Synthesis and biological evaluation of novel 5,6,7-trimethoxy flavonoid salicylate derivatives as potential anti-tumor agents

Article abstract of DOI:10.1016/j.bioorg.2020.103652

5,6,7-Trimethoxy flavonoid salicylate derivatives were designed by the joining of three important pharmacophores (TMP, flavonoid, and SA) according to the combination principle. A series of novel trimethoxy flavonoid salicylate derivatives were synthesized and their in vitro anti-tumor activities were evaluated. Among these derivatives, compound 7f exhibited excellent antiproliferative activity against HGC-27 cells and MGC-803 cells with IC₅₀ values of 10.26 ± 6.94 μM and 17.17 ± 3.03 μM, respectively. Subsequently, the effects on cell colony formation (clonogenic survival assay), cell migration (wound healing assay), cell cycle distribution (PI staining assay), cell apoptosis (Hoechst 33258 staining assay and annexin V-FITC/PI dual staining assay), lactate level (lactate measurement), microtubules disarrangement (immunofluorescence staining analysis) and docking posture (molecular docking simulation) were determined. Further western blot analysis confirmed that compound 7f could effectively down-regulate the expression of glycolysis-related proteins HIF-1α, PFKM and PKM2 and tumor angiogenesis-related proteins VEGF. Overall, these studies suggested that compound 7f, as the representative compound of those, might be a promising candidate for the treatment of gastric cancer and deserved the further studies.

Full text of DOI:10.1016/j.bioorg.2020.103652

Journal Pre-proofs
Synthesis and biological evaluation of novel 5,6,7-trimethoxy flavonoid sali-
cylate derivatives as potential anti-tumor agents
Renbo Liu, Xiangping Deng, Yijiao Peng, Wanshi Feng, Runde Xiong, Yang
Zou, Xiaoyong Lei, Xing Zheng, Zhizhong Xie, Guotao Tang
PII:
S0045-2068(19)31409-9
DOI:
https://doi.org/10.1016/j.bioorg.2020.103652
YBIOO 103652
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Bioorganic Chemistry
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28 August 2019
1 February 2020
3 February 2020
Please cite this article as: R. Liu, X. Deng, Y. Peng, W. Feng, R. Xiong, Y. Zou, X. Lei, X. Zheng, Z. Xie, G.
Tang, Synthesis and biological evaluation of novel 5,6,7-trimethoxy flavonoid salicylate derivatives as potential
anti-tumor agents, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103652
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Synthesis and biological evaluation of novel 5,6,7-trimethoxy
flavonoid salicylate derivatives as potential anti-tumor
agents
Renbo Liu,‡a,b Xiangping Deng,‡a,b Yijiao Peng,a Wanshi Feng,a Runde Xiong,a Yang Zou,a Xiaoyong Lei,a Xing
Zheng,a Zhizhong Xiea and Guotao Tang*a,b
a Institute of Pharmacy and Pharmacology, Hunan Province Cooperative Innovation Center for Molecular Target
New Drug Study, University of South China, Hengyang City, P.R. China.
bHunan Provincial Key Laboratory of tumor microenvironment responsive drug research, Hengyang City, Hunan
Province, P.R. China.
*Corresponding Author: Dr. Guotao Tang, Ph.D.
Corresponding author at：
Guotao Tang, Institute of Pharmacy and Pharmacology, University of South China, Hengyang, Hunan, 421001,
People’s Republic of China.
E-mail address: tgtzq@163.com (Guotao Tang)
‡ These authors contributed equally to this work.

A B S T R A C T
5,6,7-Trimethoxy flavonoid salicylate derivatives were designed by the joining of three important
pharmacophores (TMP, flavonoid, and SA) according to the combination principle. A series of
novel trimethoxy flavonoid salicylate derivatives were synthesized and their in vitro anti-tumor
activities were evaluated. Among these derivatives, compound 7f exhibited excellent
antiproliferative activity against HGC-27 cells and MGC-803 cells with IC50 values of 10.26 ± 6.94
μM and 17.17 ± 3.03 μM, respectively. Subsequently, the effects on cell colony formation
(clonogenic survival assay), cell migration (wound healing assay), cell cycle distribution (PI staining
assay), cell apoptosis (Hoechst 33258 staining assay and annexin V-FITC/PI dual staining assay),
lactate level (lactate measurement), microtubules disarrangement (immunofluorescence staining
analysis) and docking posture (molecular docking simulation) were determined. Further western
blot analysis confirmed that compound 7f could effectively down-regulated the expression of
glycolysis-related proteins HIF-1α, PFKM and PKM2 and tumor angiogenesis-related proteins
VEGF. Overall, these studies suggested that compound 7f, as the representative compound of those,
maybe a promising candidate for the treatment of gastric cancer and deserving the further studies.
Keywords: Flavonoid; Anti-tumor; Microtubules; HIF-1α; Glycolysis
1. Introduction

Tumor is a kind of tissue with uncontrolled and abnormal cell proliferation, and its differentiation
rate is higher than normal cells [1]. Nowadays, Cancer incidence and mortality are sharply growing
in the world [2]. Hence, the development of various drugs for cancer treatment is particularly
important. However, the traditional chemotherapy drugs are faced many obstacles including
cytotoxicity, drug resistance and lack of sensitivity, etc [3,4]. Currently, a growing number of
studies have shown that the pharmacophore combination strategy is conducive to overcome these
shortcomings. A single drug molecule designed by the pharmacophore combination can act on
dual/multi molecular targets which represent an efficient, logical and alternative approach to drug
combinations [5]. For instance, cabometyx (cabozantinib) which has been approved by US Food
and drug administration (FDA), as a small molecule dual-targeting inhibitor of the tyrosine kinases
c-Met and vascular endothelial growth factor receptor 2 (VEGFR2), has been shown to reduce tumor
growth, metastasis and angiogenesis [6,7].
During the last few decades, there has been an increasing interest in the approach of chemical
modification of natural products aimed to improve their original biological activities. Flavonoids
(Fig. 1A) as one of the vital classes of bioactive chemicals widely distribute in vegetables, fruits
and medicinal plants [8,9]. Semi-synthetic and synthetic flavonoids have been reported to exhibit
many therapeutic activities such as antioxidant [10], antiallergic [11], antibacterial [12], anti-
inflammatory [13], antidiabetic [14], anxiolytic [15], and anticancer effects [16]. Some of the
flavonoids from natural origin like silymarin, gingko flavone glycosides and naringenin, can be
found in the commercial market. Simultaneously, some natural flavonoids for the auxiliary
treatment of cancer or coronary heart diseases, including oroxylin A, wogonin and vitexin, are
undergoing phase I/II clinical trials in China [17-19]. According to our previous research, flavonoid
derivatives had performed an anti-tumor effect possibly associated with glycolysis-related proteins
[20].
Furthermore, microtubule is the cage of tubulin dimers, involves in the dynamics of
chromosomes specifically, mitotic spindle formation during cell division and serving as an
important target for anticancer therapy [21]. Microtubule-targeting agents (MTAs) disrupt
microtubule dynamic and arrest cancer cells in G2/M phase, and eventually induce apoptosis of
cancer cells, which have achieved significant success in the treatment of cancer [22,23]. Some
MTAs, as effective tubulin inhibitors, have entered clinical trials, such as colchicine analogs

ZD6126 [24] and CA4P [25]. MTAs have been substantiated to cause indirect tumor cell death
owing to going short for oxygen and nutrients by disrupting the tumor vessels and effectively
shutting off blood flow [26,27]. Another, drug resistance caused by the overexpression of the β-III
tubulin isoform can be also effectively circumvented by the colchicine binding site inhibitors
(CBSIs) [28]. CBSIs binds at the interface of ɑ-subunit and b-subunit of tubulin therefore result in
microtubule depolymerization. It has been demonstrated that trimethoxyphenyl (TMP) (Fig. 1B) as
a common moiety for many CBSIs plays a crucial role in maintaining suitable molecular
conformations for the optimal interactions with tubulin to emerge the highest-level anti-tumor
efficacy [29].
Salicylic acid (SA) (Fig. 1C) is a mono-hydroxybenzoic acid that serves as a precursor of
aspirin (ASA). ASA is a classic wonder drug and is widely used for the treatment of inflammation,
fever and pain over a century [30]. Besides, it’s reported that ASA and its metabolite SA could
selectively induce apoptosis and alter tumor glucose utilization in several colorectal carcinoma cell
lines [31-33]. Therefore, ASA or SA could be an outstanding anti-tumor active moiety for the
development of new anticancer agents.
Given these, three important pharmacophores, including flavonoid, TMP and SA, are
introduced into the design of a novel series of anti-tumor molecules according to the pharmacophore
combination principle. Specifically, the trimethoxyphenyl group is introduced into the A-ring of the
flavonoid and then SA and its derivatives are linked to the 4'-OH of the flavonoids with three lengths
of chain alkanes (Fig. 1). The molecules are expected to inhibit the assembly of tubulin, disrupt the
intracellular cytoskeleton network, and reduce the glycolytic capacity of tumor cells. Herein, the in
vitro antitumor activity of these compounds and their effects on tubulin and glycolysis-related
proteins are evaluated.

Fig. 1. The design of 5, 6, 7-trimethoxy flavonoid salicylate derivatives through the pharmacophore combination
principle.
2. Results and discussion
2.1. Chemistry
In this study, to discover new chemical entity and improve the drug-like properties, 24 flavonoid
salicylate derivatives were designed and synthesized according to the general pathway shown in
Scheme 1. The key intermediate 2-(4-Hydroxy-phenyl)-5,6,7-trimethoxy-chromen-4-one (3) was
obtained via a two-step synthesis procedure, starting with 3,4,5-trimethoxy-phenol (1), chloroacetyl
chloride and p-hydroxybenzaldehyde. First, the commercially available material 1 was used to offer
6-hydroxy-2,3,4-trimethoxy-benzoyl chloride (2) by the Friedel-crafts acylation under reflux
conditions, with aluminum trichloride as a catalyst in dichloromethane. Then base-catalyzed aldol
condensation of 2 with p-hydroxybenzaldehyde generated the chalcone, which was cyclized in the
solvent of 10% hydrochloric acid and ethanol at room temperature (RT) to afford compound 3 in
high yield with recrystallization from 95% ethanol. The compound 3 was treated with corresponding
brominated alkanes (1,2-dibromoethane, 1,3-dibromopropane, 1,4-dibromobutane), in the presence

of potassium carbonate in a polar aprotic solvent acetone, to facilitate the formation of the alkylation
product of the 4′-hydroxy group 4a-4c in good yields. Additionally, the intermediates 6a–6h with
various types substitutions could be rashly prepared by reacting 5a–5h with SOCl2 in EtOH under
reflux conditions. Eventually, etherification of the intermediates 4a–4c and 6a–6h to obtain the final
desired compounds 7a–7x under reflux conditions, in the presence of potassium carbonate in polar
aprotic solvent acetone. All of the synthetic compounds gave adequate analytical and spectroscopic
data, which agreed well with their proposed structures.
OH
MeO
MeO
OH
Cl
MeO
MeO
O
MeO
MeO
OH
i
ii
iii
OMe O
OMe O
OMe
2
1
3
Br
( )
n
Br
O
( )
n
Br
R
MeO
MeO
O
O
( )
O
n
4a n=1
4b n=2
4c n=3
MeO
MeO
O
OMe
O
O
OMe
v
4a-c
O
COOH
OMe O
iv
OMe
R
R
7a-x
OH
5a-h
OH
6a-h
Scheme 1. General synthesis of 5,6,7-trimethoxy flavonoid salicylate derivatives 7a-x. Reagents and conditions: (i)
C2H2Cl2O, AlCl3, DCM, 40 °C, Reflux, 1.5 h; (ii) (a) p-hydroxybenzaldehyde, 10% NaOH, 95% Ethanol, RT, 24 h;
(b) 10% HCl 1 h; (iii) K2CO3, KI Acetone, 60 °C, Reflux, 9 h; (iv) (a) CH3OH,SOCl2, 0 °C, 10 min; (b) 65 °C, 48
h; (v) K2CO3, KI, Acetone, Reflux, 24 h.
2.2. Cell proliferation
The anti-proliferative activity in vitro of the novel synthesized compounds 7a–7x was assayed
against a panel of human tumor cells, MGC-803 (gastric cancer cells), HCT-116 (colon cancer cells),
HGC-27 (undifferentiated gastric cancer cells), SGC-7901 (gastric carcinoma cells) and BEL-7402
(hepatoma cells), by applying 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay. The cytotoxicity results were summarized in Table 1, which were expressed in terms of IC50
values (the minimum concentration in μM that causes 50% of cell death concerning the control
culture), where effective anti-cancer agent 5-fluorouracil (5-FU) was used as the positive control.

Target compounds 7a-7x almost did not affect on SGC-7901 cells and BEL-7402 cells. Few 5,6,7-
trimethoxy flavonoid salicylate derivatives displayed a little cytotoxicity against HCT-116 cell line.
Additionally, some of these compounds showed passable to favorable cytotoxic activities against
MGC-803 cell lines. The cytotoxicity of the synthesized novel flavonoid salicylate derivatives
compounds 7a-7x against HGC-27 cells was the best out of the five cancer cell lines. We desired to
confirm whether those compounds were toxic to exhibited selective antiproliferative activities
between normal cells and cancer cells, all target compounds were screened against GES-1 cells
(human gastric epithelial cell line). As a whole, the benzene ring monosubstituted derivatives of SA
in target compounds were more active than polysubstituted derivatives. After comparison of the
biological activity data, it was observed that compounds with substitutions on the R5-position of
SA showed more remarkable inhibitory activity than the R4-positioned ones, when the SA had only
one position substituted on the benzene ring. Moreover, it was worth noting that the linkage of 2
carbons was presented stronger anti-proliferative activity than 3 carbons or 4 carbons against HGC-
27 cells when R4-position of SA was bromine. In addition, In general, compound 7f displayed the
most potent inhibitory activity, with IC50 of 17.17 ± 3.03 μM and 10.26 ± 6.94 μM on MGC-803
cells and HGC-27 cells, respectively, which was better than that of 5-FU (IC50 = 45.51 ± 13.3 μM
and 17.11 ± 2.64 μM, respectively). The IC50 of the compound 7f on GES-1 cells was almost 2-4
times that of gastric cancer cells, indicating that the compound 7f had certain selectivity for normal
cells and cancer cells. Accordingly, compound 7f should undergo further mechanism investigation
to seek for a potentially more safe and effective chemotherapy agent.
Table 1
Anti-proliferative activity results of compounds 7a-7x
R
O
( )
O
n
MeO
MeO
O
O
OMe
OMe O
7a-x
IC50 (μM)
compound
n
R
SGC-7901
MGC-803
HGC-27
HCT-116
BEL-7402
GES-1

7a
7b
7c
3
3
3
2
2
2
1
1
1
3
3
3
2
2
2
1
1
1
3
2
1
3
2
1
/
5-CH3
5-Cl
N.D
N.D
> 100
> 100
8.74 ± 1.46
> 100
50.9± 7.56
> 100
> 100
> 100
> 100
N.D
N.D
N.D
49.16 ± 6.37
> 100
5-Br
N.D
> 100
> 100
N.D
N.D
7d
7e
5-CH3
5-Cl
N.D
N.D
> 100
N.D
N.D
N.D
25.08 ± 5.07
17.17 ± 3.03
> 100
14.52 ± 9.73
10.26 ± 6.94
66.08 ± 6.79
17.32 ± 8.04
9.97 ± 4.25
> 100
N.D
9.88 ± 1.66
39.01± 3.72
> 100
7f
5-Br
N.D
N.D
7g
7h
7i
5-CH3
5-Cl
N.D
> 100
N.D
N.D
N.D
> 100
N.D
46.02± 5.28
24.48± 3.64
N.D
5-Br
N.D
22.99 ± 10.4
N.D
N.D
N.D
7j
4-CH3
4-Cl
N.D
> 100
N.D
N.D
7k
7l
N.D
N.D
> 100
N.D
N.D
4-Br
N.D
> 100
> 100
> 100
> 100
N.D
N.D
> 100
7m
7n
7o
7p
7q
7r
4-CH3
4-Cl
N.D
N.D
> 100
N.D
N.D
N.D
N.D
N.D
N.D
N.D
4-Br
N.D
N.D
> 100
> 100
> 100
>100
N.D
N.D
4-CH3
4-Cl
N.D
> 100
74.02 ± 14.07
>100
N.D
N.D
N.D
>100
N.D
75.51± 5.41
74.77± 6.37
> 100
4-Br
N.D
> 100
28.86 ± 9.72
27.15 ± 6.50
>100
> 100
37.20 ± 7.53
N.D
N.D
7s
H
N.D
87.60 ± 2.23
N.D
N.D
7t
H
N.D
N.D
N.D
7u
7v
7w
7x
5-FU
H
N.D
> 100
15.27 ± 6.84
> 100
N.D
N.D
N.D
3-Cl,5-Cl,6-Cl
3-Cl,5-Cl,6-Cl
3-Cl,5-Cl,6-Cl
/
N.D
N.D
N.D
N.D
N.D
N.D
N.D
> 100
> 100
N.D
N.D
N.D
N.D
N.D
> 100
N.D
N.D
29.27±2.60
45.51 ± 13.31
17.11 ± 2.64
31.96 ± 7.93
46.82±4.90
> 100
N.D. = not detected.
2.3. Colony formation
The colony formation rate means that the ability of a single cell-independent viability in vitro.
Therefore, to assess the inhibition efficiency of compound 7f, the HGC-27 cells were treated with

the candidate compound 7f at different consistency (0, 2, 4, 8 μM). Then the comparison of the
inhibition activity data (Fig. 2), manifested that compound 7f at a concentration of 8 μM markedly
inhibited colony formation by HGC-27 cells.
Fig. 2. Inhibition of colony formation on HGC-27 cells by compound 7f.
2.4. Cell migration
The wound healing assay is a laboratory technique to determine the capability of the cell migration
and interactions. For this assay, the HGC-27 cells were conducted with different concentrations of
7f (0, 4, 8 and 16 μM) for 48 h to investigate if compound 7f could influence the migration of the
cell line after an injury stimulus. As shown in Fig. 3, compared with the control group, compound
7f inhibited migration of HGC-27 cells in a concentration-dependent manner.

Fig. 3. (A) Inhibition of the migration of HGC-27 cells treated with compound 7f for 48 h in the wound healing
assay. (B) Wound closure rate was calculated as mean ± SD from three different fields. *p < 0.05, **p < 0.01 and
***p < 0.001 vs. control.
2.5. Cell apoptosis
The significance of apoptosis is to building a natural balance between cell death and cell renewal in
the multicellular creature. Thence, tumor cells can divide ruthlessly without programmed cell death,
when there is a lack of apoptosis [34]. To define whether the antiproliferative effect of compound
7f on tumor cells was as a result of induction of cell apoptosis, the HGC-27 cells were treated with
compound 7f at different concentrations (0, 4, 8, and 16 μM) for 48 h, Hoechst 33258 staining was
used to differentiate between the apoptotic and normal cells. As shown in Fig. 4, control cells were
consistently stained with Hoechst 33258 and presented round homogeneous nuclei, without
morphological changes and emitted blue fluorescence. While HGC-27 cells treated with compound
7f exhibited bright chromatin condensation, nuclear fragmentation and displayed stronger blue

fluorescence, which was a sign of apoptosis. The stain results certified that compound 7f induced
HGC-27 cells apoptosis.
Fig. 4. Effect of compound 7f treatment for 48 h on the morphology of the HGC-27 cells stained with Hoechst
33258.
Additionally, the percentage of apoptotic cells was determined by double-stained with FITC-
Annexin V and PI staining by the flow cytometry. As indicated in Fig. 5, the percentage of apoptotic
cells increased as the concentration of the compound increased (0 μM, 5.58%; 4 μM, 11.55%; 8
μM, 29%; 16 μM, 54.7%). The results showed that compound 7f could induce HGC-27 cells
apoptosis in a concentration-dependent manner.

Fig. 5. (A) The apoptosis of HGC-27 cells was induced by compound 7f for 48 h in a concentration-dependent
manner. (B) Flow cytometry analysis of the percentage of early apoptosis and late apoptosis in triplicate and repeated
three times. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control.
2.6. Cell cycle distribution
Besides apoptosis, inducing cell cycle arrest are an important means to control growth and
development of cancer cells [35]. Cell-cycle dysregulation is an important feature of tumor cells
[36]. To investigate the possible significance of cell cycle arrest in the HGC-27 cell death induced
by compound 7f, cell cycle proportion was measured by flow cytometry. HGC-27 cells were treated
with different concentrations of compound 7f (0, 4, 8 and 16 μM) for 48 h and stained with
propidium iodide (PI), to detect cell cycle distribution in the different phases by the flow cytometry
analysis. As given in Fig. 6, with the increase of drug concentration, the percentage of cells in G2
phase increased. Compared with the control group, the percentage of cells in S phase change of the
drug group showed a tendency to decrease. The experimental results indicated that HGC-27 cells
proliferation were suppressed mainly through induction of G2/M cell cycle arrest under the
inhibiting effect of compound 7f in a significant concentration-dependent manner.

Fig. 6. Flow cytometry analysis of cell cycle distribution for HGC-27 cells treated with compound 7f for 48 h. (A)
Representative photographs from three independent experiments were showed. (B) Cell cycle proportion are the
mean ± SD from three independent experiments. *p<0.05, **p<0.01 vs control.
2.7. Microtubules disarrangement
Microtubule network perturbation can cause subsequent mitotic arrest [37]. To investigate the
effects of compound 7f on the cellular microtubule network in HGC-27 cells, in-vitro tubulin
morphological analysis was determined with immunofluorescence staining via confocal microscopy.
As shown in Fig. 7, in the negative control, the majority of HGC-27 cells displayed the normal
distribution of microtubules. However, disordered microtubule distribution was observed around
the cells after treatment with compound 7f. Tubulin appeared agregated and disappeared in the
presence of compound 7f. 64 μM compound 7f had the most severe effect on microtubules
organization, the network almost completely disappeared. What’s more, according to the results of
cell apoptosis analysis and cell cycle analysis, it was speculated that compound 7f inhibited the
assembly of intracellular tubulin and disrupted microtubule dynamic, thereby arresting cells in

G2/M phase, eventually cancer cells underwent apoptosis [21-23,35].
Fig. 7. Cy3 proliferation of HGC-27 cells cells treated with compound 7f at 0, 4 and 64 μM for 48 h, respectively.
HGC-27 cells were treated as indicated and stained for Cy3 incorporation (red) or DAPI (blue) to highlight nuclei,
and the red and blue images were merged.
2.8. Docking studies with tubulin
The structure and ligand-based collaborative method is being applied to determine the relationship
between structure and its biological activity. Tubulin has been confirmed as a new significant anti-
cancer drug target [29]. To get a deeper insight into the inhibition of HGC-27 cells by the screened
compound 7f from for ligand-protein interactions, we conducted the molecular docking simulation
with the crystal structure of the DAMA–colchicine–tubulin complex (Protein Data Bank (PDB) ID:
1SA0). And the docking studies for reference drug (colchicine) be carried out as comparison. As
illustrated in Fig. 8, compound 7f occupied the colchicine binding site on tubulin, the binding site
existed between the α1-tubulin and β1-tubulin subunit (Fig. 8A). Furthermore, docking simulations
demonstrated that compound 7f formed strong interactions with the catalytically active residues
SER-140, GLY-146, THR-145 and ASN-101 via hydrogen-bonding. A hydrogen bond was formed

between the OH of SER-140 and oxygen atom of A-ring C5 on flavonoid structure of compound
7f. A hydrogen bond was formed between the NH2 of GLY-146 and oxygen atom of A-ring C6 on
flavonoid structure of compound 7f. Similarly, the NH2 of ASN-101 formed a hydrogen bonds with
the oxygen atom of C-ring O1 on flavonoid structure of compound 7f. In addition, the NH2 of THR-
145 formed two additional hydrogen bonds with the two oxygen atoms of A-ring C7 and A-ring C6
on flavonoid structure of compound 7f, respectively (Fig. 8B). Simultaneously. the colchicine binds
well in the colchicine-binding domain at the α/β-tubulin interface (Fig. 8C). Moreover, a hydrogen
bond was formed between the NH2 of ASN-206 and oxygen atom of one of the methoxy groups on
colchicine. And the NH2 of ASN-179 formed a hydrogen bonds with the oxygen atom of amide
bond carbonyl (Fig. 8D). The docking studies results showed that both compound 7f and the tubulin
inhibitor colchicine binds well in the colchicine-binding domain at the α/β-tubulin interface, and
formed a hydrogen bonds with the NH2 of amino acid residues at the methoxy atom positions in a
similar manner, which various hydrogen-bonding interactions appear to play a key role in the
binding mode.
Fig. 8. (A) Structure of tubulin in complex with compound 7f. (B) Hydrogen bonding interactions were displayed in
yellow dashed lines. (C) Structure of tubulin in complex with colchicine. (D) Hydrogen bonding interactions were
displayed in yellow dashed lines. Docking mode of compound 7f and colchicine with the colchicine binding domain
of tubulin (PDB code: 1SA0). The figures were engendered using PyMOL (http://www.pymol.org/).

2.9. Lactate level
Lactate usually as a bio-marker and even as a therapeutic target [38]. From a metabolic standpoint,
the most distinctive feature of cancer cells is enhanced lactate production due to the increased
glycolytic activity which correlates with high glucose uptake, regardless of oxygen availability
[39,40]. Studies have shown that, on one hand, proliferating cancer cells can enhance the production
of lactic acid. On the other hand, the lactate exported to the extracellular space contributes to the
acidification of micro-environment and remodeling of extracellular-matrix promoting tumor
dissemination, and can also be taken up by neighboring cells and used as a metabolic fuel enhancing
cell survival and proliferation [41]. Thence, to examine the ability of compound 7f to suppress
generation of lactic acid in cancer cells, the HGC-27 cell were cultured with various concentrations
(0, 4, 8 and 16 μM) of compound 7f, and then treated followed the steps in the lactic acid test kit.
As summarized in Fig. 9, the lactic acid content of the cell culture medium showed a decreasing
trend as the concentration of the target compound was increased, demonstrating that the target
compound 7f restrained the production of lactate in a manner of concentration dependence.
Fig. 9. Inhibition of lactate level of HGC-27 cells treated with compound 7f in the lactic acid test kit in triplicate
and repeated three times. ***p<0.001 vs control.
2.10. Effects on glycolysis related proteins
Tumor hypoxia is a common characteristic of many solid tumors [42]. Hypoxia-inducible factor 1
(HIF-1) is the critical transcription factor in the regulation of hypoxia. HIF-1 is closely related to
the occurrence and development of tumors, such as angiogenesis, glycolysis, metastasis, cell

survival and apoptosis. Hexokinase (HK), pyruvate kinase (PK) and phosphofructokinase (PFK) are
the key rate-limiting enzyme in glycolysis, and also are the target gene of HIF-1α regulation [43-
45]. Additionally, HIF-1α functions as the key regulator in the process of angiogenesis via the
activation of HIF-1α target genes such as VEGF [46]. So far, accumulating evidence concerns
flavonoids as HIF-1α inhibitors for drug therapy [47,48]. Corresponding research have manifested
that ASA and SA inhibited purified PFK due to the modulation of the enzyme quaternary structure
[33]. TMP is considered to be an important pharmacophoric point with tubulin binding for CBSIs.
Hence, 5,6,7-trimethoxy flavonoid salicylate derivatives were designed by the joining of three
important pharmacophores (TMP, flavonoid, and SA) according to the combination principle. The
aim was to screen out compounds that simultaneously inhibited tumor glycolysis, established blood
vessels and angiogenesis, completely cut off the energy source of tumor cells to accelerate tumor
cell death. To investigate whether compound 7f could affect the expression levels of the
aforementioned proteins, correspondent western blot assays were conducted, and subsequent results
were shown in Fig. 10. Compound 7f down-regulated the expression of HIF-1α under hypoxic
conditions, which was consistent with design ideas of 5,6,7-trimethoxy flavonoid salicylate
derivatives. What’s more, compound 7f down-regulated the expression of VEGF, PFKM and PKM2
protein apart from HK-2 in normoxia conditions. These results are basically consistent with the
goals we expect to design, but the specific mechanism of how compound 7f acts on multiple related
proteins in HGC-27 cell line needs further in-depth mechanistic studies.

Fig. 10. HIF-1α, VEGF, PFKM, PKM2 and HK2 expression was detected by Western blot analysis. (A) The effect
of compound 7f treatment on the expression of VEGF, PFKM, PKM2 and HK2 under normoxic conditions. (B)
Quantification of the Western blot result in Fig.10A was conducted by calculating the ratio of the value to the control
group. (C) CoCl2 hypoxia-mimicking conditions and serum starvation (0.1% FBS) stimulated the expression of HIF-
1α. The effect of compound 7f treatment on the expression of HIF-1α under hypoxic conditions. (D) Quantification
of the Western blot result in Fig.10C was conducted by calculating the ratio of the value to the control group. All
the data were confirmed by three repeated tests. *p < 0.05; **p < 0.01; ***p < 0.001 vs. control. ###p < 0.001 vs.
CoCl2 without compound 7f.
3. Conclusion
Up till now, 24 novel trimethoxy flavonoid salicylate derivatives were designed and synthesized
entirely and evaluated their cytotoxicity against five human cancer cell lines (MGC-803, HCT-116,
HGC-27, SGC-7901 and BEL-7402) and a normal human gastric epithelial cell line (GES-1).
According to the preliminary activity evaluation results, compound 7f seemed to be the most
prospective agents against the HGC-27 cell line. Subsequent the colony formation assay and wound
healing assay indicated that compound 7f effectively decreased the HGC-27 cell colony formation

and migration. Besides that, the Hoechst 33258 staining assay and flow cytometry also showed that
compound 7f induced the HGC-27 cells apoptosis, while it arrested HGC-27 cells in the G2/M phase
in a concentration-dependent manner. What’s more, the immunofluorescence staining analysis and
molecular docking simulation exhibited that compound 7f bound with the tubulin of cancer cells
and destroyed the microtubule network which could cause subsequent mitotic arrest. Finally, the
lactate measurements and western blot analysis findings revealed that compound 7f reduced the
production of lactic acid possibly by down-regulateing the expression of the glycolysis-related
proteins (HIF-1α, PFKM and PKM2). In addition, compound 7f decreased the level of angiogenesis-
related proteins VEGF, suggesting that compound 7f might inhibit the tumor angiogenesis. Taken
together, compound 7f as a representative compound possessed a good anti-tumor effect. And our
research might provide a new idea for design in gastric cancer medical drug. However, the specific
mechanism of action of compound 7f still needs further research.
4. Experimental protocol
4.1. Chemicals and reagents
All the starting materials and reagents (reagent grade) were purchased from commercial sources
unless otherwise and used without further purification. Thin-layer chromatography (TLC) was run
on the silica gel 60 F254 plates and visualized under UV light at 254 nm or 365 nm, which were
used to monitor reaction’s progress. The concentration of solutions from reactions and extractions
in a rotary evaporator below 60 °C at reduced pressure. Separation of the compounds by flash
column chromatography was carried out with silica gel (200 mesh size). Melting points (uncorrected)
were measured with an electrothermal digital melting point apparatus (Thermo Fisher Scientific).
ESI mass spectra were measured on a mass spectrometer (Bruker) in the indicated solvents (DHB
as a matrix). NMR spectra were carried out on a spectrometer (Bruker) at 400 MHz in the indicated
solvents (TMS as an internal standard): the values of the chemical shifts were expressed in δ values
(ppm) and the coupling constants (J) in Hz.
4.1.1. General procedure for the synthesis of 2-(4-hydroxy-phenyl)-5,6,7-trimethoxy-
chromen-4-one (compound 3)
To a solution of 3,4,5-trimethoxy-phenol (1) (0.020 mol) in dichloromethane (100 mL),
chloroacetyl chloride (0.024 mol) and a catalytic amount of aluminum trichloride were added,

followed by heating at 40 °C for 1.5 h with calcium chloride drying device，and monitored by TLC.
After refrigeration, filtration, washing and drying, obtaining a flaky white precipitate of 6-Hydroxy-
2,3,4-trimethoxy-benzoyl chloride (2) was obtained. Para-hydroxybenzaldehyde (0.016 mol) and
intermediate 2 (0.013 mol) were added together to a stirred solution of 5 mL 95% ethanol, then 10%
NaOH (aq.) solution was added dropwise into the solution for 24 h at RT. Finally, acidification with
10% HCl (aq.) gave a crude product, which was filtered off and purified by recrystallization from
95% ethanol to give compound 3 as a yellow powder.
4.1.2. General procedure for the synthesis of compounds 4a-4c
To a stirring solution of compound 3 (0.5 mol) in acetone (50 mL), a certain amount of K2CO3 at
room temperature and 1, 2 dibromoethane (2.0 mol) were added dropwise to the reaction mixture,
refluxed at 60 °C for 9 h (the reaction was monitored by TLC). After the completion of the reaction,
the solvent was evaporated under vacuum. The residue was purified by flash column
chromatography on silica gel, eluted with dichloromethane/methanol (v/v, 100 : 1) to get the
intermediate 4a as a yellow powder. Compounds 4b and 4c were prepared in the same way, except
that 1, 3 dibromopropane and 1, 4 dibromobutane were used as the reactants, respectively.
4.1.2.1. 2-[4-(2-Bromo-ethoxy)-phenyl]-5,6,7-trimethoxy-chromen-4-one(compound
4a)
Compound 4a was obtained as yellow solid; yield: 85.5%; m.p: 143.6-144.2 °C; 1H NMR (400 MHz,
CDCl3): δ (ppm) 7.88 (d, J = 8.6 Hz, 2H), 7.01 (d, J = 8.6 Hz, 2H), 6.79 (s, 1H), 6.59 (s, 1H), 4.39
(t, J = 6.2 Hz, 2H), 4.30 (s, 3H), 4.03 (s, 3H), 3.87 (s, 3H), 3.71 (t, J = 6.2 Hz, 2H). MS (ESI) m/z:
435.196 [M + H]+, calcd. for C20H19BrO6 434.037.
4.1.2.2. 2-[4-(3-Bromo-propoxy)-phenyl]-5,6,7-trimethoxy-chromen-4-one(compound
4b)
1
Compound 4b was obtained as yellow solid; yield: 82.7%; m.p: 117.0-117.9 °C; H NMR (400
MHz, CDCl3): δ (ppm) 7.89 – 7.80 (m, 2H), 7.02 – 6.95 (m, 2H), 6.77 (s, 1H), 6.57 (s, 1H), 4.28
(s, 3H), 4.23 – 4.15 (m, 2H), 4.00 (s, 3H), 3.86 (d, J = 9.8 Hz, 3H), 3.64 (t, J = 6.4 Hz, 2H), 2.44 –
2.29 (m, 2H). MS (ESI) m/z: 449.228 [M + H]+, calcd. for C21H21BrO6 448.052.
4.1.2.3. 2-[4-(4-Bromo-butoxy)-phenyl]-5,6,7-trimethoxy-chromen-4-one(compound
4c)
Compound 4c was obtained as yellow solid; yield: 79.7%; m.p: 117.2-118.2 °C; 1H NMR (400 MHz,

CDCl3): δ (ppm) 7.92 – 7.80 (m, 2H), 7.04 – 6.93 (m, 2H), 6.79 (s, 1H), 6.59 (d, J = 2.4 Hz, 1H),
4.30 (d, J = 2.6 Hz, 3H), 4.10 (dd, J = 7.6, 4.1 Hz, 2H), 4.02 (s, 3H), 3.87 (d, J = 2.5 Hz, 3H), 3.54
(dd, J = 8.5, 4.4 Hz, 2H), 2.16 – 1.98 (m, 4H). MS (ESI) m/z: 463.300 [M + H]+, calcd. for
C22H23BrO6 462.068.
4.1.3. General procedure for the synthesis of intermediates 6a-6h
Methyl 4-bromosalicylate (1 mol) was dissolved in methanol (25 mL), and thionyl chloride was
added dropwise under ice-cooling, after 10 min, The mixture was stirred at 65 °C for 48 h. and
monitored by TLC. After the reaction was completed, excess methanol was evaporated, the residue
was extracted with ethyl acetate and aqueous sodium carbonate, and the organic phase was added
to anhydrous sodium sulfate and concentrated to obtain intermediate 6a.
The compounds 6b–6h were prepared analogously.
4.1.4. General procedure for the synthesis of compounds 7a–7x.
The intermediates 6a and 4a were dissolved in acetone (30 mL), and a catalytic amount of anhydrous
potassium carbonate was added to this solution at 60 °C for 48 h (the reaction was monitored by
TLC). After completion of the reaction, the solvent was evaporated, the residue was extracted with
ethyl acetate and water, the organic phase was treated with anhydrous sodium sulfate, and then
concentrated. The final target product 7a was purified by flash column chromatography on silica
gel, and eluted with a solution of methanol and dichloromethane (v/v, 1 : 100) and recrystallized
from anhydrous ethanol.
Compounds 7b–7x were prepared similarly.
4.1.4.1.
5-Methyl-2-{4-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
butoxy}-benzoic acid methyl ester (compound 7a)
Compound 7a was obtained as yellow solid; yield: 88.9%; m.p: 113.5-114.8 °C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.86 (d, J = 8.8 Hz, 2H), 7.64 (d, J = 1.9 Hz, 1H), 7.27 (d, J = 2.2 Hz, 1H), 7.00
(d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.5 Hz, 1H), 6.79 (s, 1H), 6.59 (s, 1H), 4.30 (s, 3H), 4.14 (q, J = 5.6
Hz, 4H), 4.02 (s, 3H), 3.91 (s, 3H), 3.86 (s, 3H), 2.34 (s, 3H), 2.13 – 2.03 (m, 4H). 13C NMR (101
MHz, CDCl3): δ (ppm) 180.73 (s), 166.90 (s), 163.79 (s), 161.39 (s), 160.19 (s), 156.44 (s), 151.60
(s), 146.55 (s), 136.58 (s), 133.95 (s), 132.96 (s), 131.94 (s), 129.60 (s), 125.10 (s), 120.03 (s),
114.91 (s), 113.37 (s), 111.56 (s), 107.30 (s), 90.54 (s), 68.54 (s), 67.63 (s), 62.41 (s), 61.62 (s),

56.60 (s), 51.87 (s), 25.94 (d, J = 2.1 Hz), 20.28 (s). MS (ESI) m/z: 549.11 [M + H]+, calcd. for
C31H32O9 548.20.
4.1.4.2.
5-Chloro-2-{4-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
butoxy}-benzoic acid methyl ester (compound 7b)
Compound 7b was obtained as yellow solid; yield: 68.2%; m.p: 136.8-138.5 °C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.86 (d, J = 8.8 Hz, 2H), 7.82 (d, J = 2.7 Hz, 1H), 7.45 (dd, J = 8.9, 2.7 Hz, 1H),
6.98 (dd, J = 15.2, 8.9 Hz, 3H), 6.80 (s, 1H), 6.60 (s, 1H), 4.30 (s, 3H), 4.16 (d, J = 2.9 Hz, 4H),
4.03 (s, 3H), 3.92 (s, 3H), 3.87 (s, 3H), 2.10 (s, 4H). 13C NMR (101 MHz, CDCl3): δ (ppm) 180.74
(s), 165.42 (s), 163.79 (s), 161.41 (s), 160.10 (s), 157.15 (s), 151.60 (s), 146.58 (s), 136.59 (s),
133.11 (s), 132.96 (s), 131.38 (s), 125.19 (d, J = 2.9 Hz), 121.49 (s), 114.89 (s), 114.53 (s), 111.51
(s), 107.30 (s), 90.54 (s), 68.76 (s), 67.53 (s), 62.41 (s), 61.63 (s), 56.61 (s), 52.15 (s), 25.85 (d, J =
3.8 Hz). MS (ESI) m/z: 569.02 [M + H]+, calcd. for C30H29ClO9 568.15.
4.1.4.3.
5-Bromo-2-{4-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
butoxy}-benzoic acid methyl ester (compound 7c)
Compound 7c was obtained as yellow solid; yield: 64.8%; m.p: 137.9-139.5 °C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.96 (d, J = 2.5 Hz, 1H), 7.86 (d, J = 8.7 Hz, 2H), 7.58 (dd, J = 8.8, 2.5 Hz, 1H),
6.99 (d, J = 8.7 Hz, 2H), 6.91 (d, J = 8.9 Hz, 1H), 6.80 (s, 1H), 6.60 (s, 1H), 4.30 (s, 3H), 4.15 (d, J
= 3.4 Hz, 4H), 4.03 (s, 3H), 3.92 (s, 3H), 3.87 (s, 3H), 2.10 (s, 4H). 13C NMR (101 MHz, CDCl3):
δ (ppm) 180.71 (s), 165.27 (s), 163.78 (s), 161.40 (s), 160.09 (s), 157.63 (s), 151.59 (s), 146.57 (s),
136.59 (s), 136.03 (s), 134.24 (s), 132.96 (s), 125.16 (s), 121.91 (s), 114.90 (d, J = 3.7 Hz), 112.21
(s), 111.48 (s), 107.28 (s), 90.54 (s), 68.68 (s), 67.52 (s), 62.41 (s), 61.62 (s), 56.61 (s), 52.15 (s),
25.85 (s). MS (ESI) m/z: 612.97 [M + H]+, calcd. for C30H29BrO9 612.10.
4.1.4.4.
5-Methyl-2-{3-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
propoxy}-benzoic acid methyl ester (compound 7d)
Compound 7d was obtained as yellow solid; yield: 45.3%; m.p: 107.5-109.0 °C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.86 (d, J = 8.8 Hz, 2H), 7.64 (d, J = 2.0 Hz, 1H), 7.28 (d, J = 1.9 Hz, 1H), 7.02
(d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.5 Hz, 1H), 6.79 (s, 1H), 6.59 (s, 1H), 4.33 (t, J = 6.1 Hz, 2H),
4.30 (s, 3H), 4.26 (t, J = 5.9 Hz, 2H), 4.02 (s, 3H), 3.90 (s, 3H), 3.87 (s, 3H), 2.40 – 2.32 (m, 5H).
13C NMR (101 MHz, CDCl3): δ (ppm) 180.73 (s), 166.80 (s), 163.79 (s), 161.39 (s), 160.08 (s),
156.32 (s), 151.59 (s), 146.57 (s), 136.58 (s), 133.99 (s), 132.97 (s), 131.95 (s), 129.82 (s), 125.21

(s), 120.08 (s), 114.95 (s), 113.53 (s), 111.51 (s), 107.29 (s), 90.54 (s), 65.40 (s), 64.55 (s), 62.40
(s), 61.62 (s), 56.60 (s), 51.88 (s), 29.21 (s), 20.28 (s). MS (ESI) m/z: 535.09 [M + H]+, calcd. for
C30H30O9 534.19.
4.1.4.5.
5-Chloro-2-{3-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
propoxy}-benzoic acid methyl ester (compound 7e)
Compound 7e was obtained as yellow solid; yield: 62.8%; m.p: 154.0-154.5 °C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.86 (d, J = 8.8 Hz, 2H), 7.81 (d, J = 2.7 Hz, 1H), 7.44 (dd, J = 8.9, 2.7 Hz, 1H),
7.00 (dd, J = 16.2, 8.9 Hz, 3H), 6.79 (s, 1H), 6.58 (s, 1H), 4.32 (t, J = 6.1 Hz, 2H), 4.30 (s, 3H), 4.27
(t, J = 5.9 Hz, 2H), 4.02 (s, 3H), 3.91 (s, 3H), 3.86 (s, 3H), 2.37 (p, J = 5.9 Hz, 2H). 13C NMR (101
MHz, CDCl3): δ (ppm) 180.73 (s), 165.30 (s), 163.79 (s), 161.42 (s), 159.96 (s), 157.04 (s), 151.60
(s), 146.60 (s), 136.59 (s), 133.15 (s), 132.97 (s), 131.39 (s), 125.40 (s), 125.31 (s), 121.51 (s),
114.93 (s), 114.69 (s), 111.43 (s), 107.28 (s), 90.54 (s), 65.59 (s), 64.30 (s), 62.40 (s), 61.62 (s),
56.61 (s), 52.16 (s), 29.08 (s). MS (ESI) m/z: 555.11 [M + H]+, calcd. for C29H27ClO9 554.13.
4.1.4.6.
5-Bromo-2-{3-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
propoxy}-benzoic acid methyl ester (compound 7f)
Compound 7f was obtained as yellow solid; yield: 80.9%; m.p: 160.5-162.8 °C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.92 (d, J = 2.6 Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.55 (dd, J = 8.9, 2.6 Hz, 1H),
6.99 (d, J = 8.8 Hz, 2H), 6.90 (d, J = 8.9 Hz, 1H), 6.76 (s, 1H), 6.56 (s, 1H), 4.29 (t, J = 6.0 Hz,
2H), 4.27 (s, 3H), 4.24 (t, J = 5.9 Hz, 2H), 3.99 (s, 3H), 3.88 (s, 3H), 3.84 (s, 3H), 2.34 (p, J = 5.9
Hz, 2H).13C NMR (101 MHz, CDCl3): δ (ppm) 180.71 (s), 165.16 (s), 163.79 (s), 161.41 (s), 159.95
(s), 157.53 (s), 151.61 (s), 146.60 (s), 136.60 (s), 136.07 (s), 134.27 (s), 132.97 (s), 125.32 (s),
121.94 (s), 115.08 (s), 114.93 (s), 112.43 (s), 111.41 (s), 107.28 (s), 90.54 (s), 65.52 (s), 64.29 (s),
62.41 (s), 61.62 (s), 56.61 (s), 52.16 (s), 29.06 (s). MS (ESI) m/z: 601.76 [M + H]+, calcd. for
C29H27BrO9 598.08.
4.1.4.7.
5-Methyl-2-{2-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
ethoxy}-benzoic acid methyl ester (compound 7g)
Compound 7g was obtained as yellow solid; yield: 63.1%; m.p: 172.5-175.6 °C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.88 (d, J = 8.8 Hz, 2H), 7.65 (d, J = 1.8 Hz, 1H), 7.34 – 7.31 (m, 1H), 7.06 (d, J
= 8.8 Hz, 2H), 7.00 (d, J = 8.4 Hz, 1H), 6.80 (s, 1H), 6.59 (s, 1H), 4.46 (d, J = 8.9 Hz, 4H), 4.30 (s,
3H), 4.03 (s, 3H), 3.88 (s, 3H), 3.87 (s, 3H), 2.36 (s, 3H). 13C NMR (101 MHz, CDCl3): δ (ppm)

180.74 (s), 166.80 (s), 163.82 (s), 161.45 (s), 159.77 (s), 156.12 (s), 151.60 (s), 146.67 (s), 136.60
(s), 133.97 (s), 132.96 (s), 131.92 (s), 130.71 (s), 125.59 (s), 120.95 (s), 115.11 (s), 114.92 (s),
111.35 (s), 107.26 (s), 90.55 (s), 68.36 (s), 66.70 (s), 62.40 (s), 61.62 (s), 56.61 (s), 51.96 (s), 20.34
(s). MS (ESI) m/z: 521.07 [M + H]+, calcd. for C29H28O9 520.17.
4.1.4.8.
5-Chloro-2-{2-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
ethoxy}-benzoic acid methyl ester (compound 7h)
Compound 7h was obtained as yellow solid; yield: 67.2%; m.p: 139.0-140.5 °C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.88 (d, J = 8.8 Hz, 2H), 7.82 (d, J = 2.7 Hz, 1H), 7.47 (dd, J = 8.9, 2.7 Hz, 1H),
7.05 (d, J = 8.8 Hz, 3H), 6.79 (s, 1H), 6.59 (s, 1H), 4.46 (s, 4H), 4.30 (s, 3H), 4.02 (s, 3H), 3.88 (s,
3H), 3.87 (s, 3H). 13C NMR (101 MHz, CDCl3): δ (ppm) 180.73 (s), 165.32 (s), 163.81 (s), 161.47
(s), 159.60 (s), 156.83 (s), 151.61 (s), 146.71 (s), 136.61 (s), 133.11 (s), 132.96 (s), 131.36 (s),
126.21 (s), 125.72 (s), 122.41 (s), 115.98 (s), 115.07 (s), 111.23 (s), 107.23 (s), 90.55 (s), 68.44 (s),
66.55 (s), 62.40 (s), 61.62 (s), 56.62 (s), 52.24 (s). MS (ESI) m/z: 541.12 [M + H]+, calcd. for
C28H25ClO9 540.12.
4.1.4.9. 5-Bromo-2-{2-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-ethoxy}-
benzoic acid methyl ester (compound 7i)
Compound 7i was obtained as yellow solid; yield: 52.3%; m.p: 160.5-162.8 °C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.96 (d, J = 2.6 Hz, 1H), 7.88 (d, J = 8.8 Hz, 2H), 7.61 (dd, J = 8.8, 2.6 Hz, 1H),
7.05 (d, J = 8.8 Hz, 2H), 7.00 (d, J = 8.9 Hz, 1H), 6.80 (s, 1H), 6.59 (s, 1H), 4.46 (s, 4H), 4.30 (s,
3H), 4.03 (s, 3H), 3.88 (s, 3H), 3.87 (s, 3H). 13C NMR (101 MHz, CDCl3): δ (ppm) 180.70 (s),
165.19 (s), 163.81 (s), 161.46 (s), 159.59 (s), 157.33 (s), 151.64 (s), 146.73 (s), 136.64 (s), 136.02
(s), 134.25 (s), 132.94 (s), 125.76 (s), 122.88 (s), 116.35 (s), 115.09 (s), 113.31 (s), 111.18 (s),
107.25 (s), 90.54 (s), 68.37 (s), 66.55 (s), 62.39 (s), 61.61 (s), 56.61 (s), 52.22 (s), 29.69 (s). MS
(ESI) m/z: 584.94 [M + H]+, calcd. for C28H25BrO9 584.07.
4.1.4.10.
4-Methyl-2-{4-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
butoxy}-benzoic acid methyl ester (compound 7j)
Compound 7j was obtained as yellow solid; yield: 58.0%; m.p: 109.0-112.2°C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.82 (d, J = 8.1 Hz, 2H), 7.72 (d, J = 7.8 Hz, 1H), 6.96 (d, J = 8.1 Hz, 2H), 6.78
(t, J = 8.7 Hz, 3H), 6.55 (s, 1H), 4.26 (s, 3H), 4.12 (d, J = 4.8 Hz, 4H), 3.98 (s, 3H), 3.86 (s, 3H),
3.82 (s, 3H), 2.37 (s, 3H), 2.06 (s, 4H). 13C NMR (101 MHz, CDCl3): δ (ppm) 180.72 (s), 166.62

(s), 163.78 (s), 161.39 (s), 160.19 (s), 158.75 (s), 151.59 (s), 146.55 (s), 144.52 (s), 136.58 (s),
132.96 (s), 131.86 (s), 125.10 (s), 121.05 (s), 117.23 (s), 114.91 (s), 113.93 (s), 111.54 (s), 107.29
(s), 90.54 (s), 68.25 (s), 67.62 (s), 62.40 (s), 61.61 (s), 56.60 (s), 51.72 (s), 25.93 (s), 21.90 (s). MS
(ESI) m/z: 549.06 [M + H]+, calcd. for C31H32O9 548.20.
4.1.4.11.
4-Chloro-2-{4-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
butoxy}-benzoic acid methyl ester (compound 7k)
Compound 7k was obtained as yellow solid; yield: 73.7%; m.p: 123.5-125.5 °C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.86 (d, J = 8.8 Hz, 2H), 7.83 – 7.79 (m, 1H), 7.00 (dd, J = 7.5, 4.1 Hz, 4H), 6.80
(s, 1H), 6.59 (s, 1H), 4.30 (s, 3H), 4.16 (d, J = 1.6 Hz, 4H), 4.02 (s, 3H), 3.91 (s, 3H), 3.87 (s, 3H),
2.11 (s, 4H). 13C NMR (101 MHz, CDCl3): δ (ppm) 180.76 (s), 165.81 (s), 163.80 (s), 161.41 (s),
160.10 (s), 159.23 (s), 151.62 (s), 146.58 (s), 139.32 (s), 136.60 (s), 132.97 (s), 132.86 (s), 125.18
(s), 120.43 (s), 118.56 (s), 114.90 (s), 113.64 (s), 111.54 (s), 107.31 (s), 90.54 (s), 68.66 (s), 67.50
(s), 62.42 (s), 61.63 (s), 56.61 (s), 53.45 (s), 52.01 (s), 25.82 (d, J = 3.8 Hz). MS (ESI) m/z: 568.96
[M + H]+, calcd. for C30H29ClO9 568.15.
4.1.4.12.
4-Bromo-2-{4-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
butoxy}-benzoic acid methyl ester (compound 7l)
Compound 7l was obtained as yellow solid; yield: 58.2%; m.p: 138.0-140.0 °C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.86 (d, J = 8.8 Hz, 2H), 7.74 – 7.70 (m, 1H), 7.18 – 7.15 (m, 2H), 7.00 (d, J = 8.8
Hz, 2H), 6.79 (s, 1H), 6.59 (s, 1H), 4.30 (s, 3H), 4.16 (d, J = 1.3 Hz, 4H), 4.02 (s, 3H), 3.90 (s, 3H),
3.87 (s, 3H), 2.10 (s, 4H). 13C NMR (101 MHz, CDCl3): δ (ppm) 180.73 (s), 165.90 (s), 163.79 (s),
161.40 (s), 160.10 (s), 159.10 (s), 151.61 (s), 146.57 (s), 136.59 (s), 132.97 (s), 127.63 (s), 125.18
(s), 123.42 (s), 119.03 (s), 116.60 (s), 114.90 (s), 111.51 (s), 107.30 (s), 90.54 (s), 68.69 (s), 67.49
(s), 62.42 (s), 61.63 (s), 56.61 (s), 52.02 (s), 25.80 (s). MS (ESI) m/z: 613.03 [M + H]+, calcd. for
C30H29BrO9 612.10.
4.1.4.13.
4-Methyl-2-{3-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
propoxy}-benzoic acid methyl ester (compound 7m)
Compound 7m was obtained as yellow solid; yield: 83.5%; m.p: 105.5-109.0 °C; 1H NMR (400
MHz, CDCl3): δ (ppm) 7.81 (d, J = 8.2 Hz, 2H), 7.72 (d, J = 7.9 Hz, 1H), 6.99 (d, J = 8.3 Hz, 2H),
6.79 (d, J = 7.4 Hz, 2H), 6.75 (s, 1H), 6.54 (s, 1H), 4.30 (t, J = 6.0 Hz, 2H), 4.25 (s, 3H), 4.23 (d, J
= 5.8 Hz, 2H), 3.98 (s, 3H), 3.85 (s, 3H), 3.81 (d, J = 7.0 Hz, 3H), 2.36 (s, 3H), 2.35 – 2.29 (m, 2H).

13C NMR (101 MHz, CDCl3): δ (ppm) 180.75 (s), 166.54 (s), 163.79 (s), 161.42 (s), 160.08 (s),
158.63 (s), 151.58 (s), 146.57 (s), 144.60 (s), 136.58 (s), 132.98 (s), 131.86 (s), 125.20 (s), 121.22
(s), 117.24 (s), 114.96 (s), 114.06 (s), 111.55 (s), 107.28 (s), 90.55 (s), 65.09 (s), 64.52 (s), 62.39
(s), 61.62 (s), 56.61 (s), 51.75 (s), 29.20 (s), 21.88 (s). MS (ESI) m/z: 535.09 [M + H]+, calcd. for
C30H30O9 534.19.
4.1.4.14.
4-Chloro-2-{3-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
propoxy}-benzoic acid methyl ester (compound 7n)
Compound 7n was obtained as yellow solid; yield: 76.7%; m.p: 120.0-121.5 °C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.86 (d, J = 8.8 Hz, 2H), 7.80 (d, J = 8.3 Hz, 1H), 7.01 (dd, J = 8.4, 5.4 Hz, 4H),
6.79 (s, 1H), 6.58 (s, 1H), 4.33 (t, J = 6.0 Hz, 2H), 4.29 (s, 3H), 4.27 (d, J = 5.9 Hz, 2H), 4.02 (s,
3H), 3.90 (s, 3H), 3.86 (s, 3H), 2.37 (dd, J = 11.9, 6.0 Hz, 2H). 13C NMR (101 MHz, CDCl3): δ
(ppm) 180.75 (s), 165.69 (s), 163.80 (s), 161.42 (s), 159.95 (s), 159.11 (s), 151.61 (s), 146.60 (s),
139.36 (s), 136.59 (s), 132.98 (s), 132.87 (s), 125.32 (s), 120.59 (s), 118.56 (s), 114.93 (s), 113.76
(s), 111.47 (s), 107.29 (s), 90.54 (s), 65.44 (s), 64.23 (s), 62.41 (s), 61.63 (s), 56.61 (s), 52.01 (s),
29.00 (s). MS (ESI) m/z: 555.00 [M + H]+, calcd. for C29H27ClO9 554.13.
4.1.4.15.
4-Bromo-2-{3-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
propoxy}-benzoic acid methyl ester (compound 7o)
Compound 7o was obtained as yellow solid; yield: 59.5%; m.p: 132.2-134.2 °C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.86 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 8.3 Hz, 1H), 7.20 – 7.15 (m, 2H), 7.02 (d, J
= 8.8 Hz, 2H), 6.79 (s, 1H), 6.58 (s, 1H), 4.32 (t, J = 6.0 Hz, 2H), 4.29 (s, 3H), 4.27 (d, J = 5.9 Hz,
2H), 4.02 (s, 3H), 3.89 (s, 3H), 3.86 (s, 3H), 2.38 (p, J = 5.9 Hz, 2H). 13C NMR (101 MHz, CDCl3):
δ (ppm) 180.71 (s), 165.78 (s), 163.79 (s), 161.40 (s), 159.94 (s), 158.98 (s), 151.60 (s), 146.60 (s),
136.58 (s), 132.96 (d, J = 2.6 Hz), 127.66 (s), 125.32 (s), 123.58 (s), 119.04 (s), 116.72 (s), 114.93
(s), 111.43 (s), 107.27 (s), 90.54 (s), 65.48 (s), 64.22 (s), 62.40 (s), 61.62 (s), 56.61 (s), 52.02 (s),
29.00 (s). MS (ESI) m/z: 599.11 [M + H]+, calcd. for C29H27BrO9 598.08.
4.1.4.16.
4-Methyl-2-{2-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
ethoxy}-benzoic acid methyl ester (compound 7p)
Compound 7p was obtained as yellow solid; yield: 71.7%; m.p: 178-181.5 °C; 1H NMR (400 MHz,
CDCl3): δ (ppm) 7.84 (d, J = 8.0 Hz, 2H), 7.73 (d, J = 7.7 Hz, 1H), 7.03 (d, J = 8.1 Hz, 2H), 6.85
(d, J = 9.3 Hz, 2H), 6.76 (s, 1H), 6.55 (s, 1H), 4.43 (s, 4H), 4.26 (s, 3H), 3.98 (s, 3H), 3.83 (s, 6H),

2.39 (s, 3H). 13C NMR (101 MHz, CDCl3): δ (ppm) 180.78 (s), 166.57 (s), 163.83 (s), 161.48 (s),
159.76 (s), 158.40 (s), 151.61 (s), 146.68 (s), 144.57 (s), 136.59 (s), 132.98 (s), 131.83 (s), 125.60
(s), 121.99 (s), 118.07 (s), 115.30 (s), 115.12 (s), 111.41 (s), 107.26 (s), 90.55 (s), 67.96 (s), 66.67
(s), 62.39 (s), 61.62 (s), 56.62 (s), 51.83 (s), 21.84 (s). MS (ESI) m/z: 521.07 [M + H]+, calcd. for
C29H28O9 520.17.
4.1.4.17.
4-Chloro-2-{2-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
ethoxy}-benzoic acid methyl ester (compound 7q)
Compound 7q was obtained as yellow solid; yield: 74.9%; m.p: 148.5-150.5 °C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.88 (d, J = 8.8 Hz, 2H), 7.81 (d, J = 8.3 Hz, 1H), 7.11 (d, J = 1.8 Hz, 1H), 7.08 –
7.04 (m, 3H), 6.79 (s, 1H), 6.59 (s, 1H), 4.47 (s, 4H), 4.30 (s, 3H), 4.02 (s, 3H), 3.87 (d, J = 2.5 Hz,
6H). 13C NMR (101 MHz, CDCl3): δ (ppm) 180.74 (s), 165.74 (s), 163.82 (s), 161.46 (s), 159.57
(s), 158.87 (s), 151.62 (s), 146.72 (s), 139.22 (s), 136.61 (s), 132.97 (s), 132.83 (s), 125.75 (s),
121.29 (s), 119.36 (s), 115.08 (s), 114.84 (s), 111.24 (s), 107.24 (s), 90.54 (s), 68.16 (s), 66.47 (s),
62.40 (s), 61.62 (s), 56.62 (s), 52.09 (s). MS (ESI) m/z: 540.94 [M + H]+, calcd. for C28H25ClO9
540.12.
4.1.4.18.
4-Bromo-2-{2-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
ethoxy}-benzoic acid methyl ester (compound 7r)
Compound 7r was obtained as yellow solid; yield: 47.0%; m.p: 159.6-162.0 °C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.84 (d, J = 8.1 Hz, 2H), 7.69 (d, J = 8.3 Hz, 1H), 7.24 (s, 1H), 7.18 (d, J = 8.3 Hz,
1H), 7.01 (d, J = 8.1 Hz, 2H), 6.75 (s, 1H), 6.55 (s, 1H), 4.43 (s, 4H), 4.26 (s, 3H), 3.98 (s, 3H),
3.83 (s, 6H). 13C NMR (101 MHz, CDCl3): δ (ppm) 180.72 (s), 165.83 (s), 163.82 (s), 161.45 (s),
159.56 (s), 158.75 (s), 151.63 (s), 146.72 (s), 136.61 (s), 132.93 (d, J = 6.4 Hz), 127.46 (s), 125.76
(s), 124.30 (s), 119.86 (s), 117.85 (s), 115.08 (s), 111.22 (s), 107.24 (s), 90.54 (s), 68.23 (s), 66.49
(s), 62.41 (s), 61.62 (s), 56.62 (s), 52.11 (s), 29.71 (s). MS (ESI) m/z: 585.10 [M + H]+, calcd. for
C28H25BrO9 584.07.
4.1.4.19.
2-{4-[4-(5,6,7-Trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-butoxy}-
benzoic acid methyl ester (compound 7s)
Compound 7s was obtained as yellow solid; yield: 63.3%; m.p: 120.7-121.3 °C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.91 – 7.80 (m, 3H), 7.54 – 7.44 (m, 1H), 7.03 (dd, J = 14.0, 6.6 Hz, 4H), 6.80 (d,
J = 6.6 Hz, 1H), 6.60 (d, J = 6.6 Hz, 1H), 4.33 – 4.27 (m, 3H), 4.17 (s, 4H), 4.03 (d, J = 6.6 Hz,

3H), 3.95 – 3.90 (m, 3H), 3.89 – 3.85 (m, 3H), 2.11 (s, 4H). 13C NMR (101 MHz, CDCl3): δ (ppm)
180.74 (s), 166.77 (s), 163.79 (s), 161.40 (s), 160.18 (s), 158.48 (s), 151.59 (s), 146.55 (s), 136.58
(s), 133.46 (s), 132.97 (s), 131.68 (s), 125.11 (s), 120.30 (s), 120.20 (s), 114.91 (s), 113.11 (s),
111.56 (s), 107.30 (s), 90.55 (s), 68.30 (s), 67.61 (s), 62.41 (s), 61.63 (s), 56.61 (s), 51.91 (s), 25.92
(s). MS (ESI) m/z: 535.15 [M + H]+, calcd. for C30H30O9 534.19.
4.1.4.20.
2-{3-[4-(5,6,7-Trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-propoxy}-
benzoic acid methyl ester (compound 7t)
Compound 7t was obtained as yellow solid; yield: 65.4%; m.p: 82.5-84.5 °C; 1H NMR (400 MHz,
CDCl3): δ (ppm) 7.85 (t, J = 7.8 Hz, 3H), 7.50 (t, J = 7.9 Hz, 1H), 7.03 (dd, J = 8.0, 4.0 Hz, 4H),
6.79 (s, 1H), 6.59 (s, 1H), 4.34 (t, J = 5.9 Hz, 2H), 4.29 (d, J = 6.8 Hz, 5H), 4.02 (s, 3H), 3.91 (s,
3H), 3.86 (s, 3H), 2.38 (p, J = 5.9 Hz, 2H). 13C NMR (101 MHz, CDCl3): δ (ppm) 180.74 (s), 166.65
(s), 163.79 (s), 161.40 (s), 160.05 (s), 158.36 (s), 151.59 (s), 146.58 (s), 136.58 (s), 133.50 (s),
132.98 (s), 131.70 (s), 125.23 (s), 120.36 (d, J = 4.3 Hz), 114.95 (s), 113.25 (s), 111.51 (s), 107.29
(s), 90.55 (s), 65.13 (s), 64.48 (s), 62.41 (s), 61.63 (s), 56.61 (s), 51.92 (s), 29.15 (s). MS (ESI) m/z:
521.08 [M + H]+, calcd. for C29H28O9 520.17.
4.1.4.21.
2-{2-[4-(5,6,7-Trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-ethoxy}-
benzoic acid methyl ester (compound 7u)
Compound 7u was obtained as yellow solid; yield: 69.5%; m.p: 125.0-127.0 °C;1H NMR (400 MHz,
CDCl3): δ (ppm) 7.90 – 7.82 (m, 3H), 7.55 – 7.49 (m, 1H), 7.12 – 7.04 (m, 4H), 6.80 (s, 1H), 6.59
(s, 1H), 4.47 (s, 4H), 4.30 (s, 3H), 4.02 (s, 3H), 3.89 (s, 3H), 3.86 (s, 3H). 13C NMR (101 MHz,
CDCl3): δ (ppm) 180.73 (s), 166.66 (s), 163.81 (s), 161.45 (s), 159.73 (s), 158.14 (s), 151.60 (s),
146.68 (s), 136.60 (s), 133.44 (s), 132.96 (s), 131.66 (s), 125.63 (s), 121.15 (d, J = 5.0 Hz), 115.11
(s), 114.42 (s), 111.31 (s), 107.25 (s), 90.56 (s), 67.98 (s), 66.63 (s), 62.40 (s), 61.62 (s), 56.62 (s),
51.99 (s), 29.70 (s).MS (ESI) m/z: 507.13 [M + H]+, calcd. for C28H26O9 506.16.
4.1.4.22. 2,3,5-Trichloro-6-{4-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
butoxy}-benzoic acid methyl ester (compound 7v)
Compound 7v was obtained as yellow solid; yield: 52.5%; m.p: 70.5-72.7 °C; 1H NMR (400 MHz,
CDCl3): δ (ppm) 7.87 (d, J = 8.8 Hz, 2H), 7.59 (s, 1H), 7.00 (d, J = 8.8 Hz, 2H), 6.80 (s, 1H), 6.59
(s, 1H), 4.30 (s, 3H), 4.14 (dt, J = 13.9, 5.8 Hz, 4H), 4.02 (s, 3H), 3.99 (s, 3H), 3.86 (s, 3H), 2.10 –
1.96 (m, 4H). 13C NMR (101 MHz, CDCl3): δ (ppm) 180.74 (s), 164.49 (s), 163.80 (s), 161.41 (s),

160.10 (s), 151.61 (s), 151.45 (s), 146.59 (s), 136.59 (s), 132.98 (s), 131.83 (s), 131.68 (s), 128.85
(s), 128.23 (s), 127.33 (s), 125.23 (s), 114.89 (s), 111.49 (s), 107.29 (s), 90.53 (s), 74.91 (s), 67.52
(s), 62.41 (s), 61.63 (s), 56.61 (s), 53.14 (s), 26.64 (s), 25.71 (s). MS (ESI) m/z: 637.06 [M + H]+,
calcd. for C30H27Cl3O9 636.07.
4.1.4.23. 2,3,5-Trichloro-6-{3-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
propoxy}-benzoic acid methyl ester (compound 7w)
Compound 7w was obtained as yellow solid; yield: 54.9%; m.p: 60.5-63.0 °C; 1H NMR (400 MHz,
CDCl3): δ (ppm) 7.89 (s, 2H), 7.62 (d, J = 24.8 Hz, 1H), 7.04 (s, 2H), 6.84 (d, J = 25.3 Hz, 1H),
6.63 (d, J = 25.3 Hz, 1H), 4.34 (d, J = 25.1 Hz, 7H), 4.07 (d, J = 25.1 Hz, 3H), 3.94 (t, J = 23.4 Hz,
6H), 2.31 (s, 2H). 13C NMR (101 MHz, CDCl3): δ (ppm) 180.73 (s), 164.38 (s), 163.80 (s), 161.43
(s), 159.94 (s), 151.62 (s), 151.21 (s), 146.62 (s), 136.60 (s), 132.99 (s), 131.85 (s), 131.66 (s),
129.02 (s), 128.28 (s), 127.33 (s), 125.35 (s), 114.90 (s), 111.42 (s), 107.28 (s), 90.53 (s), 71.65 (s),
64.08 (s), 62.41 (s), 61.63 (s), 56.61 (s), 53.15 (s), 29.77 (s). MS (ESI) m/z: 622.98 [M + H]+, calcd.
for C29H25Cl3O9 622.06.
4.1.4.24. 2,3,5-Trichloro-6-{2-[4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-phenoxy]-
ethoxy}-benzoic acid methyl ester (compound 7x)
Compound 7x was obtained as yellow solid; yield: 57.0%; m.p: 152.0-154.5 °C; 1H NMR (400 MHz,
CDCl3): δ (ppm) 7.89 (d, J = 8.6 Hz, 2H), 7.62 (s, 1H), 7.04 (d, J = 8.5 Hz, 2H), 6.80 (s, 1H), 6.59
(s, 1H), 4.51 – 4.46 (m, 2H), 4.39 – 4.35 (m, 2H), 4.30 (s, 3H), 4.03 (s, 3H), 3.96 (s, 3H), 3.87 (s,
3H). 13C NMR (101 MHz, CDCl3): δ (ppm) 180.74 (s), 164.25 (s), 163.83 (s), 161.47 (s), 159.55
(s), 151.64 (s), 151.00 (s), 146.72 (s), 136.62 (s), 132.97 (s), 131.90 (s), 131.74 (s), 129.35 (s),
128.43 (s), 127.21 (s), 125.73 (s), 115.00 (s), 111.26 (s), 107.25 (s), 90.53 (s), 73.01 (s), 66.67 (s),
62.41 (s), 61.63 (s), 56.62 (s), 53.22 (s), 29.71 (s). MS (ESI) m/z: 609.10 [M + H]+, calcd. for
C28H23Cl3O9 608.04.
4.2. Cell culture and incubation procedures
The human colon cancer cells (HTC116) and the human hepatoma cancer cells (BEL-7402) were
obtained from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China.
The human gastric cancer cells (MGC-803 and HGC-27) were obtained from the National
Experimental Cell Resource Sharing Service Platform, Beijing, China. The human gastric cancer
cells (SGC-7901) and the human gastric normal epithelial cell line (GES-1) were obtained from the