Synthesis and biological evaluation of novel N-(5-phenyl-1H-pyrazol-3-yl)benzenesulfonamide derivatives as potential BRAFV600E inhibitors

Article abstract of DOI:10.1007/s00044-017-1957-z

A series of novel N-(5-phenyl-1H-pyrazol-3-yl)benzenesulfonamide derivatives (5a–5l) were synthesized and developed as potential BRAF^V600E inhibitors. Among them, compound 5l exhibited the most potent inhibitory activity with an IC₅₀

Full text of DOI:10.1007/s00044-017-1957-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1957-z
ORIGINAL RESEARCH
Synthesis and biological evaluation of novel N-(5-phenyl-1H-
pyrazol-3-yl)benzenesulfonamide derivatives as potential
V600E
BRAF
inhibitors
1 ●
1 ●
1 ●
1 ●
1 ●
Zhen-Hua Gong Jian Yao Jian-Feng Ji Jun Yang Tie Xiang
1
Chang-Kai Zhou
Received: 13 February 2017 / Accepted: 9 June 2017
©
Springer Science+Business Media, LLC 2017
Abstract A series of novel N-(5-phenyl-1H-pyrazol-3-yl)
molecule binds to its membrane receptor. One main effector
of RAS is the RAF family proteins, which is composed of
A-RAF, B-RAF, and C-RAF (also known as RAF-1) (Beck
et al. 1987; Huebner et al. 1986; Ikawa et al. 1988).
Phosphorylation of RAF stimulates its serine/threonine
activity, triggering and activating the protein kinases MEK
and ERK sequentially(Kolch 2000). Phosphorylated pro-
ducts of ERKs, such as ELK-1, are transcription factors
which directly or indirectly regulate gene expression,
cytoskeleton construction, cause prevention of apoptosis,
cell cycle arrest, and contribute to tumorigenesis (Wellbrock
et al. 2004). Therefore, kinase of this pathway such as RAF
can serve as a promising therapeutic target for anticancer
drug development.
In the RAF family proteins, because of B-RAF is easier
to be activated than A-RAF and C-RAF, it carries more
biochemical potencies(Guan et al. 2000). Currently, A
number of activating mutations of the BRAF gene have
been identified, but the most common B-RAF mutation in
human cancer is a single-base substitution at nucleotide
benzenesulfonamide derivatives (5a–5l) were synthesized
V600E
and developed as potential BRAF
inhibitors. Among
them, compound 5l exhibited the most potent inhibitory
V600E
activity with an IC50 value of 0.18 μM for BRAF
.
Antiproliferative assay results indicated that compound 5l
have higher antiproliferative activity against WM266.4 and
A375 in vitro with IC50 value of 1.58 and 2.04 μM,
respectively, which were comparable with the positive
control vemurafenib. Molecular docking of 5l into the
V600E
BRAF
active site was performed to determine the
probable binding mode.
V600E
●
●
Keywords BRAF
inhibitor Pyrazole Molecular
●
docking Antiproliferative
Introduction
600, which converts a valine to a glutamic acid (V600E;
formally identified as V599E) (Davies et al. 2002; Gray-
Schopfer et al. 2007). This single mutation leads to con-
stitutive activation of B-RAF and independent of RAS
function, resulting in unremitting proliferation of cancer
cells(Wan et al. 2004; Lee et al. 2007). Mutations in B-RAF
have been detected in approximately 8% of human tumors,
thyroid cancers(30–70%), melanomas(50%), serous low-
grade ovarian cancers(30%) and colorectal cancers(10%)
The signaling pathway RAS/RAF/MEK/ERK plays an
important role in the transduction of signals from cell sur-
face receptors to the nucleus, which regulates cell growth,
survival, differentiation and proliferation in response to
external stimuli (growth factors, cytokines or hormones)
(
Peyssonnaux and Eychène 2001; Wellbrock et al. 2004).
The RAS can be activated when an extracellular signal
(Tuveson et al. 2003; Garnett and Marais 2004; Li et al.
2
009; Tannapfel et al. 2003).
Much moleculars have been developed as BRAF inhi-
*
Jun Yang
junyang2016@126.com
bitors, such as SB-590885, AZ628, RAF265, and PLX4032
(vemurafenib) (Scheme 1) (Hwang et al. 2002; McDermott
et al. 2007; Imielinski et al. 2012; Hodi et al. 2010). Out of
1
Department of Burn and Plastic Surgery, The First People Hospital
of Nantong, Nantong 226001, Jiangsu Province, China

Med Chem Res
V600E
them, as a high selective BRAF
inhibitor, PLX4032
vemurafenib bind to the active pocket easily (Hodi et al.
2010).
Based on the statements above, a series of N-(5-phenyl-
1H-pyrazol-3-yl)benzenesulfonamide derivatives were
designed and synthesized. Upon biological evaluation,
can bind to the active conformation of the kinase pre-
ferentially (Hodi et al. 2010). It employs a common scaffold
that comprises a terminal aromatic group (phenyl or pyr-
azole) that fills the allosteric pocket created by the dis-
placement of the DFG loop and an amide linker which
couples an aryl group in the hydrophobic pocket to a hinge-
binding heterocycle. Linkage via an amide bond would
allow for rapid and efficient screening of several potential
hinge-binding groups (Suijkerbuijk et al. 2010).
Pyrazole and its derivatives are widely used in medicine
research because of their wide range of pharmacological
activities, such as antitumor, anti-inflammatory, anti-
bacterial, analgesic, fungistatic, and anti-hyperglycemic
some of the targeted compounds were found as potent
V600E
inhibitors of BRAF
. Docking simulation was per-
formed using the X-ray crystallographic structure of
V600E
BRAF
in complex with the most potent inhibitor to
explore the binding mode of the compound at the active
site.
Results and discussion
Chemistry
(
Ghorab et al. 2010; Liu et al. 2008; Li et al. 2012).
GDC-0879 containing the pyrazole skelecton is a potent
RAF small-molecule inhibitor that can inhibit BRAF
V600E
kinase activity with K of 0.13 nmol/L (Hoeflich et al.
The general synthesis of pyrazole derivatives is outlined in
Scheme 2. First of all, 2a, 2b were synthesized in tetra-
hydrofuran (THF) by treating p-substituted methyl benzoate
with acetonitrile using NaH as catalyst. Secondly, treat
compounds 2a, 2b in refluxing ethanol with hydrazine
i
2
009). A series of 5-phenyl-1H pyrazole derivatives were
V600E
reported and showed BRAF
et al. 2014). Also, sulfonamide fragment was employed in
inhibitory activity (Wang
V600E
another BRAF
inhibitor vemurafenib, which made the
Scheme 1 Chemical structures
of antitumor agents and lead
V600E
BRAF
inhibitors
Scheme 2 General synthesis of
N-(5-phenyl-1H-pyrazol-3-yl)
benzenesulfonamide derivatives
(
5a–5l). Reagents and
conditions: a CH CN, NaH,
THF, reflux, 4 h; b
NH NH ·H O, CH
3
2
2
2
3 3
SO H,
EtOH, reflux, 1 h; c TEA, DCM,
r.t., 6 h

Med Chem Res
hydrate for 1 h to get compounds 3a, 3b(Dong et al. 2013).
Lastly, the reaction between the obtained compounds 3a, 3b
and benzenesulfonyl chloride was performed in anhydrous
methylene dichloride in ice bath to get the desired com-
pounds 5a–5l. All synthetic compounds gave satisfactory
analytical and spectroscopic data which were in full
accordance with their depicted structures.
cell lines (WM266.4 and A375) showed better inhibitory
activity than wild type BRAF cell line (WM1361).
All new compounds (5a–5l) containing the N-(5-phenyl-
1H-pyrazol-3-yl)benzenesulfonamide structure were tested
V600E
for their BRAF
enzymatic activity using a solid-phase
enzyme-linked immunosorbent assay (ELISA) and the
results are presented in Table 1. All of the tested compounds
V600E
exhibited the potent BRAF
kinases inhibitory activ-
ities with IC50 values between 0.18 and 1.86 μM. Among
Antiproliferative activity and kinase inhibition assay
them, compound 5l displayed the most potent anti-
V600E
BRAF
kinase activity.
To test the anticancer activities of the synthesized com-
As shown in Table 1, structure–activity relationships
(SARs) of N-(5-phenyl-1H-pyrazol-3-yl)benzenesulfona-
mide derivatives demonstrated that compounds with
electron-donating substituents in para position of phenyl
ring A showed more potent inhibitory activities than that of
the electron-withdrawing substituents in the order of Me >
Br. What’s more, the substituents on ring B could also affect
the activities of compounds, when the substituents in phenyl
ring A was Br, the inhibitory activity of compounds with
pounds, we evaluated antiproliferative activities of com-
V600E
pounds 5a–5l against BRAF
cell lines (WM266.4 and
A375) and wild type BRAF cell line (WM1361). The
results are summarized in Table 1. The results exhibited that
N-(5-phenyl-1H-pyrazol-3-yl)benzenesulfonamide deriva-
tives owned remarkable antiproliferative effects on the
V600E
BRAF
cell lines. Out of the given compounds, it was
observed that compound 5l displayed the most potent
inhibitory activity (IC50 = 1.58 μM for WM266.4 and IC50
different substituents (R ) on ring B increased in the fol-
2
=
2.04 μM for A375) as compared to the positive control
lowing order: Br > OMe > Me > H > F > Cl; when the
substituents in phenyl ring A was Me, compounds (5a–5l)
with disubstituted groups on the ring B exhibited inhibitory
vemurafenib (IC50 = 0.05 μM for WM266.4 and IC50
=
V600E
0
.17 μM for A375, respectively). In addition, BRAF
V600E
Table 1 Structural features, inhibition (IC50) of WM266.4, A375 and WM1361 cells proliferation and inhibition (IC50) of BRAF
by
compounds 5a-5l
Compound
R
1
R
2
IC50 ± SD (μM)
IC50(μM)
V600E
WM266.4
A375
WM1361
BRAF
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
Br
H
8.82
17.44
21.03
3.81
14.32
30.87
27.62
5.44
25.56
42.18
43.91
17.34
29.48
21.37
39.63
33.14
14.88
15.68
43.42
10.12
1.64
1.39
1.81
1.86
0.61
1.06
0.93
1.62
1.44
0.32
0.87
1.79
0.18
0.06
Br
F
Br
Cl
Br
CH
Br
Br
3
7.73
8.19
Br
OCH
H
3
7.58
6.31
g
h
i
CH
CH
CH
CH
CH
CH
3
3
3
3
3
3
10.49
10.05
2.14
16.27
19.06
4.12
F
Cl
j
Br
5.97
7.67
k
l
CH
3
13.12
1.58
22.75
2.04
OCH
3
Vemurafenib
0.05
0.17

Med Chem Res
Fig. 1 Compound 5l induced apoptosis in WM266.4 cells with the concentration of 1, 2, and 4 μM. WM266.4 cells were treated for 24 h. Values
represent the mean ± S.D, n = 3. P < 0.05 vs. the control. The percentage of cells in each part is indicated
activity in the order of OMe > Cl > Br > F > H > Me.
Above all, 5l with p-Me group on the A-ring and p-OMe
group on the B-ring showed the most activity.
in Fig. 2, one oxygen atom on the sulfonyl with the
hydrogen atom on the main chain of Ser536 (angle
O–H···O = 134.8°, distance = 1.98 Å) contributes to the
hydrogen bonding interaction together, being a probable
explanation for its nice activity. Two π–π interactions are
formed between the A ring of the compound 5l and Trp531
Analysis of the apoptosis induced in cells
(
distance = 4.68 and 5.71 Å, respectively). One π–π inter-
To evaluate the biological ability of the compounds, we
performed an apoptosis experiment of compound 5l in the
WM266.4 cell line using Annexin-V and propidium iodide
action is formed between the A ring of the compound 5l and
Phe583 (distance = 4.52 Å). Meanwhile, the receptor sur-
face model showed in Fig. 2c revealed that this candidate
BRAF
binding pocket. This molecular docking result, along with
the biological assay data, suggested that compound 5l is a
potential inhibitor of BRAF
(
PI) double staining by flow cytometry and treating the
V600E
inhibitor was tightly embedded into the ATP
WM266.4 cells with 1, 2 and 4 μM of compound 5l for 24
h. The results can be seen in Fig. 1, and showed compound
5
l is very effective in the induction of apoptosis in a dose-
V600E
.
dependent manner. Moreover, the percentage of early
apoptotic cells from 13.3 to 21.4% was markedly elevated
in a dose-dependent manner, as compared to 9.5% of
apoptotic cells in the untreated control.
Experimental
Materials and methods
Molecular docking
All chemicals and reagents used in the current study were of
analytical grade. The reactions were monitored by thin-layer
chromatography on Merck pre-coated silica GF254 plates.
Melting points (uncorrected) were determined on a XT4MP
apparatus (Taike Corp., Beijing, China). ESI mass spectra
were obtained on a Mariner System 5304 mass spectro-
To gain better understanding of the SARs observed, mole-
cular docking of the most potent inhibitor 5l into the active
V600E
site cavity of BRAF
kinases was performed on the
V600E
binding model based on the BRAF
PDB code: 2FB8). The molecular docking was performed
by inserting compound 5l into the SB-590885 binding site
crystal structure
(
1
V600E
meter, and H NMR spectra were collected on a Bruker
of BRAF
. All docking runs were applied LigandFit
DPX400 spectrometer at room temperature with tetra-
methylsilane and solvent signals allotted as internal stan-
dards. Chemical shifts are reported in ppm (δ). Elemental
analyses were performed on a CHN-O-Rapid instrument,
and were within ± 0.4% of the theoretical values.
Dock protocol of Discovery Studio 4.0. The binding modes
V600E
of compound 5h and BRAF
are depicted in Fig. 2, and
the enzyme surface model is also shown in Fig. 2, which
revealed that the molecule is well filled in the active pocket.
The amino acid residues which had interaction with
V600E
BRAF
were labeled.
In the binding mode, compound 5l is nicely bound to
General procedure for the synthesis of 2a–2c
V600E
BRAF
via one hydrogen bond and three π–π interac-
tions. Meanwhile, there are also some hydrogen bonds and
π-cation interactions though they are weaker. As illustrated
p-Substituted methyl benzoate (7 mmol) and NaH (14
mmol, 0.35 g) in boiling THF (5 mL) were treated with

Med Chem Res
Fig. 2 Molecular docking modeling of compound 5l with BRAFV600E kinase. a 2D model of the interaction between compound 5l and the ATP
binding site. b 3D model of the interaction between compound 5l and the ATP binding site. c The receptor surface model with compounds 5l
dropwise addition of solution of acetonitrile (7 mmol, 0.29
g, 0.4 mL) in THF (1 mL). The mixture was heated to reflux
for 4 h and after cooling down to room temperature, it was
diluted with diethylether (15 mL) and left to stand at room
temperature for 48 h. The precipitated sodium salt was fil-
tered and washed with diethylether. The dry compound was
dissolved in water (5 mL) and acidified with HCl (1 mol/L)
to pH 2. The collected extracts were crystallized from
toluene, filtered and dried with Na SO .
General procedure for the synthesis of 3a–3c
To a stirred mixture of compound 2a–2c (5 mmol) in EtOH
(25 mL) were added NH NH ·H O (0.3 mL, 6 mmol) and
2
2
2
CH SO H (0.1 mL, 1 mmol) at room temperature and the
3
3
mixture was stirred under reflux for 1 h. The precipitate was
purified by column chromatograghy over silica gel to give
the compounds 3a–3c.
2
4

Med Chem Res
3401, 3284,
General procedure for synthesis of compounds 5a–5l
N-(5-(4-Bromophenyl)-1H-pyrazol-3-yl)-4-
bromobenzenesulfonamide (5d)
A solution of acyl chloride (0.5 mM) in dichloromethane
was added dropwise to compounds 3a or 3b (0.5 mM) in
dichloromethane containing Triethylamine (1 mM) at 0 °C
with constant stirring overnight. The reaction mixture was
then poured in excess of water and extracted with dichlor-
omethane.The extraction liquid was purified by a flash
chromatography with EtOAc/petroleum ether to give the
desired compounds 5a–5l.
Yield: 66%, m.p.: 191–193 °C; IR (KBr) v
max
1
1476, 1317, 1144; H NMR (400 MHz, DMSO): 5.86(s,
1H, ArH), 7.66–7.72(m, 4H, ArH), 7.83(m, 2H, ArH), 7.88
1
3
(m, 2H, ArH), 11.74(s, 1H, ArH), 12.79(s, 1H, ArH).
C
NMR (100 MHz, DMSO): 156.08, 152.07, 138.71, 133.87
(2), 132.91, 131.94(2), 130.12(2), 129.52(2), 126.37,
122.76, 87.12. MS (ESI): 458.22 (C H Br N O S, [M +
1
5
12
2 3 2
+
H] ). Anal. calcd for C H Br N O S: C, 39.41; H, 2.43;
1
5
11
2 3 2
N, 9.19%. Found: C, 39.42; H, 2.41; N, 9.18%.
N-(5-(4-Bromophenyl)-1H-pyrazol-3-yl)
benzenesulfonamide (5a)
N-(5-(4-Bromophenyl)-1H-pyrazol-3-yl)-4-
methylbenzenesulfonamide (5e)
Yield: 73%, m.p.: 178–180 °C; IR (KBr) v
3343, 3247,
max
1
1
486, 1307, 1142; H NMR (400 MHz, dimethyl sulfoxide
Yield: 71%, m.p.: 226–228 °C; IR (KBr) v
3371, 3194,
max
(
DMSO)): 5.86(s, 1H, ArH), 7.64(m, 2H, ArH), 7.73(m,
1
1474, 1308, 1146; H NMR (400 MHz, DMSO): 5.88(s,
2
H, ArH), 7.53–7.59(m, 2H, ArH), 7.82(m, 2H, ArH),
1
3
1H, ArH), 7.62(d, J = 6.86 Hz, 2H, ArH), 7.73(d, J = 6.86
Hz, 2H, ArH), 7.32(d, J = 7.76 Hz, 2H, ArH), 7.64(m, 2H,
ArH), 11.74(s, 1H, ArH), 12.83(s, 1H, ArH). C NMR
1
1.81(s, 1H, ArH), 12.84(s, 1H, ArH). C NMR (100
MHz, DMSO): 155.81, 151.42, 138.97, 134.12(2), 133.06,
32.39, 130.19(2), 129.43(2), 126.73(2), 122.71, 86.48. MS
1
3
1
+
(100 MHz, DMSO): 156.11, 152.11, 137.86, 137.17,
(
ESI): 379.26 (C H BrN O S, [M + H] ). Anal. calcd for
15 13 3 2
1
8
33.79(2), 132.89, 130.08(2), 129.66(2), 128.43(2), 122.61,
C H BrN O S: C, 47.63; H, 3.20; N, 11.11%. Found: C,
1
5
12
3 2
7.04, 22.14. MS (ESI): 393.28 (C H BrN O S, [M +
1
6
15
3 2
4
7.61; H, 3.24; N, 11.14%.
+
H] ). Anal. calcd for C H BrN O S: C, 48.99; H, 3.60;
1
6
14
3 2
N, 10.71%. Found: C, 49.02; H, 3.58; N, 10.73%.
N-(5-(4-Bromophenyl)-1H-pyrazol-3-yl)-4-
fluorobenzenesulfonamide (5b)
N-(5-(4-Bromophenyl)-1H-pyrazol-3-yl)-4-
methoxybenzenesulfonamide (5f)
Yield: 64%, m.p.: 192–194 °C; IR (KBr) vmax 3318, 3181,
1
1
1
462, 1311, 1148; H NMR (400 MHz, DMSO): 5.88(s,
Yield: 79%, m.p.: 235–237 °C; IR (KBr) v
3362, 3212,
max
H, ArH), 7.62(d, J = 6.72 Hz, 2H, ArH), 7.76(m, 2H,
1
1
1
2
1
1
1
486, 1317, 1152; H NMR (400 MHz, DMSO): 5.86(s,
ArH), 7.39(m, 2H, ArH), 7.88(m, 2H, ArH), 11.72(s, 1H,
H, ArH), 7.63(m, 2H, ArH), 7.72(m, 2H, ArH), 7.13(m,
H, ArH), 7.58(m, 2H, ArH), 11.76(s, 1H, ArH), 12.84(s,
1
3
ArH), 12.82(s, 1H, ArH). C NMR (100 MHz, DMSO):
65.41, 155.92, 151.47, 134.83, 133.91(2), 132.87, 130.18
2), 129.87(2), 122.84, 114.98(2), 87.14. MS (ESI): 397.31
1
13
H, ArH). C NMR (100 MHz, DMSO): 164.28, 156.07,
(
(
52.04, 133.67(2), 132.76, 132.18, 130.11(2), 126.41(2),
+
C H BrFN O S,
[M + H] ).
Anal.
calcd
for
1
5
12
3 2
22.54, 115.16(2), 87.07, 56.37. MS (ESI): 409.12
C H BrFN O S: C, 45.47; H, 2.80; N, 10.60%. Found: C,
+
1
5
11
3 2
(
C H BrN O S,
[M + H] ).
Anal.
calcd
for
1
6
15
3 3
4
5.51; H, 2.82; N, 10.58%.
C H BrN O S: C, 47.07; H, 3.46; N, 10.29%. Found: C,
1
6
14
3 3
4
7.05; H, 3.43; N, 10.31%.
N-(5-(p-tolyl)-1H-pyrazol-3-yl)benzenesulfonamide (5g)
Yield: 75%, m.p.: 164–166 °C; IR (KBr) v 3354, 3223,
N-(5-(4-Bromophenyl)-1H-pyrazol-3-yl)-4-
chlorobenzenesulfonamide (5c)
Yield: 78%, m.p.: 201–203 °C; IR (KBr) vmax 3356, 3214,
max
1
1
1
1
481, 1321, 1137; H NMR (400 MHz, DMSO): 5.86(s,
1476, 1311, 1147; H NMR (400 MHz, DMSO): 5.87(s,
1H, ArH), 7.12(d, J = 7.44 Hz, 2H, ArH), 7.52(m, 2H,
ArH), 7.56–7.61(m, 3H, ArH), 7.58(m, 2H, ArH), 11.76(s,
H, ArH), 7.61(m, 2H, ArH), 7.74(m, 2H, ArH), 7.67(d, J
=
8.12 Hz, 2H, ArH), 7.82(m, 2H, ArH), 11.69(s, 1H,
1
3
13
ArH), 12.81(s, 1H, ArH). C NMR (100 MHz, DMSO):
56.24, 151.51, 138.35, 137.98, 134.06(2), 132.98, 130.31
1H, ArH), 12.81(s, 1H, ArH). C NMR (100 MHz,
1
DMSO): 156.12, 153.12, 140.27, 132.19. 131.67, 130.14,
129.45(2), 129.18(2), 127.53(2), 125.87(2), 87.28, 22.13.
(
(
2), 129.84(2), 129.17(2), 122.74, 87.04. MS (ESI): 413.78
+
+
C H BrClN O S, [M + H] ). Anal. calcd for
MS (ESI): 314.08 (C H N O S, [M + H] ). Anal. calcd
1
5
12
3
2
16 16 3 2
C H BrClN O S: C, 43.66; H, 2.69; N, 10.18%. Found:
for C H N O S: C, 61.32; H, 4.82; N, 13.41%. Found: C,
1
5
11
3
2
16 15 3 2
C, 43.64; H, 2.71; N, 10.16%.
61.34; H, 4.83; N, 13.38%.

Med Chem Res
4
-Fluoro-N-(5-(p-tolyl)-1H-pyrazol-3-yl)
Anal. calcd for C H N O S: C, 62.36; H, 5.23; N,
17 17 3 2
benzenesulfonamide (5h)
12.83%. Found: C, 62.34; H, 5.21; N, 12.84%.
Yield: 69%, m.p.: 183–185 °C; IR (KBr) v
3348, 3198,
4-Methoxy-N-(5-(p-tolyl)-1H-pyrazol-3-yl)
benzenesulfonamide (5l)
max
1
1
1
3
1
1
1
481, 1313, 1138; H NMR (400 MHz, DMSO): 5.86(s,
H, ArH), 7.14(m, 2H, ArH), 7.61(m, 2H, ArH), 7.41(m,
H, ArH), 7.91(m, 2H, ArH), 11.77(s, 1H, ArH), 12.83(s,
Yield: 78%, m.p.: 229–231 °C; IR (KBr) v
3341, 3219,
max
1
3
1
H, ArH). C NMR (100 MHz, DMSO): 167.34, 156.08,
53.41, 134.94, 131.74, 131.27(2), 129.87, 129.67(2),
1483, 1312, 1141; H NMR (400 MHz, DMSO): 5.86(s,
1H, ArH), 7.19(d, J = 8.04 Hz, 2H, ArH), 7.68(m, 2H,
ArH), 7.15(m, 2H, ArH), 7.58(m, 2H, ArH), 11.76(s, 1H,
25.74(2), 116.17(2), 87.74, 22.07. MS (ESI): 332.32
+
13
(
C H FN O S,
[M + H] ).
Anal.
calcd
for
ArH), 12.81(s, 1H, ArH). C NMR (100 MHz, DMSO):
1
6
15
3 2
C H FN O S: C, 57.99; H, 4.26; N, 12.68%. Found: C,
164.28, 156.11, 153.71, 132.43, 131.66, 130.14, 130.11(2),
1
6
14
3 2
5
7.97; H, 4.27; N, 12.66%.
127.18(2), 125.87(2), 115.26(2), 87.68, 56.28, 22.04. MS
+
(
ESI): 328.24 (C H N O S, [M + H] ). Anal. calcd for
1
7 18 3 3
4
-Chloro-N-(5-(p-tolyl)-1H-pyrazol-3-yl)
C H N O S: C, 59.46; H, 4.99; N, 12.24%. Found: C,
17 17 3 3
benzenesulfonamide (5i)
59.48; H, 4.98; N, 12.26%.
Yield: 67%, m.p.: 197–198 °C; IR (KBr) v
3346, 3207,
Antiproliferation activity
max
1
1
1
478, 1304, 1144; H NMR (400 MHz, DMSO): 5.86(s,
H, ArH), 7.18(d, J = 6.84 Hz, 2H, ArH), 7.59(m, 2H,
WM1361, WM266.4 and A375 cell lines were cultured in
ArH), 7.64(d, J = 7.12 Hz, 2H, ArH), 7.83(m, 2H, ArH),
DMEM/10% fetal bovine serum, in 5% CO water saturated
2
1
3
1
1.78(s, 1H, ArH), 12.86(s, 1H, ArH). C NMR (100
atmosphere at 37 °C. Cell suspensions (10,000/mL) were
prepared and 100 mL/well dispensed into 96-well plates
(Costar) giving 1000 cells/well. The plates were returned to
the incubator for 24 h to allow the cells to reattach. Tested
samples at pre-set concentrations were added to six wells
with vemurafenib as positive references. After 48 h expo-
sure period, 40 μL of PBS containing 2.5 mg/mL of MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bro-
mide)) was added to each well. Four hours later, 100 μL
extraction solution (10% sodium dodecyl sulfate–5% iso-
butyl alcohol–0.01 M HCl) was added. After an overnight
incubation at 37 °C, the optical density was measured at a
wavelength of 570 nm on an ELISA microplate reader. In
all experiments three replicate wells were used for each
drug concentration. Each assay was carried out for at least
three times.
MHz, DMSO): 155.97, 153.34, 138.18, 138.15, 131.46,
1
2
30.08, 129.64(2), 129.54(2), 129.11(2), 125.71(2), 87.61,
+
1.94. MS (ESI): 348.73 (C H ClN O S, [M + H] ).
1
6
15
3 2
Anal. calcd for C H ClN O S: C, 55.25; H, 4.06; N,
1
6
14
3 2
1
2.08%. Found: C, 55.27; H, 4.04; N, 12.07%.
4
-Bromo-N-(5-(p-tolyl)-1H-pyrazol-3-yl)
benzenesulfonamide (5j)
Yield: 62%, m.p.: 201–204 °C; IR (KBr) v
3324, 3186,
max
1
1
1
469, 1318, 1134; H NMR (400 MHz, DMSO): 5.88(s,
H, ArH), 7.14(m, 2H, ArH), 7.62(d, J = 8.04 Hz, 2H,
ArH), 7.85(m, 2H, ArH), 7.87(m, 2H, ArH), 11.74(s, 1H,
1
3
ArH), 12.82(s, 1H, ArH). C NMR (100 MHz, DMSO):
56.14, 153.41, 139.27, 132.29(2), 131.68, 130.11, 129.87
2), 129.58(2), 127.13, 125.84(2), 87.54, 21.98. MS (ESI):
1
(
3
+
93.21 (C H BrN O S, [M + H] ). Anal. calcd for
Kinase assay
1
6
15
3 2
C H BrN O S: C, 48.99; H, 3.60; N, 10.71%. Found: C,
1
6
14
3 2
4
8.98; H, 3.62; N, 10.78%.
The BRAF Kinase inhibitory activity was measured using
previously methods described by J. Dietrich et al. (Dong
et al. 2013). The assay was performed in triplicate for each
4
-Methyl-N-(5-(p-tolyl)-1H-pyrazol-3-yl)
benzenesulfonamide (5k)
tested compound in this study. Briefly, 7.5 ng Mouse Full-
V600E
Length GST-tagged BRAF
(Invitrogen, PV3849) was
Yield: 80%, m.p.: 224–226 °C; IR (KBr) vmax 3337, 3241,
pre-incubated at room temperature for 1 h with 1 μL drug
and 4 μL assay dilution buffer. The kinase assay was initi-
ated when 5 μL of a solution containing 200 ng recombinant
human full length, N-terminal His-tagged MEK1 (Invitro-
1
1
1
471, 1311, 1139; H NMR (400 MHz, DMSO): 5.87(s,
H, ArH), 7.20(d, J = 7.76 Hz, 2H, ArH), 7.67(m, 2H,
ArH), 7.38(d, J = 7.92 Hz, 2H, ArH), 7.64(m, 2H, ArH),
1
3
1
1.75(s, 1H, ArH), 12.84(s, 1H, ArH). C NMR (100
gen), 200 μM ATP (0.8 μCi hot ATP), and 30 mM MgCl in
2
MHz, DMSO): 156.08, 153.61, 138.16, 137.27, 131.87,
assay dilution buffer was added. The kinase reaction was
allowed to continue at room temperature for 25 min and was
then quenched with 5 μL 5 × protein denaturing buffer
1
2
30.41(2), 130.28, 130.02(2), 129.33(2), 125.93(2), 87.74,
+
2.13, 21.83. MS (ESI): 328.24 (C H N O S, [M + H] ).
1
7 18 3 2

Med Chem Res
(
lithium dodecyl sulfate) solution. Protein was further
cell line. Among them, compound 5l demonstrated the most
V600E
denatured by heating for 5 min at 70 °C. 10 μL of 20 each
reaction was loaded into a 15-well, 4–12% precast NuPage
gel (Invitrogen) and run at 200 V, and upon completion, the
front, which contained excess hot ATP, was cut from the gel
and discarded. The gel was then dried and developed onto a
phosphor screen. A reaction that contained no active
enzyme was used as a negative control, and a reaction
without inhibitor was used as the positive control.
potent BRAF
inhibition activities (IC50 = 0.18 μM for
V600E
BRAF
) and antiproliferative activities (IC50 = 1.58
μM for WM266.4 and IC50 = 2.04 μM for A375). Docking
simulation was performed to position compound 5l into the
V600E
BRAF
protein active site to determine the potential
binding model, and the result shows compound 5l can bind
well with the BRAF active site and act as BRAF inhibitor.
In general, compound 5l along with other N-(5-phenyl-1H-
pyrazol-3-yl)benzenesulfonamide derivatives could be act
as potential anticancer agents.
Flow cytometry
5
Acknowledgements The authors are very much grateful to Nantong
University, for providing laboratory facility.
Approximately 10 cells per well were plated in a 24-well
plate and allowed to adhere. After 12 h, the medium was
replaced with fresh culture medium containing compounds
l at final concentrations of 1, 2 and 4 μM. Nontreated wells
Compliance with ethical standards
5
received an equivalent volume of ethanol (<0.1%). To
determine the optimal time course for the induction of
apoptosis, cells were harvested after 24 h. They were tryp-
sinized, washed with PBS and centrifuged at 2000 rpm for
Conflict of interest The authors declare that they have no competing
interests.
5
min. The pellet was then resuspended in 500 μL of
References
staining solution (containing 1 μL Annexin V-PE and 5 μL
PI in binding buffer), mixed gently and incubated for 15
min at room temperature (15–25 °C) in the dark. The
samples were then read in a FACSCalibur flow cytometer
Beck TW, Huleihel M, Gunnell M, Bonner TI, Rapp UR (1987) The
complete coding sequence of the human A-raf-1 oncogene and
transforming activity of a human A-raf carrying retrovirus.
Nucleic Acids Res 15:595–609
(
USA) at 488 nm excitation. A 578 nm band pass filter for
Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague
J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E,
Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kos-
midou V, Menzies A, Mould C, Parker A, Stevens C, Watt S,
Hooper S, Wilson R, Jayatilake H, Gusterson BA, Cooper C,
Shipley J, Hargrave D, Pritchard-Jones K, Maitland N, Chenevix-
Trench G, Riggins GJ, Bigner DD, Palmieri G, Cossu A, Fla-
nagan A, Nicholson A, Ho JW, Leung SY, Yuen ST, Weber BL,
Seigler HF, Darrow TL, Paterson H, Marais R, Marshall CJ,
Wooster R, Stratton MR, Futreal PA (2002) Mutations of the
BRAF gene in human cancer. Nature 417:949–954
Dong J, Li Q, Wang S, Li C, Zhao X, Qiu H et al. (2013) Synthesis,
biological evaluation and molecular docking of novel 5-phenyl-1
H-pyrazol derivatives as potential BRAF V600E inhibitors. Org
Biomol Chem 11:6328–6337
Garnett MJ, Marais R (2004) Guilty as charged: B-RAF is a human
oncogene. Cancer Cell 6:313–319
fluorescein detection and a > 600 nm filter for PI detection
were used. Analyses were performed using the software
supplied in the instrument.
Docking simulations
V600E
The crystal structures of BRAF
(PDB code: 2FB8)
was obtained from the Protein Data Bank (http://www.rcsb.
org). Molecular docking of compound 5h into the three-
V600E
dimensional X-ray structure of BRAF
was carried out
using CDOCKER Dock protocol of Discovery Studio 3.5.
All bound water and ligands were eliminated from the
protein and the polar hydrogen was added. The whole
V600E
BRAF
complex was defined as a receptor and the site
Ghorab MM, Ragab FA, Alqasoumi SI, Alafeefy AM, Aboulmagd SA
(2010) Synthesis of some new pyrazolo [3, 4-d] pyrimidine
derivatives of expected anticancer and radioprotective activity.
Eur J Med Chem 45:171–178
Gray-Schopfer V, Wellbrock C, Marais R (2007) Melanoma biology
and new targeted therapy. Nature 445:851–857
sphere was selected based on the ligand binding location of
SB-590885, then the SB-590885 molecule was removed
and 5h was placed during the molecular docking procedure.
Guan KL, Figueroa C, Brtva TR, Zhu T, Taylor J, Barber TD, Vojtek
AB (2000) Negative regulation of the serine/threonine kinase B-
Raf by Akt. J Biol Chem 275:27354–27359
Conclusions
Hodi FS, O’Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen
JB et al. (2010) Improved survival with ipilimumab in patients
with metastatic melanoma. N Engl J Med 363:711–723
Hoeflich KP, Herter S, Tien J, Wong L, Berry L, Chan J et al. (2009)
Antitumor efficacy of the novel RAF inhibitor GDC-0879 is
predicted by BRAFV600E mutational status and sustained
extracellular signal-regulated kinase/mitogen-activated protein
kinase pathway suppression. Cancer Res 69:3042–3051
In conclusion, a series of novel N-(5-phenyl-1H-pyrazol-3-
yl)benzenesulfonamide derivatives have been synthesized
and evaluated their biological activities. The results showed
that most of desired compounds exhibited good inhibitory
V600E
BRAF
activities, and owned the potent anti-
proliferative activity against WM266.4 cell line and A375

Med Chem Res
Huebner K, Griffin CA, Isobe M, Kozak C, Emanuel BS, Nagarajan L
et al. (1986) Actively transcribed genes in the raf oncogene
group, located on the X chromosome in mouse and human. Proc
Natl Acad Sci USA 83:3934–3938
Hwang K, Park K, Lee C, Kim B (2002) Novel benzoylurea deriva-
tives as potential antitumor agents; synthesis, activities and
structure-activity relationships. Arch Pharm Res 25:
substituted-4, 5-dihydropyrazol-1-yl) ethanone oxime ester deri-
vatives. Bioorg Med Chem 16:4075–4082
McDermott U, Sharma SV, Dowell L, Greninger P, Montagut C,
Lamb J et al. (2007) Identification of genotype-correlated sensi-
tivity to selective kinase inhibitors by using high-throughput
tumor cell line profiling. Proc Natl Acad Sci 104:19936–19941
Peyssonnaux C, Eychène A (2001) The Raf/MEK/ERK pathway: new
concepts of activation. Biol Cell 93:53–62
7
81–785
Ikawa S, Fukui M, Ueyama Y, Tamaoki N, Yamamoto T, Toyoshima
K (1988) B-raf, a new member of the raf family, is activated by
DNA rearrangement. Mol Cell Biol 8:2651–2654
Suijkerbuijk BM, Niculescu-Duvaz I, Gaulon C, Dijkstra HP,
Niculescu-Duvaz D, Ménard D et al. (2010) Development of
novel, highly potent inhibitors of V-RAF murine sarcoma viral
oncogene homologue B1 (BRAF): increasing cellular potency
through optimization of a distal heteroaromatic group. J Med
Chem 53:2741–2756
Tannapfel A, Sommerer F, Benicke M, Katalinic A, Uhlmann D,
Witzigmann H et al. (2003) Mutations of the BRAF gene in
cholangiocarcinoma but not in hepatocellular carcinoma. Gut
52:706–712
Imielinski M, Cha S, Rejtar T, Richardson EA, Karger BL, Sgroi DC
(
2012) Integrated proteomic, transcriptomic, and biological net-
work analysis of breast carcinoma reveals molecular features of
tumorigenesis and clinical relapse. Mol Cell Proteom 11:
M111–14910
Kolch W (2000) Meaningful relationships: the regulation of the Ras/
Raf/MEK/ERK pathway by protein interactions. Biochem J
3
51:289–305
Tuveson DA, Weber BL, Herlyn M (2003) BRAF as a potential
therapeutic target in melanoma and other malignancies. Cancer
Cell 4:95–98
Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM
et al. (2004) Mechanism of activation of the RAF-ERK signaling
pathway by oncogenic mutations of B-RAF. Cell 116:855–867
Wang S, Zhu Y, Zhu P, Makawana JA, Zhang Y, Zhao M, Lv P, Zhu
H (2014) Design, synthesis and biological evaluation of novel 5-
phenyl-1H-pyrazole derivatives as potential BRAF V600E inhi-
bitors. Bioorg Med Chem 22:6201–6208
Lee JH, Lee ES, Kim YS (2007) Clinicopathologic significance of
BRAF V600E mutation in papillary carcinomas of the thyroid.
Cancer 110:38–46
Li Q, Li C, Lu X, Zhang H, Zhu H (2012) Design, synthesis and
biological evaluation of novel (E)-α-benzylsulfonyl chalcone
derivatives as potential BRAF inhibitors. Eur J Med Chem
5
0:288–295
Li Y, Nakamura M, Kakudo K (2009) Targeting of the BRAF gene in
papillary thyroid carcinoma (Review). Oncol Rep 22:671–681
Liu X, Cui P, Song B, Bhadury PS, Zhu H, Wang S (2008) Synthesis,
structure and antibacterial activity of novel 1-(5-substituted-3-
Wellbrock C, Karasarides M, Marais R (2004) The RAF proteins take
centre stage. Nat Rev Mol Cell Bio 5:875–885