Synthesis, biological evaluation, and molecular docking studies of N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)aniline derivatives as novel anticancer agents

Article abstract of DOI:10.1016/j.bmc.2012.06.056

A series of N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)aniline derivatives (5a-8d) have been designed and synthesized, and their biological activities were also evaluated as potential antitumor and cyclin dependent kinase 2 (CDK2) inhibitors. Among all the compounds, compound 5a displayed the most potent CDK2/cyclin E inhibitory activity in vitro, with an IC₅₀ of 0.98 ± 0.06 μM. Antitumor assays indicated that compound 5a owned high antiproliferative activity against MCF-7 and B16-F10 cancer cell lines with IC₅₀ values of 1.88 ± 0.11 and 2.12 ± 0.15 μM, respectively. Docking simulation was performed to insert compound 5a into the crystal structure of CDK2 at active site to determine the probable binding model. Based on the preliminary results, compound 5a with potent inhibitory activity in tumor growth may be a potential anticancer agent.

Full text of DOI:10.1016/j.bmc.2012.06.056

Bioorganic & Medicinal Chemistry 20 (2012) 4895–4900
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation, and molecular docking studies
of N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)aniline derivatives
as novel anticancer agents
Xian-Feng Huang a, , Xiang Lu b, , Yong Zhang c, , Guo-Qiang Song a, Qi-Long He a, Qing-Shan Li b,
Xian-Hui Yang b, Yao Wei c, Hai-Liang Zhu b,
⇑
a School of Pharmaceutical Engineering & Life Science, Changzhou University, Changzhou 213164, People’s Republic of China
b State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
c Department of orthopaedics, An Hui Armed Police Corps Hospital, Hefei 230041, 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:
A series of N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)aniline derivatives (5a–8d) have been designed and
synthesized, and their biological activities were also evaluated as potential antitumor and cyclin depen-
dent kinase 2 (CDK2) inhibitors. Among all the compounds, compound 5a displayed the most potent
Received 8 June 2012
Revised 28 June 2012
Accepted 29 June 2012
Available online 10 July 2012
CDK2/cyclin E inhibitory activity in vitro, with an IC50 of 0.98 0.06
compound 5a owned high antiproliferative activity against MCF-7 and B16-F10 cancer cell lines with IC50
values of 1.88 0.11 and 2.12 0.15 M, respectively. Docking simulation was performed to insert com-
lM. Antitumor assays indicated that
l
Keywords:
pound 5a into the crystal structure of CDK2 at active site to determine the probable binding model. Based
on the preliminary results, compound 5a with potent inhibitory activity in tumor growth may be a poten-
tial anticancer agent.
N-((1,3-Diphenyl-1H-pyrazol-
4-yl)methyl)aniline derivatives
Cyclin dependent kinase 2 (CDK2)
Structure–activity relationship
Molecular docking
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
active pursuit of small molecule inhibitors of this enzyme as a
possible treatment against cancer and other hyper-proliferative
With the changes in the living habit and environment, cancer
has become the major cause of death in both developed and devel-
oping countries.1,2 All cancers are characterized by an abnormal
control of cell proliferation. This is caused by mutation or mis-reg-
ulation of cell-cycle regulatory genes and proteins.3 The progres-
sion through the cell cycle is orchestrated by a set of Ser/Thr
kinases called cyclin-dependent kinases (CDKs). These kinases
upon activation with their cyclins, phosphorylate and modulate
the activity of many target substrates such as organizational pro-
teins, transcription factors as well as proteins involved in the rep-
lication assembly and machinery of cells. In conjunction with
findings that implicate aberrant CDKs, especially CDK2 control in
the majority of cancer cases.4–9 In complex with cyclin E, CDK2
plays a paramount role during the G1/S transition of the cell cycle
while in complex with cyclin A it facilitates the progression of the S
phase of the cell cycle. Recent evidence also suggests that CDK2
may have a crucial role in the G2 phase of the cell cycle.4,10,11
The importance of CDK2 for cell cycle progression has led to an
disorders.4,12
All CDK inhibitors identified so far act by competing with ATP
for binding at the catalytic site of their kinase targets.8,13,14
Representative compounds of CDK inhibitors include flavopiridol
(1), 2-thioflavopiridol (2a), 2-oxoflavopiridol (2b), quercetin (3),
purvalanol (4), olomoucine (5) and related analogues (Fig. 1).
Among these structures, pyrazol ring is a promising skeleton that
has shown the good anti-CDK2 activity with selective and low
toxic properties, such as PNU-292137 (1), 1H-indazole-3-carbox-
ylic acid (4-sulfamoylphenyl)amide (2) and 4-amino-1H-pyra-
zole-3-carboxylic acid ethyl Ester (3) (Fig. 2). Several of them
have already undergone in vivo efficacy studies, preclinical and
clinical evaluation.8,15–20 In addition, benzamide derivates dis-
played ATP competitive inhibitory activities and low toxicities in
previous reports.21 As a continuation of our effort to optimize the
pyrazole derivatives for anticancer agents 22 and in the effort to en-
hance the affinity of CDK2, we tried to extend the CDK2 inhibitors
mutual interactions by introduction of benzamide skeletons to the
pyrazol rings. We thus went on to synthesize a group of substi-
tuted N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)aniline derivatives
to analyze their structure–activity relationships and to determine
the in vitro antiproliferative properties of these compounds against
cancer cell lines.
⇑
Corresponding author. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
These three authors equally contributed to this paper.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.056

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X.-F. Huang et al. / Bioorg. Med. Chem. 20 (2012) 4895–4900
OH
O
the synthetic compounds gave satisfactory elementary analytical
and spectroscopic data. 1H NMR and ESI-MS spectra were consis-
tent with the assigned structures.
OH
O
Cl
HO
O
X
H
HO
O
2.2. Bioactivity
OH
Cl
H
OH
To test the anticancer activities of the synthesized compounds,
we evaluated antiproliferative activities of compounds 5a–8d
against MCF-7 and B16-F10 cells. The results were summarized in
Table 1. These N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)aniline
derivatives (5a–8d) showed remarkable antiproliferative effects.
Among them, compound 5a displayed a more potent inhibitory
N
N
C(2)-Thioflavopiridol (X=S) (2a)
C(2)-Oxoflavopiridol (X=O) (2b)
Flavopiridol (1)
CO2H
activity (IC50 = 1.88 0.11
for B16-F10 cells, respectively), than the positive control olomou-
cine (IC50 = 130 5.62 for MCF-7, IC50 = 105 6.28 for
lM for MCF-7 and IC50 = 2.12 0.15 lM
OH
O
O
HN
Cl
l
M
lM
B16-F10 cells). An overview of the potency data we obtained by
screening compounds 5a–8d against the CDK2/cyclin E holoen-
zyme indicates that 5a, 5b and 5c showed strong inhibitory effect
N
N
N
OH
OH
HO
HO
N
H
N
(IC50 = 1.18 0.09 lM, 1.85 0.121 lM and 0.98 0.06 lM, respec-
tively). Compound 5a displayed the most potent anti-CDK2/cyclin E
activity (Table 1). This result indicated the anti-proliferative effect
was produced partly by connection of CDK2 protein and the
compounds.
Purvalanol (4)
Quercetin (3)
Structure–activity relationships in these N-((1,3-diphenyl-1H-
pyrazol-4-yl)methyl)aniline derivatives demonstrated that com-
pounds with para electron-withdrawing substituents showed
more potent activities than those with electron-donating substitu-
ents in the A-ring. A comparison of the para substituents on the
A-ring demonstrated that an electron-withdrawing group have
improved antiproliferative activity and the potency order is
F > Cl > Br, whereas a methyl group substituent had minimal
effects. In the case of constant A ring substituents, change of
substituents on B ring could also affect the activities of these com-
pounds. Among these compounds, compounds with para electron-
withdrawing substituted (5a–6d) showed stronger anticancer
NH
N
N
N
N
HO
N
H
Olomoucine (5)
Figure 1. Chemical structures of anti-CDK2 inhibitors.
2. Results and discussion
2.1. Chemistry
activities and the strength order was similar with
A ring:
Cl > Br > Me > MeO, followed that 8d showed the lowest activity.
Among all the compounds, 5a with para-F and Cl group in the A
ring and B ring respectively, led to the best activity.
The synthetic route of the N-((1,3-diphenyl-1H-pyrazol-4-
yl)methyl)aniline derivatives (5a–8d) are outlined in Scheme 1.
Compounds 5–8 were prepared by the simple condensation and
cyclization of phenylhydrazine and various substituted acetophe-
nones. Synthesis of N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)
aniline derivatives (5a–8d) is synthesized from 1,3-diphenyl-1H-
pyrazole-4-carbaldehyde (5–8) and substituted benzamides by
direct reductive amination using NaBH4/I2 as a reducing agent to
give the desired compounds 5a–8d (Table 1). Among these com-
pounds, 5a–6d and 8a–8d are reported for the first time. All of
To gain better understanding on the potency of the studied
compounds and guide further SAR studies, we proceeded to exam-
ine the interaction of compound 5a with CDK2 (PDB code: 1H0V).
The molecular docking was performed by simulation of compound
5a into the ATP binding site of CDK2. All docking runs were applied
LigandFit Dock protocol of Discovery Studio 3.1. The binding
modes of compound 5a and CDK2 were depicted in Figure 3. All
the amino acid residues which had interactions with CDK2 were
H
N
O
O
O
H2N
S
N
HN
O
N
H
NH
N
PNU-292137 (1)
1H-Indazole-3-carboxylic Acid (4-Sulfamoylphenyl)amide (2)
O
NH2
NH
O
N
4-Amino-1H-pyrazole-3-carboxylic Acid Ethyl Ester (3)
Figure 2. ATP-competitive inhibitors of CDK2 with pyrazol core.

X.-F. Huang et al. / Bioorg. Med. Chem. 20 (2012) 4895–4900
4897
R1
R1
CHO
N
O
CH3
H
N
a
b
CH3
NH2
N
N
NH
R1
1-4
5-8
R2
d
R2
R1
R1
R1
CHO
N
CH
N
H2C NH
c
NH2
N
N
R2
N
N
N
5a-8d
a-d
5-8
Scheme 1. General synthesis of N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)aniline derivatives (5a–8d). Reagents and conditions: (a) ethanol, 50–60 °C, 3 h; (b) DMF, POCl3,
50–60 °C, 5 h; (c) ethanol, 50–60 °C, 5 h; (d) tetrahydrofuran, NaBH4/I2 , 50–60 °C, 12 h.
Table 1
Inhibition of CDK2/cyclin E and in vitro antiproliferative activity of N-((1,3-diphenyl-
1H-pyrazol-4-yl)methyl)aniline derivatives
R1
A
N
H
N
N
R2
B
Compound
R1(p-)
R2(p-)
IC50(lM)
MCF-7
B16-F10
CDK2/cyclin E
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
F
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Me
Me
Me
Me
OMe
OMe
OMe
OMe
1.88 0.11
2.28 0.16
2.62 0.17
3.11 0.19
3.96 0.18
4.15 0.23
5.00 0.24
5.38 0.27
5.88 0.26
6.02 0.36
6.89 0.33
7.26 0.45
5.98 0.32
6.25 0.39
7.02 0.46
8.16 0.58
130 5.62
2.12 0.15
2.45 0.18
3.01 0.19
3.48 0.20
4.18 0.26
4.29 0.29
5.27 0.38
5.98 0.46
5.67 0.40
6.01 0.47
6.76 0.46
7.38 0.51
6.58 0.43
7.18 0.45
8.06 0.55
8.38 0.66
105 6.28
0.98 0.06
1.18 0.09
1.85 0.12
2.08 0.15
2.36 0.18
2.56 0.17
2.78 0.16
3.02 0.26
3.60 0.26
3.89 0.30
4.15 0.31
4.55 0.33
4.02 0.30
4.28 0.29
4.96 0.25
5.06 0.39
5.00 0.45
Cl
Br
Me
F
Cl
Br
Me
F
Cl
Br
Me
F
Cl
Br
Me
Figure 3. Compound 5a (colored by atom: carbons: gray; nitrogen: blue; oxygen:
red;) is bond into ATP binding sites of CDK2 (entry 1H0V in the Protein Data Bank).
Olomoucine
The dotted lines show the hydrogen bonds and the yellow line show the
interactions.
p-cation
exhibited. In the binding mode, compound 5a is nicely bound to
the ATP binding site of CDK2 via hydrogen bonds and -cation
p
3. Conclusion
interactions. The nitrogen atom of the amide bond formed one
hydrogen bond with the carbonyl group oxygen of ASP 86 (bond
length: ASP 86 N–HÁ Á ÁO = 1.312 Å; bond angle: ASP 86 N–
HÁ Á ÁN = 159.0°). The binding modeling also showed that there
In this study, a series of novel N-((1,3-diphenyl-1H-pyrazol-4-
yl)methyl)aniline derivatives (5a–8d) had been synthesized and
evaluated their biological activities. These compounds exhibited
potent CDK2 inhibitory activities and antiproliferative activities
against MCF-7 and B16-F10 cells. Among all of the compounds,
5a showed the most potent inhibition activity which inhibited
the growth of MCF-7 and B16-F10 cell lines with IC50 values of
was a
p–cation interaction between benzene ring of 5a and the
NH of LYS 89. Overall, these results of the molecular docking study
showed that the N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)aniline
derivatives could act synergistically to interact with the ATP bind-
ing site of CDK2, suggested that compound 5a is a potential inhib-
itor of CDK2.
1.88 0.11 and 2.12 0.15
l
M and inhibited the CDK2/cyclin E
M. Molecular
holoenzyme activities with IC50 of 0.98 0.06
l

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X.-F. Huang et al. / Bioorg. Med. Chem. 20 (2012) 4895–4900
docking was further performed to study the inhibitor-CDK2 pro-
tein interactions. After analysis of the binding model of compound
4.3.3. 4-Bromo-N-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)benzenamin (5c)
5a with CDK2, it was found that a hydrogen bond and a
p-cation
Brown powders, yield 75%, mp: 127–129 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 4.22 (s, 2H); 6.19 (s, 1H); 6.63 (d, J = 5.0 Hz, 2H);
7.22 (d, J = 4.7 Hz, 2H); 7.32 (t, J = 4.4 Hz, 1H); 7.51 (t, J = 3.6 Hz,
4H); 7.81 (d, J = 4.6 Hz, 2H); 7.87 (d, J = 4.6 Hz, 2H); 8.57 (s, 1H).
MS (ESI): 439.2 (C22H18BrClN3, [M+H]+). Anal. Calcd for
interaction with the protein residues in the ATP binding site of
CDK2 might play a crucial role in its anti-CDK2 and antiprolifera-
tive activities. The information of this work might be helpful for
the design and synthesis of a leading compound 5a toward the
development of new therapeutic agent to fight against cancer.
C22H17BrClN3: C, 60.22; H, 3.91; N, 9.58. Found: C, 60.42; H,3.90;
N, 9.55 %.
4. Experiments
4.3.4. N-((3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)-4-methylbenzenamine (5d)
4.1. Materials and measurements
Yellow powders, yield 85%, mp: 103–104 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 2.15 (s, 3H); 4.20 (d, J = 4.8 Hz, 2H); 5.70 (s, 1H);
6.59 (d, J = 8.4 Hz, 2H); 6.91 (d, J = 8.4 Hz, 2H); 7.32 (t, J = 7.3 Hz,
1H); 7.49 (d, J = 3.7 Hz, 4H); 7.51–7.88 (m, 4H); 8.57 (s, 1H). MS
(ESI): 374.5 (C23H21ClN3, [M+H]+). Anal. Calcd for C23H20ClN3: C,
73.89; H, 5.39; N, 11.24. Found: C, 73.60; H, 5.40; N, 11.28 %.
All chemicals and reagents used in current study were of analyt-
ical grade. All the 1H NMR spectra were recorded on a Bruker
DPX300 model Spectrometer in DMSO-d6 and chemical shifts were
reported in ppm (d). ESI-MS spectra were recorded on a Mariner
System 5304 Mass spectrometer. Elemental analyses were per-
formed on a CHN-O-Rapid instrument. TLC was performed on the
glassbacked 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.
4.3.5. N-((3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)-4-fluorobenzenamine (6a)
Yellow powders, yield 77%, mp: 145–147 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 4.20 (s, 2H); 5.68 (s, 1H); 6.60 (d, J = 4.9 Hz, 2H);
6.91 (d, J = 4.9 Hz, 2H); 7.27–7.37 (m, 3H); 7.51 (t, J = 4.8 Hz, 2H);
7.84–7.88 (m, 4H); 8.55 (s, 1H). MS (ESI): 423.8 (C22H18BrFN3,
[M+H]+). Anal. Calcd for C22H17BrFN3: C, 62.57; H, 4.06; N, 9.95.
Found: C, 62.33; H,4.08; N, 9.99 %.
4.2. 1,3-Diphenyl-1H-pyrazole-4-carbaldehyde (5–8)
The starting material 1,3-diphenyl-1H-pyrazole-4-carbalde-
hyde (5–8) was synthesized based on a literature method.23
1-phenyl-2-(1-phenylethylidene)hydrazine (1–4) (3.6 g, 0.015 mol)
was added to a cold solution of DMF (25 mL), then POCl3 (5 mL)
was added and the resulting mixture was stirred at 50–60 °C for
6 h. The mixture was poured into ice-cold water. A saturated
solution of sodium hydroxide was added to neutralize the mixture,
the solid precipitate was filtered, washed with water, dried and
recrystallized from ethanol.
4.3.6. N-((3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)-4-chlorobenzenamine (6b)
Brown powders, yield 85%, mp: 126–128 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 4.23 (s, 2H); 6.17 (s, 1H); 6.68 (d, J = 5.2 Hz, 2H);
7.12 (d, J = 5.2 Hz, 2H); 7.33 (t, J = 4.4 Hz, 1H); 7.52 (t, J = 4.8 Hz,
2H); 7.65 (d, J = 5.1 Hz, 2H); 7.76 (d, J = 5.0 Hz, 2H); 7.87 (d, J =
4.7 Hz, 2H); 8.58 (s, 1H). MS (ESI): 439.8 (C22H18BrClN3, [M+H]+).
Anal. Calcd for C22H17BrClN3: C, 60.22; H, 3.91; N, 9.58. Found: C,
60.45; H, 3.92; N, 9.61 %.
4.3. N-((1,3-Diphenyl-1H-pyrazol-4-yl)methyl)aniline
derivatives (5a–8d)
4.3.7. 4-Bromo-N-((3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)benzenamine (6c)
To
a solution of 1,3-diphenyl-1H-pyrazole-4-carbaldehyde
Brown powders, yield 68%, mp: 127–129 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 4.22 (s, 2H); 6.17 (s, 1H); 6.63 (d, J = 8.8 Hz, 2H);
7.22 (d, J = 8.8 Hz, 2H); 7.32 (t, J = 7.4 Hz, 1H); 7.52 (t, J = 7.9 Hz,
2H); 7.65 (d, J = 8.6 Hz, 2H); 7.75 (d, J = 8.4 Hz, 2H); 7.87 (d, J =
8.4 Hz, 2H); 8.56(s, 1H). MS (ESI): 484.1 (C22H18Br2N3, [M+H]+).
Anal. Calcd for C22H17Br2N3: C, 54.68; H, 3.55; N, 8.70. Found: C,
54.88; H, 3.56; N, 8.67 %.
(0.278 g, 0.001 mol) in 10 mL of methanol, substituted benzamides
(0.145 g, 0.001 mol) and iodine (0.051 g, 0.002 mol) were added
with stirring at room temperature. To the stirred solution, sodium
borohydride (0.055 g, 0.015 mol) was added slowly. Stirring was
continued for 12 h. The precipitate formed was filtered, washed
with water, dried and purified by column chromatography using
petroleum ether and ethyl acetate (5:1).24
4.3.8. N-((3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)-4-methylbenzenamine (6d)
4.3.1. N-((3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)-4-fluorobenzenamine (5a)
White powders, yield 70%, mp: 105–106 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 2.15 (s, 3H); 4.20 (d, J = 4.9 Hz, 2H); 5.70 (s, 1H);
6.59 (d, J = 8.2 Hz, 2H); 6.91 (d, J = 8.0 Hz, 2H); 7.32 (t, J = 7.3 Hz,
1H); 7.51(t, J = 7.8 Hz, 2H); 7.64 (d, J = 8.2 Hz, 2H); 7.77 (d, J =
8.4 Hz, 2H); 7.87 (d, J = 8.0 Hz, 2H); 8.57 (s, 1H). MS (ESI): 419.7
(C23H21BrN3, [M+H]+). Anal. Calcd for C23H20BrN3: C, 66.04; H,
4.82; N, 10.04. Found: C, 66.29; H, 4.80; N, 10.00 %.
White powders, yield 73%, mp: 136–137 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 4.19 (d, J = 4.9 Hz, 2H); 6.16 (t, J = 4.9 Hz, 1H);
6.66 (d, J = 8.9 Hz, 2H); 7.10 (d, J = 8.8 Hz, 2H); 7.25–7.34 (m,
3H); 7.50 (t, J = 7.9 Hz, 2H); 7.79–7.87 (m, 4H); 8.56 (s, 1H). MS
(ESI): 378.3 (C22H18ClFN3, [M+H]+). Anal. Calcd for C22H17ClFN3:
C, 69.93; H, 4.53; N, 11.12. Found: C, 69.75; H, 4.55; N, 11.16%.
4.3.2. 4-Chloro-N-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)benzenamine (5b)
4.3.9. 4-Fluoro-N-((1-phenyl-3-p-tolyl-1H-pyrazol-4-
yl)methyl)benzenamine (7a)
White powders, yield 88%, mp: 114–115 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 4.23 (s, 2H); 6.16 (s, 1H); 6.67 (d, J = 5.2 Hz, 2H);
7.11 (d, J = 5.2 Hz, 2H); 7.32 (t, J = 4.4 Hz, 1H); 7.49-7.52 (m, 4H);
7.82 (d, J = 5.0 Hz, 2H); 7.87 (d, J = 4.8 Hz, 2H); 8.57 (s, 1H). MS
(ESI): 395.6 (C22H18Cl2N3, [M+H]+). Anal. Calcd for C22H17Cl2N3: C,
67.01; H, 4.35; N, 10.66; Found: C, 67.25; H,4.37; N, 10.63 %.
White powders, yield 70%, mp: 75–77 °C; 1H NMR (300 MHz,
DMSO- d6, d ppm): 2.32 (s, 3H); 4.17 (d, J = 4.9 Hz, 2H); 5.87 (s,
1H); 6.59–6.66 (m, 2H); 6.92 (t, J = 9.0 Hz, 2H); 7.23–7.31 (m,
3H); 7.49 (t, J = 8.0 Hz, 2H); 7.68 (d, J = 8.0 Hz, 2H); 7.86 (d,
J = 8.4 Hz, 2H); 8.53 (s, 1H). MS (ESI): 358.5 (C23H21FN3, [M+H]+).

X.-F. Huang et al. / Bioorg. Med. Chem. 20 (2012) 4895–4900
4899
Anal. Calcd for C23H20FN3: C, 77.29; H, 5.64; N, 11.76; Found: C,
77.33; H, 5.61; N, 11.79 %.
2H); 5.68 (s, 1H); 6.59 (d, J = 8.2 Hz, 2H); 6.91 (d, J = 8.0 Hz, 2H);
7.01 (d, J = 8.8 Hz, 2H); 7.29 (t, J = 7.3 Hz, 1H); 7.50 (t, J = 7.8 Hz,
2H); 7.75 (d, J = 8.8 Hz, 2H); 7.85 (d, J = 7.8 Hz, 2H); 8.51 (s, 1H).
MS (ESI): 369.1 (C24H24N3O, [M+H]+). Anal. Calcd for C24H23N3O:
C, 78.02; H, 6.27; N, 11.37. Found: C, 78.30; H, 6.25; N, 11.39 %.
4.3.10. 4-Chloro-N-((1-phenyl-3-p-tolyl-1H-pyrazol-4-
yl)methyl)benzenamine (7b)
Yellow powders, yield 81%, mp: 68–70 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 2.31 (s, 3H); 4.19 (d, J = 4.8 Hz, 2H); 6.15 (s,
1H); 6.66 (d, J = 8.8 Hz, 2H); 7.10 (d, J = 8.8 Hz, 2H); 7.23–7.32
(m, 3H); 7.49 (t, J = 7.9 Hz, 2H); 7.66 (d, J = 8.0 Hz, 2H); 7.85 (d,
J = 7.9 Hz, 2H); 8.53 (s, 1H). MS (ESI): 374.5 (C23H21ClN3, [M+H]+).
Anal. Calcd for C23H20ClN3: C, 73.89; H, 5.39; N, 11.24. Found: C,
73.60; H, 5.42; N, 11.29 %.
4.4. Antiproliferation assay
The antiproliferative activities of the prepared compounds
against MCF-7 and B16-F10 cells were evaluated as described else-
where with some modifications.25 Target tumor cell lines were
grown to log phase in RPMI 1640 medium supplemented with
10% fetal bovine serum. After diluting to 2 Â 104 cells mLÀ1 with
4.3.11. 4-Bromo-N-((1-phenyl-3-p-tolyl-1H-pyrazol-4-
yl)methyl)benzenamine (7c)
the complete medium, 100 lL of the obtained cell suspension
was added to each well of 96-well culture plates. The subsequent
incubation was permitted at 37 °C, 5% CO2 atmosphere for 24 h be-
fore the cytotoxicity assessments. Tested samples at pre-set con-
centrations were added to six wells with Olomoucine as positive
White powders, yield 88%, mp: 77–79 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 2.34 (s, 3H); 4.21(s, 2H); 6.19 (s, 1H); 6.63 (d,
J = 8.8 Hz, 2H); 7.21–7.33 (m, 5H); 7.51 (t, J = 7.9 Hz, 2H); 7.67 (d,
J = 8.0 Hz, 2H); 7.87 (d, J = 8.0 Hz, 2H); 8.55 (s, 1H). MS (ESI):
418.1 (C23H21BrN3, [M+H]+). Anal. Calcd for C23H20BrN3: C, 66.04;
H, 4.82; N, 10.04. Found: C, 65.87; H, 4.81; N, 10.08 %.
references. After 48 h exposure period, 40 lL of PBS containing
0.5 mg mLÀ1 of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide)) was added to each well. After 4 h incuba-
tion, the optical absorbance was measured at 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.
4.3.12. 4-Methyl-N-((1-phenyl-3-p-tolyl-1H-pyrazol-4-
yl)methyl)benzenamine (7d)
White powders, yield 76%, mp: 69–71 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 2.07 (s, 3H); 2.25(s, 3H); 4.11 (d, J = 4.9 Hz,
2H); 5.62 (s, 1H); 6.51 (d, J = 8.0 Hz, 2H); 6.84 (d, J = 8.0 Hz, 2H);
7.01 (d, J = 8.8 Hz, 2H); 7.29 (t, J = 7.3 Hz, 1H); 7.42 (t, J = 7.8 Hz,
2H); 7.63 (d, J = 8.8 Hz, 2H); 7.80 (d, J = 7.9 Hz, 2H); 8.51 (s, 1H).
MS (ESI): 354.3 (C24H24N3, [M+H]+). Anal. Calcd for C24H23N3: C,
81.55; H, 6.56; N, 11.89. Found: C, 81.23; H, 6.53; N, 11.85 %.
4.5. CDK2/cyclin E inhibition assay
The ability of the test compounds 5a–8d to inhibit CDK2/cyclin
E was determined according to previously reported method
8
.
4.6. Docking simulations
4.3.13. 4-Fluoro-N-((3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazol-4-yl)methyl)benzenamine (8a)
Molecular docking of compound 5a into the three-dimensional
X-ray structure of CDK2 (PDB code: 1H0V) was carried out using
LigandFit Dock protocol of Discovery Studio 3.1.
Orange powders, yield 71%, mp: 78–80 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 3.81 (s, 3H); 4.20 (s, 2H); 6.15 (s, 1H); 6.69 (d,
J = 5.3 Hz, 2H); 7.01 (d, J = 5.2 Hz, 2H); 7.10 (d, J = 5.3 Hz, 2H);
7.32 (t, J = 4.4 Hz, 1H); 7.53 (t, J = 4.7 Hz, 2H); 7.71 (d, J = 5.2 Hz,
2H); 7.85 (d, J = 4.9 Hz, 2H); 8.55 (s, 1H). MS (ESI): 374.9
(C23H21ClN3O, [M+H]+). Anal. Calcd for C23H20FN3O: C, 73.98; H,
5.40; N, 11.25. Found: C, 73.91; H, 5.38; N, 11.21 %.
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Orange powders, yield 71%, mp: 72–74 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 3.79 (s, 3H); 4.20 (s, 2H); 6.16 (s, 1H); 6.68 (d,
J = 5.3 Hz, 2H); 7.02 (d, J = 5.2 Hz, 2H); 7.12 (d, J = 5.3 Hz, 2H);
7.30 (t, J = 4.4 Hz, 1H); 7.50 (t, J = 4.7 Hz, 2H); 7.73 (d, J = 5.2 Hz,
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pyrazol-4-yl)methyl)benzenamine (8c)
Yellow powders, yield 80%, mp: 86–88 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 3.77(s, 3H); 4.19 (d, J = 4.8 Hz, 2H); 6.19 (t,
J = 4.8 Hz, 1H); 6.62 (d, J = 8.8 Hz, 2H); 7.24 (d, J = 6.9 Hz, 2H);
7.28–7.33 (m, 3H); 7.50 (t, J = 7.9 Hz, 2H); 7.79–7.87 (m, 4H);
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4.3.16. N-((3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-
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