Biological evaluation of 4-aryl-1,4-dihydropyridines as VEGFR-2 kinase inhibitors

Article abstract of DOI:10.1134/S1070363216120574

Vascular endothelial growth factor-2 receptor (VEGFR-2) kinase is a promising target for the development of novel anticancer drugs. Molecular docking modeling was performed on a series of 4-aryl-1,4-dihydropyridines derivatives to evaluate the structural basis for VEGFR-2 inhibitory activity. Some 4-aryl-1,4-dihydropyridines were synthesized in the reaction of aromatic aldehydes and ethyl propiolate with anilines in acetic acid. The biological activities were evaluated against the cells A549, A431 and Hep-G2. The results indicated that 4-aryl-1,4-dihydropyridines could be the promising potential VEGFR-2 inhibitors.

Full text of DOI:10.1134/S1070363216120574

ISSN 1070-3632, Russian Journal of General Chemistry, 2016, Vol. 87, No. 12, pp. 2891–2899. © Pleiades Publishing, Ltd., 2016.
Biological Evaluation of 4-Aryl-1,4-dihydropyridines
as VEGFR-2 Kinase Inhibitors1
W. Sun, Z. Ma, and H. Yana*
College of Life Science and Bioengineering, Beijing University of Technology,
Pingleyuan no. 100 of Chaoyang District, Beijing, 100124 China
*e-mail: hongyan@bjut.edu.cn
Received January 26, 2016
Abstract—Vascular endothelial growth factor-2 receptor (VEGFR-2) kinase is a promising target for the
development of novel anticancer drugs. Molecular docking modeling was performed on a series of 4-aryl-1,4-
dihydropyridines derivatives to evaluate the structural basis for VEGFR-2 inhibitory activity. Some 4-aryl-1,4-
dihydropyridines were synthesized in the reaction of aromatic aldehydes and ethyl propiolate with anilines in
acetic acid. The biological activities were evaluated against the cells A549, A431 and Hep-G2. The results
indicated that 4-aryl-1,4-dihydropyridines could be the promising potential VEGFR-2 inhibitors.
Keywords: VEGFR-2 inhibitors, 4-aryl-1,4-dihydropyridines, molecular docking, biological activity
DOI: 10.1134/S1070363216120574
INTRODUCTION
known inhibitors and VEGFR-2, there was presented a
typical pharmacophore model for the inhibitors with
central nitrogen heterocycles occupying the important
region. Such nitrogen heterocycles constitute an
important scaffold for VEGFR-2 inhibitors and
provide some information about VEGFR-2 kinase
inhibitors [12–14]. Herein, we describe an on-going
effort to develop small-molecule VEGFR-2 kinase
inhibitors based on the 1,4-dihydropyridines scaffold.
Molecular docking modeling was performed for
elucidating the benefit of 4-aryl-1,4-dihydropyridines
for VEGFR-2 inhibition. A series of 4-aryl-1,4-
dihydropyridines was synthesized by the reaction of
aromatic aldehydes and ethyl propiolate with anilines
in acetic acid and evaluated for inhibitory activity
against A549 (small cell lung cancer) cells in vitro
using a MTT assay.
Vascular endothelial growth factor-2 receptor
(VEGFR-2) is an important member of the Receptor
Tyrosine Kinases (RTKs) family and widely studied in
the regulating tumor angiogenesis [1, 2]. VEGFR-2
signaling promotes several endothelial responses such
as cell proliferation, migration and survival that are
required for the formation of new blood vessels [3].
Activation of VEGFR-2 is correlated with poor
prognosis and metastasis in the majority of solid tumor
patients. Inhibition of VEGFR-2 signaling blocks the
new blood vessels formation in growing tumors and
results in stasis or suppression of tumor growth [4, 5].
The development of small-molecule VEGFR-2 kinase
inhibitors for anticancer therapy has initiated active
pharmaceutical research. At present, many structurally
diverse VEGFR-2 inhibitors have been approved for
therapeutic treatment, including Vatalanib, BMS-
794833 and Sorafenib Tosylate (Fig. 1) [6–8].
RESULTS AND DISCUSSION
Molecular docking. On the basis of the crystal
structure of VEGFR-2 complexed with a Sorafenib
tosylate inhibitor (PDB code: 4ASD), AutoDock 4.0
(Scripps Research Institute, La Jolla, CA, USA) was
used to simulate 4-aryl-1,4-dihydropyridines (1) [15].
The docking results (Table 1) and are compared with
those of the known inhibitors 1–3.
1,4-Dihydropyridines make a class of calcium
antagonists that oppose the entry of calcium into cells
and reduce the influx of calcium ions through the slow
calcium channels in the cells [9]. However, it was
considered that the 1,4-dihydropyridines have an
influence on tumor cells [10, 11]. Based on the
molecular modelling of the interactions between the
According to data presented in Table 1, compounds
1 exhibited higher binding capacity with VEGFR-2
than the known inhibitors. Free energy ΔG and
1 The text was submitted by the authors in English.
2891

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SUN et al.
F
Cl
HN
N
N
O
O
H
N
F
NH
N
H2N
O
N
Cl
BMS-794833
O
Vatalanib
Cl
CF3
O
O
N
H
N
N
N
H
H
Sorafenib Tosylate
Fig. 1. Representative VEGFR-2 kinase inhibitors.
inhibition constant values Ki of 1 were between –7 and
–5 kcal/mol and between 4 and 150 μM, respectively.
The binding capacities were similar to those obtained
from the docking of the known inhibitors (ΔG = –6.41,
–6.53, and 6.42 kcal/mol, Ki = 20.12, 16.44, and
19.62 μM, respectively). The compound 1m' (R1 =
2,3,4-triOCH3, R2 = Bn) exhibited higher binding
capacity with VEGFR-2 (ΔG = 7.34 kcal/mol, Ki =
4.19 μM). The substituents R1 and R2 probably could
influence upon the binding capacity. In cases of R1 =
OCH3 or NO2 and R2 = Bz the binding capacity
increased significantly.
The compounds 1w and 1m' were also involved in
hydrogen bonding with the enzyme backbone. The –
COOCH3 group formed two H-bonds with the
CYS1043 sulfhydryl and the –OCH3 groups in meta-
and para-positions formed other three H-bonds with
the backbone N–H of ASN921 (Figs. 3d, 3f). For the
compound 1w, the –OCH3 groups in meta-position
formed the H-bond with the CYS1043 sulfhydryl
group (Figs. 3c, 3e). The –OCH3 groups could make
the molecule ligand integrated closely with amino acid
of the receptor by the H-bond. However, 1w was
involved in the π–π interaction between the benzene
ring and the amino acid PHE916. The protein cavity
could be totally occupied by the bulky Ph group of 1w
which strengthened stability of the complex constituted
by the ligand and receptor ultimately. The above could
probably explain why 1w and 1m' exhibited the higher
binding capacity for H-bonds with the enzyme
backbone and the benzyl transforming the core of 1
into an acceptable conformation within the active site
of VEGFR-2.
The sites and modes of interactions between ligands
and VEGFR-2 were considered. For the compounds
1w and 1m' and the known inhibitors a similar binding
site to VEGFR-2 was detected and the compounds 1w
and 1m' demonstrated high overlap ratios with
Vatalanib and BMS-794833 in the binding site of the
VEGFR-2 kinase (Fig. 2), which was consistent with
the biological activities data. For the known inhibitors,
the hydrogen bonds with the enzyme backbone were
detected. In the inhibitor BMS-794833 the NH2 group
formed two H-bonds with the ARG840 carboxyl
group. The C=O group formed the third H-bond with
the backbone N–H of PHE1045 (Fig. 3a). In the
inhibitor Sorafenib Tosylate the NH group formed the
H-bond with the ASP1026 carboxyl group (Fig. 3b).
Synthesis of 4-aryl-1,4-dihydropyridines. Ac-
cording to the results of molecular docking, some of 4-
aryl-1,4-dihydropyridines were selected for the study.
Based on previously reported data [16, 17], 4-aryl-1,4-
dihydropyridines 1 were synthesized in the reaction of
aromatic aldehydes and ethyl propiolate with anilines
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BIOLOGICAL EVALUATION OF 4-ARYL-1,4-DIHYDROPYRIDINES
Table 1. Molecular docking data for 1 and the known inhibitors with VEGFR-2
2893
Comp.
no.
Comp.
no.
R1
R2 ΔGa
Kib
R1
R2
ΔGa
Kib
85.31
1a
1b
1c
1d
1e
1f
H
H
H
H
H
H
H
H
H
H
H
H
H
5.13 172.92
5.18 158.38
1w
1x
H
H
H
H
H
H
H
H
4-CH3-Ph
4-Cl-Ph
5.55
4-CH3
5.33 125.02
5.24 143.70
4-OH
5.66
70.51
1y
4-F-Ph
4-Cl
4.70 359.93
1z
4-NO2-Ph
4-OCH3-Ph
6.59
6.13
14.70
32.28
60.43
4-F
5.49
6.10
94.51
33.70
1a'
1b'
1c'
1d'
1e'
1f'
1g'
1h'
1i'
4-NO2
2,4-diOCH3-Ph 5.63
1g
1h
1i
4-CF3
4.86 274.68
5.25 141.15
4.38 613.15
4.98 224.01
5.06 196.03
4.80 303.73
3,4,5-triCH3-Ph 5.35 119.82
4-OCH3
2,4-diOCH3
2,3,4-triOCH3
2,4,5-triOCH3
3,4,5-triOCH3
H
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
6.69
6.92
6.75
6.99
6.60
7.05
6.26
6.64
6.68
7.34
7.43
7.29
6.41
6.53
6.42
12.44
8.46
4-CH3
4-OH
1j
11.29
7.57
1k
1l
4-Cl
4-F
14.40
6.74
1m
1n
1o
1p
1q
1r
1s
Ph 5.45 100.49
Ph 5.85 51.44
4-NO2
4-CF3
4-CH3
1j'
1k'
1l'
25.81
13.49
12.68
4.19
4-OH
Ph 5.24 143.70
Ph 5.37 116.36
4-OCH3
2,4-diOCH3
4-Cl
4-F
Ph 5.67
Ph 6.26
Ph 6.53
69.77
25.81
16.44
1m'
1n'
1o'
2,3,4-triOCH3 Bn
2,4,5-triOCH3 Bn
3,4,5-triOCH3 Bn
4-NO2
3.55
4-OCH3
2,4-diOCH3
4.53
1t
Ph 5.40 109.91
Vatalanib
–
–
–
–
–
–
20.12
16.44
19.62
1u
2,4,5-triOCH3 Ph 5.68
68.34
BMS-794833
1v
3,4,5-triOCH3 Ph 6.75
11.29
Sorafenib Tosylate
a Binding free energy (kcal/mol). b Inhibition constant (μM).
in acetic acid (Scheme 1). The structures of products 1
were confirmed by 1H NMR.
kinase was evaluated against A549 (lung carcinoma)
cells, A431 (epidermoid carcinoma) cells and Hep-G2
(hepatocellular carcinoma) cells using a MTT assay
[18, 19]. Taxol was used as a reference drug. The
biological activity data of 4-aryl-1,4-dihydropyridines
Biological evaluation. The preliminary biological
activity of 4-aryl-1,4-dihydropyridines on VEGFR-2
Scheme 1. Synthesis of 4-aryl-1,4-dihydropyridines 1.
R1
R1
O
O
O
HOAc
+
+ R2−NH3
EtO
OEt
OEt
CHO
N
R2
1
R1 = H, 4-CH3, 4-OH, 4-Cl, 4-F, 4-NO2, 4-CF3, 4-OCH3, 2,4-diOCH3, 2,3,4-triOCH3, 2,4,5-triOCH3, 3,4,5-triOCH3; R2 = H,
4-CH3-Ph, 4-F-Ph, 4-Cl-Ph, 4-NO2-Ph, 4-OCH3-Ph, 2,4-diOCH3-Ph, 3,4,5-triOCH3-Ph.
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SUN et al.
(b)
(a)
Vatalanib
(c)
BMS-794833
(d)
1w
1m'
(e)
(f)
1m'
1w
Fig. 2. The binding site of Vatalanib, BMS-794833, 1w, and 1m' to VEGFR-2. (a) The binding site of Vatalanib with VEGFR-2
kinase. (b) The binding site of BMS-794833 with VEGFR-2 kinase. (c) The binding site of 1w with VEGFR-2 kinase. (d) The
binding site of 1m' with VEGFR-2 kinase. (e) 3D model of the binding site of 1w with VEGFR-2 kinase. (f) 3D model of the
binding site of 1m' with VEGFR-2 kinase.
1, expressed as IC50 values, are summarized in Table 2.
The products 1 exhibited promising inhibitory active-
ties with IC50 values less than 100 μM for 1w and 1m'
that could be related to the influence of the substituents
as mentioned above. It was determined that the
introduction of the methoxy groups into the benzene
ring increased the inhibitory activity of such com-
pounds as 1s (R1 = 4-OCH3) and 1w (R1 = 3,4,5-
triOCH3). Likewise, the benzyl group at the N atom
increased the inhibitory activity of 1l' (R2 = Bn) and
1m' (R2 = Bn) as well. This was consistent with the
molecular docking data.
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BIOLOGICAL EVALUATION OF 4-ARYL-1,4-DIHYDROPYRIDINES
(b)
2895
(a)
BMS-794833
(c)
Sorafenib tosylate
(d)
1w
1m'
(e)
(f)
1w
1m'
Fig. 3. The binding mode of BMS-794833, Sorafenib tosylate, 1w, and 1m' to VEGFR-2. (a) Interactions of BMS-794833 with
VEGFR-2 kinase. (b) Interactions of Sorafenib tosylate with VEGFR-2 kinase. (c) Interactions of 1w with VEGFR-2 kinase.
(d) Interactions of 1m' with VEGFR-2 kinase. (e) 2D diagram of interactions of 1w with VEGFR-2 kinase. (f) 2D diagram of
interactions of 1m' with VEGFR-2 kinase.
EXPERIMENTAL
standard bond lengths and bond angles of the
GAUSSVIEW 3.09 software (Gaussian, Inc.,
Wallingford, CT, USA). The geometry optimization
was performed by the semi-empirical AM1 method
and the output files were then minimized using the
density functional (DFT) method by applying the B3
LYP (Becke, Lee, Yang, and Parr) correlation function
in the course of second optimization. The Gasteiger
Molecular docking modeling. Molecular docking
was performed using AutoDock 4.0 that used the
Lamarckian genetic algorithm (LGA) for exploring the
full range of ligand conformational flexibility with
partial flexibility of the receptor. Three-dimensional
structures of the ligands were constructed using
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SUN et al.
Table 2. Biological activities of 4-aryl-1,4-dihydropyridines 1 against A549, A431 and Hep-G2 cells lines in vitro
a
IC50 , μM
A431
>200
IC50a, μM
A431
>200
Comp. no.
Comp. no.
A549
>200
Hep-G2
112.62
>200
A549
165.91
81.37
89.54
Hep-G2
130.35
89.95
1w
1a'
1a
1c
1f
159.92
144.23
101.01
71.54
127.71
166.31
78.89
88.26
69.69
90.02
85.05
>200
35.24
117.69
>200
>200
1d'
129.96
133.54
>200
1h
1i
70.32
58.63
72.98
58.44
86.20
147.25
>200
1f'
>200
1i'
105.64
88.36
67.23
29.50
85.40
77.18
0.0006
120.54
60.12
31.39
20.10
50.00
43.25
7.359
1j
65.54
1k'
103.00
96.49
1k
1l
80.23
1l'
68.10
1m'
1n'
41.88
1m
1n
1s
1v
180.76
150.50
34.40
77.35
>200
1o'
Taxolb
94.60
26.56
41.00
4.855
23.09
16.20
30.23
a Values are the means of at least three independent experiments SD < 10%.
b Used as positive control.
partial charges were assigned using AutoDock Tools
General procedure for 4-aryl-1,4-dihydropyri-
[15]. The crystal structure of VEGFR-2 complexed
with sorafenib inhibitor (PDB code: 4ASD) was
retrieved from the Brookhaven Protein Data Bank. The
co-crystallized ligand was extracted from the PDB file
and AutoDock Tools were used to assign polar
hydrogens and Gasteiger charges. AutoGrid4 was used
to create affinity grid maps for all atom types. The
affinity grids enclosed a volumetric space of 50 Å
with 0.375 Å spacing, centered on the coordinates x =
–0.975, y = 37.541, z = 20.332. The docked conforma-
tions were generated using the LGA with an initial
population size of 150 structures. The rest of
parameters were set to their default values. The model
analysis was performed using the ACCELRYS DS
VISUALIZER 3.0 software (Accelrys, Inc., San
Diego, CA, U.S.A.).
dine-3,5-dicarboxylate (1). A mixture of aromatic
aldehyde (0.05 mol), ethyl propiolate (0.10 mol),
amine (0.05 mol), and 5.0 mL of acetic acid was
heated in a steam bath for 25 min [16, 17]. The product
was crystallized from methanol : water, 4 : 1 and then
recrystallized from acetone : hexane, 1 : 1.
4-Phenyl-1,4-dihydropyridine-3,5-dicarboxylate
1
(1a). Yield 51.8%. H NMR spectrum, δ, ppm: 1.19 t
(6H, CH2CH3), 4.00–4.15 m (4H, CH2CH3), 4.89 s
(1H, Ar-CH), 6.65 br.s (1H, N–H), 7.12–7.35 m (5H,
Ar-H), 7.30 d (J = 5.2 Hz, 2H, C=CH).
4-(4-Hydroxyphenyl)-1,4-dihydropyridine-3,5-
1
dicarboxylate (1c). Yield 55.4%. H NMR spectrum,
δ, ppm: 1.19 t (6H, CH2CH3), 4.03–4.16 m (4H,
CH2CH3), 4.84 s (1H, Ar-CH), 6.08 br.s (1H, N-H),
6.71 d (J = 8.5 Hz, 2H, Ar-H), 7.22 d (J = 8.5 Hz, 2H,
Ar-H), 7.33 d (J = 5.3 Hz, 2H, C=CH).
Synthesis of 1,4-bis(2,3-dihydropyrrol-5-one-4-
yl)-1,3-butadienes. All chemicals were used as
purchased without further purification. All solvents
were reagent grade and, when necessary, purified and
dried using the standard methods. Reactions progress
was monitored by TLC on 0.25 mm silica gel plates
(60GF-254) and visualized with UV light. The melting
points were determined using an X-5 apparatus (open
4-(4-Nitrophenyl)-1,4-dihydropyridine-3,5-
1
dicarboxylate (1f). Yield 67.0%. H NMR spectrum,
δ, ppm: 1.19 t (6H, CH2CH3), 4.03–4.16 m (4H,
CH2CH3), 5.04 s (1H, Ar-CH), 6.78 br.s (1H, N-H),
7.38 d (J = 5.8 Hz, 2H, Ar-H), 7.52 d (J = 5.8 Hz, 2H,
Ar-H), 8.12 d (J = 8.5 Hz, 2H, C=CH).
1
capillaries, uncorrected values). H NMR spectra were
4-(4-Methoxylphenyl)-1,4-dihydropyridine-3,5-
measured on a Bruker ARX-400 MHz spectrometer in
CHCl3 using TMS as the internal standard.
1
dicarboxylate (1h). Yield 56.8%. H NMR spectrum,
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BIOLOGICAL EVALUATION OF 4-ARYL-1,4-DIHYDROPYRIDINES
2897
δ, ppm: 1.19 t (6H, CH2CH3), 3.75 s (3H, Ar-OCH3),
4.03–4.13 m (4H, CH2CH3), 4.84 s (1H, Ar-CH), 6.28
br.s (1H, N-H), 6.77 d (J = 8.8 Hz, 2H, Ar-H), 7.24 d
(J = 8.8 Hz, 2H, Ar-H), 7.30 d (J = 5.6 Hz, 2H,
C=CH).
OCH3), 4.06–4.16 m (4H, CH2CH3), 4.91 s (1H, Ar-
CH), 6.79 d (J = 8.8 Hz, 2H, Ar-H), 7.29 d (J = 8.8 Hz,
2H, Ar-H), 7.26–7.48 m (5H, Ar-H), 7.65 s (2H, C=CH).
1-Phenyl-4-(3,4,5-trimethoxylphenyl)-1,4-di-
hydropyridine-3,5-dicarboxylate (1v). Yield 60.0%.
1H NMR spectrum, δ, ppm: 1.21 t (6H, CH2CH3), 3.80
s (6H, Ar-OCH3), 3.81 s (3H, Ar-OCH3), 4.11–4.18 m
(4H, CH2CH3), 4.92 s (1H, Ar-CH), 6.60 s (2H, Ar-H),
7.26–7.48 m (5H, Ar-H), 7.67 s (2H, C=CH).
4-(2,4-Dimethoxylphenyl)-1,4-dihydropyridine-
3,5-dicarboxylate (1i). Yield 58.0%. 1H NMR
spectrum, δ, ppm: 1.17 t (6H, CH2CH3), 3.76 s (3H, Ar-
OCH3), 3.81 s (3H, Ar-OCH3), 3.99–4.09 m (4H,
CH2CH3), 5.12 s (1H, Ar-CH), 6.27 br.s (1H, N-H),
6.39 s (1H, Ar-H), 6.40 d (J = 7.9 Hz, 1H, Ar-H), 7.16
d (J = 7.9 Hz, 2H, Ar-H), 7.26 d (J = 5.3 Hz, 2H,
C=CH).
1-(4-Methylphenyl)-4-phenyl-1,4-dihydropyri-
1
dine-3,5-dicarboxylate (1w). Yield 39.7%. H NMR
spectrum, δ, ppm: 1.17 t (6H, CH2CH3), 2.38 s (3H,
Ar-CH3), 4.04–4.15 m (4H, CH2CH3), 4.96 s (1H, Ar-
CH), 7.14–7.27 m (5H, Ar-H), 7.16 t (J = 8.4 Hz, 2H,
Ar-H), 7.35 t (J = 8.4 Hz, 2H, Ar-H), 7.62 s (2H, C=CH).
4-(2,3,4-Trimethoxylphenyl)-1,4-dihydropyri-
1
dine-3,5-dicarboxylate (1j). Yield 59.5%. H NMR
spectrum, δ, ppm: 1.19 t (6H, CH2CH3), 3.81 s (3H, Ar-
OCH3), 3.83 s (3H, Ar-OCH3), 3.94 s (3H, Ar-OCH3),
4.02–4.12(m, 4H, CH2CH3), 5.06 s (1H, Ar-CH), 6.18
br.s (1H, N-H), 6.55 s (J = 8.6 Hz, 1H, Ar-H), 6.98 s
(J = 8.6 Hz, 1H, Ar-H), 7.30 d (J = 5.2 Hz, 2H, C=CH).
1-(4-Methoxylphenyl)-4-phenyl-1,4-dihydropyri-
dine-3,5-dicarboxylate (1a'). Yield 56.4%. H NMR
spectrum, δ, ppm: 1.18 t (6H, CH2CH3), 3.83 s (3H, Ar-
OCH3), 4.02–4.16 m (4H, CH2CH3), 4.95 s (1H, Ar-
CH), 6.95 t (J = 8.8 Hz, 2H, Ar-H), 7.14–7.28 m (5H,
Ar-H), 7.36 t (J = 8.8 Hz, 2H, Ar-H), 7.55 s (2H, C=CH).
1
4-(2,4,5-Trimethoxylphenyl)-1,4-dihydropyri-
1
dine-3,5-dicarboxylate (1k). Yield 51.3%. H NMR
1-Benzyl-4-phenyl-1,4-dihydropyridine-3,5-dicar-
spectrum, δ, ppm: 1.18 t (6H, CH2CH3), 3.80 s (3H, Ar-
OCH3), 3.81 s (3H, Ar-OCH3), 3.84 s (3H, Ar-OCH3),
4.00–4.10 m (4H, CH2CH3), 5.07 s (1H, Ar-CH), 6.46
s (1H, Ar-H), 6.47 br.s (1H, N-H), 6.82 s (1H, Ar-H),
7.29 d (J = 5.4 Hz, 2H, C=CH).
1
boxylate (1d'). Yield 46.3%. H NMR spectrum, δ,
ppm: 1.17 t (6H, CH2CH3), 4.01–4.11 m (4H,
CH2CH3), 4.59 s (2H, Ar-CH2), 4.91 s (1H, Ar-CH),
7.13–7.43 m (10H, Ar-H), 7.22 s (2H, C=CH).
1-Benzyl-4(4-hydroxyphenyl)-1,4-dihydropyri-
dine-3,5-dicarboxylate (1f'). Yield 48.9%. H NMR
spectrum, δ, ppm: 1.18 t (6H, CH2CH3), 4.02–4.12 m
(4H, CH2CH3), 4.57 s (2H, Ar-CH2), 4.83 s (1H, Ar-
CH), 6.61 d (J = 8.5 Hz, 2H, Ar-H), 7.10 d (J = 8.5 Hz,
2H, Ar-H), 7.26 s (2H, C=CH), 7.28–7.41 m (5H, Ar-H).
4-(3,4,5-Trimethoxylphenyl)-1,4-dihydropyri-
dine-3,5-dicarboxylate (1l). Yield 60.5%. H NMR
spectrum, δ, ppm: 1.22 t (6H, CH2CH3), 3.80 s (3H, Ar-
OCH3), 3.81 s (6H, Ar-OCH3), 4.06–4.17 m (4H,
CH2CH3), 4.87 s (1H, Ar-CH), 6.43 br.s (1H, N-H),
6.57 s (2H, Ar-H), 7.36 d (J = 5.2 Hz, 2H, C=CH).
1
1
1-Benzyl-4(4-nitrophenyl)-1,4-dihydropyridine-
3,5-dicarboxylate (1i'). Yield 62.0%. 1H NMR
spectrum, δ, ppm: 1.10 t (6H, CH2CH3), 3.95–4.04 m
(4H, CH2CH3), 4.56 s (2H, Ar-CH2), 4.98 s (1H, Ar-
CH), 6.68 d (J = 8.5 Hz, 2H, Ar-H), 7.23 d (2H, Ar-H),
7.27 s (2H, C=CH), 7.32 t (1H, Ar-H), 7.36 d.d (2H,
Ar-H), 7.37 d (J = 8.6 Hz, 2H, Ar-H), 8.01 d (J =
8.6 Hz, 2H, Ar-H).
1-Phenyl-4-phenyl-1,4-dihydropyridine-3,5-
dicarboxylate (1m). Yield 42.5%. 1H NMR spectrum,
δ, ppm: 1.20 t (6H, CH2CH3), 4.04–4.18 m (4H,
CH2CH3), 4.97 s (1H, Ar-CH), 7.15–7.48 m (10H,
Ar-H), 7.67 s (2H, C=CH).
1-Phenyl-4-(4-methylphenyl)-1,4-dihydropyri-
1
dine-3,5-dicarboxylate (1n). Yield 44.0%. H NMR
spectrum, δ, ppm: 1.19 t (6H, CH2CH3), 2.29 s (3H,
Ar-CH3), 4.04–4.17 m (4H, CH2CH3), 4.92 s (1H, Ar-
CH), 7.05 d (J = 8.0 Hz, 2H, Ar-H), 7.27 d (J = 8.0 Hz,
2H, Ar-H), 7.24–7.48 m (5H, Ar-H), 7.65 s (2H, C=CH).
1-Benzyl-4(4-methoxylphenyl)-1,4-dihydropyri-
dine-3,5-dicarboxylate (1k'). Yield 51.3%. H NMR
1
spectrum, δ, ppm: 1.17 t (6H, CH2CH3), 3.74 s (3H, Ar-
OCH3), 4.01–4.12 m (4H, CH2CH3), 4.58 s (2H, Ar-
CH2), 4.84 s (1H, Ar-CH), 6.68 d (J = 8.5 Hz, 2H,
Ar-H), 7.03 d (J = 8.5 Hz, 2H, Ar-H), 7.22 s (2H,
C=CH), 7.29–7.34 m (5H, Ar-H).
1-Phenyl-4-(4-methoxylphenyl)-1,4-dihydropyri-
1
dine-3,5-dicarboxylate (1s). Yield 50.7%. H NMR
spectrum, δ, ppm: 1.21 t (6H, CH2CH3), 3.77 s (3H, Ar-
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 86 No. 12 2016

2898
SUN et al.
1-Benzyl-4(2,4-dimethoxylphenyl)-1,4-dihydro-
concentration of 5 mg/mL and incubated with the cells
at 37°C for 4 h. Formazan crystals were dissolved in
100 μL of DMSO for each well and the absorbance at
570 nm was measured using the multifunctional reading
machine Synergy 4. All compounds were tested in
triplicate for each of the cell lines. The results were
expressed as average IC50 values of three measure-
ments and calculated using GraphPad Prism5 software.
pyridine-3,5-dicarboxylate (1l'). Yield 56.8%. 1H NMR
spectrum, δ, ppm: 1.15 t (6H, CH2CH3), 3.70 s (3H,
Ar-OCH3), 3.76 s (3H, Ar-OCH3), 3.97–4.07 m (4H,
CH2CH3), 4.56 s (2H, Ar-CH2), 5.12 s (1H, Ar-CH),
6.64 d (J = 9.0 Hz, 1H, Ar-H), 6.65 s (1H, Ar-H), 7.07
d (J = 9.0 Hz, 1H, Ar-H), 7.23 s (2H, C=CH), 7.30–
7.44 m (5H, Ar-H).
1-Benzyl-4(2,3,4-trimethoxylphenyl)-1,4-dihydro-
pyridine-3,5-dicarboxylate (1m'). Yield 42.2%. 1H NMR
spectrum, δ, ppm: 1.17 t (6H, CH2CH3), 3.80 s (3H, Ar-
OCH3), 3.82 s (3H, Ar-OCH3), 3.84 s (3H, Ar-OCH3),
4.01–4.10 m (4H, CH2CH3), 4.58 s (2H, Ar-CH2), 5.07
s (1H, Ar-CH), 6.51 d (J = 8.6 Hz, 1H, Ar-H), 6.88 d
(J = 8.6 Hz, 1H, Ar-H), 7.23 s (2H, C=CH), 7.31–7.44
m (5H, Ar-H).
CONCLUSIONS
A series of 4-aryl-1,4-dihydropyridines as the potential
VEGFR-2 inhibitors is presented. Molecular docking
performed with the ligand binding site of VEGFR-2
indicated that 4-aryl-1,4-dihydropyridines had higher
binding capacity with VEGFR-2 compared with the
known inhibitors. Introduction of the methoxy groups
into the benzene ring and the benzyl group at the N
atom increased the binding capacity with VEGFR-2.
4-Aryl-1,4-dihydropyridines were synthesized by the
reaction of aromatic aldehydes and ethyl propiolate
with anilines in acetic acid. The biological activity of
4-aryl-1,4-dihydropyridines on VEGFR-2 kinase was
evaluated against A549 cells, A431 cells and Hep-G2
cells using an MTT assay. Compounds 1 demonstrated
promising potential inhibitory activities with IC50
values less than 100 μM. According to the accu-
mulated data 4-aryl-1,4-dihydropyridines have good
potential for further development as a new class of
VEGFR-2 inhibitors with promising antitumor potential.
1-Benzyl-4(2,4,5-trimethoxylphenyl)-1,4-dihydro-
pyridine-3,5-dicarboxylate (1n'). Yield 45.0%. 1H NMR
spectrum, δ, ppm: 1.18 t (6H, CH2CH3), 3.68 s (3H, Ar-
OCH3), 3.71 s (3H, Ar-OCH3), 3.83 s (3H, Ar-OCH3),
3.99–4.09 m (4H, CH2CH3), 4.56 s (2H, Ar-CH2), 5.11
s (1H, Ar-CH), 6.42 s (1H, Ar-H), 6.70 s (1H, Ar-H),
7.26 s (2H, C=CH), 7.32–7.42 m (5H, Ar-H).
1-Benzyl-4(3,4,5-trimethoxylphenyl)-1,4-dihydro-
1
pyridine-3,5-dicarboxylate (1o'). Yield 60.0%. H
NMR spectrum, δ, ppm: 1.21 t (6H, CH2CH3), 3.72 s
(6H, Ar-OCH3), 3.78 s (3H, Ar-OCH3), 4.05–4.16 m
(4H, CH2CH3), 4.58 s (2H, Ar-CH2), 4.86 s (1H, Ar-
CH), 6.48 s (2H, Ar-H), 7.29–7.40 m (5H, Ar-H), 7.30
s (2H, C=CH).
ACKNOWLEDGMENTS
Biological assays. The biological activities of com-
pounds 1 were evaluated against A549 (lung carcinoma)
cells, A431 (epidermoid carcinoma) cells and Hep-G2
(hepatocellular carcinoma) cells. The cells were ob-
tained from Shanghai Institute of Biochemistry and
Cell Biology (Shanghai, China). The cells were cultured
in RPMI-1640 and maintained in a Thermo incubator
(Waltham, MA) under humidified air containing 5%
CO2 and 95% air. All culture media contained 10%
fetal bovine serum (FBS) and 1% penicillin-strepto-
mycin solution (10000 units of penicillin and 10 mg of
streptomycin in 0.9% NaCl). The cancer cell lines
were cultured in minimum essential media (MEM).
Four thousand cells (per well) suspended in MEM
were plated into each well of a 96-well plate and
incubated for 24 h. The tested compounds were added
to the culture medium at the indicated final concentra-
tions and the cell cultures were stored for 72 h. Fresh
MTT was added to each well to make the final
This work was financially supported by the Key
Projects in the Beijing Municipal Natural Science
Foundation (no. KZ201510005007).
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