Discovery of N-(benzyloxy)-1,3-diphenyl-1H-pyrazole-4-carboxamide derivatives as potential antiproliferative agents by inhibiting MEK

Article abstract of DOI:10.1016/j.bmc.2016.08.002

Mitogen activated protein kinase (MAPK) signal transduction pathway has been proved to play an important role in tumorigenesis and cancer development. MEK inhibitor has been demonstrated significant clinical benefit for blocking MAPK pathway activation and possibly could block reactivation of the MAPK pathway at the time of BRAF inhibitor resistance. Twenty N-(benzyloxy)-1,3-diphenyl-1H-pyrazole-4-carboxamide derivatives have been designed and synthesized as MEK inhibitors, and their biological activities were evaluated. Among these compounds, compound 7b showed the most potent inhibitory activity with IC₅₀of 91?nM for MEK1 and GI₅₀value of 0.26?μM for A549 cells. The SAR analysis and docking simulation were performed to provide crucial pharmacophore clues that could be used in further structure optimization.

Full text of DOI:10.1016/j.bmc.2016.08.002

Accepted Manuscript
Discovery of N-(benzyloxy)-1, 3-diphenyl-1H-pyrazole-4-carboxamide deriv-
atives as potential antiproliferative agents by inhibiting MEK
Xian-Hai Lv, Zi-Li Ren, Ben-Guo Zhou, Qing-Shan Li, Ming-Jie Chu, Dao-
Hong Liu, Kai Mo, Li-Song Zhang, Xiao-Kang Yao, Hai-Qun Cao
PII:
S0968-0896(16)30595-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.08.002
BMC 13185
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
2 June 2016
24 July 2016
2 August 2016
Please cite this article as: Lv, X-H., Ren, Z-L., Zhou, B-G., Li, Q-S., Chu, M-J., Liu, D-H., Mo, K., Zhang, L-S.,
Yao, X-K., Cao, H-Q., Discovery of N-(benzyloxy)-1, 3-diphenyl-1H-pyrazole-4-carboxamide derivatives as
potential antiproliferative agents by inhibiting MEK, Bioorganic & Medicinal Chemistry (2016), doi: http://
dx.doi.org/10.1016/j.bmc.2016.08.002
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Bioorganic & Medicinal Chemistry
Discovery of N-(benzyloxy)-1, 3-diphenyl-1H-pyrazole-4-carboxamide derivatives as
potential antiproliferative agents by inhibiting MEK
a,b
b
b
c
a
a
a
Xian-Hai Lv , Zi-Li Ren , Ben-Guo Zhou , Qing-Shan Li , Ming-Jie Chu , Dao-Hong Liu , Kai Mo , Li-
a
a
b*
Song Zhang , Xiao-Kang Yao , Hai-Qun Cao
a
School of Science, Anhui Agricultural University, Hefei 230036, P.R. China;
School of Plant Protection, Anhui Agricultural University, Hefei 230036, P.R. China
School of Medical Engineering, Hefei University of Technology, Hefei 230009, P.R. China
b
c
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Mitogen activated protein kinase (MAPK) signal transduction pathway has been proved to play
an important role in tumorigenesis and cancer development. MEK inhibitor has been
demonstrated significant clinical benefit for blocking MAPK pathway activation and possibly
could block reactivation of the MAPK pathway at the time of BRAF inhibitor resistance.
Twenty N-(benzyloxy)-1,3-diphenyl-1H-pyrazole-4-carboxamide derivatives have been
designed and synthesized as MEK inhibitors, and their biological activities were evaluated.
Among these compounds, compound 7b showed the most potent inhibitory activity with IC50 of
Available online
Keywords:
anti-cancer activity
inhibitors
9
1 nM for MEK1 and GI50 value of 0.26 μM for A549 cells. The SAR analysis and docking
simulation were performed to provide crucial pharmacophore clues that could be used in further
structure optimization.
MEK
molecular docking; pyrazole
QSAR
2009 Elsevier Ltd. All rights reserved.
V600
1
. Introduction
progression-free and overall survival in patients with BRAF
mutated metastatic melanoma.
-
8-11
Although the overwhelming
In recent years, a number of key signaling pathways,
majority of patients benefit from BRAF inhibitors administration,
unfortunately the therapeutic effects are often temporary (median
membrane
receptors,
kinases
and
other
biological
macromolecules, which play an important role in tumorigenesis
and cancer development, have been identified along with the
further understanding of the pathogenesis of human cancer.
Development of small molecular drugs specially targeting these
specific macromolecules provides opportunities to overcome
human cancers. MAPK signal transduction pathway is one of the
12
time, 5 to 7 months) and limited by rapidly acquired resistance.
Multiple BRAF inhibitor resistance mechanisms in melanoma
have been identified, including emergence of mutant BRAF-
concurrent upstream RAS or downstream MEK (MAP2K)
mutations , amplification or alternative splicing of mutant
BRAF , upregulation of RTKs (receptor tyrosine kinases) and
13
14
15
16
1
-3
most important signaling pathways inside the cell.
17
COT kinase (MAP3K8) .
BRAF, which is one of the most important pro-oncogenes in
MAPK pathway, is mutated in approximately 8% of human
tumors. Especially, BRAF mutations are frequently found in 40%
As noted above, most reported resistance mechanisms were
MAPK-dependent, which induced the reactivation of the MAPK
pathways despite continued treatment with the drug.
Furthermore, MEK is the downstream effector of BRAF, so that
MEK inhibition is useful for blocking MAPK pathway activation
4, 5
to 60% of cutaneous melanoma patients.
Most of these
V600E
mutations cause substitutions of glutamic acid (BRAF
, 70-
, 10-30%) for valine, which
constitutively activates MEK/ERK pathway in cancer cells,
V600K
90%) or lysine (BRAF
and possibly could block reactivation of the MAPK pathway at
18, 19
6, 7
the time of BRAF inhibitor resistance.
Given these
leading to tumor development, invasion, and metastasis. There
is an accumulating body of experimental evidences validating
treatment of selective class I BRAF inhibitors (Vemurafenib and
Dabrafenib) as an effective therapeutic strategy for improved
observations, several trials of MEK inhibitors administered alone
or in combination with BRAF inhibitors were pursued in order to
V600E
overcome resistance of BRAF
melanomas treated with RAF
inhibitors. Indeed, several therapy approaches increased the
—
——
*
Corresponding author. Tel. & fax: +86 551 65786906, e-mail: haiquncao@163.com (H.-Q. Cao)
V600E
V600K
Abbreviations: MEK, mitogen-activated protein kinase kinase; BRAF, V-RAF murine sarcoma viral oncogene homologue B1; ERK, extracellular regulated kinase; BRAF
IC50, half maximal inhibitory concentration; GI50, the concentration that causes 50% growth inhibition; SAR, Structure-Activity Relationship.
, V600E mutant BRAF; BRAF
, V600K mutant BRAF;

magnitude and/or durability of response, resulted in more
complete and durable inhibition of MAPK as well as greater
6, 20
tumor growth inhibition.
There are several small molecule
MEK inhibitors in clinical trials, such as CI-1040, PD0325901,
U0126, PD98059, GSK1120212 and AZD2644. Trametinib
(GSK1120212), a selective class I inhibitor of MEK1/2, is well
tolerated and clinically active in a phase 3 open-label trial that
V600
patients could have been treated with BRAF
mutant
Scheme 2. Synthesis of compounds 6a-10d. Reagents and
metastatic melanoma, with the exclusion of BRAF and MEK
conditions: (vi) THF, pyridine, rt, 2 h.
11, 19
inhibitors and Ipilimumab.
Dabrafenib administrated alone, combination of Dabrafenib and
Trametinib demonstrated significant improvement in
progression-free survival in previously untreated patients who
In a phase 3 trial, compared with
The 1,3-diphenyl-1H-pyrazole-4-carbonyl chloride derivatives
(6-10) were prepared according to a previously reported synthetic
a
28, 29
route outlined in Scheme 1
. The synthetic route of 1,3-
V600E/K
21
had metastatic melanoma with BRAF
mutations. This
diphenyl-N-benzyloxy-1H-pyrazole-4-carboxamide derivatives
combined trial also could delayed the emergence of resistance
and decreased the incidence of cutaneous hyperproliferative
lesions in preclinical models. Due to MEK inhibitor has been
demonstrated significant clinical benefit to reduce the emerged
resistance， several studies are focus on the development of
selectively MEK inhibitor. In continuation of our studies on the
(6a-10d) was shown in Scheme 2. According to previously
3
0, 31
reported synthetic scheme
, the synthesis of compounds (6a-
10d) began with the interaction of substituted 1,3-diphenyl-1H-
pyrazole-4-carbonyl chloride （ 2.0 mmol ） and various
substituted O-benzyl hydroxylamine hydrochloride （2.0 mmol
） with the help of pyridine (4.4 mmol) in anhydrous
tetrahydrofuran. Then compounds 6a-10d (Table 1) were
obtained by subsequent purification with recrystallization from
an ethanol-DMF mixture (2:1).
These compounds were reported for the first time except 6a
and 8a. All of the synthesized compounds 6a-10d (Table 1) gave
satisfactory elementary analysis and spectroscopic data, which
were full accordance with their depicted structures.
2
2-25
development of anti-cancer agents targeting MAPK pathway
,
we focus on the identification of novel highly specific and
selected small molecule agents to inhibit the protein-kinase
activity of MEK.
Table 1. Structures of compounds 6a-10d.
Figure 1. Reported MEK inhibitors owing N-methoxy methanamide
moiety.
Many pyrazole derivatives have been reported to possess
potent anticancer activities in vitro and in vivo, and much
attention was paid to the discovery of Ser/Thr kinases inhibitors
based on pyrazole core. As seen in Figure.1, several studies have
shown that compounds owing N-methoxy methanamide moiety
2
6, 27
are known to exhibit attractive MEK inhibitory potency
.
Encouraged by above observations, in this thesis we designed
and synthesized a serials of novel 1,3-diphenyl-1H-pyrazole
derivatives containing derivatives N-methoxy methanamide
moiety, in the hope of obtaining novel anticancer agents through
inhibiting MEK kinase activity. Their biological activities in
vitro are evaluated and the structure-activity relationships (SAR)
are also discussed. Docking simulations were performed using
the X-ray crystallographic structure of the MEK-1 to explore the
binding modes of these compounds at the active site.
Compound
R
1
R
2
Compound
8c
R
1
R
2
6
a
H
H
OCH
OCH
Cl
Cl
Cl
Cl
F
3
3
2-Cl
2,4-Cl
H
6b
H
2-F
2-Cl
2,4-Cl
H
8d
6
c
H
9a
6d
H
9b
2-F
7
7
7
7
8
8
a
b
c
CH
CH
CH
CH
3
3
3
3
9c
2-Cl
2,4-Cl
H
2. Results and discussion
2-F
2-Cl
2,4-Cl
H
9d
2.1 Chemistry
10a
10b
10c
10d
d
a
b
F
2-F
OCH
OCH
3
3
F
2-Cl
2,4-Cl
2-F
F
2.2 Bioassays and SAR
All synthesized 1,3-diphenyl-N-benzyloxy-1H-pyrazole-4-
carboxamide derivatives 6a-10d were evaluated for their in vitro
antiproliferative activities against three tumor cell lines, which
were HeLa, MCF-7 and A549 cell lines, comparing with the
positive control. The in vitro growth inhibitory activities of target
compound were expressed as the concentration of the compound
that inhibited each human cancer cells proliferation to 50 %
Scheme 1. General synthesis of compounds 6-10. Reagents and
conditions: (i) ethanol, 50–60 ºC, 3 h; (ii) DMF, POCl , 50-60 ºC, 5
h; (iii) KMnO , 70-80 ºC, 3 h; (iv) SOCl , 70-80 ºC, 3 h.
3
4
2

(
GI50) of the control value, and the results were summarized in
respectively). Inspection of the chemical structure of all the
compounds suggested that it could be divided into two subunits:
A rings and B rings. A comparison of the para substituents on the
A-ring demonstrated that an electron-withdrawing group has
improved antiproliferation activity, in which introduction of
methyl group generated the most potent activities. For the
substituents on the ring B, compounds with ortho electron-
withdrawing substitution showed stronger activities in the
following order: F > Cl > H. Introduction of substituent to para-
position of ring B results in less active analog.
Table 2.
Table 2. In vitro antiproliferation activities of compounds 6a-10d.
a, b
GI50 (μM)
CC50 (μM)
Compd.
HeLa
MCF-7
A549
293T
6
6
a
8.83±0.64
13.38±0.72
5.00±0.26
5.94±0.34
10.29±0.62
7.35±0.44
1.14±0.06
0.26±0.02
0.38±0.03
1.08±0.06
1.94±0.09
2.16±0.14
2.45±0.16
3.83±0.20
121.61±10.16
75.72±5.82
82.05±6.60
40.22±2.55
40.81±2.67
20.57±1.48
38.69±2.65
39.62±2.94
200.7±12.25
24.56±1.68
586.45±36.37
544.75±32.84
51.93±3.62
125.64±7.82
128.64±8.04
46.27±3.24
Furthermore, the toxicity of compounds 6a-10d was
preliminary evaluated by a against human kidney epithelial cell
b
11.75±0.72 16.14±0.84
293T. The results were expressed as median cytotoxic
6
c
17.84±0.84
5.10±0.26
6.69±0.45
1.18±0.06
4.68±0.25
2.04±0.11
2.64±0.14
2.90±0.15
3.48±0.18
5.75±0.30
8.35±0.48
4.17±0.23
8.85±0.45
5.16±0.26
6.11±0.32
7.41±0.38
9.66±0.54
1.87±0.12
2.11±0.12
2.64±0.14
2.31±0.13
2.77±0.14
6.00±0.35
3.73±0.21
2.57±0.18
concentration (CC50) data obtained from the MTT assay (Table 2).
As shown, these compounds were tested at multiple doses and
demonstrated low cytotoxic activities in vitro against human
kidney epithelial cell 293T.
6d
7a
7b
7
c
7d
8a
8b
8
c
8d
9a
9b
10.78±0.61 12.98±0.69
15.10±0.81 12.06±0.63
16.10±0.92 15.45±0.86
16.18±0.92 13.12±0.72
a
Values are the average of three independent experiments run in triplicate.
Variation was generally 5–10%.
Figure 2. MEK1 inhibitory activities of selected compounds.
9
c
9
d
Table 3. pERK activity and kinase inhibitory activities against
1
0a
0b
15.30±0.87 10.38±0.65 217.04±13.53
selected kinases of compound 7b.
1
10.27±0.64 12.01±0.64 17.49±0.94
36.42±2.42 11.37±0.62 8.33±0.67
25.32±1.67 13.95±0.84
6.71±0.34 2.86±0.18
146.78±8.98
44.69±3.06
45.00±3.24
pERK
EGFR
0.610.03
1
0c
0d
Gefitinib
>25
HER-2
FAK
>25
1
7.35±0.38
1.52±0.08
15.60.9
8.300.5
a
Aurora-A
VEGFR-2
Antiproliferation activity was measured using the MTT assay. Values are the
average of three independent experiments run in triplicate.
b
>
25
Errors were in the range of 5–10% of the reported values.
The inhibitory activities are displayed as IC50 ( μM).
From the data obtained from cellular assay, it can be concluded
that
target
1,3-diphenyl-N-benzyloxy-1H-pyrazole-4-
The inhibitory activities of selected compounds against
MEK were evaluated by the Raf-MEK-ERK cascade kinase
assay using recombinant proteins. Compounds owing the most
potent activities in antiproliferation assay (serials 7 and 8), and
owing less potent activities (serials 10), were selected to perform
kinase assay. The results were summarized in Figure 2, it can be
found that compounds in serials 10 showed poor MEK inhibitory
activities while most of the compounds in serials 7 and 8
displayed potent inhibitory activities. That is to say, for tested
carboxamide derivatives showed moderate inhibitory activities
against HeLa and MCF-7 cell lines, ranging from 1.18 to 36.42
μM. Compound 7b, in which A ring was substituted by methyl
group and B ring was substituted by ortha-fluorine, owed the
most potent inhibitory activities (GI50 = 1.18 μM) against HeLa
cells. In MCF-7 cells, compound 7a, N-(benzyloxy)-3-phenyl-1-
(p-tolyl)-1H-pyrazole-4-carboxamide, in which A ring was
substituted by methyl group as well, exhibited the lower GI50
value (1.87μM).
1,3-diphenyl-N-benzyloxy-1H-pyrazole-4-carboxamide
derivatives, the SAR of MEK inhibitory activities were in
accordance with their inhibitory activities of cancer cells
proliferation. Among the tested compounds, 7b showed the most
potent inhibitory activity with IC50 of 91 nM, which was
comparable to the positive control U0126 with IC50 of 89 nM.
In comparison with HeLa and MCF-7 cells, A549 cell, which
was bearing a constitutively active MAPK pathways, was more
sensitive to most of target compounds, and showed low
micromolar GI50 values. Compounds 7b and 7c exhibited GI50
value in submicromolar range (GI50 = 0.26 μM and 0.38 μM,

The results suggested that the antiproliferative effects were
mediated by direct interaction of target compounds with MEK.
Moreover, the phosphorylation level of extracellular signal-
regulated kinase (ERK) was measured in a cell-based assay (IC50
pERK). As shown in Table 3, compound 7b exhibited obviously
inhibitory activity of ERK phosphorylation in BRAF mutant cell
line. As shown in Table 3, the results of the kinase selectivity
assay revealed that compound 7b has an excellent selectivity
profile.
Figure 3. (a). Binding model of 7b (purple) in the active site of the
MEK1 protein-kinase. The H-bond is displayed as dashed line. (b)
2D projection drawing of 7b docked into MEK1 active site.
3. Conclusion
In this study, a series of novel N-(benzyloxy)-1,3-diphenyl-
1H-pyrazole-4-carboxamide derivatives have been synthesized
and evaluated for their antitumor activities as MEK inhibitors.
These compounds exhibited potent inhibitory activities against
various human cancer cell lines, especially in A549 cells which
bears a constitutively active MAPK pathways. Compound 7b and
To gain more understanding of the interaction between target
compounds and MEK, we explored their binding modes
generated by molecular docking based on the reported
MEK1/inhibitor complex structure (PDB code: 3EQF) and
preprocessed by the DS 3.1 (Discovery Studio 3.1, Accelrys, Inc.,
7c exhibited the most potent anti-proliferative activities against
A549 cells (GI50 = 0.26 μM and 0.38 μM, respectively). In MEK
inhibitory assay, the SAR of tested compounds was in
accordance with the trend in cellular assay, among which 7b
showed the most potent inhibitory activity with IC50 of 91 nM,
which was comparable to the positive control U0126 with IC50 of
32, 33
San Diego, CA)
. Each ligand was docked as described
3
4
previously , and the pose with the highest --ECD (cdocker
interaction energy) was considered as the optimum pose for it.
The binding models of 7b with the MEK1 structure are shown in
Figure 3. Visual inspection of the pose of 7b into the MEK-
binding site revealed that it has suitable shape complementarity
with the ATP binding pocket. Especially the 1,3-diphenyl-1H-
pyrazole skeleton was deeply embedded into the pocket (Figure
89 nM. Docking simulation was performed to position compound
7b into the active site of the MEK1 kinase to probe the binding
mode. Analysis of the compound 7b binding conformation in
active site showed that it has suitable shape complementarity
with the ATP binding pocket, and the conformation was
stabilized by cation-π interaction with Lys 156 and an H-bond
with Ser 150. Above all, the results obtained from this study
3
1
a), and extensive hydrophobic interactions are formed between
,3-diphenyl-1H-pyrazole skeleton and residues Val 82, Ala 95,
Val 127, Met 143, Met 143 and Leu 197 of the ATP-binding
pocket. On the other side, different interactions formed by N-
suggest
that
N-(benzyloxy)-1,3-diphenyl-1H-pyrazole-4-
carboxamide derivatives skeleton may serve as a novel scaffold
for the further development of more potent and selective MEK
inhibitors which use as new therapeutic agent to fight against
cancer.
(benzyloxy)amide side chain of 7b with amino acid residues in
binding site were stabilified the binding mode (Figure 3b). A
cation-π interaction was established between Lys 156 and an
electron-rich π system of aromatic ring B of 7b. And an H-bond
…
was also detected in the binding model (O H-N/Ser 150, angle
…
o
4. Experimental section
O H-O = 108.4 , distance = 2.5 Å), which was simultaneously
contributed to the combination. According to the above, the
molecular docking result along with the biological assay data
suggested that compound 7b might be a potential inhibitor of the
MEK1.
4.1. General
All of the synthesized compounds were chemically
characterized by thin layer chromatography (TLC), proton
1
nuclear magnetic resonance ( H NMR) and elemental
1
13
microanalyses (CHN). H NMR and C NMR spectra were
measured on a Bruker AV-300 or a Agilent DD2 600Hz
spectrometer AV-600 spectrometer at 25 ºC and referenced to
Me4Si. Chemical shifts were reported in ppm (δ) using the
residual solvent line as internal standard. Splitting patterns were
designed as s, singlet; d, doublet; t, triplet; m, multiplet. ESI-MS
spectra were recorded on a Mariner System 5304 Mass
spectrometer. Elemental analyses were performed on a CHN-O-
Rapid instrument and were within ±0.4% of the theoretical
values. Melting points were determined on a XT4 MP apparatus
(Taike Corp., Beijing, China) and were as read. Analytic thin-
layer chromatography (TLC) was performed on the glass backed
silica gel sheets (silica gel 60Å GF254). All compounds were
detected by using UV light (254 nm or 365 nm).
4
1
.2. General method for the preparation of target compounds 6-
0
The starting material substituted 1,3-diphenyl-1H-pyrazole-4-
carbonyl chloride derivatives (6-10) was synthesized as
following: para-substituted acetophenone (1-5) (20mmol) interact
with phenylhydrazine hydrochloride (25mmol) couple with
sodium acetate (40mmol) in anhydrous ethanol to form 1-phenyl-
2
-(1-phenylethylidene) hydrazine, which was then dissolved in a
cold mixed solution of DMF (20mL) and POCl (16 mL), stirred
3
at 50-60 ºC for 5 h. The resulting mixture was poured into ice-
cold water, a saturated solution of sodium hydroxide was added
to neutralize the mixture, then the obtained solid precipitate was
oxidized to the corresponding carboxylic acids by treatment with
potassium permanganate (10mmol), stirred at 70-80 ºC for 3 h
while the transformation of acids into the appropriate acid

chlorides was accomplished with thionyl chloride in refluxing for
h ， and then thionyl chlorid was removed under reduced
pressure to give the desired compounds 6-10.
119.40, 115.58, 115.44, 71.81, 21.34. MS (ESI): 402.2
+
3
3 2 3 2
(C24H20FN O , [M+H] ). Anal. Calcd for C24H20FN O : C, 71.81;
H, 5.02; N, 10.47; Found: C, 72.00; H, 4.98; N, 10.51%.
4.3. General procedure for 1,3-diphenyl-N-benzyloxy-1H-
4.3.7 N-((2-chlorobenzyl)oxy)-3-phenyl-1-(p-tolyl)-1H-pyrazole-
pyrazole-4-carboxamide derivatives 6a-10d
4-carboxamide(7c)
White solid. Yield: 79%. Mp: 151-153 ºC. H NMR(300 MHz,
DMSO-d ): 2.40 (s, 3H); 5.10 (s, 2H); 7.18-7.51 (m, 11H); 7.75
6
(d, J=8.04Hz, 2H); 8.52 (s, 1H); 10.21 (s, 1H). C NMR (151
1
Compounds 6a-10d were synthesized from a stirring mixture
of the starting material substituted 1,3-diphenyl-1H-pyrazole-4-
carbonyl chloride derivatives (6-10) (2 mmol) and substituted O-
benzyl hydroxylamine hydrochloride (2.0mmol) with the help of
pyridine (4.4 mmol) in anhydrous tetrahydrofuran (15 mL) at the
room temperate for 2 h. The reaction mixture was poured into
three times its volume of cold water when the product as a solid.
The solid was filtered under vacuum, and then recrystallized
from ethanol-DMF to afford the pure product 6a-10d.
1
3
MHz, cdcl ) δ 150.96, 139.25, 139.17, 134.34, 133.12,
3
131.20, 131.00, 129.89, 129.61, 129.56, 129.24, 128.98,
128.70, 127.42, 126.90, 199.42, 114.97, 75.43, 21.36. MS
+
(ESI): 418.1 (C H ClN O , [M+H] ). Anal. Calcd for
2
4
20
3
2
C H ClN O : C,68.98; H, 4.82; N, 10.06; Found: C, 68.74; H,
24
20
3
2
4.89; N, 10.27%.
4
.3.1
N-(benzyloxy)-1,3-diphenyl-1H-pyrazole-4-carboxamide
4
.3.8
N-((2,4-dichlorobenzyl)oxy)-3-phenyl-1-(p-tolyl)-1H-
(6a)
pyrazole-4-carboxamide (7d)
White solid. Yield: 85%. Mp: 169-171 ºC. H NMR(500 MHz,
DMSO-d ): 2.37 (s, 3H); 5.01 (s, 2H); 7.22 (d, J=7.95Hz, 2H);
1
1
White solid. Yield: 80%. Mp: 125-127 ºC. H NMR (300
MHz, CDCl
1.21 (s, 1H). MS (ESI): 370.1 (C23
Calcd for C23 : C, 74.78; H, 5.18; N, 11.37; Found: C,
4.81; H, 5.34; N, 11.29%.
3
): 4.96 (s, 2H); 7.36-7.76 (m, 15H); 8.51 (s, 1H);
6
+
1
19 3 2
H N O , [M+H] ). Anal.
7.38 (t, J=7.30Hz, 1H); 7.47 (d, J=8.20Hz, 1H); 7.55 (t,
19 3 2
H N O
J=7.92Hz, 3H); 7.61 (d, J=7.95Hz, 1H); 7.67 (d, J=7.65Hz, 3H);
7
7
4
.87 (d, J=7.90Hz, 2H); 8.78 (s, 1H); 11.46 (s, 1H). MS (ESI):
+
2 3 2 2 3 2
52.2 (C24H19Cl N O , [M+H] ). Anal. Calcd for C24H19Cl N O :
4.3.2
N-((2-fluorobenzyl)oxy)-1,3-diphenyl-1H-pyrazole-4-
C, 63.73; H, 4.23; N, 9.29; Found: C, 63.69; H, 4.19; N, 9.57%.
carboxamide (6b)
White solid. Yield: 73%. Mp: 137-138 ºC. H NMR(300 MHz,
1
4
4
.3.9 N-(benzyloxy)-1-(4-methoxyphenyl)-3-phenyl-1H-pyrazole-
DMSO-d
6
): 4.99 (s, 2H); 7.20-7.27 (m, 2H); 7.36-7.45 (m,
-carboxamide(8a)
1
5
1
H);7.79 (d, J=5.85Hz, 2H); 7.87 (d, J=8.43Hz, 2H); 8.79 (s,
White solid. Yield: 82%. Mp: 143-145 ºC. H NMR (300 MHz,
CDCl ): 3.87 (s, 3H); 4.97 (s, 2H); 6.87 (d, J=8.58Hz, 2H); 7.36-
7.41 (m, 6H); 7.48 (t, J=8.13Hz, 4H); 7.73 (d, J=8.25Hz, 2H);
8.50 (s, 1H); 10.21 (s, 1H). C NMR (151 MHz, cdcl ) δ 155.59,
+
H); 11.48(s, 1H). MS (ESI): 388.1 (C23
H18FN
3
O
2
, [M+H] ).
3
Anal. Calcd for C23
H
18FN
3
O
2
: C, 71.31; H, 4.38; N, 10.85;
13
Found: C, 71.38; H, 4.54; N, 11.01%.
3
1
1
5
34.42, 126.95, 126.50, 125.99, 125.94, 125.41, 124.83, 122.66,
4
.3.3
carboxamide (6c)
White solid. Yield: 80%. Mp: 140-141 ºC. H NMR(300 MHz,
DMSO-d ): 5.05 (s, 2H); 7.39-7.62 (m, 10H); 7.79 (d, J=6.03Hz,
H); 7.88 (d, J=8.04Hz, 2H); 8.81 (s, 1H); 11.51 (s, 1H). MS
N-((2-chlorobenzyl)oxy)-1,3-diphenyl-1H-pyrazole-4-
19.50, 119.47, 119.39, 114.69, 110.86, 110.72, 109.58, 67.11,
+
0.58. MS (ESI): 400.2 (C24
H
21
N
3
O
3
, [M+H] ). Anal. Calcd for
1
C H N O : C, 72.16; H, 5.30; N, 10.52; Found: C, 72.38; H,
5.26; N, 10.78%.
24
21
3
3
6
2
+
(ESI): 404.1 (C23
3 2
H18ClN O , [M+H] ). Anal. Calcd for
4
1
.3.10 N-((2-fluorobenzyl)oxy)-1-(4-methoxyphenyl)-3-phenyl-
C
23
H
18ClN : C, 68.40; H, 4.49; N, 10.40; Found: C, 68.53; H,
3 2
O
H-pyrazole-4-carboxamide (8b)
1
4.47; N, 10.296%.
White solid. Yield: 70%. Mp: 144-146 ºC. H NMR(300 MHz,
6
DMSO-d ): 3.86 (s, 3H); 5.05 (s, 2H); 6.91 (d, J=8.40Hz, 2H);
7.05-7.15 (m, 2H);7.36-7.54 (m, 7H); 7.75 (d, J=7.50Hz, 2H);
4
.3.4 N-((2,4-dichlorobenzyl)oxy)-1,3-diphenyl-1H-pyrazole-4-
carboxamide(6d)
8.50 (s, 1H); 10.20 (s, 1H). MS (ESI): 418.1 (C H FN O ,
2
4
20
3
3
1
+
White solid. Yield: 87%. Mp: 167-169 ºC. H NMR(300
[
M+H] ). Anal. Calcd for C24
H20FN
3
O
3
: C, 69.05; H, 4.83; N,
MHz, CDCl
3
): 4.96 (s, 2H); 7.27-7.56 (m, 13H); 7.75 (d,
10.07; Found: C, 69.29; H, 5.00; N, 10.04%.
J=7.86Hz, 2H); 8.51 (s, 1H); 11.20 (s, 1H). MS (ESI): 438.1
+
(C
23
H
2
17Cl N
3
O
2
, [M+H] ). Anal. Calcd for (C23
2
H17Cl N
3
O
2
: C,
4.3.11 N-((2-chlorobenzyl)oxy)-1-(4-methoxyphenyl)-3-phenyl-
63.03; H, 3.91; N, 9.59; Found: C, 63.25; H, 4.01; N, 9.48%.
1H-pyrazole-4-carboxamide (8c)
White solid. Yield: 78%. Mp: 121-123 ºC. H NMR(300 MHz,
1
4.3.5
N-(benzyloxy)-3-phenyl-1-(p-tolyl)-1H-pyrazole-4-
DMSO-d
6
): 3.87 (s, 3H); 5.03 (s, 2H); 7.17-7.52 (m, 11H); 7.75
carboxamide (7a)
White solid. Yield: 79%. Mp: 173-175 ºC. H NMR (300 MHz,
CDCl
(
d, J=8.04Hz, 2H); 8.52 (s, 1H); 10.21 (s, 1H). MS (ESI): 434.1
1
+
(C
24
H
3 3 3 3
20ClN O , [M+H] ). Anal. Calcd for C24H20ClN O : C,
3
): 2.41 (s, 3H); 4.96 (s, 2H); 7.15 (d, J=7.89Hz, 2H); 7.27-
66.44; H, 4.65; N, 9.68; Found: C, 66.67; H, 4.78; N, 9.56%.
7.51 (m, 10H); 7.74 (d, J=8.22Hz, 2H); 8.51 (s, 1H); 10.20 (s,
+
1H). MS (ESI): 384.2 (C24
21
H N
3
O
2
, [M+H] ). Anal. Calcd for
4.3.12
phenyl-1H-pyrazole-4-carboxamide (8d)
White solid. Yield: 87%. Mp: 155-157 ºC. H NMR(300 MHz,
CDCl ): 3.87 (s, 3H); 5.06 (s, 2H); 6.94 (t, J=8.72Hz, 2H); 7.24
N-((2,4-dichlorobenzyl)oxy)-1-(4-methoxyphenyl)-3-
24 21 3 2
C H N O : C, 75.18; H, 5.52; N, 10.96; Found: C, 75.47; H,
1
5.56; N, 11.17%.
3
4
4
.3.6 N-((2-fluorobenzyl)oxy)-3-phenyl-1-(p-tolyl)-1H-pyrazole-
(m, 1H);7.33-7.54 (m, 7H); 7.74 (d, J=7.47Hz, 2H); 8.50 (s, 1H);
+
-carboxamide(7b)
10.19 (s, 1H). MS (ESI): 468.1 (C H Cl N O , [M+H] ). Anal.
2
4
19
2
3
3
1
White solid. Yield: 69%. Mp: 142-144 ºC. H NMR(300 MHz,
24 19 2 3 3
Calcd for C H Cl N O : C, 61.55; H, 4.09; N, 8.97; Found: C,
CDCl
J=8.22Hz, 2H); 8.52 (s, 1H); 10.21 (s, 1H). C NMR (151 MHz,
cdcl ) δ 162.16, 160.51, 139.23, 139.18, 131.62, 131.16, 130.68,
30.63, 129.60, 129.55, 128.98, 128.66, 127.41, 124.21,124.18,
3
): 2.40 (s, 3H); 5.04 (s, 2H); 7.05-7.51 (m, 11H); 7.75 (d,
61.78; H, 3.98; N, 9.23%.
13
3
4.3.13 N-(benzyloxy)-1-(4-chlorophenyl)-3-phenyl-1H-pyrazole-
1
4-carboxamide (9a) White solid. Yield: 81%. Mp: 169-171 ºC.

1
H NMR (500 MHz, DMSO-d
6
): 4.93 (s, 2H); 7.37-7.41 (m, 4H);
.45 (s, 2H); 7.51 (d, J=8.50Hz, 2H); 7.56 (t, J=7.92Hz, 2H);
.84 (d, J=8.55Hz, 2H); 7.88 (d, J=8.55Hz, 2H); 8.84 (s, 1H);
J=7.32Hz, 1H); 7.48 (d, J=7.00Hz, 1H); 7.54-7.67 (m, 4H); 7.84
7
7
1
(d, J=7.90Hz, 2H); 7.88 (d, J=7.60Hz, 2H); 8.81 (s, 1H); 11.51 (s,
+
1H). MS (ESI): 456.1 (C23
FN
3.26; N, 9.37%.
H
16Cl
2
FN
3
O
2
, [M+H] ). Anal. Calcd for
+
1.51 (s, 1H). MS (ESI): 404.1 (C23
: C, 68.40; H, 4.49; N, 10.40; Found: C,
8.65; H, 4.25; N, 10.56%.
H18ClN
3
O
2
, [M+H] ). Anal.
C
23
H
16Cl
2
3 2
O : C, 60.54; H, 3.53; N, 9.21; Found: C, 60.75; H,
3 2
Calcd for C23H18ClN O
6
4.4 Antiproliferative activity
4.3.14 1-(4-chlorophenyl)-N-((2-fluorobenzyl)oxy)-3-phenyl-1H-
The antiproliferative activities of the prepared compounds
pyrazole-4-carboxamide (9b)
White solid. Yield: 65%. Mp: 152-154 ºC. H NMR(500 MHz,
DMSO-d ): 5.03 (s, 2H); 7.24 (t, J=7.17Hz, 2H); 7.38-7.45 (m,
H); 7.50 (d, J=8.50Hz, 2H); 7.53-7.58 (m, 3H); 7.84 (d,
J=8.20Hz, 2H); 7.88 (d, J=7.90Hz, 2H); 8.83 (s, 1H); 11.53 (s,
were evaluated by using a standard (MTT)-based colorimetric
assay with some modification. Cell lines grew to log phase in
DMEM supplemented with 10% fetal bovine serum, under a
1
6
2
2
humidified atmosphere of 5% CO at 37 ˚C. Cell suspensions
were prepared and 100 μL/well dispensed into 96-well plates
+
5
1H). MS (ESI): 422.1 (C23H17ClFN
3
O
2
, [M+H] ). Anal. Calcd for
giving 10 cells/well. The plates were returned to the incubator
C
23
H
17ClFN : C, 65.49; H, 4.06; N, 9.96; Found: C, 65.54; H,
3
O
2
for 24 h to allow the cells to reattach. Subsequently, cells were
treated with the target compounds at increasing concentrations in
the presence of 10% FBS for 48 h. Then, cell viability was
assessed by the conventional 3-(4,5-dimethylthiazol- 2-yl)-2,5-
diphenyltetrazolium bromide (MTT) reduction assay and carried
out strictly according to the manufacturer instructions (Sigma).
The absorbance (OD490) was read on an ELISA reader (Tecan,
Austria).
4.03; N, 10.08%.
4.3.15 N-((2-chlorobenzyl)oxy)-1-(4-chlorophenyl)-3-phenyl-1H-
pyrazole-4-carboxamide (9c)
White solid. Yield: 78%. Mp: 177-178 ºC. H NMR(500 MHz,
DMSO-d ): 5.05 (s, 2H); 7.40 (t, J=7.32Hz, 3H); 7.50 (d,
J=8.50Hz, 3H); 7.55-7.60 (m, 3H); 7.85 (d, J=8.25Hz, 2H); 7.88
1
6
(
d, J=7.65Hz, 2H); 8.84 (s, 1H); 11.55 (s, 1H). MS (ESI): 438.1
+
(C
23
H
2
17Cl N
3
O
2
, [M+H] ). Anal. Calcd for C23
H
2
17Cl N
3
O
2
: C,
4.5 kinase assay
63.03; H, 3.91; N, 9.59; Found: C, 63.14; H, 4.03; N, 9.46%.
The MEK1 kinase assay was conducted by using a modified
35
procedure as described by Toshiyuki et al. . Tested compounds
were dissolved in DMSO and were present at increasing
concentrations. Briefly, non-phosphorylated myelin basic protein
was coated onto an ELISA plate, and the active form of BRAF
(Invitrogen) was mixed with unphosphorylated MEK1
(Millipore) and ERK2 (Invitrogen) in 10 μM ATP and 12.5 mM
4
1
.3.16 1-(4-chlorophenyl)-N-((2,4-dichlorobenzyl)oxy)-3-phenyl-
H-pyrazole-4-carboxamide (9d)
1
White solid. Yield: 81%. Mp: 172-174 ºC. H NMR(500 MHz,
DMSO-d
J=9.00Hz, 3H); 7.55-7.67 (m, 4H); 7.82 (d, J=7.90Hz, 2H); 7.88
6
): 5.02 (s, 2H); 7.40 (t, J=7.32Hz, 1H); 7.49 (t,
(
d, J=7.60Hz, 2H); 8.82 (s, 1H); 11.52 (s, 1H). MS (ESI): 472.0
2
MgCl containing MOPS buffer in the presence of various
concentrations of tested compounds. The phosphorylation of
+
(C
23
H
3
16Cl N
3
O
2
, [M+H] ). Anal. Calcd for (C23
H
3
16Cl N
3
O
2
: C,
58.44; H, 3.41; N, 8.89; Found: C, 58.57; H, 3.26; N, 9.01%.
MBP was detected by the anti-phospho-MBP antibody.
Detection of the effect of compounds on cell-based pERK1/2
activity in A549 cells was performed in ELISA kits (Invitrogen)
and strictly according to the manufacturer instructions.
4
4
.3.17 N-(benzyloxy)-1-(4-fluorophenyl)-3-phenyl-1H-pyrazole-
-carboxamide(10a)
1
White solid. Yield: 83%. Mp: 158-159 ºC. H NMR (500 MHz,
The kinase profiling of compound 7b against EGFR, HER-2,
FAK, Aurora-A, and VEGFR-2 were evaluated by the same
DMSO-d
H); 7.56 (t, J=7.95Hz, 2H); 7.85 (d, J=8.20Hz, 2H); 7.88 (d,
J=8.25Hz, 2H); 8.82 (s, 1H);11.48 (s, 1H). MS (ESI): 388.1
6
): 4.93 (s, 2H); 7.27 (t, J=9.00Hz, 2H); 7.36-7.46 (m,
36-38
6
protocol with our previously reported.
+
(C
23
H
18FN
3
O
2
, [M+H] ). Anal. Calcd for C23
3 2
H18FN O : C, 71.31;
H, 4.68; N, 10.85; Found: C, 71.39; H, 4.55; N, 11.03%.
4.6 Molecular docking
The X-ray crystal structure of the BRAF kinase domain in
an active configuration (MEK1/ K252A, PDB code: 3EQF,
Protein Data Bank) was used as the target structure in this
approach. The molecular docking procedure was performed by
using CDOCKER protocol for receptor-ligand interactions
section of DS 3.1 (Discovery Studio 3.1, Accelrys, Inc., San
4
.3.18 N-((2-fluorobenzyl)oxy)-1-(4-fluorophenyl)-3-phenyl-1H-
pyrazole-4-carboxamide (10b)
White solid. Yield: 65%. Mp: 151-153 ºC. H NMR(500 MHz,
1
DMSO-d
6
): 5.00 (s, 2H); 7.22-7.28 (m, 4H); 7.38-7.47 (m, 2H);
7
1
.56 (t, J=7.95Hz, 3H); 7.84-7.98 (m, 4H); 8.81 (s, 1H); 11.50 (s,
H). MS (ESI): 406.1 (C23
+
33
H
17
F
2
N
3
O
2
, [M+H] ). Anal. Calcd for
Diego, CA).
23 17 2 3 2
C H F N O : C, 68.14; H, 4.23; N, 10.37; Found: C, 68.27; H,
4.01; N, 10.53%.
Acknowledgment
This work was supported by National Natural Science
Foundation of China (No.21302002), Anhui Provincial Natural
Science Foundation (1408085QB33, 1508085MB33) and China
Postdoctoral Science Foundation (2014M552387).
4
.3.19 N-((2-chlorobenzyl)oxy)-1-(4-fluorophenyl)-3-phenyl-1H-
pyrazole-4-carboxamide (10c)
White solid. Yield: 80%. Mp: 135-136 ºC. H NMR(500 MHz,
DMSO-d ): 5.05 (s, 2H); 7.27 (t, J=9.00Hz, 2H); 7.40 (m, 3H);
.49-7.60 (m, 4H); 7.85 (d, J=8.20Hz, 2H); 7.88 (d, J=8.25Hz,
H); 8.83 (s, 1H); 11.53 (s, 1H). MS (ESI): 422.1
1
6
7
2
References and notes
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(C
23
H
17ClFN
3
O
2
, [M+H] ). Anal. Calcd for C23
3 2
H17ClFN O : C,
1
2.
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Graphical Abstract
Twenty
N-(benzyloxy)-1,3-diphenyl-1H-pyrazole-4-
Discovery of N-(benzyloxy)-1, 3-diphenyl-1H-pyrazole-
carboxamide derivatives have been designed and
synthesized as MEK inhibitors, compound 7b showed the
most potent inhibitory activity with IC50 of 91 nM for
MEK1 and GI50 value of 0.26 μM for A549 cells.
4
-carboxamide derivatives as potential antiproliferative
agents by inhibiting MEK
Xian-Hai Lv , Zi-Li Ren , Ben-Guo Zhou ,
a,b
b
b
c
a
a
a
a
a
Qing-Shan Li , Ming-Jie Chu , Dao-Hong Liu , Kai Mo , Li-Song Zhang , Xiao-Kang Yao , Hai-Qun
b*
Cao
a
School of Science, Anhui Agricultural University, Hefei 230036, P.R. China;
b
School of Plant Protection, Anhui Agricultural University, Hefei 230036, P.R. China
c
School of Medical Engineering, Hefei University of Technology, Hefei 230009, P.R. China