Synthesis and characterisation of (Z)-styrylbenzene derivatives as potential selective anticancer agents

Article abstract of DOI:10.1080/14756366.2018.1513925

To identify anticancer agents with high potency and low toxicity, a series of (Z)-styrylbenzene derivatives were synthesised and evaluated for anticancer activities using a panel of nine cancer cell lines and two noncancerous cell lines. Most derivatives exhibited significant anti-proliferative activities against five cancer cell lines, including MGC-803 and BEL-7402. (Z)-3-(p-Tolyl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile (6h) showed a strong inhibitory effect on MGC-803 cells (IC₅₀ < 0.01 μM) and exhibited stronger anti-proliferative activity than taxol (IC₅₀ < 0.06 ± 0.01 μM). The IC₅₀ value of 6h in L-02 cells was 10,000-fold higher than in MGC-803 cells. Compound 6h inhibited proliferation of BEL-7402 cells by arresting at the G2/M phase through up-regulation of cyclin B1 expression, down-regulation of cyclin A and D1 expression, and induction of apoptosis. In addition, 6h inhibited the migration of BEL-7402 cells and the formation of cell colonies.

Full text of DOI:10.1080/14756366.2018.1513925

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and characterisation of (Z)-
styrylbenzene derivatives as potential selective
anticancer agents
Ya-Bing Xin, Jia-Jun Li, Hong-Jian Zhang, Jun Ma, Xin Liu, Guo-Hua Gong & Yu-
Shun Tian
To cite this article: Ya-Bing Xin, Jia-Jun Li, Hong-Jian Zhang, Jun Ma, Xin Liu, Guo-Hua Gong
& Yu-Shun Tian (2018) Synthesis and characterisation of (Z)-styrylbenzene derivatives as
potential selective anticancer agents, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1,
1554-1564, DOI: 10.1080/14756366.2018.1513925
To link to this article: https://doi.org/10.1080/14756366.2018.1513925
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 33, NO. 1, 1554–1564
https://doi.org/10.1080/14756366.2018.1513925
RESEARCH PAPER
Synthesis and characterisation of (Z)-styrylbenzene derivatives as potential
selective anticancer agents
a
a
a
b
a
c,d
Ya-Bing Xin , Jia-Jun Li , Hong-Jian Zhang , Jun Ma , Xin Liu , Guo-Hua Gong and Yu-Shun Tiana
ꢀ
ꢀ
aKey Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain, Affiliated Ministry of Education, College of
Pharmacy, Yanbian University, Yanji, P.R. China; Jiangsu Hansoh Pharmaceutical Group Co., Ltd., Lianyungang, P.R. China; First Clinical Medical
College of Inner Mongolia University for Nationalities, Tongliao, P.R. China; Inner Mongolia Key Laboratory of Mongolian Medicine
b
c
d
Pharmacology for Cardio-Cerebral Vascular System, Inner Mongolia University for Nationalities, Tongliao, P.R. China
ABSTRACT
ARTICLE HISTORY
Received 8 May 2018
Revised 31 July 2018
Accepted 16 August 2018
To identify anticancer agents with high potency and low toxicity, a series of (Z)-styrylbenzene derivatives
were synthesised and evaluated for anticancer activities using a panel of nine cancer cell lines and two
noncancerous cell lines. Most derivatives exhibited significant anti-proliferative activities against five cancer
cell lines, including MGC-803 and BEL-7402. (Z)-3-(p-Tolyl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile (6h)
showed a strong inhibitory effect on MGC-803 cells (IC50 < 0.01 mM) and exhibited stronger anti-prolifera-
tive activity than taxol (IC50 < 0.06 0.01 mM). The IC50 value of 6h in L-02 cells was 10,000-fold higher
than in MGC-803 cells. Compound 6h inhibited proliferation of BEL-7402 cells by arresting at the G2/M
phase through up-regulation of cyclin B1 expression, down-regulation of cyclin A and D1 expression, and
induction of apoptosis. In addition, 6h inhibited the migration of BEL-7402 cells and the formation of
cell colonies.
KEYWORDS
Styrylbenzene; cyano;
selective toxic effect;
anticancer; synthesis
Introduction
bond, was found to have better selective toxic effect against HeLa
cells with fewer toxic effects on non-cancerous L-02 cells when
compared with the commercially available drug taxol2.
Cancer is becoming an increasingly important disease and is a
leading cause of death worldwide1. Although a variety of anti-
cancer agents are currently available, none is able to eradicate
cancer cells without having toxic effects on healthy tissues2.
Therefore, finding new anticancer agents with higher potency and
lower toxicity is a great challenge3,4. More than 60% of anticancer
drugs used in the clinic originate from natural sources5. Agents
containing a styrylbenzene structure, such as resveratrol ((E)-3,5,40-
trihydroxystilbene)6 and CA-4 ((Z)-2-methoxy-5–(3,4,5-trimethoxys-
tyryl) phenol)7, are naturally present in medicinal plants (Figure 1)
and have been shown to exert anticancer effects with high tox-
On the basis of the previous study, we retained the trimethoxy
groups on the A benzene ring, introduced a cyano group into the
vinyl bridge to maintain its Z isomer, and either changed the sub-
stituents on the B benzene ring or replaced the B benzene ring
with other heterocyclic structures to design various styrylbenzene
derivatives. We also replaced the 3,4,5-trimethoxy groups with
p-methyl groups to further study the effect of trimethoxy groups
on anticancer activity and selective toxic effects (Figure 1).
icity to normal cells6–8
.
Materials and methods
The microtubule system of eukaryotic cells is an important tar-
get for the development of anticancer agents9. Recently, much
attention has been devoted to studying microtubule colchicine-
binding sites of antimitotic agents, such as CA-4 and its ana-
logs10–13. Compounds with a structure similar to that of colchicine
and having trimethoxy groups on the A benzene ring (Figure 1)
may have potential as anticancer drugs. For example, it was
reported that compounds with O-methylation on the A ring of the
styrylbenzene structure displayed significant anticancer effects14.
Cyano is highly polar, small in size, has electron-withdrawing
properties and is able to react with key amino acid residues of
Chemistry
1H NMR and 13C NMR spectra were obtained on an AV-300
(Bruker, Switzerland), and using TMS as internal standard. The
chemical shifts are given in ppm referenced to the respective solv-
ent peak, and coupling constants (J) are reported in hertz (Hz).
High-resolution mass spectra were measured on a MALDI-TOF/TOF
mass spectrometer (Bruker Dartonik, Germany). IR spectra were
recorded (in KBr) on a FTIR1730 instrument. All reagents were
obtained commercially and used were of analytical grade.
Reaction processes were tracked by TLC on silica gel plates.
active sites on a variety of proteins15. In a previous experiment, Melting points of target compounds were determined in the open
compound 3 (Figure 1), which had a cyano group on the ethylene glass capillaries and were uncorrected.
CONTACT Yu-Shun Tian
tyshhanguo1@hanmail.net
Key Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain, Affiliated
Ministry of Education, College of Pharmacy, Yanbian University, Yanji, Jilin Province, 133002, PR China; Guo-Hua Gong
Clinical Medical College of Inner Mongolia University for Nationalities, Tongliao, Inner Mongolia, 028002, PR China
gongguohua0211@163.com
First
ꢀ
These authors contributed equally to this work and should be considered co-first authors.
Supplemental data for this article can be accessed here.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1555
Figure 1. The structures of resveratrol, compound 3, CA-4, colchicine, and the design of target compounds 6a-h, 7a-c, 13a-d, and 14a-b.
General procedure for synthesis of compounds 6a-h, 7a-c,
13a-d, and 14a-b
effect (NOE) (Figure 2). The detailed information on the structural
and physiochemical characteristics of the final target compounds
can be found in the Supplementary material.
3,4,5-Trihydroxybenzoic acid (1) and 4-methylbenzoic acid (8)
were used as starting materials to synthesise 5-(bromomethyl)-
1,2,3-trimethoxybenzene (4) and (bromomethyl) benzene (11),
respectively. Next, a mixture of 10 mmol of compound 4 or 11,
1.485 g (15 mmol) of TMSCN (trimethylsilyl cyanide), 4.725 g
(15 mmol) of TBAF (tetrabutyl-ammonium fluoride), and 30 ml of
acetonitrile was refluxed for 4–6 h with stirring. The reflux process
was monitored by TLC. The reaction mixture was cooled to room
temperature (RT), and then was added to 200 ml of ice water with
severe stirring. The mixture was filtered, washed with 50% metha-
nol, and purified by recrystallisation from 95% ethanol, yielding a
Biological evaluation
Materials
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide
(MTT) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).
The propidium iodide (PI) and Annexin V-FITC apoptosis detection
kit were purchased from Invitrogen (Eugene, OR, USA).
final product of compound 5 or 1216–20
.
Cell lines and cell culture
A mixture of 1 mmol compounds 5 or 12, 1 mmol properly sub-
stituted benzaldehyde or aromatic heterocyclic aldehyde, and
10 ml of methanol was heated to 60 ꢁC with stirring. After 30 min,
sodium methoxide (0.027 g, 0.5 mmol) was added to the mixture,
and the mixture was kept at 60 ꢁC for 4–6 h. The reaction was
monitored by TLC. Once complete, the reaction mixture was
cooled to RT, and the precipitate was separated by filtration and
recrystallised from methanol to give the target final compound
(Scheme 1). Because of a cyano-steric effect, the final structure
All human cell lines were used in this study. MGC-803 late-stage dif-
ferentiation gastric cancer cell, A549 lung cancer cell, HepG2 hepa-
toma cell, AGS gastric cancer cell, BEL-7402 liver cancer cell, HCT-
116 colorectal cancer cell, HeLa cervical cancer cell, SGC-7901 early
differentiation gastric cancer cell, L-02 normal liver cell, MCF-7
breast cancer cell, and MCF-10A normal breast cell were initially pur-
chased from American Type Culture Collection (ATCC, Manassas, VA,
USA). RPMI-1640 media, Dulbecco’s modified Eagle’s medium
(DMEM), and foetal bovine serum (FBS) were provided from Gibco
was fixed as the Z isomer, confirmed by the nuclear Overhauser Company (Grand Island, NY, USA). The cells were maintained in

1556
Y.-B. XIN ET AL.
O
O
OH
HO
O
O
O
O
O
O
OH
O
OH
Br
a
b
c
d
OH
O
O
O
1
2
3
4
CN
R1
O
O
O
R1
O
O
6a-6h
CN
e
O
CN
O
R2
O
O
R2
5
O
O
7a-7c
O
O
OH
O
OH
Br
a
b
c
d
9
10
8
11
CN
R3
O
R3
13a-13d
CN
e
CN
R4
14a-14b
13a R3 = -p-CH3
R4
O
12
6e R1 = -3,4,5-OCH3
6a R1 = -p-NO2
13c R3 = -3,4,5-OCH3
13d R3 = -p-H
13b R3 = -p-CH2CH3
6b R1 = -p-OC6H5
6c R1 = -m-OCH3
6f R1 = -p-CH2(CH3)2
6g R1 = -p-CH2CH3
6h R1 = -p-CH3
N
S
14a R4 =-
14b R4 =-
6d R1 = -p-OCH3
N
S
7c R2=-
7a R2 =-
7b R2 =-
Scheme 1. Reagents and conditions: (a) (CH3)2SO4, (CH3)2CO, K2CO3, reflux; (b) LiAlH4, THF, 0 ꢁC-rt, 4–6 h; (c) CH2Cl2, PBr3, 0 ꢁC-rt, 3–6 h; (d) CH3CN, TMSCN, TBAF,
reflux, 4–6 h; (e) CH3OH, CH3ONa, aromatic aldehydes, 4–6 h.
DMEM or RPMI-1640, supplemented with 10% FBS, 100 IU/mL peni- At least three independent experiments were conducted, and the
cillin and 100 mg/mL streptomycin (Grand Island, NY, USA), and at results were summarised in Table 1.
37 ꢁC in a humidified atmosphere containing 5% CO2.
Analysis for cell cycle by flow cytometry
Cell growth inhibition assay
BEL-7402 cells were plated 6-well plates with 5.0 ꢂ 105 cells/well
Cells were plated in 96-well plates at a density of 30–40% to ensure for overnight (O/N) and were synchronised at the G0 phase by
exponential growth throughout the experimental period, and then serum starvation (0.1% FBS in RPMI-1640) for 24 h. Then the cells
allowed to adhere for 1 day21. Next, the cells were treated with four were incubated with 0.01, 0.05, 0.1 mM of 6h and 0.1 lM of taxol,
serial concentrations (1, 10, 50, and 100 mM) of each compound. respectively. Vehicle group was treated with 0.1% DMSO.
Taxol, CA-4, and CA-4P (phosphorylated form of CA-4) were used as Following 12 h of incubation, the cells were detached with 0.05%
positive controls. After 48 h incubation, MTT solution was added to trypsin/5 mM EDTA and centrifuged at 1000 rpm for 10 min, and
each well at a final concentration of 2 mg/mL, and incubated for an fixed in 70% ethanol at ꢃ20 ꢁC for O/N. Then, the cells were sub-
additional 4 h. Next, the MTT solution was removed and 150 mL of sequently analysed the cell cycle distribution by flow cytometry
dimethyl sulphoxide (DMSO)/well was added (Sigma-Aldrich, St using a FACSCalibur flow cytometer with Cell Quest software
Louis, MO, USA). The plates were shaken vigorously at RT to ensure (Becton-Dickinson, Franklin Lakes, NJ, USA), plotting at least
complete solubilisation, and absorption at 492 nm was determined. 30,000 events per sample22. The percentage of cells in the G0/G1,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1557
Figure 2. NOE (nuclear overhauser effect) result of compound 6h.
S, and G2/M phases of the cell cycle were determined using the chambers was counted in each experiment under a phase-contrast
ModFit LT version 4.0 software package (Verity Software, microscope at 200ꢂ magnification.
Topsham, ME, USA).
Colony formation assay
Analysis of apoptosis by flow cytometry
A previously described colony formation assay was used7 with
slight modification. 1 ꢂ 104 BEL-7402 cells/well were seeded into
Apoptosis was detected using an apoptosis detection kit. BEL-
7402 cells were plated in 6-well plates (5.0 ꢂ 105 cells/well) and
6-well plates and then stimulated with the indicated concentration
incubated at 37 ꢁC overnight. Exponentially growing cells were
then incubated with 6h at either 0.01 or 0.1 mM, or taxol at
0.1 mM. Vehicle group was treated with 0.1% DMSO. Following
12 h of incubation, cells were analysed for apoptosis as previously
described23, using a FACSCalibur flow cytometer with Cell Quest
software (Becton-Dickinson, Franklin Lakes, NJ, USA).
of 6h, taxol, or DMSO. The plates were removed from incubation
when colonies were large enough to count (>50 cells). Colonies
were then fixed and stained with 1 ꢂ Giemsa solution, rinsed,
allowed to dry, and counted. All the experiments were repeated
independently at least three times. The LD50 value for 6h was
determined using SigmaPlot II.
Western blotting
Cell migration assay
BEL-7402 cells were harvested in logarithmic growth phase and
the adherent and floating cells were collected after 12 h of drug
treatment. The collected cells were resuspended in cold RIPA buf-
fer (100 mM NaCl, 100 mM Tris (pH7.4), 10% Triton X-100, 1 mM
PMSF, pH7.5) with 0.1 mM leupetin protease inhibitor as well as
1 mg/mL pepstatin and cracked on ice for 30 min. Then according
to the reference to did immunoblotting assay22.
Quantitative cell migration was performed using 24-well Boyden
chambers (Corning, NY, USA) as described7. Briefly, transwells with
8-mm pore size filters were inserted into 24-well plates. RPMI-1640
(500 mL) containing 10% FBS was added to the lower chamber,
and 100 mL of a serum-free cell suspension (1 ꢂ 105 cells) was
placed in the upper chamber. The BEL-7402 cells were pre-treated
with indicated concentrations of 6h, taxol, or DMSO. The plates
were incubated at 37 ꢁC with 5% CO2 for 24 h, and the cells on
the upside were removed using cotton swaps. After several rinses,
the cells adhering in the lower layer of insert were fixed with
Molecular modelling
methanol and stained with 1ꢂ crystal violet solution. The number The molecular docking study was performed using Discovery
of migrated cells in nine randomly selected fields from triplicate Studio 2017 Client. In this study, the structure of tubulin (PDB

1558
Y.-B. XIN ET AL.
code: 1SA0) downloaded from protein database was chosen for
docking. The 3D structures of compounds were provided using
Chem 3D, the pdbqt format files of ligand, and the autodock for-
mat of protein were provided using AutoDockTools version 1.5.6.
Results and discussion
Chemistry
As shown in Scheme 1, through series of reactions, such as esteri-
fication, reduction, bromination, substitution, and Knoevenagel
condensation, two series of derivatives were synthesised16–20. In
brief, 3,4,5-trihydroxybenzoic acid (1) or p-methylbenzoic acid (8)
as the starting material, dimethyl sulphate in the presence of acet-
one as solvent, potassium carbonate as a catalyst, compound 2 or
9 was obtained. Then, the compound was reduced respectively
under ice-cooling with lithium aluminium hydride as a reducing
agent, anhydrous tetrahydrofuran as solvent to give compound 3
or 10. The resulting compound 3 or 10 was dissolved in anhyd-
rous CH2Cl2 and replaced by PBr3 under ice bath to give com-
pound 4 or 11. Compound 5 or 12 was prepared by dissolving
compound 4 or 11 in acetonitrile, with TMSCN and TBAF as cata-
lysts. The series of target compounds were prepared by substitut-
ing aromatic aldehydes with compound 5 or 12 in the presence
of methanol as solvent and sodium methoxide as catalyst. Before
1
biological evaluation, the compounds were characterised via IR, H
NMR, and 13C NMR spectrometry as well as high-resolution mass
spectrometry, and the Z isomer was confirmed by NOE.
Biological evaluation
In vitro cytotoxicity assay and structure-activity relationship
(SAR) studies
According to standard protocol2, preliminary in vitro anti-prolifera-
tive activities of the synthesised compounds were evaluated by
MTT assay, using nine human cancer cell lines (MGC-803, A549,
HepG2, AGS, BEL-7402, HCT116, HeLa, SGC-7901, and MCF-7) and
two normal human cell lines (L-02 and MCF-10A), and compared
with those of CA-4, CA-4P, and taxol.
Results of the screening are summarised in Table 1. Most com-
pounds showed good anti-proliferative activities against cancer
cells including MGC-803, AGS, BEL-7402, HCT-116, and MCF-7.
Three alkyl-substituted compounds (6f, 6g, and 6h), p-methoxy-
substituted 6d, thiophene-2-yl derivative 7a, and naphthalene-1-yl
derivative 7c, all with 3,4,5-trimethoxy groups on the A benzene
ring, had lower IC50 values (<0.01–7.21 mM) in MGC-803. The order
of anticancer activity was p-CH3
ꢄ
p-CH2CH3 > thiophene-2-
yl > naphthalene-1-yl > p-OCH3 > p-(CH2)2CH3. Compounds 6g and
6h exhibited particularly strong inhibitory activities, with IC50 val-
ues less than 0.01 mM, making them more active than all control
agents tested. Similarly, 6d, 6f, 6g, 6h, 7a, and 7c exhibited better
anticancer activity (IC50 ¼ 0.05, 1.33, 4.38, 0.51, 8.01, 3.51 mM,
respectively) in AGS cells. In this assay, 6d exhibited activity simi-
lar to the positive control drug taxol (IC50 ¼ 0.02 mM), and stronger
activity than CA-4 (IC50 ¼ 3.42 mM) and CA-4P (IC50 ¼ 4.73 mM).
Compounds 6f, 6g, 6h, and 7c also showed stronger activity than
CA-4 and CA-4P against AGS cells. The order of anticancer
activity was p-OCH3 > p-CH3 > p-(CH2)2CH3 > naphthalene-1-yl > p-
CH2CH3 > thiophene-2-yl. Analysis using human hepatocellular car-
cinoma cells (BEL-7402) revealed that compound 6h had the best
anticancer activity (IC50 ¼ 0.15 mM), much stronger than CA-4
(IC50 ¼ 2.02 mM) and CA-4P (IC50 ¼ 5.45 mM). The high anticancer
activity of 6h was followed by compounds 6g, 6f, 6d, 7a, and 7c

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1559
Table 2. Selectivity index values of compounds relative to the effects on normal cell L-02 or MCF-10A cells.
SI
IC50
IC50
IC50
IC50
IC50
IC50
IC50
Agent
R
(L-02/MGC-803)
(L-02/A549)
(L-02/HepG-2)
(L-02/AGS)
(L-02/BEL-7402)
(L-02/HCT116)
(MCF-10A/MCF-7)
6d
6f
6g
6h
4-OCH3
4-CH2(CH3)2
4-CH2CH3
4-CH3
Thiophene-2-yl
–
–
>2.7
–
>188.6
–
–
–
–
–
–
>83.0
>75.1
>22.8
>196.0
>2.6
–
>5.5
>138.8
>153.8
>666.6
>11.6
–
>2.7
–
>1.3
–
>3.6
>3.7
>14.6
>30.3
>2.2
>13.8
>10000
>10000
>31.6
>18.3
>89.0
>1666
>588.2
>294.1
>163.9
>1.2
>6.8
>4.5
>26.7
7a
–
–
–
CA-4
CA-4P
Taxol
–
–
>227.2
>106.3
>5000
>1111
>3333
–: not suitable for calculation; SI: selectivity index, IC50 (L-02 or MCF-10A)/IC50 (cancer cell).
with IC50 values of 0.65, 0.72, 0.75, 1.82, and 2.53 mM, higher selectivity for cancer cells than for L-02 cells, including 6d
respectively. The order of anticancer activity was p-CH3 > p-
CH2CH3 > p-(CH2)2CH3 > p-OCH3 > thiophene-2-yl > naphthalene-1-
yl. Compounds 6f, 6g, and 6h exhibited better anticancer
activities (IC50 ¼ 0.17, 0.34, and 0.61 mM, respectively) than CA-4P
(IC50 ¼ 0.59 mM) on HCT116 cells. Compounds 6f, 6h, and 7c
expressed strong anticancer activity against MCF-7 (human breast
adenocarcinoma) cells. Remarkably, compound 6h was 6-fold
more active than taxol against MGC-803 cells and exhibited similar
activity to taxol against A549 and BEL-7402 cells. Compound 6h
was superior to CA-4 and CA-4P against almost all cancer cell lines
tested, including MGC-803, A549, HepG-2, AGS, and BEL-7402 cells.
However, 17 of the synthesised compounds displayed poor anti-
cancer activity against HeLa and SGC-7901 cancer cell lines. In
addition, among the compounds having 3,4,5-trimethoxy groups
on the A benzene ring, p-NO2, p-OBn, m-OCH3, and pyridine-2-yl
substituted compounds (6a, 6b, 6c, and 7b) expressed extremely
poor in vitro anticancer activities against all nine cancer cell lines,
and 3,4,5-trimethoxy substituted compound 6e, which has a heavy
steric effect, also showed weak anticancer activity against all can-
cer cell lines tested. Synthesised compounds having p-methyl
groups on the A benzene ring (13a–d and 14a–b) also demon-
strated poor anticancer activities against all cell lines tested.
Using these preliminary results, we identified the following
SARs: (1) the anticancer activity of a structure with a 3,4,5-trime-
thoxy group on the A benzene ring was greater than that of a
structure with a p-methyl group on the A benzene ring; and (2) in
structures containing 3,4,5-trimethoxy groups on the A benzene
ring, introducing a B benzene ring with small size para-electron-
donating groups, such as p-CH3, p-OCH3, and p-CH2CH3, or
exchanging B benzene with an electron-rich aromatic heterocyclic
structure, such as thiophene, significantly improves anticancer
activity. Based on these observations, we expect that in structures
containing 3,4,5-trimethoxy groups on the A benzene ring, elec-
tron density and steric hindrance effects on the B benzene ring
strongly influence the anticancer activity of the (Z)-styrylbenzene
derivatives bearing a cyano group synthesised.
for AGS, 6f and 6g for BEL-7402 and HCT116, 6h for A549, AGS,
BEL-7402, and HCT116 cells. However, none of the compounds
showed strong selectivity for MCF-7 (breast cancer) cells over
MCF-10A (normal breast) cells. The control compounds CA-4 and
CA-4P displayed poor selectivity for cancer cells over normal cells
although they induced strong cytotoxicity in cancer cells. On the
basis of these results, we speculated that 6h was the best candi-
date for further mechanism analysis. The strong toxicity of 6h for
MGC-803 cells (IC50 < 0.01 mM) is likely to introduce experimental
error even with small changes in concentration; therefore, BEL-
7402 cells, for which 6h demonstrated moderate toxicity and
selectivity, were selected for further study of 6h.
Cell cycle regulation by compound 6h and Western
blot analysis
The cell-cycle is a series of events that result in cell division, dupli-
cation, and proliferation. Numerous cytotoxic compounds exert
their anti-proliferative effect by inducing cell-cycle arrest, apop-
tosis, or both2. These mechanisms are considered effective anti-
cancer strategies21. To study the mechanism by which compound
6h reduced the viability of BEL-7402 cells, we used flow cytometry
to analyse cell-cycle distribution. As shown in Figure 3(A), 6h
increased the percentage of G2/M cell population in a concentra-
tion-dependent manner from 23.09% to 59.99% after 12 h incuba-
tion, and the trend was similar in the
S cell population
(4.44–19.35%). After treatment with 0.1 mM 6h, the cell distribution
at G0/G1, G2/M, and S phases was very similar to the 0.1 mM taxol
treatment group. This finding suggests that 6h induces cell-cycle
arrest at the S and G2/M phases.
Since entry into the S phase in the cell-cycle requires the accu-
mulation of cell-cycle activation-related cyclins7, we assessed the
expression levels of cyclins, such as cyclin A and cyclin D1 to
determine whether the decreased cell proliferation by 6h involved
these proteins. The 6h treatment group had lower expression of
cyclin A and D1 compared with the vehicle group, this reduced
expression was dose-dependent, and similar to that of the taxol
treatment group at 0.1 mM (Figure 3(B)). Cyclin B1 plays an import-
ant role in the transition from interphase to mitotic phase and
governs cell cycle progression by enhancing cell-cycle distribution
in the G2/M fraction24, and it has been reported that expression
of cyclin B1 is increased by exposure to anticancer agents25. In
our experiment, 6h treatment induced increased expression of
cyclin B1 compared with treatment with vehicle or 0.1 mM of taxol.
These results suggest that the down-regulation of cyclins A and
D1 and up-regulation of cyclin B1 are related to the decrease in
Selective inhibition of cancer cell growth
Lack of selective cytotoxicity is the main factor restricting the tol-
erated dose of most conventional chemotherapeutic agents2. To
address this, we compared the toxicity of all synthesised com-
pounds with CA-4, CA-4P, and taxol on normal human cell lines
L-02 and MCF-10A. The calculated selectivity index (SI) of each
compound is depicted in Table 2. Compounds 6g and 6h exhib-
ited 10,000-fold higher selectivity for MGC-803 cells than for nor-
mal L-02 cells, which is a much higher selectivity than displayed
by taxol. In addition, many compounds expressed significantly cell proliferation induced by 6h treatment on BEL-7402 cells.

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Y.-B. XIN ET AL.
Figure 3. Compound 6h decreases the expression of cyclin A and cyclin D1 and increases the expression of cyclin B1, thus reducing the cell cycle progression of BEL-
7402 cells. (A) Cell numbers in the phases of the cell cycle were analyzed on 12 h after stimulation. Values were obtained from three separate experiments and repre-
ꢀ
ꢀꢀ
ꢀꢀꢀ
sent the mean SD. p < 0.05. p < 0.01. p < 0.001 compared to respective control group. (B) Immunoblots were probed using antibodies directed against cyclin
A, B1, and D1, and b-actin. Whole-cell protein lysates were extracted from BEL-7402 cells cultured after 12 h. Results are representative of three separate experiments.
Figure 4. Apoptosis induction in BEL-7402 cells after treatment for 12h with (A) 0.1% DMSO (vehicle); (B) 0.01lM 6h; (C) 0.1 lM 6h; (D) 0.1 lM taxol. (E) The results
of the cell apoptosis.
Apoptotic effects by compound 6h
Cell migration and colony formation inhibition by
compound 6h
To further investigate whether 6h induces apoptosis, BEL-7402
cells were treated with vehicle, 6h (0.01 or 0.1 mM), or taxol Aggressive tumours have a strong ability to proliferate and
(0.1 mM) for 12 h, then stained with Annexin V-FITC and PI. As migrate, and in many cases, cell mobility affects cell proliferation2.
shown in Figure 4, the percentage of total apoptotic cells (ear- We used a transwell migration assay to investigate the effect of
ly þ late apoptotic cells) increased in a dose-dependent manner compound 6h on the migration of BEL-7402 cells. As shown in
with 6h treatment, but the effect was weak. Treatment with Figure 5, treatment with 0.1 or 0.5 mM of 6h resulted in migration
0.1 mM 6h resulted in a similar number of total apoptotic cells to ratios that were markedly decreased compared with the control
the taxol treatment group (10.2% and 10.7%, respectively), both group. A similar finding was observed for taxol at 0.1 mM.
of which were higher than the vehicle treatment group (4.7%).
These results indicate that apoptosis plays only a slightly import- assay with BEL-7402 cells over a relatively long exposure time
ant role in the inhibition of BEL-7402 cell proliferation by 6h. (> 10 days) to 6h. As shown in Figure 6, 6h had a dose-dependent
The LD50 of 6h was determined using a colony formation

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1561
Figure 5. Transwell assay of compound 6h showing inhibition of BEL-7402 cell migration. (A) The images of stained BEL-7402 cells adhering in the lower layer of
insert of transwell with phase-contrast microscopy (200ꢂ magnification). (B) The relative migration ratio of BEL-7402 cells. Data expressed as mean SD (n ꢅ 3).
ꢀꢀꢀ
p < 0.001 compared to control group.
Figure 6. Colony formation ability was inhibited by 6h treatment. (A) The images of stained colonies under phase-contrast microscopy. (B) The curve was fitted as the
inhibition ratio of colony formation against the concentration. Values are represented as mean SD from three independent colony formation experiments.
inhibitory effect on colony formation, with an LD50 value of and CA-4 were used as positive controls. As shown in
Figure 7 (A–C), 6h, taxol, and CA-4 interacted with tubulin resi-
dues differently. Compound 6h formed abundant chemical bonds
with the amino acid residues on the b chain of tubulin and
showed strong affinity, whereas the number of chemical bonds
formed with the amino acid residues on the a chain was sparse
and showed poor affinity. The cyano group tended to bind to the
b subunit but not the a subunit. In the case of the cis isomer of
CA-4, the entire structure made multiple contacts with the b sub-
unit. The 2D diagram effectively displays these structural features,
highlighting several binding regions on the b subunit, including
Cys241, Leu248, Ala316, and Ala354. Taxol bound between the a
and b subunits of tubulin, the three benzene rings docked into
the b subunit side while the rest of the compound docked into
the a subunit. The 2D image shows several binding sites on the a
subunit, including Val181, but most of the binding contacts are
on the b subunit. The superposition of the three compounds in
the biding pocket clearly shows the binding posture of 6h and
taxol are similar, whereas the biding location of CA-4 is com-
pletely different from them (Figure 7(D)). On the basis of the
0.056 0.0047 mM, further confirming the anticancer activity of the
compound. Compared with the dense and large cell colonies of
control group, the 6h treatment groups showed sparse and small
cell colonies which was observed in a dose-dependent manner.
Trypan blue staining was used to further investigate the cytotox-
icity of 6h. Treatment of cells with 6h at concentrations ranging
from 0.001 mM to 10 mM resulted in percentages of dead cells that
were negligible (data not shown). These results indicate that 6h
has a strong anti-proliferative effect on BEL-7402 cells in the
absence of evident cytotoxicity.
Molecular docking
To investigate the possible binding modes of the compound with
tubulin, 6h, a well-behaved and selectively toxic compound, was
used in molecular docking studies. The structure of tubulin used
in the docking study was obtained from the Protein Data Bank
(PDB code: 1SA0, https://www.rcsb.org/structure/1SA0)26. All
docked conformations are ranked based on docking scores. Taxol molecular modelling results, we suggest that 6h selectively

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Y.-B. XIN ET AL.
Figure 7. Molecular modelling of compounds in complex with tubulin (PDB: 1SA0). Shown is the proposed binding mode and interaction between tubulin and
selected compounds. (A) 6h, (B) taxol, and (C) CA-4. The compounds and important amino acids in the binding pockets are shown in stick model. (D) Superposition of
6h, taxol, and CA-4 in the pocket of tubulin in surface representation. The compounds are shown in stick model with carbon atoms in grey (6h), yellow-green (taxol),
or green (CA-4).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1563
inhibits cancer cell proliferation without toxicity to normal cell
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Conclusions
A series of (Z)-styrylbenzene derivatives, containing a cyano group,
were designed, synthesised, and evaluated for anti-proliferative
properties against nine human cancer cell lines and two non-can-
cerous human cell lines. Most derivatives exhibited significant
anticancer activity against five of the cancer cell lines, including
MGC-803 and BEL-7402 cells. SAR analysis indicated that in struc-
tures containing 3,4,5-trimethoxy groups on the A benzene ring,
the electron density and steric effect of the B benzene ring
strongly affect the anticancer activity of the derivatives. Most com-
pounds, especially 6h, exhibited notably high potency against a
set of cancer cell lines. Compound 6h possessed the highest anti-
cancer activity against MGC-803 cells, being 6-fold more potent
than taxol and CA-4. The IC50 value of 6h in L-02 cells was more
than 10,000-fold higher than in MGC-803 cells and more than
666-fold higher than in BEL-7402 cells, indicating that 6h exhibits
selective toxic effects. In addition, 6h arrested BEL-7402 cells in
the G2/M phase of the cell cycle and slightly induced apoptosis.
Mechanistic studies suggested that the blockade of G2/M phase
was associated with up-regulation of cyclin B1 and down-regula-
tion of cyclins A and D1. Molecular modelling results suggested
the selectivity of 6h to inhibit only cancer cell proliferation was
achieved by targeting both the a and b subunits of tubulin.
Compound 6h inhibited the migration of BEL-7402 cells and the
formation of cell colonies. In summary, these newly developed
compounds showed marked biological activity in vitro and have
potential for further development as a novel class of selective
anticancer agents.
12. Madadi NR, Penthala NR, Song L, et al. Preparation of 4,5
disubstituted-2H-1,2,3-triazoles from (Z)-2,3-diaryl substituted
acrylonitriles. Tetrahedron Lett 2014;55:4207–11.
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conformationally restricted b-lactam type combretastatin
analogues: synthesis, antiproliferative activity and tubulin
targeting effects. Eur J Med Chem 2010;45:5752–66.
14. Samanta S, Lim TL, Lam YL. Synthesis and in vitro evaluation
of West Nile Virus protease inhibitors based on the 2-{6-[2-
Acknowledgements
We thank Sarah Bubeck, Ph.D., from Liwen Bianji, Edanz Editing
China (www.liwenbianji.cn/ac), for editing the English text of a
draft of this manuscript.
(5-phenyl-4H-{1,2,4]
triazol-3-ylsulfanyl)acetylamino]benzo-
Disclosure statement
thiazol-2-ylsulfanyl}acetamide
scaffold.
ChemMedChem
2013;8:994–1001.
No potential conflict of interest was reported by the authors.
15. Li YT, Wang JH, Pan CW, et al. Syntheses and biological
evaluation of 1,2,3-triazole and 1,3,4-oxadiazole derivatives
of imatinib. Bioorg Med Chem Lett 2016;26:1419–27.
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Funding
This work was supported by the National Natural Science
Foundation of China [grant Nos. 81260226 and 81660837].
17. Chakraborty S, Das UK, Ben-David Y, et al. Manganese cata-
lyzed a-olefination of nitriles by primary alcohols. J Am
Chem Soc 2017;139:11710–3.
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