or colorectal cancer cells harboring overexpressed EGFR with submicromolar IC ₅₀ values. Further mechanism investigation revealed that 6a could sustainably inhibit the activation of the MAPK path way in the resistant SK-MEL-28 PR30 melanoma cancer cells and WiDr colorectal cancer cells with EGFR amplification. Our results support the hypothesis that the EGFR/B-Raf ^V600E dual inhibition might be a tractable strategy to overcome the intrinsic and acquired resistance of melanoma and/or colorectal cancers against the current B-Raf^V600E inhibitor therapy.

Full text of DOI:10.1021/jm500007h

Article
pubs.acs.org/jmc
Identification and Optimization of New Dual Inhibitors of B‑Raf and
Epidermal Growth Factor Receptor Kinases for Overcoming
Resistance against Vemurafenib
Huimin Cheng,†,∥ Yu Chang,‡,∥ Lianwen Zhang,†,∥ Jinfeng Luo,† Zhengchao Tu,† Xiaoyun Lu,†
Qingwen Zhang,‡ Jibu Lu,§ Xiaomei Ren,† and Ke Ding*,†
†Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, #190 Kaiyuan
Avenue, Guangzhou 510530, China
‡University of Macau, Avenue Padre Tomas
́
Pereira Taipa, Macau, China
§Shenyang Pharmaceutical University, #103 Wenhua Road, Shenyang 110016, China
S
* Supporting Information
ABSTRACT: Epidermal growth factor receptor (EGFR) amplification has been
demonstrated to be critical for the inherent and/or acquired resistance against current B-
RafV600E inhibitor therapy for melanoma and colorectal cancer patients. We describe the
discovery and structure−activity relationship study of a series of 1H-pyrazolo[3,4-
b]pyridine-5-carboxamide analogues as novel dual inhibitors of EGFR and B-RafV600E
mutant. One of the most promising compounds, 6a, potently inhibited both of the kinases
with IC50 values of 8.0 and 51 nM, respectively. The compound also strongly suppressed the
proliferation of a panel of intrinsic and acquired resistant melanoma and/or colorectal
cancer cells harboring overexpressed EGFR with submicromolar IC50 values. Further
mechanism investigation revealed that 6a could sustainably inhibit the activation of the
MAPK path way in the resistant SK-MEL-28 PR30 melanoma cancer cells and WiDr
colorectal cancer cells with EGFR amplification. Our results support the hypothesis that the
EGFR/B-RafV600E dual inhibition might be a tractable strategy to overcome the intrinsic and
acquired resistance of melanoma and/or colorectal cancers against the current B-RafV600E inhibitor therapy.
INTRODUCTION
■
The Raf proteins belong to a serine/threonine kinase family
and are critical components in the mitogen activated protein
kinase (MAPK) signal transduction pathway.1,2 Three isoforms
of Raf kinase, i.e., A-Raf, B-Raf, and C-Raf, have been
identified.3 Aberrant activation and/or constitutively activating
mutation of Raf kinase have been linked with multiple types of
cancers.4 In particular, B-Raf mutations have been identified in
various cancers including malignant melanoma (50−60%),
papillary thyroid (30−50%), colorectal (10−15%), and lung
(∼3%) cancers, hairy cell leukemia (∼100%), and others.5−7
The Val 600 → Glu 600 (V600E) transition is the most common
mutation and accounts for over 90% in all of the B-Raf
oncogenic mutants reported to date. The identification of B-Raf
Figure 1. Chemical structures of FDA approved B-RafV600E inhibitors
mutation in multiple human cancers promoted the extensive
efforts to develop B-Raf inhibitors as new potential antitumor
agents.8,9 Recently, two selective B-Raf inhibitors, vemurafenib
(1, PLX4032)10 and dabrafinib (2),11 were approved by the US
Food and Drug Administration (FDA) for the management of
late-stage or unresectable melanoma harboring B-RafV600E
mutation (Figure 1).12,13 The drugs have achieved remarkable
clinical benefit for melanoma patients with B-RafV600E
mutation.14 For instance, drug 1 treatment produces a 60−
80% overall response rate and a median progression-free
survival of approximately 7 months in the B-RafV600E mutated
vemurafenib (1) and dabrafinib (2), EGFR inhibitor gefitinib (3), and
the new B-Raf/EGFR dual inhibitor 4.
melanoma patients.15,16 The similar clinical efficacy has also
been reported for drug 2.17,18
Although the antitumor effects of drugs 1 and 2 in melanoma
are highly promising, intrinsic and acquired resistance limits the
Received: January 3, 2014
Published: March 3, 2014
© 2014 American Chemical Society
2692
dx.doi.org/10.1021/jm500007h | J. Med. Chem. 2014, 57, 2692−2703

Journal of Medicinal Chemistry
Article
a
Scheme 1. Syntheses of Compounds 4, 5, and 6
a
Reagents and conditions: (a) PyBOP, Et3N, THF, rt, 12 h, 46−85%; (b) H2, 10% Pd/C, CH3OH, rt, 8 h, 68−92%; (c) HATU, DIEA, DMF, rt, 12
h, 47−62%; (d) Pd(OAc)2, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, Et3N, CH3OH, CO, 100 °C, 12 h, 52−83%; (e) LiOH,
MeOH:THF:H2O (1:1:2), rt, 3.0 h, 44−89%.
therapeutic benefit of current drugs.19−31 For instance, a
majority of colorectal cancer patients display inherent resistance
against drug 1, although they were detected to harbor B-
RafV600E mutation, and the overall response rate is less than 5%
in a clinical investigation.32 The exact mechanism for the
significant difference in the efficacy of drug 1 between B-
RafV600E mutated colorectal cancer and melanomas remains
elusive. The most recent results from two independent groups
strongly suggested that the epidermal growth factor receptor
(EGFR) activation might be a critical cause of the inherent
resistance against B-RafV600E inhibition in colorectal cancer.25,26
A combinational therapy of drug 1 with an EGFR inhibitor has
been conducted to effectively overcome the resistance both in
vitro and in vivo. More encouragingly, a clinically successful case
for the off-label combination of cetuximab (an EGFR
monoclonal antibody) with sorafenib (a Raf inhibitor) to
treat the metastatic colorectal cancer patient with B-RafV600E
mutation was recently reported.33 It was also demonstrated that
upregulation of v-erb-b2 avian erythroblastic leukimia viral
congene homologue 3 (ERBB3), another member of EGFR
kinase family, contributes greatly to the adaptive resistance of
melanoma against drug 1. Combination of drug 1 with an
ERBB2/EGFR inhibitor lapatinib successfully reduced tumor
burden and extended latency of tumor growth in both cultured
cancer cells and mouse xenograft models through inactivation
of the ERBB3 signal.34 Combinational therapy of FDA
approved drugs with different mechanisms of action (MOA)
is a powerful strategy for the treatment of various cancers.35,36
However, a synergistic hepatotoxicity was observed in
melanoma patients after combinational treatment of drug 1
with another FDA approved antimelanoma antibody ipilimu-
mab, suggesting the requirement of careful protocol design for
combinational therapies to avoid the potential drug−drug
interaction.37 Thus, dual inhibition of B-Raf and EGFR by
using a single compound may provide a novel promising and
manageable strategy for the treatment of colorectal cancer
V600E
patients with B-Raf
mutation.
Several molecules have been reported to show nonselective
dual inhibition against both B-Raf and EGFR kinases.38,39
However, to the best of our knowledge, there is rarely a
successful report on the treatment of intrinsic resistant
colorectal cancers by using a B-Raf/EGFR dual inhibitor.
Herein, we would like to describe the discovery of a class of 1H-
pyrazolo[3,4-b]pyridine-5-carboxamides as novel dual inhib-
2693
dx.doi.org/10.1021/jm500007h | J. Med. Chem. 2014, 57, 2692−2703

Journal of Medicinal Chemistry
Article
V600E
Table 1. In Vitro Kinase Inhibitory Activities of the New Inhibitors against B-Raf
Effects
and EGFR and Their Antiproliferation
a
V600E
B-Raf
kinase activity assays were performed using the FRET-based Z′-Lyte assays according to the manufacturer’s instructions.44 The
compounds were incubated with the kinase reaction mixture for 1−1.5 h before measurement. Reported data are the means from 2 independent
experiments. EGFR kinase activity assays were performed using the FRET-based Z′-Lyte assays according to the manufacturer’s instructions.44 The
b
compounds were incubated with the kinase reaction mixture for 1−1.5 h before measurement. Reported data are the means from 2 independent
c
experiments. The antiproliferative activities of the compounds were evaluated using the MTS assay. Data are reported as the means
(standard deviations) from at least three independent experiments.
SDs
itors of B-RafV600E and EGFR kinases. The compounds potently
inhibited the kinase activities of both B-RafV600E and EGFR with
low nanomolar IC50 values. Furthermore, the compounds also
displayed potent inhibition on the proliferation of a panel of
vemurafenib-resistant cancer cells with high levels of EGFR,
representing new leads for further development of B-Raf/
EGFR dual inhibitors to overcome the resistance against FDA
approved drug 1.
phosphate (PyBOP) to produce the benzamides 9. Compounds
9 were hydrogenated to provide the anilines 10. Compounds
10 could react with different aromatic acids 13 or 16 to afford
the final products 4, 5a−o, and 6a−f by using another coupling
reagent 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-
[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU). It
was noteworthy that the amino group in 13a should be
protected with the butoxycarbonyl group before it could
effectively couple with the aniline 10 to afford inhibitor 5f. The
commercially unavailable intermediates 13 and 16 could be
readily prepared by using compounds 11 or 14, respectively, by
utilizing the Pd-catalyzed carbonyl insertion coupling reac-
tion,40 followed by standard hydrolysis.
CHEMISTRY
■
The synthesis of compounds 4, 5a−o, and 6a−f is illustrated in
Scheme 1. Briefly, commercially available compound 7 was
coupled with different carboxylic acids 8 under the catalysis of
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluoro-
2694
dx.doi.org/10.1021/jm500007h | J. Med. Chem. 2014, 57, 2692−2703

Journal of Medicinal Chemistry
Article
Chart 1. Strategies for the Structural Optimization of Initial “Hit” Compound 4
compound 5f as a new starting point. It was found that the
compound is well tolerated to the N-substitution for the dual
inhibition on B-Raf and EGFR. The N-methyl (5h), N-ethyl
(5i), N-iropropyl (5j), N-cyclopropyl (5k), and N-cyclopentyl
(5l) analogues displayed similar potencies on both B-RafV600E
and EGFR to that of compound 5f. Interestingly, although the
RESULTS AND DISCUSSION
■
In order to identify novel B-Raf/EGFR dual inhibitors for
overcoming the intrinsic resistance of colorectal cancers against
the current B-Raf inhibitor therapy, we conducted a random
screening on our self-constructed-kinase-inhibitor focused
library. Compound 4 was discovered to display good inhibitory
activities against both B-RafV600E and EGFR, with IC50 values of
551 nM and 550 nM, respectively (Table 1). Similar to the
reported data, drug 1 potently inhibits the kinase activity of B-
Raf V600E mutant with an IC 50 value of 26 nM,41 but it is almost
totally inactive against EGFR. However, gefitinib (3), an FDA
approved EGFR inhibitor, strongly inhibits EGFR with a 1.0
nM IC50 value,42,43 but its potency on B-Raf is minor.
N-phenyl compound 5n showed an IC50 value of 210 nM on B-
V600E
Raf
similar to that of 5f (about 410 nM), its EGFR
inhibitory effect is 10-fold more potent, and the IC50 value is
about 6.0 nM. However, the N-cyclohexyl derivative (5m)
showed about 8 times more improvement on the B-Raf
suppressing effect, while its inhibitory activity on EGFR was
almost identical to that of the lead compound 5f. The IC50
values of compound 5m on B-RafV600E and EGFR were 37 and
47 nM, respectively. Further investigation suggested that the
N,N-disubstitution is detrimental to the compound’s inhibitory
activity on EGFR kinase, although it barely affects the inhibition
on B-Raf. For instance, the N,N-dimethyl analogue (5o)
displayed an IC50 value of 300 nM on B-RafV600E, which is
almost identical to that of 5f, but its IC50 value on EGFR is over
100 times greater than that of the lead compound, highlighting
the critical role of a hydrogen bond donating moiety for the
EGFR suppressing function.
Inspired by the above investigation, compounds containing
either a pyrrolo[2,3-b]pyridinyl (6a) or 1H-pyrazolo[3,4-
b]pyridinyl (6b) motif were designed by utilizing a
conformation-constraining strategy. It was shown that both
6a and 6b displayed potencies against B-RafV600E similar to that
of 5m, with IC50 values of 51 and 48 nM, respectively, but their
activities against EGFR differentiated obviously. Compound 6a
inhibited EGFR with an IC50 value of 8 nM, which is
approximately 6-fold more potent than that of compound 5m,
while 6b displayed an IC50 value of 30 nM, which is almost
identical to that of 5m. However, when a 1H-indol-5-yl group
was introduced (6c), the resulting compound totally abolished
its inhibitory effect on EGFR, and its potency on B-RafV600E was
Extensive structural optimization of the lead compound (4)
was conducted to improve its dual inhibition against EGFR and
B-RafV600E (Chart 1) and the results are summarized in Table 1.
We first investigated the potential impact of the nitrogen atom
in aromatic ring A of compound 4 on its dual inhibition against
EGFR and B-Raf V600E kinases by altering the position of the N-
atom. It was found that the position of the N-atom displayed
diverse effects on the inhibitory activity on EGFR and B-Raf
V600E. Although the 2-pyridinylcarboxyl-derivative (5a) dis-
played similar potencies on both EGFR and B-Raf V600E to that
of compound 4, the 4-pyridinylcarboxyl-compound 5b totally
lost its suppressive effect on EGFR, while its inhibitory potency
on B-RafV600E was improved about 5-fold. Interestingly, the
benzamide compound 5c also selectively suppressed the kinase
function of B-RafV600E with an IC50 value of 240 nM, while the
inhibitory effect on EGFR was totally abolished. Pyrazine-2-
carboxamide (5d) and pyrimidine-5-carboxamide (5e) ana-
logues were also designed and synthesized. Both of the
compounds showed moderate inhibitory activities against both
B-Raf and EGFR kinases with IC50 values of 280 and 670 nM,
and 3.90 and 1.92 μM, respectively. Further investigation
revealed that the introduction of a 2-amino group (5f) in
compound 5e hardly affected its suppressing potency on B-Raf
V600E, but the inhibitory effect on EGFR was significantly
improved about 50-fold. The IC50 values of compound 5f
against B-Raf and EGFR were 410 and 43 nM, respectively,
representing a new lead compound for further structural
optimization. However, when a 6-hydroxyl group was
introduced in compound 4, the resulting compound 5g totally
failed to suppress the function of B-Raf. Its inhibitory potency
on EGFR was also obviously decreased about 3-fold.
also decreased by a factor of 4. The strongly dual inhibition of
600E
compound 6a against both EGFR and B-Raf
makes it a
highly promising lead compound for further investigation.
Further structural modification on 6a suggested that the
methyl group in ring B is optimal for the dual inhibition against
B-Raf and EGFR. When the methyl was removed, although the
resulting compound 6d maintained a strong inhibition again B-
RafV600E with an IC50 value of 48 nM, its potency on EGFR was
dramaticly decreased about 90-fold. Whereas, the replacement
of the methyl group with a relatively larger group, i.e., an ethyl
We further investigated the impact of the substituent on the
2-amino group on the B-Raf/EGFR dual inhibition by adopting
2695
dx.doi.org/10.1021/jm500007h | J. Med. Chem. 2014, 57, 2692−2703

Journal of Medicinal Chemistry
Article
Figure 2. Clinical drug 1 potently and sustainably inhibits the activation of the MAPK path way in SK-MEL-28 melanoma cancer cells, but its effect
on p-Erk is transient in the vemurafenib-resistant SK-MEL-28-PR30 cells. (A) The vemurafenib-induced resistant cancer cells harbor a high level of
EGFR as determined by Western blot analysis but not k-Ras or n-Ras mutation. (B) Drug 1 potently and sustainably inhibits the activation of the
MAPK path way in SK-MEL-28 melanoma cancer cells, but p-Erk reaccumulation was observed in the vemurafenib-resistant SK-MEL-28-PR30 cells
after 24 h of treatment of 1. Western blots were preformed after SK-MEL-28 and SK-MEL-28-PR30 cells were treated with drug 1 for the indicated
time (2, 24, and 48 h).
Table 2. Compounds 6a and 6b Potently Inhibit the Proliferation of a Panel of Colorectal Cancer Cells Harboring Different
States of EGFR and B-Raf Mutation
b
Cell Growth Inhibition (IC50, μM)
compds
cell lines
B-Raf
HT-29
V600E
++
COLO205
V600E
+
HCT116
WT
LOVO
WT
−
WiDr
V600E
+
a
EGFR
++
6a
6b
0.48 0.04
0.78 0.03
0.22 0.02
0.36 0.04
1.33 0.07
0.17 0.01
0.48 0.02
0.67 0.14
1.35 0.07
1.38 0.19
a
b
The level of EGFR were detected by immunoblotting (Supporting Information, Figure S1). The antiproliferative activities of the compounds were
evaluated using the MTS assay. Data are reported as the means SDs (standard deviations) from at least three independent experiments.
(6e) or an isopropyl (6f), induced a significant potency loss
both on B-Raf and EGFR.
of clinical drug 1, which are 0.13, 0.54, and 0.48 μM. More
significantly, they also displayed strong suppression on the
proliferation of vemurafenib-resistant SK-MEL-28-PR30 cancer
cells with IC50 values of 0.45 and 0.78 μM. It was also
noteworthy that both of the compounds displayed almost
identical potencies on the vemurafenib-sensitive and vemur-
afenib-resistant cancer cells, indicating their potential applica-
tion to treat the intrinsic and acquired resistant melanoma and/
or colorectal cancer cells with both B-RafV600E mutaition and
EGFR overexpression.
The antiproliferation effects of the new inhibitors against the
vemurafenib-induced resistant SK-MEL-28-PR30 melanoma
cancer cells (Supporting Information), which were demon-
strated to harbor the B-RafV600E mutant and overexpression of
EGFR (Figure 2A), together with the parental SK-MEL-28 cells
with B-RafV600E mutation but low level of EGFR, were also
investigated (Table 1). Highly consistent to the previous
investigation, drug 1 potently inhibited the growth of the B-
RafV600E mutated SK-MEL-28 cells with an IC50 value of 0.48
μM41 but is almost totally inactive to suppress the proliferation
of the PR30 cancer cells, which express high level of EGFR.
Whereas, the selective EGFR inhibitor 3 did not show obvious
growth inhibition on either SK-MEL-28-PR30 or SK-MEL-28
cells. Consistent with their weak dual inhibition against B-Raf
Further investigation also revealed that the compounds
indeed displayed strong inhibition against the growth of a panel
of colorectal cancer cells harboring different states of EGFR and
B-Raf mutation (Table 2). For instance, compound 6a potently
inhibited the growth of HT-29, COLO205, and WiDr
V600E
colorectal cancer cells which carry the B-Raf
mutant and
V600E
and EGFR, compounds 4 and 5a only displayed minor
a high level of EGFR with IC50 values of 0.48, 0.22, and 0.67
μM, respectively. Interestingly, the compound also strongly
suppressed the proliferation of HCT116 and LOVO colorectal
cancer cells with IC50 values of 1.33 and 0.17 μM, respectively,
although the cells harbor wild-type B-Raf.
The selectivity profile of 6a against a panel of 456 kinases
(including 395 different wild-type kinases) was also investigated
by using the KINOMEscan platform (Ambit Bioscience, San
Diego, USA) at a concentration of 1.0 μM, which was about
20−125 times its IC50 values against B-RafV600E and EGFR
kinases, respectively. It was found that, in addition to its dual
inhibition against EGFR and B-RafV600E, compound 6a also
exhibited strong suppression on ERRB2, ERRB4, and PDGFRβ
kinases, which have been demonstrated to indirectly mediate
(i.e., ERRB2 through ERRB334) or directly link (PDGFRβ23)
with the acquired resistance of melanoma. However, special
attention is also required to further investigate the potential
suppressing effect on the growth of SK-MEL-28-PR30 and/or
SK-MEL-28 cells. Not surprisingly, compounds 5b, 5d, 5e, and
5g are inactive to show an antiproliferative effect on the cancer
cells, which might be a consequent effect of their poor
inhibition against both B-Raf V600E and EGFR kinases. However,
the dual inhibitor 5f displayed moderate inhibition on both of
the cancer cell lines with IC50 values of 1.84 and 1.89 μM.
Other novel dual inhibitors, i.e., 5h, 5i, 5j, 5k, 5l, 5m, 5n, 6d,
6e, and 6f, also showed low micromolar values to suppress the
growth of both vemurafenib-sensitive and vemurafenib-resistant
melanoma cancer cells. Encouragingly, we were pleased to find
that the two most potent B-Raf V600E/EGFR dual inhibitors, 6a
and 6b, displayed the strongest inhibition against the cellular
proliferation for both of the cancer cell lines. The compounds
potently inhibited the growth of vemurafenib-sensitive SK-
MEL-28 melanoma cancer cells with similar IC50 values to that
2696
dx.doi.org/10.1021/jm500007h | J. Med. Chem. 2014, 57, 2692−2703

Journal of Medicinal Chemistry
Article
Figure 3. Inhibitor 6a dose-dependenty inhibits the activation of the MAPK path way and the phosphorylation of EGFR in SK-MEL-28 PR30, A375
melanoma cancer cells, and WiDr, HT29 colorectal cancer cells. Western blots were performed after the cells were treated with 6a for 2.0 h.
safety issue of 6a associated with its relatively broad inhibition
on a panel of other kinases (Supporting Information).
and/or colorectal cancer cells to drug 1. In order to get a better
understanding on the strong inhibition of 6a against the
proliferation of vemurafenib-resistant SK-MEL-28-PR 30 cells,
we also investigated the suppressing effect of this compound on
the activation of p-Erk at different time courses. It was shown
that compound 6a sustainably inhibited the phosphorylation of
Erk at a concentration of 1.0 μM and that p-Erk suppression
was successfully maintained after 48-h treatments. As a parallel
comparison, drug 1 only displayed a transient inhibition on the
phosphorylation of Erk at 1.0 μM, and the p-Erk was
significantly reaccumulated in 48 h, which may be due to a
consequent upregulation of erbB3 by the transphosphorylation
partner EGFR.34 A similar sustainable inhibition of p-Erk by 6a
was also observed in WiDr colorectal cancer cells. These results
strongly support the potential of these compounds to overcome
the intrinsic and acquired resistant melanoma and/or colorectal
cancer cells with EGFR overexpression.
In summary, a series of 1H-pyrazolo[3,4-b]pyridine-5-
carboxamide analogues were identified as novel dual inhibitors
of the EGFR and B-RafV600E mutant. The compounds potently
inhibited both of the kinases with low nanomolar IC50 values
and strongly suppressed the proliferation of a panel of intrinsic
and acquired resistant melanoma and/or colorectal cancer cells
harboring overexpressed EGFR with submicromolar IC50
values. Furthermore, the representative compounds 6a also
displayed potent and sustainable inhibition against the
activation of the MAPK path way in the resistant SK-MEL-28
PR30 melanoma cancer cells and WiDr colorectal cancer cells,
suggesting that the EGFR/B-RafV600E dual inhibition, at least in
part, might be a tractable strategy to overcome the intrinsic and
acquired resistance of melanoma and/or colorectal cancers
against the current B-RafV600E inhibitor therapy. Further
structural optimization to improve pharmacokinetic properties,
in vivo pharmacodynamic evaluation, and molecular mechanism
studies on this class of compounds are in progress and will be
reported in due course. However, it is also noteworthy that
EGFR amplification is not the only key player for the intrinsic
and acquired resistance of melanoma and/or colorectal cancer
cells against current B-Raf inhibitors. New medicinal chemistry
efforts based on an understanding of the extensive mechanism
will be highly worthwhile for overcoming the resistance.
In order to further validate the dual inhibitory effects of new
inhibitors, we investigated their suppressive functions on the
activation of the MAPK signal pathway and the phosporylation
of EGFR protein in self-constructed SK-MEL-28-PR30 cells
which have been demonstrated to harbor B-RafV600E mutation
and EGFR amplification (Figure 2A) by taking compound 6a as
an example. As shown in Figure 3, the treatment of compound
6a indeed caused a dose-dependent reduction of the
phosphorylation of Erk in SK-MEL-28-PR30 cells, indicating
the blockage of the MAPK signal pathway. Furthermore, the
activation of EGFR was also obviously inhibited by 6a, while
the total protein levels of EGFR, Erk, and GAPDH remained
unchanged. Further investigation demonstrated that the
compound also potently blocked the MAPK signal pathway
and suppressed the activation of EGFR in WiDr, HT29
colorectal cancer cells, and A375 melanoma cancer cells which
express a relatively high level of EGFR and B-RafV600E mutation.
Similar to the previous data obtained from WiDr colorectal
cancer cell models,25 our investigation also demonstrated that
although drug 1 induced highly potent and sustainable
suppression on the activation of MAPK signal pathway in
vemurafenib-sensitive SK-MEL-28 melanoma cancer cells, its
effect on p-Erk is transient, and significant reaccumulation of p-
Erk could be observed in the vemurafenib-resistant SK-MEL-
28-PR30 cells after 24 h (Figures 2B and 4). The unsustainable
inhibition on the MAPK pathway may contribute greatly to the
intrinsic and acquired insensitivity of B-Raf mutated melanoma
EXPERIMENTAL SECTION
General Methods for Chemistry. All reagents and solvents were
used directly as purchased from commercial sources. Flash
Figure 4. Inhibitor 6a potently and sustainably inhibits the activation
of the MAPK path way in SK-MEL-28 PR30 melanoma cancer cells
and WiDr colorectal cancer cells.
■
2697
dx.doi.org/10.1021/jm500007h | J. Med. Chem. 2014, 57, 2692−2703

Journal of Medicinal Chemistry
Article
chromatography was performed using silica gel (300 mesh). All
reactions were monitored by TLC, using silica gel plates with
DMSO-d6) δ 10.47 (s, 1 H), 10.27 (s, 1 H), 9.17 (s, 1 H), 8.79 (d, J =
3.6 Hz, 1 H), 8.34 (d, J = 7.6 Hz, 1 H), 8.20 (s, 1 H), 8.06 (d, J = 8.0
Hz, 1 H), 8.01 (s, 1 H), 7.85 (d, J = 8.0 Hz, 1 H), 7.70 (d, J = 4.4 Hz, 1
H), 7.60 (dd, J = 4.8 Hz, 7.6 Hz, 1 H), 7.48 (d, J = 8.0 Hz, 1 H), 3.56
(s, 2H), 2.40−2. 32 (m, 11 H), 2.16 (s, 3 H). 13C NMR (125 MHz,
DMSO-d6) δ 165.10, 164.19, 152.30, 148.70, 138.27, 138.23, 136.17,
135.50, 132.37, 132.03, 131.27, 130.55, 129.94, 127.54, 126.02, 125.51,
123.65, 123.57, 117.38, 117.30, 57.45, 54.68(2C), 52.62(2C), 45.63,
18.00. HRMS (ESI) calcd for C27H28F3N5O2 [M + H]+, 512.2268;
found, 512.2272. HPLC purity = 97.06%, Rt 5.82 min.
1
fluorescence F254 and UV light visualization. H NMR and 13C NMR
spectra were recorded on a Bruker AV-400 spectrometer at 400 MHz
and a Bruker AV-500 spectrometer at 125 MHz. Coupling constants
(J) are expressed in hertz (Hz). Chemical shifts (δ) of NMR are
reported in parts per million (ppm) units relative to an internal control
(TMS). Low resolution ESI-MS were recorded on an Agilent 1200
HPLC-MSD mass spectrometer and high resolution ESI-MS on an
Applied Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrom-
eter. The purity of compounds was determined by reverse-phase high
performance liquid chromatography (HPLC) analysis to be >95%.
HPLC instrument, Dionex Summit HPLC (column, Diamonsil C18,
5.0 μm, 4.6 × 250 mm (Dikma Technologies); detector, PDA-100
photodiode array; injector, ASI-100 autoinjector; pump, p-680A). A
flow rate of 1.0 mL/min was used with a mobile phase of MeOH in
H2O with a 0.1% modifier (ammonia or trifluoroacetate, v/v).
Compounds 5a−o and 6a−f were synthesized by using a similar
procedure.
N-(2-Methyl-5-((4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl) carbamoyl) phenyl)picolinamide 5a.
1
Yield: 52%. H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1 H), 10.27
(s, 1 H), 9.17 (d, J = 1.5 Hz, 1 H), 8.79 (dd, J = 4.8, 1.5 Hz, 1 H), 8.34
(d, J = 8.0 Hz, 1 H), 8.20 (d, J = 1.8 Hz, 1 H), 8.07 (d, J = 8.4 Hz, 1
H), 8.01 (s, 1 H), 7.85 (dd, J = 8.0 Hz, 1.5 Hz, 1 H), 7.70 (d, J = 8.5
Hz, 1 H), 7.60 (dd, J = 7.7 Hz, 4.8 Hz, 1 H), 7.48 (d, J = 8.1 Hz, 1 H),
3.56 (s, 2 H), 2.39−2.33 (m, 11 H), 2.18 (s, 3 H). 13C NMR (125
MHz, DMSO-d6) δ 165.16, 164.25, 152.34, 148.72, 138.32, 138.26,
136.20, 135.55, 132.40, 132.04, 131.32, 130.61, 129.97, 126.05, 125.56,
123.71(2C), 123.62, 117.19, 117.08, 57.45, 54.66(2C), 52.55(2C),
45.56, 18.03. HRMS (ESI) calcd for C27H28F3N5O2 [M + H]+,
512.2268; found, 512.2266. HPLC purity = 95.01%, Rt 6.16 min.
N-(2-Methyl-5-((4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl) carbamoyl) phenyl)isonicotinamide
4-Methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl)-3-nitrobenzamide 9a. 4-((4-Methylpi-
perazin-1-yl)methyl)-3-(trifluoromethyl)aniline 7 (7.97g, 44.0 mmol)
and 4-methyl-3-nitrobenzoic acid 8a (10.00g, 36.6 mmol) were stirred
in 20 mL of dry tetrahydrofuran (THF) at room temperature. To the
mixture, PyBOP (28.60g, 54.9 mmol) and triethylamine (8.2 mL,
58.56 mmol) were added. The resulting mixture was stirred at room
temperature overnight, and then the organic solvent was removed
under vacuum. The resulting residue was diluted with water and
extracted with ethyl acetate (2 × 50 mL). The combined organic layer
was dried over anhydrous sodium sulfate and concentrated under
vacuum and then purified by column chromatography over silica gel to
1
5b. Yield: 61%. H NMR (400 MHz, DMSO-d6) δ 10.46 (s, 1 H),
10.34 (s, 1 H), 8.81 (dd, J = 4.4 Hz, 1.6 Hz, 2 H), 8.20 (d, J = 2.0 Hz,
1 H), 8.07 (dd, J = 8.8 Hz, 1.8 Hz, 1 H), 8.01 (d, J = 1.2 Hz, 1 H),
7.92−7.90 (m, 2 H), 7.86 (dd, J = 8.0 Hz, 1.8 Hz, 1 H), 7.70 (d, J = 8.8
Hz, 1 H), 7.47 (d, J = 8.0 Hz, 1 H), 3.56 (s, 2 H), 2.50−2.33 (m, 11
H), 2.16 (s, 3 H). 13C NMR (125 MHz, DMSO-d6) δ 164.96, 164.03,
150.35(2C), 141.25, 138.22, 135.93, 132.36, 131.97, 131.17, 130.50,
127,48, 127.25, 126.00, 125.62, 123.49, 121.53(2C), 117.28, 117.23,
57.41, 54.67(2C), 52.62(2C), 45.63, 17.91. HRMS (ESI) calcd for
C27H28F3N5O2 [M + H]+, 512.2268; found, 512.2269. HPLC purity =
97.9%, Rt 5.82 min.
1
afford pure compound 9a (7.50g, yield: 50%). H NMR (400 MHz,
CDCl3) δ 9.61 (s, 1 H), 8.53 (d, J = 1.6 Hz, 1 H), 8.14 (dd, J = 9.6 Hz,
1.6 Hz, 1 H), 8.10 (d, J = 2.0 Hz, 1 H), 7.81 (dd, J = 8.4 Hz, 1.6 Hz, 1
H), 7.91 (d, J = 8.4 Hz, 1 H), 7.41 (d, J = 8.0 Hz, 1 H), 3.62 (s, 2 H),
3.31−2.23(m, 8 H), 2.83 (s, 3 H), 2.59 (s, 3 H). Compounds 9b−d
were synthesized by using a similar procedure; yield, 46−85%.
3-Amino-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl) benzamide 10a. To a solution of 9a
(7.50g, 17.2 mmol) in 30 mL of methanol, 0.75g Pd/C was added, and
the reaction flask was evacuated and backfilled with hydrogen twice.
The reaction mixture was stirred at room temperature under a
hydrogen balloon for 5 h. The reaction mixture was filtered through a
pad of Celite and concentrated under vacuum to yield 10a (5.20g,
3-Benzamido-4-methyl-N-(4-((4-methylpiperazin-1-yl)-
methyl)-3-(trifluoromethyl) phenyl) benzamide 5c. Yield: 65%.
1H NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1 H), 10.08 (s, 1 H), 8.21
(s, 1 H), 8.7 (d, J = 8.5 Hz, 1 H), 8.03 (s, 1 H), 8.01 (s, 2 H), 7.83 (d, J
= 8 Hz, 1 H), 7.70 (d, J = 8.5 Hz, 1 H), 7.63−7.53 (m, 3 H), 7.46 (d, J
= 8.0 Hz, 1 H), 3.56 (s, 2 H), 2.50−2.31 (m, 11 H), 2.15 (s, 3 H). 13C
NMR (125 MHz, DMSO-d6) δ 165.46, 165.06, 138.23, 136.58, 134.25,
132.25, 131.93, 131.63, 131.16, 130.37, 128.42, 127.61, 127.47, 127.23,
126.02, 125.40, 125.23, 123.48, 123.22, 117.26, 117.21, 57.41,
54.67(2C), 52.64(2C), 45.64, 17.96. HRMS (ESI) calcd for
C28H29F3N4O2 [M + H]+, 511.2316; found, 511.2314. HPLC purity
= 98.6%, Rt 7.23 min.
N-(2-Methyl-5-((4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl) carbamoyl) phenyl)pyrazine-2-car-
boxamide 5d. Yield: 47%. 1H NMR (400 MHz, DMSO-d6) δ
10.48 (s, 2 H), 9.33 (s, 1 H), 8.97 (d, J = 2.3 Hz, 1 H), 8.85 (s, 1 H),
8.24 (s, 1 H), 8.20 (s, 1 H), 8.07 (d, J = 8.5 Hz, 1 H), 7.83 (d, J = 7.9
Hz, 1 H), 7.70 (d, J = 8.0 Hz, 1 H), 7.47 (d, J = 8.0 Hz, 1 H), 3.56 (s, 2
H), 2.48−2.25 (m, 11 H), 2.17 (s, 3 H). 13C NMR (125 MHz,
DMSO-d6) δ 165.23, 161.80, 148.03, 144.62, 143.92, 143.52, 138.27,
136.77, 135.84, 132.53, 132.02, 131.30, 130.50, 127.58, 125.47, 125.13,
124.54, 123.59, 117.32, 57.46, 54.67(2C), 52.57(2C), 45.59, 17.78.
HRMS (ESI) calcd for C26H27F3N6O2 [M + H]+, 513.2221; found,
513.2228. HPLC purity = 99.64%, Rt 7.01 min.
N-(2-Methyl-5-((4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl) carbamoyl) phenyl)pyrimidine-5-car-
boxamid 5e. Yield: 45%. 1H NMR (400 MHz, DMSO-d6) δ 10.48(s,
1 H), 10.42 (s, 1 H), 9.40 (s, 1 H), 9.32 (s, 2 H), 8.20 (s, 1 H), 8.19−
8.03 (m, 2 H), 7.86 (dd, J = 8.0 Hz, 1.8 Hz, 1 H), 7.70 (d, J = 8.4 Hz, 1
H), 7.48 (d, J = 8.0 Hz, 1 H), 3.56 (s, 2 H), 2.39−2.35 (m, 11 H), 2.15
(s, 3 H). 13C NMR (125 MHz, DMSO-d6) δ 164.98, 162.34, 160.27,
156.23(2C), 138.18, 138.00, 135.74, 132.40, 131.99, 131.19, 130.54,
127.96, 127.24, 125.81, 125.54, 123.50, 117.28, 117.23, 57.42,
1
yield: 75%). H NMR (400 MHz, DMSO-d6) δ 10.27 (s, 1 H), 8.22
(d, J = 2.0 Hz, 1 H), 8.04 (dd, J = 8.4 Hz, 1.6 Hz, 1 H), 7.66 (d, J = 8.4
Hz, 1 H), 7.22 (d, J = 1.6 Hz, 1 H), 7.13−7.06 (m, 2 H), 5.08 (s, 2 H),
3.54 (s, 2 H), 3.41 (s, 1 H), 2.48−2.21 (m, 7 H), 2.13 (m, 6 H).
Compounds 10b−d were synthesized by using a similar procedure;
yield, 68−92%.
3-Amino-4-ethyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl)benzamide 10c. 1H NMR (500 MHz,
DMSO-d6) δ 10.29 (s, 1H), 8.21 (d, J = 1 Hz, 1H), 8.05 (d, J = 8.5 Hz,
1H), 7.67 (d, J = 8.5 Hz, 1H), 7.16 (s, 1H), 7.11 (d, J = 8.0 Hz, 1H),
7.06 (d, J = 8.0 Hz, 1H), 5.12 (s, 2H), 3.61 (s, 2H), 2.84 −2.49 (m,
8H), 2.51 (q, J = 7.5 Hz, 2H), 1.15 (t, J = 7.5 Hz, 3H); 13C NMR (125
MHz, DMSO-d6) δ 166.99, 147.41, 146.48, 139.19, 133.27, 131.73,
131.28, 131.18, 128.22, 123.67, 117.51, 115.65, 113.92, 60.15, 57.35,
54.07 (2C), 51.21, 44.07, 23.84, 13.35; MS (ESI), m/z 421 [M + H]+.
N-(2-Methyl-5-((4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl)carbamoyl) phenyl)nicotinamide 4.
To a solution of 10a (100.0 mg, 0.25 mmol) in 5 mL of N,N-
dimethylformamide (DMF), 46.4 mg of nicotinic acid (0.38 mmol),
(1-[bis(dimethylamino) methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxid hexafluorophosphate) (HATU) (140.1 mg, 0.37
mmol), and N,N-diisopropylethylamine (DIPEA) (0.13 mL, 0.75
mmol) were added. The resulting mixture was stirred at room
temperature overnight. The reaction was quenched with water and
extracted with ethyl acetate (3 × 20 mL). The combined organic layer
was dried over anhydrous sodium sulfate, concentrated under vacuum,
and then purified by column chromatography over silica gel to afford
pure compound 4 (72.9 mg) (yield: 57%). 1H NMR (400 MHz,
2698
dx.doi.org/10.1021/jm500007h | J. Med. Chem. 2014, 57, 2692−2703

Journal of Medicinal Chemistry
Article
54.68(2C), 52.65(2C), 45.66, 17.96. HRMS (ESI) calcd for
C26H27F3N6O2 [M + H]+, 513.2221; found, 513.2229. HPLC purity
= 98.70%, Rt 5.90 min.
H), 8.20 (d, J = 2.0 Hz, 1 H), 8.08−8.04 (m, 2 H), 7.99 (d, J = 1.6 Hz,
1 H), 7.82 (dd, J = 7.9, 1.6 Hz, 1 H), 7.70 (d, J = 8.6 Hz, 1 H), 7.45 (d,
J = 8.1 Hz, 1 H), 3.56 (s, 2 H), 2.82 (tq, J = 7.5, 3.9 Hz, 1 H), 2.40−
2.31 (m, 11 H), 2.15 (s, 3 H), 0.75−0.70 (m, 2 H), 0.55−0.51 (m, 2
H). 13C NMR (125 MHz, DMSO-d6) δ 165.06, 164.03, 162.96,
158.05, 158.03, 138.21, 138.01, 136.36, 132.23, 131.93, 131.17, 130.38,
125.81, 125.09, 123.48, 117.26, 117.21, 116.50, 57.41, 54.68 (2C),
52.65 (2C), 45.66, 23.95, 17.99, 6.24 (2C). HRMS (ESI) calcd for
C29H32F3N7O2 [M + H]+, 528.2643; found, 528.2640. HPLC purity =
96.68%, Rt 6.80 min.
2-(Cyclopentylamino)-N-(2-methyl-5-((4-((4-methylpipera-
zin-1-yl)methyl)-3-(trifluoromethyl) phenyl)carbamoyl)-
phenyl)pyrimidine-5-carboxamide 5l. Yield: 52%. 1H NMR
(400 MHz, DMSO-d6) δ 10.45 (s, 1 H), 9.84 (s, 1 H), 8.85 (s, 2
H), 8.20 (s, 1 H), 8.06 (d, J = 8.0 Hz, 1 H), 7.99 (s, 1 H), 7.90 (d, J =
7.2 Hz, 1 H), 7.81 (d, J = 7.6 Hz, 1 H), 7.69 (d, J = 8.4 Hz, 1 H), 7.43
(d, J = 8.0 Hz, 1 H), 4.27 (t, J = 6.6 Hz, 1 H), 3.56 (s, 2 H), 2.50−2.31
(m, 11 H), 2.18 (s, 3 H), 1.92 (d, J = 3.6 Hz, 2 H), 1.70 (s, 2 H), 1.54
(s, 4 H). 13C NMR (125 MHz, DMSO-d6) δ 165.09, 163.01, 162.56,
158.08, 138.24, 138.02, 136.41, 132.23, 131.88, 131.17, 130.36, 127.48,
125.84, 125.06, 123.48, 123.22, 117.26, 117.22, 115.75, 57.39,
54.61(2C), 52.53(2C), 52.34, 45.53, 32.04(2C), 23.38(2C), 18.00.
HRMS (ESI) calcd for C31H36F3N7O2 [M + H]+, 596.2956; found,
596.2959. HPLC purity = 96.00%, Rt 9.32 min.
2-Amino-N-(2-methyl-5-((4-((4-methylpiperazin-1-yl)-
methyl)-3-(trifluoromethyl) phenyl) carbamoyl)phenyl)-
1
pyrimidine-5-carboxamide 5f. Yield: 38%. H NMR (400 MHz,
DMSO-d6) δ 10.47 (s, 1 H), 9.88 (s, 1 H), 8.83 (s, 2 H), 8.20 (s, 1 H),
8.06 (d, J = 8.4 Hz, 1 H), 7.98 (s, 1 H), 7.81 (d, J = 7.6 Hz, 1 H), 7.69
(d, J = 8.4 Hz, 1 H), 7.44 (d, J = 8.0 Hz,1 H) 7.34 (s, 2 H), 3.56 (s, 2
H), 2.49−2.30 (m, 11 H), 2.20 (s, 3 H). 13C NMR (125 MHz,
DMSO-d6) δ 165.73, 164.74, 163.64, 158.81(2C), 138.67, 138.50,
136.61, 132.59, 132.35, 131.73, 128.00, 127.77, 126.29, 125.76, 125.64,
124.02, 123.58, 117.79, 116.69, 57.69, 54.78(2C), 52.57(2C), 45.59,
18.33; HRMS (ESI) calcd for C26H28F3N7O2 [M + H]+, 528.2330;
found, 528.2332. HPLC purity = 97.80%, Rt 5.15 min.
6-Hydroxy-N-(2-methyl-5-((4-((4-methylpiperazin-1-yl)-
methyl)-3-(trifluoromethyl)phenyl) carbamoyl) phenyl)-
1
nicotinamide 5g. Yield: 56%. H NMR (400 MHz, DMSO-d6) δ
12.10 (s, 1 H), 10.47 (s, 1 H), 9.82 (s, 1 H), 8.21−8.19 (m, 2 H), 8.11
(d, J = 8.4 Hz, 1 H), 7.97 (dd, J = 9.6, 2.8 Hz, 1 H), 7.94 (d, J = 1.6
Hz, 1 H), 7.81 (dd, J = 8.0, 1.6 Hz, 1 H), 7.70 (d, J = 8.8 Hz, 1 H),
7.44 (d, J = 8.4 Hz, 1 H), 6.43 (d, J = 9.6 Hz, 1 H), 3.65 (s, 2 H),
3.30−2.50 (br, 8 H), 2.73 (s, 3 H), 2.29 (s, 3 H). 13C NMR (125
MHz, DMSO-d6) δ 165.49, 163.15, 162.77, 139.61, 138.65, 138.58,
138.55, 136.85, 132.66, 132.38, 131.61, 130.81, 127.91, 127.67, 126.40,
125.85, 125.55, 123.92, 123.67, 119.45, 117.65, 112.49, 57.87,
55.13(2C), 53.10(2C), 46.12, 18.43; HRMS (ESI) calcd for
C27H28F3N5O3 [M + H]+, 528.2217; found, 528.2220. HPLC purity
= 97.72%, Rt 4.43 min.
2-(Cyclohexylamino)-N-(2-methyl-5-((4-((4-methylpiperazin-
1-yl)methyl)-3-(trifluoromethyl) phenyl)carbamoyl)phenyl)-
1
pyrimidine-5-carboxamide 5m. Yield: 66%. H NMR (400 MHz,
DMSO-d6) δ 10.45 (s, 1 H), 9.83 (s, 1 H), 8.84 (s, 2 H), 8.20 (d, J =
1.8 Hz, 1 H), 8.06 (d, J = 8.4 Hz, 1 H), 7.99 (d, J = 1.2 Hz, 1 H), 7.82
(s, 1 H), 7.80 (s, 1 H), 7.70 (d, J = 8.6 Hz, 1 H), 7.44 (d, J = 8.1 Hz, 1
H), 3.82−3.79 (m, 1 H), 3.56 (s, 2 H), 2.47−2. 30 (m, 11 H), 2.15 (s,
3 H), 1.89 (s, 2 H), 1.73 (s, 2 H), 1.60 (d, J = 12.0 Hz, 1 H), 1.30 (dd,
J = 16.0, 7.9 Hz, 4 H), 1.18−1.12 (m, 1 H). 13C NMR (125 MHz,
DMSO-d6) δ 165.06, 162.95, 162.19, 158.17, 138.20, 137.99, 136.39,
132.21, 131.92, 131.16, 130.36, 127.22, 125.80, 125.39, 125.05, 123.47,
117.25, 117.20, 115.72, 57.41, 54.68 (2C), 52.65 (2C), 49.45, 45.66,
32.17 (2C), 25.23, 24.68 (2C), 17.98. HRMS (ESI) calcd for
C32H38F3N7O2 [M + H]+, 610.3112; found, 610.3114. HPLC purity =
99.42%, Rt 11.84 min.
N-(2-Methyl-5-((4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl)carbamoyl) phenyl)-2-(methylamino)-
1
pyrimidine-5-carboxamide 5h. Yield: 55%. H NMR (400 MHz,
DMSO-d6) δ 10.45 (s, 1 H), 9.86 (s, 1 H), 8.90 (s, 1 H), 8.83 (s, 1 H),
8.20 (s, 1 H), 8.06 (d, J = 8.4 Hz, 1 H), 7.99 (s, 1 H), 7.82−7.80 (m, 2
H), 7.70 (d, J = 8.5 Hz, 1 H), 7.44 (d, J = 7.9 Hz, 1 H), 3.56 (s, 2 H),
2.88 (d, J = 4.6 Hz, 3 H), 2.39−2.30 (m, 11 H), 2.16 (s, 3 H). 13C
NMR (125 MHz, DMSO-d6) δ 165.06, 163.34, 162.98, 158.13 (2C),
138.21, 138.05, 136.37, 132.21, 131.92, 131.17, 130.39, 127.38, 125.84,
125.11, 123.48, 117.25, 117.21, 115.85, 57.40, 54.67 (2C), 52.62 (2C),
45.63, 27.86, 18.01. HRMS (ESI) calcd for C27H30F3N7O2 [M + H]+,
542.2486; found, 542.2486. HPLC purity = 95.62%, Rt 6.35 min.
2-(Ethylamino)-N-(2-methyl-5-((4-((4-methylpiperazin-1-yl)-
methyl)-3-(trifluoromethyl)phenyl) carbamoyl)phenyl)-
N-(2-Methyl-5-((4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl)carbamoyl) phenyl)-2-(phenylamino)-
1
pyrimidine-5-carboxamide 5n. Yield: 81%, H NMR (400 MHz,
DMSO-d6) δ 10.48 (s, 1 H), 10.20 (s, 1 H), 10.04 (s, 1 H), 9.04 (s, 2
H), 8.20 (s, 1 H), 8.05−8.02 (m, 2 H), 7.80−7.78 (m, 3 H), 7.71 (s, 1
H), 7.46 (d, J = 6.4 Hz, 1 H), 7.34 (s, 2 H), 7.03 (s, 1 H), 3.56 (s, 2
H), 2.49−2.33 (m, 11 H), 2.18 (s, 3 H). 13C NMR (125 MHz,
DMSO-d6) δ 165.61, 163.17, 161.27, 158.48(2C), 139.84, 138.58,
138.55, 136.58, 132.68, 132.37, 131.69, 130.95, 129.02(2C), 126.26,
125.70, 123.99, 123.02, 120.21(2C), 118.69, 117.71, 57.77, 54.93(2C),
52.78(2C), 45.79, 18.40; HRMS (ESI) calcd for C32H32F3N7O2 [M +
H]+, 604.2643; found, 604.2642. HPLC purity = 99.42%, Rt 8.29 min.
2-(Dimethylamino)-N-(2-methyl-5-((4-((4-methylpiperazin-
1-yl)methyl)-3-(trifluoromethyl) phenyl)carbamoyl)phenyl)-
1
pyrimidine-5-carboxamide 5i. Yield: 30%. H NMR (400 MHz,
DMSO-d6) δ 10.46 (s, 1 H), 9.85 (s, 1 H), 8.88 (s, 1 H), 8.83 (s, 1 H),
8.20 (s, 1 H), 8.07 (d, J = 8.1 Hz, 1 H), 7.98 (s, 1 H), 7.89 (t, J = 5.7
Hz, 1 H), 7.81 (d, J = 7.9 Hz, 1 H), 7.73−7. 65 (m, 2 H), 7.44 (d, J =
8.1 Hz, 1 H), 3.57 (s, 2 H), 3.38 (q, J = 7.0 Hz, 2 H), 2.40 (s, 7 H),
2.31 (s, 4 H), 2.20 (s, 3 H), 1.15 (t, J = 7.1 Hz, 3 H). 13C NMR (125
MHz, DMSO-d6) δ 167.02, 165.21, 163.13, 162.84, 158.24, 138.29,
138.15, 136.44, 132.28, 131.94, 131.30, 130.49, 128.69, 125.92, 125.18,
123.59, 123.29, 117.33, 115.91, 57.41, 54.58(2C), 52.41(2C), 45.42,
35.60, 18.06, 14.51. HRMS (ESI) calcd for C28H32F3N7O2 [M + H]+,
556.2643; found, 556.2644. HPLC purity = 99.38%, Rt 6.73 min.
2-(Iisopropylamino)-N-(2-methyl-5-((4-((4-methylpiperazin-
1-yl)methyl)-3-(trifluoromethyl) phenyl)carbamoyl)phenyl)-
1
pyrimidine-5-carboxamide 5o. Yield: 71%. H NMR (400 MHz,
DMSO-d6) δ 10.47 (s, 1 H), 9.90 (s, 1 H), 8.91 (s, 2 H), 8.20 (s, 1 H),
8.06 (d, J = 7.6 Hz, 1 H), 7.99 (s, 1 H), 7.81 (d, J = 7.6 Hz, 1 H), 7.69
(d, J = 7.6 Hz, 1 H), 7.40 (d, J = 7.6 Hz, 1 H), 3.56 (s, 2 H), 3.21 (s, 6
H), 2.50−2.31 (m, 11 H), 2.19 (s, 3 H). 13C NMR (125 MHz,
DMSO-d6) δ 165.63, 163.58, 162.54(2C), 138.58, 136.69, 132.62,
132.29, 131.70, 130.91, 126.27, 125.61, 123.98, 117.72, 115.47, 57.72,
54.83(2C), 52.61(2C), 45.61, 37.21(2C), 18.37; HRMS (ESI) calcd
for C28H32F3N7O2 [M + H]+, 556.2643; found, 556.2641. HPLC
purity = 97.80%, Rt 7.51 min.
1
pyrimidine-5-carboxamide 5j. Yield: 52%. H NMR (400 MHz,
DMSO-d6) δ 10.46 (s, 1 H), 9.84 (s, 1 H), 8.84 (s, 2 H), 8.20 (s, 1 H),
8.06 (d, J = 8.2 Hz, 1 H), 7.98 (s, 1 H), 7.80 (t, J = 7.8 Hz, 2 H), 7.70
(d, J = 8.5 Hz, 1 H), 7.44 (d, J = 7.8 Hz, 1 H), 4.17−4.11 (m, 1 H),
3.56 (s, 2 H), 2.41−2. 30 (m, 11 H), 2.16 (s, 3 H), 1.18 (d, J = 6.3 Hz,
6 H). 13C NMR (125 MHz, DMSO-d6) δ 165.07, 162.97, 162.19,
158.16, 158.14, 138.22, 138.01, 136.40, 132.22, 131.92, 131.17, 130.37,
125.82, 125.05, 123.48, 123.22, 117.25, 117.20, 115.72, 57.41, 54.66
(2C), 52.61 (2C), 45.63, 42.25, 22.13 (2C), 18.00. HRMS (ESI) calcd
for C29H34F3N7O2 [M + H]+, 570.2799; found, 570.2798. HPLC
purity = 98.64%, Rt 7.63 min.
N-(2-Methyl-5-((4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl)carbamoyl) phenyl)-1H-pyrrolo[2,3-
1
b]pyridine-5-carboxamide 6a. Yield: 63%. H NMR (400 MHz,
DMSO-d6) δ 11.99 (s, 1 H), 10.47 (s, 1 H), 10.09 (s, 1 H), 8.88 (d, J =
2.4 Hz, 1 H), 8.61 (d, J = 1.6 Hz, 1 H), 8.21 (d, J = 2.0 Hz, 1 H), 8.07
(d, J = 8.4 Hz, 1 H), 8.04 (d, J = 1.3 Hz, 1 H), 7.83 (dd, J = 7.9, 1.6
Hz, 1 H), 7.70 (d, J = 8.5 Hz, 1 H), 7.62−7.60 (m, 1 H), 7.47 (d, J =
8.0 Hz, 1 H), 6.62 (dd, J = 3.4, 1.6 Hz, 1 H), 3.57 (s, 2 H), 2.40−2.34
2-(Cyclopropylamino)-N-(2-methyl-5-((4-((4-methylpipera-
zin-1-yl)methyl)-3-(trifluoromethyl) phenyl)carbamoyl)-
phenyl)pyrimidine-5-carboxamide 5k. Yield: 48%. 1H NMR
(400 MHz, DMSO-d6) δ 10.46 (s, 1 H), 9.88 (s, 1 H), 8.88 (s, 2
2699
dx.doi.org/10.1021/jm500007h | J. Med. Chem. 2014, 57, 2692−2703

Journal of Medicinal Chemistry
Article
(m, 11 H), 2.19 (s, 3 H). 13C NMR (125 MHz, DMSO-d6) δ 165.29,
165.11, 160.91, 149.69, 142.77, 138.24, 138.13, 136.72, 132.22, 131.86,
131.18, 130.37, 128.02, 127.74, 127.47, 125.96, 125.09, 123.48, 122.04,
118.71, 117.26, 117.21, 100.97, 57.37, 54.59 (2C), 52.49 (2C), 45.49,
18.02. HRMS (ESI) calcd for C29H29F3N6O2 [M + H]+, 551.2377;
found, 551.2375. HPLC purity = 96.06%, Rt 6.60 min.
123.48, 121.97, 118.73, 117.25, 117.20, 100.98, 57.40, 54.65(2C),
52.60(2C), 45.60, 27.76, 22.89(2C). HRMS (ESI) calcd for
C31H33F3N6O2 [M + H]+, 579.2690; found, 579.2689. HPLC purity
= 97.27%, Rt 7.10 min.
Methyl 2-(methylamino)pyrimidine-5-carboxylate 12b. To a
solution of 11b (1.05g, 5.58 mmol) in 6 mL of DMF, Pd(OAc)2 (62.0
mg, 0.28 mmol), Xantphos (194.3 mg, 0.34 mmol), triethylamine (1.4
mL, 10.0 mmol), and 1.0 mL og methanol were added. The reaction
flask was evacuated and backfilled with CO twice. The mixture was
heated to 100 °C and stirred overnight under a CO balloon. Then, the
reaction mixture was cooled to room temperature and diluted with
water and extracted with ethyl acetate (3 × 50 mL). The combined
organic layer was dried over anhydrous sodium sulfate, concentrated
under vacuum, and was used in the next step without further
purification. Compounds 12c−i were synthesized by a similar
procedure.
N-(2-Methyl-5-((4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl)carbamoyl) phenyl)-1H-pyrazolo[3,4-
1
b]pyridine-5-carboxamide 6b. Yield: 42%. H NMR (400 MHz,
DMSO-d6) δ 13.98 (s, 1 H), 10.48 (s, 1 H), 10.25 (s, 1 H), 9.12 (d, J =
1.9 Hz, 1 H), 8.88 (d, J = 1.9 Hz, 1 H), 8.35 (s, 1 H), 8.20 (s, 1 H),
8.08−8.03 (m, 2 H), 7.84 (d, J = 7.9 Hz, 1 H), 7.70 (d, J = 8.5 Hz, 1
H), 7.48 (d, J = 8.1 Hz, 1 H), 3.56 (s, 2 H), 2.48−2.23 (m, 11 H), 2.15
(s, 3 H). 13C NMR (125 MHz, DMSO-d6) δ 165.09, 164.52, 152.57,
148.71, 138.26, 138.13, 136.46, 134.65, 132.32, 131.87, 131.20, 130.46,
130.37, 127.51, 127.27, 125.95, 125.41, 123.50, 123.23, 117.29, 117.24,
113.62, 57.36, 54.55(2C), 52.41(2C), 45.40, 18.03. HRMS (ESI) calcd
for C28H28F3N7O2 [M + H]+, 552.2330; found, 552.2331. HPLC
purity = 97.48%, Rt 5.97 min.
N-(2-Methyl-5-((4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl)carbamoyl) phenyl)-1H-indole-5-car-
boxamide 6c. Yield: 57%, 1H NMR (400 MHz, DMSO-d6) δ
13.36 (s, 1 H), 10.47 (s, 1 H), 10.07 (s, 1 H), 8.53 (s, 1 H), 8.28 (s, 1
H), 8.21 (d, J = 1.6 Hz, 1 H), 8.07 (dd, J = 8.4 Hz, 1.6 Hz, 1 H), 8.03
(d, J = 1.2 Hz, 1 H), 8.00 (dd, J = 8.8 Hz, 1.2 Hz, 1 H), 7.83 (dd, J =
8.0 Hz, 1.6 Hz, 1 H), 7.70 (d, J = 8.4 Hz, 1 H), 7.66 (d, J = 8.8 Hz, 1
H), 7.46 (d, J = 8.0 Hz, 1 H), 3.57 (s, 2 H), 2.50−2.34 (m, 11 H), 2.20
(s, 3 H). 13C NMR (125 MHz, DMSO) δ 165.77, 165.13, 160.92,
141.06, 138.26, 138.16, 136.88, 134.92, 132.22, 131.84, 131.19, 130.35,
127.48, 126.66, 125.98, 125.48, 125.05, 123.48, 122.37, 121.24, 117.27,
109.95, 57.36, 54.56(2C), 52.43(2C), 45.42, 18.02. HRMS (ESI) calcd
for C30H30F3N5O2 [M + H]+, 550.2425; found, 550.2425. HPLC
purity = 98.24%, Rt 6.12 min.
Methyl 2-(methylamino)pyrimidine-5-carboxylate 13b. To a
solution of 12b (446.0 mg, 0.27 mmol) in 15 mL of mixture solvent
(MeOH/THF/H2O = 3:3:9), LiOH (224.2 mg, 5.34 mmol) was
added, and the resulting mixture was stirred at room temperature for 3
h. The organic solvent was removed under vacuum, and the resulting
residue was diluted with water and extracted with ethyl acetate (2 × 50
mL). Then, the aqueous solution was acidified with dilute hydrochloric
acid (pH about.4), and the precipitate was filtered and washed with
water and petroleum ether to give the pure product 13b (297.4 mg;
yield, 73%). 1H NMR (400 MHz, DMSO-d6) δ 8.74 (d, J = 2.0 Hz, 1
H), 8.65 (d, J = 2.4 Hz, 1 H), 7.90 (d, J = 4.8 Hz, 1 H), 2.86 (d, J = 4.8
Hz, 3 H). MS (ESI), m/z 154 [M + H]+.
2-(Ethylamino)pyrimidine-5-carboxylic Acid 13c. 1H NMR
(500 MHz, DMSO-d6) δ 12.68 (s, 1 H), 8.72 (s, 1 H), 8.66 (s, 1 H),
7,99 (t, J = 5 Hz, 1 H), 3.38−3.32 (m, 2 H), 1.12 (t, J = 7 Hz, 3 H).
MS (ESI), m/z 168 [M + H]+.
1
2-(Cyclopropylamino)pyrimidine-5-carboxylic Acid 13e. H
N - ( 3 - ( ( 4 - ( ( 4 - M e t h y l p i p e r a z i n - 1 - y l ) m e t h y l ) - 3 -
(trifluoromethyl)phenyl)carbamoyl)phenyl)-1H-pyrrolo[2,3-b]-
pyridine-5-carboxamide 6d. Yield: 85%. 1H NMR (400 MHz,
DMSO-d6) δ 12.02 (s, 1 H), 10.58 (s, 1 H), 10.52 (s, 1 H), 8.87 (d, J =
1.2 Hz, 1 H), 8.62 (s, 1 H), 8.39 (s, 1 H), 8.22 (s, 1 H), 8.08−8.04 (m,
2 H), 7.72 (s, 1 H), 7.70 (s, 1 H), 7.61 (t, J = 2.8 Hz, 1 H), 7.54 (t, J =
8.0 Hz, 1 H), 6.63 (d, J = 1.6 Hz, 1 H), 3.57 (s, 2 H), 2.39 (s, 8 H),
2.17 (s, 3 H). 13C NMR (125 MHz, DMSO) δ 166.28, 165.92, 150.13,
143.28, 140.01, 138.66, 135.56, 132.46, 131.65, 129.11, 128.52, 128.23,
127.88(q, J = 30 Hz), 124.77(q, J = 274 Hz), 123.94, 120.28, 119.12,
117.64, 101.47, 57.87, 55.13(2C), 53.09(2C), 46.10; HRMS (ESI)
calcd for C28H27F3N6O2 [M + H]+, 537.2221; found, 537.2220. HPLC
purity = 99.14%, Rt 6.66 min.
N-(2-Ethyl-5-((4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl)carbamoyl)phenyl)-1H-pyrrolo[2,3-b]-
pyridine-5-carboxamide 6e. Yield: 42%. 1H NMR (400 MHz,
DMSO-d6) δ 12.00 (s, 1 H), 10.49 (s, 1 H), 10.10 (s, 1 H), 8.88 (s, 1
H), 8.60 (s, 1 H), 8.21 (s, 1 H), 8.07 (d, J = 8.4 Hz, 1 H), 7.99 (s, 1
H), 7.89 (d, J = 7.6 Hz, 1 H), 7.70 (d, J = 8.4 Hz, 1 H), 7.61 (s, 1 H),
7.49 (d, J = 8.0 Hz, 1 H), 6.63 (s, 1 H), 3.56 (s, 2 H), 2.74 (dd, J =
14.4 Hz, 7.2 Hz, 2 H), 2.50−2.38 (m, 8 H), 2.16 (s, 3 H), 1.19 (t, J =
7.2 Hz, 3 H). 13C NMR (125 MHz, DMSO) δ 166.32, 165.66, 150.04,
144.66, 143.12, 138.57, 136.41, 132.63, 132.39, 131.70, 129.17, 128.50,
128.23, 127.97, 127.74, 127.35, 126.20, 124.00, 123.63, 119.24, 117.72,
101.54, 57.77, 54.93(2C), 52.80(2C), 45.82, 24.47, 14.26; HRMS
(ESI) calcd for C30H31F3N6O2 [M + H]+, 565.2534; found, 565.2533.
HPLC purity = 95.37%, Rt 6.60 min.
NMR (400 MHz, DMSO-d6) δ 12.74 (s, 1 H), 8.77 (s, 1 H), 8.67 (s, 1
H) 8.16 (d, J = 3.6 Hz, 1 H), 2.83−2.78 (m, 1 H), 0.73−0.68 (m, 2
H), 0.54 −0.50(m, 2 H); MS (ESI), m/z 180 [M + H]+.
2-(Cyclopentylamino)pyrimidine-5-carboxylic Acid 13f. 1H
NMR (500 MHz, DMSO-d6) δ 12.67 (s, 1 H), 8.71 (s, 1 H), 8.65 (s, 1
H), 8.03 (d, J = 7.5 Hz, 1 H), 4.25−4.21 (m, 1 H), 1.91−1.86 (m, 2
H), 1.71−1.1.65 (m, 2 H), 1.57−1.50 (m, 4 H); MS (ESI), m/z 208
[M + H]+.
Compounds 13a−i and 16a−c45 were synthesized by the same
procedure; yield, 44−89%.
1H-Pyrrolo[2,3-b]pyridine-5-carboxylic Acid 16a. 1H NMR
(400 MHz, DMSO-d6) δ 12.01 (s, 1 H), 8.78 (d, J = 1.8 Hz, 1 H), 8.50
(d, J = 1.8 Hz, 1 H), 7.58 (dd, J = 3.2 Hz, 1.8 Hz, 1 H), 6.60 (dd, J =
3.2 Hz, 1.8 Hz, 1 H); MS (ESI), m/z 163 [M + H]+.
1
1H-Pyrazolo[3,4-b]pyridine-5-carboxylic Acid 16b. H NMR
(400 MHz, DMSO-d6) δ 13.99 (s, 1H), 9.02 (d, J = 2.0 Hz, 1 H), 8.79
(d, J = 2.0 Hz, 1 H), 8.30 (s, 1 H); MS (ESI), m/z 164 [M + H]+.
Cell Culture. The human colorectal adenocarcinoma cell lines HT-
29, HCT-116, Colo205, LOVO, and WiDr and malignant melanoma
cell lines A375 and SK-MEL-28 were purchased from ATCC. HT-29
and HCT116 were maintained in McCoy’s 5a with 10% FBS, Colo205,
LOVO, and A375 were maintained in RPMI-1640, F12K, and DMEM
with 10% FBS, respectively, while WiDr and SK-MEL-28 were grown
in Eagle’s minimum essential medium with 10% FBS. The drug 1
resistant cell line SK-MEL-28-PR30 was selected in our laboratory by
exposing the parental SK-MEL-28 cells to increasing concentrations of
drug 1 for 2 months (Supporting Information).
N-(2-Isopropyl-5-((4-((4-methylpiperazin-1-yl)methyl)-3-
In Vitro Enzymatic Activity Assay. EGFR, B-RafV600E (as B-
RafV599E in supplier’s catalogue), and the Z′-Lyte Kinase Assay Kit
were purchased from Invitrogen. The experiments were performed
according to the instructions of the manufacturer. The concentrations
of different kinases were determined by optimization experiments, and
the respective concentrations were EGFR (PV3872, Invitrogen), 0.43
μg/μL; and BRAFV600E (PV4173, Invitrogen), 0.22 μg/μL. First, the
compounds were diluted 3-fold from 5.1 × 10−9 M to 1 × 10−4 M in
DMSO, and a 4× compound solution was prepared (4 μL of
compound dissolved in 96 μL of water). Second, a 40 μM ATP
(trifluoromethyl)phenyl)carbamoyl) phenyl)-1H-pyrrolo[2,3-
1
b]pyridine-5-carboxamide 6f. Yield: 28%. H NMR (400 MHz,
DMSO-d6) δ 12.00 (s, 1 H), 10.48 (s, 1 H), 10.13 (s, 1 H), 8.89 (d, J =
2.0 Hz, 1 H), 8.62 (d, J = 1.6 Hz, 1 H), 8.21 (d, J = 1.6 Hz, 1 H), 8.07
(d, J = 8.4 Hz, 1 H), 7.94−7.92 (m, 2 H), 7.70 (d, J = 8.4 Hz, 1 H),
7.61 (t, J = 2.8 Hz, 1 H), 7.56 (d, J = 8.8 Hz, 1 H), 6.63 (dd, J = 3.2
Hz, 1.6 Hz, 1 H), 3.56 (s, 2 H), 3.33−3.27 (m, 1 H), 2.50−2.38 (m, 8
H), 2.16 (s, 3 H), 2.21 (d, J = 6.8 Hz, 6 H). 13C NMR (125 MHz,
DMSO-d6) δ 165.96, 165.04, 149.70, 149.11, 142.75, 138.23, 135.31,
132.05, 131.92, 131.18, 127.96, 127.74, 127.51, 127.24, 126.19, 125.91,
2700
dx.doi.org/10.1021/jm500007h | J. Med. Chem. 2014, 57, 2692−2703

Journal of Medicinal Chemistry
Article
solution in 1.33× kinase buffer was prepared. Third, a kinase/peptide
mixture containing 2× kinase and 4 μM Tyr 4 peptide (Invitrogen,
PV3193) was prepared right before use.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR and 13C NMR for final compounds, biological data,
and assay details. This material is available free of charge via the
Internet at http://pubs.acs.org.
For both EGFR and B-Raf assays, 10 μL of kinase reactions were
made at first (including 2.5 μL of compound solution, 5 μL of kinase/
peptide mixture, and 2.5 μL of ATP solution). The plate was mixed
thoroughly and incubated for 1 h at room temperature. Then 5 μL of
development solution was added to each well, and the plate was
incubated for 1 h at room temperature; the nonphospho-peptides were
cleaved at this time. In the end, 5 μL of stop reagent was loaded to
stop the reaction. For the control setting, 5 μL of phospho-peptide
solution instead of the kinase/peptide mixture was used as 100% of the
phosphorylation control. 2.5 μL of 1.33 × kinase buffer instead of ATP
solution was used as 100% inhibition control, and 2.5 μL of 4% DMSO
instead of compound solution was used as the 0% inhibitor control.
The plate was measured on an EnVision Multilabel Reader (Perkin-
Elmer). Curve fitting and data presentations were performed using
Graph Pad Prism, version 5.0. Every experiment was repeated at least 2
times.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +86-20-32015276. Fax: +86-20-32015299. E-mail: ding_
ke@gibh.ac.cn.
Author Contributions
∥H.C., Y.C., and L.Z. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the National Natural Science Foundation
(#21302186) and the Key Project on Innovative Drug of
Guangdong Province (#2012A080201014) for their financial
support.
For the B-RafV600E assay, the kinase/peptide mixture was prepared
by diluting the Z′-LYTE Ser/Thr3 peptide (Invitrogen, PV3176) and
three kinases (B-Raf, MAP2K1/MEK1 (Invitrogen, P3093), MAPK1/
ERK2 (Invitrogen, PV3314)) in 1× kinase buffer, and a 0.2 μM Ser/
Thr3 phospho-peptide solution was made by adding Z′-LYTE Ser/
Thr3 Phospho-peptide to 1× kinase buffer. The final 10 μL reaction
consists of 0.002 ng of B-Raf, 10 ng of inactive MAP2K1 (MEK1),100
ng of inactive MAPK1 (ERK2), and 2 μM Ser/Thr3 peptide in 1×
kinase buffer.
Cell Proliferation and Growth Inhibition Assay. WiDr, A375,
HT-29, SK-MEL-28, SK-MEL-28-PR30, HCT-116, and LOVO cells
were cultured with the respective growth medium. Cells of log phase
were used. One thousand to 3000 cells/well were seeded in 96-well
plates with a 100 μL volume, and 6 parallels and 8 rows were designed.
Compounds were dissolved to 10 μM with DMSO, and a 5-fold serial
dilution of the compounds from 1 × 10−5 M to 0.64 × 10−9 M was
performed. Two microliters of compound solution was added to 998
μL of growth medium, the mixture was vortexed sufficiently. One
hundred microliters of the mixture was correspondingly added to the
96-well plate. Two microliters of DMSO instead of compound
solution was used as the 0% inhibitor control. After coincubation for
68 h, 20 μL of MTT (5 mg/mL) was added. Four hours later, the
supernatant was discarded completely, and 150 μL of DMSO was
added. After shaking for 10 min, the plates were read in the Synergy
HT (Bio Tek) at 570 nm. The data was calculated using Graph Pad
Prism, version 4.0. The IC50 were fitted using a nonlinear regression
model with a sigmoidal dose−response.
ABBREVIATIONS USED
■
MAPK, mitogen-activated protein kinase signaling pathway;
EGFR, epidermal growth factor receptor; Erk, extracellular
signal-regulated kinase; PDGFR, Platelet-derived growth factor;
Val (V), valine; Glu (E), glutamic acid; rt, room temperature;
DIEA, N,N-diisopropylethylamine; PyBOP, benzotriazol-1-yl-
oxytripyrrolidinophosphonium hexafluorophosphate; HATU,
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxid hexafluorophosphate; MOA, mechanisms of
action; FDA, Food and Drug Administration; IC50, the half
maximal (50%) inhibitory concentration (IC) of a substance;
THF, tetrahydrofuran; DMSO, dimethyl sulfoxide; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; DMF, N,N-dime-
thylformamide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide
REFERENCES
■
(1) Peyssonnaux, C.; Eychene, A. The Raf/MEK/ERK pathway: new
concepts of activation. Biol. Cell 2001, 93, 53−62.
(2) Dhillon, A. S.; Hagan, S.; Rath, O.; Kolch, W. MAP kinase
signalling pathways in cancer. Oncogene 2007, 26, 3279−3290.
(3) Marais, R.; Marshall, C. J. Control of the ERK MAP kinase
cascade by Ras and Raf. Cancer Surv. 1996, 27, 101−125.
Western Blot. Cells (1 ×106) of WiDr, A375, HT-29, SK-MEL-28,
and SK-MEL-28-PR30 were seeded into 6-cm dishes overnight. The
medium was changed, and 3, 1, 0.3 μM 6a was added the next day;
medium with 1‰ DMSO was used as the control. Cells were exposed
to treatment for different times. The dishes were washed twice using
precold PBS, and 400 μL of RIPA was added then. After incubating
plates on ice for 15 min, cells were scraped carefully and centrifuged
for 10 min at 14,000g at 4 °C immediately, and lysates (the
supernantant) were maintained at −70 °C. A BCA protein assay kit
(23227, Thermo) was used to quantitate the cell lysates. Proper 5×
loading buffer was loaded before use, and the samples were denatured
by boiling. The same amount of quantitated sample was loaded, and
proteins were transferred to the PVDF membrane (Milipore) then.
After blocking for 1.0 h at room temperature, diluted primary antibody
EGFR (CST, 2232), phospho-EGFR (Tyr1068) (CST, 2234), ERK
(CST, 9102), phospho-ERK (t202/y204) (CST, 9101), and GAPDH
(KC-5G5, KangChen) were added. A second antibody with horse-
radish peroxidase (HRP, sigma) conjugated was used then. Blots were
developed by enhanced chemiluminescence (Thermo).
(4) Garnett, M. J.; Marais, R. Guilty as charged: B-RAF is a human
oncogene. Cancer Cell 2004, 6, 313−319.
(5) Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins, S.;
Clegg, S.; Teague, J.; Woffendin, H.; Garnett, M. J.; Bottomley, W.;
Davis, N.; Dicks, N.; Ewing, R.; Floyd, Y.; Gray, K.; Hall, S.; Hawes,
R.; Hughes, J.; Kosmidou, V.; Menzies, A.; Mould, C.; Parker, A.;
Stevens, C.; Watt, S.; Hooper, S.; Wilson, R.; Jayatilake, H.; Gusterson,
B. A.; Cooper, C.; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.;
Maitland, N.; Chenevix-Trench, G.; Riggins, G. J.; Bigner, D. D.;
Palmieri, G.; Cossu, A.; Flanagan, A.; Nicholson, A.; Ho, J. W. C.;
Leung, S. Y.; Yuen, S. T.; Weber, B. L.; Siegler, H. F.; Darrow, T. L.;
Paterson, H.; Marais, R.; Marshall, C. J.; Wooster, R.; Stratton, M. R.;
Futreal, P. A. Mutations of the BRAF gene in human cancer. Nature
2002, 417, 949−954.
(6) Yuen, S. T.; Davies, H.; Chan, T. L.; Ho, J. W.; Bignell, G. R.;
Cox, C.; Stephens, P.; Edkins, S.; Tsui, W. W.; Chan, A. S.; Futreal, P.
A.; Stratton, M. R.; Wooster, R.; Leung, S. Y. Similarity of the
phenotypic patterns associated with BRAF and KRAS mutations in
colorectal neoplasia. Cancer Res. 2002, 62, 6451−6455.
2701
dx.doi.org/10.1021/jm500007h | J. Med. Chem. 2014, 57, 2692−2703

Journal of Medicinal Chemistry
Article
(7) Kimura, E. T.; Nikiforova, M. N.; Zhu, Z. W.; Knauf, J. A.;
Nikiforov, Y. E.; Fagin, J. A. High prevalence of BRAF mutations in
thyroid cancer: genetic evidence for constitutive activation of the
RET/PTC-RAS-BRAF signaling pathway in papillary thyroid
carcinoma. Cancer Res. 2003, 63, 1454−1457.
A. COT drives resistance to RAF inhibition through MAP kinase
pathway reactivation. Nature 2010, 468, 968−972.
(20) Nazarian, R.; Shi, H. B.; Wang, Q.; Kong, X. J.; Koya, R. C.; Lee,
H.; Chen, Z. G.; Lee, M. K.; Attar, N.; Sazegar, H.; Chodon, T.;
Nelson, S. F.; McArthur, G.; Sosman, J. A.; Ribas, A.; Lo, R. S.
Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or
N-RAS upregulation. Nature 2010, 468, 973−977.
(21) Villanueva, J.; Vultur, A.; Lee, J. T.; Somasundaram, R.;
Fukunaga-Kalabis, M.; Cipolla, A. K.; Wubbenhorst, B.; Xu, X. W.;
Gimotty, P. A.; Kee, D.; Santiago-Walker, A. E.; Letrero, R.; D’Andrea,
K.; Pushparajan, A.; Hayden, J. E.; Brown, K. D.; Laquerre, S.;
McArthur, G. A.; Sosman, J. A.; Nathanson, K. L.; Herlyn, M.
Acquired resistance to BRAF inhibitors mediated by a RAF kinase
switch in melanoma can be overcome by cotargeting MEK and IGF-
1R/PI3K. Cancer Cell 2010, 18, 683−695.
(22) Poulikakos, P. I.; Persaud, Y.; Janakiraman, M.; Kong, X. J.; Ng,
C.; Moriceau, G.; Shi, H. B.; Atefi, M.; Titz, B.; Gabay, M. T.; Salton,
M.; Dahlman, K. B.; Tadi, M.; Wargo, J. A.; Flaherty, K. T.; Kelley, M.
C.; Misteli, T.; Chapman, P. B.; Sosman, J. A.; Graeber, T. G.; Ribas,
A.; Lo, R. S.; Rosen, N.; Solit, D. B. RAF inhibitor resistance is
mediated by dimerization of aberrantly spliced BRAF(V600E). Nature
2011, 480, 387−390.
(23) Shi, H. B.; Kong, X. J.; Ribas, A.; Lo, R. S. Combinatorial
treatments that overcome PDGFR beta-driven resistance of melanoma
cells to B-V600E-RAF inhibition. Cancer Res. 2011, 71, 5067−5074.
(24) Wagle, N.; Emery, C.; Berger, M. F.; Davis, M. J.; Sawyer, A.;
Pochanard, P.; Kehoe, S. M.; Johannessen, C. M.; Macconaill, L. E.;
Hahn, W. C.; Meyerson, M.; Garraway, L. A. Dissecting therapeutic
resistance to RAF inhibition in melanoma by tumor genomic profiling.
J. Clin. Oncol. 2011, 29, 3085−3096.
(25) Corcoran, R. B.; Ebi, H.; Turke, A. B.; Coffee, E. M.; Nishino,
M.; Cogdill, A. P.; Brown, R. D.; Della Pelle, P.; Dias-Santagata, D.;
Hung, K. E.; Flaherty, K. T.; Piris, A.; Wargo, J. A.; Settleman, J.;
Mino-Kenudson, M.; Engelman, J. A. EGFR-mediated re-activation of
MAPK signaling contributes to insensitivity of BRAF mutant
colorectal cancers to RAF inhibition with vemurafenib. Cancer
Discovery 2012, 2, 227−235.
(26) Prahallad, A.; Sun, C.; Huang, S. D.; Di Nicolantonio, F.;
Salazar, R.; Zecchin, D.; Beijersbergen, R. L.; Bardelli, A.; Bernards, R.
Unresponsiveness of colon cancer to BRAF(V600E) inhibition
through feedback activation of EGFR. Nature 2012, 483, 100−103.
(27) Shi, H.; Moriceau, G.; Kong, X.; Koya, R. C.; Nazarian, R.;
Pupo, G. M.; Bacchiocchi, A.; Dahlman, K. B.; Chmielowski, B.;
Sosman, J. A.; Halaban, R.; Kefford, R. F.; Long, G. V.; Ribas, A.; Lo,
R. S. Preexisting MEK1 exon 3 mutations in V600E/KBRAF
melanomas do not confer resistance to BRAF inhibitors. Cancer
Discovery 2012, 2, 414−424.
(28) Shi, H.; Moriceau, G.; Kong, X.; Lee, M. K.; Lee, H.; Koya, R.
C.; Ng, C.; Chodon, T.; Scolyer, R. A.; Dahlman, K. B.; Sosman, J. A.;
Kefford, R. F.; Long, G. V.; Nelson, S. F.; Ribas, A.; Lo, R. S.
Melanoma whole-exome sequencing identifies (V600E)B-RAF
amplification-mediated acquired B-RAF inhibitor resistance. Nat.
Commun. 2012, 3, 724.
(8) Wellbrock, C.; Hurlstone, A. BRAF as therapeutic target in
melanoma. Biochem. Pharmacol. 2010, 80, 561−567.
(9) Tuveson, D. A.; Weber, B. L.; Herlyn, M. BRAF as a potential
therapeutic target in melanoma and other malignancies. Cancer Cell
2003, 4, 95−98.
(10) Tsai, J.; Lee, J. T.; Wang, W.; Zhang, J.; Cho, H.; Mamo, S.;
Bremer, R.; Gillette, S.; Kong, J.; Haass, N. K.; Sproesser, K.; Li, L.;
Smalley, K. S. M.; Fong, D.; Zhu, Y. L.; Marimuthu, A.; Nguyen, H.;
Lam, B.; Liu, J.; Cheung, I.; Rice, J.; Suzuki, Y.; Luu, C.; Settachatgul,
C.; Shellooe, R.; Cantwell, J.; Kim, S. H.; Schlessinger, J.; Zhang, K. Y.
J.; West, B. L.; Powell, B.; Habets, G.; Zhang, C.; Ibrahim, P. N.; Hirth,
P.; Artis, D. R.; Herlyn, M.; Bollag, G. Discovery of a selective inhibitor
of oncogenic B-Raf kinase with potent antimelanoma activity. Proc.
Natl. Acad. Sci. U.S.A. 2008, 105, 3041−3046.
(11) Rheault, T. R.; Stellwagen, J. C.; Adjabeng, G. M.; Hornberger,
K. R.; Petrov, K. G.; Waterson, A. G.; Dickerson, S. H.; Mook, R. A.;
Laquerre, S. G.; King, A. J.; Rossanese, O. W.; Arnone, M. R.;
Smitheman, K. N.; Kane-Carson, L. S.; Han, C.; Moorthy, G. S.; Moss,
K. G.; Uehling, D. E. Discovery of dabrafenib: a selective inhibitor of
Raf kinases with antitumor activity against B-Raf-driven tumors. ACS
Med. Chem. Lett. 2013, 4, 358−362.
(12) Bollag, G.; Tsai, J.; Zhang, J. Z.; Zhang, C.; Ibrahim, P.; Nolop,
K.; Hirth, P. Vemurafenib: the first drug approved for BRAF-mutant
cancer. Nat. Rev. Drug Discovery 2012, 11, 873−886.
(13) Ballantyne, A. D.; Garnock-Jones, K. P. Dabrafenib: first global
approval. Drugs 2013, 73, 1367−1376.
(14) Chapman, P. B.; Hauschild, A.; Robert, C.; Haanen, J. B.;
Ascierto, P.; Larkin, J.; Dummer, R.; Garbe, C.; Testori, A.; Maio, M.;
Hogg, D.; Lorigan, P.; Lebbe, C.; Jouary, T.; Schadendorf, D.; Ribas,
A.; O’Day, S. J.; Sosman, J. A.; Kirkwood, J. M.; Eggermont, A. M.;
Dreno, B.; Nolop, K.; Li, J.; Nelson, B.; Hou, J.; Lee, R. J.; Flaherty, K.
T.; McArthur, G. A. Improved survival with vemurafenib in melanoma
with BRAF V600E mutation. N. Engl. J. Med. 2011, 364, 2507−2516.
(15) Flaherty, K. T.; Puzanov, I.; Kim, K. B.; Ribas, A.; McArthur, G.
A.; Sosman, J. A.; O’Dwyer, P. J.; Lee, R. J.; Grippo, J. F.; Nolop, K.;
Chapman, P. B. Inhibition of mutated, activated BRAF in metastatic
melanoma. N. Engl. J. Med. 2010, 363, 809−819.
(16) Sosman, J. A.; Kim, K. B.; Schuchter, L.; Gonzalez, R.; Pavlick,
A. C.; Weber, J. S.; McArthur, G. A.; Hutson, T. E.; Moschos, S. J.;
Flaherty, K. T.; Hersey, P.; Kefford, R.; Lawrence, D.; Puzanov, I.;
Lewis, K. D.; Amaravadi, R. K.; Chmielowski, B.; Lawrence, H. J.; Shyr,
Y.; Ye, F.; Li, J.; Nolop, K. B.; Lee, R. J.; Joe, A. K.; Ribas, A. Survival in
BRAF V600-mutant advanced melanoma treated with vemurafenib. N.
Engl. J. Med. 2012, 366, 707−714.
(17) Falchook, G. S.; Long, G. V.; Kurzrock, R.; Kim, K. B.; Arkenau,
T. H.; Brown, M. P.; Hamid, O.; Infante, J. R.; Millward, M.; Pavlick,
A. C.; O’Day, S. J.; Blackman, S. C.; Curtis, C. M.; Lebowitz, P.; Ma,
B.; Ouellet, D.; Kefford, R. F. Dabrafenib in patients with melanoma,
untreated brain metastases, and other solid tumours: a phase 1 dose-
escalation trial. Lancet 2012, 379, 1893−1901.
(18) Hauschild, A.; Grob, J. J.; Demidov, L. V.; Jouary, T.; Gutzmer,
R.; Millward, M.; Rutkowski, P.; Blank, C. U.; Miller, W. H., Jr.;
Kaempgen, E.; Martin-Algarra, S.; Karaszewska, B.; Mauch, C.;
Chiarion-Sileni, V.; Martin, A. M.; Swann, S.; Haney, P.; Mirakhur,
B.; Guckert, M. E.; Goodman, V.; Chapman, P. B. Dabrafenib in
BRAF-mutated metastatic melanoma: a multicentre, open-label, phase
3 randomised controlled trial. Lancet 2012, 380, 358−365.
(29) Straussman, R.; Morikawa, T.; Shee, K.; Barzily-Rokni, M.; Qian,
Z. R.; Du, J. Y.; Davis, A.; Mongare, M. M.; Gould, J.; Frederick, D. T.;
Cooper, Z. A.; Chapman, P. B.; Solit, D. B.; Ribas, A.; Lo, R. S.;
Flaherty, K. T.; Ogino, S.; Wargo, J. A.; Golub, T. R. Tumour micro-
environment elicits innate resistance to RAF inhibitors through HGF
secretion. Nature 2012, 487, 500−504.
(30) Antony, R.; Emery, C. M.; Sawyer, A. M.; Garraway, L. A. C-
RAF mutations confer resistance to RAF inhibitors. Cancer Res. 2013,
73, 4840−4851.
(31) Lito, P.; Rosen, N.; Solit, D. B. Tumor adaptation and resistance
to RAF inhibitors. Nat. Med. 2013, 19, 1401−1409.
(19) Johannessen, C. M.; Boehm, J. S.; Kim, S. Y.; Thomas, S. R.;
Wardwell, L.; Johnson, L. A.; Emery, C. M.; Stransky, N.; Cogdill, A.
P.; Barretina, J.; Caponigro, G.; Hieronymus, H.; Murray, R. R.; Salehi-
Ashtiani, K.; Hill, D. E.; Vidal, M.; Zhao, J. J.; Yang, X. P.; Alkan, O.;
Kim, S.; Harris, J. L.; Wilson, C. J.; Myer, V. E.; Finan, P. M.; Root, D.
E.; Roberts, T. M.; Golub, T.; Flaherty, K. T.; Dummer, R.; Weber, B.
L.; Sellers, W. R.; Schlegel, R.; Wargo, J. A.; Hahn, W. C.; Garraway, L.
(32) Kopetz, S.; Desai, J.; Chan, E.; Hecht, J. R.; O’Dwyer, P. J.; Lee,
R. J.; Nolop, K. B.; Saltz, L. PLX4032 in metastatic colorectal cancer
patients with mutant BRAF tumors. J. Clin. Oncol. 2010, 28 (Suppl),
a3534.
2702
dx.doi.org/10.1021/jm500007h | J. Med. Chem. 2014, 57, 2692−2703

Journal of Medicinal Chemistry
Article
(33) Al-Marrawi, M. Y.; Saroya, B. S.; Brennan, M. C.; Yang, Z.;
Dykes, T. M.; El-Deiry, W. S. Off-label use of cetuximab plus sorafenib
and panitumumab plus regorafenib to personalize therapy for a patient
with V600E BRAF-mutant metastatic colon cancer. Cancer Biol. Ther.
2013, 14, 703−710.
(34) Abel, E. V.; Basile, K. J.; Kugell, C. H., III; Witkiewicz, A. K.; Le,
K.; Amaravadi, R. K.; Karakousis, G. C.; Xu, X.; Xu, W.; Schuchter, L.
M.; Lee, J. B.; Ertel, A.; Fortina, P.; Aplin, A. E. Melanoma adapts to
RAF/MEK inhibitors through FOXD3-mediated upregulation of
ERBB3. J. Clin. Invest. 2013, 123, 2155−2168.
(35) DeVita, V. T.; Schein, P. S. The Use of drugs in combination for
the treatment of cancer. N. Engl. J. Med. 1973, 288, 998−1006.
(36) Pegram, M. D.; Konecny, G. E.; O’Callaghan, C.; Beryt, M.;
Pietras, R.; Slamon, D. J. Rational combinations of trastuzumab with
chemotherapeutic drugs used in the treatment of breast cancer. J. Natl.
Cancer Inst. 2004, 96, 739−749.
(37) Ribas, A.; Hodi, F. S.; Callahan, M.; Konto, C.; Wolchok, J.
Hepatotoxicity with combination of vemurafenib and ipilimumab. N.
Engl. J. Med. 2013, 368, 1365−1366.
(38) Rowbottom, M. W.; Faraoni, R.; Chao, Q.; Campbell, B. T.; Lai,
A. G.; Setti, E.; Ezawa, M.; Sprankle, K. G.; Abraham, S.; Tran, L.;
Struss, B.; Gibney, M.; Armstrong, R. C.; Gunawardane, R. N.;
Nepomuceno, R. R.; Valenta, I.; Hua, H. L.; Gardner, M. F.; Cramer,
M. D.; Gitnick, D.; Insko, D. E.; Apuy, J. L.; Jones-Bolin, S.; Ghose, A.
K.; Herbertz, T.; Ator, M. A.; Dorsey, B. D.; Ruggeri, B.; Williams, M.;
Bhagwat, S.; James, J.; Holladay, M. W. Identification of 1-(3-(6,7-
dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-(1,1,1-trifluoro-2-methyl-
propan-2-yl)isoxazol-3-yl)urea hydrochloride (CEP-32496), a highly
potent and orally efficacious inhibitor of V-RAF murine sarcoma viral
oncogene homologue B1 (BRAF) V600E. J. Med. Chem. 2012, 55,
1082−1105.
(39) Zhang, Q. W.; Diao, Y. Y.; Wang, F.; Fu, Y.; Tang, F.; You, Q.
D.; Zhou, H. Y. Design and discovery of 4-anilinoquinazoline ureas as
multikinase inhibitors targeting BRAF, VEGFR-2 and EGFR.
MedChemComm 2013, 4, 979−986.
(40) Heffron, T. P.; Wei, B. Q.; Olivero, A.; Staben, S. T.; Tsui, V.;
Do, S.; Dotson, J.; Folkes, A. J.; Goldsmith, P.; Goldsmith, R.;
Gunzner, J.; Lesnick, J.; Lewis, C.; Mathieu, S.; Nonomiya, J.;
Shuttleworth, S.; Sutherlin, D. P.; Wan, N. C.; Wang, S. M.;
Wiesmann, C.; Zhu, B. Y. Rational design of phosphoinositide 3-
kinase alpha inhibitors that exhibit selectivity over the phosphoinosi-
tide 3-kinase beta isoform. J. Med. Chem. 2011, 54, 7815−7833.
(41) Joseph, E. W.; Pratilas, C. A.; Poulikakos, P. I.; Tadi, M.; Wang,
W. Q.; Taylor, B. S.; Halilovic, E.; Persaud, Y.; Xing, F.; Viale, A.; Tsai,
J.; Chapman, P. B.; Bollag, G.; Solit, D. B.; Rosen, N. The RAF
inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation
in a V600E BRAF-selective manner. Proc. Natl. Acad. Sci. U.S.A. 2010,
107, 14903−14908.
(42) Wakeling, A. E.; Guy, S. P.; Woodburn, J. R.; Ashton, S. E.;
Curry, B. J.; Barker, A. J.; Gibson, K. H. AZD1839 (Iressa): An orally
active inhibitor of epidermal growth factor signaling with potential for
cancer therapy. Cancer Res. 2002, 62, 5749−5754.
(43) Muhsin, M.; Graham, J.; Kirkpatrick, P. Gefitinib. Nat. Rev. Drug
Discovery 2003, 2, 515−516.
(44) Jares-Erijman, E. A.; Jovin, T. M. FRET imaging. Nat. Biotechnol.
2003, 21, 1387−1395.
(45) Zheng, X. Z.; Bair, K. W.; Bauer, P.; Baumeister, T.; Bowman, K.
K.; Buckmelter, A. J.; Caligiuri, M.; Clodfelter, K. H.; Feng, Y. Z.; Han,
B. S.; Ho, Y. C.; Kley, N.; Li, H.; Liang, X. R.; Liederer, B. M.; Lin, J.;
Ly, J.; O’Brien, T.; Oeh, J.; Oh, A.; Reynolds, D. J.; Sampath, D.;
Sharma, G.; Skelton, N.; Smith, C. C.; Tremayne, J.; Wang, L. L.;
Wang, W. R.; Wang, Z. G.; Wu, H. X.; Wu, J. S.; Xiao, Y.; Yang, G. X.;
Yuen, P. W.; Zak, M.; Dragovich, P. S. Identification of amides derived
from 1H-pyrazolo [3,4-b]pyridine-5-carboxylic acid as potent inhib-
itors of human nicotinamide phosphoribosyltransferase (NAMPT).
Bioorg. Med. Chem. Lett. 2013, 23, 5488−5497.
2703
dx.doi.org/10.1021/jm500007h | J. Med. Chem. 2014, 57, 2692−2703