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

Article abstract of DOI:10.1039/c7md00578d

A series of 3′,4′,5′-trimethoxy flavonoids with benzimidazole linked by different chain alkanes have been designed and synthesized. The potential activity of these compounds as anti-tumor agents was evaluated by cytotoxicity assay in MGC-803 (human gastri

Full text of DOI:10.1039/c7md00578d

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DOI: 10.1039/C7MD00578D
Design, synthesis and biological evaluation of 3’,4’,5’-trimethoxy
flavonoid benzimidazole derivatives as potential anti-tumor agents
a,1
a,1
a
a
a
a
Zhe Wang , Xiangping Deng , Runde Xiong , Shujuan Xiong , Juan Liu , Xuan Cao ,
a
b
a,
a,
Xiaoyong Lei , Yanming Chen , Xing Zheng *, Guotao Tang *
a
Institute of Pharmacy and Pharmacology, Hunan Province Cooperative Innovation
Center for Molecular Target New Drug Study, University of South China, Hengyang,
China
b
Mu Dan Jiang You Bo Pharmacertical Co.Ltd, Mudanjiang, China
*
Corresponding Author: Dr. Guotao Tang, Ph.D. Dr .Xing Zheng, Ph.D
Corresponding author at：
Guotao Tang, Xing Zheng, Institute of Pharmacy and Pharmacology, University of
South China, Hengyang, Hunan, 421001,People’s Republic of China.
E-mail adress: tgtzq@163.com (Guotao Tang), 2393628359@qq.com (Xing Zheng)
1
These authors contributed equally to this work.

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DOI: 10.1039/C7MD00578D
Abstract
A series of 3’,4’,5’-trimethoxy flavonoids with benzimidazole linked by different chain alkanes have been
designed and synthesized. The potential activity of these compounds as anti-tumor agents was evaluated by
cytotoxicity assay in MGC-803 (Human gastric cancer cell line), MCF-7 (Human breast cancer cell line), HepG-2
(
Human hepatoma cell line) and MFC (Mouse gastric cancer cell line) tumor cell lines. Among them, compound 15
-(3-(2-chloro-1H-benzo[d]imidazol-1-yl)propoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one displayed the
most potent antiproliferative activity with IC50 values of 20.47 ± 2.07, 43.42 ± 3.56, 35.45 ± 2.03 ꢀM and 23.47 ±
.59 ꢀM, respectively. The flow cytometry (FCM) results showed that the compound 15 caused the cell cycle to
be arrested in G1 phase and induced apoptosis of MFC cells in a dose dependent manner. In addition, compound
5 exhibited a significant inhibitory effect on tumor growth in vivo. All the results outlined the great potential of
7
3
1
compound 15 for further exploitation as anti-tumor agent.
Keywords: Flavonoids; benzimidazoles; synthesis; anti-tumor
1
. Introduction
Tumor is one of the most major threats to human life in the world. There is an urgent need for seeking safer
and more effective chemotherapy drugs which will improve the survival rates of various types of tumor patients.
1
It is well known that many anti-tumor drugs are derived from the structural of natural products at present .
Flavonoid is a class of natural products that are widely found in nature and exhibit a wide range of biological
2
-5
6
activities , and they tend to be used as dominant parental structure in drug discovery . Over the past decade, a
large number of natural, semi-synthetic, synthetic flavonoids have been exploited and evaluated in a variety of
7
8
9, 10
therapeutic activities, such as anti-inflammatory, anti-bacterial , anti-allergy, anti-oxidation , anti-tumor
.
Particularly, their potential therapeutic effects and reliable safety in the tumors treatment have received
11-13
extensive attention
.
Benzene trimethoxy has special value in the design of antineoplastic drugs, with particular concern in the
design of vascular blockers. At present, the vascular disrupting agents in the clinical research stage are mainly
targeted at the tubulin to inhibit the formation of microtubules, such as CAP4 and its analogues OXi4503,
1
4-17
18
19
20
AVE8062
, colchicine analogs ZD6126 , BNC-105 and CKD-516 (Fig. 1). Interestingly, these compounds
contain benzene trimethoxy. In addition, we also note that benzimidazoles have been widely used in the design
2
1-25
and development of anticancer drugs
, such as some benzimidazoles and their derivatives exhibited
significant anti-tumor effect by inhibiting tumor cell mitosis and proliferation, mediating tumor cell apoptosis or
26-28
inhibiting the expression of hypoxia inducible factor (HIF)
.

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In this study, we attempted to introduce the trimethoxy group into the B-ring of flavonoid and link
benzimidazole and its derivatives to 7-OH with different chain alkanes. We are eager to discover new flavonoid
derivatives with potential anti-tumor activity. In this study, we synthesized a series of 3’,4’,5’-trimethoxy
flavonoid benzimidazole derivatives 5-28, and described the anti-tumor activity screening results of these
compounds. We successfully screened the compound 15 with notable anti-tumor activity in vitro, and further
demonstrated in vivo. The inhibitory effect of compound 15 on expression of tubulin or hypoxia inducible factor
(
HIF) requires further investigation.
2
. Results and discussion
2
.1. Chemistry
In this study, we synthesized twenty four new 3’,4’,5’-trimethoxy flavonoid benzimidazole derivatives. The
synthetic route was shown in Scheme 1. The key intermediate 3 was prepared in a three-step synthesis starting
2
9
.
with resorcinol, 2-chloroacetonitrile and 3,4,5-trimethoxybenzaldehyde according to a literature procedure
Houben-Hoesch reaction of resorcinol under the catalysis of zinc chloride in ether at 0 ℃ to provide
4
2
5
-(2-chloro-1-iminoethyl)benzene-1,3-diol
hydrochloride
(1)
and
then
be
turned
into
-chloro-1-(2,4-dihydroxyphenyl)ethanone (2) by hydrolization. Base-catalyzed aldol condensation of 2 with 3, 4,
-trimethoxybenzaldehyde afforded the chalcone. The chalone was cyclized in the solvent of 10% hydrochloric
acid and ethanol at room temperature to provide the flavonoid 3 in high yield with recrystallization from 95%
ethanol.
New analogues 4a-4c were conveniently synthesized in high yields (83-92%) by etherification reaction of the
key intermediate 3 with different chain length substituted alcohols in the presence of K CO in acetone. Alkylation
2
3
2 3
of the bromide intermediates 4a-4c and corresponding benzimidazoles in the presence of K CO in acetone
introduced basic functionalities providing final compounds 5-28 in 50-80% yields. All of the synthetic compounds
gave satisfactory analytical and spectroscopic data, which were in full accordance with their depicted structures.
2
2
.2. Biological activity
.2.1. In vitro antiproliferative assay
The in vitro antiproliferative activity of 3’,4’,5’-trimethoxy flavonoid benzimidazole derivatives 5-28 and the
positive control 5-Fu on MGC-803, MCF-7, HepG-2 and MFC cells was studied by MTT colorimetric assay. The
compounds were tested in a concentration range of 2 to 256 ꢀM and the calculated IC50 values were reported in
Table 1.
In general, some of the synthesized novel flavonoid derivatives exhibited excellent antiproliferative activity.

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DOI: 10.1039/C7MD00578D
Among them, compounds with substitutions on the R
1
-position of benzimidazole derivatives showed better
antiproliferative activity than the R
H atom at R
antiproliferative activity compared to the substitutions of electron-donation groups (6, 14, 22). For compounds 8,
6, 24 introduced nitro on the R - position, the antiproliferative activity was also decreasing compared to the
electron-donation groups on R - position. Besides, since the number of carbon atoms on the three linkages is
similar, the difference in antiproliferative activity of the three chain length derivatives is not significant.
It is clear that the synthesized compounds with chlorine atom substitutions in R -position on the
2
- and R
3
- position ones. For the R
1
- position substituted ones, replacement of
1
- position by electron-withdrawing groups (7, 11, 15, 19, 23, 27) resulted in the improving of their
1
3
3
1
benzimidazole derivatives (7, 15, 23) showed higher activity than others, Specially, compound 15 displayed the
most potent inhibitory activity with IC50 of 20.47 ± 2.07 ꢀM and 23.47 ± 3.59 ꢀM on MGC-803 cells and MFC cells,
respectively, which was better that of 5-Fu (IC50 = 74.39 ± 2.03 and 78.52 ± 3.92, respectively). Therefore, the IC50
value suggested that compound 15 may be a promising lead for the further development of novel anti-tumor
agents, and we chose the MFC cell lines to carry out cell assay, apoptosis assay and anti-tumor efficacy assay in
vivo.
In addition, it is worth noting that the three benzimidazole derivatives (6-nitrobenzimdazole,
5
-methylbenzimidazole and 5-methoxybenzimidazole) we selected will produce isomers during the last step of
30
the reaction . Since we did not find any significant anti-tumor activity advantage among them during the
previous active screening, we did not further disassemble the isomers of these compounds (compound
8
,10,12,16,18,20,24,26,28). However, from the point of view of drug molecule synthesis design, we believe that
isomers of the active compounds obtained by alkylation with 4-substituted or 5-substituted benzimidazoles may
have to be isolated and purified prior to studying their further biological activity.
2
.2.2. Apoptosis assay
Since compound 15 exhibited excellent activity against cell growth in MGC-803 cells and MFC cells, we plan
to use MFC cells to establish mouse tumor models for preliminary study of the anti-tumor effect in vivo of the
candidate compounds. Prior to this, in order to further verify the antitumor activity of candidate compounds in
vitro and to explore its mechanism of action, MFC cells were also selected for apoptosis study. Induction of
apoptosis is an effective anti-tumor strategy. The FCM assay determined that compound 15 could induce the
apoptosis of MFC cells in a dose dependent manner. As shown in Fig.2, MFC cells were treated with 10, 20, 40 ꢀM
of compound 15 for 24 h. The compound increased the percentage of apoptosis by Annexin V-FITC/PI staining in a
dose-dependent manner. The result indicated that compound 15 induced apoptosis of MFC cells.

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2
.2.3. Cell cycle assay
Cell cycle regulation is a necessary process to maintain cell proliferation and development. However, the
main feature of tumor cells is cell cycle regulation disorders leading to cell differentiation and apoptosis block, cell
3
1
proliferation out of control . We also chose MFC cell lines for cell cycle experiments. Studies were performed by
flow cytometry analysis to determine the effects of the compound 15 on cell cycle progression. As shown from
the cell cycle detection (Fig. 3), there was significant up-regulation of G1 phase in dose dependent manner after
treatment with three different concentrations of compound 15. Most of the tumor cells are often shortened in
the G1 phase due to the abnormal expression of Cyclin, CDK (Cyclin-dependent kinase) and CKI (cyclin-dependent
32
kinase inhibitor), thus rapidly entering the S phase and leading to excessive proliferation . Thus, the
experimental results showed that compound 15 could effectively upregulate G1 phase, thereby inducing
apoptosis of tumor cells. However, which regulatory molecule was affected by the compound is not clear, further
study is currently in progress.
2
.2.4. In vivo anti-tumor efficacy
Gastric carcinoma was chosen as a model cancer to investigate the anti-tumor efficacy of the three different
doses of compound 15. The anti-tumor growth effect in vivo was evaluated by measuring tumor volume following
treatment with different doses of compound 15 (10 mg/kg, 20 mg/kg, 40 mg/kg), 5-Fu (25 mg/kg) and saline. As
illustrated in Fig. 4, different doses of compound 15 and the positive control 5-Fu inhibited the tumor growth
remarkably as compared with the saline. The tumor growth inhibition of compound 15 was dose-independent.
The middle-dose group and high-dose group exhibited stronger tumor grouth inhibition with TIR of 67.8% and
8
0.2%, respectively, compared with the 5-Fu (TIR of 60.8%) (Fig.5). Fig. 6 illustrated the growth in body weight
during the 18 day experimental period. The gradual increase in body weight among all of the treat groups was
consistent with that of the control group, which represented the natural growth in body weight of tumor-bearing
mice. The animal experiment in vivo showed that the compound 15 was associated with stronger efficiency in
suppressing tumor growth compared with 5-Fu and exhibited less toxic side effects. Based on the results of
preliminary pharmacodynamic studies in mice above, we will conduct pharmacodynamic studies in nude mice in
order to further validate the anti-tumor activity of this compound.
3
. Conclusions
In summary, a series of 3’,4’,5’-trimethoxy flavonoid benzimidazole derivatives were firstly synthesized and
evaluated for their antiproliferative activities against MGC-803, MCF-7, HepG-2 and MFC tumor cell lines. It is
worth noting that compound 15 displayed the most outstanding antiproliferative activities in vitro with IC50 of

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2
0.47 ± 2.07, 43.42 ± 3.56, 35.45 ± 2.03 ꢀM and 23.47 ± 3.59 ꢀM, respectively. In addition, the preliminary
mechanism of compound 15 inhibition effects was detected by flow cytometry, and the compound exert
antiproliferative activity via inducing the apoptosis of tumor cells in a dose dependent manner. Flow cytometry
1
analysis also revealed that compound 15 arrested the cell cycle of MFC cells in the G phase with a
concentration-dependent effect. Furthermore, the animal experiment in vivo showed that compound 15
exhibited stronger efficiency in suppressing tumor growth compared with 5-Fu. All the results outlined the great
potential of compound 15 for further exploitation as anti-tumor drug.
4
. Experimental Section
4
.1. Chemistry
All chemicals (reagent grade) used were purchased from commercial source and used without further
purification unless otherwise stated. Separation of the compounds by column chromatography was carried out
with silica gel (100-200 mesh size). TLC was run on the silica gel 60 F254 plates and visualized under UV light at
2
54 nm or iodine vapour. Melting points (uncorrected) were determined on a Thermo Scientific Electrothermal
1
Digital Melting Point Apparatus. ESI mass spectra were obtained on a Waters GCT mass spectrometer, and H
NMR spectra were measured on a Bruker AV-400 model Spectrometer with TMS and solvent signals slotted as
internal standards. Chemical shifts were reported in ppm (δ).
4
.1.1. 7-hydroxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (3)
To a solution of resorcinol (2.5 g, 0.023 mol) and 2-chloroacetonitrile (2 mL, 0.032 mol) in absolute ether (30
2
mL) with catalytic amount of ZnCl at 0 ℃ and freshly prepared hydrogen chloride gas was bubbled slowly and
continuously to the reaction mixture for 2 h. After standing the reaction flask at low temperature for overnight,
lots of yellow precipitate was prepared after filtrating, washing and drying. To reflux the yellow precipitate in
water solvent, a precipitate was produced. The milky white precipitate2-chloro-1-(2,4-dihydroxyphenyl)ethanone
was filtered off, washed with water, and dried in vacuo to give
a milky white precipitate.
3
,4,5-trimethoxybenzaldehyde (2.0 g, 0.01 mol) and 2-chloro-1-(2,4-dihydroxyphenyl)ethanone (2.2 g, 0.012 mol)
were added in one portion to a stirred solution of 5 mL ethanol, the drop 10% NaOH aqueous solution into the
solution. The reaction mixture was stirred at room temperature for 24 h, and acidification with 10% HCl (aq) gave
a crude product, which was filtered off and purified by recrystallized from 95% ethanol to give the compound 3 as
yellow powder.
4
.1.2. General method of synthesis 4a-4c
To a stirred solution of compound 3 (1.0 mol) in acetone (30 ml) with certain amount of K CO at room
2
3

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temperature and different chain length substituted alcohols (4.0 mol) was added to the reaction mixture. The
mixture was refluxed for 5 h (the reaction was monitored by TLC). After cooling, the crude product was filtered off,
washed with water and acetone, dried in vacuo. The crude product was purified by column chromatography on
silica gel, eluting with dichloromethane/methanol (50:1) to give a viridescent powder.
3
3
4
.1.2.1. 7-(2-bromoethoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (4a).
Yellow powder, yield: 95%.
1
Mp:142-145℃ H NMR (400 MHz, CDCl
3
) δ 7.62 (dd, J = 20.0, 9.8 Hz, 1H), 7.07 (s, 2H), 6.84 – 6.55 (m, 3H), 4.30
1
3
(
(
dt, J = 20.6, 4.8 Hz, 2H), 3.87 (t, J = 9.4 Hz, 9H), 3.65 (dt, J = 11.1, 8.1 Hz, 2H); C NMR (101 MHz, CDCl ) δ 182.76
3
s), 168.05 (s), 165.61 (s), 153.32 (s), 147.21 (s), 127.71 (s), 126.08 (s), 115.51 (s), 112.49 (s), 112.18 (s), 108.81 (s),
+
9
4
7.62 (s), 68.43 (s), 61.03 (s), 56.25 (s), 29.71 (s), 28.21 (s). MS (ESI):434.0 (C20
H
19BrO
6
, [M+H ]).
33
.1.2.2. 7-(3-bromopropoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (4b).
1
Yellow powder, yield: 94%. Mp:168-170℃ H NMR (400 MHz, CDCl
3
) δ 7.72 (d, J = 8.3 Hz, 1H), 7.16 (s, 2H), 6.77
1
3
(
d, J = 10.0 Hz, 3H), 4.25 (t, J = 5.4 Hz, 2H), 3.94 (d, J = 11.6 Hz, 9H), 3.63 (t, J = 6.0 Hz, 2H), 2.52 – 2.29 (m, 2H); C
3
NMR (101 MHz, CDCl ) δ 182.78 (s), 168.22 (s), 166.36 (s), 153.30 (s), 147.29 (s), 139.81 (s), 127.79 (s), 125.88 (s),
1
15.07 (s), 112.43 (s), 112.26 (s), 108.70 (s), 97.24 (s), 66.22 (s), 61.03 (s), 56.22 (s), 31.91 (s), 29.54 (s). MS
+
(
6
ESI):448.1 (C21H21BrO , [M+H ]).
4
.1.2.3. 7-(4-bromobutoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (4c).
1
Yellow powder, yield: 95%. Mp:159-160℃ H NMR (400 MHz, CDCl ) δ 7.71 (d, J = 8.5 Hz, 1H), 7.16 (s, 2H), 6.75 (t,
3
13
J = 8.3 Hz, 3H), 4.13 (t, J = 5.2 Hz, 2H), 3.93 (d, J = 10.9 Hz, 9H), 3.51 (t, J = 6.0 Hz, 2H), 2.26 – 1.86 (m, 4H);
C
NMR (101 MHz, CDCl
3
) δ 182.75 (s), 168.24 (s), 166.59 (s), 153.29 (s), 147.32 (s), 139.78 (s), 127.81 (s), 125.83 (s),
1
14.89 (s), 112.41 (s), 112.14 (s), 108.69 (s), 97.14 (s), 67.86 (s), 61.02 (s), 56.21 (s), 33.17 (s), 29.24 (s), 27.58 (s).
+
MS (ESI):462.1 (C22
H
6
23BrO , [M+H ]).
4
.1.3. General method of synthesis 5-28
Equimolar quantities of the bromide intermediates (4a-4c), corresponding benzimidazoles were dissolved in
acetone (30 mL), and certain amount of K CO was also added to the solution. The solution was then refluxed for
2
3
approximately 12 h. The product was extracted by dichloromethane. Then evaporated of the solvent, the residue
was purified by column chromatography on silica gel, eluting with dichloromethane/methanol (30:1) to give a
yellow powder.
4
.1.3.1.7-(2-(1H-benzo[d]imidazol-1-yl)ethoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (5). Yellow powder,
1
yield: 64%. Mp:209-210℃ H NMR (400 MHz, CDCl
3
) δ 8.03 (s, 1H), 7.82 (d, J = 7.0 Hz, 1H), 7.68 (d, J = 8.6 Hz, 1H),
7
.47 (d, J = 7.3 Hz, 1H), 7.32 (dq, J = 7.2, 6.0 Hz, 2H), 7.11 (s, 2H), 6.74 – 6.70 (m, 2H), 6.70 (d, J = 2.1 Hz, 1H), 6.65

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1
3
(
d, J = 2.0 Hz, 1H), 4.64 (t, J = 5.2 Hz, 2H), 4.41 (t, J = 5.1 Hz, 2H), 3.91 (d, J = 7.6 Hz, 9H); C NMR (101 MHz, CDCl
3
)
δ 182.66 (s), 167.85 (s), 165.22 (s), 153.28 (s), 147.09 (s), 143.84 (s), 143.45 (s), 139.88 (s), 133.63 (s), 127.65 (s),
1
6
4
26.02 (s), 123.27 (s), 122.49 (s), 120.69 (s), 115.64 (s), 112.58 (s), 111.87 (s), 109.31 (s), 108.74 (s), 97.56 (s),
+
6.85 (s), 61.03 (s), 56.21 (s), 44.08 (s). MS (ESI):472.1 (C27
H
24
N
2
O
6
, [M+H ]).
.1.3.2.7-(2-(2-methyl-1H-benzo[d]imidazol-1-yl)ethoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
(6).
1
Orange yellow powder, yield: 53%. Mp:192-193℃ H NMR (400 MHz, CDCl
3
) δ 7.71 – 7.67 (m, 1H), 7.66 (s, 1H),
7
6
.37 – 7.32 (m, 1H), 7.27 – 7.25 (m, 2H), 7.10 (s, 1H), 6.73 (s, 1H), 6.68 (d, J = 2.0 Hz, 1H), 6.66 (d, J = 2.1 Hz, 1H),
13
.60 (d, J = 2.0 Hz, 1H), 4.59 (t, J = 5.3 Hz, 2H), 4.39 (t, J = 5.2 Hz, 2H), 3.92 (t, J = 8.8 Hz, 9H), 2.72 (s, 3H); C NMR
(
101 MHz, CDCl
3
) δ 182.69 (s), 167.90 (s), 165.30 (s), 153.28 (s), 152.04 (s), 147.09 (s), 142.65 (s), 139.89 (s),
1
1
4
34.82 (s), 127.63 (s), 126.01 (s), 122.38 (s), 122.30 (s), 119.36 (s), 115.55 (s), 112.63 (s), 111.98 (s), 108.83 (s),
+
08.75 (s), 97.29 (s), 66.71 (s), 61.02 (s), 56.22 (s), 42.97 (s), 14.11 (s). MS (ESI):486.1 (C28
H N
26 2
O
6
, [M+H ]).
.1.3.3.7-(2-(2-chloro-1H-benzo[d]imidazol-1-yl)ethoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (7). Yellow
1
powder, yield: 78%. Mp:153-156℃ H NMR (400 MHz, CDCl
3
) δ 7.70 (d, J = 7.1 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H),
7
1
.44 (d, J = 7.4 Hz, 1H), 7.37 – 7.26 (m, 2H), 7.11 (s, 1H), 6.73 (s, 1H), 6.68 (d, J = 2.1 Hz, 1H), 6.66 (d, J = 2.1 Hz,
13
H), 6.63 (d, J = 2.0 Hz, 1H), 4.66 (t, J = 5.4 Hz, 2H), 4.41 (dd, J = 10.6, 5.1 Hz, 2H), 3.92 (t, J = 8.7 Hz, 9H); C NMR
(
(
(
101 MHz, CDCl
3
) δ 182.69 (s), 167.92 (s), 165.23 (s), 153.31 (s), 147.11 (s), 141.74 (s), 140.49 (s), 140.14 – 139.67
m), 135.28 (s), 127.65 (s), 126.03 (s), 123.52 (s), 123.09 (s), 119.73 (s), 115.62 (s), 112.60 (s), 112.06 (s), 109.55
+
s), 108.77 (s), 97.34 (s), 66.46 (s), 61.03 (s), 56.24 (s), 43.51 (s). MS (ESI):507.1 (C27
H
23ClN
2
O
6
, [M+H ]).
4
.1.3.4.7-(2-(6(5)-nitro-1H-benzo[d]imidazol-1-yl)ethoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
(8).
1
Orange yellow powder, yield: 74%. Mp:207-209℃ H NMR (400 MHz, CDCl ) δ 8.74 (s, 1H), 8.54 (d, J = 2.0 Hz,
3
1
1
2
H), 8.30 (d, J = 11.2 Hz, 1H), 8.27 – 8.20 (m, 1H), 7.88 (d, J = 9.0 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.57 (d, J = 9.0 Hz,
H), 7.11 (d, J = 2.1 Hz, 1H), 6.70 (dd, J = 16.3, 7.7 Hz, 2H), 4.72 (dt, J = 9.9, 4.7 Hz, 2H), 4.45 (dd, J = 9.8, 5.0 Hz,
1
3
3
H), 3.92 (dd, J = 9.0, 7.4 Hz, 9H); C NMR (101 MHz, CDCl ) δ 182.58 (s), 167.78 (s), 164.84 (s), 153.30 (s), 147.87
(
s), 147.01 (s), 146.72 (s), 144.05 (s), 143.92 (s), 127.56 (s), 126.14 (s), 120.78 (s), 119.05 (s), 118.25 (s), 117.34 (s),
1
15.89 (s), 112.76 (s), 111.74 (s), 109.64 (s), 108.81 (s), 108.76 (s), 107.00 (s), 97.67 (s), 67.02 (s), 66.88 (s), 61.03
+
(
s), 56.22 (s), 44.82 (s), 44.62 (s). MS (ESI):517.1 (C27
H
23
N
3
O
8
, [M+H ]).
4
.1.3.5.7-(2-(5,6-dimethyl-1H-benzo[d]imidazol-1-yl)ethoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
(9).
1
Yellow powder, yield: 68%. Mp:235-237℃ H NMR (400 MHz, CDCl
3
) δ 7.91 (s, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.57 (s,
1
2
H), 7.21 (s, 1H), 7.11 (s, 2H), 6.73 (s, 1H), 6.70 (dd, J = 8.6, 2.0 Hz, 1H), 6.65 (d, J = 2.0 Hz, 1H), 4.58 (t, J = 5.1 Hz,
1
3
H), 4.38 (t, J = 5.2 Hz, 2H), 3.91 (d, J = 7.8 Hz, 9H), 2.38 (d, J = 13.9 Hz, 6H); C NMR (101 MHz, CDCl ) δ 182.47
3

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(
s), 167.68 (s), 165.32 (s), 153.32 (s), 147.15 (s), 142.69 (s), 139.89 (s), 132.47 (s), 131.42 (s), 127.68 (s), 120.67 (s),
1
2
4
15.63 (s), 112.59 (s), 111.91 (s), 109.45 (s), 108.76 (s), 97.62 (s), 66.81 (s), 61.04 (s), 56.23 (s), 44.04 (s), 20.67 (s),
+
0.25 (s), 0.01 (s). MS (ESI):500.1 (C29
H
28
N
2
O
6
, [M+H ]).
.1.3.6.7-(2-(5(6)-methyl-1H-benzo[d]imidazol-1-yl)ethoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (10).
1
Yellow powder, yield: 62%. Mp:208-210℃ H NMR (400 MHz, CDCl
3
) δ 8.01 (d, J = 6.5 Hz, 1H), 7.73 (d, J = 8.5 Hz,
1
2
H), 7.66 (d, J = 12.8 Hz, 1H), 7.39 (d, J = 8.5 Hz, 1H), 7.24 – 7.10 (m, 3H), 6.80 – 6.72 (m, 2H), 6.70 (s, 1H), 4.64 (s,
13
H), 4.44 (s, 2H), 3.96 (t, J = 9.0 Hz, 9H), 2.54 (d, J = 13.7 Hz, 3H); C NMR (101 MHz, CDCl ) δ 182.68 (s), 167.87
3
(
s), 165.27 (s), 153.28 (s), 147.11 (s), 144.20 (s), 143.39 (s), 143.03 (s), 141.95 (s), 139.88 (s), 133.79 (s), 133.29 (s),
1
1
32.21 (s), 131.71 (s), 127.65 (s), 126.02 (s), 124.73 (s), 124.08 (s), 120.41 (s), 120.17 (s), 115.61 (s), 112.57 (s),
11.89 (s), 109.18 (s), 108.72 (s), 97.56 (s), 66.82 (d, J = 9.0 Hz), 61.02 (s), 56.20 (s), 43.95 (s), 21.88 (s), 21.51 (s).
+
MS (ESI):486.1 (C28
H
26
N
2
O
6
, [M+H ]).
4
.1.3.7.7-(2-(2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)ethoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
1
(
11). Yellow powder, yield: 77%. Mp:207-208℃ H NMR (400 MHz, CDCl
3
) δ 7.91 (d, J = 8.0 Hz, 1H), 7.67 (t, J = 8.3
Hz, 2H), 7.51 (t, J = 7.5 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H), 7.12 (s, 2H), 6.74 (s, 1H), 6.71 – 6.58 (m, 2H), 4.82 (s, 2H),
1
3
4
1
1
3
.47 (s, 2H), 3.94 (t, J = 8.2 Hz, 9H); C NMR (101 MHz, CDCl ) δ 182.70 (s), 167.93 (s), 165.11 (s), 153.32 (s),
47.08 (s), 141.08 (s), 139.97 (s), 135.82 (s), 127.62 (s), 126.04 (s), 125.71 (s), 124.07 (s), 121.84 (s), 115.66 (s),
12.69 (s), 112.11 (s), 110.89 (s), 108.81 (s), 97.20 (s), 66.95 (s), 61.03 (s), 56.25 (s), 44.08 (s). MS (ESI):540.1
+
(
C
28
H
23
F
3
N
2
O
6
, [M+H ]).
4
.1.3.8.7-(2-(5(6)-methoxy-1H-benzo[d]imidazol-1-yl)ethoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (12).
1
Yellow powder, yield: 71%. Mp:204-206℃ H NMR (400 MHz, CDCl ) δ 7.96 (d, J = 17.8 Hz, 1H), 7.69 (dd, J = 16.8,
3
9
4
.5 Hz, 2H), 7.12 (s, 2H), 6.97 (dd, J = 22.2, 11.3 Hz, 2H), 6.81 – 6.61 (m, 3H), 4.60 (d, J = 4.8 Hz, 2H), 4.41 (s, 2H),
1
3
3
.04 – 3.79 (m, 12H); C NMR (101 MHz, CDCl ) δ 182.67 (s), 167.87 (s), 165.24 (s), 157.03 (s), 156.37 (s), 153.28
(
s), 147.10 (s), 144.68 (s), 143.57 (s), 142.67 (s), 139.88 (s), 138.29 (s), 134.31 (s), 128.22 (s), 127.65 (s), 126.03 (s),
1
9
21.10 (s), 115.63 (s), 113.51 (s), 112.60 (s), 111.92 (s), 111.42 (s), 109.74 (s), 108.73 (s), 102.54 (s), 97.54 (s),
3.22 (s), 66.94 (s), 66.84 (s), 61.03 (s), 56.21 (s), 55.97 (s), 55.82 (s), 44.22 (s), 43.99 (s). MS (ESI):502.1
+
(
C
28
H
26
N
2
O
7
, [M+H ]).
4
.1.3.9.7-(3-(1H-benzo[d]imidazol-1-yl)propoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
(13).
Yellow
1
powder, yield: 70%. Mp:185-187℃ H NMR (400 MHz, CDCl
3
) δ 7.87 (d, J = 17.7 Hz, 1H), 7.82 (d, J = 3.3 Hz, 1H),
7
4
.74 (d, J = 8.5 Hz, 1H), 7.41 (s, 1H), 7.36 – 7.21 (m, 4H), 6.78 (d, J = 9.3 Hz, 2H), 6.66 (s, 1H), 4.49 (t, J = 6.3 Hz, 2H),
13
.00 (dd, J = 13.4, 8.4 Hz, 2H), 3.92 (d, J = 7.2 Hz, 9H), 2.52 – 2.32 (m, 2H); C NMR (101 MHz, CDCl ) δ 182.76 (s),
3

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1
1
68.15 (s), 165.93 (s), 153.31 (s), 147.21 (s), 143.93 (s), 143.10 (s), 139.87 (s), 133.67 (s), 127.73 (s), 126.02 (s),
23.19 (s), 122.36 (s), 120.61 (s), 115.35 (s), 112.45 (s), 112.31 (s), 109.43 (s), 108.74 (s), 97.23 (s), 64.79 (s), 61.03
+
(
s), 56.23 (s), 41.29 (s), 29.18 (s). MS (ESI):486.1 (C28
H
26
N
2
O
6
, [M+H ]).
4
.1.3.10.7-(3-(2-methyl-1H-benzo[d]imidazol-1-yl)propoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
(14).
1
Yellow powder, yield: 57%. Mp:169-171℃ H NMR (400 MHz, CDCl
3
) δ 7.62 (t, J = 8.8 Hz, 2H), 7.19 (t, J = 12.3 Hz,
2
7
H), 7.11 (td, J = 14.2, 6.7 Hz, 3H), 6.67 (d, J = 11.4 Hz, 2H), 6.56 (s, 1H), 4.30 (t, J = 6.3 Hz, 2H), 3.90 (dd, J = 12.8,
13
.5 Hz, 2H), 3.83 (d, J = 3.5 Hz, 9H), 2.44 (d, J = 42.5 Hz, 2H), 2.28 (dd, J = 11.8, 5.7 Hz, 2H); C NMR (101 MHz,
CDCl
3
) δ 182.74 (s), 168.14 (s), 165.86 (s), 153.29 (s), 151.52 (s), 147.19 (s), 142.44 (s), 139.89 (s), 134.88 (s),
1
9
4
27.69 (s), 126.02 (s), 122.32 (s), 122.15 (s), 119.12 (s), 115.32 (s), 112.50 (s), 112.32 (s), 109.01 (s), 108.76 (s),
+
7.19 (s), 64.76 (s), 61.00 (s), 56.21 (s), 40.07 (s), 28.84 (s). MS (ESI):500.1 (C H N O , [M+H ]).
2
9 28 2 6
.1.3.11.7-(3-(2-chloro-1H-benzo[d]imidazol-1-yl)propoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
(15).
1
Yellow powder, yield: 66%. Mp:140-142℃ H NMR (400 MHz, CDCl
3
) δ 7.72 (t, J = 7.8 Hz, 1H), 7.56 (s, 1H), 7.37 –
7
.29 (m, 2H), 7.14 (s, 2H), 7.07 (s, 1H), 6.76 (d, J = 6.8 Hz, 1H), 6.65 (s, 1H), 4.49 (t, J = 5.7 Hz, 2H), 4.09 (d, J = 20.6
1
3
Hz, 2H), 3.93 (d, J = 6.5 Hz, 9H), 2.40 (s, 2H); C NMR (101 MHz, CDCl
3
) δ 182.89 (s), 168.18 (s), 165.98 (s), 153.31
(
s), 147.24 (s), 141.59 (s), 140.51 (s), 139.87 (s), 135.03 (s), 128.92 (s), 127.73 (s), 126.04 (s), 123.52 (s), 123.10 (s),
1
23.03 (s), 119.53 (s), 115.29 (s), 112.59 (s), 112.36 (s), 109.27 (s), 108.77 (s), 97.22 (s), 64.90 (s), 61.05 (s), 56.25
+
(
s), 41.00 (s), 29.28 (s), 28.76 (s). MS (ESI):521.1 (C H ClN O , [M+H ]).
2
8
25
2
6
4
.1.3.12.7-(3-(6(5)-nitro-1H-benzo[d]imidazol-1-yl)propoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (16).
1
Yellow powder, yield: 54%. Mp:225-228℃ H NMR (400 MHz, CDCl
3
) δ 8.50 (d, J = 113.5 Hz, 1H), 8.23 – 8.01 (m,
2
5
H), 7.62 (ddd, J = 88.9, 65.5, 8.9 Hz, 2H), 7.05 (s, 2H), 6.76 – 6.54 (m, 3H), 4.61 – 4.38 (m, 2H), 4.01 (dd, J = 11.2,
1
3
3
.5 Hz, 2H), 3.86 (t, J = 10.2 Hz, 9H), 2.40 (dt, J = 11.4, 5.6 Hz, 2H); C NMR (101 MHz, CDCl ) δ 182.66 (s), 168.06
(
s), 165.60 (s), 153.31 (s), 148.18 (s), 147.54 (s), 147.13 (s), 146.46 (s), 143.96 (s), 143.85 (s), 143.23 (s), 139.95 (s),
1
1
2
4
37.82 (s), 133.07 (s), 127.65 (s), 126.11 (s), 120.78 (s), 118.99 (s), 118.16 (s), 117.34 (s), 115.55 (s), 112.60 (s),
12.53 (s), 112.08 (s), 109.48 (s), 108.79 (s), 106.62 (s), 97.28 (s), 64.68 (s), 61.03 (s), 56.23 (s), 53.47 (s), 41.97 (s),
+
9.23 (s). MS (ESI):531.1 (C28
H
25
N
3
O
8
, [M+H ]).
.1.3.13.7-(3-(5,6-dimethyl-1H-benzo[d]imidazol-1-yl)propoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (17).
1
Yellow powder, yield: 63%. Mp:222-224℃ H NMR (400 MHz, CDCl ) δ 7.73 (dd, J = 23.4, 14.8 Hz, 2H), 7.56 (s,
3
1
1
1
H), 7.14 (s, 3H), 6.78 (d, J = 12.1 Hz, 2H), 6.65 (s, 1H), 4.42 (s, 2H), 3.99 (s, 2H), 3.92 (d, J = 6.9 Hz, 9H), 2.38 (d, J =
1
3
4.7 Hz, 6H), 2.31 (s, 2H); C NMR (101 MHz, CDCl
3
) δ 182.74 (s), 168.17 (s), 166.01 (s), 153.30 (s), 147.22 (s),
42.35 (s), 139.84 (s), 132.36 (s), 132.25 (s), 131.23 (s), 127.73 (s), 125.96 (s), 120.47 (s), 115.25 (s), 112.41 (s),

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1
09.66 (s), 108.71 (s), 97.21 (s), 64.77 (s), 61.02 (s), 56.21 (s), 41.12 (s), 29.26 (s), 20.52 (s), 20.24 (s). MS
+
(
ESI):514.1 (C30
H
30
N
2
O
6
, [M+H ]).
4
.1.3.14.7-(3-(5(6)-methyl-1H-benzo[d]imidazol-1-yl)propoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (18).
1
Yellow powder, yield: 58%. Mp:154-156℃ H NMR (400 MHz, CDCl ) δ 7.85 (d, J = 6.4 Hz, 1H), 7.74 (t, J = 9.0 Hz,
3
1
H), 7.70 (d, J = 8.3 Hz, 1H), 7.62 (s, 1H), 7.22 – 7.09 (m, 3H), 6.85 – 6.75 (m, 2H), 6.67 (s, 1H), 4.46 (s, 2H), 4.01 (d,
1
3
J = 4.7 Hz, 2H), 3.94 (d, J = 7.3 Hz, 9H), 2.50 (s, 2H), 2.46 – 2.38 (m, 3H); C NMR (101 MHz, CDCl
3
) δ 182.78 (s),
1
1
1
4
4
68.17 (s), 165.98 (s), 153.30 (s), 147.22 (s), 144.21 (s), 143.03 (s), 142.63 (s), 141.91 (s), 139.85 (s), 133.92 (s),
33.22 (s), 132.10 (s), 131.74 (s), 127.73 (s), 126.01 (s), 124.69 (s), 123.97 (s), 120.26 (s), 120.00 (s), 115.28 (s),
12.70 – 112.18 (m), 109.36 (s), 108.96 (s), 108.73 (s), 97.21 (s), 64.78 (d, J = 3.4 Hz), 61.02 (s), 56.22 (s), 41.29 (s),
+
1.09 (s), 29.24 (s), 21.76 (s), 21.50 (s). MS (ESI):500.1 (C H N O , [M+H ]).
2
9
28
2
6
.1.3.15.7-(3-(2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)propoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-o
1
ne (19). Yellow powder, yield: 72%. Mp:175-178℃ H NMR (400 MHz, CDCl
3
) δ 7.91 (d, J = 6.4 Hz, 1H), 7.75 (d, J =
8
4
1
1
.4 Hz, 1H), 7.40 (dd, J = 15.9, 8.3 Hz, 3H), 7.15 (s, 2H), 6.78 (d, J = 6.8 Hz, 2H), 6.68 (s, 1H), 4.62 (t, J = 6.8 Hz, 2H),
1
3
3
.14 (s, 2H), 3.95 (t, J = 10.9 Hz, 9H), 2.45 (d, J = 5.6 Hz, 2H); C NMR (101 MHz, CDCl ) δ 182.89 (s), 168.17 (s),
65.94 (s), 153.31 (s), 147.23 (s), 141.00 (s), 139.88 (s), 135.45 (s), 127.72 (s), 126.03 (s), 125.69 (s), 123.99 (s),
23.64 (s), 121.72 (s), 115.32 (s), 112.64 (s), 112.31 (s), 110.30 (s), 108.78 (s), 97.22 (s), 65.17 (s), 61.04 (s), 56.21
+
(
s), 41.80 (s), 29.57 (s). MS (ESI):554.1 (C H F N O , [M+H ]).
2
9 25 3 2 6
4
.1.3.16.7-(3-(5(6)-methoxy-1H-benzo[d]imidazol-1-yl)propoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
1
(
20). Yellow powder, yield: 67%. Mp:129-131℃ H NMR (400 MHz, CDCl
3
) δ 7.81 (d, J = 15.8 Hz, 1H), 7.74 (d, J =
8
1
.4 Hz, 1H), 7.69 (d, J = 8.9 Hz, 1H), 7.28 (s, 1H), 7.14 (s, 2H), 6.93 (t, J = 8.6 Hz, 1H), 6.86 – 6.74 (m, 2H), 6.67 (s,
13
H), 4.43 (d, J = 6.0 Hz, 2H), 4.07 – 3.98 (m, 2H), 3.98 – 3.69 (m, 12H), 2.51 – 2.29 (m, 2H); C NMR (101 MHz,
CDCl
3
) δ 182.77 (s), 168.17 (s), 165.95 (s), 156.98 (s), 156.30 (s), 153.31 (s), 147.20 (s), 142.27 (s), 139.90 (s),
1
1
4
4
38.30 (s), 134.39 (s), 127.70 (s), 126.05 (s), 121.04 (s), 115.34 (s), 113.51 (s), 112.57 (s), 112.38 (s), 111.64 (s),
09.82 (s), 108.76 (s), 102.42 (s), 97.21 (s), 92.91 (s), 64.73 (s), 61.03 (s), 56.24 (s), 55.82 (s), 55.77 (s), 41.40 (s),
+
1.05 (s), 29.24 (s). MS (ESI):516.1 (C29
28
H N
2
O
7
, [M+H ]).
.1.3.17.7-(4-(1H-benzo[d]imidazol-1-yl)butoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
(21).
Yellow
1
powder, yield: 59%. Mp:177-179℃ H NMR (400 MHz, CDCl ) δ 7.95 (s, 1H), 7.83 (d, J = 6.8 Hz, 1H), 7.70 (t, J =
3
9
.8 Hz, 1H), 7.43 (d, J = 7.1 Hz, 1H), 7.31 (dd, J = 19.0, 13.9 Hz, 4H), 6.72 (dd, J = 19.2, 10.4 Hz, 3H), 4.31 (t, J = 6.7
1
3
Hz, 2H), 4.09 (t, J = 5.5 Hz, 2H), 4.02 – 3.84 (m, 9H), 2.22 – 2.07 (m, 2H), 2.00 – 1.83 (m, 2H); C NMR (101 MHz,
CDCl ) δ 182.77 (s), 168.19 (s), 166.38 (s), 153.30 (s), 147.28 (s), 139.83 (s), 127.78 (s), 125.92 (s), 123.02 (s),
3

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1
22.24 (s), 120.56 (s), 115.04 (s), 112.26 (s), 109.58 (s), 108.73 (s), 97.20 (s), 68.07 (s), 61.03 (s), 56.23 (s), 44.75
+
(
s), 26.70 (s), 26.33 (s). MS (ESI):500.1 (C29
28
H N
2
O
6
, [M+H ]).
4
.1.3.18.7-(4-(2-methyl-1H-benzo[d]imidazol-1-yl)butoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
(22).
1
Yellow powder, yield: 61%. Mp:160-163℃ H NMR (400 MHz, CDCl ) δ 7.71 (d, J = 8.2 Hz, 2H), 7.31 (s, 1H), 7.25
3
(
d, J = 3.2 Hz, 2H), 7.15 (s, 2H), 6.77 – 6.65 (m, 3H), 4.23 (t, J = 6.9 Hz, 2H), 4.10 (dd, J = 14.1, 8.6 Hz, 2H), 3.94 (t, J
1
3
=
1
1
3
10.4 Hz, 9H), 2.64 (s, 3H), 2.15 – 1.99 (m, 2H), 1.89 (d, J = 6.3 Hz, 2H); C NMR (101 MHz, CDCl ) δ 182.77 (s),
68.20 (s), 166.39 (s), 153.32 (s), 151.32 (s), 147.29 (s), 142.71 (s), 135.06 (s), 127.78 (s), 125.95 (s), 122.10 (s),
21.93 (s), 119.23 (s), 115.07 (s), 112.27 (s), 112.20 (s), 109.04 (s), 108.76 (s), 97.21 (s), 68.17 (s), 61.04 (s), 56.25
+
(
s), 43.49 (s), 26.60 (s), 26.45 (s), 14.05 (s). MS (ESI):514.1 (C30
H
30
N
2
O
6
, [M+H ]).
4
.1.3.19.7-(4-(2-chloro-1H-benzo[d]imidazol-1-yl)butoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
(23).
1
Yellow powder, yield: 60%. Mp:163-166℃ H NMR (400 MHz, CDCl
3
) δ 7.61 (q, J = 8.0 Hz, 3H), 7.33 – 7.12 (m, 4H),
6
–
1
1
3
4
.75 – 6.50 (m, 3H), 4.24 (t, J = 6.7 Hz, 2H), 4.01 (t, J = 5.5 Hz, 2H), 3.85 (t, J = 9.3 Hz, 9H), 2.13 – 1.92 (m,2H), 1.89
1
3
3
1.74 (m,2H); C NMR (101 MHz, CDCl ) δ 182.78 (s), 168.19 (s), 166.40 (s), 153.31 (s), 147.29 (s), 141.79 (s),
40.45 (s), 139.85 (s), 134.94 (s), 127.78 (s), 125.93 (s), 123.27 (s), 122.80 (s), 119.63 (s), 115.04 (s), 112.26 (s),
12.23 (s), 109.35 (s), 108.75 (s), 97.21 (s), 69.53 (s), 68.06 (s), 61.03 (s), 56.23 (s), 53.80 (s), 53.47 (s), 44.11 (s),
+
1.76 (s), 29.28 (s), 26.24 (s), 26.18 (s). MS (ESI):534.1(C29
H
2
27ClN O
6
, [M+H ]).
.1.3.20.7-(4-(6(5)-nitro-1H-benzo[d]imidazol-1-yl)butoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
(24).
1
Yellow powder, yield: 62%. Mp:140-142℃ H NMR (400 MHz, CDCl
3
) δ 8.56 (d, J = 113.5 Hz, 1H), 8.21 (dd, J =
2
4
3.4, 12.4 Hz, 2H), 7.98 – 7.38 (m, 2H), 7.12 (s, 2H), 6.70 (dd, J = 8.7, 5.1 Hz, 3H), 4.39 (dt, J = 14.0, 7.1 Hz, 2H),
13
.22 – 4.03 (m, 2H), 3.92 (d, J = 6.2 Hz, 9H), 2.36 – 2.06 (m, 2H), 1.91 (dd, J = 14.0, 7.0 Hz, 2H); C NMR (101 MHz,
CDCl
3
) δ 182.69 (s), 168.12 (s), 166.22 (s), 153.28 (s), 148.18 (s), 147.37 (s), 147.20 (s), 146.31 (s), 143.86 (s),
1
1
43.73 (s), 143.17 (s), 139.85 (s), 137.83 (s), 133.06 (s), 127.70 (s), 125.91 (s), 120.66 (s), 118.84 (s), 118.05 (s),
17.23 (s), 115.08 (s), 112.33 (s), 112.18 (s), 112.11 (s), 109.66 (s), 108.73 (s), 106.74 (s), 97.28 (s), 97.17 (s), 67.93
+
(
s), 67.87 (s), 61.01 (s), 56.21 (s), 45.27 (s), 26.84 (s), 26.25 (s), 26.19 (s). MS (ESI):545.2 (C29
H
27
N
3
O
8
, [M+H ]).
4
.1.3.21.7-(4-(5,6-dimethyl-1H-benzo[d]imidazol-1-yl)butoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (25).
1
Yellow powder, yield: 73%. Mp:204-207℃ H NMR (400 MHz, CDCl
3
) δ 7.82 (s, 1H), 7.69 (t, J = 8.7 Hz, 1H), 7.57 (s,
1
9
1
1
H), 7.16 (d, J = 11.1 Hz, 3H), 6.82 – 6.61 (m, 3H), 4.25 (t, J = 6.7 Hz, 2H), 4.12 – 3.99 (m, 2H), 3.94 (t, J = 10.1 Hz,
1
3
3
H), 2.38 (d, J = 7.1 Hz, 6H), 2.22 – 2.02 (m, 2H), 1.83 (d, J = 19.3 Hz, 2H); C NMR (101 MHz, CDCl ) δ 182.74 (s),
68.18 (s), 166.42 (s), 153.29 (s), 147.28 (s), 142.58 (s), 142.17 (s), 139.80 (s), 132.15 (s), 131.10 (s), 127.78 (s),
25.85 (s), 120.45 (s), 114.96 (s), 112.21 (s), 109.77 (s), 108.70 (s), 97.16 (s), 68.08 (s), 61.02 (s), 56.21 (s), 44.67

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+
(
32 2 6
s), 26.54 (s), 26.29 (s), 20.64 (s), 20.26 (s). MS (ESI):528.1 (C31H N O , [M+H ]).
4
.1.3.22.7-(4-(5(6)-methyl-1H-benzo[d]imidazol-1-yl)butoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (26).
1
Yellow powder, yield: 54%. Mp:159-163℃ H NMR (400 MHz, CDCl
3
) δ 7.88 (d, J = 7.1 Hz, 1H), 7.75 – 7.58 (m, 2H),
7
3
.30 (d, J = 8.3 Hz, 1H), 7.22 – 7.08 (m, 3H), 6.78 – 6.64 (m, 3H), 4.27 (d, J = 3.1 Hz, 2H), 4.08 (d, J = 5.2 Hz, 2H),
13
.93 (d, J = 9.0 Hz, 9H), 2.50 (d, J = 6.4 Hz, 3H), 2.11 (dd, J = 23.2, 16.2 Hz, 2H), 1.84 (t, J = 19.2 Hz, 2H); C NMR
(
101 MHz, CDCl
3
) δ 182.76 (s), 168.19 (s), 166.40 (s), 153.30 (s), 147.29 (s), 142.84 (s), 142.47 (s), 127.79 (s),
1
6
25.92 (s), 124.51 (s), 123.85 (s), 120.27 (s), 115.02 (s), 112.24 (s), 109.45 (s), 109.09 (s), 108.73 (s), 97.19 (s),
8.08 (s), 61.03 (s), 56.23 (s), 44.76 (s), 44.62 (s), 26.65 (s), 26.32 (s), 21.88 (s), 21.53 (s). MS (ESI):514.1
+
(
C
30
H
30
N
2
O
6
, [M+H ]).
4
.1.3.23.7-(4-(2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)butoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-on
1
e (27). Yellow powder, yield: 63%. Mp:153-156℃ H NMR (400 MHz, CDCl
3
) δ 7.92 (d, J = 7.8 Hz, 1H), 7.72 (t, J =
8
4
.4 Hz, 1H), 7.48 (q, J = 8.0 Hz, 2H), 7.42 (dd, J = 14.8, 7.5 Hz, 1H), 7.17 (s, 2H), 6.81 – 6.68 (m, 2H), 5.32 (s, 1H),
13
.47 (t, J = 7.3 Hz, 2H), 4.15 (t, J = 5.1 Hz, 2H), 3.96 (t, J = 10.3 Hz, 9H), 2.25 – 2.09 (m, 2H), 1.98 (s, 2H); C NMR
) δ 182.79 (s), 168.20 (s), 166.33 (s), 153.32 (s), 147.28 (s), 141.21 (s), 139.87 (s), 135.31 (s),
27.78 (s), 125.96 (s), 125.46 (s), 123.78 (s), 121.87 (s), 115.13 (s), 112.29 (s), 112.22 (s), 110.42 (s), 108.76 (s),
(
101 MHz, CDCl
3
1
9
27 3 2 6
7.22 (s), 68.03 (s), 61.03 (s), 56.23 (s), 53.45 (s), 44.77 (s), 26.86 (s), 26.26 (s). MS (ESI):568.2 (C30H F N O ,
+
[
M+H ]).
4
.1.3.24.7-(4-(5(6)-methoxy-1H-benzo[d]imidazol-1-yl)butoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (28).
1
Yellow powder, yield: 56%. Mp:87-90℃ H NMR (400 MHz, CDCl
3
) δ 7.86 (d, J = 17.0 Hz, 1H), 7.76 – 7.64 (m, 2H),
7
2
1
1
1
4
4
4
.15 (s, 2H), 6.95 (t, J = 10.7 Hz, 1H), 6.85 (s, 1H), 6.79 – 6.63 (m, 3H), 4.25 (t, J = 6.7 Hz, 2H), 4.08 (d, J = 4.7 Hz,
1
3
3
H), 3.93 (dd, J = 23.4, 14.4 Hz, 12H), 2.20 – 2.04 (m, 2H), 1.88 (s, 2H); C NMR (101 MHz, CDCl ) δ 182.79 (s),
68.19 (s), 166.39 (s), 156.83 (s), 153.31 (s), 147.28 (s), 144.72 (s), 142.99 (s), 142.16 (s), 139.85 (s), 138.41 (s),
34.32 (s), 127.78 (s), 125.95 (s), 120.96 (s), 115.04 (s), 113.35 (s), 112.31 (s), 112.22 (s), 111.27 (s), 109.97 (s),
08.75 (s), 108.52 (s), 102.37 (s), 97.20 (s), 96.29 (s), 93.35 (s), 68.09 (s), 61.03 (s), 56.24 (s), 55.94 (s), 55.82 (s),
+
4.89 (s), 44.64 (s), 26.67 (s), 26.49 (s), 26.30 (s). MS (ESI):530.1 (C30
H
30
N
2
O
7
, [M+H ]).
.2. Biological assay
.2.1. Cell culture
Human gastric tumor cell line MGC-803, breast tumor cell line MCF-7, liver tumor cell line HepG-2 and
murine gastric tumor cell line MFC were obtained from the Type Culture Collection of the Chinese Academy of
Sciences, Shanghai, China and cells were cultured in DMEM medium (Gibco, NY, USA) which was supplemented

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2
with 10% fetal bovine serum (FBS) (sigma). The cultures were incubated at 37 ℃ in humidified 5% CO incubator.
When experiments were performed in the absence of PBS, PBS was eliminated 24 h before initiating the
experiments.
4
.2.2. In vitro antiproliferative assay
The antiproliferative activities of compounds 5-28 were determined using a standard (MTT)-based
3
colorimetric assay (Sigma). The cells were seeded at a density of approximately 8×10 cells/well in 96-well
microtiter plates (Costar) and maintained at 37 ℃ in 95% humidity and 5% CO for 48 h. Different concentration
2
(
256, 128, 64, 32, 16, 8, 4, 2 ꢀM) of compounds were treated. After 48 h, the cells washed twice with PBS, and 20
L of the MTT solution (5 mg/mL in PBS) added to each well and plate was incubated at 37 ℃ . After 4 h, 100 ꢀL of
ꢀ
DMSO was added to each well to dissolve the formazan crystals and absorbance was measured at 570 nm on
aWellscanMK-2 microplate reader. The results are compared with the standard drug 5-Fu as positive control drug
against all tested cell lines. IC50 values were determined form replicates of 96-well from three independent
experiments.
4
.2.3. Apoptosis assay
The MFC cells were incubated for 48 h after different concentration of compound 15 were added and then
stained with both Annexin V-FITC (fluorescein isothiocyanate) and propidium iodide (PI). Then samples were
analyzed by FACSCalibur flow cytometer.
4
.2.4. Cell cycle assay
The MFC cells were incubated with different concentrations of compound 15 (0,10,
20,
40
ꢀ
M) in a six-well plate. After 48 h the cells were harvested by trypsinization, washed in PBS, and fixed in 70%
ice-cold (4°C) ethanol for 12 h. Then added 500 of proppidium staining to each tube. Gently vortex the cells and
incubated for 30 min at 37°C in the dark. Analyzed by flow cytometry.
4
.2.5. In vivo anti-tumor efficacy
All experiments with animals were performed in compliance with the relevant laws and institutional
guidelines of China animal experiments, including Laboratory Animal Management Regulations and Guidance on
the Treatment of Experimental Animals. The Institutional Animal Care and Committee of University of South
China [permit number: SYXK (Xiang) 2015-0001] have approved the experiments.
Gastric tumor cell line MFC was chosen as a model tumor to investigate the anti-tumor efficacy of the three
doses of compound 15 (10 mg/kg, 20 mg/kg, 40 mg/kg). Gastric tumor-bearing mice were established by
7
subcutaneous injection of MFC cells in the ventral part that suspended in saline to 1×10 /0.2 ml/mouse (defined

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as day 0). On day 1, the mice were randomly divided into five groups (n = 6) and treated with three doses of
compound 15, 5-Fu (positive control 25 mg/kg) and saline (control) via a intraperitoneal injection on days 2, 5, 8,
1
1 and 14. From the 6th day on, tumor volumes were monitored every other day by measuring two perpendicular
ꢃ
diameters using Vernier caliper and calculated using the formula: Volume = 0.5ꢀ × Lengthꢀ × ꢁWidthꢂ . Body
weights were recorded every other day. On day 18, the animals were sacrificed and the tumor mass was dissected,
weight, and photographed. The tumor inhibition ratio (TIR) was calculated using the formula: TIRꢁ%ꢂ =
ꢅꢆ
ꢄ1 − ꢈ × 100%, where Wt and Ws represent the average tumor weight of the treatment and saline groups,
ꢅꢇ
respectively.
Acknowledgements
This work was financially supported the Construct Program of the key Discipline in Hunan province and Hun
an Province Cooperative Innovation Center for Molecular Target New Drug Study.
Conflict of interest
The authors declare that there are no conflicts of interest.
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HO
OH
HO
OH
HO
ZnCl2
Cl
+
N
Cl
NHHCl
HCl(g)
1
OCH3
OCH3
OH
O
OCH3
H O
OCH3 a)10%NaOH
HO
O
O
2
OCH3
+
Cl
b)10%HCl
OHC
OCH3
2
3
OCH3
OCH3
Br(CH ) Br
Br(CH ) O
2
n
2 n
O
OCH3
K CO ,acetone
2
3
O
4a n=2
4b
c
n=3
n=4
4
a-4c
4
R2
R3
N
K CO ,acetone
R1
2
3
N
H
OCH3
OCH3
R1
N
N
O
O
O
n
OCH3
R2
R3
5-28
5
6
7
8
9
1
1
1
n=2 R =H,R =H,R =H
13 n=3 R1=H,R2=H,R3=H
14 n=3 R1=CH3,R2=H,R3=H
15 n=3 R1=Cl,R2=H,R3=H
16 n=3 R1=H,R2=H,R3=NO2
17 n=3 R1=H,R2=CH3,R3=CH3
18 n=3 R1=H,R2=CH3,R3=H
19 n=3 R1=CF3,R2=H,R3=H
20 n=3 R1=H,R2=OCH3,R3=H
21 n=4 R1=H,R2=H,R3=H
22 n=4 R1=CH3,R2=H,R3=H
23 n=4 R1=Cl,R2=H,R3=H
24 n=4 R1=H,R2=H,R3=NO2
25 n=4 R1=H,R2=CH3,R3=CH3
26 n=4 R1=H,R2=CH3,R3=H
27 n=4 R1=CF3,R2=H,R3=H
28 n=4 R1=H,R2=OCH3,R3=H
1
2
3
n=2 R =CH ,R =H,R =H
1
3
2
3
n=2 R =Cl,R =H,R =H
1
2
3
n=2 R =H,R =H,R =NO
2
1
2
3
n=2 R =H,R =CH ,R =CH
3
1
2
3
3
0 n=2 R =H,R =CH ,R =H
1
2
3
3
1 n=2 R =CF ,R =H,R =H
1
3
2
3
2 n=2 R =H,R =OCH ,R =H
1
2
3
3
Scheme 1 General synthesis of compounds 5-28.

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-
+
O Na
O
H CO
H
3
CO
CO
3
-
O Na+
P
H CO
O
P
H
3
O
3
O Na+
-
-
O Na+
-
-
OCH3
OCH
3
P
O
O Na+
O
O Na+
OCH3
OCH
3
CA4P
OXi4503(CAIP)
H CO
3
H CO
3
NHAc
H CO
O
3
H CO
3
OCH3
OCH3
OPO H
2 2
N
OH
NH HCl
H
OCH3
2
AVE8062(AC7700)
ZD6126
N
N
O
O
N
O
H CO
3
OH
H CO
3
O
NH HCl
2
N
S
H CO
3
OCH3
N
H CO
3
OCH3
H
OCH3
BNC-105
CKD-516
Fig. 1 The structures of CA4P, OXi4503, AVE8062, ZD6126, BNC-105 and CKD-516.
Fig. 2 MFC cells were cultured with various concentration of compound 15 for 24 h. Cells were
stained by Annexin V-FICT/PI and apoptosis was analyzed by flow cytometry.

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Fig. 3 Flow cytometry analysis of cell cycle distribution for MFC cells treated with compound 15
0, 10, 20, 40 μmol/L) for 24 h.
(
1
1
200
000
Saline
40mg/kg
20mg/kg
10mg/kg
5-Fu
800
600
400
200
0
6
8
10
12
14
16
18
Time (days)
Fig. 4 Tumor volume growth curves of the mice after treatment with saline, different
concentrations of compound 15 (10, 20, 40 mg/kg) and 5-Fu. Data were showed as a mean ± SD
(
(
n = 6). The difference of tumor volume growth among five groups was statistically significant
P<0.05).

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Fig. 5 Photographs of the harvested tumors from the mice of 18 days (left); Average tumor
weight of the mice of 18 days (right).
35
30
25
20
15
10
saline
10mg/kg
20mg/kg
40mg/kg
5-Fu
0
2
4
6
8
10 12 14 16 18 20
Time (days)
Fig. 6 Body weight evolution curves of the mice after treatment with saline, different
concentrations of compound 15 (10, 20, 40 mg/kg) and 5-Fu. Data were showed as a mean ± SD
(
n = 6). No significant body weight loss was observed compared with the saline group.

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Table 1 Anti-proliferative activity of compounds against the tumor cell lines
IC50 (µM) ±SD
compounds
MGC-803
>100
MCF-7
>100
HepG-2
>100
MFC
>100
5
6
7
8
9
94.47±5.21
75.12±3.47
91.54±4.26
>100
>100
84.32±3.54
82.53±3.28
87.53±3.28
N.D
87.68±5.47
87.34±4.39
95.17±3.21
N.D
72.56±2.83
90.73±5.14
N.D
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
84.58±3.42
75.43±3.12
>100
>100
>100
>100
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
84.71±4.17
N.D
>100
80.37±3.62
>100
>100
N.D
N.D
N.D
>100
77.52±3.71
20.47±2.07
83.14±2.52
N.D
91.46±4.36
43.42±3.56
90.47±5.32
>100
>100
88.46±3.16
23.47±3.59
88.43±3.63
N.D
35.45±2.03
>100
N.D
97.38±5.54
77.27±4.76
>100
>100
N.D
>100
92.37±3.62
N.D
86.83±4.73
N.D
82.24±3.81
90.32±2.17
>100
94.38±4.76
73.62±4.73
63.51±2.64
54.86±4.28
N.D
N.D
N.D
81.43±2.57
78.34±3.53
>100
>100
77.42±4.37
67.15±4.26
62.37±5.14
>100
76.82±5.39
72.48±3.51
>100
N.D
95.62±5.34
76.27±3.74
84.73±5.12
74.39±2.03
87.46±4.17
62.37±2.35
>100
>100
90.01±3.23
58.43±4.43
>100
>100
N.D
5
-Fu
57.09±3.17
63.37±2.52
78.52±3.92
SD=standard deviation
N.D=not detected

Page 23 of 23
MedChemComm
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DOI: 10.1039/C7MD00578D