Design, synthesis and pharmacological evaluation of novel 2-chloro-3-(1: H -benzo [d] imidazol-2-yl)quinoline derivatives as antitumor agents: In vitro and in vivo antitumor activity, cell cycle arrest and apoptotic response

Article abstract of DOI:10.1039/c8ra04640a

A series of novel 2-chloro-3-(1H-benzo[d]imidazol-2-yl)quinoline derivatives (3a₁-3d₆) were designed and synthesized as antitumor agents. In vitro antitumor assay results showed that some compounds exhibited moderate to high inhibitory activities against HepG2, SK-OV-3, NCI-H460 and BEL-7404 tumor cell lines, and most compounds exhibited much lower cytotoxicities against HL-7702 normal cell line compared to 5-FU and cisplatin. In vivo antitumor assay results showed that the representative compound 3a₁ exhibited effective inhibition on tumor growth in the HepG2 xenograft mouse model. Mechanistic studies suggested that 3a₁ may exert antitumor activity by the up-regulation of Bax, intracellular Ca²⁺ release, ROS generation, p21, p27 and p53, downregulation of Bcl-2, activation of caspase-9 and caspase-3 and subsequent cleavage of PARP, and inhibition of CDK activity.

Full text of DOI:10.1039/c8ra04640a

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PAPER
Design, synthesis and pharmacological evaluation
of novel 2-chloro-3-(1H-benzo[d]imidazol-2-yl)
quinoline derivatives as antitumor agents: in vitro
and in vivo antitumor activity, cell cycle arrest and
apoptotic response†
Cite this: RSC Adv., 2018, 8, 24376
ab
c
b
b
a
Wen-Bin Kuang, Ri-Zhen Huang, Yi-Lin Fang, Gui-Bin Liang, Chen-Hui Yang,
a
abd
Xian-Li Ma* and Ye Zhang
*
A series of novel 2-chloro-3-(1H-benzo[d]imidazol-2-yl)quinoline derivatives (3a
1
ꢀ3d
6
) were designed and
synthesized as antitumor agents. In vitro antitumor assay results showed that some compounds exhibited
moderate to high inhibitory activities against HepG2, SK-OV-3, NCI-H460 and BEL-7404 tumor cell lines,
and most compounds exhibited much lower cytotoxicities against HL-7702 normal cell line compared to
5
-FU and cisplatin. In vivo antitumor assay results showed that the representative compound 3a
1
exhibited
Received 31st May 2018
Accepted 19th June 2018
effective inhibition on tumor growth in the HepG2 xenograft mouse model. Mechanistic studies suggested
2+
that 3a
1
may exert antitumor activity by the up-regulation of Bax, intracellular Ca
release, ROS
DOI: 10.1039/c8ra04640a
generation, p21, p27 and p53, downregulation of Bcl-2, activation of caspase-9 and caspase-3 and
subsequent cleavage of PARP, and inhibition of CDK activity.
rsc.li/rsc-advances
present in a number of natural and synthetic agents: chlor-
oquinoline and benzimidazole.
1
. Introduction
As one of the second leading causes of mortality, cancer is
a great concern to mankind. Due to negative lifestyle and food biological activities including inhibition of cellular prolifera-
Quinoline and its derivatives display a variety of powerful
5–7
habits, which seriously increase the cancer risk, more and more tion and developmental changes, which make the quinoline
1,2
people around the globe are diagnosed with this disease.
ring a valuable versatile synthetic scaffold for new antitumor
Therefore, the discovery and development of promising new agents. Among the various existing active scaffolds of quino-
antitumor agents with high efficiency and low toxicity to healthy lines, chloroquinoline is a type of alkaloid, which is as abun-
cells and tissues have become urgent and key focuses of phar- dant as quinoline. Previous studies have indicated that
maceutical chemists worldwide. To realize this goal, a great deal chloroquinoline derivatives display preferable biological activ-
8,9
of attention is being focused on molecules that act synchro- ities such as anticancer and antiproliferation activities.
nously on multiple targets by combining two/more active Chloroquinoline scaffold is thus chosen in the present study as
pharmacophores covalently in a single-hybrid molecule with an active antitumor pharmacy core, and some structural
3,4
dual/multiple antitumor effects. This combination principle modications are designed to explore the antitumor activity. In
strategy encouraged us to design, synthesize and evaluate a new addition, a bicyclic compound, namely, benzimidazole, which
set of hybrid molecules by combining two pharmacophores is the fusion of benzene and imidazole, is a pharmacophore
10
possessing a wide range of biological activities. Some benz-
imidazole derivatives have been found to display potent anti-
a
School of Pharmacy, Guilin Medical University, North Ring 2nd Road 109, Guilin
11–13
tumor activities.
Accordingly, the benzimidazole nucleus
5
41004, PR China. E-mail: zhangye81@126.com; mxl78@glmc.edu.cn; Fax: +86-
73-5895132; Tel: +86-773-5895132
has been considered as an important pharmacophore in anti-
tumor chemistry. It is thus expected that combining the chlor-
7
b
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal
Resources (Ministry of Education of China), School of Chemistry and oquinolone scaffold with the benzimidazole skeleton may lead
Pharmaceutical Sciences of Guangxi Normal University, Guilin 541004, PR China
to potent antitumor activity. Therefore, under the combination
principle, a set of 2-chloroquinoline-benzimidazole derivatives
c
Department of Pharmaceutical Engineering, School of Chemistry and Chemical
Engineering, Southeast University, Nanjing 211189, PR China
3
(3a ꢀ3d ) were synthesized, and their antitumor activities
1
6
d
Department of Chemistry & Pharmaceutical Science, Guilin Normal College, Guangxi
were evaluated. Moreover, previous studies have indicated that
both quinoline and benzimidazole derivatives exert antitumor
activities through modulation of cell cycle arrest and apoptotic
5
41001, PR China
†
Electronic supplementary information (ESI) available: The procedures for
1
13
biological assays, H NMR, C NMR and HR-MS. See DOI: 10.1039/c8ra04640a
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responses.
Therefore, mechanistic studies focused on 2.2. In vitro antiproliferative activity
apoptosis and cycle arrest induced by the representative
The in vitro antiproliferative activities of the compounds
compound 3a were also performed.
1
3
(3a
1
ꢀ3d
6
) were evaluated against 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, using the
2. Results and discussion
2.1 Chemistry
15,17
methylthiazol tetrazolium (MTT) assay.
Two commercial
2
3
-Chloro-3-(1H-benzo[d]imidazol-2-yl)quinoline
derivatives
anticancer drugs 5-uorouracil (5-FU) and cisplatin were used
as positive controls. The results are shown in Table 1. Table 1
indicates that many compounds showed evident anti-
proliferative activities against HepG2, SKOV3, NCI-H460 and
BEL-7404 cell lines, and some displayed better antiproliferative
activities than 5-FU and cisplatin while exhibiting lower cyto-
toxicities against the HL-7702 normal cell line. In the HepG2
(3a ꢀ3d ) were synthesized as shown in Scheme 1. First, 2-
1
6
chloro-quinoline-3-carbaldehyde derivatives 2(2aꢀ2d) were
synthesized through the Vilsmeier–Haack–Arnold reaction,
which involves the combination of acetanilide derivatives
1
of
(1aꢀ1d) with N,N-dimethylformamide (DMF) in the presence
1
5
phosphorus
oxychloride.
ꢀ3d
2-Chloro-quinoline-3-
) were then obtained in
benzimidazole derivatives 3(3a
1
6
cell line assay, compounds 3a
1
, 3a
3 4 5 3 4 5
, 3a , 3a , 3c , 3c , 3c and
moderate yields by the condensation reactions of 2(2aꢀ2d) with
o-phenylenediamine derivatives in the presence of methanol.
The structures of compounds 3(3a ꢀ3d ) were conrmed using
3c6 exhibited higher antiproliferative activities than 5-FU (IC50
¼
31.98 mM), with IC50 values in the range of 7.54–28.24 mM.
1
6
Compounds 3a and 3a5 exhibited better antiproliferative
1
1
13
various spectroscopic methods including H NMR, C NMR
and high-resolution mass spectrometry (HR-MS) (Part 2 of
activities than cisplatin (IC50 ¼ 10.12 mM). In the SKOV-3 cell
1 3 5 3 5 6
line assay, compounds 3a , 3a , 3a , 3c , 3c and 3c showed
1
ESI†). In H NMR spectra, the hydrogen of NH fused in benz-
imidazole and the aromatic hydrogens (H-Ar) in quinolone and
benzimidazole were identied by chemical shis (d) 12.66–
higher antiproliferative activities than 5-FU (IC50 ¼ 26.34 mM),
with IC50 in the range of 8.83–23.00 mM. Compounds 3a , 3a
and 3c displayed better antiproliferative activities than
1
3
3
1
3.26 and 7.14–8.79, respectively. In addition, the hydrogens of
cisplatin (IC50 ¼ 15.60 mM). In the NCI-H460 cell line assay,
methyl groups were also identied at d 3.93, 3.84, 2.54 and 2.34,
compounds 3a , 3a , 3a , 3a , 3c , 3c , 3c and 3c5 showed
1
3
4
5
1
3
4
whereas the hydrogens of O–CH –O group were found around
d 6.30. In C NMR spectra, carbons in carbonyl and O–CH –O
2
groups were identied at d 161.04–161.99 and around 95.40,
respectively, whereas the carbons of methyl groups were found
at d 20.59–20.94 and 55.94–56.09. The signals (d) at 99.47–
2
higher antiproliferative activities than 5-FU (IC50 ¼ 45.44 mM),
with IC50 in the range of 14.03–38.25 mM. Compounds 3a and
displayed higher antiproliferative activities than cisplatin
IC50 ¼ 20.36 mM). In the BEL-7404 cell line assay, compounds
, 3a , 3a , 3a , 3c , 3c , 3c , 3c and 3c exhibited higher
1
3
1
3a
5
(
3a
1
3
4
5
1
3
4
5
6
1
48.19 were assigned to carbons in C]C and C]N groups as
antiproliferative activities than 5-FU (IC50 ¼ 40.21 mM), with
well as the aromatic carbons in quinolone and benzimidazole.
IC50 values in the range of 9.06–39.94 mM. The compound 3c4
HR-MS provided exact formula weights and compositions of
exhibited higher antiproliferative activity than cisplatin (IC50
14.72 mM). Based on the antiproliferation results shown in
Table 1, it can be concluded that for compounds 3(3a ),
the absence of substitution and the methoxy substitution at 6
position of quinolone moiety as well as dimethyl, chloro and
bromo substitutions in the benzimidazole moiety had positive
effects on the antiproliferative activity.
¼
1
3
(3a ꢀ3d ), which were well consistent with the results of H
1
6
1
3
NMR and C NMR and the structures shown in Scheme 1, thus
1
ꢀ3d
6
indicating that the structures of compounds 3(3a
conrmed.
1
6
ꢀ3d ) were
Among the compounds 3(3a ꢀ3d ), the compound 3a showed
1
6
1
the best potential antiproliferative activity against these cancer cell
lines. Thus, 3a was selected as a representative compound for in
1
vivo antiproliferative activity and mechanistic studies.
2.3. Preliminary safety evaluation of 3a
1
The in vivo toxicity of compound 3a was evaluated in Kun-ming
1
(KM) mice. Through intraperitoneal injection, the mice received
3
a
1
ꢀ
(dissolved in 5% v/v DMSO/saline solvent) at a dose of 50 mg
1
kg per day for 8 consecutive days; then, for the next 6 days, the
1
mice were treated with compound 3a at the same dose once
every two days. The body weights of the animals were monitored
daily and used as a parameter of systemic toxicity.
No adverse effects were observed in the KM mice injected with
3
a1 during the 14 day monitoring period. As shown in Fig. 1, the
Scheme 1 Synthetic routes of compounds 3(3a
1
ꢀ3d
6
). Reagents and
average body weights of the mice treated with 3a showed similar
growth rates to those of the control group. It is also worth noting
1
3
conditions: (a) POCl , DMF; (b) o-phenylenediamine derivatives,
methanol.
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a
a
Table 1 IC50 values of compounds 3(3a
1
6
ꢀ3d ) toward four selected tumor cell lines and a normal cell line
IC50 (mM)
Compounds
HepG2
SKOV-3
NCI-H460
BEL-7404
HL-7702
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
5
a1(3c)
a2(3m)
a3(3q)
a4(3i)
a5(3e)
a6(3u)
b1(3d)
b2(3n)
b3(3r)
b4(3j)
b5(3f)
b6(3v)
c1(3a)
c2(3o)
c3(3s)
c4(3k)
c5(3g)
c6(3w)
d1(3b)
d2(3p)
d3(3t)
d4(3l)
d5(3h)
d6(3x)
-FU
7.54 ꢁ 0.77
>100
11.52 ꢁ 0.89
28.24 ꢁ 1.24
7.59 ꢁ 1.64
>100
>100
>100
>100
>100
9.40 ꢁ 0.69
>100
10.40 ꢁ 1.34
34.14 ꢁ 1.82
21.35 ꢁ 0.88
>100
>100
>100
>100
>100
14.03 ꢁ 0.94
>100
22.64 ꢁ 1.02
31.40 ꢁ 1.47
15.11 ꢁ 0.57
>100
>100
>100
>100
>100
15.40 ꢁ 0.85
>100
22.20 ꢁ 0.76
29.99 ꢁ 0.59
19.39 ꢁ 1.43
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
37.81 ꢁ 1.05
36.92 ꢁ 2.01
38.25 ꢁ 1.58
39.94 ꢁ 1.18
>100
>100
>100
>100
14.10 ꢁ 1.55
12.15 ꢁ 1.05
16.68 ꢁ 1.19
22.35 ꢁ 1.25
>100
>100
>100
>100
>100
8.83 ꢁ 2.10
32.66 ꢁ 0.70
17.88 ꢁ 1.79
23.00 ꢁ 1.57
>100
>100
>100
>100
>100
23.84 ꢁ 0.84
26.79 ꢁ 1.06
21.14 ꢁ 1.41
>100
>100
>100
>100
>100
>100
>100
26.69 ꢁ 0.39
11.29 ꢁ 1.18
21.06 ꢁ 0.99
>100
>100
>100
>100
>100
>100
>100
>100
41.57
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 a
IC50 values are presented as mean ꢁ SD (standard error of the mean) from three repeated experiments.
ꢀ
1
that, as shown in Fig. 1, the injection of 3a
1
even led to extra and high doses (20 mg kg ) were determined to be 31.3% and
weight gain in the KM mice, indicating that no adverse effects 19.5% (p < 0.01), respectively, at day 41. Paclitaxel at the 5 mg
ꢀ
1
were found aer the treatment of these mice with 3a1.
kg dose showed a T/C value of 19.0%, which was almost equal
ꢀ1
to that of 3a at 20 mg kg dose; the result indicated that 3a (at
0 mg kg dose) showed almost equal in vivo antitumor activity
with paclitaxel (at 5 mg kg dose) in the HepG2 mouse model.
In addition, no evident changes in the body weight and no other
1
1
ꢀ
1
2
ꢀ1
2.4. In vivo antiproliferative activity of 3a1
The potential in vitro antiproliferative activity and the absence of
adverse effect of 3a1 encouraged us to perform in vivo anti-
proliferative assays to further evaluate its antitumor activity. The
HepG2 xenogra model was selected in this study, and paclitaxel
was used as the positive control. SPF BALB/c nude mice were
adverse effects were found in the mice treated with 3a
1
(at 20 mg
ꢀ
1
kg dose) (Fig. S1-1†), suggesting that 3a
1
may be a good
candidate for an antitumor agent.
randomly divided into vehicle control group, low dose of 3a
treated group, high dose of 3a -treated group, and positive control
group. The mice in the vehicle control group were injected with 5%
DMSO saline solution, and the mice in low and high dose of 3a
1
-
1
1
-
treated group were injected with 3a (in 5% DMSO saline solution),
1
whereas the mice in the positive control group were injected with
paclitaxel (in 5% DMSO saline solution). Tumor size of mice was
tested twice in a week in two dimensions, and the volume was
measured to evaluate in vivo antitumor activity.
As shown in Fig. 2, compared with the observations for
1
control, the treatment with 3a and paclitaxel signicantly
inhibited the increase in the tumour volume in mice, indicating
that compound 3a exhibited potential antitumor effect. The
1
ꢀ
1
treatment of high dose of 3a (20 mg kg ) exhibited similar
1
inhibition effect on the tumour volume as that of paclitaxel
Fig.
1 Changes in the body weight of KM mice injected with
ꢀ1
ꢀ1
(
5 mg kg ). The T/C values of 3a
1
injected in low (10 mg kg
)
compound 3a
1
. There are six mice in each experimental group.
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ꢀ1
1 1
Fig. 2 In vivo anticancer activity of 3a in the HepG2 xenograft model. Effect of 3a (at doses of 10 and 20 mg kg ), paclitaxel (at dose of 5 mg
ꢀ
1
3
kg ) and vehicle control (5% DMSO in saline, v/v) on tumor growth. Tumor growth was monitored by mean tumor volume (mm ) ꢁ SD (n ¼ 6) (A)
and calculated as relative tumor increment rate (T/C, %) (B).
1 1
Fig. 3 Cell cycle analysis of HepG2 cells treated with 3a . The HepG2 cells were treated with compound 3a at different concentrations, i.e., (a)
0
mM, (b) 6 mM, (c) 8 mM and (d) 10 mM for 48 h.
1
Fig. 4 Effects of compounds 3a on the expression levels of relevant proteins in HepG2 cells. western blot analysis was performed with anti-
bodies against cyclin B1, CDK1, CDC25C, p21, p23, and p53. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading
standard.
2.5. Study on cell cycle distribution
treatment of HepG2 cells with 3a promoted cell cycle arrest at
1
the G2 phase, which led to a clear increase in the G -phase
population (34.80% at 6 mM, 40.65% at 8 mM and 53.33% at 10
mM) compared with the observations in the control group
2
As signicant control mechanisms, cell cycle checkpoints
control and ensure the generation and performance of cell cycle
18
events. To study the potential role of cell cycle arrest in tumor
(
19.04%). Consequently, the G
of the HepG2 cells decreased. The results indicated that
compound 3a clearly arrested the cell cycle of HepG2 cells at
1
-phase and S-phase populations
cell death induced by compounds 3, HepG2 cells were treated
with the representative compound 3a at different concentra-
1
1
tions (0, 6, 8 and 10 mM) for 48 h. As shown in Fig. 3, the
the G /M stage in a concentration-dependent manner.
2
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1
Fig. 5 Effects of the compound 3a on the morphology of HepG2 cells. The cells were stained with the Hoechst 33 258 dye.
Cyclin B1/CDK1(CDC2) kinase complexes are main regulators
of G /M transition, and their generation is usually monitored by
the positive mediator cell division cyclin 25 homolog C (CDC25C)
2
19,20
phosphatase.
CDC25C mediates the phosphorylation of CDK1
and consequently leads to the activation of cyclin B1/CDK1
1
9,20
complexes and execution of G
tion of CDC25C accompanied by DNA damage and cellular
activities resulting in G /M arrest causes its degradation and
sequestration to the cytoplasm, which consequently leads to the
2
/M transition;
phosphoryla-
2
21
destabilization of the cyclin–CDK complexes. The p53, p27 and
p21 genes encoded by some antioncogenes are also involved in
the G /M arrest through increasing the accumulation of the
2
22
dormant cyclin B–CDK complex. Therefore, in the present
study, the expression levels of cyclin B1, CDK1, CDC25C, p53, p27
and p21 in the HepG2 cells were assayed using western blot. As
shown in Fig. 4, compared to the observations for the control, the
1
lysates of the HepG2 cells treated with 3a down-regulated the
levels of cyclin B1, CDK1 and CDC25C while up-regulating the
levels of p53, p21 and p27. Thus, compound 3a induced G /M
1
2
Fig. 6 Compound 3a
visualized via AO/EB staining coupled with fluorescence microscopy.
1
induced apoptosis in HepG2 cells. The cells were phase cell cycle arrest through inhibition of CDK activity and
increase in the expression levels of p53, p27 and p21.
2.6. Apoptosis assays
Apoptosis is another important mechanism in cancer treat-
23,24
ment, and it is a key pathway leading to cell death.
Thus, the
Fig. 7 Apoptosis ratios of HepG2 cells. (a) Negative control, (b) treated
with 6 mM 3a , (c) treated with 8 mM 3a , (d) treated with 10 mM 3a . The Fig. 8 Compound 3a
cells were analyzed using the Annexin V/PI assay in a flow cytometer. HepG2 cells.
1
1
1
1
affected the levels of intracellular ROS in
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Fig. 7, the treatment of HepG2 cells with compound 3a at
different concentrations (6, 8 and 10 mM) led to signicant
increase in the apoptotic cell population in a concentration-
dependent manner. The results indicated that compound 3a1
induced apoptosis of HepG2 cells in a dose-dependent manner.
2.7. ROS generation
A previous study proved that the generation of intracellular
25
reactive oxygen species (ROS) can induce apoptosis. To
investigate the role of ROS in 3a -induced apoptosis in the
1
HepG2 cells, intracellular ROS levels were determined through
0

uorescence microscopy using 4 ,6-diamidino-2-phenylindole
dihydrochloride (DAPI) and 2,7-dichlorouorescein diacetate
DCFH-DA) as uorescent probes. Fig. 8 demonstrates that both
(
control cells and HepG2 cells treated with compound 3a dis-
1
played blue uorescence in the cytoplasm in the DAPI staining
assay, whereas the HepG2 cells treated with compound 3a1
exhibited stronger green uorescence signals compared to the
control in the DCFH-DA staining assay, indicating that 3a
up-regulate the levels of intracellular ROS. These results indi-
cated that the generation of ROS induced by 3a may be an
1
can
1
Fig. 9 Compound 3a caused an increase in the level of intracellular
Ca in HepG2 cells.
2+
1
24
underlying cause for apoptosis.
1
apoptosis-inducing effect of compound 3a was investigated by
staining with the uorescent probes Hoechst 33 258 and acri-
dine orange/ethidium bromide (AO/EB). In the Hoechst 33 258
staining assay (Fig. 5), normal HeGp2 cells emitted blue uo-
2
+
2
.8. Intracellular Ca release
It is known that intracellular calcium takes part in cell
apoptosis, and an overload of calcium can induce apoptosis.
rescence, whereas the cells treated with 3a for 12 h exhibited
26
1
stronger blue uorescence and displayed characteristic
Thus, the role of calcium signaling in cellular apoptosis
apoptotic morphologies. This phenomenon indicated that
1
induced by 3a was studied. The HepG2 cells were treated with
21
2+
compound 3a
AO/EB staining assay, the morphology of 3a
1
induced the apoptosis of HeGp2 cells. In the
-treated HeGp2
3a1 for 24 h, and the intracellular Ca level was assayed by
1

uorescence microscopy using DCFH-DA as a calcium probe.
caused
an increase in the intracellular Ca concentration, indicating
that 3a -induced apoptosis was related to the up-regulation of
intracellular Ca level.
cells exhibited evident changes (Fig. 6). The cell nuclei were
stained yellow/orange, and the morphology changes suggested
pycnosis, membrane blebbing and cell budding, all of which are
Fig. 9 shows that the treatment of HepG2 cells with 3a
1
2
+
1
21
2+
clear characteristics of apoptosis. Therefore, on the basis of
the above-mentioned observations, it can be concluded that the
compound 3a
The apoptosis ratios of HepG2 cells treated with 3a
then quantitatively assayed by ow cytometry. As shown in
1
induced apoptosis of HeGp2 cells.
2
.9. Caspase-dependent apoptosis in HepG2 cells
1
were
As well-known anti- and pro-apoptotic proteins, Bcl-2/Bax
family proteins play important roles in the inhibition or
1
Fig. 10 Effect of compound 3a on the expression levels of Bax, Bcl-2, cytochrome c, caspase-3, caspase-9 and PARP in HepG2 cells. GAPDH
was used as a loading standard.
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27,28
promotion of cell death.
A previous study proved that the obtained on a BRUKER ESQUIRE HCT spectrometer. The
ratio of Bcl-2/Bax expression is vital in the regulation of GelRed nucleic acid stain was purchased from Biotium.
29
apoptosis. Bcl-2/Bax family proteins mainly participate in
apoptosis by interfering with caspases, which are the pivotal
30,31
4.2. General synthesis procedure for compounds 3(3a
1
ꢀ3d
)
6
effectors of programmed cell death.
Caspases are a family of
cysteinyl aspartate specic proteases, which participate in 2-Chloro-quinoline-3-carbaldehyde derivatives 2(2aꢀ2d) were
apoptosis, and they can be divided into initiators (caspases 8, 9 synthesized by condensation of acetanilide derivatives 1(1aꢀ1d)
3
0,31
and 10) and executioners (caspases 3, 6 and 7).
The caspase with DMF in the presence of phosphorus oxychloride, according
15
cascade is mediated by the proteolysis of inactive procaspases to our previous study. 2-Chloro-quinoline 3-carbaldehyde
and propagated by the cleavage of downstream caspases and derivatives 2(2aꢀ2d) (1 mmol), o-phenylenediamine derivatives
21
substrates including poly(ADP-ribose) polymerase (PARP). To (1 mmol) and 3 mL methanol were mixed in a pressure tube and
ꢂ
investigate the mechanism of 3a -induced apoptosis in cancer heated at 90 C for 4 h. Aer the reaction, the mixture was
1
cells and to nd if caspases were involved in 3a
apoptosis, the expression levels of Bax, Bcl-2, cytochrome c, as yellow powder. If necessary, the target compounds
caspase-9, caspase-3 and PARP in HepG2 cells treated with 3a
3(3a ꢀ3d ) were puried by column chromatography (silica
were assayed by western blot. As shown in Fig. 10, the treatment 300–400 mesh) with acetic ether–petroleum benzine (v/v: 1/5) as
of HepG2 cells with 3a led to evident increase in the expression an eluent.
levels of Bax, caspase-9, caspase-3 and PARP and reduction in
3a . Yield, 64.8%; H NMR (500 MHz, DMSO-d ) d 12.68 (s,
1
-induced cooled to room temperature and ltered to obtain the product
1
1
6
1
1
1
6
the expression level of Bcl-2 accompanied by the release of 1H, NH), 8.97 (s, 1H, H-Ar), 8.20 (s, 1H, H-Ar), 8.06 (s, 1H, H-Ar),
mitochondrial cytochrome c into the cytosol compared to the 7.92 (d, J ¼ 8.4 Hz, 1H, H-Ar), 7.75 (d, J ¼ 8.1 Hz, 1H, H-Ar), 7.45
1
3
observations for control. These results indicated that (s, 2H, H-Ar), 2.36 (s, 6H, –CH ); C NMR (126 MHz, DMSO-d )
3
6
1
compound 3a could down-regulate the expression of Bcl-2, d 147.68, 147.21, 141.58, 132.36, 129.01, 128.44, 128.16, 126.87,
leading to the release of cytochrome c, activation of caspase-9 125.14, 20.50; ESI-HRMS, calculated m/z for C H ClN [M +
1
8
14
3
+
and induction of apoptosis, thus demonstrating that H] : 308.0955, found: 308.0972.
1
compound 3a
1
exerted pro-apoptotic effects through a mito-
3a
2
. Yield, 57.3%; H NMR (500 MHz, DMSO-d
6
) d 13.08 (s,
chondrial-mediated pathway and caspase cascade.
1H, NH), 9.01 (s, 1H, H-Ar), 8.20 (d, J ¼ 7.6 Hz, 1H, H-Ar), 8.07 (d,
J ¼ 8.5 Hz, 1H, H-Ar), 7.95 (d, J ¼ 8.4 Hz, 1H, H-Ar), 7.77 (s, 1H,
H-Ar), 7.70 (s, 1H, H-Ar), 7.50 (s, 1H, H-Ar), 7.16 (d, J ¼ 8.5 Hz,
1
3
3. Conclusions
1
6
H, H-Ar); C NMR (126 MHz, DMSO-d ) d 147.48, 147.36,
1
41.99, 132.66, 129.11, 128.58, 128.19, 126.79, 124.57; ESI-
In summary, we designed and synthesized a set of 2-chloro-3-
1H-benzo[d]imidazol-2-yl)quinoline derivatives and evaluated
+
HRMS, calculated m/z for C H ClFN [M + H] : 298.0547,
1
6
9
3
(
found: 298.0543.
. Yield, 56.9%; H NMR (400 MHz, DMSO-d
H, NH), 9.03 (s, 1H, H-Ar), 8.21 (d, J ¼ 8.0 Hz, 1H, H-Ar), 8.08 (d,
J ¼ 8.4 Hz, 1H, H-Ar), 7.95 (s, 1H, H-Ar), 7.77 (s, 2H, H-Ar), 7.70
the antiproliferative activities of these compounds against four
cancer cell lines (HepG2, SKOV-3, NCI-H460 and BEL-7404). We
identied some compounds exhibiting high in vitro anticancer
activities and low toxicities against normal HL-7702 cells. In vivo
antitumor activity study and animal toxicology test showed that
1
3a
3
6
) d 13.17 (s,
1
1
3
(
6
s, 1H, H-Ar), 7.32 (s, 1H, H-Ar); C NMR (101 MHz, DMSO-d )
d 147.43, 147.40, 142.16, 132.75, 129.15, 128.61, 128.20, 126.77,
3a
1
exhibited potent antitumor activity in the HepG2 xenogra
ꢀ
1
3
24.41; ESI-HRMS, calculated m/z for C H Cl N [M ꢀ H] :
1
6
9
2 3
mouse model. Mechanistic studies showed that 3a caused
1
12.0096, found: 312.0098.
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
1
3a
4
. Yield, 55.4%; H NMR (500 MHz, DMSO-d
6
) d 13.18 (s,
1
H, NH), 9.02 (s, 1H, H-Ar), 8.21 (d, J ¼ 7.7 Hz, 1H, H-Ar), 8.07 (d,
J ¼ 7.9 Hz, 1H, H-Ar), 7.95 (s, 1H, H-Ar), 7.91 (s, 1H, H-Ar), 7.77
2
+
Ca release and ROS regulation. Cell cycle studies indicated
that compound 3a induced cell cycle arrest at the G /M phase
1
3
(
s, 1H, H-Ar), 7.67 (s, 1H, H-Ar), 7.44 (s, 1H, H-Ar); C NMR (126
MHz, DMSO-d ) d 147.44, 147.42, 142.14, 132.75, 129.14, 128.61,
9
28.20, 126.76, 124.38; ESI-HRMS, calculated m/z for C16H -
1
2
6
by inhibition of CDK activity and activation of the p53 protein.
This study suggests that the rational design of 2-chloro-3-(1H-
benzo[d]imidazol-2-yl)quinoline derivatives under the combi-
nation principle is a feasible method to discover new antitumor
therapeutics.
1
ꢀ
BrClN [M ꢀ H] : 355.9590, found: 355.9625.
3
1
3
a . Yield, 55.1%; H NMR (400 MHz, DMSO-d ) d 13.30 (s,
5
6
1
H, NH), 9.03 (s, 1H, H-Ar), 8.21 (d, J ¼ 7.6 Hz, 1H, H-Ar), 8.08 (d,
13
J ¼ 8.4 Hz, 2H, H-Ar), 7.96 (s, 2H, H-Ar), 7.77 (s, 1H, H-Ar);
C
6
NMR (101 MHz, DMSO-d ) d 150.82, 147.47, 147.32, 142.32,
1
32.89, 129.19, 128.67, 128.21, 126.71, 124.03; ESI-HRMS,
4
. Experimental
ꢀ
calculated m/z for C16
45.9747.
3a . Yield, 63.2%; H NMR (400 MHz, DMSO-d ) d 12.97 (s,
H
8
Cl
3
N
3
[M ꢀ H] : 345.9706, found:
4.1. Materials
3
1
All chemicals of reagent grade were commercially available and
6
6
used without further purication. NMR spectra were assayed on 1H, NH), 9.01 (s, 1H, H-Ar), 8.21 (d, J ¼ 7.4 Hz, 1H, H-Ar), 8.08 (d,
a BRUKER AVANCE AV 400/500 spectrometer using tetramethyl J ¼ 8.0 Hz, 1H, H-Ar), 7.94 (s, 1H, H-Ar), 7.77 (s, 2H, H-Ar), 7.67
1
3
6
silane (TMS) as the internal standard. Mass spectra were (s, 1H, H-Ar), 7.30 (s, 2H, H-Ar); C NMR (101 MHz, DMSO-d )
24382 | RSC Adv., 2018, 8, 24376–24385
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11
d 148.21, 147.63, 147.31, 141.97, 132.58, 129.09, 128.55, 128.19, 124.66, 106.76, 56.23; ESI-HRMS, calculated m/z for C17H -
+
1
26.83, 124.92; ESI-HRMS, calculated m/z for C H ClN [M ꢀ ClFN O [M + H] : 328.0653, found: 328.0640.
1
6
10
3
3
ꢀ
1
H] : 278.0485, found: 278.0489.
3c . Yield 55.1%, H NMR (400 MHz, DMSO-d ) d 13.12 (s,
3 6
1
3
b . Yield, 58.1%; H NMR (500 MHz, DMSO-d ) d 12.66 (s, 1H, NH), 8.88 (s, 1H, H-Ar), 7.98 (d, J ¼ 9.0 Hz, 1H, H-Ar), 7.76 (s,
1
6
1
7
H, NH), 8.84 (s, 1H, H-Ar), 7.94 (s, 2H, H-Ar), 7.84 (s, 1H, H-Ar), 1H, H-Ar), 7.70 (s, 1H, H-Ar), 7.60 (s, 1H, H-Ar), 7.58 (d, J ¼
13
.46 (s, 2H, H-Ar), 2.54 (s, 3H, –CH
3
), 2.36 (s, 6H, –CH
3
);
C
9.0 Hz, 1H, H-Ar), 7.32 (d, J ¼ 8.6 Hz, 1H, H-Ar), 3.93 (s, 3H,
13
NMR (126 MHz, DMSO-d
6
) d 147.32, 146.78, 145.84, 140.84, –OCH
3
); C NMR (101 MHz, DMSO-d
6
) d 158.74, 149.73, 144.65,
1
38.21, 134.49, 127.90, 127.54, 126.88, 125.09, 21.62, 20.50; ESI- 143.45, 140.73, 129.68, 128.10, 125.02, 124.51, 106.81, 56.26;
+
ꢀ
HRMS, calculated m/z for C19
H
16ClN
3
[M + H] : 322.1111, found: ESI-HRMS m/z calc. for C17
H
11Cl
2
N
3
O [M ꢀ H] : 342.0201;
322.1129.
found: 342.0205.
1
1
3
b . Yield, 64.6%; H NMR (400 MHz, DMSO-d ) d 12.14 (s,
3c . Yield, 57.1%; H NMR (400 MHz, DMSO-d ) d 13.14 (s,
4
2
6
6
1
1
H, NH), 8.87 (s, 1H, H-Ar), 7.96 (d, J ¼ 8.6 Hz, 1H, H-Ar), 7.94 (s, 1H, NH), 8.88 (s, 1H, H-Ar), 7.98 (d, J ¼ 9.1 Hz, 1H, H-Ar), 7.90 (s,
H, H-Ar), 7.77 (d, J ¼ 8.6 Hz, 1H, H-Ar), 7.70 (s, 1H, H-Ar), 7.51 1H, H-Ar), 7.68 (s, 1H, H-Ar), 7.60 (s, 1H, H-Ar), 7.58 (d, J ¼
1
3
(s, 1H, H-Ar), 7.16 (s, 1H, H-Ar), 2.54 (s, 3H, –CH
3
); C NMR (101 9.1 Hz, 1H, H-Ar), 7.43 (d, J ¼ 8.4 Hz, 1H, H-Ar), 3.93 (s, 3H,
13
MHz, DMSO-d
6
) d 146.57, 145.98, 141.23, 138.35, 134.76, 127.92, –OCH
3
); C NMR (101 MHz, DMSO-d
6
) d 158.73, 144.64, 143.45,
1
C
27.61, 126.79, 124.52, 21.62; ESI-HRMS, calculated m/z for 140.75, 129.68, 128.10, 125.04, 124.47, 106.80, 56.26; ESI-HRMS,
+
ꢀ
17
H
11ClFN
3
[M + H] : 312.0704, found: 312.0702.
calculated m/z for C17
H
11Cl
2
N
3
O [M ꢀ H] : 385.9696, found:
1
3b . Yield, 55.9%; H NMR (400 MHz, DMSO-d ) d 13.13 (s, 385.9715.
3
6
1
1
1
H, NH), 8.88 (s, 1H, H-Ar), 7.97 (d, J ¼ 8.7 Hz, 1H, H-Ar), 7.95 (s,
3c . Yield, 57.5%; H NMR (400 MHz, DMSO-d ) d 13.26 (s,
5 6
H, H-Ar), 7.78 (d, J ¼ 8.6 Hz, 2H, H-Ar), 7.70 (s, 1H, H-Ar), 7.32 1H, NH), 8.88 (s, 1H, H-Ar), 7.97 (s, 3H, H-Ar), 7.60 (s, 2H, H-Ar),
1
3
13
(
d, J ¼ 8.6 Hz, 1H, H-Ar), 2.54 (s, 3H, –CH
3
); C NMR (101 MHz, 3.93 (s, 3H, –OCH
3 6
); C NMR (101 MHz, DMSO-d ) d 158.77,
DMSO-d
6
) d 146.53, 146.04, 141.38, 138.40, 134.86, 127.94, 150.93, 144.53, 143.52, 140.87, 129.69, 128.05, 125.15, 124.12,
1
C
27.64, 126.77, 124.36, 21.64; ESI-HRMS, calculated m/z for 106.85, 56.27; ESI-HRMS, calculated m/z for C17
H
11Cl
2
N
3
O [M ꢀ
ꢀ
ꢀ
17
H11Cl
2
N
3
[M ꢀ H] : 326.0252, found: 326.0255.
H] : 375.9811, found: 378.9859.
1
1
3b
4
. Yield, 61.0%; H NMR (400 MHz, DMSO-d
6
) d 13.16 (s,
3c
6
. Yield, 65.1%; H NMR (400 MHz, DMSO-d
6
) d 12.68 (s,
1
1
H, NH), 8.88 (s, 1H, H-Ar), 7.97 (d, J ¼ 8.8 Hz, 1H, H-Ar), 7.95 (s, 1H, NH), 9.10 (s, 1H, H-Ar), 7.75 (s, 1H, H-Ar), 7.66 (s, 1H, H-Ar),
H, H-Ar), 7.78 (d, J ¼ 8.6 Hz, 1H, H-Ar), 7.73 (s, 1H, H-Ar), 7.68 7.52 (s, 1H, H-Ar), 7.39 (d, J ¼ 9.0 Hz, 1H, H-Ar), 7.29 (s, 1H, H-
1
3
(
s, 1H, H-Ar), 7.43 (d, J ¼ 8.3 Hz, 1H, H-Ar), 2.54 (s, 3H, –CH ); Ar), 7.22 (s, 1H, H-Ar), 7.20 (s, 1H, H-Ar), 3.84 (s, 3H, –OCH );
C
3
3
1
3
C NMR (101 MHz, DMSO-d
6
6
) d 146.53, 146.04, 141.40, 138.41, NMR (101 MHz, DMSO-d ) d 160.79, 155.14, 148.34, 143.27,
1
34.87, 127.94, 127.65, 126.77, 124.34, 21.64; ESI-HRMS, 139.05, 134.88, 133.84, 122.73, 122.36, 121.76, 120.64, 120.25,
ꢀ
calculated m/z for C17
H
11BrClN
3
[M ꢀ H] : 369.9747, found: 118.77, 117.06, 113.27, 110.27, 56.00; ESI-HRMS, calculated m/z
+
3
69.9774.
. Yield, 59.9%; H NMR (500 MHz, DMSO-d
H, NH), 8.88 (s, 1H, H-Ar), 7.96 (d, J ¼ 8.6 Hz, 2H, H-Ar), 7.94 (s, 1H, NH), 8.75 (s, 1H, H-Ar), 7.54 (s, 1H, H-Ar), 7.42 (s, 3H, H-Ar),
for C17
3
H12ClN O [M + H] : 310.0747, found: 310.0741.
1
1
3b
5
6
) d 13.26 (s,
3d . Yield, 64.5%; H NMR (400 MHz, DMSO-d ) d 12.59 (s,
1
6
1
2
1
3
13
H, H-Ar), 7.78 (s, 1H, H-Ar), 2.54 (s, 3H, –CH ); C NMR (126 6.29 (s, 2H, O–CH –O), 2.34 (s, 6H, –CH ); C NMR (101 MHz,
3
2
3
MHz, DMSO-d ) d 150.92, 146.41, 146.14, 141.46, 138.44, 134.94, DMSO-d ) d 152.93, 149.06, 147.43, 145.92, 145.06, 140.02,
6
6
1
27.94, 127.66, 126.71, 123.99, 21.61; ESI-HRMS, calculated m/z 124.16, 122.81, 104.52, 103.46, 103.22, 20.54; ESI-HRMS,
ꢀ
+
for C17
H
11BrClN
3
[M ꢀ H] : 359.9862, found: 359.9903.
3 2
calculated m/z for C19H14ClN O [M + H] : 352.0853, found:
1
3b
6
. Yield, 61.7%; H NMR (400 MHz, DMSO-d
6
) d 12.96 (s, 352.0880.
3d . Yield, 58.9%; H NMR (500 MHz, DMSO-d
H, H-Ar), 7.77 (d, J ¼ 8.6 Hz, 1H, H-Ar), 7.69 (s, 2H, H-Ar), 7.29 1H, NH), 8.77 (s, 1H, H-Ar), 7.68 (s, 1H, H-Ar), 7.54 (s, 1H, H-Ar),
1
1
1
H, NH), 8.87 (s, 1H, H-Ar), 7.97 (d, J ¼ 8.7 Hz, 1H, H-Ar), 7.95 (s,
2
6
) d 12.96 (s,
1
3
(d, J ¼ 6.0 Hz, 2H, H-Ar), 2.54 (s, 3H, –CH ); C NMR (101 MHz, 7.47 (s, 1H, H-Ar), 7.43 (s, 1H, H-Ar), 7.14 (s, 1H, H-Ar), 6.30 (s,
3
13
DMSO-d ) d 148.32, 146.73, 145.95, 141.19, 138.32, 134.69, 2H, O–CH –O); C NMR (126 MHz, DMSO-d ) d 153.14, 149.15,
6
2
6
1
27.93, 127.60, 126.83, 124.87, 21.64; ESI-HRMS, calculated m/z 146.17, 144.91, 140.25, 124.12, 122.24, 104.55, 103.52, 103.28,
+
+
for C17
H
12ClN
3
[M + H] : 294.0798, found: 294.0790.
100.00; ESI-HRMS, calculated m/z for C17
H
9
ClFN
3
O
2
[M + H] :
1
3c
1
. Yield, 59.0%; H NMR (500 MHz, DMSO-d
6
) d 12.78 (s, 342.0446, found: 342.0429.
1
1
1
H, NH), 8.84 (s, 1H, H-Ar), 7.96 (d, J ¼ 9.2 Hz, 1H, H-Ar), 7.60 (s,
H, H-Ar), 7.55 (d, J ¼ 9.1 Hz, 1H, H-Ar), 7.46 (s, 2H, H-Ar), 3.93 1H, NH), 8.79 (s, 1H, H-Ar), 7.78 (s, 1H, H-Ar), 7.65 (s, 1H, H-Ar),
3d . Yield, 59.1%; H NMR (400 MHz, DMSO-d ) d 13.05 (s,
3
6
1
3
(s, 3H, –OCH
3
), 2.36 (s, 6H, –CH
3
); C NMR (126 MHz, DMSO- 7.56 (s, 1H, H-Ar), 7.44 (s, 1H, H-Ar), 7.30 (d, J ¼ 8.1 Hz, 1H, H-
13
d6) d 158.70, 147.27, 144.89, 143.27, 140.29, 131.65, 129.64, Ar), 6.30 (s, 2H, O–CH –O); C NMR (101 MHz, DMSO-d )
2
6
1
28.16, 125.06, 124.67, 106.81, 56.24, 20.49; ESI-HRMS, calcu- d 153.22, 149.19, 146.25, 144.87, 140.40, 124.12, 122.08, 104.57,
+
lated m/z for C H ClN O [M + H] : 338.1060, found: 338.1076. 103.54, 103.32; ESI-HRMS, calculated m/z for C H Cl N O [M
1
9
16
3
17
9
2 3 2
1
ꢀ
3c
2
. Yield, 56.4%; H NMR (500 MHz, DMSO-d
6
) d 13.05 (s, ꢀ H] : 355.9994, found: 355.9999.
1
1
1
H, NH), 8.87 (s, 1H, H-Ar), 7.97 (d, J ¼ 9.1 Hz, 1H, H-Ar), 7.76 (s,
3d
4
. Yield, 68.6%; H NMR (400 MHz, DMSO-d
6
) d 13.04 (s,
H, H-Ar), 7.59 (s, 1H, H-Ar), 7.57 (d, J ¼ 9.1 Hz, 1H, H-Ar), 7.44 1H, NH), 8.79 (s, 1H, H-Ar), 7.87 (s, 1H, H-Ar), 7.64 (s, 1H, H-Ar),
1
3
(s, 1H, H-Ar), 7.15 (s, 1H, H-Ar), 3.93 (s, 3H, –OCH
3
); C NMR 7.55 (s, 1H, H-Ar), 7.44 (s, 1H, H-Ar), 7.41 (d, J ¼ 8.6 Hz, 1H, H-
13
(126 MHz, DMSO-d
6
2 6
) d 158.70, 143.38, 129.65, 128.11, 124.92, Ar), 6.30 (s, 2H, O–CH –O); C NMR (101 MHz, DMSO-d )
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d 153.22, 149.18, 146.25, 144.86, 140.40, 124.11, 122.04, 104.57, and the State Key Laboratory Cultivation Base for Chemistry and
1
ꢀ
03.54, 103.31; ESI-HRMS, calculated m/z for C H BrClN O [M Molecular Engineering of Medicinal Resources (Ministry of
1
7
9
3 2
ꢀ
H] : 399.9489, found: 399.9508.
Science and Technology of China, No. CMEMR2014-B14).
1
3
d . Yield, 60.5%; H NMR (500 MHz, DMSO-d ) d 13.15 (s,
5
6
1
7
H, NH), 8.79 (s, 1H, H-Ar), 7.96 (s, 2H, H-Ar), 7.56 (s, 1H, H-Ar),
1
3
2
.44 (s, 1H, H-Ar), 6.30 (s, 2H, O–CH –O); C NMR (126 MHz,
Notes and references
DMSO-d
6
) d 153.32, 151.05, 149.23, 146.38, 144.79, 140.46,
1
31.96, 129.12, 124.07, 121.69, 104.58, 103.57, 103.33; ESI-
1 World Cancer Report (World Health Organization, International
Agency for Cancer Research), ed. B. Stewart and P. Kleihues,
IARC Press, Lyon, 2003.
2 D. M. Parkin, Global cancer statistics in the year 2000, Lancet
Oncol., 2001, 2, 533–543.
3 A. Anighoro, J. Bajorath and G. Rastelli, Polypharmacology:
challenges and opportunities in drug discovery, J. Med.
Chem., 2014, 57, 7874–7887.
4 S. Fortin and G. Berube, Advances in the development of
hybrid anticancer drugs, Expert Opin. Drug Discovery, 2013,
ꢀ
HRMS, calculated m/z for C17
found: 389.9649.
H
8
Cl
3
N
3
O
2
[M ꢀ H] : 389.9604,
1
3
d . Yield, 62.2%; H NMR (400 MHz, DMSO-d ) d 12.96 (s,
6
6
1
7
H, NH), 8.78 (s, 1H, H-Ar), 7.67 (s, 2H, H-Ar), 7.55 (s, 1H, H-Ar),
1
3
.44 (s, 1H, H-Ar), 7.27 (s, 2H, H-Ar), 6.30 (s, 2H, O–CH
) d 153.07, 149.12, 148.42, 146.10,
45.04, 140.27, 124.14, 122.89, 122.55, 104.55, 103.50, 103.27;
2
–O);
C
NMR (101 MHz, DMSO-d
1
6
+
ESI-HRMS, calculated m/z for C17
found: 324.0532.
H10ClN
3
O
2
[M + H] : 324.0540,
8
, 1029–1047.
4
.3. Biological assays
The procedures for biological assays are described in the ESI†
Part 1). The materials, instrumentation, and methods for the
cytotoxicity assay, cell cycle analysis, cell apoptosis analysis and
5 S. Kumar, S. Bawa and H. Gupta, Biological activities of
quinoline derivatives, Mini-Rev. Med. Chem., 2009, 9, 1648–
1654.
(
6
7
C. H. Tseng, C. C. Tzeng, C. L. Yang, P. J. Lu, H. L. Chen,
H. Y. Li, et al., Synthesis and antiproliferative evaluation of
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ˇ´ˇ
´
1
Bax
PI
Bcl2-associated X protein
Propidium iodide
EB
Ethidium bromide
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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
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Cell division cyclin
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Cyclin-dependent kinase
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CDC25C Cell division cyclin 25 homolog C
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Acknowledgements
This study was supported by the National Natural Science 14 C.-C. Chiu, H.-L. Chou, B.-H. Chen, K.-F. Chang, C.-H. Tseng,
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