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

DOI: 10.1039/c8nj04533j

Source and publish data:

New Journal of Chemistry p. 1874 - 1884 (2019)

Update date:2022-08-04

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Authors:

Deng, Xiangping

Liu, Renbo

Li, Junjian

Li, Zhongli

Liu, Juan

Xiong, Runde

Lei, Xiaoyong

Zheng, Xing

Xie, Zhizhong

Tang, Guotao

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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.^10–12Representative 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;¹³flavopiridol,

To improve the survival rates of cancer patients, significant which is in clinical trial phase III;¹⁴apigenin, which is in clinical

attention has been focused on the design and synthesis of new trial phase III,^15,16and so on. The diversiform structures,^17,18

anti-tumor drugs with high eﬃciency and low toxicity. During multiple targets, and varied pharmacological activities^2,19make

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.^20–22In 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.¹According 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.^4,23However, 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,^2–4anti-angiogenic,⁵anti-inflammatory,^6,7and anti- years, its properties as anti-tumor drugs have been observed.^24,25

oxidative^8,9effects. 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,²⁶breast

effects. Accordingly, much attention has been focused on their cancer,²⁷colon cancer,^28,29and cervical cancer cells.³⁰SA 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.⁴The 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 K₂CO₃in 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 SOCl₂in 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 IC₅₀value of 21.13 ꢀ 4.57 mM. The cytotoxicity of compound

K₂CO₃in 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.³²Compound 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)₂Br₂or (CH₂)₃Br₂or (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

MGC-803

MCF-7

HepG-2

A549

HELA

HCT-116

HUVEC

—

4-CH₃

5-CH₃

4-Br

5-Br

4-Cl

5-Cl

N.D.

4100

N.D.

91.48 ꢀ 4.99 4100

52.34 ꢀ 2.78 59.02 ꢀ 8.31

N.D.

4100

N.D.

4100

66.31

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.

4100

N.D.

4100

N.D.

4100

N.D.

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

N.D.

4100

N.D.

4100

N.D.

4100

N.D.

4100

N.D.

4100

N.D.

4100

N.D.

4100

N.D.

4100

N.D.

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-CH₃

5-CH₃

4-Br

5-Br

4-Cl

5-Cl

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.

4100

N.D.

4100

N.D.

4100

N.D.

4100

N.D.

4100

N.D.

4100

N.D.

4100

N.D.

4100

N.D.

4-CH₃

5-CH₃

4-Br

5-Br

4-Cl

5-Cl

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

N.D.

4100

N.D.

21.13 ꢀ 4.57 54.74 ꢀ 3.67

N.D.

4100

N.D.

4100

N.D.

3-Cl,5-Cl,6-Cl N.D.

—

4100

N.D.

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.^39,40The 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.^41,42Over 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.³³To 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.^43–46Also, 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)^34–36In addition, HK-2 and PFK are the key rate-limiting enzymes in

and hexokinase-2 (HK-2)^37,38can 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. CoCl₂hypoxia-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

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 ZnCl₂was 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 K₂CO₃was 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)₃Br₂and (CH₂)₄Br₂were 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 K₂CO₃were 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 mL^ꢁ1RNase A and 50 mg mL^ꢁ1PI, 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 10³cells 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 mL^ꢁ1in 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.²⁹In 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

New J. Chem.

View Article Online

Paper

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.

13 P. Garcıa-Maceira and J. Mateo, Oncogene, 2009, 28,

313–324.

14 M. V. Blagosklonny, Cell Cycle, 2004, 3, 1537–1542.

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and J. C. Pelling, Mol. Carcinog., 2008, 47, 686–700.

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Conflicts of interest

22 M. J. Perez-Perez, E. M. Priego, O. Bueno, M. S. Martins,

M. D. Canela and S. Liekens, J. Med. Chem., 2016, 59,

8685–8711.

No potential conflicts of interest were disclosed.

23 X. Deng, Z. Wang, J. Liu, S. Xiong, R. Xiong, X. Cao, Y. Chen,

X. Zheng and G. Tang, RSC Adv., 2017, 7, 38171–38178.

24 J. Cuzick, F. Otto, J. A. Baron, P. H. Brown, J. Burn,

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25 I. Phillips, R. Langley, D. Gilbert and A. Ring, Clin. Oncol.,

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Acknowledgements

This work was financially supported by Hunan Province Coop-

erative Innovation Center for Molecular Target New Drug Study

(0223-0002-0002000-49) and Innovation Platform Open Foun-

dation of Education, Department of Hunan province (contract

grant number 16K079, 17K082).

26 M. Gao, Q. Kong, H. Hua, Y. Yin, J. Wang, T. Luo and

Y. Jiang, Oncotarget, 2016, 7, 16349–16361.

27 S. Saha, S. Mukherjee, P. Khan, K. Kajal, M. Mazumdar,

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