Discovery of novel 4(1H)-quinolone derivatives as potential antiproliferative and apoptosis inducing agents

Article abstract of DOI:10.1016/j.bmcl.2017.07.005

A series of novel 4(1H)-quinolone derivatives was synthesized and evaluated for antiproliferative activity in vitro. The results showed that these compounds exhibited more potent antiproliferative effect against a panel of human tumor cell lines than the lead compound 7-chloro-4(1H)-quinolone 1. Compound 7e was found to be the most potent antiproliferative agent and to exhibit selective cytotoxic activity against HepG2 cell lines with IC₅₀ value lower than 1.0 μM. Annexin V/FITC-PI assay showed that compound 7e induced apoptosis in HepG2 cells with a dose-dependent manner. Western blotting analysis indicated that compound 7e induced cell cycle arrest in G2/M phase by p53-depedent pathway.

Full text of DOI:10.1016/j.bmcl.2017.07.005

Accepted Manuscript
Discovery of novel 4(1H)-quinolone derivatives as potential antiproliferative
and apoptosis inducing agents
Ping Zhou, Linsheng Huang, Jie Zhou, Bin Jiang, Yanmei Zhao, Xuehua Deng,
Qin Zhao, Fei Li
PII:
S0960-894X(17)30700-X
http://dx.doi.org/10.1016/j.bmcl.2017.07.005
BMCL 25118
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 May 2017
29 June 2017
1 July 2017
Please cite this article as: Zhou, P., Huang, L., Zhou, J., Jiang, B., Zhao, Y., Deng, X., Zhao, Q., Li, F., Discovery
of novel 4(1H)-quinolone derivatives as potential antiproliferative and apoptosis inducing agents, Bioorganic &
Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.07.005
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Discovery of novel 4(1H)-quinolone derivatives as
potential antiproliferative and apoptosis inducing agents
Ping Zhoua,1, Linsheng Huanga,1, Jie Zhoub, Bin Jianga, Yanmei Zhaoa,
b
Xuehua Dengb, Qin Zhaob and Fei Li*,
a Department of Hepatopancreatobiliary Surgery, Taihe Hospital, Hubei University of Medicine,
Hubei, China;
b School of Pharmaceutical Sciences, Hubei University of Medicine, Hubei, China
Abstract
A series of novel 4(1H)-quinolone derivatives was synthesized and evaluated
for antiproliferative activity in vitro. The results showed that these compounds
exhibited more potent antiproliferative effect against a panel of human
tumor cell lines than the lead compound 7-chloro-4(1H)-quinolone 1. Compound 7e
was found to be the most potent antiproliferative agent and to exhibit selective
cytotoxic activity against HepG2 cell lines with IC50 value lower than 1.0 μM.
Annexin V/FITC-PI assay showed that compound 7e induced apoptosis in HepG2
cells with a dose-dependent manner. Western blotting analysis indicated that
compound 7e induced cell cycle arrest in G2/M phase by p53-depedent pathway.
Keywords: Synthesis; Quinolone; Antiproliferative Activity; Apoptosis.
*Corresponding authors Tel: 0719-8843101, Fax: 0719-8843101, E-mail addressed:
piaopodexinlifei@163.com
1

Quinolones represent a large group of naturally occurring and synthetic alkaloids.
Since the discovery of nalidixic acid by Lesher in 1962, numerous quinolones have
been prepared in search of more potent antibacterial agents and the structure-activity
relationships of quinolones have been extensively investigated1-3. Quinolones were
initially discovered to be specific inhibitors of the type II bacterial topoisomerase such
as DNA gyrase3-5. Recently, topoisomerase IV6 and penicillin-binding proteins
(PBPs)7 were also found to be the pharmacological target of this class of compounds.
Due to structural and functional similarities between bacterial DNA gyrase and
mammalian topoisomerase II8, in the past decade, considerable attention was attracted
to the antitumor potencies of quinolones and numerous investigations on structural
modification and structure-activity relationships of quinolone derivatives as promising
antitumor agents have been reported9-14. Consequently, quinolones have become
potential lead compounds in the development of cancer chemotherapeutic agents.
An initial lead compound that interested us was the 7-chloro-4(1H)-quinolone 1
(Figure 1), which exerted antitumor effect by blocking DNA synthesis and damaging
DNA template of tumor cell15. Its capsule was approved by the SFDA (State Food and
Drug Administration of China) as antitumor drug for late mammary cancer and
non-small cell lung cancer treatment in 200316. However, 1 has limited utility for
O
Cl
N
H
1
Figure 1 The chemical structure of the lead compound 7-chloro-4(1H)-quinolone
2

cancer therapy because of its poor solubility, which has only moderate solubility even
in DMSO. To circumvent the solubility problem in the 7-chloro-4(1H)-quinolone 1
and find congeners more potent as potential antitumor agents, in the present
investigation, we prepared five quinolone derivatives incorporating an alkyl
substituent at position-1 as well as bearing a flexible amino side chain in position-3.
We report herein the preparation of novel 7-chloro-4(1H)-quinolone derivatives, their
cytotoxic activities and their underlying mechanism of action as antitumor agents.
The synthetic routes of novel 4(1H)-quinolone derivatives 7a-e are outlined
Scheme 1. The intermediates 4-6 were prepared according to already published
Scheme 1 Synthesis of compounds 7a-e. Reagents and conditions: (i) stirred at 100 ℃；（ ii）
diphenyl ether, reflux; (iii) DMF, NaH, alkyl halogenide, stirred at RT；（ iv）NH2CH2CH2CH2NH2,
microwave irradiation, 150 ℃, 15 min.
3

methods17. Amination of carboxylates 6a-e with an excess of diamines without
solvent by subjecting to microwave irradiation (150℃, 15 min) provided the targeted
compounds 7a-e in 45-70% yield. The chemical structure of all the newly synthesized
compounds was characterized by MS, HRMS, 1H NMR and 13C NMR spectra.
The antiproliferative effects of novel quinolone derivatives 7a-e were
investigated and compared with the reference drugs adriamycin, cisplatin and
7-chloro-4(1H)-quinolone 1 by MTT assay against a panel of tumor cell lines. In
order to enhance water solubility, all compounds were prepared in the form of
hydrochloride salt before use. The tumor cell line panel was consisted of human
laryngeal carcinoma (Hep-2), breast carcinoma (MCF-7), gastric carcinoma
(BGC-823), liver carcinoma (HepG2), colorectal carcinoma (HCT-8), cervical
carcinoma (Hela) and prostate carcinoma (PC-3). The results were summarized in
Table 1. As shown in Table 1, 7-chloro-4(1H)-quinolone 1 failed to display cytotoxic
activities against seven human tumor cell lines at the concentration of 50.0 μM, and
the poor water-soluble property might be contributed to its weak activity. While
compounds 7a-e bearing a flexible amino side chain displayed potent cytotoxic
potencies against seven human tumor cell lines with IC50 values lower than 50.0 μM.
Interestingly, compound 7e bearing a (3-phenyl) propyl group in N1 of quinolone was
found to be the most potent cytotoxic agent with IC50 values lower than 10.0 μM
against seven human tumor cell lines investigated. In addition, compound 7e exhibited
selective cytotoxic activity with IC50 values of lower than 1.0 μM against HepG2 cell
lines. These results indicated that the bulky substituent at position-1 might facilitate
4

Table 1 Cytotoxic activities of quinolones derivatives 7a-e in vitro
IC50 (μM)±SD a
Compds
ClogP c
Hep-2 b
28.2±2.6
36.5±4.8
21.7±2.4
8.31±1.2
3.24±0.42
>50.0
MCF-7
35.5±4.6
31.6±3.2
12.6±2.1
5.02±0.68
3.12±0.44
>50.0
BGC-823
30.2±2.8
20.0±3.2
16.6±1.8
6.71±1.0
3.20±0.65
>50.0
HCT-8
31.6±4.2
34.7±3.5
22.3±3.2
14.5±1.8
6.03±0.58
>50.0
HepG2
13.8±2.0
17.0±2.8
5.23±0.48
5.42±0.82
<1.0
HeLa
20.0±1.8
22.4±3.4
15.0±2.0
4.54±0.36
2.31±0.42
>50.0
PC-3
12.6±1.6
13.2±1.4
12.0±2.2
9.12±1.0
6.92±0.82
>50.0
7a
1.148
1.393
2.206
3.177
3.095
1.053
-
7b
7c
7d
7e
1
>50.0
Adriamycin
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
Cisplatin
10.8±1.8
9.62±1.2
8.88±0.72
10.2±1.6
6.68±0.58
12.4±1.4
8.92±0.61
-
aCytotoxicity as IC50 for each cell line, is the concentration of compound which reduced by 50% the optical density of treated cells with respect to untreated cells using the MTT assay.
bCell lines include human laryngeal carcinoma (Hep-2), breast carcinoma (MCF-7), gastric carcinoma (BGC-823), liver carcinoma (HepG2), colon carcinoma (HCT-8), cervical carcinoma
(Hela), prostate carcinoma (PC-3).
c ClogP represent the calculated n-octanol/water partition coefficient (log Pow), and the values produced by Chemdraw software.
5

antiproliferative potency.
It is noted that compound 1 having ClogP values of 1.05 failed to exhibit
antiproliferative activity at the concentration of 50 μM, and compound 7a, 7b and 7c
with low ClogP values also exhibited weaker antiproliferative potency, while
compound 7d and 7e having slightly high ClogP values showed more potent
antiproliferative effect. These results suggested that the ClogP values of the whole
molecule seemed to be a very important determinant factor of the antiproliferative
properties of this class of compounds.
Figure 2 Compound 7e inhibited the viability of HepG2 cell lines. (A) HepG2 cells were seeded
in 96 well plate, after 24 h, the cells were treated with indicated concentration of compound 7e for
24 h. The cell viability was measured with CCK-8 assay. (B)1000 HepG2 cells were seeded in 96
well plate for 24 h, treated indicated time with 10 μM compound 7e. The cell viability was
measured with CCK-8 assay. (C and D) The number of colonies of HepG2 cells was counted after
the treatment with indicated concentration of compound 7e for 24 h.
6

We also examined the inhibitory effect of the selected compound 7e on growth of
HepG2 using the Cell Counting Kit-8(CCK-8) assay. Compared with the vehicle
treated cells, compound 7e inhibited efficiently the proliferation of HepG2 in the
time-dependent manner (Figure 2A). To investigate whether compound 7e affected
tumorigenicity-correlated responses in vitro，then plate colony formation assay was
performed. The data indicated that the single HepG2 cell were more sensitive to
compound 7e at the concentration of 10 μM (Figure 2 and 2C). These results
suggested that compound 7e not only inhibited proliferation of HepG2, but also
induced cell death.
Figure 3 Compound 7e induced apoptosis in HepG2 cells. (A) HepG2 cells were treated with
indicated concentration of compound 7e for 24 h and then binding with Annexin V/FITC-PI. The
results were analyzed with flow cytometry. (B) The protein levels of Bak, PARP and Caspase-3
were detected by western blot, β-actin was used as an internal control.
7

To evaluate which type of cell death induced by compound 7e, we performed
Annexin V/FITC-PI assay. Obviously, compound 7e induced apoptosis in HepG2 cell
lines with a dose-dependent manner (Figure 3A). We detected the level of maker
protein about apoptosis, the level of Bak, a pro-apoptotic protein, increased by
stimulating of compound 7e, However, the quantity of PARP (poly ADP-ribose
polymerase) and Caspase-3 increased instead of decreasing in a dose-dependent
manner (Figure 3B). Possibly, PARP is thought to be a receptor for DNA damage,
which is activated by identifying structural damage of DNA. Thus, PARP plays a
important role in repairing the DNA which is suffered damage from compound 7e.
Stimulated with different concentrations of compound 7e, the cell cycle of
HepG2 was detected by PI staining, the results demonstrated that the phrase of G2/M
was arrested dramatically with increasing concentration of compound 7e (Figure 4A).
Subsequently, we screened the 10 signaling pathways purchased from Qiagen using
luciferase assay in HepG2 cell lines after compound 7e treatment. The results
indicated that the luciferase activities of several pathways were altered with different
levels. Particularly, the activity of p53 increased by about 1.5 times (Figure 4B). p53
plays an important regulation role in phase of G2/M. Western blotting analysis was
used to evaluate the activity of p53 and G2/M phase related protein kinases after
compound 7e treatment, p53 and p21, a downstream gene of p53, increased
dramatically in protein level, and Cyclin B1 and CDK1 decreased in a dose-dependent
manner (Figure 4C). These results suggested that compound 7e induced cell cycle
arrest in G2/M phase by p53-depedent pathway possibly.
8

Figure 4 Compound 7e induced cell cycle arrest in G2/M phase. (A) HepG2 cells were treated
with indicated concentration of compound 7e for 24 h and cell cycle was assayed with PI staining.
The results were analyzed with flow cytometry. (B) Luciferase activity of different signaling
pathway reporters after treatment with compound 7e. HepG2 cells were transfected with the
indicated pathway reporter for 24 h, treated with 4 μM of compound 7e for another 24 h and then
performed the dual-luciferase assay. (C) The protein levels of p53, p21, Cyclin B1 and CDK1
were detected by western blot, β-actin was used as an internal control.
In conclusion, we have synthesized a series of novel quinolones incorporating
an alkyl substituent at position-1 as well as a flexible amino side chain in position-3.
These compounds exhibited more potent antitumor activities against a panel of human
tumor cell lines than the lead compound 7-chloro-4(1H)-quinolone 1. And compound
7e was found to be the most potent antitumor agent and exhibit selective cytotoxic
9

activity against HepG2 cell lines with IC50 value lower than 1.0 μM. Compound 7e
induced apoptosis with a dose-dependent manner in HepG2 cells. Western blotting
analysis indicated that compound 7e induced cell cycle arrest in G2/M phase by
p53-depedent pathway. Undoubtedly, to acquire more information about the structural
requirements for improving cytotoxic activities, the synthesis of more new quinolones
with different substituent at other positions is needed.
Acknowledgments
This work was supported by the National Natural Science Foundation of China
grant 81402994, the Natural Science Foundation of Hubei Province of China grant
2014CFB652, the Fund of Hubei 2011 Cooperative Innovation Center grant
2011JH-2014CXTT08, the Foundation of Health and Family planning Commission of
Hubei Province grant WJ2017F071, the Foundation of Hubei Engineering Research
Center for Comprehensive Utilization of Medicinal Plants grant GC2015202，the
Initial Project for Post-Graduates of Hubei University of Medicine grant
2014QDJZR03, and the Foundation of Development funds of Hubei University of
Medicine grant FDFR201610.
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Graphic abstract
The representative compound 7e could induce apoptosis in HepG2 cells with a
dose-dependent manner and cell cycle arrest in G2/M phase by p53-depedent
pathway. .
12