Synthesis, molecular modeling and biological evaluation of cinnamic acid derivatives with pyrazole moieties as novel anticancer agents

Article abstract of DOI:10.1039/c4ra05257a

A series of pyrazole derivatives (1e-30e) has been designed and synthesized, and their biological activities were evaluated for EGFR and HER-2 inhibition and tumor cell antiproliferation. Among the compounds synthesized, compound 30e exhibited excellent enzyme inhibitory activity (IC₅₀ = 0.21 ± 0.05 μM for EGFR and IC₅₀ = 1.08 ± 0.15 μM for HER-2). Compound 30e also showed the most potent antiproliferative activity, which inhibited the growth of MCF-7 and B16-F10 cell lines with IC₅₀ values of 0.30 ± 0.04 and 0.44 ± 0.05 μM, respectively. The molecular docking study was performed to analyze the probable binding models and the 3D-QSAR models were built for the rational design of EGFR/HER-2 inhibitors. Based on the results obtained, compound 30e with potent EGFR and HER-2 inhibitory activity may be a potential anticancer agent. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra05257a

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Synthesis, molecular modeling and biological
evaluation of cinnamic acid derivatives with
pyrazole moieties as novel anticancer agents
Cite this: RSC Adv., 2014, 4, 37197
Wei-Ming Zhang,†ab Man Xing,†ab Ting-Ting Zhao,ab Yu-Jia Ren,ab Xian-Hui Yang,ab
ab
ab
Yu-Shun Yang,ab Peng-Cheng Lv
and Hai-Liang Zhu
*
*
A series of pyrazole derivatives (1e–30e) has been designed and synthesized, and their biological activities
were evaluated for EGFR and HER-2 inhibition and tumor cell antiproliferation. Among the compounds
synthesized, compound 30e exhibited excellent enzyme inhibitory activity (IC50 ¼ 0.21 ꢀ 0.05 mM for
EGFR and IC50 ¼ 1.08 ꢀ 0.15 mM for HER-2). Compound 30e also showed the most potent
antiproliferative activity, which inhibited the growth of MCF-7 and B16-F10 cell lines with IC50 values of
0.30 ꢀ 0.04 and 0.44 ꢀ 0.05 mM, respectively. The molecular docking study was performed to analyze
the probable binding models and the 3D-QSAR models were built for the rational design of EGFR/HER-2
inhibitors. Based on the results obtained, compound 30e with potent EGFR and HER-2 inhibitory activity
may be a potential anticancer agent.
Received 11th June 2014
Accepted 1st August 2014
DOI: 10.1039/c4ra05257a
www.rsc.org/advances
intracellular tyrosine kinase domain to block the auto-phos-
phorylation of tyrosine kinase (TK), disrupt the activation of
Introduction
signal-transduction cascades, and then interfere cell prolifera-
tion, angiogenesis, invasion and metastasis, has accordingly
and historically been a successful area of research for the
advancement of cancer drugs.12,13 Getinib and erlotinib (Fig. 1)
are the representative drugs for this kind of inhibitors and have
been approved by US-FDA in these years.14,15
Although extensive efforts have been applied to co-deliver
anticancer therapeutics by nanoparticles to induce synergistic
anticancer effects, conventional anticancer therapy using cyto-
toxic drugs lacks selectivity and is prone to toxicity and drug
resistance.1,2 Therefore, targeted therapies to minimize toxicity
to healthy tissues have been attracting extensive attention, such
as using small molecules, peptides or aptamers.3,4 Actually,
epidermal growth factor receptor (EGFR) kinase, playing a key
role in signal transduction pathways that regulate cell division,
differentiation and migration, has been proposed as a prom-
ising target for drug design. Thus, EGFR inhibitors were widely
used to treat human cancers.5–7 The EGFR family comprises the
four members of EGFR/erbB-1/HER-1, erbB-2/Neu/HER-2, erbB-
3/HER-3, and erbB-4/HER-4, which belong to the type I receptor
tyrosine kinase (RTK) class. They play an important role in the
mediating growth factor signaling.8,9 The abnormal signaling of
these pathways results in disregulated cell proliferation, evasion
from apoptosis, angiogenesis, migration, and metastasis.
Especially, the overexpression of EGFR and HER-2 was observed
in many human cancers including bladder, breast, colon, and
lung cancers.10,11 As reported, using small molecules to inhibit
kinase activity of EGFR and HER-2 by binding the ATP site of the
Apart from the above two agents, several other compounds
(Fig. 1 and 2), compounds containing pyrazole and cinnamyl
moiety, for example, compound A and compound E, were found
to possess high inhibitory activity against EGFR and HER-2.16–21
Pyrazole motif displays a variety of pharmacological activities,
such as anti-inammatory,22 antibacterial–antifungal,23 inhibi-
tion against cyclooxigenase-2,24 p38 MAP kinase25 and CDK2/
Cyclin A.26,27 Therefore, the pyrazole ring is an advantageous
choice for the synthesis of pharmaceutical compounds with
different biological activities and good safety proles.28–30 Cin-
namoyl moiety was also demonstrated to possess a wide range
of pharmacological activities. Particularly, cinnamic acid ester
derivatives showed potential antitumor activity.31,32 For
instance, Qian et al. described the SAR of the novel series of
cinnamic acid metronidazole ester derivatives, and the biolog-
ical activity evaluation indicated that cinnamic acid ester
derivatives are potent inhibitors of EGFR and HER-2.20 More-
over, other studies also demonstrated that the incorporation of
cinnamoyl moiety could contribute to the binding of target
compounds with EGFR and HER-2 active site.33
aNanjing Institute for the Comprehensive Utilization of Wild Plant, Nanjing 210042,
People's Republic of China. E-mail: zhuhl@nju.edu.cn; Fax: +86-25-8359 2672; Tel:
+86-25-8359 2572
bState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, People's Republic of China. E-mail: pclv@nju.edu.cn
† These two authors equally contributed to this paper.
Encouraged by the above mentioned research, herein we per-
formed the synthesis and biological evaluation of a series of novel
pyrzole derivatives possessing pyrazole–cinnamyl moiety. Enzyme
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prepared by treating different substituted acetophenone (a)
with phenylhydrazine hydrochloride in the presence of sodium
acetate. Secondly, 1,3-diphenyl-1H-pyrazole-4-carbaldehyde (c)
was afforded by treating compound b with POCl3. Thirdly,
different substituted (1,3-diphenyl-1H-pyrazol-4-yl)methanol (d)
was gotten by the reaction of compound c with sodium boro-
hydride in DCM. Finally, compound d was coupled with
different substituted cinnamic acid in the presence of DMAP
and K2CO3. Twenty-seven out of the thirty target compounds
(1e–30e) were reported for the rst time. All of the synthetic
compounds gave satisfactory elementary analytical and spec-
troscopic data. 1H NMR and ESI-MS spectra were consistent
with the assigned structures.
Results and discussion
The synthesized compounds were evaluated for their anti-
proliferative activities against MCF-7 and B16-F10 cells. The
results were summarized in Table 2. As shown in Table 2, pyr-
azole derivatives bearing the pyrazole–cinnamyl moiety exhibi-
ted remarkable antiproliferative effects. Among them,
compound 30e displayed the most potent antiproliferative
activity (IC50 ¼ 0.30 ꢀ 0.04 mM for MCF-7 and IC50 ¼ 0.44 ꢀ 0.05
mM for B16-F10). Subsequently, structure-activity relationships
(SAR) studies were performed by modication of the parent
compound to determine how the substituents of the subunits
affect the antiproliferative activities. Inspection of the chemical
structures of the compounds 1e–30e suggested that they could
be divided into two subunits: A-ring and B-ring (Table 1).
As shown in Table 2, it was revealed that different substitutes
on the A-ring led to different antiproliferative activities. A
comparison of the para substituents on the A-ring demon-
strated that an electron-donating group could improve their
antiproliferative activity, and the potency order was OMe > Me.
Similarly, change of substituents on the B-ring could also affect
the antiproliferative activities. Among them, compounds with
electron-donating substituent at para position showed higher
antiproliferative activity and the potency order was similar with
A-ring: OMe > Me, while compounds with electron-withdrawing
substituents led to a drop for the antiproliferative activity. The
potency order of para-substituents compounds is that: OMe >
Me > H > Br > Cl > F. Among all the compounds tested,
compound 30e that has para-OMe group on both A-ring and B-
ring showed the best antiproliferative activity.
Fig. 1 Chemical structures of antimitotic agents and lead EGFR
inhibitors.
A solid-phase ELISA assay was performed with the aim to
examine whether this kind of compounds could inhibit the
autophosphorylation of EGFR and HER-2 kinases. As shown
in Table 2, it was observed that pyrazolyl–cinnamyl derivatives
showed good inhibitory activities against EGFR and HER-2.
Among them, compound 30e which has the best anti-
proliferative activity, also displayed the most potent inhibi-
tory activity (IC50 ¼ 0.21 ꢀ 0.05 mM for EGFR and IC50 ¼ 1.08 ꢀ
0.15 mM for HER-2). Subsequently, using Discovery Studio 3.1
(Discovery Studio 3.1, Accelrys, Inc. San Diego, CA), a molec-
ular modeling study was performed to explore the interaction
Fig. 2 Hybrid analogue 30e from compound A and compound E.
inhibitory results show that some of them are potential EGFR and
HER-2 inhibitors. Molecular modeling studies were also per-
formed to understand the binding mode between this kind of
inhibitor and the active site of EGFR/HER-2 crystal structures.
Chemistry
The synthetic route for pyzazole (1e–30e) is outlined in Scheme between the synthesized compounds and the active site of
1. Firstly, 1-phenyl-2-(1-phenylethylidene)hydrazine (b) was EGFR/HER-2. In the present study, the crystal structures of
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Scheme 1 General synthesis of compounds 1e–30e. Reagents and conditions: (i) ethanol, sodium acetate, 50–60 ꢁC, 3h; (ii) POCl3, DMF,
50–60 ꢁC, 6 h; (iii) sodium borohydride, DCM, RT, 8 h. (iv) DMAP, K2CO3, DCM, RT, 8 h.
EGFR (PDB Code: 1M17.pdb) and HER-2 (PDB Code:
3PP0.pdb) were chosen, which were obtained from the RCSB
protein data bank (http://www.pdb.org). Aer preparing
Table 1 Structure of compounds 1e–30e
the receptor and ligands, the site sphere was selected based
on the ligand binding location. The binding models of the
most potent compounds with corresponding target were
depicted in Fig. 3 (30e with 1M17) and Fig. 4 (30e with 3PP0),
respectively.
As shown in Fig. 3, compound 30e was nicely bound to the
ATP binding site of EGFR via hydrophobic interactions and
binding is stabilized by a hydrogen bond and a Pi-Sigma
interaction. The oxygen atom of the OMe group in A-ring
formed one hydrogen bond with the amino hydrogen of Lys A:
ꢁ
˚
721 (N–H/O ¼ 2.477 A; 130.6 ). A Pi-Sigma interaction was also
formed between Leu694 and the benzene ring of compound 30e
Cpd
R1
R2
Cpd
R1
R2
˚
(bond length: 2.48 A), which could enhance the binding action
1e
2e
3e
4e
5e
6e
7e
8e
H
H
H
H
H
H
F
F
F
F
F
H
F
Cl
Br
Me
OMe
H
F
Cl
Br
Me
OMe
H
F
Cl
16e
17e
18e
19e
20e
21e
22e
23e
24e
25e
26e
27e
28e
29e
30e
Cl
Cl
Cl
Me
Me
Me
Me
Me
Br
between receptor EGFR and compound 30e. Similarly, it can be
seen from Fig. 4 that compound 30e can bind to the active site
of HER2 via a hydrogen bond. The oxygen atom of the OMe
group in A-ring formed one hydrogen bond with the amino
Me
OMe
H
F
Cl
ꢁ
˚
hydrogen of Met A: 801 (N–H/O ¼ 2.278 A; 145.8 ).
Br
Finally, in order to give a systematic evaluation on pyrazole
derivatives as antitumor agents and to discover more potent
and selective dual EGFR/HER-2 inhibitors, a 3D-QSAR model
was initiated by using a novel 3D-QSAR protocol of Discovery
Studio 3.1. Thirty compounds with denite IC50 values against
EGFR and HER-2 inhibitors were selected as the model
dataset. By convention, we used the pIC50 scale (ꢂlg IC50), in
Me
OMe
H
F
Cl
Br
Me
OMe
9e
Me
10e
11e
12e
13e
14e
15e
OMe
OMe
OMe
OMe
OMe
OMe
F
Cl
Cl
Cl
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Table 2 Inhibition (IC50) of MCF-7 and B16-F10 cells proliferation and
inhibition of EGFR and HER-2 by compounds 1e–30e
IC50 ꢀ SD (mM)
Compound MCF-7a
B16-F10a
EGFRb
HER-2b
1e
2e
3e
4e
5e
6e
7e
8e
4.05 ꢀ 0.37
3.51 ꢀ 0.17
7.51 ꢀ 0.45
5.34 ꢀ 0.26
4.84 ꢀ 0.32
2.98 ꢀ 0.17
1.82 ꢀ 0.12
6.46 ꢀ 0.42
5.45 ꢀ 0.47
6.33 ꢀ 0.53
7.45 ꢀ 0.67
5.14 ꢀ 0.51
4.74 ꢀ 0.41
3.51 ꢀ 0.31
2.11 ꢀ 0.18
6.34 ꢀ 0.47
9.05 ꢀ 0.68 16.85 ꢀ 1.42
6.47 ꢀ 0.72
6.68 ꢀ 0.53
4.26 ꢀ 0.48
3.24 ꢀ 0.49
9.26 ꢀ 0.89
9.97 ꢀ 0.78
6.24 ꢀ 0.72
4.63 ꢀ 0.28
8.15 ꢀ 0.58 10.52 ꢀ 0.94
10.37 ꢀ 0.87 11.47 ꢀ 1.27 14.22 ꢀ 1.16 19.13 ꢀ 1.62
9e
8.57 ꢀ 0.79
8.22 ꢀ 0.65
4.64 ꢀ 0.30
3.87 ꢀ 0.18
6.17 ꢀ 0.46
9.63 ꢀ 0.89
9.82 ꢀ 0.74 18.04 ꢀ 1.36
10e
11e
12e
13e
14e
15e
16e
17e
18e
19e
20e
21e
22e
23e
24e
25e
26e
27e
28e
29e
30e
Erlotinib
8.21 ꢀ 0.44 10.68 ꢀ 0.86 18.55 ꢀ 1.27
4.51 ꢀ 0.23
3.27 ꢀ 0.22
5.86 ꢀ 0.25
5.94 ꢀ 0.37
4.87 ꢀ 0.53
8.12 ꢀ 0.75
6.23 ꢀ 0.79
7.95 ꢀ 0.68 10.35 ꢀ 1.03
9.05 ꢀ 0.61 10.59 ꢀ 0.92 12.46 ꢀ 0.92 19.01 ꢀ 0.16
7.96 ꢀ 0.69
7.03 ꢀ 0.51
4.35 ꢀ 0.44
2.75 ꢀ 0.25
1.27 ꢀ 0.12
2.81 ꢀ 0.21
2.35 ꢀ 0.14
1.50 ꢀ 0.16
0.67 ꢀ 0.04
0.32 ꢀ 0.05
0.92 ꢀ 0.09
2.02 ꢀ 0.17
1.97 ꢀ 0.11
1.15 ꢀ 0.10
0.56 ꢀ 0.04
0.30 ꢀ 0.04
7.93 ꢀ 0.76
7.31 ꢀ 0.58
4.48 ꢀ 0.24
2.35 ꢀ 0.13
1.13 ꢀ 0.09
2.75 ꢀ 0.22
2.16 ꢀ 0.11
1.27 ꢀ 0.13
0.74 ꢀ 0.08
0.53 ꢀ 0.07
0.85 ꢀ 0.11
1.55 ꢀ 0.19
1.42 ꢀ 0.14
1.03 ꢀ 0.09
0.66 ꢀ 0.05
0.44 ꢀ 0.05
8.35 ꢀ 0.78 16.41 ꢀ 1.73
8.59 ꢀ 0.62 12.44 ꢀ 0.97
5.33 ꢀ 0.47 7.060.84
Fig.
3
The molecular docking of compound 30e into EGFR.
Compound 30e (colored by atom: carbons: gray; nitrogen: blue;
oxygen: red) is nicely bound into EGFR (entry 1M17 in the Protein Data
Bank). The dotted lines show the hydrogen bond and the yellow line
show the Pi-Sigma interactions.
3.58 ꢀ 0.28
1.68 ꢀ 0.23
4.78 ꢀ 0.31
3.37 ꢀ 0.25
2.05 ꢀ 0.18
0.98 ꢀ 0.10
0.44 ꢀ 0.09
1.39 ꢀ 0.21
2.29 ꢀ 0.19
2.35 ꢀ 0.31
1.37 ꢀ 0.20
0.62 ꢀ 0.08
0.21 ꢀ 0.05
5.12 ꢀ 0.62
3.52 ꢀ 0.27
5.86 ꢀ 0.53
4.86 ꢀ 0.59
3.64 ꢀ 0.37
2.53 ꢀ 0.31
1.85 ꢀ 0.24
2.74 ꢀ 0.21
4.13 ꢀ 0.35
3.85 ꢀ 0.38
3.12 ꢀ 0.25
2.23 ꢀ 0.19
1.08 ꢀ 0.15
0.08 ꢀ 0.006 0.12 ꢀ 0.011 0.03 ꢀ 0.002 0.14 ꢀ 0.02
a
Inhibition of the growth of tumor cell lines. b Inhibition of EGFR and
HER-2.
which higher value indicates exponentially greater potency, to
measure the inhibitory activity. The training set and test set
were chosen by the Diverse Molecules method in Discovery
Studio 3.1. To ensure a good alignment, we chose the align-
ment conformation of each molecule with lowest energy in the
docked results of CDOCKER protocol. Before building the
QSAR model, we applied the alignment by the substructure (E)-
(1,3-diphenyl-1H-pyrazol-4-yl)methyl 3-phenylacrylate. The
correlation coefficient r2 between observed activity of testing
set and training set was found to be 0.819 for the EGFR model
and 0.861 for the HER-2 model, which conrm that the QSAR
models built are both acceptable. The molecules aligned with
the iso-surfaces of the 3D QSAR model coefficients on elec-
trostatic potential grids and van der Waals grids are shown in
Fig. 5. Electrostatic map indicates red contours around
regions where high electron density (negative charge) is
expected to increase activity, and blue contours represent
areas where low electron density (partial positive charge) is
expected to increase activity. Similarly, steric map indicates
areas where steric bulk is predicted to increase (green) or
Fig.
4 The molecular docking of compound 30e into HER-2.
Compound 30e (colored by atom: carbons: gray; nitrogen: blue;
oxygen: red) is nicely bound into EGFR (entry 3PP0 in the Protein Data
Bank).
decrease (yellow) activity. As for EGFR, on the ring A, a slightly
bulk and low negative charged group on the para-position
would facilitate the inhibitory activity, which is in good
agreement with the fact that OMe-substituent compounds
display more potent activity than that of F- or Cl-substituent.
Meanwhile, on ring B, it is suggested that compounds with low
negative charged and slightly bulk R2 group on the para-
position might make the efficiency better. Similar trends were
observed for HER-2 model. The 3D-QSAR models correlate
well with the experimental data and could provide insights
on further discovery of more potent EGFR and HER-2
inhibitors.
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General synthetic procedure of (1,3-diphenyl-1H-pyrazol-4-yl)
methanol
A mixture of para-substituted acetophenone (a) (20 mmol) and
phenylhydrazine hydrochloride (25 mmol) coupled with sodium
acetate (40 mmol) was dissolved in anhydrous ethanol to yield
compounds b. Then compounds b and POCl3 (8 mL) was added
to a cold solution of DMF (10 mL), respectively. The mixture was
stirred at 50–60 ꢁC for 6 h. Aer cooling, the mixture was
basied to pH 8 by the dropwise addition of 50% NaOH solution
under ice bath, the precipitate was ltered and recrystallized
from ethanol to form compounds c. Then, compounds c and 1.5
eq. sodium borohydride were dissolved in anhydrous CH2Cl2
solution and reuxed for 8 h to gain the desired compound d.
General synthetic procedure of target compounds 1e–30e
Compounds 1e–30e were synthesized by coupling compounds d
with cinnamic acids (1 eq.), with the help of DMAP (1.2 eq.) and
K2CO3 (3 eq.). The mixture was reuxed in anhydrous CH2Cl2
for 8–10 h. The products were extracted with ethyl acetate. The
extract was washed successively with 10% HCl, saturated
NaHCO3 and water, respectively, then dried over anhydrous
Na2SO4, ltered and evaporated. The residue was recrystallized
from ethanol to gain the desired compounds.
Fig. 5 3D-QSAR model pyrazole derivatives for EGFR (A and B) and for
HER-2 (C and D). Red contours mean high electron density and are
expected to increase activity while blue contours mean low electron
density is better. Green areas mean steric bulk is better while yellow
areas mean small groups are helpful.
Conclusion
Spectral properties of pyrazole derivatives
(E)-(1,3-Diphenyl-1H-pyrazol-4-yl)methyl 3-phenylacrylate (1e).
In the present work, a series of novel potential anticancer agents
(1e–30e) was synthesized and evaluated for their inhibitory
activity against EGFR and HER-2 and antiproliferative activities
against MCF-7 cell lines and B16-F10 cell lines. Compound 30e
demonstrated the most potent inhibitory activity against EGFR
and HER-2 with IC50 of 0.21 ꢀ 0.05 mM and 1.08 ꢀ 0.15 mM,
which can also inhibit the growth of MCF-7 and B16-F10 cells
with IC50 values of 0.30 ꢀ 0.04 and 0.44 ꢀ 0.05 mM respectively.
Molecular docking showed that compound 30e bound nicely to
the ATP binding site by hydrogen bonds which might play a
crucial role in its EGFR/HER-2 inhibition and antiproliferative
activities. 3D-QSAR models were also built to provide more
pharmacophore understanding that could be useful for the
design and discovery of novel anticancer agents with dual EGFR
and HER-2 inhibitory activity.
1
White powders, yield 77%, mp: 93–94 ꢁC; H NMR (300 MHz,
CDCl3, d ppm): 5.32 (s, 2H); 6.50 (d, J ¼ 15.90 Hz, 1H); 7.31 (d, J ¼
7.50 Hz, 1H); 7.37–7.51 (m, 10H); 7.72 (d, J ¼ 16.08 Hz, 1H); 7.77
(d, J ¼ 7.68 Hz, 2H); 7.83 (d, J ¼ 6.96 Hz, 2H); 8.15 (s, 1H). MS
(ESI): 381.4 (C25H21N2O2, [M + H]+). Anal. calcd for C25H20N2O2: C,
78.93; H, 5.30; N, 7.36; found: C, 78.86; H, 5.31; N, 7.33%. 13C
NMR (CDCl3, 100.6 MHz): 167.32(C11), 150.12(C7), 144.21(C13),
139.88(C100), 136.22(C10), 133.82(C1), 129.82(C300), 129.71(C500),
129.51(C5), 129.43(C3), 128.75(C4), 128.31(C30), 128.28(C50),
128.01(C60), 127.92(C20), 127.82(C40), 127.43(C2), 127.39(C6),
126.51(C400), 123.21(C9), 119.54(C200), 119.49(C600), 118.21(C12),
117.52(C8), 59.12(C10).
(E)-(1,3-Diphenyl-1H-pyrazol-4-yl)methyl 3-(4-uorophenyl)-
acrylate (2e). White powders, yield 83%, mp: 95–96 ꢁC; 1H NMR
(300 MHz, CDCl3, d ppm): 5.32 (s, 2H); 6.42 (d, J ¼ 15.90 Hz, 1H);
7.08 (t, J ¼ 8.69 Hz, 2H); 7.31 (t, J ¼ 7.40 Hz, 1H); 7.38–7.54 (m,
7H); 7.68 (d, J ¼ 16.08 Hz, 1H); 7.76–7.85 (m, 4H); 8.14 (s, 1H).
MS (ESI): 399.4 (C25H20FN2O2, [M + H]+). Anal. calcd for
Experimental part
Chemistry
C
25H19FN2O2: C, 75.36; H, 4.81; N, 7.03; found: C, 75.40; H, 4.80;
All chemicals and reagents used in the current study were of N, 7.01%. 13C NMR (CDCl3, 100.6 MHz): 167.12(C11),
analytical grade. Melting points (uncorrected) were determined 163.84(C40), 149.88(C7), 143.66(C13), 139.99(C100), 133.81(C1),
on an XT4 MP apparatus (Taike Corp., Beijing, China). All the 130.92(C10), 130.56(C60), 130.54(C20), 129.65(C300), 129.62(C500),
1H NMR spectra were recorded on a Bruker DPX300 model 129.43(C5), 129.39(C3), 128.53(C4), 127.99(C2), 127.95(C6),
Spectrometer in DMSO-d6 and chemical shis were reported in 126.47(C400), 123.11(C9), 119.95(C200), 119.89(C600), 118.01(C12),
ppm (d). ESI-MS spectra were recorded on a Mariner System 117.82(C8), 114.98(C50), 114.93(C30), 57.98(C10).
5304 Mass spectrometer. Elemental analyses were performed on
(E)-(1,3-Diphenyl-1H-pyrazol-4-yl)methyl 3-(4-chlorophenyl)-
a CHN–O-Rapid instrument. TLC was performed on the glass acrylate (3e). White powders, yield 81%, mp: 99–100 ꢁC; 1H
backed silica gel sheets (Silica Gel 60 GF254) and visualized in NMR (300 MHz, CDCl3, d ppm): 5.32 (s, 2H); 6.46 (d, J ¼ 16.08
UV light (254 nm).
Hz, 1H); 7.33–7.51 (m, 10H); 7.65 (d, J ¼ 16.11 Hz, 1H); 7.75–7.83
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(m, 4H); 8.14 (s, 1H). MS (ESI): 415.9 (C25H20ClN2O2, [M + H]+). 126.32(C400), 123.21(C9), 120.21(C200), 120.15(C600), 118.91(C12),
Anal. calcd for C25H19ClN2O2: C, 72.37; H, 4.62; N, 6.75; found: 117.43(C8), 116.29(C5), 116.12(C3), 58.87(C10).
C, 72.40; H, 4.60; N, 6.71%. 13C NMR (CDCl3, 100.6 MHz):
(E)-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl3-
165.97(C11), 149.83(C7), 143.87(C13), 139.88(C100), 133.75(C40), (4-uorophenyl)acrylate (8e). White powders, yield 89%, mp:
133.64(C10), 133.02(C1), 129.99(C300), 129.87(C500), 129.77(C3), 136–137 ꢁC, 1H NMR (300 MHz, CDCl3, d ppm): 5.29 (s, 2H); 6.41
129.65(C5), 129.21(C20), 129.01(C60), 128.65(C4), 128.12(C30), (d, J ¼ 15.90 Hz, 1H); 7.08 (t, J ¼ 8.6 Hz, 2H); 7.17 (d, J ¼ 8.69 Hz,
128.01(C50), 127.98(C6), 127.87(C2), 126.55(C400), 123.10(C9), 2H); 7.32 (d, J ¼ 7.68 Hz, 1H); 7.44–7.54 (m, 4H); 7.65 (d, J ¼ 15.9
119.77(C200), 119.73(C600), 119.01(C12), 117.36(C8), 59.01(C10).
Hz, 1H); 7.74–7.83 (m, 4H); 8.13 (s, 1H). ESI-MS: 417.4
(E)-(1,3-Diphenyl-1H-pyrazol-4-yl)methyl 3-(4-bromophenyl)- (C25H19F2N2O2, [M + H]+). Anal. calcd for C25H18F2N2O2: C,
acrylate (4e). White powders, yield 81%, mp: 101–102 ꢁC, 1H 72.11; H, 4.36; N, 6.73; found: C, 72.02; H, 4.37; N, 6.75%. 13C
NMR (300 MHz, CDCl3, d ppm): 5.32 (s, 2H); 6.48 (d, J ¼ 16.08 NMR (CDCl3, 100.6 MHz): 164.87(C11), 163.12(C4), 162.34(C40),
Hz, 1H); 7.31–7.53 (m, 10H); 7.64 (d, J ¼ 15.93 Hz, 1H); 7.75–7.84 150.21(C7), 142.25(C13), 139.26(C100), 135.23(C10), 131.81(C6),
(m, 4H); 8.14 (s, 1H). ESI-MS: 459.8 (C25H20BrN2O2, [M + H]+). 131.74(C2), 129.57(C60), 129.51(C20), 128.81(C500), 128.72(C300),
Anal. calcd for C25H19BrN2O2: C, 65.37; H, 4.17; N, 6.10; found: 128.65(C1), 126.71(C400), 123.04(C9), 120.02(C200), 119.91(C600),
C, 65.31; H, 4.18; N, 6.02%. 13C NMR (CDCl3, 100.6 MHz): 118.21(C12), 117.34(C8), 116.12(C3), 116.04(C5), 115.87(C30),
166.97(C11), 151.01(C7), 144.43(C13), 139.91(C100), 134.32(C10), 115.76(C50), 59.21(C10).
133.01(C1), 131.65(C50), 131.62(C30), 129.53(C300), 129.52(C500),
(E)-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl3-
129.01(C5), 128.91(C3), 128.76(C4), 128.44(C60), 128.32(C20), (4-chlorophenyl)acrylate (9e). White powders, yield 78%, mp:
127.67(C2), 127.65(C6), 126.76(C400), 123.06(C9), 122.43(C40), 149–151 ꢁC, 1H NMR (300 MHz, CDCl3, d ppm): 5.29 (s, 2H); 6.46
121.12(C200), 121.03(C600), 118.74(C12), 117.43(C8), 54.99(C10).
(d, J ¼ 16.08 Hz, 1H); 7.17 (t, J ¼ 8.69 Hz, 2H); 7.28–7.37 (m, 3H);
(E)-(1,3-Diphenyl-1H-pyrazol-4-yl)methyl 3-(p-tolyl)acrylate (5e). 7.47 (t, J ¼ 7.86 Hz, 4H); 7.66 (d, J ¼ 15.90 Hz, 1H); 7.74–7.83 (m,
White powders, yield 82%, mp: 98–99 ꢁC, 1H NMR (300 MHz, 4H); 8.13 (s, 1H). ESI-MS: 433.9 (C25H19ClFN2O2, [M + H]+). Anal.
CDCl3, d ppm): 2.37 (s, 3H); 5.32 (s, 2H); 6.45 (d, J ¼ 15.90 Hz, 1H); calcd for C25H18ClFN2O2: C, 69.37; H, 4.19; N, 6.47; found: C,
7.19 (d, J ¼ 7.68 Hz, 2H); 7.41–7.49 (m, 8H); 7.70 (d, J ¼ 16.08 Hz, 69.33; H, 4.17; N, 6.49%. 13C NMR (CDCl3, 100.6 MHz):
1H); 7.77 (d, J ¼ 7.86 Hz, 2H); 7.83 (d, J ¼ 6.93 Hz, 2H); 8.14 (s, 1H). 166.56(C11), 163.43(C4), 150.64(C7), 145.32(C13), 139.99(C100),
ESI-MS: 395.5 (C26H23N2O2, [M + H]+). Anal. calcd for C26H22N2O2: 133.56(C40), 133.34(C10), 130.76(C2), 130.73(C6), 129.54(C300),
C, 79.16; H, 5.62; N, 7.10; found: C, 79.11; H, 5.63; N, 7.02%. 13
C
129.51(C500), 129.01(C60), 128.98(C20), 128.33(C50), 128.21(C30),
NMR (CDCl3, 100.6 MHz): 166.79(C11), 148.92(C7), 143.76(C13), 128.01(C1), 126.78(C400), 123.03(C9), 119.87(C200), 119.83(C600),
139.87(C100), 137.72(C40), 133.32(C1), 132.53(C10), 129.87(C300), 117.73(C12), 117.01(C8), 116.21(C5), 116.07(C3), 59.01(C10).
129.86(C500), 129.44(C3), 129.23(C5), 128.81(C50), 128.76(C30),
(E)-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl3-
128.54(C4), 128.04(C60), 128.01(C20), 127.65(C2), 127.43(C6), (4-bromophenyl)acrylate (10e). White powders, yield 86%, mp:
126.45(C400), 123.04(C9), 119.82(C200), 119.78(C600), 118.43(C12), 156–157 ꢁC, 1H NMR (300 MHz, CDCl3, d ppm): 5.29 (s, 2H); 6.47
117.47(C8), 58.97(C10), 21.33(CH3).
(d, J ¼ 16.11 Hz, 1H); 7.17 (t, J ¼ 8.13 Hz, 2H); 7.33 (d, J ¼ 7.11
(E)-(1,3-Diphenyl-1H-pyrazol-4-yl)methyl 3-(4-methoxyphenyl)- Hz, 1H); 7.39 (d, J ¼ 8.4 Hz, 2H); 7.45–7.54 (m, 4H); 7.64 (d, J ¼
1
ꢁ
acrylate (6e). White powders, yield 76%,mp: 96–97 C, H NMR 15.93 Hz, 1H); 7.77 (t, J ¼ 9.0 Hz, 4H); 8.13 (s, 1H). ESI-MS:
(300 MHz, CDCl3, d ppm): 3.84 (s, 3H); 5.31 (s, 2H); 6.37 (d, J ¼ 478.33 (C25H19BrFN2O2, [M + H]+). Anal. calcd for C25H18
-
15.90 Hz, 1H); 6.90 (d, J ¼ 8.76 Hz, 2H); 7.40–7.49 (m, 8H); 7.68 (d, BrFN2O2: C, 62.91; H, 3.80; N, 5.87; found: C, 62.83; H, 3.82; N,
J ¼ 15.90 Hz, 1H); 7.76–7.85 (m, 4H); 8.14 (s, 1H). ESI-MS: 411.5 5.84%. 13C NMR (CDCl3, 100.6 MHz): 167.32(C11), 162.99(C4),
(C26H23N2O3, [M + H]+). Anal. calcd for C26H22N2O3: C, 76.08; H, 150.02(C7), 144.19(C13), 138.96(C100), 135.01(C10), 131.87(C50),
5.40; N, 6.82; found: C, 76.15; H, 5.38; N, 6.88%. 13C NMR (CDCl3, 131.84(C30), 130.98(C2), 130.89(C6), 129.46(C300), 129.41(C500),
100.6 MHz): 167.21(C11), 160.21(C40), 150.21(C7), 143.87(C13), 128.73(C1), 128.56(C60), 128.51(C20), 126.76(C400), 123.43(C9),
139.88(C100), 133.32(C1), 130.43(C20), 130.30(C60), 129.76(C300), 122.54(C40), 119.65(C200), 119.58(C600), 118.15(C12), 117.19(C8),
129.65(C500), 129.12(C3), 129.04(C5), 128.87(C4), 127.76(C10), 115.82(C5), 115.78(C3), 58.91(C10).
127.54(C2), 127.43(C6), 126.43(C400), 123.32(C9), 120.19(C200),
120.12(C600), 118.32(C12), 117.43(C8), 114.65(C30), 114.43(C50), (p-tolyl)acrylate (11e). White powders, yield 83%, mp: 133–
58.12(C10), 54.81(OCH3).
135 ꢁC, 1H NMR (300 MHz, CDCl3, d ppm): 2.37 (s, 3H); 5.29 (s,
(E)-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl3-
(E)-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl 3- 2H); 6.44 (d, J ¼ 15.90 Hz, 1H); 7.17 (t, J ¼ 8.97 Hz, 4H); 7.32 (d, J
phenylacrylate (7e). White powders, yield 88%, mp: 126–128 ꢁC, ¼ 7.50 Hz, 1H); 7.41–7.49 (m, 4H); 7.67–7.84 (m, 5H); 8.13 (s,
1H NMR (300 MHz, CDCl3, d ppm): 5.29 (s, 2H); 6.49 (d, J ¼ 15.93 1H). ESI-MS: 413.46 (C26H22FN2O2, [M + H]+). Anal. calcd for
Hz, 1H); 7.17 (t, J ¼ 8.69 Hz, 2H); 7.31 (t, J ¼ 7.50 Hz, 1H); 7.39 (t, C26H21FN2O2: C, 75.71; H, 5.13; N, 6.79; found: C, 75.61; H, 5.12;
J ¼ 3.29 Hz, 3H); 7.44–7.55 (m, 4H); 7.70–7.84 (m, 5H); 8.14(s, N, 6.77%. 13C NMR (CDCl3, 100.6 MHz): 166.76(C11),
1H). ESI-MS: 399.4 (C25H20FN2O2, [M + H]+). Anal. calcd for 163.21(C4), 151.22(C7), 144.29(C13), 140.11(C100), 137.87(C40),
C
25H19FN2O2: C, 75.36; H, 4.81; N, 7.03; found: C, 75.27; H, 4.82; 132.11(C10), 130.78(C2), 130.71(C6), 129.46(C300), 129.43(C500),
N, 7.06%. 13C NMR (CDCl3, 100.6 MHz): 170.01(C11), 128.96(C50), 128.90(C30), 128.54(C1), 128.33(C60), 128.21(C20),
164.93(C4), 149.21(C7), 144.21(C13), 139.21(C100), 135.93(C10), 126.42(C400), 123.08(C9), 118.95(C200), 118.89(C600), 118.12(C12),
130.87(C6), 130.54(C2), 129.65(C300), 129.54(C500), 128.98(C1), 117.09(C8), 116.16(C3), 116.12(C5), 57.12(C10), 22.01(CH3).
128.87(C50), 128.78(C30), 128.51(C60), 128.48(C20), 128.26(C40),
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(E)-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl3-
(4-methoxyphenyl)acrylate (12e). White powders, yield 85%,
mp: 102–103 ꢁC, 1H NMR (300 MHz, CDCl3, d ppm): 3.84 (s, 3H);
5.28 (s, 2H); 6.36 (d, J ¼ 15.93 Hz, 1H); 6.90 (d, J ¼ 8.58 Hz, 2H);
7.17 (t, J ¼ 8.69 Hz, 2H); 7.32 (d, J ¼ 7.11 Hz, 1H); 7.47 (t, J ¼ 8.13
Hz, 4H); 7.68 (d, J ¼ 15.9 Hz, 1H); 7.74–7.84 (m, 4H); 8.13 (s, 1H).
ESI-MS: 429.45 (C26H22FN2O3, [M + H]+). Anal. calcd for
RSC Advances
161–163 ꢁC; 1H NMR (300 MHz, CDCl3, d ppm): 5.30 (s, 2H); 6.47
(d, J ¼ 16.08 Hz, 1H); 7.29–7.54 (m, 9H); 7.64 (d, J ¼ 16.29 Hz,
1H); 7.77 (s, 4H); 8.13 (s, 1H). MS (ESI): 494.8 (C25H19BrClN2O2,
[M + H]+). Anal. calcd for C25H18BrClN2O2: C, 60.81; H, 3.67; N,
5.67; found: C, 60.88; H, 3.65; N, 5.62%. 13C NMR (CDCl3, 100.6
MHz): 166.78(C11), 150.32(C7), 144.85(C13), 139.74(C100),
134.56(C4), 134.29(C10), 131.68(C30), 131.61(C50), 131.27(C1),
129.63(C300), 129.59(C500), 129.48(C3), 129.46(C5), 128.89(C2),
128.85(C6), 128.54(C60), 128.48(C20), 126.36(C400), 123.25(C9),
122.48(C40), 120.01(C200), 119.98(C600), 118.37(C12), 117.35(C8),
56.73(C10).
(E)-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl3-
(p-tolyl)acrylate (17e). White powders, yield 80%, mp: 131–132
ꢁC; 1H NMR (300 MHz, CDCl3, d ppm): 2.37 (s, 3H); 5.29 (s, 2H);
6.44 (d, J ¼ 15.90 Hz, 1H); 7.19 (d, J ¼ 8.04 Hz, 2H); 7.31 (t, J ¼
7.31 Hz, 1H); 7.41–7.50 (m, 6H); 7.66–7.80 (m, 5H); 8.13 (s, 1H).
MS (ESI): 429.9 (C26H22ClN2O2, [M + H]+). Anal. calcd for
C
26H21FN2O3: C, 72.88; H, 4.94; N, 6.54; found: C, 72.76; H, 4.95;
N, 6.57%. 13C NMR (CDCl3, 100.6 MHz): 167.12(C11),
162.12(C4), 159.96(C40), 150.21(C7), 143.52(C13), 139.45(C100),
130.76(C6), 130.71(C2), 130.28(C60), 130.21(C20), 129.54(C300),
129.50(C500), 128.97(C1), 127.56(C10), 126.84(C400), 123.32(C9),
120.01(C200), 119.98(C600), 118.12(C12), 117.45(C8), 116.23(C5),
116.19(C3), 114.12(C50), 114.08(C30), 59.01(C10), 55.76(OCH3).
(E)-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl3-
phenylacrylate (13e). White powders, yield 81%, mp: 104–
105 ꢁC; 1H NMR (300 MHz, CDCl3, d ppm): 5.30 (s, 2H); 6.49 (d,
J ¼ 15.90 Hz, 1H); 7.33 (d, J ¼ 7.50 Hz, 1H); 7.39 (t, J ¼ 3.29 Hz,
3H); 7.46 (d, J ¼ 8.43 Hz, 4H); 7.50–7.55 (m, 2H); 7.69–7.80 (m,
5H); 8.14 (s, 1H). MS (ESI): 415.9(C25H20ClN2O2, [M + H]+). Anal.
calcd for C25H19ClN2O2: C, 72.37; H, 4.62; N, 6.75; found: C,
72.40; H, 4.61; N, 6.71%. 13C NMR (CDCl3, 100.6 MHz):
167.31(C11), 150.10(C7), 144.29(C13), 139.65(C100), 135.43(C10),
134.83(C4), 131.59(C1), 129.77(C300), 129.74(C500), 129.43(C3),
129.39(C5), 128.87(C2), 128.84(C6), 128.43(C50), 128.34(C30),
128.12(C20), 128.11(C60), 127.86(C40), 126.32(C400), 123.31(C9),
119.76(C200), 119.72(C600), 118.37(C12), 117.10(C8), 59.21(C10).
(E)-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl3-
(4-uorophenyl)acrylate (14e). White powders, yield 76%, mp:
129–130 ꢁC; 1H NMR (300 MHz, CDCl3, d ppm): 5.30 (s, 2H); 6.41
(d, J ¼ 15.90 Hz, 1H); 7.08 (t, J ¼ 8.60 Hz, 2H); 7.32 (t, J ¼ 7.23
Hz, 1H); 7.44–7.54 (m, 6H); 7.65 (t, J ¼ 13.53 Hz, 1H); 7.77 (t, J ¼
8.22 Hz, 4H); 8.13 (s, 1H). MS (ESI): 433.9 (C25H19ClFN2O2, [M +
H]+). Anal. calcd for C25H18ClFN2O2: C, 69.37; H, 4.19; N, 6.47;
found: C, 69.40; H, 4.21; N, 6.45%. 13C NMR (CDCl3, 100.6
MHz): 166.52(C11), 162.33(C40), 150.03(C7), 143.69(C13),
140.11(C100), 134.33(C4), 131.26(C1), 131.29(C10), 130.54(C60),
130.51(C20), 129.65(C300), 129.61(C500), 129.47(C5), 129.38(C3),
128.78(C6), 128.75(C2), 126.43(C400), 123.32(C9), 119.21(C200),
119.07(C600), 118.26(C12), 117.37(C8), 115.62(C30), 115.59(C50),
59.21(C10).
C
26H21ClN2O2: C, 72.81; H, 4.94; N, 6.53; found: C, 72.88; H,
4.95; N, 6.52%. 13C NMR (CDCl3, 100.6 MHz): 166.93(C11),
149.97(C7), 145.43(C13), 139.98(C100), 137.98(C40), 134.54(C4),
133.01(C10), 131.43(C1), 129.76(C300), 129.73(C500), 129.45(C5),
129.41(C3), 128.74(C50), 128.71(C30), 128.23(C2), 128.18(C6),
128.13(C60), 128.09(C20), 126.32(C400), 123.03(C9), 119.76(C200),
119.72(C600), 118.43(C12), 117.29(C8), 58.32(C10), 22.35(CH3).
(E)-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl3-
(4-methoxyphenyl)acrylate (18e). White powders, yield 75%,
mp: 121–122 ꢁC; 1H NMR (300 MHz, CDCl3, d ppm): 3.84 (s, 3H);
5.29 (s, 2H); 6.35 (d, J ¼ 15.90 Hz, 1H); 6.90 (d, J ¼ 8.76 Hz, 2H);
7.31 (t, J ¼ 7.13 Hz, 1H); 7.47 (t, J ¼ 7.77 Hz, 6H); 7.67 (d, J ¼
16.08 Hz, 1H); 7.77 (t, J ¼ 8.78 Hz, 4H); 8.13 (s, 1H). MS (ESI):
445.9 (C26H22ClN2O3, [M + H]+). Anal. calcd for C26H21ClN2O3:
C, 70.19; H, 4.76; N, 6.30; found: C, 70.13; H, 4.75; N, 6.32%. 13
C
NMR (CDCl3, 100.6 MHz): 165.73(C11), 159.94(C40), 150.13(C7),
143.76(C13), 139.87(C100), 134.54(C4), 131.43(C1), 130.57(C60),
130.53(C20), 129.65(C300), 129.62(C500), 129.32(C3), 129.28(C5),
128.94(C2), 128.91(C6), 127.62(C10), 126.49(C400), 123.82(C9),
119.43(C200), 119.41(C600), 118.24(C12), 117.37(C8), 114.35(C50),
114.31(C30), 57.32(C10), 55.21(OCH3).
(E)-(1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)methyl 3-phenyl-
acrylate (19e). White powders, yield 87%,mp: 96–97 ꢁC; 1H NMR
(300 MHz, CDCl3, d ppm): 2.28 (s, 3H); 5.19 (s, 2H); 6.37 (d, J ¼
16.11 Hz, 1H); 7.10–7.18 (m, 3H); 7.23 (t, J ¼ 2.93 Hz, 3H); 7.31
(t, J ¼ 7.77 Hz, 2H); 7.38 (t, J ¼ 2.75 Hz, 2H); 7.61–7.64 (m, 5H);
7.96 (d, J ¼ 15.00 Hz, 1H). MS (ESI): 395.5 (C26H23N2O2, [M +
H]+). Anal. calcd for C26H22N2O2: C, 79.16; H, 5.62; N, 7.10;
found: C, 79.10; H, 5.61; N, 7.13%. 13C NMR (CDCl3, 100.6
MHz): 164.21(C11), 148.32(C7), 144.32(C13), 139.93(C100),
135.22(C10), 131.87(C4), 130.21(C1), 129.65(C5), 129.61(C3),
129.44(C300), 129.41(C500), 128.73(C50), 128.71(C30), 128.32(C60),
128.29(C20), 127.99(C40), 126.34(C400), 125.83(C2), 125.80(C6),
123.03(C9), 119.87(C200), 119.83(C600), 118.36(C12), 117.31(C8),
59.32(C10), 23.10(CH3).
(E)-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl3-
(4-chlorophenyl)acrylate (15e). White powders, yield 83%, mp:
153–154 ꢁC; 1H NMR (300 MHz, CDCl3, d ppm): 5.30 (s, 2H); 6.45
(d, J ¼ 15.90 Hz, 1H); 7.26–7.37 (m, 3H); 7.46 (d, J ¼ 8.40 Hz, 6H);
7.65 (d, J ¼ 16.08 Hz, 1H); 7.77 (t, J ¼ 7.68 Hz, 4H); 8.13 (s, 1H).
MS (ESI): 450.3 (C25H19Cl2N2O2, [M + H]+). Anal. calcd for
C
25H18Cl2N2O2: C, 66.83; H, 4.04; N, 6.23; found: C, 66.80; H,
4.05; N, 6.22%. 13C NMR (CDCl3, 100.6 MHz): 164.37(C11),
149.98(C7), 143.69(C13), 139.89(C100), 134.54(C4), 133.78(C40),
133.53(C10), 131.32(C1), 129.76(C300), 129.74(C500), 129.45(C5),
129.34(C3), 129.03(C20), 129.01(C60), 128.87(C2), 128.83(C6),
128.65(C30), 128.62(C50), 126.43(C400), 123.21(C9), 119.98(C200),
119.95(C600), 118.21(C12), 117.34(C8), 59.32(C10).
(E)-(1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)methyl3-(4-uorophenyl)-
acrylate (20e). White powders, yield 80%, mp: 102–104 ꢁC; 1H NMR
(300 MHz, CDCl3, d ppm): 2.41 (s, 3H); 5.31 (s, 2H); 6.42 (d, J ¼ 16.08
Hz, 1H); 7.08 (t, J ¼ 8.60 Hz, 2H); 7.26–7.32 (m, 3H); 7.44–7.54 (m,
4H); 7.65–7.78 (m, 5H); 8.12 (s, 1H). MS (ESI): 413.5 (C26H22FN2O2,
(E)-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl3-
(4-bromophenyl)acrylate (16e). White powders, yield 82%, mp:
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[M + H]+). Anal. calcd for C26H21FN2O2: C, 75.71; H, 5.13; N, 6.79; 130.32(C60), 130.29(C20), 130.02(C1), 129.76(C5), 129.74(C3),
found: C, 75.78; H, 5.11; N, 6.81%. 13C NMR (CDCl3, 100.6 MHz): 129.43(C300), 129.32(C500), 127.64(C10), 126.32(C400), 125.87(C6),
167.82(C11), 162.31(C40), 150.12(C7), 143.52(C13), 139.52(C100), 125.81(C2), 123.04(C9), 119.32(C200), 119.31(C600), 118.64(C12),
131.78(C4), 130.26(C10), 130.01(C60), 129.99(C20), 129.89(C1), 117.32(C8), 114.54(C50), 114.52(C30), 58.32(C10), 55.65(OCH3),
129.67(C5), 129.65(C3), 129.33(C300), 129.30(C500), 126.35(C400), 21.44(CH3).
125.87(C2), 125.83(C6), 123.01(C9), 120.05(C200), 120.01(C600),
118.21(C12), 117.34(C8), 115.65(C50), 115.62(C30), 58.59(C10), phenylacrylate (25e). White powders, yield 78%, mp: 101–
21.37(CH3).
103 ꢁC, 1H NMR (300 MHz, CDCl3, d ppm): 3.86 (s, 3H); 5.31 (s,
(E)-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methyl3-
(E)-(1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)methyl3-(4-chloro- 2H); 6.50 (d, J ¼ 16.11 Hz, 1H); 7.02 (d, J ¼ 8.76 Hz, 2H); 7.30 (d, J
phenyl)acrylate (21e). White powders, yield 76%, mp: 128– ¼ 7.32 Hz, 1H); 7.38–7.46 (m, 5H); 7.51 (d, J ¼ 11.7 Hz, 2H); 7.74
129 ꢁC; 1H NMR (300 MHz, CDCl3, d ppm): 2.41 (s, 3H); 5.31 (s, (t, J ¼ 12.8 Hz, 5H); 8.12 (s, 1H). ESI-MS: 411.46 (C26H23N2O3, [M
2H); 6.46 (d, J ¼ 15.90 Hz, 1H); 7.26–7.37 (m, 5H); 7.46 (d, J ¼ + H]+). Anal. calcd for C26H22N2O3: C, 76.08; H, 5.40; N, 6.82;
8.22 Hz, 4H); 7.63–7.78 (m, 5H); 8.12 (s, 1H). MS (ESI): 429.5 found: C, 76.01; H, 5.42; N, 6.85%. 13C NMR (CDCl3, 100.6
(C26H22ClN2O2, [M + H]+). Anal. calcd for C26H21ClN2O2: C, MHz): 166.43(C11), 160.21(C4), 150.41(C7), 143.76(C13),
72.81; H, 4.94; N, 6.53; found: C, 72.85; H, 4.93; N, 6.55%. 13C 139.54(C100), 135.62(C10), 129.43(C300), 129.41(C500), 128.76(C30),
NMR (CDCl3, 100.6 MHz): 165.32(C11), 149.32(C7), 143.73(C13), 128.73(C500), 128.43(C20), 128.42(C60), 128.22(C6), 128.19(C2),
139.87(C100), 133.76(C40), 133.43(C10), 131.98(C4), 130.21(C1), 127.93(C4 ), 126.17(C400), 125.52(C1), 123.01(C9), 119.87(C200),
129.64(C5), 129.61(C3), 129.36(C300), 129.32(C500), 129.03(C60), 119.83(C600), 118.32(C12), 117.37(C8), 114.65(C3), 114.54(C5),
129.01(C20), 128.73(C30), 128.69(C50), 126.43(C400), 125.76(C2), 58.21(C10), 55.32(OCH3).
125.73(C6), 123.01(C9), 120.04(C200), 120.01(C600), 118.93(C12),
117.32(C8), 57.32(C10), 21.43(CH3).
(E)-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methyl3-
(4-uorophenyl)acrylate (26e). White powders, yield 81%, mp:
(E)-(1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)methyl3-(4-bromo- 106–107 ꢁC, 1H NMR (300 MHz, CDCl3, d ppm): 3.86 (s, 3H); 5.30
phenyl)acrylate (22e). White powders, yield 85%, mp: 129– (s, 2H); 6.42 (d, J ¼ 15.93 Hz, 1H); 7.00–7.10 (m, 4H); 7.30 (d, J ¼
1
130 ꢁC; H NMR (300 MHz, CDCl3, d ppm): 2.41 (s, 3H); 5.31 7.5 Hz, 1H); 7.43–7.54 (m, 4H); 7.67 (d, J ¼ 15.93 Hz, 1H); 7.76
(s, 2H); 6.48 (d, J ¼ 15.93 Hz, 1H); 7.28 (d, J ¼ 12.27 Hz, 3H); 7.38 (d, J ¼ 8.4 Hz, 4H); 8.11 (s, 1H). ESI-MS: 429.45 (C26H22FN2O3,
(d, J ¼ 8.43 Hz, 2H); 7.44–7.53 (m, 4H); 7.61–7.77 (m, 5H); 8.12 [M + H]+). Anal. calcd for C26H21FN2O3: C, 72.88; H, 4.94; N, 6.54;
(s, 1H). MS (ESI): 474.4 (C26H22BrN2O2, [M + H]+). Anal. calcd for found: C, 72.79; H, 4.92; N, 6.57%. 13C NMR (CDCl3, 100.6
C
26H21BrN2O2: C, 65.97; H, 4.47; N, 5.92; found: C, 65.93; H, MHz): 166.75(C11), 163.21(C40), 160.43(C4), 151.64(C7),
4.45; N, 5.95%. 13C NMR (CDCl3, 100.6 MHz): 166.75(C11), 143.64(C13), 139.33(C100), 130.93(C10), 130.44(C20), 130.41(C60),
149.65(C7), 143.56(C13), 139.87(C100), 134.32(C10), 131.69(C4), 129.54(C300), 129.51(C500), 128.65(C6), 128.62(C2), 126.64(C400),
131.43(C30), 131.41(C50), 130.21(C1), 129.67(C3), 129.65(C5), 125.63(C1), 123.33(C9), 119.32(C200), 119.21(C600), 118.03(C12),
129.21(C300), 129.09(C500), 128.68(C60), 128.63(C20), 126.32(C400), 117.43(C8), 115.63(C50), 115.61(C30), 114.54(C5), 114.51(C3),
125.87(C2), 125.86(C6), 123.05(C9), 122.54(C40), 119.74(C200), 58.31(C10), 55.91(OCH3).
119.71(C600), 118.42(C12), 11.32(C8), 58.75(C10), 21.43(CH3).
(E)-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methyl3-
(E)-(1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)methyl 3-(p-tolyl)- (4-chlorophenyl)acrylate (27e). White powders, yield 84%, mp:
acrylate (23e). White powders, yield 78%, mp: 99–100 ꢁC; 1H 124–126 ꢁC, 1H NMR (300 MHz, CDCl3, d ppm): 3.87 (s, 3H); 5.30
NMR (300 MHz, CDCl3, d ppm): 2.39 (d, J ¼ 12.24 Hz, 6H); 5.31 (s, 2H); 6.46 (d, J ¼ 15.90 Hz, 1H); 7.02 (d, J ¼ 8.76 Hz, 2H); 7.29–
(s, 2H); 6.45 (d, J ¼ 15.90 Hz, 1H); 7.19 (d, J ¼ 7.50 Hz, 2H); 7.26 7.37 (m, 3H); 7.46 (t, J ¼ 6.5 Hz, 4H); 7.66 (d, J ¼ 15.9 Hz, 1H);
(s, 3H); 7.43 (d, J ¼ 7.86 Hz, 4H); 7.69 (d, J ¼ 16.11 Hz, 5H); 8.13 7.76 (d, J ¼ 8.58 Hz, 4H); 8.11 (s, 1H). ESI-MS: 445.9
(s, 1H). MS (ESI): 409.5 (C27H25N2O2, [M + H]+). Anal. calcd for (C26H22ClN2O3, [M + H]+). Anal. calcd for C26H21ClN2O3: C,
C
27H24N2O2: C, 79.39; H, 5.92; N, 6.86; found: C, 79.33; H, 5.91; 70.19; H, 4.76; N, 6.30; found: C, 70.08; H, 4.77; N, 6.33%. 13C
N, 6.89%. 13C NMR (CDCl3, 100.6 MHz): 166.43(C11), NMR (CDCl3, 100.6 MHz): 166.43(C11), 160.32(C4), 150.42(C7),
150.01(C7), 143.76(C13), 139.94(C100), 137.84(C40), 132.43(C10), 143.21(C13), 139.92(C100), 133.76(C40), 133.32(C10), 129.42(C300),
131.76(C4), 130.32(C1), 129.76(C3), 129.72(C5), 129.35(C300), 129.41(C500), 129.02(C60), 129.01(C20), 128.66(C30), 128.62(C50),
129.31(C500), 128.83(C50), 128.79(C30), 128.53(C60), 128.51(C20), 128.22(C2), 128.19(C6), 126.11(C400), 125.44(C1), 123.11(C9),
126.43(C400), 125.65(C2), 125.61(C6), 123.00(C9), 119.54(C200), 119.33(C200), 119.29(C600), 118.21(C12), 117.32(C8), 114.52(C5),
119.39(C600), 118.21(C12), 117.32(C8), 58.21(C10), 21.22(CH3), 114.51(C3), 58.33(C10), 55.32(OCH3).
21.09(CH3).
(E)-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methyl3-
(E)-(1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)methyl3-(4-methoxy- (4-bromophenyl)acrylate (28e). White powders, yield 85%, mp:
phenyl)acrylate (24e). White powders, yield 77%, mp: 94–95 ꢁC; 138–139 ꢁC, 1H NMR (300 MHz, CDCl3, d ppm): 3.86 (s, 3H); 5.30
1H NMR (300 MHz, CDCl3, d ppm): 2.41 (s, 3H); 3.84 (s, 3H); 5.30 (s, 2H); 6.48 (d, J ¼ 15.90 Hz, 1H); 7.01 (d, J ¼ 8.79 Hz, 2H); 7.30
(s, 2H); 6.37 (d, J ¼ 15.90 Hz, 1H); 6.90 (d, J ¼ 8.76 Hz, 2H); 7.29 (d, J ¼ 7.32 Hz, 1H); 7.37–7.53 (m, 6H); 7.64 (d, J ¼ 16.08 Hz, 1H);
(d, J ¼ 8.04 Hz, 3H); 7.46 (t, J ¼ 9.05 Hz, 4H); 7.65–7.78 (m, 5H); 7.76 (d, J ¼ 8.97 Hz, 4H); 8.11 (s, 1H). ESI-MS: 490.4
8.12 (s, 1H). MS (ESI): 425.5 (C27H25N2O3, [M + H]+). Anal. calcd (C26H22BrN2O3, [M + H]+). Anal. calcd for C26H21BrN2O3: C,
for C27H24N2O3: C, 76.39; H, 5.70; N, 6.60; found: C, 76.36; H, 63.81; H, 4.33; N, 5.72; found: C, 63.87; H, 4.32; N, 5.69%. 13C
5.71; N, 6.58%. 13C NMR (CDCl3, 100.6 MHz): 166.32(C11), NMR (CDCl3, 100.6 MHz): 166.32(C11), 160.32(C4), 149.87(C7),
159.93(C40), 150.21(C7), 143.65(C13), 139.95(C100), 131.72(C4), 143.54(C13), 139.83(C100), 134.41(C10), 131.52(C50), 131.50(C30),
37204 | RSC Adv., 2014, 4, 37197–37207
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129.54(C300), 129.52(C500), 128.76(C60), 128.73(C20), 128.43(C2), times. The results were summarized in Table 2. All data are
128.41(C6), 126.21(C400), 125.57(C1), 123.41(C9), 122.41(C40), presented as mean ꢀ SD unless otherwise noted.
119.43(C200), 119.39(C600), 118.73(C12), 117.13(C8), 114.54(C5),
114.51(C3), 58.43(C10), 55.32(OCH3).
Enzyme inhibitory assay
(E)-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methyl3-
(p-tolyl)acrylate (29e). White powders, yield 76%, mp: 76–77 ꢁC,
1H NMR (300 MHz, CDCl3, d ppm): 2.37 (s, 3H); 3.86 (s, 3H); 5.30
(s, 2H); 6.45 (d, J ¼ 16.08 Hz, 1H); 7.02 (d, J ¼ 8.58 Hz, 2H); 7.19
(d, J ¼ 7.86 Hz, 2H); 7.30 (d, J ¼ 7.32 Hz, 1H); 7.41–7.48 (m, 4H);
7.67–7.78 (m, 5H); 8.12 (s, 1H). ESI-MS: 425.5 (C27H25N2O3, [M +
H]+). Anal. calcd for C27H24N2O3: C, 76.39; H, 5.70; N, 6.60;
found: C, 76.31; H, 5.72; N, 6.63%. 13C NMR (CDCl3, 100.6
MHz): 167.43(C11), 160.76(C4), 148.94(C7), 143.65(C13),
139.56(C100), 137.85(C40), 132.54(C10), 129.68(C300), 129.65(C500),
128.96(C500), 128.93(C30), 128.65(C2), 128.63(C6), 128.42(C20),
128.23(C6 ), 126.32(C400), 125.62(C1), 123.03(C9), 120.10(C200),
120.06(C600), 118.43(C12), 117.16(C8), 114.32(C3), 114.31(C5),
58.21(C10), 55.37(OCH3), 21.43(CH3).
A 1.6 kb cDNA encoded for the EGFR cytoplasmic domain
(EGFR-CD, amino acids 645–1186) and 1.7 kb cDNA encoded for
human HER-2 cytoplasmic domain (HER-2-CD, amino acids
676–1245) were cloned into baculoviral expression vectors
pBlueBacHis2B and pFASTBacHTc (Huakang Company, China),
respectively. A sequence that encodes (His)6 was located at the
50 upstream to the EGFR and HER-2 sequences. Sf-9 cells were
infected for three days for protein expression. Sf-9 cell pellets
were solubilized at 0 ꢁC in a buffer at pH 7.4 containing 50 mM
HEPES, 10 mM NaCl, 1% Triton, 10 mM ammonium molybdate,
100 mM sodium vanadate, 10 mg mLꢂ1 aprotinin, 10 mg mLꢂ1
leupeptin, 10 mg mLꢂ1 pepstatin, and 16 mg mLꢂ1 benzamidine
HCl for 20 min followed by 20 min centrifugation. Crude extract
supernatant was passed through an equilibrated Ni-NTA
superow packed column and washed with 10 mM and then
100 mM imidazole to remove nonspecically bound material.
Histidine tagged proteins were eluted with 250 and 500 mM
imidazole and dialyzed against 50 mM NaCl, 20 mM HEPES,
10% glycerol, and 1 mg mLꢂ1 each of aprotinin, leupeptin, and
pepstatin for 2 h. The entire purication procedure was per-
formed at 4 ꢁC or on ice.
Both EGFR and HER-2 kinase assays were set up to assess the
level of autophosphorylation based on DELFIA/Time-Resolved
Fluorometry. Compounds 1e–30e were dissolved in 100%
DMSO and diluted to the appropriate concentrations with 25
mM HEPES at pH 7.4. In each well, 10 mL compound was
incubated with 10 mL (5 ng for EGFR or 12.5 ng for HER-2)
recombinant enzyme (1 : 80 dilution in 100 mM HEPES) for 10
min at room temperature. Then, 10 mL of 5 mM buffer (con-
taining 20 mM HEPES, 2 mM MnCl2, 100 mM Na3VO4, and 1 mM
DTT) and 20 mL of 0.1 mM ATP–50 mM MgCl2 were added for 1
h. Positive and negative controls were included in each plate by
incubation of enzyme with or without ATP-MgCl2. At the end of
incubation, liquid was aspirated, and plates were washed three
times with wash buffer. A 75 mL (400 ng) sample of europium
labeled anti-phosphotyrosine antibody was added to each well
for another 1 h of incubation. Aer washing, enhancement
solution was added and the signal was detected by Victor
(Wallac Inc.) with excitation at 340 nm and emission at 615 nm.
The percentage of autophosphorylation inhibition by the
compounds was calculated using the following formula: 100%
ꢂ [(negative control)/(positive control ꢂ negative control)]. The
IC50 was obtained from curves of percentage inhibition with
eight concentrations of compound. As the contaminants in the
enzyme preparation are fairly low, the majority of the signal
detected by the anti-phosphotyrosine antibody is from EGFR or
HER-2. All data are presented as mean ꢀ SD unless otherwise
noted.
(E)-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methyl3-
(4-methoxyphenyl)acrylate (30e). White powders, yield 82%,
1
ꢁ
mp: 73–74 C, H NMR (300 MHz, CDCl3, d ppm): 3.85 (s, 3H);
3.86 (s, 3H); 5.29 (s, 2H); 6.37 (d, J ¼ 15.93 Hz, 1H); 6.90 (d, J ¼
8.61 Hz, 2H); 7.01 (d, J ¼ 8.25 Hz, 2H); 7.30 (d, J ¼ 7.50 Hz, 1H);
7.46 (t, J ¼ 9.33 Hz, 4H); 7.67 (d, J ¼ 15.93 Hz, 1H); 7.76 (d, J ¼
6.21 Hz, 4H); 8.11 (s, 1H). ESI-MS: 441.5 (C27H25N2O4, [M + H]+).
Anal. calcd for C27H24N2O4: C, 73.62; H, 5.49; N, 6.36; found: C,
73.67; H, 5.47; N, 6.39%. 13C NMR (CDCl3, 100.6 MHz):
164.26(C11), 161.61(C4), 159.87(C40), 149.59(C7), 143.76(C13),
139.98(C100), 130.32(C20), 130.31(C60), 129.46(C300), 129.44(C500),
128.56(C2), 128.52(C6), 127.51(C10), 126.39(C400), 125.37(C1),
123.08(C9), 119.75(C200), 119.69(C600), 118.06(C12), 117.34(C8),
114.56(C3), 114.53(C5), 114.32(C30), 114.31(C50), 58.53(C10),
55.76(OCH3), 55.65(OCH3).
Biological evaluation
Antiproliferation assay
The antiproliferative activities of the prepared compounds
against MCF-7 and B16-F10 cells were evaluated as described in
the literature34,35 with some modications. Target tumor cell
lines were grown to log phase in RPMI 1640 medium supple-
mented with 10% fetal bovine serum. Aer diluting to 2 ꢃ 104
cells per mL with the complete medium, 100 mL 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 before the cytotoxicity assessments. Tested
samples at pre-set concentrations were added to six wells with
Erlotinib co-assayed as positive references. Aer 48 h exposure
period, 40 mL of PBS containing 2.5 mg mLꢂ1 of MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was
added to each well. Four hours later, 100 mL extraction solution
(10% SDS–5% isobutyl alcohol–0.01 M HCl) was added. Aer an
overnight incubation at 37 ꢁC, the optical density was measured
at a wavelength of 570 nm on an ELISA microplate reader. In all
Docking simulations
experiments three replicate wells were used for each drug The three-dimensional structures of the aforementioned
concentration. Each assay was carried out for at least three compounds were constructed using Chem. 3D ultra 12.0
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Paper
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Among all the 30 compounds, 80% (i.e., 24) were utilized as a
training set for QSAR (quantitative structure–activity relation-
ship) modeling. The remaining 20% (i.e., 6) were chosen as an
external test subset for validating the reliability of the QSAR
model by the Diverse Molecules protocol in Discovery Studio
3.1. The selected test compounds were: 5e, 8e, 10e, 15e, 28e and
29e. The inhibitory activity of the compounds in literature [IC50
(mol Lꢂ1)] was initially changed into the minus logarithmic
scale [pIC50 (mol Lꢂ1)] and then used for subsequent QSAR
analysis as the response variable. In Discovery Studio, the
CHARMm force eld is applied and the electrostatic potential
together with the van der Waals potential are treated as separate
terms. As the electrostatic potential probe, A + le point change is
used while distance-dependent dielectric constant is used to
mimic the solvent effect. As for the van der Waals potential, a
˚
carbon atom with a radius of 1.73 A 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 3.1.
Acknowledgements
27 P. Pevarello, D. Fancelli, A. Vulpetti, R. M. Amici, V. Villa,
This work was supported by Natural Science Foundation of
Jiangsu Province (no. BK20130554).
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