Synthesis and pharmacological evaluation of 2,3-diphenyl acrylonitriles-bearing halogen as selective anticancer agents

Article abstract of DOI:10.1111/cbdd.13180

Eighteen novel 2,3-diphenyl acrylonitrile derivatives bearing halogens were designed, synthesized, and evaluated for biological activity. Preliminary in vitro results indicated that the majority of the compounds with a para-substituted halogen had considerable antiproliferative activity against five human cancer cell lines, including MGC-803, AGS, and BEL-7402, with IC₅₀ values in the range of 0.46–100?μm. No significant toxic effects on the non-cancerous human liver cell line L-02 were observed. The selective inhibitory activities against cancer cells were significantly better than that of the control lead compound CA-4 and CA-4P. Particularly, potent activities were found for the derivatives of 3-(4-halogen phenyl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile, such as 5c (4-fluoro), 5f (4-bromo), 5h (4-chloro), and 5k (4-trifluoro- methyl), for AGS with IC₅₀ values of 0.75?±?0.24, 0.68?±?0.21, 0.41?±?0.05, and 1.49?±?0.92?μm, respectively. The antiproliferative effects of 5f were attributed to cell-cycle arrest in the G₂/M phase, induction of cellular apoptosis, suppression of cell migration, and inhibition of cell colony formation in AGS cells.

Full text of DOI:10.1111/cbdd.13180

PROFESSOR YU-SHUN TIAN (Orcid ID : 0000-0001-5013-788X)
Article type : Research Article
Synthesis and Pharmacological Evaluation of 2,3-Diphenyl
Acrylonitrile Bearing Halogen as Selective Anticancer Agents
Jia-Jun Lia, Jun Mab, Ya-Bing Xina, Zhe-Shan Quana*, 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, Jilin Province 133002, PR China
bJiangsu Hansoh Pharmaceutical Group Co., Ltd. lianyungang, jiangsu Province 222000, PR China
Abstract
Eighteen novel 2,3-diphenyl acrylonitrile derivatives bearing halogens were designed, synthesized and evaluated for biological
activity. Preliminary in vitro results indicated that the majority of the compounds with a para-substituted halogen had considerable
anti-proliferatory activity against five human cancer cell lines, including MGC-803, AGS, and BEL-7402, with IC50 values in the
range of 0.46–100 µM. No significant toxic effects on the non-cancerous human liver cell line L-02 were observed. The selective
inhibitory activities against cancer cells were significantly better than that of the control lead compound CA-4 and CA-4P.
Particularly potent activities were found for the derivatives of 3-(4-halogen phenyl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile, such as
5c(4-fluoro), 5f(4-bromo), 5h(4-chloro), and 5k(4-trifluoro- methyl), for AGS with IC50 values of 0.75±0.24, 0.68±0.21, 0.41±0.05,
and 1.49±0.92 µM, respectively. The antiproliferation effects of 5f were attributed to cell-cycle arrest in the G2/M phase, induction
of cellular apoptosis, suppression of cell migration, and inhibition of cell colony formation in AGS cells.
Keywords: 2,3-Diphenyl acrylonitrile; Cyano group; Halogen; Anticancer; Selective inhibition
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13180
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1
Abbreviations
CA-4: combretastatin A-4; CA-4P: combretastatin A-4 phosphate; FBS: fetal bovine serum; MTT:
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]; DMSO: dimethyl sulphoxide
At present, cancer is the second most life-threatening disease, after cardiovascular disease, affecting more than 6
million people per year worldwide [1]. The basic method of treatment for solid tumors is radical surgery, which can be
preceded by chemotherapy and/or radiotherapy aimed at reducing the tumor size [2]. However, because of the toxic
effects of chemotherapy drugs on healthy tissues, the development of new anticancer agents that enable more selective
treatment strategies is the focus of research around the world [3].
CA-4 is a tubulin inhibitor found in plants from South America [4]. The structure of CA-4 consists of two rings (A
and B) linked by an ethene bridge (Figure 1); the three methoxy groups on the A ring are required to maintain
activity [5-6]. The cis configuration is more active than the trans, and the mechanism of action is mainly through
inhibiting the angiogenesis of tumor cells [7]. A phosphate derivative, CA-4P, is currently in clinical trials, but it has
been found to have very serious side effects, such as tumor pain, hypertension, and hematotoxicity [8]. Hence, there
is a need to investigate further structural modifications to obtain safer and more specific anticancer drugs.
A cyano group consists of a carbon and nitrogen atom joined by a triple bond. This structure confers a high level
of polarity and electron-withdrawing properties, with a volume that is substantially smaller than that of a methyl
group [9]. These characteristics enable cyano groups to bind to the key amino acid residues in the active site of
various enzymes by forming hydrogen bonds. Cyano-containing drugs in clinical use, include the phosphodiesterase
inhibitor milrinone [10] for the treatment of heart failure and the calcium channel antagonist verapamil [11], which
is indicated for a variety of different clinical conditions. Introducing a cyano group to small-molecule drugs can alter
the physicochemical properties of the drug [1,3,4], in particular increasing the stability of the compound by fixing
the cis-trans isomerism [3].
1*Corresponding author: Tel.: +864332436028
Fax: +864332435026
Present address: 977Gongyuan Road, Yanji, Jilin province, P.R. China.
E-mail address: ystian@ybu.edu.cn (Y.-S. Tian)
zsquan@ybu.edu.cn (Z.S Quan)
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Scheme 1. The synthetic route of target compounds 5a-k and 10a-g.
Reagents and conditions: (i): (CH3)2SO4, (CH3)2CO, K2CO3, reflux, 4 h; LiAlH4, THF, 0°C-rt, 4-6 h; (ii) CH2Cl2, PBr3, 0°C-rt, 3-6 h; (iii): CH3CN,
TMSCN, TBAF, reflux, 4-6 h; (iv): CH3OH, CH3ONa, aromatic aldehydes, 4-6 h.
Halides are widely found in nature, especially in the metabolites of marine organisms. To date, more than 3800
naturally occurring halogenated compounds have been found, and many of these have good biological activity [12].
Therefore, halogens are often introduced into compounds to obtain new drugs with enhanced biological activity
[10,13].
On this basis, we were interested in designing and synthesizing a number of novel compounds combining CA-4,
cyano, and halogen moieties. To obtain more structure-activity relationship (SAR) information, we also synthesized
another novel series of derivatives, which contain a p-methyl group on the A ring (Figure 1 and Scheme 1).
Methods and Materials
All solvents and chemicals were ACS pure, used without further purification, and purchased from commercial
1
sources. NMR measurements were performed on a Bruker Avance 300 (at 300 MHz for H and 75 MHz for 13C; TMS,
internal standard; Bruker Corp., Billerica, MA, USA). The IR spectra were recorded (in KBr) using an FTIR1730
spectrometer (Perkin-Elmer, Waltham, MA, USA). Mass spectrometry was performed using an Agilent 1100
HPLC/MSD mass spectrometer (Hewlett-Packard Co., Palo Alto, CA, USA).
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All cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). The RPMI-1640
and DMEM culture media, and FBS were purchased from Gibco Laboratories (Gaithersberg, MD, USA). The
absorbance was detected using SILFTA microplate reader (Perten Instruments, USA).
1. Synthetic procedures
1.1 Synthesis of the series of 5a-5k [3]
3,4,5-Trihydroxybenzoic acid was used as a starting material to synthesized 3. Then, a mixture of 2.08 g (10
mmol) of 3, 1.485 g (15 mmol) of TMSCN (trimethylsilyl cyanide), 4.725 g (15 mmol) of TBAF (tetrabutylammonium
fluoride), and 30 mL of acetonitrile was refluxed with stirring. After completion of the reaction, precipitation with ice
water, separation, and recrystallization from 95% ethanol to afford 4. A mixture of 0.208 g (1 mmol) of compound 4,
1 mmol of halogen substituted aromatic 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 was maintained the temperature for 4-6
h. After completion of the reaction, the reaction mixture was cooled to RT, and the precipitate was separated by
filtration and recrystallized from methanol to give 5a-5k.
1.2 Synthesis of the series of 10a-10g
4-Methylbenzoic acid was used as a starting material to synthesize 8. Then a mixture of 1.84 g (10 mmol) of
compound 8, 1.485 g (15 mmol) of TMSCN, 4.725 g (15 mmol) of TBAF, and 30 mL of acetonitrile was refluxed for 4-6
h with stirring. After completion of the reaction, precipitation with ice water, separation, and recrystallization from
95% ethanol to afford 9. Afterward, a mixture of 0.131 g (1 mmol) of compound 9, 1 mmol of halogen substituted
aromatic 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 keep the temperature for 4-6 h. After completion of the reaction, the
reaction mixture was cooled to RT, and the precipitate was separated by filter and recrystallized from methanol to
give 10a-10g.
2. General biological analysis
2.1 In vitro anticancer activity assay (MTT assay)
According to the reference [3] to do MTT assays. The drug-containing media (0.1, 1, 10, 50 and 100 μM) with
triplicate wells per concentration, while equal treatment volumes of DMSO (0.1%) were used as vehicle control. CA-4
and CA-4P were used the references.
2.2 Apoptosis assay
According to the references method [14], apoptosis was determined by staining AGS cells with annexin V-FITC
and PI labeling and were analyzed by a FACScalibur (BD Biosciences, FACSCanto II).
2.3 Cell cycle analysis by FACS
According to the references method to do the assay [3,15].
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2.4 Cell migration assay
Quantitative cell migration was performed using 24-well Boyden chambers (Corning, NY, USA) as described
previously [16]. The cells were stimulated with 0.1, 1, 10 μM of compound 5f and 10 μM of taxol. Control group was
treated with 0.1% DMSO.
2.5 Colony formation assay
According to the reference method to do colony formation assay by slight modification [16]. The 1X104 AGS
cells/well were seeded into 6-well plates, and exposed to 5f at different concentrations for 10 days to form colonies.
The plates were removed from incubation when colonies were large enough to count (>50 cells). The colonies were
then fixed and stained with 1X Giemsa solution, rinsed, allowed to dry, and counted. The control group was treated
with 0.1% DMSO, and the treatment conditions were the same as those experimental group.
2.6 Trypan blue staining assay
AGS cells were seeded in 6-well plates at density of 2X105 cells/well and adhered for overnight. Then the cells
were exposed to increasing concentrations of 5f for 48 h. Afterward, cell suspension was mixed with 0.4% trypan
blue solution at a 1:9 ratio. The number of viable cells (clear) vs dead cells (blue) was determined.
3. Statistical analysis
The data are represented as the mean ± standard deviation (SD) for the number of experiments indicated.
Other differences between treated and control groups were analyzed using the Student’s t-test. A p-value <0.01 and
<0.001 were considered statistically extremely significant.
Results and Discussions
According to published methods [3], we synthesized two novel series of halogenated 2,3-diphenyl acrylonitrile
derivatives using a safe and simple method. The concise synthetic route used to synthesize these compounds is
outlined in Scheme 1. The detailed synthetic method and information on the structural and physicochemical
characteristics of the compounds can be found in the supplementary material. Owing to the existence of a
cyano-steric effect, the final structure was fixed as the Z isomer, confirmed by NOE (nuclear overhauser effect)
results (Fig. S1). All compounds were evaluated for their in vitro antitumor activities against a panel of human tumor
cell lines and normal human liver cells (L-02) using an MTT assay, with CA-4 and CA-4P as reference agents. The
panel consisted of human late stage differentiation gastric cancer (MGC-803), human lung epithelial calcinoma
(A549), human liver hepatocellular carcinoma (HepG2), human stomach cancer (AGS), human liver cancer (BEL-7402),
human colorectal cancer (HCT-116), human cervical cancer (HeLa) and human early stage differentiation gastric
cancer (SGC-7901) cells. On the basis of data from a preliminary MTT assay, the IC50 values of a range of 2,3-diphenyl
acrylonitrile derivatives against the nine cell lines were investigated. The substituent structures and cytotoxicity (IC50)
values are listed in Table 1 and the selectivity index values are listed in Table 2, for more detailed inhibition rates,
see Table S1.
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To explore the SAR to develop more potent and selective antiproliferative effects, we substituted the methoxy
groups on the A benzene ring with a methyl group and substituted the B ring with different halogen moieties,
including fluoride, chloride, bromine, and trifluoromethyl groups. All of the tested compounds did not have
significant antiproliferative activity in three cancer cell lines, such as HepG2, HeLa, and SGC-7901 cells, but it still had
selective antitumor activity against some tumor cell lines, such as MGC-803 cells, without toxic to L-02 normal cells.
Eleven compounds had toxic effects for cancer cells, with less toxic effects observed with the non-cancerous human
liver cell line L-02. Against AGS cells, the antiproliferative activities of ten compounds, 5a–k and 10a–g, were higher
than 50%. In particular, the inhibitory activities of the four compounds 5c (4-F), 5f (4-Br), 5h (4-Cl), and 5k (4-CF3)
were particularly prominent, with IC50 values of 0.75 ± 0.24, 0.68 ± 0.21, 0.41 ± 0.05, and 1.49 ± 0.92 μM,
respectively, and were significantly stronger than that of the control lead compound CA-4 and its phosphate
derivative CA-4P. More importantly, CA-4 and CA-4P expressed very toxic effects against L-02 normal cell and had no
selective inhibitory activities on A549, AGS, and BEL-7402 cancer cells. The difference in the selective anti-tumor cell
proliferation activity may be due to the different binding modes to the target protein tubulin, analyzed by protein
docking methods (Fig. S2).
Although a few of the ortho- and meta-substituted compounds in the 5a–k series, including 5g and 5e, showed
good anticancer activities, most of the compounds had poor activities. Interestingly, the inhibitory activity of
compound 5k on the late stage differentiation human gastric cancer cell line MGC-803 was much higher than that
on the early stage differentiation human gastric cancer cell line SGC-7901. Hence, it is necessary to further study
the relationship between compound 5k and the development of human gastric cancer. Against MGC-803 cells, the
IC50 value of compound 5k reached 0.49 ± 0.02 μM. When the A ring has a p-methyl substituent (series 10a–g),
p-halogen substituents on the B ring, 10a (4-F), 10c (4-Cl), 10f (4-Br), and 10g (4-CF3) resulted in anticancer activity
against MGC-803 and AGS gastric cancer cell lines, and ortho- or meta-substituents on the B benzene ring didn’t
produce significant anticancer activity in any of the cell lines. By comparing the activities of these two series of
compounds (5a–k and 10a–g), we found that the 3,4,5-trimethoxy groups on A ring was more effective for
anticancer activity than methyl group and the general effect of substituents on B benzene ring was para > meta >
ortho, although this trend was not found for the L-02 normal liver cell line. Several of these agents expressed a
significantly higher selectivity index (SI) values than CA-4 and CA-4P, indicating that the agents can be considered
good candidate anticancer compounds (Table 2). In particular, compounds 5h and 5k demonstrated SI values higher
than 243.9 and 204.1 in AGS and MGC-803 cells, respectively, relative to L-02 cells, suggesting that 5h and 5k have
potential as treatments for human gastric cancer, with a low risk of cytotoxic effects on liver cells. The
gastrointestinal cancer cells, such as MGC-803, AGS, and HCT-116, are more sensitive to these compounds than
cells from other cancer sites. Therefore, these compounds have the potential to be developed into highly selective
drugs for the treatment of human digestive system cancers. It is important to investigate the influence of the
para-methoxy groups on the A benzene ring on the anticancer activity of 2,3-diphenyl acrylonitrile compounds
containing a halogenated B benzene ring. The compound containing p-F on the B benzene ring and
3,4,5-substituted methoxy groups on the A ring (5c) has strong inhibitory activity against HCT-116 cells, but when
the A ring has 3,5-substituted methoxy groups [3], the activity was substantially decreased. However, against HeLa
cells, the structures which contain p-Br (5f) and p-Cl (5h) on the B ring and a 3,4,5-trimethoxy substituted A ring,
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showed very low activity, but p-Br and p-Cl compounds with a 3,5-dimethoxy substituted A ring [3], had excellent
inhibitory activity, superior to that of the positive control drug taxol. It can be concluded that the anticancer activity
of compounds containing a p-methoxy group on the A benzene ring in this series of compounds are different for
different cell types.
The cell cycle involves a series of events, resulting in cell division, duplication, and proliferation. Compounds 5f
and 5h had excellent anti-proliferative effects against AGS cells (Tables 1 and 2). However, compound 5f was less
toxic to L-02 normal cells than 5h (Table S1), so we chose 5f for further study. To determine whether the
suppression of AGS cancer cell growth using 5f was a result of inhibition of cell-cycle progression, a cellular
proliferation assay involving propidium iodide staining was used to quantify the percentages of AGS cells at
different stages of the cell cycle. Percentages of cells at each stage were quantified using flow cytometric analysis.
The extent of cell-cycle inhibition in cells treated with taxol or vehicle (control) were determined under the same
conditions (Fig. 2). The detailed experimental method can be found in the supplementary material. As shown in Fig.
2, at a low concentration (1 µM), compound 5f didn’t produce significant effects, but at 10 μM, significant
inhibition of AGS cellular proliferation was observed, resulting in accumulation of cells at the G2/M stage of the cell
cycle. The percentage of cells in the G2/M phase increased from 16.83% ± 1.82% under control conditions to
70.67% ± 0.50% in the presence of 5f, an effect very similar to that of the reference drug taxol (75.47% ± 1.83%). A
decrease in the percentage of cells in the G0/G1 phase was observed, from 65.91% ± 3.18% under control conditions
to 15.28% ± 0.85% in the presence of 10 µM 5f. The cellular population in the S-phase of the cell cycle was 17.24%
± 1.46% under control conditions and 14.05% ± 1.83% in the presence of 5f. From these results, the mechanisms of
inhibition of AGS cell proliferation by compound 5f appears similar to those of the anticancer agent taxol at 10 μM
concentration. Hence, 5f induces a marked arrest of the cell cycle at the G2/M phase and subsequent cell death.
To determine whether cancer cell apoptosis affects cell proliferation as well as cell-cycle progression [3], an
apoptosis assay was carried out in AGS cells (Fig. 3). Similar to taxol, when treated with 10 μM 5f, there was a
marked increase in early apoptotic cells, but there was no significant change at a low concentration of 5f, compared
with the control group. The early and late percentages of apoptotic cells were 12.6% and 6.3%, respectively, on
treatment with 10 μM 5f. These results indicate that apoptosis also plays an important role in the inhibition of AGS
cell proliferation by 5f.
Proliferation of tumor cells is closely related to tumor-cell migration [17]. We investigated compound 5f, which
has excellent antiproliferative effects on AGS cells, as verified using an MTT assay (Tables 1 and 2). The effects
of compound 5f on AGS cell migration were investigated using the transwell migration assay (Fig. 4 and
supplementary material). As shown in Fig. 4, compared to control groups, treatment with 1 and 10 μM of 5f,
resulted in migration ratios that were all decreased markedly, and a similar finding was observed for taxol in
this assay. In addition, the tumor chemosensitivity of 5f was evaluated by the colony formation assay [16]
against the AGS cell line over a relatively long exposure time (10 days). Compound 5f had a significant
inhibitory effect on colony formation in a dose-dependent manner, and afforded an LD50 value of 0.020 ± 0.002
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μM (Fig. 5A and B), further confirming the anticancer activity. Compared with the dense and large cell
colonies of the control group, the 5f treatment groups showed loose and small cell colonies, and the severity of
this change was dose dependent. Trypan blue staining was further used to investigate the cytotoxicity of 5f. In
the presence of 5f at concentrations ranging from 0.001 μM to 10 μM, the percentages of dead cells were
negligible (Fig. 5C and D). These results indicated that 5f had a strong antiproliferative activity on AGS cells in
the absence of evident cytotoxicity.
Conclusions
In summary, eighteen novel derivatives of 2,3-diphenyl acrylonitrile, bearing halogen substituents on the B
benzene ring, were designed and synthesized via a simple and safe method and the biological activities were
evaluated. Preliminary SAR analyses indicated requirements for a cyano group on the ethene bridge,
3,4,5-trimethoxy groups on the A benzene ring, and a para-halogen substituent on the B benzene ring to achieve
satisfactory selective antitumor activity (Table 1, series 5a–k). When the 3,4,5-substituted methoxy groups were
replaced by a p-methyl group on the A benzene ring, the anticancer activities were substantially decreased (Table 1,
series 10a–g). Compared with the lead compounds CA-4 and CA-4P, which are very toxic to normal cell L-02, the
selective inhibitory activities of 5c, 5f, 5h, and 5k against the AGS gastric cancer cell line, relative to that observed in
L-02 normal cells, were high, may be due to the differences of binding points and modes with tubulin (Fig. S2), which
suggests that these agents have potential as treatments for gastric cancer, with a low risk of hepatotoxic adverse
effects. Further investigation revealed that the anti-proliferative effects of 5f are a result of cell-cycle arrest at the
G2/M phase (Fig. 2) and induction of the AGS cell early apoptosis pathway (Fig. 3). In particular, the anticancer
activity of 5f was shown to occur through suppression of cell migration (Fig. 4) and inhibition of AGS cell colony
formation ability (Fig. 5). Further investigation is required to identify the cell-cycle and apoptosis-related proteins or
genes that are modulated, and thus the mechanism of action of 5f. The potential effects of these agents include
modulating cell-cycle related proteins, such as cyclin A, cyclin D1, p21, p27 and apoptosis-related proteins, such as
Bax, PARP, and caspase 3/9 [15,18]. These data suggest that the halogenated 2,3-diphenyl acrylonitrile derivatives,
especially compounds 5c, 5f, 5h, and 5k, can be used as candidates for specific, potent, and safe anticancer agents.
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Table 1. Cytotoxic effects in vitro of 5a–k and 10a–g on the human normal cell line L-02 and eight cancer cell lines
Agent
5a
R
MGC-803
A549
AGS
BEL-7402
HCT116
HepG2
HeLa
SGC-7901
L-02
2-F
>100
>100
>100
>100
55.6±19.9
>100
>100
>100
96.25±43.3
3-F
4-F
>100
>100
89.76±2.35
0.75±0.24
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
5b
5c
5.10±2.08
77.21±12.5
91.53±1.30
2.26±1.39
2-Br
3-Br
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
5d
5e
99.20±8.96
98.01±7.31
47.5±0.098
57.21±9.60
4-Br
2-Cl
>100
97.21±5.28
98.31±2.56
>100
0.68±0.21
68.62±6.54
0.41±0.05
>100
29.99±2.3
50.28±31.6
24.99±5.22
>100
1.843±1.69
11.26±3.57
1.71±1.49
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
72.20±4.6
>100
>100
>100
>100
>100
>100
>100
>100
>100
5f
5g
>100
4-Cl
11.35±1.20
>100
5h
2-CF3
3-CF3
4-CF3
4-F
>100
5i
>100
>100
>100
>100
>100
5j
0.49±0.02
>100
1.49±0.92
>100
9.00±0.50
>100
5.17±3.15
>100
5k
96.43±14.30 >100
>100 >100
85.54±11.14 >100
10a
10b
10c
10d
10e
2-Cl
>100
>100
>100
4-Cl
>100
>100
>100
2-Br
3-Br
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4-Br
4-CF3
83.03±2.3
>100
>100
>100
43.51±5.8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
10f
10g
100.0±12.65 >100
0.06±0.02
0.03±0.02
>100
>100
3.42±0.03
4.73±0.22
2.02±0.03
0.16±0.00
0.59±0.22
>100
>100
>100
>100
0.30±0.00
0.12±0.03
1.10±0.03
2.67±0.61
CA-4
5.45±2.04
CA-4P
.
IC50 value of compounds against each cell line, which was defined as the concentration (µM) that caused 50% inhibition of cell growth in
vitro.
CA-4 and CA-4P (fosbretabulin) were used as the reference agents.
The purity (%) of tested compound exceeded 95%.
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Table 2. Selectivity index values of compounds relative to the effects on L-02 cells.
SI
Agent
IC50(L-02/MGC-803)
IC50(L-02/A549)
IC50(L-02/AGS)
> 1.11
IC50(L-02/BEL-7402)
-
IC50(L-02/HCT-116)
-
5b
3-F
-
-
5c
4-F
> 19.61
> 1.30
> 133.33
> 1.02
> 1.09
> 2.11
> 3.33
> 1.44
> 4.00
> 11.11
-
> 44.30
> 1.75
> 54.26
> 6.39
> 58.48
> 19.34
-
5e
3-Br
4-Br
2-Cl
4-Cl
-
> 1.00
5f
-
> 1.03
> 147.06
> 1.05
5j
-
-
5h
> 8.81
-
> 243.90
> 67.11
> 2.30
5k
4-CF3 > 204.08
-
10f
CA-4
CA-4P
4-Br
> 1.20
> 15.71
> 89.0
-
> 0.011
> 0.027
> 0.322
> 0.564
> 0.545
> 0.490
> 6.875
> 4.525
-: not suitable for calculation.
SI: selectivity index, IC50 (L-02) / IC50 (cancer cell)
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Figure 1. Structures of CA-4 and (Z)-2-(3,5-dimethoxyphenyl)-3-(4-ethoxyphenyl)acrylonitrile, and the structural design of compounds 5a-k
and 10a-g.
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Figure 2. Compound 5f induces AGS cells by increasing G2/M phase cell cycle arrest. Synchronized AGS cells were treated for 12 h with
0.1% DMSO as vehicle (A), 5f 1 μM (B), 5f 10 μM (C), and taxol 10 μM (D) for 12 h. (E) The cell numbers in the phases of the cell cycle.
Values were obtained at least three separate cultures and represent the mean ± SD (n≥3). **, p < 0.01, ***, p < 0.001 when the
experiment and control groups were compared. FACS analysis was performed to determine the cell distributions at each phase of the cell
cycle.
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Figure 3. Effects of 5f and taxol on annexin V-FITC and PI fluorescence in AGS cells. The cells were treated with 5f 1 µM (A), 10 μM (B),
taxol 10 μM (D), and DMSO (0.1%, control) (C) for 12 h. Annexin V-FITC and PI staining cells were measured with a FACS calibur flow
cytometer.
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Figure 4. Transwell assay of compound 5f showing inhibition of AGS cell migration. (A) The images of stained AGS cells adhering in the
lower layer of insert of transwell with phase-contrast microscopy (X200 magnification). (B) The relative migration ratio of AGS cells. Data
expressed as mean ± SD (n≥3). ***, p < 0.001 compared to control group.
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Figure 5. Growth inhibitory effects of 5f on AGS cell lines by colony formation assay and trypan blue staining. (A) Representative stained
colony formation images on AGS cells after exposed to 5f for 10 days under phase-contrast microscopy (×100 magnification). (B) The curve
was fitted as the inhibition ratio of colony formation against the concentration. The cytotoxicity of 5f on AGS cell was estimated by trypan
blue exclusion. After exposed to 5f for 48 h, AGS cells were harvested and stained with trypan blue solution. Arrow indicated the dead
cells. The results were expressed as a percentage based on the ratio of the number of viable treated cells to that of vehicle control. Data
are presented as mean ± SD from three independent experiments.
Acknowledgments
The work was supported by the National Science Foundation of China (grant number 81260226).
Supplementary data
Supplementary data (synthetic procedures, structural characterizations and biological experiments) associated with
this article can be found in the online version at supplementary materials.csv.
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