Design, synthesis and biological evaluation of flavonoid salicylate derivatives as potential anti-tumor agents

Article abstract of DOI:10.1039/c7ra07235j

A series of flavonoid salicylate derivatives containing trimethoxybenzene and a series of chrysin salicylate derivatives were synthesized for use as anti-tumor agents, and evaluated for antiproliferative activity using three human tumor cells: MCF-7 (breast carcinoma cells), HepG2 (liver carcinoma cells), MGC-803 (gastric carcinoma cells) and the mice tumor cells MFC (forestomach carcinoma cells). A substituent group of a suitable size and the trimethoxybenzene had a certain influence on the bioactivity of the flavonoid salicylate derivatives. Compound 2 and its salicylate derivatives 7a-7g containing the trimethoxybenzene exhibited more antiproliferative activity. Among them, compound 7g displayed the most potent antiproliferative activity against MGC-803 cells and MFC cells with the concentration causing 50% inhibition of cell growth (IC₅₀) values of 11.05 ± 1.58 μM and 13.73 ± 2.04 μM, respectively. The flow cytometry results showed that compound 7g caused the cell cycle to be arrested in the G0/G1 phase and induced apoptosis of MFC cells in a dose-dependent manner. Furthermore, compound 7g showed good anti-tumor activity in vivo. These results suggested that compound 7g could be a new, potent anti-tumor candidate which should be optimized and evaluated further.

Full text of DOI:10.1039/c7ra07235j

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PAPER
Design, synthesis and biological evaluation of
flavonoid salicylate derivatives as potential anti-
tumor agents†
Cite this: RSC Adv., 2017, 7, 38171
Xiangping Deng,‡a Zhe Wang,‡a Juan Liu,a Shujuan Xiong,a Runde Xiong,a Xuan Cao,a
a
Yanming Chen,b Xing Zhenga and Guotao Tang
*
A series of flavonoid salicylate derivatives containing trimethoxybenzene and a series of chrysin salicylate
derivatives were synthesized for use as anti-tumor agents, and evaluated for antiproliferative activity
using three human tumor cells: MCF-7 (breast carcinoma cells), HepG2 (liver carcinoma cells), MGC-803
(gastric carcinoma cells) and the mice tumor cells MFC (forestomach carcinoma cells). A substituent
group of a suitable size and the trimethoxybenzene had a certain influence on the bioactivity of the
flavonoid salicylate derivatives. Compound
2 and its salicylate derivatives 7a–7g containing the
trimethoxybenzene exhibited more antiproliferative activity. Among them, compound 7g displayed the
most potent antiproliferative activity against MGC-803 cells and MFC cells with the concentration
causing 50% inhibition of cell growth (IC50) values of 11.05 ꢀ 1.58 mM and 13.73 ꢀ 2.04 mM, respectively.
The flow cytometry results showed that compound 7g caused the cell cycle to be arrested in the G0/G1
phase and induced apoptosis of MFC cells in a dose-dependent manner. Furthermore, compound 7g
showed good anti-tumor activity in vivo. These results suggested that compound 7g could be a new,
potent anti-tumor candidate which should be optimized and evaluated further.
Received 30th June 2017
Accepted 20th July 2017
DOI: 10.1039/c7ra07235j
rsc.li/rsc-advances
inhibiting the hypoxia-inducible factor 1-alpha (HIF-1a) protein
level by increasing its degradation and decreasing its stability.10
Introduction
Substantial studies have been conducted to identify diverse
structurally modied avonoids with biological benets supe-
rior to their natural counterparts.11,12
Tumors are the most common disease in the world and cause
serious harm to human health. There is a critical need for
discovering new and reliable anti-tumor drugs to improve the
survival rates of tumor patients. Natural products have played
an important role in the drug discovery and development
because of their wide range of bioactivities, low toxicity and
hardly any side effects.1–3
During the last few decades, a growing number of studies
have indicated that avonoids have important effects in tumor
treatment.4 Flavonoids are attractive natural plant products
which widely exist in nature and have good biological activities,
including anti-tumor, anti-inammatory, anti-angiogenic,
cardiovascular protective and anti-mutagenic effects.5–8 As the
rst discovered avonoid vascular disrupting agent, avonoid
acetic acid (FAA) has shown great anti-tumor activity in the
animal model.9 Acacetin signicantly inhibited ovarian cancer,
cell-induced angiogenesis and tumor growth in vivo by
Acetylsalicylic acid (ASA) and salicylic acid (SA) which are
well known as anti-inammatory drugs also show anti-tumoral
properties, induction of apoptosis13,14 and changing tumor
glucose utilization.15 The anti-tumor effect of ASA and SA are
because of their effects on cyclooxygenase (COX)16 and 6-
phosphofructo-1-kinase (PFK).15 It has been demonstrated that
ASA and SA modulated PFK quaternary structure and decreased
tumor cells' glucose consumption and viability, suggesting that
ASA and SA could be used as an anti-tumoral agent.15
The structure of the trimethoxybenzene group, which exists
in many tumor vascular disrupting agents (Fig. 1), such as CA4P
and its analogues OXi4503, AVE8062,17–20 the colchicine analog
ZD6126,21 BNC-105 (ref. 22) and CKD-516,23 has an important
research value in tumor drug design.
Based on the previous considerations, it was proposed that
introducing trimethoxybenzene groups and SA and its diverse
derivatives into a avonoid scaffold might be an effective
strategy for seeking novel avonoid derivatives with potential
anti-tumor activity. Therefore, a series of avonoid salicylate
derivatives were synthesized which contained trimethox-
ybenzene and a series of chrysin salicylate derivatives, and these
were evaluated for anti-tumor bioactivities in vitro and in vivo.
aInstitute of Pharmacy and Pharmacology, Hunan Province Cooperative Innovation
Center for Molecular Target New Drug Study, University of South China, Hengyang,
Hunan, 421001, China. E-mail: tgtzq@163.com
bMudanjiang Youbo Pharmaceutical Co. Ltd, Mudanjiang, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra07235j
‡ These authors contributed equally to this work.
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Fig. 1 The structures of CA4P, OXi4503, AVE8062, ZD6126, BNC-105 and CKD-516. These tumor vascular disrupting agents all have a trime-
thoxybenzene group.
chloride (ZnCl2) as catalyst in a mixture of absolute ether and
Results and discussion
Chemistry
chloroform or absolute ether at 0 ꢁC. The key intermediates 1 or
3 were reacted with an aromatic aldehyde to give avonoid 2 or 4
with recrystallization from 95% ethanol. As shown in Scheme 2,
hydroxyl of salicylic acids 5a–5h were methylation and then
carboxyl were acylating chlorination to obtain 6a–6h. The three
groups of the nal target compounds 7a–7g, 8a–8g and 9a–9g
were synthesized as shown in Scheme 3 and the values of the
concentration causing 50% inhibition of cell growth (IC50) for
them are listed in Table 1.
In this study, two avonoids and 21 avonoid salicylate deriv-
atives were synthesized and satisfactory analytical and spec-
troscopic data was obtained, which agreed well with their
expected structures. The synthesis of compounds 2 and 4 are
shown in Scheme 1. Commercially available 3,4,5-trimethox-
yphenol or 1,3,5-trihydroxybenzene were used to produce
compound 1 or 3, respectively, by the Hoesch reaction with zinc
Scheme 1 Synthetic route of flavonoids 2 and 4.
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Scheme 2 Synthetic route of compounds 6a–6h.
Scheme 3 Synthetic route of compounds 7a–7h, 8a–8h and 9a–9h.
Aer comparison of the bioactivity data, compound 2 and its
salicylate derivatives 7a–7g, and chrysin and its salicylate
derivatives 9a–9g showed better inhibition activity than
compound 4 and its salicylate derivatives or 8a–8g with the
larger sized substituents, suggesting that a suitable size of
substituent group for avonoids was favorable. As a whole,
compound 2 and its salicylate derivatives 7a–7g displayed
higher inhibitory activity, when compared to chrysin and its
salicylate derivatives 9a–9g, implying that the scaffold hopping
strategy was feasible. Compounds 7f, 8f and 9f with the 5-Cl
substituent showed a lower inhibitory activity. At the same time,
Biological activities
In vitro antiproliferative activity assay. The antiproliferative
activity in vitro of compounds 2, 4, 7a–7g, 8a–8g and 9a–9g were
tested on the human tumor cells: MCF-7 (breast carcinoma
cells), HepG2 (liver carcinoma cells), MGC-803 (gastric carci-
noma cells) and on MFC mice tumor cells (forestomach carci-
noma)
using
the
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. Most compounds
exhibited moderate to good antiproliferative activity against
MGC-803 cell lines and MFC cell lines.
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Table 1 IC50 values (mM) of flavonoid salicylate derivatives towards
in the S phrase and G2 (G2/M) phrase were decreased. These
results suggested that the target compound 7g mainly arrested
MFC cells in the G0/G1 phase in a dose-dependent manner.
Apoptosis assay. The ow cytometry assay determined the
effect of compound 7g on apoptosis in MFC cells. The cells were
treated with compound 7g at different concentrations (0, 10, 20,
and 40 mM) for 24 h, and were collected, stained with Annexin V-
uorescein isothiocyanate (FITC)/PI, and then examined. The
percentage of apoptotic cells included an early apoptotic pop-
ulation and a late apoptotic population. As shown in Fig. 3, the
percentage of apoptotic cells obviously increased as the
concentration of the compound increased (10 mM, 10.12%; 20
mM, 12.77%; 40 mM, 25.70%), suggesting that there was a dose-
dependent effect. The results showed that target compound 7g
induced apoptosis MFC cells in a dose-dependent manner.
In vivo anti-tumor efficacy assay. In the in vitro anti-tumor
activity assay, it was observed that compound 7g had an
inhibition impact on the growth of the tumor cells. In order to
further conrm the effect, the MFC models were evaluated
using the anti-tumor activity assay. The anti-tumor growth
effect in vivo was evaluated by measuring tumor volume
following treatment with uorouracil (5-Fu, 25 mg kgꢂ1),
different doses of compound 7g (10, 20, 40 mg kgꢂ1) and
saline. As illustrated in Fig. 4, the tumors treated with the
middle dose and the high dose of compound 7g, exhibited
slower tumor growth, with a tumor inhibition ratio (TIR) of
77.14% and 69.54%, respectively, compared with the tumor
treated with 5-Fu (TIR of 61.14%). Furthermore, the inhibition
of the tumor growth by compound 7g was remarkably dose-
dependent. The differences between the last measured
four selected tumor cell lines, for 48 ha
IC50 (mM)
Compound MGC-803
MCF-7
HepG-2
MFC
2
4
31.19 ꢀ 6.48 44.23 ꢀ 3.77 >100
>100 ND
50.27 ꢀ 3.71
78.36 ꢀ 5.11 >100
7a
7b
7c
7d
7e
27.08 ꢀ 5.91 >100
52.33 ꢀ 6.92 >100
63.78 ꢀ 5.01 >100
21.59 ꢀ 5.75 ND
27.53 ꢀ 3.93 >100
89.31 ꢀ 6.05 ND
>100
ND
>100
34.62 ꢀ 3.64
64.48 ꢀ 5.27
89.22 ꢀ 4.97
65.44 ꢀ 4.63 25.38 ꢀ 5.13
>100
40.46 ꢀ 3.95
7f
99.72 ꢀ 7.41 >100
7g
8a
8b
8c
8d
8e
11.05 ꢀ 1.58 55.70 ꢀ 3.75 60.43 ꢀ 8.06 13.73 ꢀ 2.04
>100
>100
>100
ND
>100
ND
>100
>100
53.35 ꢀ 8.92 84.97 ꢀ 6.98 76.27 ꢀ 4.27 91.26 ꢀ 6.09
>100
ND
ND
ND
78.21 ꢀ 5.98 >100
ND
>100
77.24 ꢀ 6.72
8f
>100
>100
ND
>100
8g
9a
9b
9c
>100
87.9 ꢀ 5.61 >100
28.28 ꢀ 4.34 48.54 ꢀ 3.29 62.34 ꢀ 6.18 31.06 ꢀ 2.35
84.68 ꢀ 11.03 >100
>100
ND
73.45 ꢀ 5.82
>100 ND
ND
9d
9e
9f
9g
5-FU
Chyrsin
37.53 ꢀ 6.81 53.54 ꢀ 4.16 64.75 ꢀ 3.15 32.37 ꢀ 4.13
36.1 ꢀ 7.89
51.49 ꢀ 3.67 84.37 ꢀ 5.02 44.69 ꢀ 2.98
>100
>100
>100
>100
ND
25.95 ꢀ 3.72
21.77 ꢀ 6.53 ND
70.11 ꢀ 3.08 60.43 ꢀ 2.95 68.17 ꢀ 3.29 79.26 ꢀ 4.55
87.21 ꢀ 4.37 68.83 ꢀ 5.14 >100
91.48 ꢀ 4.99
a
ND ¼ not detected.
it was noted that compounds 7a and 9a with 4-CH3 substituent tumor volume in the compound 7g treatment groups, 5-Fu
on the R3 position, and compounds 7d and 9d with 4-Cl groups and saline group were statistically signicant (P < 0.05).
substituent on the R3 position displayed good antiproliferative These results conrmed that compound 7g had great anti-
activity against MGC-803 cells and MFC cells. Furthermore, tumor activity that was superior to that of 5-Fu. No signi-
compounds 7g and 9g with the 3,5,6-trichloro substituent were cant body weight loss was observed compared with the saline
more active than derivatives with other substituents. Out of group, which represented the natural growth in body weight of
these compounds, compound 7g bearing the 3,5,6-trichloro MFC tumor-bearing mice (Fig. 5).
substituent and the trimethoxybenzene showed the most potent
antiproliferative activity against MGC-803 cells and MFC cells
with IC50 values of 11.05 ꢀ 1.58 mM and 13.73 ꢀ 2.04 mM,
Materials and methods
Chemicals
respectively. Therefore, compound 7g was selected for further
evaluation for anti-tumor activity.
The Kunming male mice were chosen for the anti-tumor All the starting materials, solvents and reagents were reagent
efficacy assay in vivo. The biological activity of compound 7g grade and purchased from commercial sources unless other-
against MGC-803 cells and MFC cells was found to be similar. wise stated. Silica gel column chromatography was performed
Considering all these factors, the MFC cell lines were chosen for with silica gel 100–200 mesh size. Thin-layer chromatography
the cell cycle assay, the apoptosis assay, and the anti-tumor was carried out on silica gel 60 F254 plates and visualized under
efficacy assay in vivo.
(ultraviolet) UV light at 254 nm. Melting points (uncorrected)
Cell cycle assay. A cell cycle assay was conducted to check the were measured using an electrothermal digital melting point
distribution of MFC cells, treated with compound 7g at different apparatus (Thermo Fisher Scientic). Proton-nuclear magnetic
concentrations (0, 10, 20, and 40 mM) for 24 h, in the different resonance (1H-NMR) and 13C-NMR spectra were measured on
phases. Aer treatment the cells were gathered, stained with a spectrometer (Bruker) at 400 MHz and 101 MHz, respectively.
propidium iodide (PI), and the cell cycle distribution was Mass spectra (MS) were obtained using GCT time-of-ight mass
determined using ow cytometry. As indicated in Fig. 2, the spectrometer (Waters). Chemical shis and coupling constants
1
proportion of cells in G1 (G0/G1) phrase were signicantly are reported in ppm (d) and Hz (J), respectively. The H-NMR,
increased (37.5%, 42.5%, 46.2% and 55.2% for concentrations 13C-NMR and electrospray ionization (ESI)-MS spectral data of
of 0, 10, 20, and 40 mM, respectively), and the proportion of cells the compounds are supplied as ESI.†
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Fig. 2 Investigation of cell cycle distribution of compound 7g using flow cytometry. Cells were treated with compound 7g at concentrations of
10, 20, or 40 mM for 24 h. Cells treated with vehicle (DMSO) were used for comparison. (A) Representative photographs from three independent
experiments are shown. (B) Cell cycle proportion in (A) was quantitated. Data presented are the mean ꢀ SD of three independent experiments.
was produced. The light yellow precipitate, 2-chloro-1-(6-
hydroxy-2,3,4-trimethoxyphenyl)ethan-1-one (1) was ltered
off, washed with water (H2O), and dried in vacuo to give a light
yellow precipitate. 4-Hydroxybenzaldehyde (0.01 mol) and 1
(0.012 mol) were added together to a stirred solution of 3 mL
ethanol, then 10% sodium hydroxide [NaOH (aq)] was added
dropwise into the solution. The reaction mixture was stirred
at room temperature (rt) for 48 h, and acidication with 10%
HCl (aq) gave a crude product, which was ltered off and
puried by recrystallization from 95% ethanol to give
compound 2 as golden powder.
Synthesis of 5,7-dihydroxy-2-(3,4,5-trimethoxyphenyl)-4H-
chromen-4-one (2)
Freshly prepared dry hydrogen chloride (HCl) gas was
bubbled slowly and continuously into a reaction mixture of
a solution of 3,4,5-trimethoxyphenol (0.012 mol) and 2-
chloroacetonitrile (0.024 mol) in a mixed solvent of absolute
ether and chloroform (30 mL) with a catalytic amount of
ZnCl2 at 0 ꢁC, for 3 h. Aer standing the reaction ask at 4 ꢁC
for 3 d, a yellow precipitate was prepared aer ltration,
washing and drying. The yellow precipitate was dissolved in
hot water and heated to reux, and a light yellow precipitate
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Fig. 3 Evaluation of concentration dependent cellular apoptosis of compound 7g by flow cytometry. Cells were treated with compound 7g at
10, 20, or 40 mM for 24 h, respectively. Cells treated with vehicle (DMSO) were used for comparison. Representative photographs from three
independent experiments are shown.
absolute ether (30 mL) with a catalytic amount of ZnCl2 at 0 ꢁC,
for 3 h. Aer standing the reaction ask at 4 ꢁC for 3 d, an
Synthesis of 2-(4-hydroxyphenyl)-5,6,7-trimethoxy-4H-
chromen-4-one (4)
orange precipitate was prepared aer ltration, washing and
drying. The orange precipitate was dissolved in hot water and
heated to reux, and a red-brown precipitate was produced. The
Freshly prepared dry HCl gas was bubbled slowly and continu-
ously into a reaction mixture of a solution of 1,3,5-trihydroxy-
benzene (0.023 mol) and 2-chloroacetonitrile (0.032 mol) in
red-brown
precipitate,
2-chloro-1-(2,4,6-trihydroxyphenyl)
ethan-1-one (3) was ltered off, washed with water, and dried
in vacuo to give a light red-brown precipitate. 4-Hydrox-
ybenzaldehyde (0.01 mol) and 3 (0.012 mol) were added
together to a stirred solution of 3 mL ethanol, then 10% NaOH
(aq) was added dropwise into the solution. The reaction mixture
was stirred at rt for 48 h, and acidication with 10% HCl (aq)
gave a crude product, which was ltered off and puried by
recrystallization from 95% ethanol to give compound 4 as
reddish brown powder.
Fig. 4 (A) Tumor volume growth curves of the mice after treatment
with 5-Fu (25 mg kgꢂ1, dissolved in saline mixed with 5% Tween 80),
different doses of compound 7g (10, 20, 40 mg kgꢂ1, dissolved in saline
mixed with 5% Tween 80) and saline (mixed with 5% Tween 80). Data
are shown as a mean ꢀ SD (n ¼ 6). The differences of the last measured
tumor volume among five groups are statistically significant (P < 0.05).
(B) The TIR (%) of different doses of compound 7g.
Fig. 5 Body weight evolution curves of the mice after treatment with
5-Fu (20 mg kgꢂ1, dissolved in saline mixed with 5% Tween 80),
different doses of compound 7g (10, 20, 40 mg kgꢂ1, dissolved in saline
mixed with 5% Tween 80) and saline (mixed with 5% Tween 80). Data
are shown as a mean ꢀ SD (n ¼ 6).
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General method of synthesis for 6a–6g
In vitro anti-tumor efficacy
A stirred solution of 4-methylsalicylic acid (0.005 mol) in In vitro anti-tumor efficacy of the compounds were evaluated
ꢁ
distilled water (10 mL), was heated at 37 C. The solution was using the human tumor cells MCF-7, HepG2, MGC-803 and the
adjusted to pH 10 with 30% NaOH. Aer stirring for 30 min, mice tumor cells MFC. Each tested compound was dissolved in
dimethyl sulfate (2 mL) was slowly added and the pH of the dimethyl sulfoxide (DMSO). Cells were plated in 96-well
solution was adjusted to 7, and then 30% NaOH to adjust the microtiter plates at a density of 8 ꢃ 103 cells per well and
pH of the solution to 10. The reaction was carried out for 2 h and incubated in a humidied atmosphere with 5% CO2 at 37 ꢁC for
ꢁ
the solution was hydrolyzed at 80 C for 15 min. Aer cooling, 48 h. Test compounds of different concentrations (256, 128, 64,
the solution was adjusted to pH 5 with 10% HCl, and a white 32, 16, 8, 4, 2 mM) were added into triplicate wells and DMSO
precipitate was obtained. The crude product was ltered off, was used as the control. Aer 48 h, 20 mL of MTT solution (5 mg
washed with water, and dried in vacuo to give a white powder. mLꢂ1) was added to each well and incubation was continued for
The white precipitate, thionyl chloride and dichloromethane an additional 4 h. Formazan was dissolved in 150 mL of DMSO
were le with stirring at 40 ꢁC for 30 min. The methylene and added to the wells. The absorbance (optical density, OD)
chloride was evaporated under reduced pressure to give a clear was monitored on a Wellscan MK-2 microplate reader (Lab-
oily liquid (6a). Compounds 6b–6g were prepared in a similar Systems) at 490 nm. The IC50 values was determined using the
way.
logit method. All experiments were performed three times.
General method of synthesis for 7a–7g
Cell cycle analysis
The MFC cells were plated in six-well plates (2 ꢃ 104 cells per
well). Next day, the cells were incubated with different concen-
trations of compound 7g (0, 10, 20, 40 mM). Aer 24 h of
culturing, the cells were washed twice with cold phosphate
buffered saline (PBS) and then resuspended with cold PBS.
Next, the cells were xed and then stored in 70% cold ethanol
for 12 h at 4 ꢁC. Then 500 mL of PI stain was added to the cells in
each tube, and the cells were gently vortex mixed and then
incubated for 30 min at 37 ꢁC in the dark. The cells were
analyzed using ow cytometry within 24 h. All the experiments
were performed in triplicate.
A solution of 6a (0.005 mol) and compound 2 (0.004 mol) in
acetone (15 mL) with triethylamine (0.5 mL) at was stirred at rt
for 1 h and a yellow precipitate was obtained. The yellow
precipitate was ltered off, washed with acetone and dried in
vacuo. The crude product was puried using column chroma-
tography on silica gel, and eluted with dichloromethane/
methanol (50 : 1) to give a yellow powder (7a).
Compounds 7b–7g were prepared in a similar way.
General method of synthesis for 8a–8g
A solution of 6a (0.005 mol) and compound 4 (0.004 mol) in
acetone (15 mL) with triethylamine (0.5 mL) was stirred at rt for
1 h and a yellow precipitate was obtained. The yellow precipitate
was ltered off, washed with acetone and dried in vacuo. The
crude product was puried using column chromatography on
silica gel, and eluted with dichloromethane/methanol (50 : 1) to
give a yellow powder (8a).
Cell apoptosis assay
The MFC cells were plated in six-well plates (2 ꢃ 104 cells per
well). Next day, the cells were incubated with different concen-
trations of compound 7g (0, 10, 20, 40 mM). Aer 24 h of
culturing, the cells were washed twice with cold PBS and then
resuspended in 1ꢃ binding buffer. Cells were transferred to a 5
mL culture tube, and then 5 mL of FITC Annexin V and 5 mL of PI
were added to the cells. Next, the cells were gently vortex mixed
Compounds 8b–8g were prepared in a similar way.
General method of synthesis for 9a–9g
ꢁ
and incubated for 15 min at 25 C in the dark. Then 400 mL of
A solution of 6a (0.005 mol) and chrysin (0.004 mol) in acetone
(15 mL) with triethylamine (0.5 mL) was stirred at rt for 1 h and
a yellow precipitate was obtained. The yellow precipitate was
ltered off, washed with acetone and dried in vacuo. The crude
product was puried using column chromatography on silica
gel, and eluted with dichloromethane/methanol (50 : 1) to give
a light yellow powder (9a).
1ꢃ binding buffer was added to each tube. The cells were
analyzed using ow cytometry within 1 h. All the experiments
were performed in triplicate.
In vivo anti-tumor efficacy
All experiments with animals were performed in compliance
with the relevant laws and Chinese institutional guidelines for
animal experiments, and the Institutional Animal Care and
Committee of University of South China [permit number: SYXK
Compounds 9b–9g were prepared in a similar way.
Cell culture
Various human tumor cells including MCF-7, HepG2, MGC-803 (Xiang) 2015-0001] approved the experiments. Kunming male
and the mice MFC carcinoma cells were purchased from the mice (University of South China, China) were used for the in vivo
Type Culture Collection of the Chinese Academy of Sciences, experiments. Gastric carcinoma was chosen as a model tumor
Shanghai, China. Cells were cultured in Dulbecco's Modied to evaluate the anti-tumor efficacy of compound 7g. Gastric
Eagle's Medium (DMEM, Gibco, NY, USA) containing 10% fetal tumor bearing mice were established by subcutaneously
bovine serum at 37 ꢁC in a humidied incubator with 5% injecting 1 ꢃ 107 MFC cells per 0.2 mL into the right ank
carbon dioxide (CO2).
region of the mice (dened as day 0). On day 1, the mice were
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randomly divided into ve groups (n ¼ 6). The mice were treated
with different doses of compound 7g (10, 20, 40 mg kgꢂ1, dis-
Notes and references
solved in saline mixed with 5% Tween 80) and 5-Fu (25 mg kgꢂ1
,
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synthesized and these compounds were evaluated for their anti-
tumor activities. These compounds exhibited moderate to good
antiproliferative activity against MGC-803 cell lines and MFC
cell lines. An appropriate size of substituent group and the tri-
methoxybenzene had a certain inuence on the bioactivity of
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ylate derivatives 7a–7g containing the trimethoxybenzene
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activity against MGC-803 cells and MFC cells with IC50 values of
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Conflict of interest
The authors declare that there are no conicts of interest.
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Acknowledgements
This work was supported in part by the Construct Program of
the Key Discipline in Hunan Province and Hunan Province
Cooperative Innovation Center for Molecular Target New Drug
[Hunan Provincial Education Department document (Approval
number: 2014-405)]. The authors wish to thank the Department
of Laboratory Animal Science for support with the animal
experiments.
38178 | RSC Adv., 2017, 7, 38171–38178
This journal is © The Royal Society of Chemistry 2017