Novel tertiary sulfonamide derivatives containing benzimidazole moiety as potent anti-gastric cancer agents: Design, synthesis and SAR studies

Article abstract of DOI:10.1016/j.ejmech.2019.111731

With the expectation to find out new anti-gastric cancer agents with high efficacy and selectivity, a series of novel tertiary sulfonamide derivatives were synthesized and the anti-cancer activity was studied in three selected cancer cell lines (MGC-803, PC-3, MCF-7) in vitro. Some of the synthesized compounds could significantly inhibit the proliferation of these tested cancer cells and were more potent than the positive control (5-Fu). The structure-activity relationship of tertiary sulfonamide derivatives was explored in this report. Among the tested compounds, compound 13g containing benzimidazole moiety showed the best anti-proliferation activities against MGC-803 cells (IC₅₀ = 1.02 μM), HGC-27 cells (IC₅₀ = 1.61 μM), SGC-7901 (IC₅₀ = 2.30 μM) cells as well as the good selectivity between the cancer and normal cells. Cellular mechanism studies elucidated compound 13g inhibited the colony formation of gastric cancer cell lines. Meanwhile, compound 13g arrested cell cycle at G2/M phase and induced cell apoptosis. Mechanistically, compound 13g markedly decreased p-Akt and p-c-Raf expression, which revealed that compound 13g targeted gastric cancer cell lines via interfering with AKT/mTOR and RAS/Raf/MEK/ERK pathways. All the findings suggest that compound 13g might be a valuable lead compound for the anti-gastric cancer agents.

Full text of DOI:10.1016/j.ejmech.2019.111731

European Journal of Medicinal Chemistry 183 (2019) 111731
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel tertiary sulfonamide derivatives containing benzimidazole
moiety as potent anti-gastric cancer agents: Design, synthesis and SAR
studies
,
c
,
,
c
,
,
,
,
, c
Jian-Song b 1, Qiu-Lei Gao b 1, Bo-Wen Wu b c, Dong Li b c, Lei Shi b c, Ting Zhu b
,
Jian-Feng Lou b c, Cheng-Yun Jin b c, Yan-Bing Zhang b c, Sai-Yang Zhang a
,
,
,
,
, b, d, *
Hong-Min Liu b
,
c
a School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, 450001, China
b School of Pharmaceutical Sciences & Collaborative Innovation Center of New Drug Research and Safety Evaluation, Zhengzhou University, Zhengzhou,
450001, China
c Key Laboratory of Advanced Drug Preparation Technologies (Ministry of Education), Zhengzhou University, Zhengzhou, 450001, China
d Henan Institute of Advanced Technology, Zhengzhou University, Zhengzhou, 450001, China
a r t i c l e i n f o
a b s t r a c t
Article history:
With the expectation to find out new anti-gastric cancer agents with high efficacy and selectivity, a series
of novel tertiary sulfonamide derivatives were synthesized and the anti-cancer activity was studied in
three selected cancer cell lines (MGC-803, PC-3, MCF-7) in vitro. Some of the synthesized compounds
could significantly inhibit the proliferation of these tested cancer cells and were more potent than the
positive control (5-Fu). The structure-activity relationship of tertiary sulfonamide derivatives was
explored in this report. Among the tested compounds, compound 13g containing benzimidazole moiety
Received 10 August 2019
Received in revised form
8 September 2019
Accepted 21 September 2019
Available online 25 September 2019
showed the best anti-proliferation activities against MGC-803 cells (IC50 ¼ 1.02
mM), HGC-27 cells
Keywords:
Gastric cancer
(IC50 ¼ 1.61 M), SGC-7901 (IC50 ¼ 2.30 M) cells as well as the good selectivity between the cancer and
m
m
normal cells. Cellular mechanism studies elucidated compound 13g inhibited the colony formation of
gastric cancer cell lines. Meanwhile, compound 13g arrested cell cycle at G2/M phase and induced cell
apoptosis. Mechanistically, compound 13g markedly decreased p-Akt and p-c-Raf expression, which
revealed that compound 13g targeted gastric cancer cell lines via interfering with AKT/mTOR and RAS/
Raf/MEK/ERK pathways. All the findings suggest that compound 13g might be a valuable lead compound
for the anti-gastric cancer agents.
Tertiary sulfonamide
Benzimidazole
Anti-gastric cancer activity
Apoptosis
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
therapeutic use [4,5]. Thus, it is highly desirable to develop newly
targeted therapy for gastric cancer with minimal side-effects and
Gastric cancer (GC), a very aggressive tumor, is the fifth most
common malignancy with almost one million new cases diagnosed
globally each year and remains one of the deadliest malignant tu-
mors among Asians [1e3]. Although surgery procedures and
chemotherapy have improved greatly in recent years, undesirable
drug-induced toxicities and deleterious side effects owing to
insufficient selectivity against healthy mammalian cells limit their
new class of anti-cancer agents with the excellent selectivities be-
tween cancer and normal cells. Akt/mTOR and RAS/Raf/MEK/ERK
pathways play an important role in cellular functions, and they are
often overexpressed and uncontrolled in many human cancer cells
such as gastric cancer cells, breast cancer cells, and prostate cancer
cells [6e8]. Akt and mTOR are 2 pathways have cross-talks and
compensations with MAPK pathway. Moreover, The Akt/mTOR and
Ras/Raf/MEK/ERK pathways have been identified as promising
therapeutic targets for cancer therapy including gastric cancer
[9e11]. Therefore, targeting Akt/mTOR and Ras/Raf/MEK/ERK
pathways is one of the effective ways to gastric tumors.
* Corresponding author. School of Basic Medical Sciences, Zhengzhou University,
Zhengzhou, 450001, China.
The sulfonamide derivatives are known for their numerous
pharmacological activities in medicinal chemistry and modern
E-mail address: saiyangz@zzu.edu.cn (S.-Y. Zhang).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2019.111731
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
Jian-Song et al. / European Journal of Medicinal Chemistry 183 (2019) 111731
drug discovery, such as anti-bacterial [12e14], anti-tumor [15e21],
anti-HIV [22e24] and anti-inflammatory activities [25e27].
Recently, the tertiary sulfonamide fragment was frequently utilized
to design novel anticancer agents to enhance the biological efficacy,
such as tubulin polymerization inhibitors [15,16,19,28], carbonic
anhydrase inhibitors [18,20,29], HIF-1 pathway inhibitors [30], cell
cycle inhibitors [31] and MEK inhibitors [32,33]. Styryl-pyridine N-
oxide sulfonamide 1 [28] demonstrated effective inhibition of
tubulin polymerization and were found to be potent antimitotic
agents. Compound 2 [15] showed excellent activity as tubulin
0.44 mM against murine L1210 cell line (Fig. 1). Based on the above
findings mentioned, sulfonamide moiety was chosen as a core for
designing tertiary sulfonamide derivatives with anti-gastric cancer
activity.
Recently, our group has reported several series of novel tertiary
amide derivatives that exhibited excellent antiproliferative activity
against gastrointestinal track tumor cell lines [34e36]. In one of the
studies mentioned above, tertiary amide derivative containing
benzimidazole moiety (TRF-7, in Fig. 2) showed potent anti-gastric
cancer cells (MGC-803) with an IC50 value of 1.23 mM but weak
polymerization inhibitor with an IC50 value of 1.1
the antiproliferative activity with an IC50 value of 0.086
HT29 cells. Tertiary sulfonamide derivatives of pyridyl-indole based
heteroaryl chalcone 3 [20] exhibited the significant inhibition of
m
M and exhibited
activities against human prostate cancer cell line (PC-3) and human
esophageal cancer cell line (EC-109) [34]. In addition, benzimid-
azole and its derivatives occupy an important place in medicinal
chemistry due to their synthetic utility and diversified biological
activities [37]. Benzimidazole derivatives have been reported to
have potent anti-proliferative effect against many types of cancer
and benzimidazole moiety was important for anticancer activity
[38e40]. Therefore, benzimidazole moiety was introduced into the
designed hybrids to optimize for better efficacy and lower toxicity.
The molecular hybridization approach is a widely used strategy
in drug discovery of rational design new molecular entities by
incorporating two or more bioactive substructures through suitable
mM against
hCA IX activity with an IC50 value of 0.13 mM. Compound 4 was
observed strong selectivity toward tumor-associated hCA IX,
without inhibiting the off target hCA II with an IC50 value of 4.2 nM
against hCA IX [29]. Compound 5 inhibited HIF activity in an HRE-
dependent cell-based assay with an IC50 value of approximate
590 nm and displayed promising antitumor activity in animal
models [30]. 4-Methoxy-N-(3-chloro-7-indazolyl) benzenesulfo-
namide 6 [31] was identified as the most potent with an IC50 of
Fig. 1. Structures of tertiary sulfonamide derivatives with anticancer activity.
Fig. 2. Designed tertiary sulfonamide derivative 13g in this work.

Jian-Song et al. / European Journal of Medicinal Chemistry 183 (2019) 111731
3
linkages. As the continuation of our studies on the development of
anti-gastric cancer agents, we designed and synthesized a series of
tertiary sulfonamide derivatives containing benzimidazole moiety
by the scaffold hopping and molecular hybridization approaches.
The structure-activity relationships of tertiary sulfonamide de-
rivatives containing benzimidazole moiety were further discussed.
Finally, the mechanistic study of compound 13g containing benz-
imidazole moiety was investigated (Fig. 2).
acetone to give the secondary amine 9. The intermediate com-
pound 9 reacted with different benzenesulfonyl chloride de-
rivatives in the presence of triethylamine in DMF to obtain the
tertiary
sulfonamide
derivatives
10a-10e.
And,
3,4,5-
trimethoxyaniline 7 reacted with benzyl chlorides 11a-11g in the
presence of K2CO3 in acetone to give the secondary amines 12a-12g.
The 12a-12g reacted with 4-toluene sulfonyl chloride to obtain the
tertiary sulfonamide derivatives 13a-13g. The intermediate com-
pound 12g reacted with different benzene sulfonyl chloride de-
rivatives or different acyl chloride derivatives to obtain the tertiary
sulfonamide derivatives 14a-14f and the tertiary amides 18a-18f,
respectively. The amines 15 reacted with 11g obtain the tertiary
sulfonamide derivatives 17a-17f. Characterization of all the com-
pounds was carried out by means of 1H, 13C NMR and HREI-mass
2. Chemistry
The synthetic routes of target tertiary sulfonamide derivatives
were shown in Scheme 1. First, 3,4,5-trimethoxyaniline 7 reacted
with 4-methoxybenzyl chloride 8 in the presence of K2CO3 in
Scheme 1. Reagents and conditions: a) K2CO3, acetone, reflux, 6 h; b) benzenesulfonyl chloride derivatives, TEA, DMF, rt; c) acyl chloride derivatives, TEA, DMF, rt.

4
Jian-Song et al. / European Journal of Medicinal Chemistry 183 (2019) 111731
Table 3
spectra. The detailed physical and analytical data are given in the
experimental part.
In vitro anti-proliferative activity of tertiary sulfonamide derivatives 14a-14f against
human cancer cell lines (MGC-803, PC-3, and MCF-7 cells).
3. Results and discussion
3.1. Anti-proliferative activity and SAR discussion
The anticancer activity of synthesized compounds was evalu-
ated against human gastric cancer MGC-803 cell line, PC-3 human
prostate cancer cell line and MCF-7 human breast adenocarcinoma
cell line using MTT assay and the well-known anticancer drug 5-
fluorouracil as reference drug. The preliminary results are listed
in Table 1, Table 2, Table 3, Table 4 and Table 5. The anticancer
Compound
R3
IC50 (m
M)a
MGC-803
PC-3
MCF-7
14a
14b
14c
14d
14e
14f
H
4-F
4-Br
2-Cl
4-C(CH3)3
3,4-diOCH3
e
12.38 0.99
5.68 0.86
2.19 0.07
19.12 2.19
24.10 0.14
22.51 2.14
6.82 1.17
25.49 2.73
7.99 1.24
6.89 0.46
34.52 1.94
29.81 1.93
30.37 1.34
18.42 1.73
27.29 2.34
9.38 1.08
7.74 0.39
38.12 2.19
33.64 1.18
47.73 1.96
17.11 2.94
Table 1
In vitro anti-proliferative activity of tertiary sulfonamide derivatives 10a-10e against
human cancer cell lines (MGC-803, PC-3, and MCF-7 cells).
5-Fu
a
Antiproliferative activity was assayed by exposure for 48 h.
activity of compound 13g was also evaluated against human gastric
cancer cell lines (MGC-803 and SGC-7901), human cell line derived
from the metastatic lymph node of gastric cancer (HGC-27) and
gastric epithelial cell line (GES-1) as shown in Table 6. Based on the
obtained data, structure-activity relationship was analyzed and
summarized in Fig. 3.
Compound
R1
IC50 (m
M)a
MGC-803
PC-3
MCF-7
Compounds 10a-10e were initially evaluated for their anti-
proliferative activities against MGC-803, PC-3, and MCF-7 cancer
cell lines to research the effect of tertiary sulfonamide derivatives
with 5-Fu as the positive control. As shown in Table 1, the majority
of the synthesized compounds exhibited moderate to potent ac-
tivity against the tested cancer cell lines. The potency of the com-
pounds varies with respect to substitutions on the simple phenyl
ring (C ring). The benzene sulfonyl group of C ring displayed weak
cytotoxicity(10b). To our delight, the compound 10a with 4-
methylbenzene sulfonyl group of C ring showed the most potent
activity against MGC-803, PC-3, and MCF-7 cells with IC50 values of
10a
10b
10c
10d
10e
5-Fu
4-CH3
H
4-C(CH3)3
3,4-diOCH3
2,4,6-triCH3
e
4.34 0.67
>50
10.31 1.93
8.31 2.67
6.44 1.58
6.82 1.17
8.22 1.08
>50
28.56 2.04
20.14 2.28
14.82 2.04
18.42 1.73
10.14 1.12
>50
32.72 3.27
22.71 1.42
18.65 1.58
17.11 2.94
a
Antiproliferative activity was assayed by exposure for 48 h.
Table 2
In vitro anti-proliferative activity of tertiary sulfonamide derivatives 13a-13g against
human cancer cell lines (MGC-803, PC-3, and MCF-7 cells).
4.74 mM, 8.22 mM and 10.14 mM, respectively.
As illustrated above with Table 1, the 4-methylbenzene sulfonyl
group of C ring(10a) showed the most potent activity against MGC-
803 cells, PC-3 cells and MCF-7 cells. To further investigate the in-
fluence of A ring in activity, compounds 13a-13g containing
different aryl substituents on A ring with the 4-methyl benzene
sulfonyl group of C ring and a 3,4,5-trimethoxy group of B ring at
the N-1 position were synthesized and the anti-proliferative ac-
tivities were listed in Table 2. The results indicated that most of the
synthesized compounds displayed high anti-proliferative activities
against selected cancer cell lines, especially for MGC-803 cells. The
potency of the compounds varies with respect to substitutions on
the A ring. Compounds 13a-13c, with electron-withdrawing groups
on the A ring, showed improved inhibitory activity against MGC-
803 cells compared to both its electron-donating groups on the A
ring (compounds 10a and 13d) and unsubstituted phenyl group on
the A ring (compound 13e). Compared with compound 13e, the
electron-donating group substituted compound 10a and com-
pound 13d showed a slightly lower activity. What’s more, com-
pounds 13f-13g, with heterocyclic groups on the A ring showed
improved inhibitory activity against MGC-803 cells with IC50 values
Compound
R2
IC50 (m
M)a
MGC-803
PC-3
MCF-7
13a
13b
13c
13d
13e
13f
4-Cl
4-F
4-Br
3-CH3
H
1.72 0.26
3.75 0.48
1.61 0.36
4.34 2.19
3.57 0.61
3.45 0.29
14.59 1.12
20.71 2.01
4.68 0.68
8.58 1.24
7.81 0.72
15.25 1.02
18.64 1.08
19.20 1.61
10.89 1.47
14.28 1.82
10.22 1.06
8.95 0.74
13g
1.02 0.03
6.82 1.17
3.34 0.09
5.40 0.51
of 1.02 0.03
13g showed the most potent activities against MGC-803, PC-3 and
MCF-7 cells with IC50 values of 1.02 0.03 M, 3.34 0.09 M, and
mM and 3.45 0.29 mM, respectively. And, compound
5-Fu
e
18.42 1.73
17.11 2.94
m
m
a
Antiproliferative activity was assayed by exposure for 48 h.

Jian-Song et al. / European Journal of Medicinal Chemistry 183 (2019) 111731
5
Table 4
In vitro anti-proliferative activity of tertiary sulfonamide derivatives 17a-17f against human cancer cell line (MGC-803, PC-3, and MCF-7 cells).
Compound
R5
IC50 (m
M)a
MGC-803
PC-3
MCF-7
17a
17b
17c
17d
17e
17f
4-F
4-Cl
H
4-CH3
4-OCH3
3,4-diOCH3
6.82 1.17
34.62 1.41
24.77 2.28
45.18 1.39
41.25 2.64
>50
44.96 1.84
42.19 2.41
>50
48.91 2.08
>50
>50
38.29 2.04
49.67 1.04
>50
>50
>50
>50
18.42 1.73
>50
17.11 2.94
5-Fu
e
a
Antiproliferative activity was assayed by exposure for 48 h.
Table 5
with respect to substitutions on the C ring. MGC-803 cells were also
more sensitive to the compounds 14a-14f than PC-3 and MCF-
7 cells. Compound 13g with the methyl group on the C ring showed
the most potent activity against MGC-803 cells with an IC50 value of
In vitro anti-proliferative activity of tertiary amides 18a-18g against human cancer
cell lines (MGC-803, PC-3, and MCF-7 cells).
1.02 0.03 mM, which was higher than compound 14a with
hydrogen atom and 14b, 14c, 14d with halogen atoms on the C ring.
Compound 14c exhibited slightly lower activity compared to 13g
against MGC-803 cells with an IC50 value of 2.19 mM. The relation-
ships between the halogen substituents and the antiproliferative
activities were 4-Br>4-F>2-Cl. In addition, compounds 14e and 14f
were less potent activity than compound 10e.
In this series of derivatives, we mainly explored the significance
of the 3,4,5-trimethoxy group of phenyl ring (B ring) at the N-1
position. These inhibitory activity results indicated that the 3,4,5-
trimethoxy group of B ring at the N-1 position was very crucial
for the antitumor activity. When the 3,4,5-trimethoxy group was
replaced by other groups (4-trimethoxy, 3,4-dimethoxy, 4-methyl,
halogen substituents, and hydrogen atom), compounds 17a-17h
Compound
R6
IC50 (m
M)a
MGC-803
PC-3
MCF-7
18a
18b
18c
18d
18e
18f
H
4-F
4-Br
3-Cl
4-CH3
4-C(CH3)3
3,4-diOCH3
e
>50
>50
>50
45.23 5.78
>50
34.86 2.62
49.25 1.42
48.81 1.32
>50
23.55 1.17
14.55 2.21
>50
30.47 2.48
>50
21.86 1.89
19.93 1.63
>50
42.04 1.00
46.04 1.00
34.86 2.62
18.42 1.73
displayed very weak activity with almost IC50 values of >50 mM
18g
5-Fu
22.38 1.08
6.82 1.17
against three selected cancer cell lines. Based on the inhibitory
activity results, the 3,4,5-trimethoxy group of B ring displayed an
important effect for the antiproliferative activity. Other substituent
groups may significantly impair anti-proliferative activity.
17.11 2.94
a
Antiproliferative activity was assayed by exposure for 48 h.
In order to complete the structure activity relationships, a series
of tertiary amide derivatives were synthesized and evaluated for
their antiproliferative activity. As shown in Table 5, some of these
compounds retain the cytotoxic activity(18a-18g) but displayed
weak activity against three selected cancer cell lines compared to
tertiary sulfonamide derivatives (13g and 14a-14f). All these
modifications and the inhibitory results revealed that the sulfonyl
groups were important for their inhibitory activity which can
maintain or enhance the anti-proliferative activity against three
tested human cancer cells.
Furthermore, MGC-803 cells were more sensitive to the com-
pounds than PC-3 cells, and MCF-7 cells. What’s more, as part of our
group’s research focus and continuation of our studies on the
development of anti-gastric cancer agents, we chose two other
common gastric cancer cell lines (HGC-27 and SGC-7901), so
compound 13g was examined for its activity against gastric cancer
cells (MGC-803, HGC-27 and SGC-7901) and non-cancer cell lines
(GES-1). As shown in Table 6, compounds 13g exhibited high ac-
tivity against MGC-803 cells, HGC-27 cells and SGC-7901 cells with
Table 6
In vitro anti-proliferative activity of 13g against gastric cancer cells (MGC-803, HGC-
27 and SGC-7901) and non-cancer cell lines (GES-1).
Compd.
IC50
(
m
M)a
Fold selectivity
MGC-803
HGC-27
SGC-7901
GES-1
A
B
C
13g
5-Fu
1.02
6.82
1.61
4.28
2.30
5.54
15.22
10.21
14.9
1.49
9.4
2.4
6.6
1.84
A ¼ IC50 (GES-1)/IC50 (MGC-803).
B ¼ IC50 (GES-1)/IC50 (HGC-27).
C ¼ IC50 (GES-1)/IC50 (SGC-7901).
5.40 0.51 mM respectively. This observation suggests that the
electron deficiency or richness of the A ring system may affect the
antiproliferative activity to a certain extent and benzimidazole or
benzothiophene motifs enhanced anticancer activity.
With the identification of benzimidazole region established
based on the SAR analysis, the focus shifted to the optimization of C
ring. As shown in Table 3, The potency of the compounds varies

6
Jian-Song et al. / European Journal of Medicinal Chemistry 183 (2019) 111731
Fig. 3. Summary of the structure-activity relationships.
IC50 values of 1.02
m
M, 1.61
m
M and 2.30
m
M, respectively. Com-
for MGC-803, Fig. 4B for HGC-27, Fig. 4C for SGC-7901). After
treatment for 48 h, compound 13g limited three cancer cells
growth lower than 2 folds. 3 gastric cancer cell lines and 1 normal
cell line were next treated with different concentration of com-
pound 13g, compound 13g inhibited cell viability of gastric cancer
cells in a dose-dependent manner but had a low cytotoxicity
against normal cell (Fig. 4D). These results indicated that com-
pound 13g selectively inhibited gastric cancer cells.
pounds 13g exhibited moderate activity against GES-1 cells with an
IC50 value of 15.22 M. Notably, compounds 13g exhibited highest
m
activity against three test human cancer cells. What’s more, com-
pounds 13g also displayed the high selectivity between the gastric
cancer cells and non-cancer cell lines (around 14.9-, 9.4- and 6.6-
fold selectivity to MGC-803 cells, HGC-27 cells and SGC-7901 cells
over GES-1 cells, respectively).
Based on above analysis of the structure-activity relationship,
we can conclude that the sulfonyl groups are important for the
activity. The electron deficiency or richness of the A ring system
may affect the antiproliferative activity to a certain extent.
Benzimidazole or benzothiophene motifs may enhance selec-
tivity to the tested cancer cell lines. The 3,4,5-trimethoxy group of B
ring at the N-1 position displayed an important effect for the
antiproliferative activity. The potency of the compounds varies
with respect to substitutions of the C ring. The summary illustration
for SAR studies of target derivatives was shown in Fig. 3.
3.3. Compound 13 g inhibited the proliferation of gastric cancer
cells
To detect the activity of compound 13g influencing cell prolif-
eration, colony formation assay was performed using three gastric
cancer cells. The colony numbers were evidently reduced by low
dose (0.5
(1.5 mol/L), there was barely colony obtained (Fig. 5C). It was
demonstrated that compound 13g inhibited the proliferation of
MGC-803, HGC-27 and SGC-7901 cells in dose-dependent
mmol/L) treatment. In mid dose (1 mmol/L) and high dose
m
In view of an effective growth inhibitory effect on both of the
tested cancer cells, compound 13g was chosen to further evaluate
its possible anticancer mechanism of action against MGC-803,
HGC-27 and SGC-7901 cells in all the subsequent experiments.
a
manner. Next, cell cycles of treated cells with different concentra-
tions of compound 13g or DMSO were analyzed. As shown in Fig. 5A
and B, gastric cancer cells were significantly arrested in G2/M phase
after 48 h treatment with compound 13g. The G2/M related pro-
teins were detected, CyclinB and CDK1 are two members of com-
plex molecules which promote cell through G2 phase to M phase.
After treatment with compound 13g for 48 h, CDK1 and CyclinB
was clearly decreased (Fig. 5D) which would result in down-
regulation of CyclinB/CDK1 complex and lead to the G2 phase
arrestment. As the cell cycle analysis cannot distinguish between
G2 and M phases, the M phase arrest biomarker p-Histone3 was
checked. As a result the up-regulation of p-Histone3 indicated the
compound 13g treatment also caused a M phase arrestment
(Fig. 5D). To sum up, compound 13g treatment lead to G2 and M
phase arrestment resulting in proliferation inhibition against 3
gastric cancer cells.
3.2. Compound 13 g selectively inhibited the cell viability of gastric
cancer cells
Compound 13g was selected to do the mechanism study based
on the results of cell viability. It showed a highest activity against
MGC-803 cells of compound 13g, and also, compound 13g inhibited
two other gastric cancer cells with potent activities (HGC-27 and
SGC-7901). We detected three gastric cancer cells in the manner
way to determine whether the mechanisms by which compound
13g inhibited three human gastric cell lines are the same. For this
reason, two other gastric cancer cell lines HGC-27, SGC-7901 were
selected to detect the activity of compound 13g inhibiting gastric
cancer cells. Normal gastric epithelial cell GES-1 was used as a
control group to detect the selective inhibition against cancer cells.
As shown in Fig. 4E, the IC50 values of three cancer cell lines were
much lower than IC50 value of normal cell GES-1. The IC50 values
3.4. Compound 13 g inhibited the Akt/mTOR and Ras/Raf/MEK/ERK
signaling pathways in gastric cancer cells
were 1.02
L(SGC-7901), respectively, while the IC50 value of GES-1 was
15.22 mol/L. As shown in Fig. 4, compound 13g exhibited the
proliferation inhibition against three gastric cancer cell lines(Fig. 4A
m
mol/L (MGC-803), 1.61
m
mol/L (HGC-27), 2.30
m
mol/
The Ras/Raf/MEK/ERK signaling pathway acts a critical role in
cell survival and proliferation, this pathway promotes cell survival
and proliferation through a cascade of RAS-Raf-MEK-ERK. The in-
hibition of Ras/Raf/MEK/ERK signaling pathway will lead to cell
m

Jian-Song et al. / European Journal of Medicinal Chemistry 183 (2019) 111731
7
Fig. 4. (A) Growth Curve of MGC-803 cancer cells treated with different concentration of compound 13g. (B) Growth Curve of HGC-27 cancer cells treated with different con-
centration of compound 13g. (C) Growth Curve of SGC-7901 cancer cells treated with different concentration of compound 13g. (D) Four gastric cells viabilities after treated with
different concentration of compound 13g. (E) IC50 values of compound 13g against three gastric cell lines and normal cell line.
proliferation inhibition or cell apoptosis. Here as the Fig. 6A shown,
compound 13g did not show effect on RAS, but had an obvious
influence on its down streams. The activation (phosphonate) of c-
Raf, MEK1, ERK1/2 were evidently down-regulated in a dose-
dependent manner. The transcription factors FoxO3a and c-Myc
which regulated by Ras/Raf/MEK/ERK signaling pathway were also
down-regulated (Fig. 6A). Akt and mTOR are 2 pathways have
cross-talks and compensations with MAPK pathway, biomarkers of
both pathway, p-Akt (473Ser) and p-mTOR (2448Ser), were both
downregulated at same time which suggested compound 13g
influenced all these pathways (Fig. 6A). To detect the relationship of
compound 13g activity and Ras/Raf/MEK/ERK signaling pathway,
combination treatment with another Ras/Raf/MEK/ERK signaling
pathway inhibitor piperlongumine was performed [41,42]. As
shown in Fig. 6B, the combination index (CI) was greater than 1,
which exhibited the antagonism between compound 13g and
piperlongumine. The CI analysis verified that compound 13g might
inhibit Ras/Raf/MEK/ERK signaling pathway as piperlongumine did.
These results indicated compound 13g regulated 3 gastric cancer
cells via the AKT/mTOR and Ras/Raf/MEK/ERK signaling pathway
and down-regulated the level of FoxO3a and c-Myc.
down-regulated (Fig. 7D). These data suggested that compound 13g
induced MGC-803, HGC-27 and SGC-7901 cells apoptosis.
4. Conclusions
In summary, a series of novel tertiary sulfonamide derivatives
containing benzimidazole moiety were synthesized and evaluated
for their antiproliferative activity. All the targeted compounds were
first reported in this work except compounds 13a, 13b and 13e.
Among these compounds, in comparison of 5-FU, compound 13g
showed better activity against the tested cells (MGC-803, PC-3 and
MCF-7) with IC50 values of 1.02 mM, 3.34 mM and 5.40 mM, respec-
tively. Moreover, compound 13g not only had the high anti-
proliferative activity against MGC-803 cells, HGC-27 cells and
SGC-7901 cells with IC50 values of 1.02 mM, 1.61 mM and 2.30 mM,
respectively, but also it displayed the highly selectivity between the
tested gastric cancer cell lines and normal cells (14.9-fold selec-
tivity to the MGC-803 cancer cells over the GES-1 normal cells, 9.9-
fold selectivity to the HGC-27 cancer cells over the GES-1 normal
cells and 6.9-fold selectivity to the SGC-7901 cells over the GES-1
normal cells, respectively). Further results suggested that potent
compound 13g inhibited the colony formation in vitro, caused a cell
cycle arrestment at G2/M phase and induced cell apoptosis in 3
gastric cancer cells. Importantly, Western blot analysis indicated
that compound 13g markedly decreased p-Akt and p-c-Raf
expression, which revealed that compound 13g targeted gastric
cancer cell lines via interfering with Akt/mTOR and RAS/Raf/MEK/
ERK pathways. All the findings suggested that tertiary sulfonamide
derivatives containing benzimidazole moiety may offer significant
potentiality for the discovery of a new class anti-gastric cancer
agents with the ability to induce apoptosis and inhibit Akt/mTOR
and Ras/Raf/MEK/ERK signaling pathways.
3.5. Compound 13 g induced apoptosis of gastric cancer cells
Since the Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways
were inhibited, the induction of cell apoptosis by compound 13g
was detected. After 48 h treatment, compound 13g induced cell
apoptosis in the dose-dependent manner (Fig. 7A and B). Mean-
while, compound 13g induced cell nucleus’ concentration and DNA
fragment of 3 gastric cancer cells which were believed as a mark of
cell apoptosis (Fig. 7C). Some apoptosis related proteins were then
checked, the apoptosis executer Caspase-7 and its substrate PARP
were cleaved. Pro-apoptosis protein Noxa was up-regulated, while
the anti-apoptosis proteins Mcl-1, Bcl-2, c-IAP1 and XIAP were
Starting from the compound 10a, the 4-methylbenzene sulfonyl
group of C ring and benzimidazole group of A ring have been

8
Jian-Song et al. / European Journal of Medicinal Chemistry 183 (2019) 111731
Fig. 5. (A) Effect of compound 13g on cell cycle in three gastric cancer cells, three gastric cancer cells were treated with different concentration of compound 13g for 48 h. (B)
Quantitative analysis of the percentage of three gastric cancer cells cycle phase distribution. (C) Three gastric cancer cells were treated with different concentration of compound
13g for 7 days and stained with Crystal Violet to determine cell colony formatting activity. (D) G2/M phase related proteins were detected using western blotting, three gastric
cancer cells were treated with different concentration of compound 13g for 48 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the
Web version of this article.)
optimized, which led to a gradually improved anti-gastric cancer
cells activity. In addition, preliminary structure-activity relation-
ship research revealed that the sulfonyl groups were important for
the activity; incorporation of benzimidazole to the hybrids was the
best optimal for bioactivity; 3,4,5-trimethoxy group of B ring at the
N-1 position displayed a vital role for the antiproliferative activity.
All of these findings suggested that the compound 13g containing
benzimidazole moiety may be a promising skeleton for the devel-
opment of anti-gastric cancer agents with improved efficacy and
fewer side effects.
(Qingdao Haiyang Chemical, China). Silica gel chromatography was
carried out on 200e300 mesh gel. Anhydrous solvents and reagents
were dried by routine protocols. Melting points were determined
on an X-5 micromelting apparatus and are uncorrected. 1H NMR
and 13C NMR spectra were recorded on a Bruker 400 MHz and
100 MHz spectrometer, respectively. High resolution mass spectra
(HRMS) of all derivatives were recorded on a Waters Micromass Q-T
of Micromass spectrometer by electrospray ionization (ESI).
5.2. General procedure for the synthesis of compound 9, 12a-12 g
and 16a-16f
5. Experimental section
A solution of various aromatic amines (0.5 mmol, 1.0eq), benzyl
chlorides derivatives (0.6 mmol, 1.2eq) and K2CO3 (0.75 mmol,
1.5eq) were added into DMF(5 ml). Then the mixture was stirred at
room temperature for 5 h. Upon completion, ethyl acetate (20 ml)
and H2O (5 ml) were added. The aqueous layer was extracted with
5.1. General
All commercial materials were used without further purifica-
tion. TLC analysis was carried out on silica gel plates GF254

Jian-Song et al. / European Journal of Medicinal Chemistry 183 (2019) 111731
9
Fig. 6. The effect of compound 13g on the Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways. (A) The Akt/mTOR and Ras/Raf/MEK/ERK signaling pathway related proteins were
detected using western blotting, three gastric cancer cells were treated with different concentration of compound 13g for 48 h. (B) Combination Index (CI) analysis of compound 13g
and piperlongumine, the concentration of 13g is 1.5 mmol/L, the horizontal axis shows the concentration of piperlongumine.
Fig. 7. (A) Cell apoptosis analysis were performed using flow cytometry, three gastric cancer cells were treated with different concentration of compound 13g for 48 h. (B)
Quantitative analysis of the apoptotic cells of three gastric cancer cells. (C) Images of 48 h treated gastric cells’ nucleus. (D) Apoptosis related proteins were detected using western
blotting, three gastric cancer cells were treated with different concentration of compound 13g for 48 h.
ethyl acetate for several times, the combined organic layers were
washed with H2O for several times, and then washed with brine,
dried over MgSO4 and evaporated to give the products. The residue
was purified with column chromatography (PE: EA ¼ 2:1e3:1) to
obtain compound 9, 12a-12g, and 16a-16f.

10
Jian-Song et al. / European Journal of Medicinal Chemistry 183 (2019) 111731
5.3. General procedure for the synthesis of compound 10a-10e, 13a-
13 g, 14a-14f, 17a-17f and 18a-18 g
found 486.1950.
5.3.6. N-(4-chlorobenzyl)-4-methyl-N-(3,4,5-trimethoxyphenyl)
benzenesulfonamide(13a)
A
solution of compound 9, 12a-12g or 16a-16g (1.0eq),
White solid, yield, 69%, m.p. 162e163 ꢀC. 1H NMR (400 MHz,
substituted sulfonyl chloride (1.1eq) or substituted acyl chlor-
ide(1.1eq) and TEA(1.5eq) were added into DMF(5 ml. And then the
mixture was stirred at room temperature for 6 h. Upon completion,
ethyl acetate (20 ml) and H2O (5 ml) were added. The aqueous layer
was extracted with ethyl acetate for several times, the combined
organic layers were washed with H2O for several times, and then
washed with brine, dried over MgSO4 and evaporated to give the
products. The crude residue was purified by silica gel column
chromatography eluted with a mixture of petroleum ether and
ethyl acetate (2:1) to obtain target compound.
DMSO‑d6)
4H), 6.26 (s, 2H), 4.73 (s, 2H), 3.59 (d, J ¼ 10.3 Hz, 9H), 2.42 (s, 3H).
13C NMR (100 MHz, DMSO‑d6)
152.40, 143.71, 137.01, 135.56,
d
7.60 (d, J ¼ 7.7 Hz, 2H), 7.44 (d, J ¼ 7.9 Hz, 2H), 7.33 (s,
d
134.80, 134.24, 131.97, 130.00, 129.68, 128.27, 127.61, 106.42, 59.98,
55.88, 53.02, 20.99. HRMS (ESI) m/z calcd for C23H24ClNO5S [MþNa]
þ 484.0961, found 484.0962.
5.3.7. N-(4-fluorobenzyl)-4-methyl-N-(3,4,5-trimethoxyphenyl)
benzenesulfonamide(13b)
White solid, yield, 69%, m.p. 153e154 ꢀC. 1H NMR (400 MHz,
5.3.1. N-(4-methoxybenzyl)-4-methyl-N-(3,4,5-trimethoxyphenyl)
benzenesulfonamide(10a)
DMSO‑d6)
d
7.61 (d, J ¼ 7.9 Hz, 2H), 7.45 (d, J ¼ 7.8 Hz, 2H), 7.33 (dd,
J ¼ 14.3, 7.3 Hz,1H), 7.20e7.01 (m, 3H), 6.28 (s, 2H), 4.75 (s, 2H), 3.59
White solid, yield, 64%, m.p. 156e157 ꢀC. 1H NMR (400 MHz,
(d, J ¼ 9.5 Hz, 9H), 2.42 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
DMSO‑d6)
2H), 4.65 (s, 2H), 3.69 (s, 3H), 3.58 (d, J ¼ 15.1 Hz, 9H), 2.42 (s, 3H).
13C NMR (100 MHz, DMSO‑d6)
158.53, 152.32, 143.55, 136.88,
135.06, 134.29, 129.64, 129.56, 128.10, 127.58, 113.64, 106.47, 59.98,
55.84, 54.95, 53.19, 20.99; HRMS (ESI) m/z calcd for C24H27NO6S
[MþNa]þ480.1457, found 480.1456.
d
7.52 (d, J ¼ 54.3 Hz, 4H), 7.17 (s, 2H), 6.83 (s, 2H), 6.22 (s,
d
163.22, 160.80, 152.41, 143.74, 139.55, 139.48, 137.02, 134.76,
134.31, 130.28, 129.68, 127.63, 124.15, 124.13, 114.86, 114.29, 106.38,
59.98, 55.88, 53.18, 20.98. HRMS (ESI) m/z calcd for C23H24FNO5S
[MþNa] þ 468.1257, found 468.1255.
d
5.3.8. N-(4-bromobenzyl)-4-methyl-N-(3,4,5-trimethoxyphenyl)
benzenesulfonamide(13c)
5.3.2. N-(4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl) benzene
sulfonamide(10b)
White solid, yield, 69%, m.p. 156e157 ꢀC. 1H NMR (400 MHz,
DMSO)
(d, J ¼ 8.1 Hz, 2H), 6.26 (s, 2H), 4.71 (s, 2H), 3.59 (d, J ¼ 10.0 Hz, 9H),
2.42 (s, 3H). 13C NMR (101 MHz, DMSO)
152.40, 143.71, 136.99,
d
7.59 (d, J ¼ 7.9 Hz, 2H), 7.46 (dd, J ¼ 12.2, 8.4 Hz, 4H), 7.26
White solid, yield, 58%, m.p. 161e162 ꢀC. 1H NMR (400 MHz,
DMSO‑d6)
d
7.82e7.75 (m, 3H), 7.70 (t, J ¼ 7.5 Hz, 2H), 7.24 (d,
d
J ¼ 8.0 Hz, 2H), 6.89 (d, J ¼ 7.8 Hz, 2H), 6.26 (s, 2H), 4.74 (s, 2H), 3.75
135.99, 134.76, 134.24, 131.19, 130.34, 129.68, 127.62, 120.51, 106.39,
59.98, 55.87, 53.05, 20.99. HRMS(ESI) m/z calcd for C23H24BrNO5S
[MþNa] þ 528.0456, found 528.0458.
(s, 3H), 3.66 (s, 3H), 3.61 (s, 6H). 13C NMR (100 MHz, DMSO‑d6)
d
158.56, 152.34, 137.90, 136.93, 134.15, 133.15, 129.59, 129.25,
128.05, 127.50, 113.66, 106.49, 59.98, 55.82, 54.96, 53.36. HRMS
(ESI) m/z calcd for C23H25NO6S [MþNa] þ 466.1300, found 466.1300.
5.3.9. 4-Methyl-N-(3-methylbenzyl)-N-(3,4,5-trimethoxyphenyl)
benzenesulfonamide (13d)
5.3.3. 4-(tert-butyl)-N-(4-methoxybenzyl)-N-(3,4,5-
trimethoxyphenyl) benzenesulfonamide(10c)
White solid, yield, 51%, m.p. 121e122 ꢀC. 1H NMR (400 MHz,
White solid, yield, 49%, m.p. 138e139 ꢀC. 1H NMR (400 MHz,
DMSO‑d6)
d
7.60 (d, J ¼ 7.9 Hz, 2H), 7.44 (d, J ¼ 8.0 Hz, 2H), 7.17 (d,
J ¼ 7.7 Hz, 2H), 7.07 (d, J ¼ 7.7 Hz, 2H), 6.24 (s, 2H), 4.68 (s, 2H), 3.58
DMSO‑d6)
J ¼ 8.2 Hz, 2H), 6.20 (s, 2H), 4.73 (s, 2H), 3.75 (s, 3H), 3.65 (s, 3H),
3.59 (s, 6H), 1.38 (s, 9H). 13C NMR (100 MHz, DMSO‑d6)
158.54,
156.24, 152.30, 136.93, 134.99, 134.29, 129.57, 128.17, 127.44, 126.01,
113.64, 106.55, 59.98, 55.71, 54.96, 53.50, 34.90, 30.75. HRMS (ESI)
m/z calcd for C27H33NO6S [MþNa] þ 522.1926, found 522.1927.
d
7.69 (q, J ¼ 8.4 Hz, 4H), 7.24 (d, J ¼ 8.2 Hz, 2H), 6.88 (d,
(d, J ¼ 13.7 Hz, 9H), 2.42 (s, 3H), 2.23 (s, 3H). 13C NMR (100 MHz,
DMSO‑d6)
d 152.33, 143.57, 136.88, 136.50, 134.99, 134.35, 133.29,
d
129.64, 128.82, 128.14, 127.58, 106.41, 59.96, 55.83, 53.46, 20.98.
þ
HRMS (ESI) m/z calcd for C24H27NO5S [MþH] 442.1688, found
442.1689.
5.3.10. N-benzyl-4-methyl-N-(3,4,5-trimethoxyphenyl)
benzenesulfonamide(13e)
5.3.4. 3,4-Dimethoxy-N-(4-methoxybenzyl)-N-(3,4,5-
trimethoxyphenyl) benzenesulfonamide(10d)
White solid, yield, 58%, m.p. 194e195 ꢀC. 1H NMR (400 MHz,
White solid, yield, 67%, m.p. 126e127 ꢀC. 1H NMR (400 MHz,
DMSO‑d6)
(m, 5H), 6.25 (s, 2H), 4.73 (s, 2H), 3.57 (d, J ¼ 13.7 Hz, 10H), 2.42 (s,
3H). 13C NMR (100 MHz, DMSO‑d6)
152.36, 143.62, 136.95, 136.43,
d
7.61 (d, J ¼ 7.8 Hz, 2H), 7.45 (d, J ¼ 7.8 Hz, 2H), 7.32e7.18
DMSO‑d6)
d
7.29 (d, J ¼ 8.4 Hz, 1H), 7.22e7.15 (m, 3H), 7.03 (s, 1H),
6.83 (d, J ¼ 8.4 Hz, 2H), 6.26 (s, 2H), 4.65 (s, 2H), 3.86 (s, 3H), 3.75 (s,
3H), 3.69 (s, 3H), 3.60 (d, J ¼ 9.4 Hz, 9H).13C NMR (100 MHz, DMSO-
d
135.02, 134.38, 129.66, 128.25, 128.15, 127.59, 127.36, 106.46, 59.97,
55.84, 53.80, 20.98. HRMS (ESI) m/z calcd for C23H25NO5S [MþNa] þ
450.1351, found 450.13.
d6)
d 158.51, 152.53, 152.30, 148.50, 136.92, 134.51, 129.58, 129.15,
128.22, 121.45, 113.63, 111.23, 110.18, 106.53, 59.97, 55.91, 55.87,
55.69, 54.93, 52.98. HRMS (ESI) m/z calcd for C25H29NO8S [MþNa] þ
526.1512, found 526.1511.
5.3.11. N-((5-chlorobenzo[b]thiophen-3-yl) methyl)-4-methyl-N-
(3,4,5-trimethoxyphenyl) benzenesulfonamide(13f)
5.3.5. N-(4-methoxybenzyl)-2,4,6-trimethyl-N-(3,4,5-
trimethoxyphenyl) benzenesulfonamide(10e)
White solid, yield, 58%, m.p. 140e141 ꢀC. 1H NMR (400 MHz,
White solid, yield, 67%, m.p. 116e117 ꢀC. 1H NMR (400 MHz,
DMSO‑d6)
J ¼ 29.0, 8.3 Hz, 3H), 6.21 (s, 2H), 4.98 (s, 2H), 3.59 (s, 3H), 3.52 (s,
6H), 2.44 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
152.30, 143.78,
d
7.98 (d, J ¼ 8.0 Hz, 2H), 7.66 (d, J ¼ 7.3 Hz, 3H), 7.44 (dd,
DMSO‑d6)
d
7.15 (d, J ¼ 8.1 Hz, 2H), 7.04 (s, 2H), 6.83 (d, J ¼ 8.1 Hz,
2H), 6.25 (s, 2H), 4.81 (s, 2H), 3.69 (s, 3H), 3.55 (d, J ¼ 22.5 Hz, 9H),
d
3.36 (s, 3H), 2.42 (s, 6H), 2.26 (s, 3H).13C NMR (100 MHz, DMSO‑d6)
139.08, 138.19, 137.08, 134.54, 134.08, 130.10, 129.71, 129.40, 129.22,
127.75, 124.56, 124.45, 121.46, 106.48, 59.97, 5þ5.80, 48.09, 21.02.
HRMS (ESI) m/z calcd for C23H25NO5S [MþNa] 540.0682, found
540.0682.
d
158.57, 152.35, 142.62, 139.73, 136.90, 133.71, 132.40, 131.65,
129.85, 128.33, 113.65, 106.66, 60.03, 55.76, 54.94, 52.24, 22.42,
20.34. HRMS (ESI) m/z calcd for C26H31NO6S [MþH] 486.1950,
þ

Jian-Song et al. / European Journal of Medicinal Chemistry 183 (2019) 111731
11
5.3.12. N-((3a,7a-dihydro-1H-benzo[d]imidazol-2-yl) methyl)-4-
5.3.18. N-((1H-benzo[d]imidazol-2-yl)methyl)-3,4-dimethoxy-N-
(3,4,5-trimethoxypheyl)benzenesulfonamide (14f)
methyl-N-(3,4,5- trimethoxyphenyl) benzenesulfonamide (13 g)
White solid, Yield, 73%, m.p. 204e205 ꢀC. 1H NMR (400 MHz,
White solid, m.p. 202e203 ꢀC, Yield, 66%. 1H NMR (400 MHz,
DMSO‑d6)
d
12.46 (s, 1H), 7.62 (d, J ¼ 8.3 Hz, 2H), 7.49 (d, J ¼ 8.2 Hz,
DMSO‑d6)
d
12.39 (s,1H), 7.46e7.37 (m,2H), 7.21 (dd, J ¼ 8.5, 2.1 Hz,
2H), 7.44 (d, J ¼ 8.2 Hz, 2H), 7.14 (s, 2H), 6.48 (s, 2H), 4.99 (s, 2H),
1H), 7.07 (dd, J ¼ 7.9, 5.3 Hz, 4H), 6.45 (s,2H), 4.93 (s,2H), 3.77 (s,3H),
3.58 (d, J ¼ 9.5 Hz, 9H), 2.42 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
3.67 (s,3H), 3.51 (d, J ¼ 4.2 Hz, 9H).13C NMR (100 MHz, DMSO‑d6)
d
152.37,149.81, 143.91, 137.15, 134.70, 134.33, 129.64, 127.77, 121.68,
d152.72,152.35, 151.56,150.01, 148.52,137.12,134.93, 128.42, 121.76,
106.42, 59.94, 55.80, 49.48, 21.01. HRMS (ESI) m/z calcd for
C
111.11, 110.28, 106.42, 59.94, 55.91, 55.83, 55.68, 49.23. HRMS (ESI)
24H25N3O5S [MþH] þ 468.1593, found 468.1590.
m/z calcd for C25H27N3O7S [MþH] þ 514.1648, found 514.1649.
5.3.19. N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(4-fluorophenyl)-
4-methylbenzenesulfonamide(17a)
5.3.13. N-((3a,7a-dihydro-1H-benzo[d]imidazol-2-yl)methyl)-N-
(3,4,5-trimethoxyphenyl) benzenesulfonamide (14a)
White solid, yield, 57%, m.p. 183e184 ꢀC. 1H NMR (400 MHz,
White solid, Yield, 52%, m.p. 181e182 ꢀC. 1H NMR (400 MHz,
DMSO‑d6)
d
12.53 (s, 1H), 7.59e7.37 (m, 6H), 7.28e7.08 (m, 6H), 5.01
162.35, 159.91,
DMSO‑d6)
d 12.49 (s, 1H),7.79e7.71 (m, 3H),7.68e7.59 (m, 2H),7.50
(s, 2H), 2.41 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
d
(t, J ¼ 8.4 Hz, 2H),7.14 (dt, J ¼ 15.1, 6.6 Hz, 2H), 6.47 (s, 2H),5.03 (s,
149.35, 143.94, 135.13, 135.10, 133.99, 130.43, 130.34, 129.79, 127.52,
2H),3.57 (d, J ¼ 15.0 Hz, 9H).13C NMR (100 MHz, DMSO‑d6)
d 152.41,
121.75, 115.81, 115.59, 48.97, 21.03. HRMS (ESI) m/z calcd for
149.73, 142.97,137.29,134.52,133.37, 129.21, 127.70, 122.10, 121.20,
C
21H18FN3O2S [MþH] þ 396.1182, found 396.1181.
118.51, 111.40,106.54,59.95,55.82,49.58. HRMS (ESI) m/z calcd for
C
23H23N3O5S [MþH] þ 454.1437, found, 454.1436.
5.3.20. N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(4-
chlorophenyl)-4-methylbenzenesulfonamide(17b)
White solid, yield, 59%, m.p. 190e191 ꢀC. 1H NMR (400 MHz,
5.3.14. N-((3a,7a-dihydro-1H-benzo[d]imidazol-2-yl) methyl)-4-
fluoro-N-(3,4,5-trimethoxyphenyl) benzenesulfonamide (14b)
Yellow solid, m.p. 192e193 ꢀC; Yield, 60%. 1H NMR (400 MHz,
DMSO‑d6)
d
12.54 (s, 1H), 7.54 (d, J ¼ 8.0 Hz, 2H), 7.44 (dd, J ¼ 16.9,
5.6 Hz, 4H), 7.36 (d, J ¼ 8.5 Hz, 2H), 7.22 (d, J ¼ 8.5 Hz, 2H), 7.15e7.09
(m, 2H), 5.02 (s, 2H), 2.41 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
DMSO‑d6)
(td, J ¼ 14.7, 6.4 Hz, 2H), 6.53 (s, 2H), 5.02 (s, 2H), 3.59 (d, J ¼ 5.2 Hz,
9H).13C NMR (100 MHz, DMSO‑d6)
166.01, 163.50,152.48,149.65,
d 12.48 (s, 1H), 7.89e7.72 (m, 2H), 7.59e7.38 (m, 4H), 7.14
d
149.25, 144.05, 137.84, 133.86, 132.45, 129.84, 129.79, 128.90,
127.51, 121.92, 114.96, 48.59, 21.04. HRMS (ESI) m/z calcd for
d
C
21H18ClN3O2S [MþH] þ 412.0887, found 412.0888.
142.96, 137.37, 134.48, 133.74, 133.71, 130.90, 130.81, 122.11, 121.20,
118.50, 116.46, 116.24,111.39,106.62,59.95, 55.89, 49.51. HRMS (ESI)
m/z calcd for C23H22FN3O5S [MþH] þ 472.1342, found, 472.1341.
5.3.21. N-((1H-benzo[d]imidazol-2-yl)methyl)-4-methyl-N-
phenylbenzenesulfonamide(17c)
White solid, yield, 69%, m.p. 160e161 ꢀC. 1H NMR (400 MHz,
5.3.15. 4-Bromo-N-((3a,7a-dihydro-1H-benzo[d]imidazol-2-yl)
methyl)-N-(3,4,5- trimethoxyphenyl) benzenesulfonamide (14c)
Yellow solid, m.p. 187e188 ꢀC, Yield 70%. 1HNMR (400 MHz,
DMSO‑d6)
7.30e7.16 (m, 5H), 7.12 (s, 2H), 5.02 (s, 2H), 2.41 (s, 3H).13C NMR
(100 MHz, DMSO‑d6) 149.49,143.85,138.94,134.21,129.73,128.81,
d
12.50 (s, 1H), 7.53 (d, J ¼ 8.0 Hz, 2H), 7.49e7.38 (m, 4H),
d
DMSO‑d6)
d
12.48 (s, 1H),7.84 (d, J ¼ 8.6 Hz, 2H),7.66 (d, J ¼ 8.6 Hz,
128.04, 127.89, 127.49, 121.71, 48.86, 21.02. HRMS (ESI) m/z calcd for
C
21H19N3O2S [MþH] þ 378.1276, found 378.13.
2H),7.49 (dd, J ¼ 5.3, 3.9 Hz, 2H), 7.14 (dd, J ¼ 6.0, 3.1 Hz, 2H), 6.56 (s,
2H), 5.03 (s, 2H), 3.60 (d, J ¼ 4.4 Hz, 9H).13C NMR (100 MHz,
DMSO‑d6) d152.50, 149.59, 138.37, 137.43, 136.61, 134.36, 132.26,
5.3.22. N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(4-
methoxyphenyl)-4-methylbenzenesulfonamide (17d)
White solid, yield, 69%, m.p. 121e122 ꢀC. 1H NMR (400 MHz,
131.82, 130.56, 129.71, 127.75, 127.39, 121.83, 114.92, 106.67, 59.96,
þ
55.89, 49.45. HRMS (ESI) m/z calcd for C23H22BrN3O5S [MþH]
532.0542, found 532.0541.
DMSO‑d6) d 12.49 (s, 1H), 7.56e7.35 (m, 6H), 7.18e7.02 (m, 4H), 6.79
(d, J ¼ 8.7 Hz, 2H), 4.97 (s, 2H), 3.66 (s, 3H), 2.42 (s, 3H). 13C NMR
(100 MHz, DMSO‑d6) d 158.55, 149.56, 143.71, 134.33, 131.22, 129.71,
5.3.16. 2-Chloro-N-((3a,7a-dihydro-1H-benzo[d]imidazol-2-yl)
methyl)-N-(3,4,5- trimethoxyphenyl) benzenesulfonamide (14d)
Yellow solid, m.p. 163e164 ꢀC, Yield, 52%. 1H NMR (400 MHz,
129.52, 127.53, 121.62, 113.96, 55.15, 49.20, 21.03. HRMS (ESI) m/z
calcd for C22H21N3O3S [MþH] þ 408.1382, found 408.1381.
DMSO‑d6)
d
12.42 (s, 1H), 7.89 (dd, J ¼ 7.9, 1.5 Hz, 1H), 7.76 (dd,
5.3.23. N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(3,4-
dimethoxyphenyl)-4-methylbenzenesulfonamide(17e)
White solid, yield, 69%, m.p. 112e113 ꢀC. 1H NMR (400 MHz,
J ¼ 8.0,1.0 Hz,1H), 7.68 (td, J ¼ 7.7,1.5 Hz,1H), 7.55 (d, J ¼ 7.7 Hz,1H),
7.53e7.45 (m, 2H), 7.20e7.10 (m, 2H), 6.54 (s, 2H), 5.27 (s, 2H), 3.54
(d, J ¼ 18.7 Hz, 9H).13C NMR (100 MHz, DMSO‑d6)
d 152.50,149.91,
DMSO‑d6)
d
12.49 (s, 1H), 7.59 (d, J ¼ 7.9 Hz, 2H), 7.52e7.40 (m, 4H),
137.32,135.97,134.77,133.59,132.35,132.08,131.11,127.70,121.78,115.04,
106.51,59.95,55.79,50.04. HRMS (ESI) m/z calcd for C23H22ClN3O5S
[MþH] þ 488.1047, found 488.1046.
7.19e7.09 (m, 2H), 6.80 (d, J ¼ 9.2 Hz, 1H), 6.68 (s, 2H), 4.98 (s, 2H),
3.67 (s, 3H), 3.53 (s, 3H), 2.42 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
d
149.67, 148.39, 148.12, 143.76, 134.38, 131.36, 129.66, 127.66,
121.63, 120.87, 112.34, 111.08, 55.37, 55.33, 49.41, 21.01. HRMS (ESI)
5.3.17. N-((1H-benzo[d]imidazol-2-yl)methyl)-4-(tert-butyl)-N-
(3,4,5-trimethoxyphenyl) benzenesulfonamide (14e)
m/z calcd for C23H23N3O4S [MþH] þ 438.1488, found 438.1488.
White solid, m.p. 192e193 ꢀC, Yield, 78%. 1H NMR (400 MHz,
5.3.24. N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(3,4,5-
trimethoxyphenyl) benzamide(18a)
DMSO‑d6)
7.06 (dd, J ¼ 6.0, 3.2 Hz, 2H), 6.35 (s,2H), 4.94 (s,2H), 3.51 (s, 3H),
3.46 (s, 6H), 1.23 (s, 9H).13C NMR (100 MHz, DMSO‑d6)
156.58,
d
12.43 (s,1H), 7.56 (s,4H), 7.41 (dd, J ¼ 5.8, 3.4 Hz, 2H),
Yellow solid, m.p. 100e101 ꢀC, Yield, 62%. 1H NMR (400 MHz,
d
DMSO‑d6)
d
12.38 (s, 1H), 7.57 (d, J ¼ 7.5 Hz, 1H), 7.50 (d, J ¼ 7.5 Hz,
152.35, 149.76, 138.42, 137.17, 134.65, 134.11, 127.66, 126.01, 125.27,
124.28, 121.79, 114.96, 106.47, 59.94, 55.66, 49.65, 3þ4.91, 31.04,
30.70. HRMS (ESI) m/z calcd for C27H31N3O5S [MþH] 510.2063,
found 510.2062.
1H), 7.41 (d, J ¼ 6.8 Hz, 2H), 7.35e7.23 (m, 3H), 7.20e7.10 (m, 2H),
6.63 (s, 2H), 5.29 (s, 2H), 3.53 (d, J ¼ 10.6 Hz, 9H).13C NMR (100 MHz,
DMSO‑d6)
d 169.78, 152.46, 150.92, 143.00, 138.56, 136.06, 135.98,
134.34, 129.53, 128.03, 127.65, 121.99, 121.15, 118.50, 111.25, 105.79,

12
Jian-Song et al. / European Journal of Medicinal Chemistry 183 (2019) 111731
59.97, 55.82, 47.71. HR-MS (ESI) m/z calcd for C24H23N3O4 [MþH] þ
147.41, 145.03, 139.16, 136.17, 129.18, 127.67, 123.16, 122.10, 121.35,
112.34, 111.98, 111.00, 110.59, 105.94, 90.55, 60.10, 55.95, 55.54,
418.1767, found: 418.1767.
þ
55.43, 55.30. HR-MS (ESI) m/z calcd for C26H27N3O6 [MþH]
5.3.25. N-((1H-benzo[d]imidazol-2-yl)methyl)-4-fluoro-N-(3,4,5-
trimethoxy phenyl) benzamide (18b)
478.1978, found 478.1980.
White solid, m.p.152e153 ꢀC, Yield 74%.1H NMR (400 MHz,
5.4. Cell culture
DMSO‑d6)
d 12.41 (s,1H), 7.62e7.45 (m, 4H), 7.20e7.08 (m,4H), 6.67
(s,2H), 5.30 (s,2H), 3.56 (d, J ¼ 9.0 Hz, 9H).13C NMR (100 MHz,
All the cancer cells were cultured at 37 ꢀC in an atmosphere with
5% CO2. And using RPMI-1640 medium with 10% fetal bovine serum
(FBS), 100 U/ml penicillin and 0.1 mg/ml streptomycin as culture
medium.
DMSO‑d6)
d 168.77, 163.64, 161.18, 152.57, 150.88, 143.03, 138.55,
136.13, 134.37, 132.46, 132.42, 130.75, 130.66, 121.98, 121.18, 118.50,
114.77, 114.55, 111.24, 105.80, 59.98, 55.85, 47.88. HR-MS (ESI) calcd
for C24H22FN3O4 [MþH] þ m/z:436.1673, found: 436.1674.
5.3.26. N-((1H-benzo[d]imidazol-2-yl)methyl)-4-bromo-N-(3,4,5-
trimethoxyphenyl) benzamide (18c)
5.5. MTT assay
White solid, m.p. 172e173 ꢀC, Yield,70%. 1H NMR (400 MHz,
Cells were seeded 5 ꢁ 103 per well in 96-well plates and incu-
DMSO‑d6)
d
12.39 (s, 1H), 7.57 (d, J ¼ 7.6 Hz, 1H), 7.50 (dd, J ¼ 7.3,
bated for 24 h, then treated with different concentrations of com-
5.5 Hz, 3H), 7.36 (d, J ¼ 7.6 Hz, 2H), 7.20e7.12 (m,2H), 6.66 (s,2H),
pound as shown in the figures. After 48 h, 20
m
L MTT solution each
L DMSO was
5.27 (s,2H), 3.54 (d, J ¼ 8.5 Hz, 9H).13C NMR (100 MHz, DMSO‑d6)
well was added, and incubated at 37 ꢀC for 4 h 150
m
d
168.78, 152.55, 150.76, 143.01, 138.27, 136.19, 135.27, 134.33,
added to each well to dissolve the formazan after discarding the
supernatant liquid, the absorbance was determined at 490 nm IC50
values are calculated with SPSS version 10.0 (SPSS, Inc., Chicago, IL,
USA).
130.72, 130.16, 123.00, 122.01, 121.16, 118.51, 111.22, 105.86, 60.00,
þ
55.88, 47.74. HR-MS (ESI) m/z calcd for C24H22BrN3O4 [MþH]
496.0872, found 496.0872.
5.3.27. N-((1H-benzo[d]imidazol-2-yl)methyl)-2-chloro-N-(3,4,5-
trimethoxyphenyl) benzamide (18d)
5.6. Colony formation assay
White solid, m.p. 141e142 ꢀC, Yield, 60%. 1H NMR (400 MHz,
5000 per well MGC-803 cells, SGC-7901 cells and HGC-27 cells
were seeded in a 6-well plate and incubated at 37 ꢀC in 5% CO2 for
24 h, then treated with different concentrations of 10e. After 7 days,
remove the culture medium, the cells were washed with PBS twice,
fixed with 4% paraformaldehyde and stained with 0.1% crystal vi-
olet. Cells’ images were captured with camera.
DMSO‑d6)
d
12.45 (s,1H), 7.59 (d, J ¼ 7.6 Hz, 1H), 7.52 (d, J ¼ 7.5 Hz,
1H), 7.48 (dd, J ¼ 5.9, 3.3 Hz, 1H), 7.33 (ddd, J ¼ 10.5, 5.8, 4.4 Hz, 1H),
7.29e7.22 (m,2H), 7.21e7.11 (m,2H), 6.67 (s,2H), 5.27 (s, 2H), 3.53
(d, J ¼ 8.2 Hz, 6H), 3.51 (d, J ¼ 4.5 Hz, 3H). 13C NMR (100 MHz,
DMSO‑d6)
d 167.18, 152.19, 150.34, 143.00, 136.90, 136.41, 136.24,
134.39, 130.22, 129.38, 129.04, 128.98, 126.56, 122.09, 121.18, 118.61,
111.33, 105.69, 59.91, 55.75, 46.59. HR-MS (ESI) m/z calcd for
C
24H22ClN3O4 [MþH] þ 452.1377, found: 452.1376.
5.7. Cell cycle distribution assay
5.3.28. N-((1H-benzo[d]imidazol-2-yl)methyl)-4-methyl-N-(3,4,5-
trimethoxyphenyl) benzamide (18e)
MGC-803 cells, SGC-7901 cells and HGC-27 cells were seeded in
6-well culture plate, then treated with certain concentration of
compound 13g for 48 h. Then cells were harvested and fixed at 4 ꢀC
with 70% cold ethanol for 8 h. The fixed cells were washed and
resuspended with PBS containing 10 mg/mL RNaseA and 50 mg/mL
PI for 0.5 h in the dark. Then, samples were analyzed for DNA
content with flow cytometry (Becton, Dickinson and Company, NJ).
White solid, M.p. 156e157 ꢀC, Yield, 65%. 1H NMR (400 MHz,
DMSO)
d
12.39 (s, 1H), 7.58 (d, J ¼ 6.9 Hz, 1H), 7.51 (d, J ¼ 7.0 Hz, 1H),
7.33 (d, J ¼ 7.6 Hz, 2H), 7.17 (d, J ¼ 7.4 Hz, 2H), 7.08 (d, J ¼ 7.9 Hz, 2H),
6.65 (s,2H), 5.29 (s,2H), 3.54 (d, J ¼ 12.7 Hz, 9H), 2.23 (s,3H).13
C
NMR (100 MHz, DMSO)
d 169.78, 152.51, 151.01, 143.02, 139.32,
138.81, 136.00, 134.37, 133.12, 128.24, 128.16, 121.96, 121.14, 118.49,
111.25, 105.78, 59.97, 55.82, 47.91, 20.81. HR-MS (ESI) calcd for
C
5.8. Cell apoptosis assay
25H25N3O4 [MþH]þm/z:432.1923, found: 432.1922.
MGC-803 cells, SGC-7901 cells and HGC-27 cells were seeded in
6-well culture plate and treated with certain concentrations of
compound 13g for 48 h. Then cells were harvested and suspended
in binding buffer containing Annexin V-FITC (0.5 mg/mL) and PI
(0.5 mg/mL). After that, samples were incubated for 0.5 h in dark
and analyzed with flow cytometry (Becton, Dickinson and Com-
pany, NJ).
5.3.29. N-((1H-benzo[d]imidazol-2-yl)methyl)-4-(tert-butyl)-N-
(3,4,5-trimethoxyphenyl)benzamide (18f)
Yellow solid, M.p. 102e103 ꢀC, Yield, 85%. 1H NMR (400 MHz,
DMSO‑d6)
8.2 Hz, 4H), 7.15 (s,2H), 6.62 (s,2H), 5.29 (s,2H), 3.53 (d, J ¼ 16.1 Hz,
9H), 1.21 (s,9H). 13C NMR (100 MHz, DMSO‑d6)
169.66, 152.50,
d
12.35 (s, 1H), 7.58e7.46 (m,2H), 7.32 (dd, J ¼ 18.0,
d
152.38, 138.72, 136.12, 133.10, 129.16, 128.07, 125.29, 124.32, 121.93,
121.12, 118.49, 111.26, 105.91, 59.99, 59.70, 55.89,þ47.80, 34.41, 30.82.
HR-MS (ESI) m/z calcd for C28H31N3O4 [MþH] 474.2393, found:
474.2394.
5.9. Western blot analysis
Human gastric cancer cell lines (MGC-803, SGC-7901 and HGC-
27) were treated with different concentrations of compound 13g or
DMSO for 48 h. And then cells were harvested and lysed. Protein
lysates were denatured and resolved by SDS-PAGE and transferred
to nitrocellulose membrane. The membranes were incubated with
appropriate antibodies at 4 ꢀC for 8 h after blocking with 5%
skimmed milk. After conjugated with secondary antibodies, the
detection of proteins was carried out with an ECL kit.
5.3.30. N-((1H-benzo[d]imidazol-2-yl)methyl)-3,4-dimethoxy-N-
(3,4,5-trimethoxypheyl)benzamide (18 g)
Yellow solid, M.p. 80e81 ꢀC, Yield, 70%. 1H NMR (400 MHz,
DMSO‑d6)
d
12.34 (s,1H), 7.51 (s,2H), 7.11 (dd, J ¼ 28.3, 4.0 Hz, 2H),
6.03 (t, J ¼ 20.3 Hz, 2H), 5.19 (t, J ¼ 44.5 Hz, 1H), 4.59 (d, J ¼ 98.3 Hz,
2H), 3.85e3.60 (m,6H), 3.61e3.39 (m,9H). 13C NMR (100 MHz,
DMSO‑d6)
d 169.17, 153.63, 153.37, 152.65, 151.02, 149.98, 148.31,

Jian-Song et al. / European Journal of Medicinal Chemistry 183 (2019) 111731
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Data from three independent experiments are presented as
mean SD. IC50 values and SD values were calculated by SPSS
version 10.0 (SPSS, Inc., Chicago, IL, USA).
Competing financial interests
The authors declare that they have no conflicts of interest.
Acknowledgement
This work was supported by the National Natural Sciences
Foundations of China (No. 81703541, 81673322) and China Post-
doctoral Science Foundation (2018M632812).
Appendix A. Supplementary data
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