Design, synthesis and biological evaluation of novel 5-phenyl-1H-pyrazole derivatives as potential BRAFV600E inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.08.029

A series of novel 5-phenyl-1H-pyrazole derivatives (5a-5u) containing niacinamide moiety were synthesized and evaluated for biological activity as potential BRAF^V600E inhibitors. Among them, compound 5h exhibited the most potent inhibitory activity with an IC₅₀ value of 0.33 μM for BRAF^V600E. Antiproliferative assay results indicated that compound 5h has better antiproliferative activity against WM266.4 and A375 in vitro with IC₅₀ value of 2.63 and 3.16 μM, respectively, being comparable with the positive control vemurafenib. Molecular docking of 5h into the BRAF^V600E active site was performed to determine the probable binding mode. Furthermore, molecular docking and 3D QSAR study by means of DS 3.5 (Discovery Studio 3.5, Accelrys, Co. Ltd) explored the binding modes and the structure and activity relationship (SAR) of these derivatives.

Full text of DOI:10.1016/j.bmc.2014.08.029

Bioorganic & Medicinal Chemistry 22 (2014) 6201–6208
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of novel
5-phenyl-1H-pyrazole derivatives as potential BRAFV600E inhibitors
Shu-Fu Wang , Yin-Ling Zhu , Ping-Ting Zhu, Jigar A. Makawana, Ya-Liang Zhang, Meng-Yue Zhao,
⇑
⇑
Peng-Cheng Lv , Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 June 2014
Revised 31 July 2014
Accepted 21 August 2014
Available online 6 September 2014
A series of novel 5-phenyl-1H-pyrazole derivatives (5a–5u) containing niacinamide moiety were synthe-
sized and evaluated for biological activity as potential BRAFV600E inhibitors. Among them, compound 5h
exhibited the most potent inhibitory activity with an IC50 value of 0.33
assay results indicated that compound 5h has better antiproliferative activity against WM266.4 and A375
l
M for BRAFV600E. Antiproliferative
in vitro with IC50 value of 2.63 and 3.16 lM, respectively, being comparable with the positive control vemu-
rafenib. Molecular docking of 5h into the BRAFV600E active site was performed to determine the probable
binding mode. Furthermore, molecular docking and 3D QSAR study by means of DS 3.5 (Discovery Studio
3.5, Accelrys, Co. Ltd) explored the binding modes and the structure and activity relationship (SAR) of these
derivatives.
Keywords:
BRAFV600E inhibitor
Pyrazole
Niacinamide
Molecular docking
3D-QSAR
Ó 2014 Published by Elsevier Ltd.
1. Introduction
human cancer is a single base substitution at nucleotide 600 which
converts a valine to a glutamic acid (V600E; formally identified as
The RAS–RAF–MEK–ERK signaling pathway plays a critical role
in human oncogenesis.1–3 It normally acts as a medium to trans-
duct signals from cell surface receptors to the nucleus and give rise
to the expression of genes that regulate cell growth, survival and
differentiation.2 The RAS will be activated when an extracellular
signal molecule binds to its membrane receptor. One main effector
of RAS is the RAF family proteins, a series of cytoplasmic serine/
threonine protein kinases, which is composed of A-RAF, B-RAF,
and C-RAF (also known as RAF-1).4–7 It mediates intracellular sig-
nal transduction by the phosphorylation and activation of MEKs
after which the downstream kinases, extracellular signal-regulated
kinases (ERKs), were triggered.8 Phosphorylated products of ERKs
are some transcription factors which directly or indirectly regulate
gene expression, cytoskeleton construction, cause prevention of
apoptosis, cell cycle arrest and contribute to tumorigenesis. There-
fore, kinase of this pathway such as RAF can serve as a promising
therapeutic target for anticancer drug development.
V599E).10 This single mutation leads to constitutive activation of
B-RAF, independent of RAS function, resulting in unremitting prolif-
eration of cancer cells.11 Mutations in B-RAF have been detected in
approximately 8% of human tumors, melanomas (50%), thyroid
cancers (30–70%), serous low-grade ovarian cancers (30%) and
colorectal cancers (10%).11,12
Inhibitors targeting BRAF have been developed such as
SB-590885, AZ628, RAF265 and PLX4032 (vemurafenib)13–16
(Scheme 1). Among of these, PLX4032 is a highly specific BRAFV600E
inhibitor that binds preferentially to the active conformation of the
kinase.17 It employs a common scaffold that includes a meta or
para substituted aromatic system inserting into the lipophilic
DFG-out pocket and a terminal aromatic group (phenyl or pyra-
zole) that fills the allosteric pockets created by the displacement
of the DFG loop. A linker is needed to interact with the salt bridge
formed between residues Glu501 and Lys483.18
Pyrazole derivatives are widely used in medicine research for
their officinal potencies such as antitumor, anti-inflammatory,
antibacterial, analgesic, fungistatic, and anti-hyperglycemic.19–22
GDC-0879 contained the pyrazole ring is a highly selective and
potent RAF small-molecule inhibitor that can inhibit BRAFV600E
kinase activity with Ki of 0.13 nM.23 In addition, nicotinamide,
which has various pharmacological effects when doses in excess
of nutritional requirements are given, is known as a radiation sen-
sitizer and used clinically in cancer therapy.24,25 Its analogues were
Among the RAF family proteins, B-RAF has more biochemical
potencies than A-RAF and C-RAF because it is easier to be activated.9
Up to now, a great number of activating mutations have been
detected in B-RAF gene, but the most common B-RAF mutation in
⇑
Corresponding authors. Tel.: +86 25 83592572; fax: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.08.029
0968-0896/Ó 2014 Published by Elsevier Ltd.

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S.-F. Wang et al. / Bioorg. Med. Chem. 22 (2014) 6201–6208
N
O
N
N
N
HN
N
O
O
NH
N
H
N
N
HO
AZ628
N
SB-590885
F
H
N
Cl
O
O
N
S
O
O
N
F3C
F
H
N
N
H
N
N
H
N
CF3
PLX4032
RAF265
Scheme 1. Chemical structures of antitumor agents and lead BRAFV600E inhibitors.
found to induce an efficient and specific inactivation of the exoge-
nous transforming genes such as RAF.26 Recently, compounds con-
tained niacinamide moiety have been synthesized and showed
greater BRAFV600E inhibitory activity.27,28
catalyst. Secondly, compounds 2a–2c and hydrazine hydrate were
dissolved in ethanol and refluxed for 1 h to get compounds 3a–3c.
Lastly, the coupling reaction between the obtained compounds
3a–3c and nicotinic acids was performed by using 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDCl) and
N-hydroxybenzotriazole (HOBt) in anhydrous methylene dichlo-
ride under reflux condition to get the desired compounds 5a–5u.
All synthesized compounds were given satisfactory analytical and
spectroscopic data which were in full accordance with their
depicted structures.
Recent years, our group have reported a number of novel
pyrazol derivatives as potent inhibitors targeting BRAF and some
of them showed potent antitumor activity.29–31 Based on the state-
ments above and to extend our research, a series of 5-phenyl-1H-
pyrazole derivatives contained niacinamide moiety were designed
and synthesized. Upon biological evaluation, some of the synthe-
sized compounds were found as potent inhibitors of BRAFV600E
.
Docking simulation was performed using the X-ray crystallo-
graphic structure of BRAFV600E in complex with the most potent
inhibitor to explore the binding mode of the compound at the
active site. Furthermore, QSAR model was built to study the struc-
ture–activity relationship that guides the further study.
2.2. Biological activity
2.2.1. Antiproliferative activity
To test the anticancer activities of the synthesized compounds,
we evaluated antiproliferative activities of compounds 5a–5u
against BRAFV600E cell lines (WM266.4 and A375) and wild type
BRAF cell line (WM1361). The results were summarized in Table 1.
5-Phenyl-1H-pyrazole derivatives beared niacinamide moiety
showed remarkable antiproliferative effects on the BRAFV600E cell
lines. Among them, compound 5h displayed the most potent inhib-
2. Results and discussion
2.1. Chemistry
itory activity (IC50 = 2.63
for A375) which was compared to the positive control
vemurafenib (IC50 = 0.06 lM for WM266.4 and IC50 = 0.19 lM for
lM for WM266.4 and IC50 = 3.16 lM
The general synthesis of pyrazol derivatives is outlined in
Scheme 2. First of all, 2a–2c were synthesized in THF by treating
p-substituted methyl benzoate with acetonitrile using NaH as
R1
O
O
CN
a
b
O
NH2
HN
N
R1
R1
3a-3c
2a-2c
1a-1c
R1
R1
R2
N
A
COOH
c
B
NH
R2
NH2
HN
N
N
HN
N
O
4a-4g
3a-3c
5a-5w
Scheme 2. General synthesis of 5-phenyl-1H-pyrazole derivatives (5a–5u). Reagents and conditions: (a) CH3CN, NaH, THF, reflux, 4 h; (b) NH2NH2ÁH2O, CH3SO3H, EtOH,
reflux, 1 h; (c) EDCl, HOBt, dichloromethane, reflux, 1 h.

S.-F. Wang et al. / Bioorg. Med. Chem. 22 (2014) 6201–6208
6203
Table 1
Structure activity relationships of 5-phenyl-1H-pyrazole deriv-
atives demonstrated that compounds with para electron-donating
substituents on phenyl ring A showed more potent inhibitory
activities than those with electron-withdrawing substituents and
the potency order is Me > H > Br. In the case of constant A ring sub-
stituents, change of substituents on B-ring could also affect the
activities of compounds and the inhibitory activities of compounds
with different substituents on ring B increased in the following
order: 2-OMe < 6-OMe < 6-Br < 5-Br < 6-Cl < 2-Cl < 2-F. This result
indicated that the strong electronic-withdrawing substituents on
phenyl ring B were beneficial for the activity. Among all the
compounds, 5h with p-Me group on the A-ring and 2-F group on
the B-ring showed the best activity.
Structural features, inhibition (IC50) of WM266.4, A375 and WM1361 cells prolifer-
ation by compounds 5a–5u
Compound
R1
R2
IC50 SD (
lM)
WM266.4a
A375a
WM1361b
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
H
H
H
H
H
H
H
Br
Br
Br
Br
Br
Br
Br
2-F
2-Cl
2-OCH3
5-Br
6-Br
6-Cl
6-OCH3
2-F
2-Cl
2-OCH3
5-Br
3.77 0.75
6.22 0.56
8.75 0.32
6.88 0.86
6.73 1.02
6.43 0.55
8.48 0.58
2.63 0.84
5.35 0.36
7.76 0.45
6.45 0.48
6.78 0.62
5.72 0.26
7.35 0.37
4.68 1.22
6.99 0.54
4.88 0.74 14.55 0.44
7.34 0.85 17.34 0.56
9.94 1.02 25.77 0.27
8.12 1.24 19.28 0.68
8.87 0.98 19.43 0.66
7.85 0.85 18.98 0.31
9.56 0.64 24.65 0.46
3.16 0.84 13.25 0.55
6.44 0.75 15.77 0.68
8.73 0.35 25.78 0.34
7.05 0.49 17.87 0.25
7.67 0.73 18.56 0.64
6.88 0.55 16.74 0.39
8.42 0.64 24.86 0.41
5.42 0.28 15.66 0.68
8.22 0.37 18.45 0.46
11.12 0.64 30.98 1.05
9.42 0.56 20.34 0.34
9.91 0.24 20.56 1.22
8.89 0.29 19.33 0.65
8.95 0.69 28.45 0.94
The results of BRAFV600E kinases inhibitory activities of the test
compounds were corresponding to the structure activity relation-
ships (SAR) of their antitumor activities. It demonstrated that the
potent antitumor activities of the synthesized compounds were
probably correlated to their BRAFV600E kinases inhibitory activities.
6-Br
6-Cl
6-OCH3
CH3 2-F
CH3 2-Cl
CH3 2-OCH3 10.25 0.62
CH3 5-Br
CH3 6-Br
CH3 6-Cl
CH3 6-OCH3
5q
5r
5s
5t
2.3. Molecular docking
8.23 0.27
8.56 0.88
7.47 0.71
9.12 0.24
0.06 0.008
To gain better understanding of the structure–activity relation-
ships observed, molecular docking of the most potent inhibitor 5h
into the active site cavity of BRAFV600E kinases was performed on
the binding model based on the BRAFV600E crystal structure (PDB
code: 2FB8). The molecular docking was performed by inserting
compound 5h into the SB-590885 binding site of BRAFV600E. All
docking runs were applied LigandFit Dock protocol of Discovery
Studio 3.5. The binding modes of compound 5h and BRAFV600E
were depicted in Figure 1, and the enzyme surface model was
shown in Figure 2, which revealed that the molecule is well filled
in the active pocket. The amino acid residues which had interaction
with BRAFV600E were labeled.
5u
Vemurafenibc
0.19 0.02
1.87 0.15
a
b
c
BRAFV600E cell lines.
Wild type BRAF cell line.
Used as a positive control.
A375, respectively). Also, the compounds showed better inhibitory
activity to BRAFV600E cell lines (WM266.4 and A375) than to wild
type BRAF cell line (WM1361).
2.2.2. BRAF inhibitory activity
In the binding mode, compound 5h is nicely bound to BRAFV600E
via one hydrogen bond and three p–p interactions. As illustrated in
Figure 1, one fluorine atom on the nicotinamide ring and the
hydrogen atom on the main chain of Thr529 (angle N–HÁ Á ÁF =
129.2°, distance = 2.41 Å) contribute to the hydrogen bonding
interaction together, being a probable explanation for its nice
The BRAFV600E kinases inhibitory potency of the synthesized
pyrazol derivatives was examined using a solid-phase ELISA assay
and the results were summarized in Table 2. All of the tested com-
pounds showed potent BRAFV600E kinases inhibitory activities with
IC50 values between 0.33 and 1.88
lM. Among them, compound 5h
displayed the most potent anti-BRAFV600E kinase activity with IC50
activity. One
of the compound 5h and Trp531. Two
p
–
p
interaction is formed between the benzene rings
interactions are formed
of 0.33 lM.
p–p
between the pyrazolyl ring of the compound 5h and Trp531 and
Phe583 respectively. Meanwhile, the receptor surface model
showed in Figure 2 revealed that this candidate BRAFV600E inhibitor
was tightly embedded into the ATP binding pocket. This molecular
docking result, along with the biological assay data, suggested that
Table 2
BRAFV600E inhibitory activity of synthetic compounds
Compound
R1
R2
IC50 SD (lM)
compound 5h is a potential inhibitor of BRAFV600E
.
5a
H
2-F
0.56 0.63
0.75 0.55
1.54 0.36
0.98 0.64
1.21 0.27
0.84 0.82
1.50 0.23
0.33 0.35
0.69 0.44
1.32 0.52
0.82 0.36
0.86 0.67
0.77 0.64
1.15 0.31
0.78 0.44
0.84 0.24
1.88 0.87
0.95 0.71
1.02 0.33
0.85 0.61
1.79 0.94
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
H
H
H
H
H
H
Br
Br
Br
Br
Br
Br
2-Cl
2-OCH3
5-Br
6-Br
6-Cl
6-OCH3
2-F
2-Cl
2-OCH3
5-Br
6-Br
6-Cl
6-OCH3
2-F
2-Cl
2-OCH3
5-Br
6-Br
6-Cl
6-OCH3
2.3.1. 3D-QSAR
In order to acquire a systematic SAR profile on the synthesized
compounds and to explore the more powerful and selective
BRAFV600E inhibitors, 3D-QSAR model was built to choose activity
conformation of the designed molecular and reasonably evaluated
the designed molecules by using the corresponding pIC50 values
which were converted from the obtained IC50 (lM) values of
BRAFV600E inhibition and performed by built-in QSAR software of
DS 3.5 (Discovery Studio 3.5, Accelrys, Co. Ltd). By convention,
the pIC50 scale (ÀlogIC50), in which higher values indicate expo-
nentially greater potency, is used as a method to measure inhibi-
tory activity. The training and test set was chosen by the Diverse
Molecules method in Discovery Studio. Considering a good align-
ment is very important for the analysis of molecular fields, we
applied CDOCKER protocol to explore each molecule with lowest
energy before alignment conformation. 5-Phenyl-1H-pyrazole
was selected as substructure to build alignment conformation
before building the QSAR model.
Br
CH3
CH3
CH3
CH3
CH3
CH3
CH3
5r
5s
5t
5u
Vemurafeniba
0.03 0.005
a
Used as a positive control.

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S.-F. Wang et al. / Bioorg. Med. Chem. 22 (2014) 6201–6208
Table 3
Experimental, predicted inhibitory activity of compounds 5a–5u by 3D-QSAR models
based upon active conformation achieved by molecular docking
Compounda
BRAFV600E
Predicted pIC50
Residual error
Actual pIC50
5a
5b
5c
6.252
6.125
5.812
6.009
6.157
6.158
5.863
6.026
0.095
À0.033
À0.051
À0.017
5d
5e
5f
5g
5h
5.917
6.076
5.824
6.481
6.161
5.956
5.977
5.783
6.463
6.176
À0.039
0.099
0.041
0.018
À0.015
À0.021
À0.060
0.064
0.009
0.000
0.006
5i
5j
5k
5l
5m
5n
5.879
6.086
6.066
6.114
5.939
6.108
5.900
6.146
6.002
6.105
5.939
6.102
5o
6.076
6.086
À0.010
À0.069
À0.099
0.010
0.143
À0.010
5p
5q
5r
5s
5t
5.726
6.022
5.991
6.071
5.747
5.795
6.121
5.981
5.928
5.757
5u
a
Underlined compounds were selected as the test sets while the rest ones were
in the training sets.
The correlation coefficient r2 between observed and predicted
activity of training set was found to be 0.879, while that of test
set was found to be 0.973, which proved that the QSAR model built
by us was acceptable. Predicted pIC50 values and residual errors of
21 compounds by this QSAR model had been given in Table 3. The
well agreement between predicted pIC50 value and experimental
pIC50 value for both test sets and training sets are shown in
Figure 3.
Also the molecules aligned with the iso-surfaces of the 3D-QSAR
model coefficients on electrostatic potential grids (Fig. 4a) and van
der Waals grids (Fig. 4b) are listed. Electrostatic map indicates red
contours around regions where high electron density (negative
charge) is expected to increase activity, and blue contours repre-
sent areas where low electron density (partial positive charge) is
expected to increase activity. Similarly, steric map indicates areas
Figure 1. Binding mode of compound 5h with BRAFV600E kinase. For clarity, only
interacting residues were labeled. Hydrogen bonding interactions are shown in
green dotted lines. This figure was made using PyMol.
Figure 2. Binding mode of compound 5h with BRAFV600E kinase. The enzyme is
shown as surface; while 5h docked structures are shown as sticks. This figure was
made using PyMol.
Figure 3. Plot of experimental versus predicted BRAFV600E inhibitory activities of
training set and test set.

S.-F. Wang et al. / Bioorg. Med. Chem. 22 (2014) 6201–6208
6205
Figure 4. (a) 3D-QSAR model coefficients on electrostatic potential grids. Blue represents positive coefficients; red represents negative coefficients. (b) 3D-QSAR model
coefficients on van der Waals grids. Green represents positive coefficients; yellow represents negative coefficients.
where steric bulk is predicted to increase (green) or decrease
(yellow) activity.
silica gel used was 50–100 times the weight charged on the col-
umn. Melting points (uncorrected) were determined on a XT4MP
apparatus (Taike Corp., Beijing, China). All the Proton nuclear mag-
netic resonance (1H NMR) spectra were recorded on a Bruker
PX400 or DPX300 model Spectrometer in DMSO-d6 at 25 °C with
TMS and chemical shifts were reported in parts per million (d).
The solvent signals allotted as internal stands. Elemental analyses
were performed on a CHN-O-Rapid instrument. All the compounds
gave satisfactory chemical analyses ( 0.4%).
According to the maps, it suggested that the compound with
high negative charged and small R2 group, would show higher
activity, validating that halogen substituted group being a better
choice than methoxy substituted group and F substituent better
than Cl as well as Cl substituent better than Br. Whereas, a low
negative charged and bulky R1 group would help obtain sound
activity, validating the 4-Me substituent could be more effective.
As a result, data summarized above demonstrated that compounds
5h, the most potent BRAFV600E inhibitor (IC50 = 0.33
lM), contain-
4.2. General procedure for the synthesis of 2a–2c
ing suitable substituents had an outstanding activity. Thus, this
promising model would provide a guideline to design and optimize
more effective BRAFV600E inhibitors and pave the way for us in the
further study.
Mixed p-substituted methyl benzoate (7 mmol) with NaH
(14 mmol, 0.35 g) in boiling tetrahydrofuran (5 mL) and then fol-
lowed by dropwise addition of solution of acetonitrile (7 mmol,
0.29 g, 0.4 mL) in tetrahydrofuran (1 mL). The resulting mixture
was refluxed for 4 h and then cooling down to room temperature,
after which the solution was diluted with diethylether (15 mL) and
left to stand at room temperature for 48 h. The precipitated sodium
salt was filtered 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 Na2SO4.
3. Conclusion
In this study, a series of novel BRAF inhibitors beared pyrazole
core and niacinamide moiety have been synthesized and evaluated
their biological activities. Most of these compounds displayed good
inhibitory BRAFV600E activities and potent antiproliferative activity
against WM266.4 cell lines and A375 cell lines. Among them, com-
pound 5h exhibited the most potent BRAFV600E inhibition activities
(IC50 = 0.33
(IC50 = 2.63
l
l
M
for BRAFV600E
M for WM266.4 and IC50 = 3.16
)
and antiproliferative activities
M for A375). Dock-
4.3. General procedure for the synthesis of 3a–3c
l
ing simulation was performed to put compound 5h into the
BRAFV600E protein active site to determine the potential binding
model and found that several interactions with the protein resi-
dues in the BRAFV600E protein active site might play an important
role in its antiproliferative activities. QSAR model was built to
provide a reliable tool for reasonable design of novel BRAFV600E
inhibitors in future.
To a stirred mixture of compound 2a–2c (5 mmol) in EtOH
(25 mL) were added NH2NH2ÁH2O (0.3 mL, 6 mmol) and CH3SO3H
(0.1 mL, 1 mmol) at room temperature and the mixture was stirred
under reflux for 1 h. The precipitate was purified by column chro-
matography over silica gel to give the compound 3a–3c.
4.4. General procedure for the synthesis of 5-phenyl-1H-
pyrazole derivatives (5a–5u)
4. Experiments
Compounds 5a–5w were synthesized by coupling substituted
3a–3c with nicotinic acids, using 1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide hydrochloride (EDCl) and N-hydroxybenzotria-
zole (HOBt) as condensing agent. The mixture was refluxed in
anhydrous CH2Cl2 for 1–3 h. The products were extracted with
ethyl acetate. The extract was washed successively with 5% HCl,
then evaporated and purified by column chromatography over
silica gel to give the compound.
4.1. Materials and measurements
All chemicals and reagents used in current study were analyti-
cal grade. TLC was performed on the glass-backed silica gel sheets
(Silica Gel 60 GF254) and visualized in UV light (254 nm). Column
chromatography was performed using silica gel (200–300 mesh)
eluting with ethyl acetate and petroleum ether. The quantity of

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S.-F. Wang et al. / Bioorg. Med. Chem. 22 (2014) 6201–6208
4.4.1. 2-Fluoro-N-(5-phenyl-1H-pyrazol-3-yl)nicotinamide (5a)
Yellow powder, yield: 76%. Mp 176–177 °C. 1H NMR (300 MHz,
DMSO, d ppm): 5.90 (s, 1H); 6.93 (s, 2H); 7.38–7.40 (m, 3H); 7.55–
7.59 (m, 1H); 7.65–7.67 (m, 2H); 8.35–8.39 (m, 1H); 8.48 (d,
J = 3.0 Hz, 1H). MS (ESI): 283.45 [M+H]+. Anal. Calcd for C15H11FN4O:
C, 63.83; H, 3.93; N, 19.85; Found: C, 68.71; H, 3.78; N, 19.62.
FN4O: C, 64.86; H, 4.42; N, 18.91; Found: C, 64.71; H, 4.48; N,
18.81.
4.4.9. 2-Chloro-N-(5-(p-tolyl)-1H-pyrazol-3-yl)nicotinamide (5i)
Yellow power, yield: 60%. Mp 146–147 °C. 1H NMR (300 MHz,
DMSO, d ppm): 2.29 (s, 3H); 5.84 (s, 1H); 6.89 (s, 2H); 7.18 (d,
J = 7.9 Hz, 2H); 7.50 (d, J = 8.1 Hz, 2H); 7.61 (dd, J1 = 4.9 Hz,
J2 = 7.5 Hz, 1H); 8.20 (dd, J1 = 1.7 Hz, J2 = 7.5 Hz, 1H); 8.59 (dd,
J1 = 1.7 Hz, J2 = 4.7 Hz, 1H). MS (ESI): 314.62 [M+H]+. Anal. Calcd
for C16H13ClN4O: C, 61.44; H, 4.19; N, 17.91; Found: C, 61.39; H,
4.22; N, 17.87.
4.4.2. 2-Chloro-N-(5-phenyl-1H-pyrazol-3-yl)nicotinamide (5b)
Yellow powder, yield: 82%. Mp 134–135 °C. 1H NMR (300 MHz,
DMSO, d ppm): 5.87 (s, 1H); 6.91 (s, 2H); 7.36–7.38 (m, 3H); 7.59–
7.63 (m, 3H); 8.21 (dd, J1 = 1.8 Hz, J2 = 7.5 Hz, 1H); 8.59 (dd,
J1 = 1.8 Hz, J2 = 4.8 Hz, 1H). MS (ESI): 299.67 [M+H]+. Anal. Calcd
for C15H11ClN4O: C, 60.31; H, 3.71; N, 18.76; Found: C, 60.21; H,
3.68; N, 18.82.
4.4.10. 2-Methoxy-N-(5-(p-tolyl)-1H-pyrazol-3-yl)nicotinamide
(5j)
Yellow powder, yield: 58%. Mp 145–146 °C. 1H NMR (300 MHz,
DMSO, d ppm): 2.30 (s, 3H); 5.81 (s, 1H); 6.82 (s, 2H); 7.07 (s, 1H);
7.40–7.43 (m, 5 h); 8.16–8.18 (m, 2H). MS (ESI): 309.23 [M+H]+.
Anal. Calcd for C17H16N4O2: C, 66.22; H, 5.23; N, 18.17; Found: C,
66.01; H, 5.48; N, 18.32.
4.4.3. 2-Methoxy-N-(5-phenyl-1H-pyrazol-3-yl)nicotinamide
(5c)
Yellow powder, yield: 68%. Mp 187–188 °C. 1H NMR (300 MHz,
DMSO, d ppm): 3.87 (s, 3H); 5.85 (s, 1H); 6.85 (s, 2H); 7.14–7.17
(m, 1H); 7.37 (d, J = 4.8 Hz, 3H); 7.61–7.63 (m, 2H); 7.96–7.98
(m, 1H); 8.36 (dd, J1 = 1.4 Hz, J2 = 3.8 Hz, 1H). MS (ESI): 295.44
[M+H]+. Anal. Calcd for C16H14N4O2: C, 65.30; H, 4.79; N, 19.04;
Found: C, 65.21; H, 4.68; N, 18.92.
4.4.11. 5-Bromo-N-(5-(p-tolyl)-1H-pyrazol-3-yl)nicotinamide
(5k)
Yellow powder, yield: 78%. Mp 191–192 °C. 1H NMR (300 MHz,
DMSO, d ppm): 5.88 (s, 1H); 6.90 (s, 2H); 7.24 (d, J = 8.0 Hz, 2H);
7.63 (d, J = 8.0 Hz, 2H); 8.64 (s, 1H); 8.94 (s, 1H); 9.14 (s, 1H). MS
(ESI): 358.43 [M+H]+. Anal. Calcd for C16H13BrN4O: C, 53.80; H,
3.67; N, 15.68; Found: C, 53.77; H, 3.68; N, 15.56.
4.4.4. 5-Bromo-N-(5-phenyl-1H-pyrazol-3-yl)nicotinamide (5d)
Yellow powder, yield: 64%. Mp 130–131 °C. 1H NMR (300 MHz,
DMSO, d ppm): 5.92 (s, 1H); 6.92 (s, 2H); 7.43 (d, J = 7.2 Hz, 3H);
7.73–7.76 (m, 2H); 8.65 (s, 1H); 8.95 (d, J = 2.2 Hz, 1H); 9.15
(d, J = 1.5 Hz, 1H). MS (ESI): 344.26 [M+H]+. Anal. Calcd for
4.4.12. 6-Bromo-N-(5-(p-tolyl)-1H-pyrazol-3-yl)nicotinamide
(5l)
C15H11BrN4O: C, 52.50; H, 3.23; N, 16.33; Found: C, 52.40; H,
3.33; N, 16.51.
Yellow powder, yield: 64%. Mp 178–179 °C. 1H NMR (300 MHz,
DMSO, d ppm): 2.34 (s, 3H); 5.88 (s, 1H); 6.91 (s, 2H); 7.24 (d,
J = 6.1 Hz, 2H); 7.66 (d, J = 6.1 Hz, 2H); 7.88 (d, J = 6.3 Hz, 1H);
8.36–8.38 (m, 1H); 9.03 (d, J = 1.8 Hz, 1H). MS (ESI): 358.61
[M+H]+. Anal. Calcd for C16H13BrN4O: C, 53.80; H, 3.67; N, 15.68;
Found: C, 53.71; H, 3.58; N, 15.82.
4.4.5. 6-Bromo-N-(5-phenyl-1H-pyrazol-3-yl)nicotinamide (5e)
Yellow powder, yield: 75%. Mp 156–157 °C. 1H NMR (300 MHz,
DMSO, d ppm): 5.92 (s, 1H); 6.93 (s, 2H); 7.43 (d, J = 5.8 Hz, 3H);
7.76–7.78 (m, 2H); 7.89 (d, J = 6.2 Hz, 1H); 8.36–8.37 (m, 1H);
9.02 (d, J = 1.5 Hz, 1H). MS (ESI): 344.68 [M+H]+. Anal. Calcd for
C
15H11BrN4O: C, 52.50; H, 3.23; N, 16.33; Found: C, 52.71; H,
4.4.13. 6-Chloro-N-(5-(p-tolyl)-1H-pyrazol-3-yl)nicotinamide
(5m)
3.48; N, 16.42.
Yellow power, yield: 67%. Mp 152–153 °C. 1H NMR (300 MHz,
DMSO, d ppm): 2.33 (s, 3H); 5.88 (s, 1H); 6.89 (s, 2H); 7.24 (d,
J = 7.9 Hz, 2H); 7.66 (d, J = 8.0 Hz, 2H); 7.34 (d, J = 8.4 Hz, 1H);
8.49 (dd, J1 = 2.2 Hz, J2 = 8.2 Hz, 1H); 9.06(d, J = 2.0 Hz, 1H). 13C
NMR (300 MHz, DMSO-d6) d: 167.1, 155.2, 153.5, 152.2, 142.4,
139.3, 129.7, 129.7, 129.5, 129.3, 128.7, 126.4, 126.4, 124.1,
85.3, 21.4. MS (ESI): 314.52 [M+H]+. Anal. Calcd for C16H13ClN4O:
C, 61.44; H, 4.19; N, 17.91; Found: C, 61.40; H, 4.08; N, 17.88.
4.4.6. 2-Chloro-N-(5-phenyl-1H-pyrazol-3-yl)nicotinamide (5f)
Yellow powder, yield: 83%. Mp 259–261 °C. 1H NMR (300 MHz,
DMSO, d ppm): 7.04 (s, 1H); 7.36 (d, J = 4.2 Hz, 1H); 7.44 (t,
J = 9.0 Hz, 2H); 7.67 (d, J = 4.9 Hz, 1H); 7.75 (d, J = 4.6 Hz, 2H);
8.38 (m, 1H); 8.98 (d, J = 1.3 Hz, 1H); 11.17 (s, 1H); 13.00 (s, 1H).
13C NMR (300 MHz, DMSO-d6) d: 167.2, 155.7, 152.9, 151.4,
146.6, 139.2, 139.0, 131.8, 129.7, 129.7, 129.4, 126.4, 126.4,
123.2, 85.5, 21.4. MS (ESI): 299.84 [M+H]+. Anal. Calcd for
C
15H11ClN4O: C, 60.31; H, 3.71; N, 18.76; Found: C, 60.19; H,
4.4.14. 6-Methoxy-N-(5-(p-tolyl)-1H-pyrazol-3-yl)nicotinamide
(5n)
3.13; N, 18.32.
Yellow powder, yield: 59%. Mp 125–126 °C. 1H NMR (300 MHz,
DMSO, d ppm): 2.33 (s, 3H); 3.98 (s, 3H); 5.87 (s, 1H); 6.84 (s,2H);
6.99 (d, J = 8.8 Hz, 1H); 7.24 (d, J = 8.0 Hz, 2H); 7.68 (d, J = 8.0 Hz,
2H); 8.73 (s, 1H); 9.03 (d, J = 2.4 Hz, 1H). MS (ESI): 309.44
[M+H]+. Anal. Calcd for C17H16N4O2: C, 66.22; H, 5.23; N, 18.17;
Found: C, 66.02; H, 5.03; N, 18.25.
4.4.7. 6-Methoxy-N-(5-phenyl-1H-pyrazol-3-yl)nicotinamide
(5g)
Yellow powder, yield: 66%. Mp 102–103 °C. 1H NMR (300 MHz,
DMSO, d ppm): 3.93 (s, 3H); 5.91 (s, 1H); 6.87 (s, 2H); 7.00 (d,
J = 6.4 Hz, 1H); 7.46 (d, J = 5.4 Hz, 2H); 7.76–7.80 (m, 3H); 8.45
(dd, J1 = 1.8 Hz, J2 = 6.6 Hz, 1H); 9.04 (d, J = 1.6 Hz, 1H). MS (ESI):
295.42 [M+H]+. Anal. Calcd for C16H14N4O2: C, 65.30; H, 4.79; N,
19.04; Found: C, 65.21; H, 4.68; N, 18.82.
4.4.15. N-(5-(4-Bromophenyl)-1H-pyrazol-3-yl)-2-
fluoronicotinamide (5o)
Yellow powder, yield: 66%. Mp 146–147 °C. 1H NMR (300 MHz,
DMSO, d ppm): 5.91 (s, 1H); 6.96 (s, 2H); 7.61 (s, 4H); 7.70–7.76
(m, 1H); 8.34–8.38 (m, 1H); 8.48 (d, J = 3.2 Hz, 1H). 13C NMR
(300 MHz, DMSO-d6) d: 165.8, 154.4, 153.1, 150.5, 142.4, 132.6.
132.1, 132.1, 131.4, 128.4, 128.4, 128.3, 123.0, 122.3, 118.3, 85.5.
MS (ESI): 362.37 [M+H]+. Anal. Calcd for C15H10BrFN4O: C, 49.88;
H, 2.79; N, 15.51; Found: C, 50.01; H, 2.68; N, 15.62.
4.4.8. 2-Fluoro-N-(5-(p-tolyl)-1H-pyrazol-3-yl)nicotinamide
(5h)
Yellow powder, yield: 58%. Mp 146–147 °C. 1H NMR (300 MHz,
DMSO, d ppm): 2.30 (s, 3H); 5.86 (s, 1H); 6.90 (s, 2H); 7.20 (d,
J = 6.0 Hz, 2H); 7.55 (d, J = 6.1 Hz, 3H); 8.33–8.38 (m, 1H); 8.47
(d, J = 2.5 Hz, 1H). MS (ESI): 297.45 [M+H]+. Anal. Calcd for C16H13

S.-F. Wang et al. / Bioorg. Med. Chem. 22 (2014) 6201–6208
6207
4.4.16. N-(5-(4-Bromophenyl)-1H-pyrazol-3-yl)-2-
chloronicotinamide (5p)
used for each drug concentration. Each assay was carried out for
at least three times.
Yellow powder, yield: 85%. Mp 171–172 °C. 1H NMR (300 MHz,
DMSO, d ppm): 5.90 (s, 1H); 6.98 (s, 2H); 7.58 (s, 4H); 7.60–7.63
(m, 1H); 8.22 (dd, J1 = 1.4 Hz, J2 = 5.7 Hz, 1H); 8.60 (dd,
J1 = 1.4 Hz, J2 = 3.6 Hz, 1H). MS (ESI): 377.62 [M+H]+. Anal. Calcd
for C15H10BrClN4O: C, 47.71; H, 2.67; N, 14.84; Found: C, 47.59;
H, 2.58; N, 14.62.
4.6. Kinase assay
The BRAF Kinase inhibitory activity was measured using
previously methods described by J. Dietrich et al.32 The assay
was performed in triplicate for each tested compound in this study.
Briefly, 7.5 ng Mouse Full-Length GST-tagged BRAFV600E (Invitro-
gen, PV3849) was pre-incubated at room temperature for 1 h with
4.4.17. N-(5-(4-Bromophenyl)-1H-pyrazol-3-yl)-2-
methoxynicotinamide (5q)
1
l
L drug and 4
tiated when 5
human full length, N-terminal His-tagged MEK1 (Invitrogen),
200 M ATP (0.8 Ci hot ATP), and 30 mM MgCl2 in assay dilution
buffer was added. The kinase reaction was allowed to continue at
room temperature for 25 min and was then quenched with 5
5Â protein denaturing buffer (LDS) solution. Protein was further
denatured by heating for 5 min at 70 °C. 10 L of 20 each reaction
l
L assay dilution buffer. The kinase assay was ini-
Yellow powder, yield: 73%. Mp 207–208 °C. 1H NMR (300 MHz,
DMSO, d ppm): 3.86 (s, 3H); 5.86 (s, 1H); 6.88 (s, 2H); 7.13–7.16
(m, 1H); 7.57 (s, 4H); 7.95–7.98 (m, 1H); 8.35–8.37 (m, 1H). MS
(ESI): 374.46 [M+H]+. Anal. Calcd for C16H13BrN4O2: C, 51.49; H,
3.51; N, 15.01; Found: C, 51.31; H, 3.68; N, 14.82.
lL of a solution containing 200 ng recombinant
l
l
lL
4.4.18. 5-Bromo-N-(5-(4-bromophenyl)-1H-pyrazol-3-
yl)nicotinamide (5r)
l
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.
Yellow powder, yield: 59%. Mp 227–228 °C. 1H NMR (300 MHz,
DMSO, d ppm): 5.93 (s, 1H); 7.65–7.71 (m, 6H); 8.61 (s, 1H); 8.94
(s, 1H); 9.14 (s, 1H). MS (ESI): 423.19 [M+H]+. Anal. Calcd for
C15H10Br2N4O: C, 42.68; H, 2.39; N, 13.27; Found: C, 42.33;
H, 2.45; N, 13.22.
4.4.19. 6-Bromo-N-(5-(4-bromophenyl)-1H-pyrazol-3-
yl)nicotinamide (5s)
4.7. Docking simulations
Yellow powder, yield: 86%. Mp 205–206 °C. 1H NMR (300 MHz,
DMSO, d ppm): 5.91 (s, 1H); 7.72–7.83 (m, 6H); 8.59 (s, 1H); 8.88
(s, 1H); 9.12 (s, 1H). MS (ESI): 422.38 [M+H]+. Anal. Calcd for
The crystal structures of BRAFV600E (PDB code: 2FB8) was
obtained from the Protein Data Bank (http://www.rcsb.org).
Molecular docking of compound 5h into the three-dimensional
X-ray structure of BRAFV600E 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 BRAFV600E complex was defined as a receptor and the
site 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.
C15H10Br2N4O: C, 42.68; H, 2.39; N, 13.27; Found: C, 42.51;
H, 2.16; N, 13.75.
4.4.20. N-(5-(4-Bromophenyl)-1H-pyrazol-3-yl)-6-
chloronicotinamide (5t)
Yellow powder, yield: 58%. Mp 178–179 °C. 1H NMR (300 MHz,
DMSO, d ppm): 5.93 (s, 1H); 6.95 (s, 2H); 7.63 (d, J = 8.6 Hz, 2H);
7.71–7.75 (m, 3H); 8.46–8.50 (m, 1H); 9.05 (d, J = 2.2 Hz, 1H). MS
(ESI): 378.77 [M+H]+. Anal. Calcd for C15H10BrClN4O: C, 47.71; H,
2.67; N, 14.84; Found: C, 47.66; H, 2.75; N, 14.94.
4.8. QSAR model
Among all the 21 compounds, 81% (that is 17) were utilized as a
training set for QSAR modeling and the remaining 19% (that is 4)
were chosen as an external test subset for validating the reliability
of the QSAR model by the Diverse Molecules protocol in Discovery
Studio 3.5. The selected test compounds were: 5d, 5i, 5o, 5p.
The inhibitory activity of the compounds in literatures [IC50
(mol/L)] was initially changed into the minus logarithmic scale
[IC50 (mol/L)] and then used for subsequent QSAR analysis as the
response variable.
4.4.21. N-(5-(4-Bromophenyl)-1H-pyrazol-3-yl)-6-
methoxynicotinamide (5u)
Yellow powder, yield: 78%. Mp 217–218 °C. 1H NMR (300 MHz,
DMSO, d ppm): 2.41 (s, 3H); 5.92 (s, 1H); 6.89 (s, 2H); 6.99 (d,
J = 8.8 Hz, 1H); 7.64 (d, J = 8.6 Hz, 2H); 7.75 (d, J = 8.5 Hz, 2H);
8.40–8.44 (m, 1H); 9.02 (d, J = 2.1 Hz, 1H). MS (ESI): 374.35
[M+H]+. Anal. Calcd for C16H13BrN4O2: C, 51.49; H, 3.51; N, 15.01;
Found: C, 51.38; H, 3.64; N, 14.94.
In Discovery Studio, the CHARMm force field is used and the
electrostatic potential and the van der Waals potential are treated
as separate terms. A+1e point charge is used as the electrostatic
potential probe and distance-dependent dielectric constant is used
to mimic the solvation effect. For the van der Waals potential a
carbon atom with a 1.73 Å radius is used as a probe.
A Partial Least-Squares (PLS) model is built using energy grids
as descriptors. QSAR models were built by using the Create 3D
QSAR Model protocol in Discovery Studio 3.5.
4.5. Antiproliferation activity
WM1361, WM266.4 and A375 cell lines were cultured in
DMEM/10% fetal bovine serum, in 5% CO2 water saturated atmo-
sphere 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 con-
centrations were added to six wells with vemurafenib as positive
references. After 48 h exposure period, 40 lL of PBS containing
2.5 mg/mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
Acknowledgements
razolium bromide)) was added to each well. Four hours later,
This work was supported by Natural Science Foundation of
Jiangsu Province (No. BK20130554) and supported by Major
Projects on Control and Rectification of Water Body Pollution
(No. 2011ZX07204-001-2214 004).
100 lL extraction solution (10% SDS–5% isobutyl 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

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S.-F. Wang et al. / Bioorg. Med. Chem. 22 (2014) 6201–6208
15. Roberts, P. J.; Der, C. J. Oncogene 2007, 26, 3291.
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