Design, synthesis, and preliminary biological evaluation of 3′,4′,5′-trimethoxy flavonoid salicylate derivatives as potential anti-tumor agents

Article abstract of DOI:10.1039/c8nj04533j

According to the pharmacophore combination principle, a set of new 3′,4′,5′-trimethoxy flavonoid salicylate derivatives were designed, synthesized, and evaluated for biological activity. The cytotoxicity evaluation revealed that compound 10v exhibited higher potency than 5-Fu against HCT-116 cells. Preliminary biological activity studies showed that compound 10v could inhibit the colony formation and migration of HCT-116 cells. Besides, the Hoechst 33258 staining assay and flow cytometry revealed that treatment with compound 10v induced the apoptosis of HCT-116 cells in a concentration-dependent manner, while it had no effect on their cell cycle. The WB analysis suggested that HIF-1α, tubulin, HK-2, and PFK might be the potential pharmacophore targets of compound 10v. Tubulin was a potential drug target for compound 10v, which was explained by analyzing the crystal structure of compound 10v complexed with tubulin. These results indicated that compound 10v might be a promising anti-tumor agent candidate, deserving further optimization and evaluation.

Full text of DOI:10.1039/c8nj04533j

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Design, synthesis, and preliminary biological
evaluation of 30,40,50-trimethoxy flavonoid salicylate
Cite this:DOI: 10.1039/c8nj04533j
derivatives as potential anti-tumor agents†
Xiangping Deng,‡ Renbo Liu,‡ Junjian Li, Zhongli Li, Juan Liu, Runde Xiong,
Xiaoyong Lei, Xing Zheng, Zhizhong Xie and Guotao Tang
*
According to the pharmacophore combination principle, a set of new 30,40,50-trimethoxy flavonoid
salicylate derivatives were designed, synthesized, and evaluated for biological activity. The cytotoxicity
evaluation revealed that compound 10v exhibited higher potency than 5-Fu against HCT-116 cells.
Preliminary biological activity studies showed that compound 10v could inhibit the colony formation and
migration of HCT-116 cells. Besides, the Hoechst 33258 staining assay and flow cytometry revealed that
treatment with compound 10v induced the apoptosis of HCT-116 cells in a concentration-dependent
manner, while it had no effect on their cell cycle. The WB analysis suggested that HIF-1a, tubulin, HK-2,
and PFK might be the potential pharmacophore targets of compound 10v. Tubulin was a potential drug
target for compound 10v, which was explained by analyzing the crystal structure of compound 10v
complexed with tubulin. These results indicated that compound 10v might be a promising anti-tumor
agent candidate, deserving further optimization and evaluation.
Received 8th September 2018,
Accepted 14th December 2018
DOI: 10.1039/c8nj04533j
rsc.li/njc
potential anti-tumor effect for decades.10–12 Representative anti-
tumor agents of flavonoids which have been discovered include
1. Introduction
Cancer is one of the main causes of mortality globally. the following: silibinin, which is an approved drug;13 flavopiridol,
To improve the survival rates of cancer patients, significant which is in clinical trial phase III;14 apigenin, which is in clinical
attention has been focused on the design and synthesis of new trial phase III,15,16 and so on. The diversiform structures,17,18
anti-tumor drugs with high efficiency and low toxicity. During multiple targets, and varied pharmacological activities2,19 make
the last few decades, a growing number of studies have flavonoids ideal parental structures in anti-tumor drug design.
proposed pharmacophore combination strategy, which combines Furthermore, TMP of most microtubule-targeting agents (MTAs)
two or more active pharmacophores covalently in a single-hybrid have been discovered as an important pharmacophoric point for
molecule with dual/multiple anti-tumor functions, so as to design tubulin binding for cancer therapy.20–22 In our previous work,
new anti-tumor molecules that act synchronously on more than the introduction of the TMP moiety into the flavonoid scaffold
one target.1 According to the pharmacophore combination was found to be an effective strategy for preparing novel flavo-
principle, we have designed a new class of anti-tumor molecules, noids with potential anti-tumor activity.4,23 However, to the best
by the joining three important pharmacophores: trimethoxyphenyl of our knowledge, 30,40,50-trimethoxy flavonoid salicylate deriva-
(TMP), flavonoid, and salicylic acid (SA) (Fig. 1).
tives have not been thoroughly explored.
Flavonoid, a kind of natural product which widely exists,
Aspirin (ASA) is widely prescribed as a non-steroidal anti-
shows many promising biological activities, including anti- inflammatory drug, a platelet anti-aggregating agent, and in recent
tumor,2–4 anti-angiogenic,5 anti-inflammatory,6,7 and anti- years, its properties as anti-tumor drugs have been observed.24,25
oxidative8,9 effects. The relevant research data have indicated There is growing evidence which has shown that aspirin inhibits
that flavonoids have useful chemopreventive and chemotherapeutic many different cancer cells, including human liver cancer,26 breast
effects. Accordingly, much attention has been focused on their cancer,27 colon cancer,28,29 and cervical cancer cells.30 SA is the
major metabolite and active component of aspirin.31
Institute of Pharmacy and Pharmacology, Hunan Province Cooperative Innovation
Based on the above considerations, we have proposed the
introduction of TMP moiety into B-ring of the flavonoid scaf-
fold, and joining this with SA and its derivatives. Herein, we have
Center for Molecular Target New Drug Study, University of South China, Hengyang,
Hunan, 421001, China. E-mail: tgtzq@163.com
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
described the detailed synthetic routes and anti-tumor activity
screening results of a set of new 30,40,50-trimethoxy flavonoid
c8nj04533j
‡ These authors contributed equally to this work.
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Fig. 1 The design idea of 30,40,50-trimethoxy flavonoid salicylate derivatives via the combination principle.
salicylate derivatives. Also, we have successfully identified com- in Scheme 1. In this study, two synthetic methods were used to
pound 10v as a promising candidate for anti-tumor activity.
join flavonoid and SA. The first synthetic route is outlined
in Scheme 2. Methylation of SA and its derivatives 5a–5h
with dimethyl sulfate was carried out, followed by hydrolysis
reaction and acetylation to obtain the intermediates 6a–6h.
The intermediates 6a–6h and 30,40,50-trimethoxy flavonoid (4)
produced the target compounds 7a–7h, by the esterification
reaction. The second synthetic route is described in Scheme 3.
The intermediates 8a–8c were prepared by the reaction of
30,40,50-trimethoxy flavonoid (4) and brominated alkanes with
2. Results and discussion
2.1. Chemistry
The key intermediate, 30,40,50-trimethoxy flavonoid (4) was
prepared via a three-step synthetic procedure, following the
previously reported procedures.4 The synthetic route is shown
Scheme 1 Synthesis of 7-hydroxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (4). Reagents and conditions: (a) chloroacetonitrile, ZnCl2, HCl (g),
anhydrous diethyl ether, 0 1C, 4 h; (b) H2O, 90 1C, 1 h; (c) (i) 3,4,5-trimethoxybenzaldehyde, 10% NaOH, 5% ethanol, 24 h; (ii) 10% HCl, 1 h.
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Scheme 2 Synthesis of compounds 4a–h. Reagents and conditions: (a) (i) 30% NaOH, dimethyl sulfate, 37 1C, 3 h; (ii) 80 1C, 15 min; (iii) thionyl chloride,
dichloromethane, 40 1C, 1 h; (b) 7-hydroxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (4) triethylamine, acetone, RT, 2 h.
different chain lengths under reflux conditions, in the presence cancer cell lines. In the cytotoxicity evaluation with the HCT-116
of K2CO3 in acetone. Moreover, the intermediates 9a–9h with cell line, compounds 7a–7h without alkane chain showed good
different substitutions could be readily obtained by reacting anti-proliferative activity. Compared to 5-Fu, compound 10v
5a–5h with SOCl2 in EtOH under reflux conditions. Finally, the displayed higher cytotoxicity against HCT-116 cells with the
intermediates 8a–8c and 9a–9h were refluxed in presence of IC50 value of 21.13 ꢀ 4.57 mM. The cytotoxicity of compound
K2CO3 in acetone, followed by etherification reaction to obtain 10v on normal HUVEC cells was similar to that of 5-Fu. Thus,
the target compounds 10a–10x. The structures of the target compound 10v was selected as the representative compound for
compounds 7a–7h and 10a–10x were confirmed using spectro- further investigation. In addition, some of these compounds
scopic methods, including 1H NMR and ESI mass spectra, displayed higher cytotoxicity against these tumor cell lines
which were in full accordance with their proposed structures.
compared to the 30,40,50-trimethoxy flavonoid (4) and SA, suggest-
ing that the pharmacophore combination principle was an effec-
tive strategy in designing new anti-tumor drug candidates.
2.2. Cytotoxicity evaluation
The methyl thiazoltetrazolium (MTT) assay was conducted to
evaluate cytotoxicity of target compounds 7a–7h and 10a–10x
against the MGC-803 (human gastric cancer cells), MCF-7
(human breast cancer cells), HepG-2 (human liver cancer cells),
A549 (human lung cancer cells), HELA (human cervical cancer
cells), HCT-116 (human colon cancer cells), and HUVEC
(human umbilical vein endothelial cell) cell lines. 5-Fu was
used for positive control, which is effective against colon
cancer, esophageal cancer, stomach cancer, pancreatic cancer,
breast cancer, and cervical cancer. The cytotoxicity results
are shown in Table 1. As shown in Table 1, target compounds
2.3. Clonogenic assay
The clonogenic or colony formation assay is used for measuring
the ability of a single cell to form a colony in vitro. This assay
was conducted in order to determine the inhibition ability of
our candidate compound against clone-forming ability of
tumor cells. Fig. 2 indicated that compound 10v at a concen-
tration of 10 mM dramatically inhibited colony formation by
HCT-116 cells, and was superior in its inhibition activity to 5-Fu.
2.4. Cell migration
7a–7h and 10a–10x had little effect on MGC-803, MCF-7, and The cell migration ability of HCT-116 cells was investigated by
HepG-2 cells. Few 30,40,50-trimethoxy flavonoid salicylate deriva- the wound healing assay. A wound healing assay is a laboratory
tives exhibited cytotoxicity against the A549 and HELA cell technique used to study cell migration and cell–cell inter-
lines. The cytotoxicity of the target compounds 7a–7h and action.32 Compound 10v suppressed migration of HCT-116 cells in
10a–10x against HCT-116 cells was the best amongst the six a concentration-dependent manner, compared to the control group.
Scheme 3 Synthesis of compounds 10a–x. Reagents and conditions: (a) (CH2)2Br2 or (CH2)3Br2 or (CH2)4Br2, K2CO3, acetone, 60 1C, 9 h; (b) (i) CH3OH,
SOCl2, 0 1C, 10 min; (ii) 65 1C, 48 h; (c) K2CO3, acetone, reflux, 48 h.
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Table 1 Cytotoxicity results of compounds 7a–7h, 10a–10x
IC50 (mM)
Compound
n
R
MGC-803
MCF-7
HepG-2
A549
HELA
HCT-116
HUVEC
7a
7b
7c
7d
7e
7f
7g
7h
—
—
—
—
—
—
—
—
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
—
—
—
4-CH3
5-CH3
4-Br
5-Br
4-Cl
5-Cl
H
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
4100
N.D.
N.D.
N.D.
N.D.
91.48 ꢀ 4.99 4100
52.34 ꢀ 2.78 59.02 ꢀ 8.31
N.D.
4100
4100
N.D.
4100
66.31
4100
4100
4100
4100
97.7 ꢀ 6.95
N.D.
99.1 ꢀ 11.8
4100
90.29 ꢀ 6.21 4100
79.08 ꢀ 7.26 68.48 ꢀ 4.01 68.03 ꢀ 9.79
N.D.
N.D.
4100
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
4100
4100
4100
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
4100
N.D.
N.D.
4100
4100
4100
N.D.
4100
N.D.
80.34 ꢀ 11.3
90.26 ꢀ 5.89
4100
73.66 ꢀ 3.31 N.D.
3-Cl,5-Cl,6-Cl 4100
4100
N.D.
4100
4100
N.D.
N.D.
N.D.
N.D.
4100
N.D.
4100
4100
N.D.
N.D.
4100
N.D.
4100
N.D.
4100
4100
N.D.
4100
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
4100
N.D.
N.D.
4100
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
4100
4100
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
10s
10t
10u
10v
10w
10x
4
4-CH3
5-CH3
4-Br
5-Br
4-Cl
5-Cl
H
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
41.78 ꢀ 4.38
N.D.
4100
N.D.
4100
46.88 ꢀ 2.36 4100
4100 4100
83.57 ꢀ 3.15 19.09 ꢀ 3.60
3-Cl,5-Cl,6-Cl N.D.
N.D.
N.D.
4100
N.D.
4100
N.D.
4100
N.D.
4100
N.D.
4100
N.D.
4100
N.D.
4100
N.D.
N.D.
N.D.
N.D.
N.D.
4100
4100
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
4-CH3
5-CH3
4-Br
5-Br
4-Cl
5-Cl
H
N.D.
N.D.
N.D.
N.D.
N.D.
4100
N.D.
3-Cl,5-Cl,6-Cl N.D.
4-CH3
5-CH3
4-Br
5-Br
4-Cl
5-Cl
H
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
4100
4100
N.D.
21.13 ꢀ 4.57 54.74 ꢀ 3.67
N.D.
4100
N.D.
N.D.
N.D.
4100
N.D.
3-Cl,5-Cl,6-Cl N.D.
—
—
—
4100
N.D.
SA
5-Fu
4100
58.34 ꢀ 3.25 60.43 ꢀ 2.95 68.17 ꢀ 3.29 4100
72.80 ꢀ 4.66 34.57 ꢀ 3.94 57.79 ꢀ 2.83
N.D. = not detected.
Fig. 2 Inhibition effects of compound 10v on HCT-116 cells colony formation.
2.5. Cell apoptosis analysis
lesser wound closure rates than 5-Fu (40 mM) (Fig. 3). Compound 10v Cell apoptosis is a form of programmed cell death that occurs
suppressed the migration of MGC-803 cells better than 5-Fu. in multicellular organisms, and is one of the main events that
Compound 10v at different concentrations (10, 20, and 40 mM) had
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Fig. 3 (A) Treatment with compound 10v or 5-Fu inhibited the migration of HCT-116 cells for 24 h in the wound healing assay. (B) Wound closure rate
was calculated as mean ꢀ SD from three different fields. **p o 0.01 vs. control. &p o 0.05, &&p o 0.01 vs. 5-Fu.
induces cell deaths and pyknosis. We conducted Hoechst 33258
staining as well as Annexin V-FITC/PI dual staining to
determine if the growth-inhibitory effects of compound 10v
were caused by apoptotic death of HCT-116 cells. Hoechst
33258 staining was used to differentiate between the apoptotic
and normal cells. In the Hoechst 33258 staining assay,
untreated HCT-116 cells emitted blue fluorescence. In contrast,
the cells treated with compound 10v for 24 h displayed stronger
blue fluorescence and presented characteristic morphologies
of early apoptotic cells (Fig. 4).
Annexin V-FITC/PI staining assays divided cells into four
groups: live population (annexin Vꢁ/PIꢁ), early apoptotic
population (annexin V+/PIꢁ), late apoptotic population
(annexin V+/PI+), and necrotic population (annexin Vꢁ/PI+).
The cell apoptotic population includes the early and late
apoptotic population. As shown in Fig. 5, the treatment of the
HCT-116 cells with 10v at different concentrations (10, 20, and
40 mM) caused an increase in the apoptotic cell population.
These results demonstrated that compound 10v induced
apoptosis of the HCT-116 cells, in a concentration-dependent
manner.
Fig. 4 Effect of compound 10v treatment for 24 h on the morphology of
the HCT-116 cells. The cells were stained with the Hoechst 33258 dye.
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Fig. 5 (A) Treatment with compound 10v for 24 h induced the apoptosis of HCT-116 cells in a concentration-dependent manner. (B) The percentage of
early apoptosis and late apoptosis of three independent tests were quantified using flow cytometry. *p o 0.05, **p o 0.01 vs. control.
2.6. Cell cycle distribution
many cancers. Recent studies have revealed that ASA and SA can
modulate PFK quaternary structure, and inhibit tumor cells’
glucose consumption and viability.39,40 The potential targets of
30,40,50-trimethoxy flavonoid salicylate derivatives were identified
by a simple screening test, using the western blotting (WB)
method. As shown in Fig. 6, HCT-116 cells treated with com-
pound 10v exhibited depressed expression of tubulin, HIF-1a,
HK-2, and PFK compared to the control group. These results
suggest that tubulin, HIF-1a, HK-2, and PFK could be the
potential pharmacophore targets of compound 10v. Tumor
blood vessels provide oxygen and nutrients to tumor growth and
survival.41,42 Over the last decade, it has been discovered that
numerous TMP based tubulin inhibitors were potent as anti-
cancer agents, and lead to microtubule and actin depolymeriza-
tion. This depolymerization destroys the cellular structural
framework and disrupts intercellular junctions, resulting in
Cell cycle regulation is a necessary process to maintain cell
proliferation and development. However, the tumor cells primarily
cause disruption of the cell cycle, which in turn blocks cell
differentiation and apoptosis, leading to uncontrolled cell
proliferation.33 To explore the possible role of cell cycle arrest
in the HCT-116 cell death induced by compound 10v, cell cycle
distribution was measured by flow cytometry. As shown in
Fig. S1 (ESI†), the G0/G1-phase, S-phase, and G2/M population
of the HCT-116 cells had no obvious and regular change
compared to the control group. The result demonstrated that
the treatment with compound 10v did not affect the cell cycle of
HCT-116 cells.
2.7. Screening of potential targets
According to the combination principle, 30,40,50-trimethoxy collapse of the tumor vessels.43–46 Also, tumor glycolysis provides
flavonoid salicylate derivatives were designed by the joining energy for rapid tumor growth and promotes cancer metastasis.
of three important pharmacophores: TMP, flavonoid, and SA. It HIF-1a transcription factor plays major roles in tumor glycolysis.
has been reported that hypoxia-inducible factor-1a (HIF-1a)34–36 In addition, HK-2 and PFK are the key rate-limiting enzymes in
and hexokinase-2 (HK-2)37,38 can serve as targets of flavonoid in glycolysis. Compound 10v may cause vascular disrupting and
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Fig. 6 Effect of compound 10v on the expression levels of tubulin, HIF-1a, HK-2, and PFK on the HCT-116 cells. CoCl2 hypoxia-mimicking conditions
(100 mM) and serum starvation (0.1% FBS) stimulated the expression of HIF-1a. Quantification of the Western blot results was conducted by calculating
the ratio of the value to the control group. Representative photographs from three independent experiments are displayed. *p o 0.05, **p o 0.01 vs.
control. &&&p o 0.001 vs. CoCl2 (100 mM) without compound 10v.
glycolysis inhibition, by down-regulating tubulin and glycolysis TYR-224 (Fig. 7D). The results of the docking study provided
related proteins. The specific mechanism describing how com- us with a new possible modification for further structural
pound 10v acts synchronously on multiple targets in HCT-116 optimization.
cell line, can be further studied.
2.8. Docking studies with tubulin
3. Experimental
3.1. Chemicals
As expected, compound 10v (Fig. 7A) occupied the colchicine
binding site on tubulin (Fig. 7B). The binding site exists
between the a2-tubulin and b2-tubulin subunit. To gain a better All material and reagents (reagent grade) were purchased from
understanding of the binding between the colchicine binding commercial sources and unless otherwise stated, were used
site of tubulin and compound 10v, we conducted the molecular without further purification. Separation of the compounds by
docking study with the crystal structure of the DAMA–colchicine– column chromatography was carried out with silica gel (200 mesh
tubulin complex (PDB ID: 1SA0). As presented in Fig. 7C, com- size). Thin layer chromatography (TLC) was run on the silica gel 60
pound 10v formed hydrophobic interactions with surrounding F254 plates and visualized under UV light at 254 nm or 365 nm,
residues, such as ALA-316, GLN-11, GLU-71, and ASN-101. More- which were used to monitor reactions. Melting points were deter-
over, the carbonyl group on salicylic acid structure of compound mined on a Thermo Scientific electrothermal digital melting point
10v formed a hydrogen bond with the amine group of GLY-144. apparatus and were uncorrected. ESI mass spectra were obtained
1
The oxygen atom of A-ring C7 on flavonoid structure of com- on a Waters GCT mass spectrometer, and H NMR spectra were
pound 10v formed a hydrogen bond with the hydroxyl group of measured on a Bruker AV-400 model spectrometer. Chemical shifts
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Fig. 7 (A) The structure of compound 10v. (B) Structure of tubulin in complex with compound 10v. The a-units are shown in grey; the b-units are shown
in black, and RB3 is shown in red. (C) Compound 10v formed hydrophobic interactions with surrounding residues. (D) Hydrogen bonding interactions are
displayed in yellow dashed lines. Docking mode of compound 10v with the colchicine binding domain of tubulin (PDB code: 1SA0). The figures were
engendered using PyMOL (http://www.pymol.org/).
were reported in ppm (d), and referenced with tetramethylsilane
(TMS). The purity of the compounds was verified by HPLC.
A mixture of 6a (0.0005 mol), 7-hydroxy-2-(3,4,5-trimethoxy-
phenyl)-4H-chromen-4-one (4) (0.0003 mol) was reacted with a
3.1.1. General procedure for the synthesis of 7-hydroxy-2- catalytic amount of triethylamine in acetone (20 mL) at RT for
(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (4). Resorcinol (1) 2 h, and monitored by TLC. After the completion of the reaction,
(0.023 mol) and 2-chloroacetonitrile (0.032 mol) were dissolved the solvent was evaporated under vacuum. The residue was
in absolute ether (50 mL). A catalytic amount of ZnCl2 was purified on silica gel using column chromatography, and eluted
added to this solution in an ice/water bath, and freshly pre- with a solution of methanol and dichloromethane (v/v, 1 : 200) to
pared hydrogen chloride gas was bubbled though this mixture give the target compound 7a.
slowly and continuously for 4 h. After filtration, washing and
The compounds 7b–7h were prepared analogously.
drying, the yellow precipitate (2) was hydrolyzed to obtain a milky
3.1.3. General procedure for the synthesis of compounds
white precipitate of 2-chloro-1-(2,4-dihydroxyphenyl) ethanone 10a–10x. 7-Hydroxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-
(3). 3,4,5-Trimethoxybenzaldehyde (0.01 mol) and intermediate one (4) (0.02 mol) and (CH2)2Br2 (0.08 mol) were dissolved in
3 (0.012 mol) were dissolved in 5 mL 95% ethanol, then 10% acetone (100 mL). A catalytic amount of K2CO3 was added to
NaOH aqueous solution was added dropwise into the solution this solution at 60 1C for 9 h, and monitored by TLC. After the
for 24 h at room temperature (RT). Finally, acidification with completion of the reaction, the solvent was evaporated under
10% HCl (aq.) gave a crude product, which was filtered off and vacuum. The residue was purified on silica gel using column
purified by recrystallization from 95% ethanol to give 7-hydroxy- chromatography, and eluted with a solution of methanol and
2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (4) as a yellow dichloromethane (v/v, 1 : 200) to give the intermediate 8a. This
powder.
procedure was also applied for the preparation of compound 8b
3.1.2. General procedure for the synthesis of compounds and 8c, except that (CH2)3Br2 and (CH2)4Br2 were used as the
7a–7h. 4-Methylsalicylic acid (5a) (0.001 mol) was dissolved in reactants, respectively.
water (10 mL). The pH of the solution was adjusted to 10 using
4-Methylsalicylic acid (5a) (0.01 mol) was dissolved in methanol,
30% NaOH. The reaction mixture was stirred at 37 1C for 30 min, and thionyl chloride was added to this solution in an ice/water
and dimethyl sulfate (2 mL) was slowly added. Whenever the pH bath. After 10 min, the reaction mixture was stirred at 65 1C for
of the solution reached 7, 30% NaOH was added to adjust the 48 h. After completion of the reaction, the solvent was evaporated,
solution pH to 10. Dimethylsulfate (1 mL) and 30% NaOH were and the residue was treated with dichloromethane and water.
added once more according to the above procedure. The reaction
After concentration of the organic phase, the obtained residue
was carried out for 3 h, and then the solution was hydrolyzed at (9a) (0.6 mmol), the intermediate 8a (0.0003 mol) and a catalytic
80 1C for 15 min. After cooling, the pH was adjusted to 5 using amount of K2CO3 were reacted in acetone (30 mL) at 60 1C for 48 h
10% HCl, and a white precipitate was obtained. The white powder and monitored by TLC. After the completion of the reaction, the
was filtered, washed and dried. The mixture of the white powder solvent was evaporated under vacuum. The residue was purified
(0.0008 mol), thionyl chloride (0.001 mol), and dichloromethane on silica gel using column chromatography, and eluted with a
(30 mL) was stirred at 40 1C for 1 h. The methylene chloride was solution of methanol and dichloromethane (v/v, 1 : 200) to give the
evaporated under reduced pressure to obtain a white solid (6a).
target compound 10a.
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The compounds 10b–10x were prepared analogously.
Paper
3.7. Annexin V-FITC/PI dual staining
Apoptosis was determined by Annexin V-FITC and PI dual
staining. Cells were treated with different concentrations of
10v (10, 20, 40 mM) for 24 h. The cells were incubated with
500 mL 1ꢂ binding buffer, followed by the addition of 5 mL
Annexin V-FITC and 5 mL PI, and then incubated for 15 min
at 25 1C in the dark. The apoptosis assay was carried out by
the FACSCalibur flow cytometry (BD Biosciences, USA) and
analyzed using FlowJo software.
3.2 Cell culture
MGC-803 cells were grown in Roswell Park Memorial Institute
medium (RPMI-1640), supplemented with 10% FBS and 1%
penicillin–streptomycin. MCF-7, HepG2, A549, HELA, and HUVEC
cells were cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM), supplemented with 10% FBS and 1% penicillin–
streptomycin. HCT-116 cells were cultured in DMEM supple-
mented with 15% FBS and 1% penicillin–streptomycin.
All of the cells were cultured in a humidified incubator at
37 1C with 5% CO2.
3.8. Cell cycle analysis
Cells were treated with the indicated concentrations of 10v
(10, 20, 40 mM) for 24 h and collected by trypsinization. The
cells were fixed in ice-cold alcohol (70%) at 4 1C overnight.
Then, the cells were stained with 500 mL PI staining solution,
containing 200 mg mLꢁ1 RNase A and 50 mg mLꢁ1 PI, at 37 1C
for 30 min in the dark. Cell cycle analysis was performed on the
FACSCalibur flow cytometry (BD Biosciences, USA).
3.3. Cell viability assay (MTT assay)
MTT method was used to evaluate the cytotoxicity of the
compounds on tumor cells by examining cell survival after
exposure to the compounds for 48 h. 5 ꢂ 103 cells per well were
planted in 96-well plate and grown for 24 h. Cells were then
treated with either 0.1% DMSO or various concentrations
(2, 4, 8, 16, 32, 64, 128, 256 mM) of the tested compounds. After
incubation for 48 h, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) solution (5 mg mLꢁ1 in PBS)
was added to each well, and the plate was incubated for 4 h.
The formazan dye was dissolved in 150 mL DMSO, and the
optical density (OD) was measured at 490 nm using Wellscan
MK-2 microplate reader. The concentration causing 50%
inhibition of cell growth (IC50) was calculated using GraphPad
Prism 6.0 software. All experiments were independently performed
at least three times.
3.9. Western blotting
The expression of the protein was analyzed by western blotting
as described elsewhere.29 In brief, 8% SDS-PAGE was per-
formed by loading equal amount of protein (50 mg) per lane.
Gels were then transferred to PVDF membranes by semi-drying
at 15 V for 60 min. After blocking with 5% non-fat milk in TBST
at room temperature for 2 h, the membranes were incubated with
tubulin (1 : 1000) (Absin, China), HIF-1a (1 : 300) (CST, USA), HK-2
(1 : 1000) (CST, USA), PFK (1 : 500) (CST, USA), or b-actin (1 : 1000)
(Proteintech, USA) overnight at 4 1C, and then later incubated with
an anti-rabbit horseradish peroxidase-conjugated secondary anti-
body (1:4000) (Proteintech, USA) for 1 h at room temperature. The
protein was visualized using an enhanced chemiluminescence
system (Tanon 6200, China).
3.4. Clonogenic assay
1000 HCT-116 cells per well were seeded in a 6-well plate and
incubated for 24 h. Different concentrations (5, 10 mM) of 10v or
5-Fu (10 mM) in fresh medium containing 10% FBS were added.
The medium was changed every three days. After 9 days of
treatment, the colonies were fixed with 4% paraformaldehyde
for 15 min and were then stained using 0.1% crystal violet for
15 min. After two washes with PBS, the colonies in plates were
photographed for studies.
3.10. Molecular docking
Molecular docking was conducted using SYBYL-X 2.0. The 3D
structure of the tubulin was downloaded from the PDB (http://
www.rcsb.org/, PDB code 1SA0). For docking simulation, molecule
10v was built with ChemDraw Ultra 7.0. The figures were engen-
dered using PyMOL (http://www.pymol.org/).
3.5. Wound healing assay
HCT-116 cells were cultured in a 6-well plate for 24 h. The cell
surface was scratched using a 10 mL pipette tip. Then, the cells
were treated with different concentrations of 10v (10, 20, 40 mM)
or 5-Fu (40 mM). After incubation for 24 h, the wound was
photographed on an inverted-phase microscope.
3.11. Statistical analysis
All data were displayed as mean ꢀ SD from three independently
repeated determinations. Statistical analysis was conducted
using Origin 8.0 and Prism 6.0 GraphPad softwares. Student
t-test was employed for determining comparisons between the two
groups. One-way ANOVA was used for multiple group compari-
sons. P values o0.05 were considered statistically significant.
3.6. Cell morphological assessment
HCT-116 cells were plated in 6-well plate and incubated for
24 h. Then, the cells were treated with different concentrations
of 10v (10, 20, 40 mM) for 24 h. Cells were fixed with 4%
paraformaldehyde for 15 min, washed with PBS, and then
4. Conclusion
incubated with 1 mL Hoechst 33258 for 10 min in the dark. A set of new 30,40,50-trimethoxy flavonoid salicylate derivatives
After washing with PBS, the cells were observed by a fluores- were designed, synthesized, and evaluated for biological activity.
cence microscope (Olympus, Japan) at a peak excitation wave- With in vitro cytotoxicity studies against six cancer cell lines, we
length of 350 nm.
identified that compound 10v showed high cytotoxicity against
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NJC
12 W. Ren, Z. Qiao, H. Wang and L. Zhang, Med. Res. Rev.,
2003, 23, 519–534.
HCT-116 cells. Preliminary biological activity studies showed
that compound 10v could inhibit the colony formation and
migration of HCT-116 cells. Besides, the Hoechst 33258 staining
assay and flow cytometry revealed that treatment of compound
10v induced the apoptosis of HCT-116 cells in a concentration-
dependent manner, whereas it had no effect on the cell cycle
of HCT-116 cells. Also, WB analysis further confirmed that
HCT-116 cells treated with compound 10v exhibited depressed
expression of tubulin, HIF-1a, HK-2, and PFK compared to the
control group, suggesting that tubulin, HIF-1a, HK-2, and PFK
could be the potential pharmacophore targets of compound 10v.
Results from the molecular docking study further suggest that
tubulin is a potential target for compound 10v. Our studies
suggest that compound 10v could be further optimized and
evaluated as a new anti-tumor agent.
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No potential conflicts of interest were disclosed.
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(0223-0002-0002000-49) and Innovation Platform Open Foun-
dation of Education, Department of Hunan province (contract
grant number 16K079, 17K082).
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