European Journal of Medicinal Chemistry 157 (2018) 139e150

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Design, synthesis and pharmacological evaluation of new 3-(1H-

benzimidazol-2-yl)quinolin-2(1H)-one derivatives as potential
antitumor agents
Wen-Bin Kuang a, b, 1, Ri-Zhen Huang c, 1, Jiao-Lan Qin b, Xing Lu b, Qi-Pin Qin b,
Bi-Qun Zou b, d, Zhen-Feng Chen b, **, Hong Liang b, ***, Ye Zhang a, b, d, *
a
School of Pharmacy, Guilin Medical University, Guilin 541004, China
b
State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmacy, Guangxi Normal University,
Guilin 541004, China
c
Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China
d
Department of Chemistry & Pharmaceutical Science, Guilin Normal College, Xinyi Road 15, Guangxi 541001, China

a r t i c l e i n f o a b s t r a c t

Article history: A series of new 3-(1H-benzimidazol-2-yl)quinolin-2(1H)-one derivatives (5a1
5d6) were designed and
Received 17 January 2018 synthesized as antitumor agents. In vitro antitumor assay results showed that some compounds
Received in revised form exhibited moderate to high inhibitory activity against HepG2, SK-OV-3, NCI-H460 and BEL-7404 tumor
24 July 2018
cell lines, and most compounds exhibited much lower cytotoxicity against the HL-7702 normal cell line
Accepted 29 July 2018
Available online 30 July 2018
compared to 5-FU and cisplatin. In vivo antitumor assay results demonstrated that 5a3 exhibited effective
inhibition on tumor growth in the NCI-H460 xenograft mouse model and that 5d3 displayed excellent
antiproliferative activity in the BEL-7402 xenograft model. These results suggested that both 5a3 and 5d3
Keywords:
3-(1H-benzimidazol-2-yl)quinolin-2(1H)-
could be used as anticancer drug candidates. Mechanistic studies suggested that compounds 5a3 and 5d3
one derivatives exerted their antitumor activity by up-regulation of Bax, intracellular Ca2þ release, ROS generation,
Synthesis downregulation of Bcl-2, activation of caspase-9 and caspase-3 and subsequent cleavage of PARP, inhi-
Antitumor activity bition of CDK activity and activation of the p53 protein.
Action mechanism © 2018 Elsevier Masson SAS. All rights reserved.
p53

1. Introduction
As a leading cause of death worldwide, cancer is a global health
problem. To combat with cancer, a lot of attention has been focused
Abbreviation: FDA, Food and Drug Administration; Bax, Bcl-2-associated X
protein; PI, propidium iodide; EB, ethidium bromide; MTT, 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide; CDC, cell division cyclin; CDK, cyclin-
dependent kinase; CDC25C, cell division cyclin 25 homolog C.
* Corresponding author. School of Pharmacy, Guilin Medical University, Guilin
541004, China.
** Corresponding author. State Key Laboratory for Chemistry and Molecular En-
gineering of Medicinal Resources, School of Chemistry and Pharmacy, Guangxi
Normal University, Guilin 541004, China.
*** Corresponding author. State Key Laboratory for Chemistry and Molecular En-
gineering of Medicinal Resources, School of Chemistry and Pharmacy, Guangxi
Normal University, Guilin 541004, China.
E-mail addresses: chenzf@gxnu.edu.cn (Z.-F. Chen), hliang@gxnu.edu.cn
(H. Liang), zhangye81@126.com (Y. Zhang).
1
These authors contributed equally to this work.
on the design and synthesis of new antitumor drugs with high ef-
ficiency and low toxicity to healthy cells and tissues. To achieve
these goals, combination principle strategy has been widely applied
to design new antitumor molecules that act synchronously on
multiple targets, by combining two/more active pharmacophores
covalently in a single-hybrid molecule with dual/multiple anti-
tumor funtion [1]. The combination principle inspires us to design,
synthesize and evaluate a new kind of heterocyclic antitumor
molecules, by the joining of two important pharmacophores: 2-
oxo-quinoline and benzimidazole (Fig. 1).
As a kind of alkaloid which widely exist as same as quinolone, 2-
oxo-quinoline derivatives exhibit numerous promising biological
activities [2e4], including antioxidation [5,6], antiproliferation [7]
and antitumor effect [8e10]. 2-Oxo-quinoline derivatives could
exert the antitumor effect through inducing cell apoptosis and
arresting the cell cycle, by the activation of caspases, p53 and Bax,
inhibition of Bcl-2, and modulation of cytochrome c and some
related genes/proteins [9,10]. The potential antitumor activity, easy
modification of structure and no evident toxicity [6] of 2-oxo-

https://doi.org/10.1016/j.ejmech.2018.07.066
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.
140 W.-B. Kuang et al. / European Journal of Medicinal Chemistry 157 (2018) 139e150
Fig. 1. Design of 3-(1H-benzimidazol-2-yl)quinolin-2(1H)-one derivatives using the
combination principle.
quinoline renders itself an ideal template for designing new che-
mopreventive agents. In our previous work, 2-oxo-quinoline has
been chose as pharmacophore to successfully design and synthe-
size some Schiff's-bases [5,6] and aminophosphate derivatives [8,9]
with potential antioxidant and antitumor activities, by the joining
2-oxo-quinoline-3-carbaldehyde with amines or/and diethyl
phosphite. In addition, benzimidazole, which consists of the fusion
of benzene and imidazole, is also a bicyclic pharmacophore.
Benzimidazole and its derivatives exhibits a wide range of biolog-
ical activities [11], containing antioxidant [12] and antitumor aci-
tivity [13e16], which make it to be a valuable synthetic scaffold for
designing new antitumor agents. Benzimidazole derivatives could
exert the antitumor effect through modulation of cell cycle arrest
and apoptotic responses [17]. Since that the antioxidants have been
forged as the antitumor drug candidates and the commercial
anticancer drug bleomycin exerting the antitumor activity is
related with free radical scavenging [18], it is thus to consider that
screening for new antitumor compounds based on the combining
of 2-oxo-quinoline and benzimidazole may be a feasible way.
Owing to the synthetic and antitumor values of 2-oxo-quinoline
and benzimidazole derivatives, it is highly possible that the com-
bination of the 2-oxo-quinoline skeleton with benzimidazole
pharmacophore may lead to potent antitumor activity. Further-
more, previous work has also indicated that some 3-(1H-benzi-
midazol-2-yl)quinolin-2(1H)-one derivatives exhibit potential
antitumor activity [19]. However, to the best of our knowledge, the
synthetic quantity and scale of 3-(1H-benzimidazol-2-yl)quinolin-
2(1H)-one derivatives, as well as their antitumor activities and
mechanisms have not been thoroughly explored. So a set of new
imidazol-2-yl-1H-quinolin-2-ones derivatives (5a1
5d6) with
different substituent group in quinolone/benzimidazo moiety were
designed and synthesized under the combination principle
(Scheme 1), and their antitumor activity was then evaluated. The
in vivo cytotoxicity of representative compounds (i.e., 5a3 and 5d3)
was also evaluated. Mechanistic studies focused on apoptosis, cycle
arrest and activation of p53 induced by the representative com-
pounds 5a3 and 5d3 was further performed. We aimed to provide
information for understanding the chemopreventive mechanism of
3-(1H-benzimidazol-2-yl)quinolin-2(1H)-one derivatives and a
combination principle strategy for designing antioxidant-based
cancer chemoprevention agents.
2. Results and discussion
2.1. Chemistry
3-(1H-Benzimidazol-2-yl)quinolin-2(1H)-one compounds 5
(5a1
5d6) were synthesized as shown in Scheme 1. First, phenyl
acetamide derivatives 2 (2a
2d) were obtained by the acylation
reaction of compounds 1 (1a
1d) [11]. 2-Chloro-quinoline-3-
carbaldehyde derivatives 3 (3a
3d) were then synthesized
through the Vilsmeier-Haack-Arnold reaction which involved the
combination of acetanilide derivatives 2 (2a
2d) with N,N-
dimethylformamide (DMF) in the presence of phosphorus oxy-
chloride [11,20]. 2-Oxo-quinoline 3-carbaldehyde derivatives
4(4a
4d) were synthesized in decent yields by the hydrolytic re-
actions of 3 (3a
3d) in an acetic acid (70%) aqueous solution [11]. 2-
Oxo-quinoline 3-benzimidazole derivatives 5 (5a1
5d6) were
finally obtained in moderate yields through coupling reactions of 4
(4a
4d) with o-phenylenediamine derivatives in the presence of
methanol. The structures of compounds 5 (5a1
5d6) were then
confirmed using various spectroscopic methods, including 1H NMR,
13
C NMR and high-resolution mass spectrometry (HR-MS)(Part 1.1
of Supplementary Data). In 1H NMR spectra, the chemical shift (d)
around 8.91e8.97 and 11.91e12.82 were attributed to the hydro-
gens of NH groups fused in quinolone/benzimidazole, and d in the
range of 8.90e9.12 were mainly attributed to the hydrogens of
HC¼C contained in quinolone ring, while d in the range of 6.16e7.93
were attributed to the aromatic hydrogens (H-Ar) in quinolone and
benzimidazole. In addition, d at 6.06, 2.38 and 2.32 were attributed
to the hydrogens of methyl group, respectively. For compounds
5d1
5d6, d in the range of 6.06e6.14 were attributed to the hy-
drogens of O-CH2-O group. In 13C NMR spectra, d in the range of
161.04e161.99 were attributed to the carbons in carbonyl groups,
and d around 95.40 was attributed to the carbons of O-CH2-O
groups, while d in the range of 20.59e20.94 and 55.94e56.09 were
attributed to the carbons of methyl group, respectively. d in the
range of 99.47e148.19 were attributed to the carbons in C¼C and
C¼N groups and the aromatic carbons in in quinolone and benz-
imidazole. In addition, HR-MS offered the precise molecular weight
and composition of 5 (5a1
5d6), which were well consistent with
structures in Scheme 1. Moreover, to better understand the struc-
ture of 5a1
5d6, the crystal structures of 5b1 and 5d6 were deter-
mined by X-ray diffraction. As shown in Fig. 1-S97 and 1-S98, the
crystal structures of 5b1 and 5d6 were well consistent with the
structures determined by 1H NMR, 13C NMR and HR-MS. So the
structures of compounds 5 (5a1
5d6) were completely confirmed.
2.2. Biology
2.2.1. In vitro cytotoxicity
The in vitro cytotoxicity of the 3-(1H-benzimidazol-2-yl)quino-
lin-2(1H)-one derivatives 5(5a1
5d6) were determined using the
methylthiazoltetrazolium (MTT) assay [11,21] against the HepG2
(human liver cancer cells), SK-OV-3 (human ovarian cancer cells),
NCI-H460 (Human large cell lung cancer cells), BEL-7404 (Human
liver cancer cells) and HL-7702 (Human liver normal cells) cell
lines. For comparison, two FDA-approved anticancer drugs 5-
fluorouracil (5-FU) and cisplatin were used as positive controls.
The cytotoxicity test results were shown in Table 1. As shown in
Table 1, many 3-(1H-benzimidazol-2-yl)quinolin-2(1H)-one de-
rivatives exhibited cytotoxicity against the HepG2, SKOV3, NCI-
H460 and BEL-7404 cell lines. Compared to the 5-FU and
cisplatin, some of these compounds displayed higher cytotoxicity
against these tumor cell lines but lower cytotoxicity against the HL-
7702 normal cell line. In the cytotoxicity assay with the HepG2 cell
line, compounds 5a2, 5a3, 5a4, 5a6, 5b3, 5d1, 5d2, 5d3 and 5d6
exhibited higher cytotoxicity than 5-FU (IC50 ¼ 31.98 mM), with
IC50values in the range of 7.54e29.63 mM. Notably, compound 5a3
even exhibited higher cytotoxicity than cisplatin (IC50 ¼ 10.12 mM).
In the cytotoxicity assay with the SKOV-3 cell line, compounds 5a2,
5a3, 5a4, 5a6, 5d1 and 5d3 showed higher cytotoxicity than 5-FU
(IC50 ¼ 26.34 mM), with IC50 in the range of 8.83e24.69 mM. Com-
pounds 5a3, 5a4, 5a6 and 5d1 even exhibited higher cytotoxicity
than cisplatin (IC50 ¼ 15.60 mM). In the cytotoxicity assay with the
NCI-H460 cell line, compounds 5a2, 5a3, 5a4, 5b3, 5d1 and 5d3
 W.-B. Kuang et al. / European Journal of Medicinal Chemistry 157 (2018) 139e150 141

Scheme 1. Synthetic routes of compounds 5(5a1
5d6). Reagents and conditions: (a) acetic anhydride; (b) POCl3, DMF; (c) acetic acid (70%) aqueous solution; (d) o-phenyl-
enediamine derivatives, methanol.

Table 1
a
IC50 values of 3-(1H-benzimidazol-2-yl)quinolin-2(1H)-one derivatives 5(5a1
5d6) towards four selected tumor cell lines and a normal cell line.

Compounds IC50 (mM)

HepG2 SKOV-3 NCI-H460 BEL-7404 HL-7702

5a1 >100 >100 >100 >100 >100

5a2 15.21 ± 1.17 18.34 ± 1.46 24.18 ± 1.38 27.45 ± 1.47 >100
5a3 8.45 ± 1.63 10.52 ± 1.83 14.72 ± 1.64 16.54 ± 1.45 >100
5a4 10.81 ± 1.04 11.40 ± 0.78 16.79 ± 1.98 14.46 ± 0.77 >100
5a5 >100 >100 >100 >100 >100
5a6 29.63 13.80 >100 >100 >100
5b1 >100 >100 >100 >100 >100
5b2 >100 >100 >100 >100 >100
5b3 26.59 ± 1.79 33.02 ± 1.52 36.53 ± 1.12 38.95 ± 1.27 >100
5b4 >100 >100 >100 >100 >100
5b5 >100 >100 >100 >100 >100
5b6 >100 >100 >100 >100 >100
5c1 >100 >100 >100 >100 >100
5c2 >100 >100 >100 >100 >100
5c3 >100 >100 >100 >100 >100
5c4 >100 >100 >100 >100 >100
5c5 >100 >100 >100 >100 >100
5c6 >100 >100 >100 >100 >100
5d1 18.66 ± 1.56 10.62 ± 1.37 23.75 ± 1.72 15.71 ± 0.89 >100
5d2 12.43 ± 0.91 >100 >100 10.35 ± 1.35 >100
5d3 10.56 ± 1.38 24.69 ± 1.22 30.36 ± 1.07 9.06 ± 1.36 >100
5d4 >100 >100 >100 >100 >100
5d5 >100 >100 >100 >100 >100
5d6 25.56 32.17 >100 >100 >100
5-FU 31.98 ± 0.56 26.34 ± 0.57 45.44 ± 0.94 40.21 ± 1.98 58.74 ± 2.31
Cisplatin 10.12 ± 0.71 15.60 ± 1.7 20.36 ± 0.50 14.72 ± 0.66 15.67 ± 0.32
a
IC50 values are presented as mean ± SD (standard error of the mean) from three repeating experiments.

exhibited higher cytotoxicity than 5-FU (IC50 ¼ 45.44 mM), with IC50
in the range of 14.03e38.25 mM. Compounds 5a3 and 5a4 even
displayed higher cytotoxicity than cisplatin (IC50 ¼ 20.36 mM). In
the cytotoxicity assay with the BEL-7404 cell line, compounds 5a2,
5a3, 5a4, 5b3, 5d1, 5d2 and 5d3 exhibited higher cytotoxicity than 5-
FU (IC50 ¼ 40.21 mM), with IC50 values in the range of
9.06e39.94 mM. Compounds 5a4, 5d2 and 5d3 even displayed
higher cytotoxicity than cisplatin (IC50 ¼ 14.72 mM).
By the respective comparison of compounds 5a1, 5b1, 5c1 with
5d1, it should be found that compound 5d1 exhibited better
antiproliferative activity than other compounds, indicating that the
benzodioxole substitution in quinolone moiety may have positive
influence on the antiproliferative activity. The respective compar-
ison of 5a2, 5b2 and 5c2 showed that 5a2 exhibited the best anti-
proliferative effect among them, implying that no(H) substitution
in 6 position of quinolone moiety may also have active influence on
the antiproliferative activity. In addition, the respective comparison
of 5a2, 5a3, 5a4 with other compounds in 5a(5a1
5a6) kind, as well
as that of 5d2, 5d3 with other compounds in 5d(5d1
5d6) kind,
demonstrated that halide-substitution in benzimidazole moiety
142 W.-B. Kuang et al. / European Journal of Medicinal Chemistry 157 (2018) 139e150
exhibited positive effect on their antiproliferative activity. The or-
der listed as follow: chlorine > bromine > fluorine. In brief, based
on the above observation, it could be concluded that, for com-
pounds 5(5a1
5d6), no(H) substitution in 6 position and the ben-
zodioxole substitution in 6,7 position in quinolone moiety, as well
as methyl, fluor, chlor and bromo substitutions in benzimidazole
moiety may have positive influence on the antiproliferative activity.
Among all the 3-(2-benzimidazolyl)quinolone derivatives,
compounds 5a3 and 5d3 showed the highest antiproliferation ac-
tivity against the cancer cell lines. They were thus selected as
representative compounds for the mechanistic studies.
2.2.2. Preliminary safety evaluation of the selected compounds
The in vivo toxicity of compounds 5a3 and 5d3 was then evalu-
ated in Kun-ming (KM) mice. Through intraperitoneal injection, the
mice received 5a3 or 5d3 (dissolved in a 5% v/v DMSO/saline sol-
vent) at a dose of 50 mg/kg per day for 14 consecutive days. The
body weight of the animals was monitored daily and used as a
parameter of systemic toxicity.
Notably, no adverse effects were observed in the KM mice
injected with the selected compounds during the 14-day moni-
toring period. As shown in Fig. 2 (5a3) and Fig. 3-S1 (5d3) (Part 3 of
Supplementary Data), the average body weight of the mice treated
with the two compounds showed similar growth rates to that of the
control group.
The pathological changes of different organs of the KM mice
treated with 5a3 and 5d3 were also characterized. The organs,
including kidney, heart, and liver, from the mice treated 5a3 and
5d3 were harvested and used to produce pathological slices for
microscopy characterization. As shown in Fig. 3-S2 (Part 3 of Sup-
plementary Data), compared to the control groups, there were no
signs of peritonitis or damage to these organs in the mice treated
with 5a3 and 5d3. These observations indicated that 5a3 and 5d3
had no significant toxicity to the KM mice at the dose of 50 mg/kg,
suggesting that they may be good candidates as antitumor drugs.
2.2.3. In vivo antitumor activity of 5a3 and 5d3
In vivo antiproliferation experiments were performed to further
evaluate the antitumor activity of 5a3 and 5d3. The NCI-460 and
BEL-7402 xenograft models were selected in this study. In the NCI-
460 tumor model, cisplatin was used as the positive control,
whereas 5-FU was used as the positive control in the BEL-7402
tumor model. The SPF BALB/c nude mice were randomly divided
Fig. 2. Changes in the body weight of the KM mice injected with compound 5a3. There
are six mice in each experimental group.
into the vehicle control groups, the experimental groups, and the
positive control groups.
As shown in Fig. 3A, both of the low dose (25 mg/kg) and high
dose (50 mg/kg) of 5a3 exhibited important inhibition on NCI-460
tumor growth, and the relative tumor increment rates (T/C) were
calculated as 40.6% and 30.7%, respectively, based on the tumor
volume. In comparison, cisplatin at the 2 mg/kg dose exhibited a T/
C value of 29.9%. The tumors were harvested and weighted on day
14, and the inhibitory rates on the growth of tumor weight were
calculated. Fig. 3C showed that 5a3 (at the 50 mg/kg dose) displayed
significant antitumor activity in the NCI-460 model with an
inhibitory rate of 66.9%, greater than that of cisplatin (63.2%,
P < 0.01). These in vivo results indicated that 5a3 (at the 50 mg/kg
dose) exhibited higher antitumor effect than cisplatin (at the 2 mg/
kg dose) in the NCI-460 mouse model, consistent with the results of
the in vitro cytotoxicity assay. It should be noted that no significant
change in body weight and no other adverse effects were observed
among the mice treated with 5a3, suggesting that 5a3 exhibited no
significant toxicity to the mice within the 13-day period of treat-
ment (Fig. 3B) and thus could be good candidate for antitumor drug
design.
The in vivo antitumor activity of 5d3 was also investigated in the
NCI-460 mouse model (Fig. 3-S3) (Part 3 of Supplementary Data).
The T/C values were determined to be 48.8% (at the 25 mg/kg dose)
and 38.1% (at the 50 mg/kg dose). The inhibitory rates were deter-
mined to be 46.2% (at the 25 mg/kg dose) and 55.1% (at the 50 mg/
kg dose). Notably, no significant change in body weight and no
other adverse effects were observed among the mice treated with
5d3, suggesting that 5d3 exhibited no significant toxicity to the
mice within the 13-day period of treatment (Fig. 3-S3B).
5-FU is the most common chemotherapeutic drug used for the
treatment of hepatocellular carcinoma. Thus it was used as the
positive control in the in vivo experiments with the BEL-7402
mouse model. As shown in Fig. 3-S4A (Part 3 of Supplementary
Data), both of the low (25 mg/kg) and high dose (50 mg/kg) of
5d3 displayed evident antiproliferative activity on the tumor
growth compared with the negative control group. The T/C values
of these two compounds were determined to be 51.5% and 44.8%
(p < 0.01), respectively. It was worth noting that 5-FU at the 25 mg/
kg dose showed a T/C value of 56.1%, higher than that of 5d3. In
addition, Fig. 3-S4C showed that the inhibitory rates on the growth
of tumor results weight were determined to be 41.2 and 44.9%
(p < 0.01) for the low and high dose of 5d3, respectively. In com-
parison, the inhibitory rate of 5-FU (at the 25 mg/kg dose) was
34.5%. These results indicated that 5d3 (both at 25 mg/kg and
50 mg/kg dose) showed more potent in vivo antitumor activity than
5-FU (at 25 mg/kg dose) in the BEL-7402 mouse model, consistent
with the result of the in vitro cytotoxicity assay. Moreover, no sig-
nificant changes in body weight and no other adverse effects were
found in the mice treated with 5d3 (Fig. 3-S4B), suggesting that 5d3
may be good candidate for antitumor drug design.
Pathological slices of the tumors harvested in the mice treated
with 5a3 and 5d3 were generated and characterized using micro-
scopy. As shown in Fig. 3-S5 (Part 3 of Supplementary Data), it was
evident that the treatment of 5a3 and 5d3 led to necrosis of the
tumor cells (NCI-460 and BEL-7402) compared with the vehicle
samples, indicating potent antiproliferative activity of 5a3 and 5d3.
2.3. Action mechanism
2.3.1. Investigation of cell cycle distribution
It is well known that cell cycle checkpoints are significant con-
trol mechanisms that ensure the performance of cell cycle events
[22]. To investigate the possible role of cell cycle arrest in the tumor
cell death induced by 3-(1H-benzimidazol-2-yl)quinolin-2(1H)-
 W.-B. Kuang et al. / European Journal of Medicinal Chemistry 157 (2018) 139e150 143

Fig. 3. In vivo anticancer activity of 5a3 in the NCI-460 xenograft model. (A) Effect of 5a3 (at the doses of 50 and 25 mg/kg), cisplatin (at the dose of 2 mg/kg), or vehicle (5% DMSO in
saline, v/v) on tumor growth. Tumor growth was monitored by the mean tumor volume (mm3) ± SD (n ¼ 6) and calculated as the relative tumor increment rate (T/C, %). (B) Body
weight change of the mice treated with 5a3. (C) Tumor weight of the mice. The tumors were collected in the mice at day 14. The “**” signs represent p < 0.01 (versus the vehicle
control group). (D) Photographs of the harvested tumors from the mice.

one derivatives, HepG2 cells were treated with the 5a3 at different
concentrations (0, 6, 8 and 10 mM) for 48 h. For comparison, BEL-
7404 cells were treated with compound 5d3 (9 mM). As shown in
Fig. 4, the treatment of HepG2 cells with compound 5a3 also
increased cell cycle arrest at the G2 phase, resulting in an obvious
increase in the G2-phase population (39.00% at 6 mM, 39.71% at
8 mM and 49.10% at 10 mM) compared with the control group
(19.04%). As a result, the G1-phase and S-phase population of the
HepG2 cells were reduced. These results demonstrated that com-
pound 5a3 arrested the cell cycle of the HepG2 cells at the G2/M
stage in a concentration-dependent manner. Fig. 3-S6 (Part 3 of
Supplementary Data) showed that treating the BEL-7404 cells with
5d3 (9 mM) also caused cell cycle arrest at the G2 phase with the
population of 30.58%, higher than that of the control group
(18.84%). Thus, compound 5d3 also arrested the cell cycle of the
BEL-7404 cells in the G2/M stage, consistent with the scenarios of
5a3.
G2/M transition is mainly regulated by timed activation of cyclin
B1/CDK1(CDC2) complexes whose activity is mainly mediated by
the positive mediator cell division cyclin 25 homolog C (CDC25C)
phosphatase [23,24]. CDC25C modulates the phosphorylation of
CDK1 leading to the activation of cyclin B1/CDK1 complexes and
execution of G2/M transition [23,24]. Responding to DNA damage
and cellular activities resulting in G2/M arrest, phosphorylation of
CDC25C leads to its degradation and sequestration to the cyto-
plasm, and finally results in the destabilization of the cyclin-CDK

Fig. 4. Cell cycle analysis of the HepG2 cells treated with 5a3. The HepG2 cells were treated with compound 5a3 at different concentrations i.e., (a) 0 mM, (b) 6 mM, (c) 8 mM and (d)
10 mM, for 48 h.
144 W.-B. Kuang et al. / European Journal of Medicinal Chemistry 157 (2018) 139e150
complexes [25]. Furthermore, the proteins encoded by some anti-
oncogenes, including p53, p27 and p21, also play an important role
in G2/M arrest through promoting the accumulation of the dormant
cyclin B-CDK complex [26]. In this work, the expression levels of
cyclin B1, CDK1, CDC25C, p53, p27 and p21 in the HepG2 cells were
determined using western blot. As shown in Fig. 5, compared to the
negative control, the lysates of the HepG2 cells treated with 5a3
exhibited decreased levels of cyclin B1, CDK1 and CDC25C as well
increased levels of p53, p21 and p27. Therefore, it could be
concluded that 5a3 induced G2/M phase cell cycle arrest through
inhibition of CDK activity by increased expression levels of p53, p27
and p21.
The in vivo expression levels of cyclin B1, CDK1 and CDC25C, as
well as p53, p27 and p21 in the tumor tissues of the NCI-460 and
BEL-7402 xenograft mouse models were also investigated by
western blot assays. As shown in Fig. 3-S7 (Part 3 of Supplementary
Data), it was evident that the in vivo expression levels of p53 in the
tumor tissues increased upon treating the mice with 5a3. Taken
together, it can be concluded that compounds 5a3 may be p53
activator.
2.3.2. Apoptosis assays
As a key pathway leading to cell death, apoptosis is deemed to
be an effective mechanism in cancer treatment [27,28]. We studied
the apoptosis-inducing effect of compound 5a3 using a fluorescent
probe Hoechst 33258 as well as acridine orange/ethidium bromide
(AO/EB) staining. In the Hoechst 33258 staining test, the normal
HeGp 2 cells could emit blue fluorescence, whereas the cells treated
with 5a3 for 12 h displayed stronger blue fluorescence and
exhibited characteristic apoptotic morphologies (Fig. 6). These ob-
servations revealed that compound 5a3 induced apoptosis of the
HeGp 2 cells [25]. In the AO/EB staining experiment, the
morphology of 5a3-treated HeGp 2 cells exhibited significant
change (Fig. 7). The cell nuclei were stained as yellow/green or
orange, and the morphology changes indicated pycnosis, mem-
brane blebbing and cell budding, all of which are characteristic of
apoptosis [25]. Taken together, it could be concluded that com-
pound 5a3 induced apoptosis in the HeGp 2 cells.
The apoptosis ratios in the HepG2 cells treated with 5a3 were
quantitatively determined by flow cytometry and the results were
shown in Fig. 8. Fig. 8 showed that the treatment of the HepG2 cells
with compound 5a3 at different concentrations (6, 8 and 10 mM)
caused an increase of the apoptotic cell population in a
Fig. 5. Effect of compound 5a3 on the expression levels of relevant proteins in HepG2 cells. Western blot analysis was performed with antibodies against cyclin B1, CDK1, CDC25C,
p21, p23, and p53. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading standard.
concentration dependent manner. These results demonstrated that
compound 5a3 induced apoptosis of the HepG2 cells in a dose-
dependent manner.
2.3.3. ROS generation
It is known that production of intracellular reactive oxygen
species (ROS) can induce apoptosis [29]. In order to study the role of
ROS in 5a3-induced apoptosis in the cancer cells, the intracellular
ROS levels were evaluated using fluorescence microscopy using
40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) and 2,7-
dichlorofluorescein diacetate (DCFH-DA) as fluorescent probes. As
shown in Fig. 9, in the DAPI staining assay, the HepG2 cells treated
with compound 5a3 as well as the control cells exhibited blue
fluorescence in the cytoplasm. For the DCFH-DA staining assay, the
HepG2 cells treated with compound 5a3 displayed stronger green
fluorescence signals compared to the control cells, suggesting that
5a3 upregulated the levels intracellular of ROS. Thus, ROS genera-
tion induced by the 3-(1H-benzimidazol-2-yl)quinolin-2(1H)-one
derivatives might be the underlying cause for apoptosis [28].
2.3.4. Intracellular Ca2þ release
Previous studies demonstrated that intracellular calcium levels
participate in cell apoptosis and calcium overload could induce
apoptosis [30]. We investigated the role of calcium signalling in the
cellular apoptosis induced by 5a3. The HepG2 cells were treated
with 5a3 for 24 h, and the intracellular Ca2þ concentration was
determined by fluorescence microscopy using DCFH-DA as a cal-
cium probe. As shown in Fig. 10, treating HepG2 cells with 5a3
resulted in an increase of intracellular Ca2þ concentration. The re-
sults indicated that 5a3-induced apoptosis was associated with up-
regulation of the intracellular Ca2þ level.
2.3.5. Caspase-dependent apoptosis in the HepG2 cells
In vitro assay. Previous work has shown that cancer cells can
restrict or escape from apoptosis in many ways, which often involve
the perturbation of the Bcl-2 intrinsic apoptotic pathway [31-35].
The Bcl-2 family proteins play significant regulating roles both as
pro- and anti-apoptotic proteins and a slight change in the dynamic
balance of these proteins may result in either inhibition or pro-
motion of cell death [31,34]. Accordingly, the ratio of Bcl-2/Bax
expression is more important than the individual levels of each
Bcl-2 family protein in the regulation of apoptosis [35]. To study the
mechanism of 5a3-induced apoptosis in cancer cells, the expression
 W.-B. Kuang et al. / European Journal of Medicinal Chemistry 157 (2018) 139e150
Fig. 6. Effects of compound 5a3 on the morphology of the HepG2 cells. The cells were stained with the Hoechst 33258 dye.
Fig. 7. Compound 5a3 induced apoptosis in the HepG2 cells. The cells were visualized
via the AO/EB staining coupled with fluorescence microscopy.
levels of Bax, Bcl-2 and cytochrome c in the HepG2 cells treated
with 5a3 were determined by western blotting. Fig. 11 shows that
treating the HepG2 cells with 5a3 resulted in an increase in the
expression level of Bax and a decrease in the expression level of Bcl-
2 accompanied by the release of mitochondrial cytochrome c into
the cytosol.
Bcl-2 family proteins play a key role in apoptosis by interfering
with caspases, which are pivotal effectors of programmed cell
death [36,37]. As a family of cysteinyl aspartate specific proteases
participate in apoptosis, caspases are divided into groups of initi-
ators (caspases 8, 9 and 10) and executioners (caspases 3, 6 and 7)
[36,37]. The caspase cascade is initiated by the proteolysis of
inactive procaspases and propagated by the cleavage of down-
stream caspases and substrates such as poly(ADP-ribose) poly-
merase (PARP) [24]. To evaluate whether caspases were activated in
5a3-induced apoptosis, the expression levels of caspase-9, caspase-
3 and PARP was determined by western blot. Fig. 11 showed that
the HepG2 cells treated with 5a3 exhibited elevated expression
levels of caspase-9, caspase-3 and PARP, compared to the control
samples. These results demonstrated that compounds 5a3 exerted
pro-apoptotic effects through a mitochondrial mediated pathway
and caspase cascade.
In vivo assay. The expression levels of Bax, Bcl-2, cytochrome c,
caspase-9, caspase-3 and PARP, as well as apoptotic protease acti-
vating facter-1 (Apaf-1), in the tumor tissues of the NCI-460 and
BEL-7402 xenograft mouse models were also investigated using
western blot. As shown in Fig. 3-S8 (Part 3 of Supplementary Data),
145
Fig. 8. Apoptosis ratio of the HepG2 cells. (a) Negative control; (b) treated with 6 mM
5a3, (c) treated with 8 mM 5a3, (d) treated with 10 mM 5a3. The cells were analyzed
using the Annexin V/PI assay in a flow cytometer.
the expression level of Bax increased in the 5a3-treated NCI-
460xenograft model, but remained relatively unchanged in the
5d3-treated group in the BEL-7402xenograft model. The expression
level of Bcl-2 decreased upon the treatment of 5a3 and 5d3 in the
NCI-460 xenograft model. The expression level of cytochrome c
increased upon the treatment of 5a3 in the NCI-460xenograft
model. There was no significant change in the expression level of
caspase-3 in both mouse models. The expression level of caspase-9
increased upon the treatment with both 5a3 and 5d3 in the NCI-460
xenograft model but decreased upon the 5d3 treatment in BEL-
7402xenograft model. The expression level of PARP increased
upon the 5a3 treatment in the NCI-460 xenograft model, but
decreased upon the 5d3 treatment in BEL-7402xenograft model.
The 5a3 treatment led to an increase in the expression level of Apaf-
1 in the NCI-460 model, whereas in BEL-7402 model the 5d3
treatment resulted in a decrease in the expression level of Apaf-1.
Through comparison of the in vitro and in vivo studies of the
expression levels of these proteins, it can be concluded that com-
pound 5a3 may inhibit the expression of Bcl-2, lead to the release of
cytochrome c and Apaf-1, and finally activate caspase-9 and induce
apoptosis.
146 W.-B. Kuang et al. / European Journal of Medicinal Chemistry 157 (2018) 139e150
Fig. 9. Compound 5a3 affected the levels of intracellular ROS in the HepG2 cells.
2þ
Fig. 10. Compound 5a3 caused an increase in the level of intracellular Ca
HepG2 cells.
2.3.6. P53 activation
p53 is a pivotal tumor suppressor protein with a key role in
many cellular processes and one of the most relevant cellular
components related to cancer growth [38,39]. According to the
intracellular context, accumulation of p53 in response to various
stresses including DNA damage leads to cell cycle arrest, senes-
cence and apoptosis [40]. Since it can be functionally activated to
eradicate tumors, p53 has been considered as an attractive target of
anticancer therapy [38,39]. To better understand whether the
expression of p53 was regulated by 5a3, the HepG 2 cells expressing
a mutant form of the p53 protein were selected. Western blot
analysis showed an obvious increase of p53 expression in the HepG
2 cells treated with 5a3 compared with the negative control sample
(5a6, Fig. 12). These results demonstrated that upregulated p53
expression may be involved in the mechanism of 5a3-induced G2/M
cell cycle arrest, confirming that 5a3 was a p53 activator.
To further confirm whether p53 was involved in the antitumor
property of the HepG2 cells, disruption of p53 expression using
siRNRs was performed. The results in Fig. 13 showed that the S117,
S118 and S119 fragments can significantly reduce the p53 mRNA
level. It was evident that the S119 fragment exhibited the best
interference effect and was selected for further studies. As shown in
Fig. 3-S9 (Part 3 of Supplementary Data), treatment of the HepG2
cells with both S119 and compound 5a3 caused cell cycle arrest in
the G2/M stage, consistent with the result obtained by treating the
cells with 5a3 alone. A combinational treatment with S119 and 5d3
caused cell cycle arrest at the G1 stage, different from the result
obtained by treating the cells with 5d3 alone. Fig. 3-S10 (Part 3 of
Supplementary Data) showed that treatment of the cells with both
S119 and compound 5a3 led to an evident increase of the apoptotic
cell population, i.e., from 14% to 19.12%, and a combinational
treatment with S119 and compound 5d3 also caused an increase of
the apoptotic cell population, i.e., from 14.59% to 19.16%. Taken
together, it can be concluded that compounds 5a3 and 5d3 may
induce apoptosis through the activation of the tumor suppressor
p53.
3. Conclusion
In summary, we designed and synthesized a series of 3-(1H-
benzimidazol-2-yl)quinolin-2(1H)-one derivatives and evaluated
the cytotoxicity of these compounds against four cancer cell lines
(HepG2, SKOV-3, NCI-H460 and BEL-7404). We identified several
compounds exhibiting high in vitro anticancer activity and low
toxicity against normal HL-7702 cells. In vivo antitumor activity
study and animal toxicology test showed that 5a3and 5d3 exhibited
potent antitumor activity in different mice models. Mechanistic
studies showed that 5a3 and 5d3 caused apoptosis of tumor cells
possibly through regulation of the expression levels of apoptotic
proteins (such as Bax, Bcl-2, cytochrome c, caspase-9, caspase-3,
and PARP), intracellular Ca2þ release and ROS regulation. Cell cycle
studies indicated that compounds 5a3 and 5d3 induced cell cycle
arrest at the G2/M phase. The observed cell cycle arrest resulted
from the inhibition of CDK activity and activation of the p53 pro-
tein. This work may provide information for understanding the
chemopreventive mechanism of 3-(1H-benzimidazol-2-yl)quino-
lin-2(1H)-one derivatives and a combination principle strategy for
designing antioxidant-based cancer chemoprevention agents.
4. Experimental
level in the
4.1. Chemistry
All chemicals of reagent grade were commercially available and
used without further purification. NMR spectra were recorded on a
BRUKER AVANCE AV 400/500 spectrometer using tetramethylsilane
(TMS) as the internal standard. The mass spectra were collected on
a BRUKER ESQUIRE HCT spectrometer. The GelRed nucleic acid stain
was purchased from Biotium.
4.1.1. General synthesis procedure for compounds 5(5a1
5d6)
2-Oxo-quinoline 3-carbaldehyde derivatives 4(4a¡4d)
(1 mmol), o-phenylenediamine derivatives (1.0 mmol) and 3 mL
methanol were mixed in a pressure tube and heated at 90  C for 4 h.
After the reaction, the mixture was cooled to room temperature
and filtered to yield the product as yellow powder.
5a1: Yield, 71.4%; 1H NMR (500 MHz, DMSO‑d6) d 12.42 (s, 2H,
 W.-B. Kuang et al. / European Journal of Medicinal Chemistry 157 (2018) 139e150
Fig. 11. Effect of compound 5a3 on the expression levels of Bax, Bcl-2, cytochrome c, caspase-3, caspase-9 and PARP in the HepG2 cells. GAPDH was used as a loading standard.
Fig. 12. Effect of compounds 5a3 and 5a6 on the expression of p53. GAPDH was used as
the loading standard.
Fig. 13. Inhibition of p53 expression using siRNAs into the HepG2 cells.
NH), 9.05 (s, 1H, HC¼C), 7.94 (d, J ¼ 7.2 Hz, 1H, H-Ar), 7.60 (t,
J ¼ 8.4 Hz, 1H, H-Ar), 7.44 (br, 3H, H-Ar), 7.28 (t, J ¼ 8.0 Hz, 1H, H-Ar),
2.32 (s, 6H, -CH3); 13C NMR (126 MHz, DMSO‑d6) d 161.28 (C¼O),
147.31 (C¼N), 138.99, 138.83 (C¼C), 131.80 (C¼C), 129.38, 123.07,
120.75, 119.70, 115.68, 20.59 (CH3); ESI-HRMS, calculated m/zfor
C18H15N3O [MþH]þ: 290.1293, found: 290.1286.
5a2: Yield, 71.7%; 1H NMR (500 MHz, DMSO‑d6) d 12.75 (d,
J ¼ 12.1 Hz, 1H, NH), 12.49 (s, 1H, NH), 9.10 (d, J ¼ 10.8 Hz, 1H,
HC¼C), 7.96 (d, J ¼ 7.9 Hz, 1H, H-Ar), 7.74e7.65 (m, 1H, H-Ar),
7.64e7.58 (m, 1H, H-Ar), 7.49 (dd, J ¼ 9.3, 2.6 Hz, 1H, H-Ar), 7.44 (d,
147
J ¼ 7.8 Hz, 1H, H-Ar), 7.30 (t, J ¼ 7.5 Hz, 1H, H-Ar), 7.11e7.04 (m, 1H,
H-Ar); 13C NMR (126 MHz, DMSO‑d6) d 161.23 (C¼O), 161.15, 160.06,
158.25, 158.19, 149.87 (C¼N), 149.18, 139.89, 139.19 (C¼C), 132.14
(C¼C), 129.55, 119.56, 115.75, 110.56, 103.89, 99.47, 99.26; ESI-
HRMS, calculated m/z for C16H10FN3O [M
H]-: 278.0730, found:
278.0740.
5a3: Yield, 72.2%; 1H NMR (500 MHz, DMSO‑d6) d 12.52 (s, 1H,
NH), 9.12 (s, 1H, HC¼C), 7.96 (s, 1H, H-Ar), 7.74 (s, 1H, H-Ar), 7.70 (d,
J ¼ 8.6 Hz, 1H, H-Ar), 7.63 (s, 1H, H-Ar), 7.45 (d, J ¼ 8.2 Hz, 1H, H-Ar),
7.30 (s, 1H, H-Ar), 7.23 (s, 1H, H-Ar); 13C NMR (126 MHz, DMSO‑d6)
d 161.14 (C¼O), 149.52 (C¼N), 140.16, 139.31 (C¼C), 132.37 (C¼C),
129.65, 126.98, 123.20, 122.90, 119.90, 119.52, 115.78; ESI-HRMS,
calculated m/z for C16H10ClN3O [M
H]-: 294.0434, found:
294.0446.
5a4: Yield, 73.2%; 1H NMR (500 MHz, DMSO‑d6) d 12.52 (s, 1H,
NH), 11.91 (s, 1H, NH), 9.12 (s, 1H, HC¼C), 7.98 (s, 1H, H-Ar), 7.88 (s,
1H, H-Ar), 7.66 (s, 1H, H-Ar), 7.64 (d, J ¼ 6.0 Hz, 1H, H-Ar), 7.45 (d,
J ¼ 8.3 Hz, 1H, H-Ar), 7.35 (s, 1H, H-Ar), 7.30 (s, 1H, H-Ar); 13C NMR
(126 MHz, DMSO‑d6) d 161.14(C¼O), 149.35 (C¼N), 140.21, 139.32
(C¼C), 132.38 (C¼C), 129.66, 125.47, 123.20, 119.88, 119.53, 115.78,
114.89; ESI-HRMS, calculated m/z for C16H10BrN3O [M
H]-:
337.9929, found: 337.9947.
5a5: Yield, 69.1%; 1H NMR (500 MHz, DMSO‑d6) d 12.18 (s, 1H,
NH), 8.97 (s, 1H, NH), 8.93 (s, 1H, HC¼C), 7.93 (s, 1H, H-Ar), 7.81 (d,
J ¼ 7.3 Hz, 1H, H-Ar), 7.60 (t, J ¼ 7.2 Hz, 1H, H-Ar), 7.37 (d, J ¼ 8.2 Hz,
1H, H-Ar), 7.27 (t, J ¼ 7.5 Hz, 1H, H-Ar), 6.89 (s, 1H, H-Ar), 6.79 (d,
J ¼ 8.8 Hz, 1H, H-Ar); 13C NMR (126 MHz, DMSO‑d6) d 161.99 (C¼O),
154.36, 151.57 (C¼N), 140.35, 139.27 (C¼C), 136.45, 134.44, 132.52
(C¼C), 129.92, 126.85, 125.01, 123.01, 119.47, 115.80, 113.37, 113.24;
ESI-HRMS, calculated m/zfor C16H10N4O3 [M
H]-: 305.0675, found:
305.0694.
5a6: Yield, 80.2%; 1H NMR (400 MHz, DMSO‑d6) d 12.66 (s, 1H,
NH), 12.48 (s, 1H, NH), 9.13 (s, 1H, HC¼C), 7.97 (d, J ¼ 7.3 Hz, 1H, H-
Ar), 7.73 (s, 1H, H-Ar), 7.67 (s, 1H, H-Ar), 7.65e7.59 (m, 1H, H-Ar),
7.45 (d, J ¼ 8.2 Hz, 1H, H-Ar), 7.30 (t, J ¼ 7.1 Hz, 1H, H-Ar), 7.24e7.19
(m, 2H, H-Ar); 13C NMR (101 MHz, DMSO‑d6) d 161.26 (C¼O), 148.19
(C¼N), 143.24, 139.56 (C¼C), 139.15, 134.90, 132.08 (C¼C), 129.53,
123.13, 122.75, 122.39, 120.48, 119.62, 118.76, 115.72, 113.26; ESI-
HRMS, calculated m/zfor C16H11N3O [MþH]þ: 262.0980, found:
262.0986.
5b1: Yield, 72.1%; 1H NMR (400 MHz, DMSO‑d6) d 12.36 (s, 2H,
NH), 8.96 (s, 1H, HC¼C), 7.71 (s, 1H, H-Ar), 7.45 (s, 2H, H-Ar), 7.42 (s,
148 W.-B. Kuang et al. / European Journal of Medicinal Chemistry 157 (2018) 139e150
1H, H-Ar), 7.34 (d, J ¼ 8.4 Hz, 1H, H-Ar), 2.38 (s, 3H, -CH3), 2.32 (s,
6H, -CH3); 13C NMR (101 MHz, DMSO‑d6) d 161.17 (C¼O), 147.44
(C¼N), 138.52 (C¼C), 137.07, 133.21, 132.14 (C¼C), 128.62, 120.65,
119.66, 118.79, 115.62, 113.05, 20.94 (CH3), 20.62 (CH3); ESI-HRMS,
calculated m/z for C19H17N3O [MþH]þ: 304.1450, found: 304.1455.
5b2: Yield, 71.9%; 1H NMR (500 MHz, DMSO‑d6) d 12.74 (d,
J ¼ 11.7 Hz, 1H, NH), 12.42 (s, 1H, NH), 9.00 (d, J ¼ 11.0 Hz, 1H,
HC¼C), 7.72 (q, J ¼ 5.3 Hz, 2H, H-Ar), 7.49 (dd, J ¼ 10.5, 1.3 Hz, 1H, H-
A), 7.47e7.41 (m, 2H, H-A), 7.35 (d, J ¼ 8.4 Hz, 1H, H-A), 2.38 (s, 3H,
-CH3); 13C NMR (126 MHz, DMSO‑d6) d 161.11 (C¼O), 161.03, 150.00
(C¼N), 139.56 (C¼C), 137.32, 133.64, 132.24 (C¼C), 131.64, 128.80,
120.02, 119.53, 115.68, 113.92, 110.71, 104.07, 99.46, 20.91 (CH3); ESI-
HRMS, calculated m/z for C17H12FN3O [MþH]þ: 294.1043, found:
294.1053.
5b3: Yield, 68.7%; 1H NMR (500 MHz, DMSO‑d6) d 12.79 (d,
J ¼ 27.0 Hz, 1H, NH), 12.43 (s, 1H, NH), 9.02 (s, 1H, HC¼C), 7.73e7.72
(m, 1H, H-Ar), 7.70 (s, 1H, H-Ar), 7.66 (d, J ¼ 8.6 Hz, 1H, H-Ar), 7.46
(d, J ¼ 8.4 Hz, 1H, H-Ar), 7.35 (d, J ¼ 8.4 Hz, 1H, H-Ar), 7.24e7.20 (m,
1H, H-Ar), 2.38 (s, 3H, -CH3); 13C NMR (126 MHz, DMSO‑d6) d 161.04
(C¼O), 149.52 (C¼N), 144.15, 139.82 (C¼C), 137.37, 133.74, 132.27
(C¼C), 128.86, 126.82, 122.83, 120.00, 119.50, 118.04, 115.70, 114.57,
112.92, 20.92 (CH3); ESI-HRMS, calculated m/z for C17H12ClN3O
[MþH]þ: 310.0747, found: 310.0752.
5b4: Yield, 71.8%; 1H NMR (500 MHz, DMSO‑d6) d 12.80 (d,
J ¼ 23.9 Hz, 1H, NH), 12.44 (s, 1H, NH), 9.03 (s, 1H, HC¼C), 7.88 (d,
J ¼ 28.0 Hz, 1H, H-Ar), 7.74 (s, 1H, H-Ar), 7.65 (dd, J ¼ 28.6, 8.5 Hz,
1H, H-Ar), 7.47 (d, J ¼ 8.3 Hz, 1H, H-Ar), 7.37e7.32 (m, 2H, H-Ar),
2.39 (s, 3H, -CH3); 13C NMR (126 MHz, DMSO‑d6) d 161.05 (C¼O),
149.69 (C¼N), 144.71, 142.33, 139.86 (C¼C), 137.40, 136.14, 133.75,
132.29 (C¼C), 128.87, 125.40, 120.44, 119.89, 119.51, 115.71, 115.05,
20.92 (CH3); ESI-HRMS, calculated m/zfor C17H12BrN3O [MþH]þ:
354.0242, found: 354.0254.
5b5: Yield, 66.2%; 1H NMR (500 MHz, DMSO‑d6) d 12.09 (s, 1H,
NH), 8.91 (s, 1H, NH), 8.89 (s, 1H, HC¼C), 7.92 (t, J ¼ 2.6 Hz, 1H, H-
Ar), 7.58 (s, 1H, H-Ar), 7.43 (d, J ¼ 8.4 Hz, 1H, H-Ar), 7.27 (d,
J ¼ 8.4 Hz, 1H, H-Ar), 6.87 (s, 1H, H-Ar), 6.79 (d, J ¼ 8.8 Hz, 1H, H-Ar),
2.37 (s, 3H, -CH3); 13C NMR (126 MHz, DMSO‑d6) d 161.87 (C¼O),
154.43, 151.54 (C¼N), 138.98 (C¼C), 138.44, 136.47, 134.45, 133.87,
131.95 (C¼C), 129.18, 126.76, 124.94, 119.42, 115.72, 113.34, 113.17,
20.85 (CH3); ESI-HRMS, calculated m/zfor C17H12N4O3 [M
H]-:
319.0831, found: 319.0860.
5b6: Yield, 79.2%; 1H NMR (400 MHz, DMSO‑d6) d 12.67 (s, 1H,
NH), 12.42 (s, 1H, NH), 9.04 (s, 1H, HC¼C), 7.73 (s, 2H, H-Ar), 7.67 (s,
1H, H-Ar), 7.46 (d, J ¼ 8.4 Hz, 1H, H-Ar), 7.36 (d, J ¼ 8.4 Hz, 1H, H-Ar),
7.22 (s, 1H, H-Ar), 7.20 (s, 1H, H-Ar), 2.39 (s, 3H, -CH3); 13C NMR
(101 MHz, DMSO‑d6) d 161.15 (C¼O), 148.32 (C¼N), 143.24, 139.24
(C¼C), 137.23, 134.88, 133.50, 132.22 (C¼C), 128.76, 122.73, 122.37,
120.36, 119.57, 118.75, 115.66, 113.25, 20.95 (CH3); ESI-HRMS,
calculated m/z for C17H13N3O [MþH]þ: 276.1137, found: 276.1144.
5c1: Yield, 68.3%; 1H NMR (400 MHz, DMSO‑d6) d 12.44 (s, 1H,
NH), 12.36 (s, 1H, NH), 9.02 (s, 1H, HC¼C), 7.51 (s, 1H, H-Ar), 7.45 (s,
2H, H-Ar), 7.37 (d, J ¼ 9.0 Hz, 1H, H-Ar), 7.27 (d, J ¼ 2.8 Hz, H-Ar),
3.83 (s, 3H, -OCH3), 2.32 (s, 6H, -CH3); 13C NMR (101 MHz,
DMSO‑d6) d 160.78 (C¼O), 155.11, 147.42 (C¼N), 138.36 (C¼C),
133.64 (C¼C), 121.49, 120.90, 120.32, 117.00, 110.17, 55.99, 55.84
(OCH3), 20.63 (CH3); ESI-HRMS, calculated m/z for C19H17N3O2
[MþH]þ: 320.1399, found: 320.1407.
5c2: Yield, 72.6%; 1H NMR (500 MHz, DMSO‑d6) d 12.76 (d,
J ¼ 11.3 Hz, 1H, NH), 12.40 (s, 1H, NH), 9.06 (d, J ¼ 10.8 Hz, 1H,
HC¼C), 7.68 (ddd, J ¼ 36.0, 8.6, 5.0 Hz, 1H, H-Ar), 7.54e7.40 (m, 2H,
H-Ar), 7.38 (d, J ¼ 9.0 Hz, 1H, H-Ar), 7.28 (d, J ¼ 8.8 Hz, 1H, H-Ar),
7.07 (t, J ¼ 8.4 Hz, 1H, H-Ar), 3.83 (s, 3H, -OCH3); 13C NMR (126 MHz,
DMSO‑d6) d 160.75 (C¼O), 155.18, 150.03 (C¼N), 140.00, 139.38
(C¼C), 138.95, 133.92, 131.63 (C¼C), 121.92, 120.20, 117.09, 114.03,
110.78, 110.36, 104.07, 99.47, 56.03 (OCH3); ESI-HRMS, calculated m/
z for C17H12FN3O2 [MþH]þ: 310.0992, found: 310.0999.
5c3: Yield, 67.7%; 1H NMR (500 MHz, DMSO‑d6) d 12.82 (s, 1H,
NH), 12.42 (s, 1H, NH), 9.08 (s, 1H, HC¼C), 7.80e7.62 (m, 2H, H-Ar),
7.52 (d, J ¼ 2.6 Hz, 1H, H-Ar), 7.38 (d, J ¼ 9.0 Hz, 1H, H-Ar), 7.29 (dd,
J ¼ 9.0, 2.8 Hz, 1H, H-Ar), 7.22 (dd, J ¼ 8.6, 2.0 Hz, 1H, H-Ar), 3.83 (s,
3H, -OCH3); 13C NMR (126 MHz, DMSO‑d6) d 160.69 (C¼O), 155.19,
149.70, 139.56 (C¼C), 134.00, 126.89, 122.78, 122.02, 120.18, 117.11,
110.40, 56.03 (OCH3); ESI-HRMS, calculated m/z for C17H12ClN3O2
[MþH]þ: 326.0696, found: 326.0703.
5c4: Yield, 73.3%; 1H NMR (500 MHz, DMSO‑d6) d 12.82 (s, 1H,
NH), 12.41 (s, 1H, NH), 9.08 (s, 1H, HC¼C), 7.87 (s, 1H, H-Ar), 7.65 (s,
1H, H-Ar), 7.51 (s, 1H, H-Ar), 7.38 (d, J ¼ 8.9 Hz, 1H, H-Ar), 7.34 (d,
J ¼ 8.5 Hz, 1H, H-Ar), 7.28 (d, J ¼ 8.9 Hz, 1H, H-Ar), 3.82 (s, 3H,
-OCH3); 13C NMR (126 MHz, DMSO‑d6) d 160.68 (C¼O), 155.19,
149.55 (C¼N), 139.61 (C¼C), 134.01 (C¼C), 125.36, 122.03, 120.17,
120.13, 117.11, 114.81, 110.39, 56.03 (OCH3); ESI-HRMS, calculated m/
z for C17H12BrN3O2 [MþH]þ: 370.0191, found: 370.0189.
5c5: Yield, 67.7%; 1H NMR (500 MHz, DMSO‑d6) d 12.09 (s, 1H,
NH), 8.93 (s, 2H, NH, HC¼C), 7.93 (s, 1H, H-Ar), 7.31 (d, J ¼ 8.8 Hz,
2H, H-Ar), 7.26 (d, J ¼ 8.9 Hz, 1H, H-Ar), 6.87 (s, 1H, H-Ar), 6.79 (d,
J ¼ 8.6 Hz, 1H, H-Ar), 3.82 (s, 3H, -OCH3); 13C NMR (126 MHz,
DMSO‑d6) d 161.56 (C¼O), 154.94, 154.40, 151.58 (C¼N), 138.81
(C¼C), 136.47, 135.11, 134.39 (C¼C), 127.08, 124.99, 122.25, 120.01,
117.16, 113.37, 113.19, 110.44, 55.94 (OCH3); ESI-HRMS, calculated m/
z for C17H12N4O4 [M
H]-: 335.0781, found: 335.0807.
5c6: Yield, 78.7%; 1H NMR (400 MHz, DMSO‑d6) d 12.68 (s, 1H,
NH), 12.40 (s, 1H, NH), 9.10 (s, 1H, HC¼C), 7.70 (s, 2H, H-Ar), 7.52 (s,
1H, H-Ar), 7.39 (d, J ¼ 9.0 Hz, 1H, H-Ar), 7.29 (d, J ¼ 9.0 Hz, 1H, H-Ar),
7.22 (d, J ¼ 6.1 Hz, 2H, H-Ar), 3.83 (s, 3H, -OCH3); 13C NMR
(101 MHz, DMSO‑d6) d 160.79 (C¼O), 155.14, 148.34 (C¼N), 139.06
(C¼C), 133.84 (C¼C), 122.55, 121.77, 120.63, 120.25, 117.06, 110.27,
56.00 (OCH3); ESI-HRMS, calculated m/z for C17H13N3O2 [MþH]þ:
292.1086, found: 292.1081.
5d1: Yield, 73.9%; 1H NMR (500 MHz, DMSO‑d6) d 12.33 (s, 2H,
NH), 8.92 (s, 1H, HC¼C), 7.43 (s, 1H, H-Ar), 7.41 (s, 2H, H-Ar), 6.91 (s,
1H, H-Ar), 6.14 (s, 2H, O-CH2-O), 2.31 (s, 6H, -CH3); 13C NMR
(126 MHz, DMSO‑d6) d 161.00 (C¼O), 151.67, 147.83 (C¼N), 144.44,
138.55 (C¼C), 136.61, 130.77 (C¼C), 117.50, 114.31, 106.12, 102.57,
95.42 (CH2), 20.56 (CH3); ESI-HRMS, calculated m/z for C19H15N3O3
[MþH]þ: 334.1192, found: 334.1188.
5d2: Yield, 73.1%; 1H NMR (400 MHz, DMSO‑d6) d 12.73 (s, 1H,
NH), 12.44 (s, 1H, NH), 8.99 (s, 1H, HC¼C), 7.69 (s, 3H, H-Ar), 7.47 (s,
1H, H-Ar), 6.92 (s, 1H, H-Ar), 6.16 (s, 2H, O-CH2-O); 13C NMR
(101 MHz, DMSO‑d6) d 154.96 (C¼O), 153.51, 151.55, 149.05 (C¼N),
147.94, 147.28, 137.62 (C¼C), 136.40, 134.00 (C¼C), 125.50, 124.56,
113.71, 113.47, 111.90, 104.69, 103.79, 103.35 (CH2); ESI-HRMS,
calculated m/z for C17H10FN3O3 [M
H]-: 322.0628, found: 322.0657.
5d3: Yield, 68.3%; 1H NMR (400 MHz, DMSO‑d6) d 12.73 (s, 1H,
NH), 12.44 (s, 1H, NH), 8.99 (s, 1H, HC¼C), 7.69 (s, 3H, H-Ar), 7.47 (s,
1H, H-Ar), 6.92 (s, 1H, H-Ar), 6.16 (s, 2H, O-CH2-O); 13C NMR
(101 MHz, DMSO‑d6) d 160.87 (C¼O), 152.11 (C¼N), 144.54, 137.09,
126.63, 122.54, 116.66, 114.21, 106.26, 102.71, 95.40 (CH2); ESI-
HRMS, calculated m/z for C17H10ClN3O3 [M
H]-: 338.0333, found:
338.0359.
5d4: Yield, 73.8%; 1H NMR (500 MHz, DMSO‑d6) d 12.72 (s, 1H,
NH), 12.42 (s, 1H, NH), 8.99 (s, 1H, HC¼C), 7.86 (s, 1H, H-Ar), 7.63 (s,
1H, H-Ar), 7.46 (s, 1H, H-Ar), 7.31 (d, J ¼ 8.5 Hz, 1H, H-Ar), 6.92 (s,
1H, H-Ar), 6.16 (s, 2H, O-CH2-O); 13C NMR (126 MHz, DMSO‑d6)
d 160.89 (C¼O), 152.12 (C¼N), 144.55, 139.72, 137.12, 125.12, 114.56,
114.22, 106.25, 102.69, 95.43 (CH2); ESI-HRMS, calculated m/z for
C17H10BrN3O3 [MþH]þ: 383.9984, found: 383.9977.
5d5: Yield, 67.9%; 1H NMR (400 MHz, DMSO‑d6) d 12.48 (s, 1H,
NH), 11.82 (s, 1H, NH), 7.66 (s, 1H, HC¼C), 7.50 (d, J ¼ 8.7 Hz, 1H, H-
Ar), 7.15 (s, 1H, H-Ar), 7.08 (s, 1H, H-Ar), 6.84 (s, 1H, H-Ar), 6.60 (d,
J ¼ 8.7 Hz, 1H, H-Ar), 6.06 (s, 2H, O-CH2-O); 13C NMR (101 MHz,
 W.-B. Kuang et al. / European Journal of Medicinal Chemistry 157 (2018) 139e150
DMSO‑d6) d 161.88 (C¼O), 150.02 (C¼N), 144.57, 143.70, 137.51
(C¼C), 135.89, 135.22, 134.37 (C¼C), 127.17, 116.56, 113.78, 111.56,
105.62, 104.70, 102.09, 95.41 (CH2); ESI-HRMS, calculated m/z for
C17H10N4O5 [M
H]-: 349.0573, found: 349.0597.
5d6: Yield, 81.9%; 1H NMR (400 MHz, DMSO‑d6) d 12.58 (s, 1H,
NH), 12.41 (s, 1H, NH), 9.00 (s, 1H, HC¼C), 7.69 (s, 1H, H-Ar), 7.62 (s,
1H, H-Ar), 7.47 (s, 1H, H-Ar), 7.17 (s, 2H, H-Ar), 6.93 (s, 1H, H-Ar),
6.16 (s, 2H, O-CH2-O); 13C NMR (101 MHz, DMSO‑d6) d 160.98
(C¼O), 151.89, 148.69 (C¼N), 144.48, 143.28, 139.21 (C¼C), 136.85,
134.81 (C¼C), 122.43, 122.21, 118.51, 117.19, 114.25, 113.11, 106.21,
102.65, 95.40 (CH2); ESI-HRMS, calculated m/z for C17H11N3O3
[MþH]þ: 306.0879, found: 306.0876.
4.2. Biological assays
The procedures for biological assays were described in the
Supplementary Data (Part 2). The materials, instrumentation, and
methods for the cytotoxicity assay, cell cycle analysis, cell apoptosis
analysis, western blot and p53-transfection assays were reported
previously [41e43].
Acknowledgments
This study was supported by the National Natural Science
Foundation of China (No. 21501032), the Innovative Team &
Outstanding Talent Program of Colleges and Universities in Guangxi
(2017-38), Guangxi Natural Science Foundation (Nos.
2016GXNSFAA380300 and 2014GXNSFBA118050), Guangxi New
Century Ten, Hundred and Thousand Talents Project ([2017]42) and
the State Key Laboratory Cultivation Base for Chemistry and Mo-
lecular Engineering of Medicinal Resources (Ministry of Science and
Technology of China, No. CMEMR2014-B14).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.07.066.
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