N-(3-ethynyl-2,4-difluorophenyl)sulfonamide derivatives as selective Raf inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.5b00039

A series of N-(3-ethynyl-2,4-difluorophenyl)sulfonamides were identified as new selective Raf inhibitors. The compounds potently inhibit B-Raf^V600E with low nanomolar IC₅₀ values and exhibit excellent target specificity in a selectivity profiling investigation against 468 kinases. They strongly suppress proliferation of a panel of human cancer cell lines and patient-derived melanoma cells with B-Raf^V600E mutation while being significantly less potent to the cells with B-Raf^WT. The compounds also display favorable pharmacokinetic properties with a preferred example (3s) demonstrating significant in vivo antitumor efficacy in a xenograft mouse model of B-Raf^V600E mutated Colo205 human colorectal cancer cells, supporting it as a promising lead compound for further anticancer drug discovery.

Full text of DOI:10.1021/acsmedchemlett.5b00039

Letter
pubs.acs.org/acsmedchemlett
N‑(3-Ethynyl-2,4-difluorophenyl)sulfonamide Derivatives as Selective
Raf Inhibitors
Yingjun Li,†,‡ Huimin Cheng,† Zhang Zhang,†,§ Xiaoxi Zhuang,† Jinfeng Luo,† Huoyou Long,†
Yang Zhou,†,‡ Yong Xu,† Rana Taghipouran,∥ Dan Li,∥ Adam Patterson,∥,⊥ Jeff Smaill,∥,⊥ Zhengchao Tu,†
Donghai Wu,† Xiaomei Ren,*,† and Ke Ding*,†
†State Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, #190
Kaiyuan Avenue, Guangzhou 510530, China
‡University of Chinese Academy of Sciences, #19 Yuquan Road, Beijing 100049, China
§Biotechnological Institute of Chinese Materia Medica and Department of Pharmacology, Jinan University, #601 Huangpu Avenue
West, Guangzhou 510632, China
⊥
∥Auckland Cancer Society Research Centre and Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland,
#92019 Private Bag, Auckland 1142, New Zealand
S
* Supporting Information
ABSTRACT: A series of N-(3-ethynyl-2,4-difluorophenyl)-
sulfonamides were identified as new selective Raf inhibitors.
The compounds potently inhibit B-RafV600E with low nano-
molar IC50 values and exhibit excellent target specificity in a
selectivity profiling investigation against 468 kinases. They
strongly suppress proliferation of a panel of human cancer cell
lines and patient-derived melanoma cells with B-RafV600E
mutation while being significantly less potent to the cells
with B-RafWT. The compounds also display favorable
pharmacokinetic properties with a preferred example (3s)
demonstrating significant in vivo antitumor efficacy in a xenograft mouse model of B-RafV600E mutated Colo205 human colorectal
cancer cells, supporting it as a promising lead compound for further anticancer drug discovery.
KEYWORDS: B-Raf, colon cancer, melanoma, kinase inhibitor, targeted therapy
colorectal cancer, papillary thyroid carcinoma, and metastatic
nonsmall cell lung cancer.12
he Raf serine/threonine kinases are key components of
1
Tmitogen-activated protein kinase (MAPK) cascade.
However, intrinsic or acquired resistance against current B-
Raf inhibitor therapies becomes a major challenge.13 For
instance, a majority of colorectal cancer patients display
inherent resistance against vemurafenib, although they were
detected to harbor B-RafV600E mutation.14 The exact mechanism
for the resistance remains elusive, but development of new
inhibitors with differentiated chemical scaffolds may be a
valuable strategy to overcome or delay the occurrence of this
challenging medical dilemma. Herein, we describe the design
and optimization of N-(3-ethynyl-2,4-difluorophenyl)-
sulfonamide derivatives as new selective Raf inhibitors.
An X-ray crystallographic analysis reveals that vemurafenib
binds to B-Raf with a DFG-in and αC-helix-out inactive
conformation.11 The 1H-pyrrolo[2,3-b]pyridine core forms two
pairs of hydrogen bonds with the backbone amide of Cys532
and the backbone carbonyl of Gln530, respectively (Figure 1B).
The sulfonamide may form two extra hydrogen bonds with
Among three isoforms of the family (i.e., A-Raf, B-Raf, and
C-Raf), mutations of B-Raf are the most frequently detected in
human cancers,2 including 50−80% of melanoma,3 ∼100% of
hairy cell leukemia,4 45−50% of papillary thyroid carcinoma,5
11% of colorectal cancer,3 and others. Notably, over 90% of B-
Raf mutations are the substitution of valine to glutamate at
residue 600 (V600E). This substitution mimics phosphoryla-
tion of the activation loop, elevating the in vitro kinase activity
by up to 500−700-fold compared with its wide-type counter-
part.6 Therefore, selectively inhibiting B-Raf becomes an
attractive strategy for the clinical treatment of B-RafV600E-driven
human cancers.7
Several classes of B-Raf inhibitors have been discovered,8,9
among which vemurafenib (1, Figure 1) and dabrafenib (2,
Supporting Information) have been approved by US FDA for
the treatment of metastatic and unresectable melanoma
harboring B-Raf mutations.10,11 Many other clinical inves-
tigations of B-Raf inhibitors alone or in combination with other
kinase inhibitors, immunotherapies, or conventional chemo-
therapies, are ongoing, for the treatment of metastatic
Received: January 27, 2015
Accepted: March 18, 2015
© XXXX American Chemical Society
A
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Letter
Figure 1. Design of new Raf inhibitors (A). Predicted binding model of 3a (blue) superimposed to vemurafenib (gray) in a crystal structure of B-Raf
(PDB: 3OG7) (B). Predicted binding model of 3o with B-Raf (C).
Asp594 and Gly596, respectively, and the propyl group is
critical for selectivity by occupying a small lipophilic pocket
enlarged by an outward shift of αC-helix of the protein. On the
basis of these observations, we rationally designed a series of N-
(3-ethynyl-2, 4-difluorophenyl)sulfonamides (3) as new B-Raf
inhibitors, in which a druglike scaffold 1H-pyrazolo[3,4-
b]pyridine was utilized as the crucial hinge-binding moiety; a
conformation and distance favorable ethynyl linker was
introduced between the hinge-binding core and a difluor-
ophenyl group; and the sulfonamide moiety was preserved to
form the important hydrogen bonding networks with the
protein. Instead of using an alkyl substituent in the sulfonamide
(i.e., a propyl group in vemurafenib), an aryl group was
introduced to capture a potential CH−π interaction with
Phe595 (Figure 1B).
a standard nitration, a stannous chloride mediated reduction, a
modified Sonogashira coupling, and a detrimethylsilylation.15
The hinge-binding 1H-pyrazolo-[3,4-b]pyridine was then
introduced via another Sonogashira reaction. A direct
arylsulfonylation of 7 with different arylsulfonyl chlorides
yielded the final sulfonamides 3a−3e with good to moderate
yields. Compounds 3f−3s were prepared through similar
protocols (Supporting Information).
A preliminary computational study suggested that 3a might
bind to the DFG-in B-RafV600E with a similar mode to that of
vemurafenib (Figure 1B). A biological evaluation showed that
3a indeed inhibited B-RafV600E kinase with an IC50 value of
0.149 μM in an FRET-based Z′Lyte assay.16 It also selectively
suppresses the proliferation of Colo205 human colorectal
cancer cells harboring B-RafV600E mutation with an IC50 value of
2.103 μM but is obviously less potent against the growth of
HCT116 colorectal carcinoma cells with B-RafWT (IC50 > 10
μM) (Table 1). These results support that 3a may serve as a
lead compound for new B-RafV600E inhibitor discovery. An
extensive structure−activity relationship investigation was then
conducted, and the results revealed that an F substitution at
different positions of R1-phenyl ring in 3a could achieve diverse
impact on the B-RafV600E inhibitory activity. For instance, ortho-
F substituted analogue 3b displays a similar B-RafV600E
inhibitory activity to that of 3a, while the meta-fluoro
compound 3c is 2−3-fold more potent, with an IC50 value of
0.068 μM. However, when an F atom was introduced in the
para-position, the resulting compound 3d was 6-fold less
potent. Further study showed that the meta-fluoro phenyl
group in 3c could be replaced by a 3-pyridyl moiety (3f) to
display almost identical potency against B-RafV600E, with an IC50
value of 0.051 μM. However, the 2-pyridyl analogue (3e) is
about 10-times less potent. It is hypothesized that 3c and 3f
may induce a conformational rearrangement of the protein to
generate additional hydrogen bonds between the F or N atom
and the backbone of the DFG motif. A future cocrystallo-
graphic investigation will be highly valuable to demonstrate the
precise interactions. Our docking study has suggested that the
two pairs of hydrogen bonds formed by 1H-pyrazolo[3,4-
b]pyridine in 3f with Cys532 contribute greatly to the
compound’s B-RafV600E inhibition. Indeed, compound 3h
lacking a hydrogen bond acceptor showed an IC50 value
against B-RafV600E of 4.31 μM, which was 85-times less potent
than 3f. However, the removal of a hydrogen bond donor (3i)
The designed compounds were synthesized by using
Sonogashira coupling as the key steps (Scheme 1). Briefly,
the intermediate 6 was readily prepared from 2-bromo-1,3-
difluorobenzene (4) through consequent procedures including
a
Scheme 1. Synthesis of Compounds 3a−3s
a
Reagents and conditions: (a) con. H2SO4, KNO3, rt, 2.0 h, 91%; (b)
SnCl2, con. HCl, EtOH, reflux, 93%; (c) (i) Pd(dba)2, CuI,
ethynyltrimethylsilane, t-(Bu)3P, K2CO3, dry THF, 120 °C, 48 h;
(ii) n-(Bu)4NF, THF, rt, 1 h, 46%; (d) 6-bromo-3H-pyrazolo[3,4-
b]pyridine, Pd(dba)2, CuI, t-(Bu)3P, K2CO3, dry THF, 120 °C, 24 h,
63%; (e) R1Cl, pyridine, dry DCM, rt, overnight, 39−90%; (f)
pyridine-3-sulfonyl chloride, pyridine, dry DCM, rt, overnight, 93%;
(g) R2Br, Pd(dba)2, CuI, t-(Bu)3P, K2CO3, dry THF, 120 °C, 24 h,
47−70%.
B
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ACS Medicinal Chemistry Letters
Letter
placed by a similar bivalent 1H-pyrrolo[2,3-b]pyridine (3g)
without obviously affecting the B-RafV600E inhibitory potency.
Further investigation revealed that 3-position of the 1H-
pyrazolo[3,4-b]pyridine core could be substituted by a
methoxyl (3j), methyl (3l), cyclopropyl (3m), or phenyl
(3n) group to achieve 2−5-fold potency improvement.
However, the 3-ethoxyl compound 3k is 2-times less potent
than 3f. Encouragingly, when the position was substituted by a
4-pyridyl group, the resulting compound 3o displayed an IC50
value of 3.0 nM against B-RafV600E, which is 10-times more
potent than vemurafenib in a parallel comparison. Compound
3o also displayed a 4-time stronger antiproliferative activity,
with an IC50 value of 78 nM against B-RafV600E mutated
Colo205 cancer cells and favorable selectivity with >10 μM IC50
value against HCT116 cancer cells with B-RafWT. It was
predicted an extra hydrogen bond between N atom of the 4-
pyridinyl group and the side chain of Ser536 may contribute to
this potency improvement (Figure 1C). The para-fluorophenyl
analogue 3s also displayed a slightly increased potency, whereas
the ortho-Cl or meta-Cl substituted compounds 3p and 3q are
8- and 7-fold less potent than 3n, respectively. Thus, 3o and 3s
were selected as the representatives for further investigations.
The antiproliferative activity of 3o and 3s were examined
against a panel of cancer cell lines and primary cancer cells
derived from New Zealand metastatic melanoma patients
harboring differing status of B-Raf.17,18 It was shown that 3s
displayed similar antiproliferative effects against B-RafV600E
mutated cancer cells to that of vemurafenib, whereas compound
3o is moderately more potent (Table 2). In contrast, none of
Table 1. Enzymatic and Cellular Activities of Compounds
3a−3s
Table 2. Antiproliferative Activities of the Compounds
against a Panel of Cancer Cells with Different Status of B-
Raf
antiproliferation (IC50, μM)
cell lines
3o
3s
1
a
Lovo
SK-MEL-2
>10
>10
>10
>10
>10
>10
>10
>10
8.33
a
ac
,
NZM09
NZM40
ac
,
4.44 1.09
4.35 1.31
3.01 0.31
b
HT29
0.263 0.02
0.202 0.07
0.075 0.03
0.046 0.02
0.002 0.001
0.384 0.07
0.448 0.07
0.257 0.11
0.101 0.05
0.023 0.004
0.024 0.006
0.601 0.08
1.499 0.04
0.381 0.15
0.079 0.02
0.024 0.003
b
SK-MEL-1
b
SK-MEL-28
b
A375
bc
,
NZM20
bc
,
NZM07
0.003 0.001
0.036 0.008
a
b
c
The cells harbor B-RafWT
.
The cells express B-Raf V600E. Primary
cell lines derived from New Zealand metastatic melanoma patients.
The IC50 values were determined using MTTa,b or sulforhodamine Bc
assay. Data are mean values
independent experiments.
standard deviation (SD) of three
the compounds showed obvious growth inhibition against cell
lines expressing B-RafWT. It is also noteworthy that the patient-
sourced melanoma cells with B-RafV600E mutation are more
sensitive to the inhibitors. Both 3o and 3s inhibit the growth of
NZM20 and NZM07 cells with low nanomolar IC50 values.
The B-Raf inhibition of 3o and 3s was further validated by
investigating their suppressive abilities on the activation of
MAPK signal pathway in NZM20 primary melanoma cancer
cells expressing B-RafV600E and the corresponding NZM40 cells
with B-RafWT (Figure 2). Both compounds displayed dose-
dependent inhibition against the phosphorylation of ERK in
a
Kinase activity assays were performed by a FRET-based Z′-Lyte
b
assay. The antiproliferative activities were evaluated using a MTT
assay. Data are means of three independent experiments, and the
variations are <20%.
also caused a significant loss of potency. Not surprisingly, 1H-
pyrazolo[3,4-b]pyridine hinge-binding moiety could be re-
C
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Letter
tumor growth inhibition (TGI) values are 47.3% and 77.1% at
dosages of 50 and 100 mg/kg/day, respectively. More
significantly, when the animals were treated with 100 mg/kg
of 3s twice a day, 3 out of 6 mice achieved significant tumor
regression, suggesting that sustained exposure of B-Raf
inhibitor may be an efficient approach to treat B-RafV600E
mutated human colorectal cancer.
In summary, a series of N-(3-ethynyl-2,4-difluorophenyl)-
sulfonamides were designed and synthesized as new selective B-
Raf inhibitors. The compounds potently inhibit B-RafV600E
kinase with low nanomolar IC50 values and selectively suppress
the proliferation of a panel of human cancer cell lines with B-
RafV600E mutation. One of the most promising compounds 3s
demonstrates favorable pharmacokinetic properties and induces
significant tumor regressions in a xenograft mouse model of B-
RafV600E mutated Colo205 human colorectal cancer cells
without significant sign of toxicity. This compound may serve
as a new lead compound for further drug discovery targeting B-
RafV600E mutation driven human cancers.
Figure 2. Compounds 3o and 3s dose-dependently inhibit the
activation of ERK in NZM20 cells with B-RafV600E (A,C) but elevate
WT
the pERK levels in NZM40 harboring B-Raf
(B,D).
NZM20 cancer cells by Western blot analysis. Alternately, an
elevation of p-ERK level was observed for the compounds in
NZM40 cells harboring B-RafWT and NRASN61H activating
mutation, suggesting that inhibition of B-RafWT in this cell line
may result in activation of a compensatory positive feedback
loop, leading to increased ERK p44/p42 phosphorylation, as
previously observed for vemurafenib, a phenomena that may be
related to the appearance of benign skin lesions.19 The MAPK
signal inhibition of the compounds was also validated in a pair
of human colorectal cancer cells including B-RafV600E mutated
Colo205 cells and HCT-116 cells with B-RafWT (Supporting
Information).
Compound 3s possesses favorable pharmacokinetic profiles
with high oral exposures (AUC(0‑∞) of over 30,000 μg/L·h and
F% of 54.0% at a 25 mg/kg oral dose) and acceptable half-life
(Supporting Information). It also displayed excellent target
specificity in a kinase profiling investigation against 468 kinases
with a selectivity score (S10) of 0.012 at 1.0 μM, which is about
48-fold of its Kd value against B-RafV600E (Supporting
Information).20
The in vivo antitumor efficacy of 3s was further evaluated
using a xenograft mouse model of Colo205 cancer cells. The
animals were repeatedly administrated with 3s once or twice
daily via oral gavage for 14 consecutive days. Vemurafenib was
used as a positive control (Figure 3). Compound 3s exhibited
dose-dependent antitumor efficacy and was well tolerated in all
of the tested groups with no mortality or significant loss of
body weight (<5% relative to the vehicle-matched controls)
observed during treatment (Supporting Information). The
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures for the syntheses, H NMR and 13C
1
NMR for final compounds, kinase selectivity, and details of in
vitro and in vivo assays. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*Tel: +86-20-32015276. Fax: +86-20-32015299. E-mail: ding_
ke@gibh.ac.cn.
*E-mail: ren_xiaomei@gibh.ac.cn.
Funding
We thank financial support from Ministry of Science and
Technology of China (#2014DFG32100), the National Natural
Science Foundation (#81425021, #21302186), and the Health
Research Council of New Zealand (13/1020).
Notes
The authors declare no competing financial interest.
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