Synthesis and biological evaluation of quinoxaline derivatives as tubulin polymerization inhibitors that elevate intracellular ROS and triggers apoptosis via mitochondrial pathway

Article abstract of DOI:10.1111/cbdd.13459

A series of novel quinoxaline derivatives were synthesized and evaluated for their antiproliferative activity in three human cancer cell lines. Compound 12 exhibited the most potent antiproliferative activity with IC₅₀ in the range of 0.19–0.51

Full text of DOI:10.1111/cbdd.13459

Received: 11 July 2018
Revised: 19 November 2018
Accepted: 24 November 2018
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DOI: 10.1111/cbdd.13459
R E S E A R C H A R T I C L E
Synthesis and biological evaluation of quinoxaline derivatives as
tubulin polymerization inhibitors that elevate intracellular ROS
and triggers apoptosis via mitochondrial pathway
Jianguo Qi1
Jing Huang1
Zhijie Yan1
Xiaomin Zhou1
Wen Luo1
Jiaxin Xie1
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Linqiang Niu1
Yahong Zhang1
Yang Luo1
Yuhui Men1
Yanan Chen2
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Jianhong Wang1
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1Key Laboratory of Natural Medicine
and Immuno-Engineering of Henan
Province, Henan University Jinming
Campus, Kaifeng, Henan, China
Abstract
A series of novel quinoxaline derivatives were synthesized and evaluated for their
antiproliferative activity in three human cancer cell lines. Compound 12 exhibited
the most potent antiproliferative activity with IC50 in the range of 0.19–0.51 μM. The
compound inhibited tubulin polymerization and disrupted the microtubule network,
leading to G2/M phase arrest. Furthermore, compound 12 induced ROS production
and malfunction of mitochondrial membrane potential. Compound 12 led to cancer
cells apoptosis in a dose-dependent manner. Western blot analysis showed that com-
pound 12 induced up-regulation of p21 and affected the expression of cell cycle-
related proteins. The binding mode was also probed by molecular docking.
2Institute of Behavior and
Psychology, Henan University Jimming
Campus, Kaifeng, Henan, China
Correspondence
Yahong Zhang and Jianhong Wang, Key
Laboratory of Natural Medicine and
Immuno-Engineering of Henan Province,
Henan University Jinming Campus,
Kaifeng, Henan, China.
Emails: zhangyahong_131@163.com and
jhworg@126.com
K E Y W O R D S
cell apoptosis, G2/M phase arrest, quinoxaline derivatives, reactive oxygen species, tubulin
polymerization inhibitors
Funding information
Program for Science Technology Innovation
Talents in Universities of Henan Province,
Grant/Award Number: 16HASTIT029;
Key Scientific Research Program of
the Higher Education Institutions of
Henan Province, Grant/Award Number:
15A350005; National Natural Science
Foundation of China, Grant/Award Number:
21272056, U1704176 and U1404819;
Henan University, Grant/Award Number:
2015YBZR043
(MDAs) in tubulin: taxanes, vinca alkaloids, laulimalide, and
colchicine (Bhalla, 2003; Dumontet & Jordan, 2010; Jordan
1
INTRODUCTION
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Microtubules, which are dynamic polymers of α/β-tubulin,
are elements of the cellular cytoskeleton and involved in
numerous cellular functions such as cell movement, organ-
elle localization, and cell division. Microtubules have been
considered an ideal target for cancer chemotherapy because
of the important role they play in mitosis. There are four
well-characterized sites for microtubule-disrupting agents
& Wilson, 2004). Currently, all the United States Food and
Drug Administration (FDA) approved MDAs for cancer ther-
apy target either taxanes (e.g., paclitaxel, docetaxel) or vinca
alkaloids (e.g., vinblastine, vincristine). However, the clinical
efficacy of the drugs is often limited by occurrence and evo-
lution of resistance mechanisms that is further compounded
by a narrow therapeutic index (Du et al., 2015; Gigant et al.,
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Chem Biol Drug Des. 2019;1–11.
wileyonlinelibrary.com/journal/cbdd
© 2019 John Wiley & Sons A/S
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QI et al.
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FIGURE 1 Microtubule-disrupting agents inhibiting tubulin polymerization
2005; Kavallaris, 2010; Perez, 2009; Wang, Chen, Miller, &
Li, 2014; Wang et al., 2012). Recently, some colchicine bind-
ing tubulin inhibitors exhibit significant ability to overcome
multidrug resistance (Bacher et al., 2001; Banerjee et al.,
2018; Bueno et al., 2018; Cui et al., 2017; Dohle et al., 2018;
Gangjee et al., 2010; Lauria et al., 2018; Li et al., 2018;
Ohsumi et al., 1998; Pang et al., 2017; Suman et al., 2015).
Even some drug candidates have already entered the clinical
trial stage (Figure 1). However, the potential clinical applica-
tions of tubulin inhibitors have been restricted by low solu-
bility, low bioavailability, and lack of tumor selectivity (Lu,
Chen, Xiao, Li, & Miller, 2012; Stanton, Gernert, Nettles, &
Aneja, 2011). Therefore, there are ongoing research efforts
to identify novel scaffolds that target the site of colchicine
binding that display improved physicochemical properties
and selectivity toward cancer cells.
inhibition of tubulin polymerization and topoisomerase I
(Diana et al., 2008). Previously, we have synthesized a series
of 3,4-dihydroquinoxalin-2(1H)-one derivatives targeting the
colchicine binding site on tubulin (Qi et al., 2018).
The series of quinoxaline derivatives include many
scaffolds, such as quinoxaline, 3,4-dihydroquinoxa
lin-2(1H)-one,
quinoxaline-2,3(1H,4H)-dione,
and
1,2,3,4-tetrahydroquinoxaline. Based on the previous
research, the 3,4-dihydroquinoxalin-2(1H)-one scaf-
fold was replaced with quinoxaline-2,3(1H,4H)-dione or
1,2,3,4-tetrahydroquinoxaline scaffold. Herein, we report
the synthesis and biological evaluation of quinoxaline-
2,3(1H,4H)-dione
and
1,2,3,4-tetrahydroquinoxaline
scaffolds derivatives to develop tubulin polymerization in-
hibitors as antitumor agents (Figure 2).
Reactive oxygen species (ROS), oxygen-free radicals pro-
duced in biological systems, play an important role in che-
motherapy. High intrinsic levels of ROS and up-regulation of
cellular antioxidant machinery are defining changes in tumor
cells exhibiting glycolysis(Szatrowski & Nathan, 1991). In
general, the mechanisms by which antioxidants are produces
in the body are performing at maximum capacity due to a
higher basal level of ROS. By taking advantage of this fact,
chemical agents that cause additional oxidative stress results
in cancer cells that are more vulnerable to small molecule
chemotherapy, thus providing a mechanism for selective tar-
geting of cancer cells (Asby et al., 2016; Hirota et al., 1999;
Huang, Feng, Oldham, Keating, & Plunkett, 2000; Lecane
et al., 2005; Raj et al., 2011).
Quinoxaline, present in many bioactive compounds, dis-
plays adequate physicochemical properties and drug-like
properties (Ajani, 2014; Tariq, Somakala, & Amir, 2018).
Numerous synthetic quinoxaline derivatives have shown in-
teresting antineoplastic activity. For instance, the substitution
of the benzene ring by quinoxaline in Combretastatin-A4
improved both the physicochemical properties and biolog-
ical activity (Pérez-Melero et al., 2004). The novel fusion
of 3,4-dihydroquinoxalin-2(1H)-one with 2H-isoindole
results in a series of compounds which showed dual
2
METHODS AND MATERIALS
Chemistry
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2.1
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All commercially obtained reagents and solvents were used
as received. HRMS spectra were acquired on a Thermo
Scientific LTQ Orbitrap XL mass spectrometer, and all the
errors were <3 ppm. H NMR spectra, and 13C NMR spec-
1
tra were acquired on a Bruker DRX 300 NMR spectrome-
ter using CDCl3 or DMSO-d6 as solvent (unless otherwise
stated). Chemical shifts were reported in parts per million
(ppm, δ) relative to the solvent peak (1H, CDCl3 δ 7.26 ppm,
DMSO-d6 δ 2.50 ppm; 13C, CDCl3 δ 77.0 ppm, DMSO-d6
δ 39.5 ppm). Coupling constants (J) were measured in hertz
(Hz). The synthesis and chemical characterization of the
compounds are in the Supporting Information Table S1.
2.2
Biological evaluation
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2.2.1
Cell lines and culture conditions
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The human epithelial cervical cancer cell line HeLa, the
human hepatoma cell line SMMC-7721, and the human in-
testinal epithelial cell line HT-29 were cultured in RPMI

QI et al.
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FIGURE 2 Quinoxaline derivatives
and different quinoxaline scaffolds
1640 medium (HyClone Co., USA), supplemented with 10%
fetal bovine serum (Sijiqing Biotechnology Co., China), in
5% CO2 humidified air at 37°C. All the cell lines were ob-
tained from the American Type Culture Collection (ATCC,
USA).
Triton X-100 in PBS for 5 min at 4°C. After then, the cells
were processed for immunofluorescence with the primary
antibody (Catalog No. BM1452, Boster Co. Ltd., China,
1:100 dilution in 2% BSA-PBS) and anti-mouse secondary
antibody conjugated with FITC for 30 min at 37°C (Catalog
No. BA1101, Boster Co. Ltd., China, 1: 100 dilution in 1%
BSA-PBS) separately. The nuclei were stained with Hoechst
33342, and immunofluorescence was detected using a fluo-
rescence microscope (Leica TCS SP8, Germany).
2.2.2
In vitro antiproliferative activity
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The MTT assay was used to determine the antiproliferative
effects of the synthesized compounds. Cells were seeded
into 96-well plates with the density of 4,000–6,000 cells
per well. After 24 hr of growth to allow attachment of
cells to the well, test compounds were added at five dif-
ferent concentrations. After incubation for 72 hr, 50 μl of
MTT solution (10 mg/ml in PBS) was added. After 4 hr of
incubation in the dark, the medium was removed and re-
placed with 100 μM DMSO. After shaking for 10 min, the
optical density was measured at a wavelength of 490 nm
using a microplate reader (Powerwave XS, Biotek, USA).
Experiments were performed in triplicate and repeated at
least three times.
2.2.5
Flow cytometric analysis of cell
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cycle and apoptosis
Cell cycle distribution was analyzed after incubation with PI,
and apoptosis was analyzed after staining with annexin V-
FITC in combination with PI. The samples were analyzed
using the flow cytometer (BD FACSVerse, USA).
2.2.6
ROS production
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HeLa cells were treated with vehicle or different concentra-
tions of the test compound for 24 hr. Then, the cells were
treated with ROS indicator DHE (Beyotime Biotechnology
Co. Ltd., China). The cells were detected using the fluores-
cence microscope or analyzed by the flow cytometer de-
scribed above.
2.2.3
Tubulin polymerization assay
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Tubulin polymerization assay was conducted using a
fluorescence-based tubulin polymerization assay kit
(BK011P, Cytoskeleton, USA) according to the manufac-
turer’s protocol. The final reaction mixture contained 2 mg/
ml tubulin, 2.0 mM MgCl2, 0.5 mM EGTA, 1.0 mM GTP,
15% glycerol, 80 mM PIPES, and test compound. The flu-
orescence intensity was measured for 1 hr at 1 min inter-
vals in CLARIOstar microplate reader (BMG Labtech Inc.,
Germany). Experiments were performed in duplicate.
2.2.7
Measurement of mitochondrial
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membrane potential
HeLa cells were treated with vehicle or different concentra-
tions of the test compound for 24 hr. Then, the cells were
incubated with the mitochondrial membrane potential probe
(JC-1, Beyotime Biotechnology Co. Ltd., China) for 30 min.
The cells were detected using the fluorescence microscope or
analyzed by the flow cytometer described above.
2.2.4
Immunofluorescence microscopy
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HeLa cells were seeded in a 96-well plate at 4,000 cells per
well and incubated for 24 hr. Then, the cells were treated
with vehicle or different concentrations of test compound.
After incubation for 24 hr, cells were fixed in 4% paraform-
aldehyde for 10 min at 4°C and permeabilized with 0.1%
2.2.8
Western blot analysis
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HeLa cells were incubated with vehicle or different concen-
trations of the test compound for 24 hr. Then, the cells were

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QI et al.
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SCHEME 1 Reagents and conditions:
a) benzyl chlorides, KI, K2CO3, CH3CN,
70°C; b) 10% Pd/C, H2, MeOH, rt; c)
3,4,5-trimethoxybenzyl chloride, KI,
K2CO3, CH3CN, 70°C; d) dimethyl
oxalate, 140°C; e) NaOH, THF, H2O, rt;
f) Boc anhydride, ammonium bicarbonate,
pyridine, 1,4-dioxane, rt; g) phosphorus
oxychloride, CHCl3, reflux; h) borane,
THF, rt
collected, centrifuged, and washed with ice-cold phosphate-
buffered saline (PBS). The pellet was resuspended in lysis
buffer and incubated on ice for 30 min, and then the lysates
were centrifuged at 15,000 g at 4°C for 10 min. The protein
concentration was determined using the Bio-Rad protein
assay (Bio-Rad Laboratories, Mississauga, Canada). Equal
amounts of protein were analyzed by SDS-PAGE and then
transferred to PVDF membrane. Membranes were blocked
with a 5% bovine serum albumin solution and then incubated
with the specific primary antibodies. After washing, the
membrane was incubated with peroxidase-conjugated sec-
ondary antibodies for 1 hr.
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RESULTS AND DISCUSSION
Chemistry
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3.1
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The synthesis of the compounds was depicted in Scheme 1.
Reaction of compound 4 with substituted benzyl chloride
provided intermediate 5 which upon further reduction yielded
intermediate 7, except for 7h, which was prepared by the reac-
tion of o-phenylenediamine (6) with 3,4,5-trimethoxybenzyl
chloride. Compound 8 was prepared by reaction of inter-
mediate 7 with dimethyl oxalate. Hydrolysis of compound
8 afforded compound 9. Compound 10 was synthesized by
the reaction of compound 9e with ammonium bicarbonate.
Compound 11 was prepared by dehydration of compound 10.
Reduction of compound 8e provided compound 12.
2.3
Molecular modeling
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The docking study was carried out using sybyl-x 2.1. The
crystalstructureoftubulinincomplexwithDAMA-colchicine
was retrieved from PDB (1SA0). The 3D structure of 12 was
built using sybyl-x 2.1 and minimized using tripos force
field and Gasteiger–Huckel charges. Powell’s method was
employed with gradient convergence criteria of 0.005 kcal/
mol. The DAMA-colchicine structure was extracted, and the
tubulin structure was prepared and minimized with Amber7
F99 force field. The binding site was constructed by the li-
gand protomol of surflex dock. Compound 12 was docked
into the DAMA-colchicine binding site.
3.2
Biological evaluation
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3.2.1
In vitro antiproliferative activity
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The antiproliferative activities of the synthesized target com-
pounds were evaluated against three distinct human cancer
cell lines, including HeLa (human epithelial cervical cancer),
SMMC-7721(human hepatoma cancer), and HT29 (human
intestinal epithelial cancer) cell lines using an MTT assay.
Indibulin, the tubulin inhibitor that was explored in clinical
trials for cancer therapy was used as reference compound.

QI et al.
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The results are summarized in Table 1. The antiproliferative
activities of the compounds were expressed as the concentra-
tion of compounds required for 50% growth inhibition of the
cells (IC50). IC50 values were calculated from at least five
different concentrations of test compounds.
As shown in Table 1, the comparison of antiproliferative
activities of 8a–8f demonstrated that 3,4,5-trimethoxybenzyl
substitution on the nitrogen atom of methyl 2,3-oxo-1,2,3,4-te
trahydroquinoxaline-6-carboxylate (8e) showed improved ac-
tivity. When the methyl carboxylate (8a–8f) was hydrolyzed
to carboxylic acid (9a–9f), the antiproliferative activities de-
creased. Compound 8g exhibited better activities than 8e, 8h,
10, and 11, indicating that 6-methoxy exhibited better activity
than 6-carboxylate, 6-H, 6-carboxamide, and 6-carbonitrile
in the substituted 2,3-oxo-1,2,3,4-tetrahydroquinoxaline.
Compound 12, the reduction product of 8e, showed potent
antiproliferative activities. It exhibited better activity than
indibulin. This indicated that 1,2,3,4-tetrahydroquinoxaline
was a privileged scaffold, and compound 12 could potentially
be a novel lead structure for further extensive optimization.
3.2.2
Effects on tubulin polymerization
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Microtubules are crucial components of mitotic spindle in the
process of mitosis, and as such has become an important tar-
get for cancer therapy. Most microtubule-targeted antitumor
agents either stabilize or destabilize tubulin polymerization.
In this study, a tubulin polymerization assay was performed
to confirm the interference of compound 12 in tubulin polym-
erization. As shown in Figure 3, the fluorescence intensity
increased gradually when the purified and unpolymerized
tubulin was incubated at 37°C and this demonstrated that tu-
bulin polymerization had occurred (control sample). When
tubulin was incubated with 12 or indibulin, the increased
tendency of fluorescence intensity was decreased. This re-
sult clearly shows that compound 12 exhibited potent tubulin
polymerization inhibiting activity, which interestingly dis-
played a higher potency than indibulin.
TABLE 1 The in vitro antiproliferative activities of synthesized
target compounds (8a–8h, 9a–9f, 10, 11, 12) doxorubicin and indibulin
against three human cancer cell lines (HeLa, SMMC-7721 and HT29)
Antiproliferative activity IC50 (μM)
Compound HeLa
SMMC-7721
HT29
8a
8b
8c
8d
8e
8f
8g
8h
9a
9b
9c
9d
9e
>50
43.5 4.5
39.6 4.8
34.7 2.6
43.4 2.1
8.02 0.62
34.3 3.3
1.60 0.37
28.8 3.4
46.2 2.7
>50
43.7 5.9
40.2 5.1
47.2 5.5
15.0 1.2
3.71 0.26
38.4 7.6
1.48 0.22
23.0 1.5
49.0 4.3
>50
33.3 7.8
35.1 6.1
15.3 4.1
3.42 1.02
29.9 4.7
0.693 0.185
28.1 2.4
45.6 8.0
>50
3.2.3
Antimicrotubule effects in HeLa cells
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Since tubulin polymerization inhibitors have an effect on
microtubule network through interference with microtubule
dynamics causing microtubule catastrophe, we performed
immunofluorescence staining of α-tubulin in HeLa cells to
study the effect of compound 12 on the microtubule network.
As shown in Figure 4, the cells without compound exhibited
normal filamentous microtubules arrays, with microtubules
extending to the peripheral region of the cells, while com-
pound 12 clearly triggered a microtubule catastrophe. From
tubulin polymerization and immunofluorescence assay,
we conclude that compound 12 is a novel microtubule-
depolymerizing agent.
42.8 7.9
39.0 5.9
34.1 5.6
>50
>50
38.4 1.4
>50
38.2 2.3
47.9 4.2
>50
9f
10
11
12
>50
>50
39.6 5.5
>50
38.1 1.7
>50
45.2 2.4
>50
0.194 0.013
0.532 0.055
0.427 0.054 0.510 0.036
0.621 0.058 0.633 0.081
Indibulin
FIGURE 3 Effects of 12 (5, 10 μM)
on tubulin polymerization. Indibulin (5 μM)
was used as reference drug, 0.1% DMSO
as control

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FIGURE 4 Fluorescence microscopic images of HeLa cells stained with Hoechst 33342 or anti-α-tubulin-FITC antibody after treatment with
0.1% DMSO, 12 (0.2, 0.4, 0.8 μM) for 24 hr
FIGURE 5 (a) Flow cytometry analysis of HeLa cells treated with 0.1% DMSO, 0.2, 0.4, or 0.8 μM compound 12 for 24 hr. (b) The
statistical graph of cell cycle distribution

QI et al.
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FIGURE 6 (a) Bi-parametric histograms of HeLa cells treated with 0.1% DMSO, 0.2, 0.4, or 0.8 μM compound 12 for 24 hr by flow
cytometry after staining (annexin V and PI). (b) Percentage of apoptotic cells
3.2.4
Cell cycle analysis
3.2.5
Effects on cell apoptosis
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progression
Tubulin polymerization inhibitors can interfere with cell divi-
sion resulting in cell cycle arrest; we next evaluated the cell
cycle distribution of compound 12-treated cells by flow cy-
tometry. As shown in Figure 5, compound 12 induced accu-
mulation of HeLa cells losses in the G1 phase. This suggests
that compound 12 induced HeLa cell cycle arrest by affecting
cell division. In addition, compound 12 induced an increase in
the percentage of cells in the sub-G1 (hypodiploid DNA con-
tent peak) phase which may indicate that HeLa cells treated
with compound 12 display apoptosis.
The annexin V/propidium iodide (PI) double-staining
assay was performed to investigate the effect of compound
12 on cell apoptosis. As shown in Figure 6, the four quad-
rants represented live cells (annexin V−/PI−), early apop-
totic cells (annexin V+/PI−), late apoptotic cells (annexin
V+/PI+), and necrotic cells (annexin V−/PI+). Compound
12 induced an accumulation of annexin V positive cells
in a dose-dependent manner suggested that compound 12
induced apoptosis.
FIGURE 7 (a) Fluorescent images of HeLa cells treated with 0.1% DMSO, 0.2, 0.4, or 0.8 μM compound 12 for 24 hr staining with
JC-1. (b) Flow cytometry analysis of HeLa cells. (c) The statistical graph of mitochondrial malfunction cells

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FIGURE 8 (a) Fluorescent images of HeLa cells treated with 0.1% DMSO, 0.2, 0.4, or 0.8 μM compound 12 for 24 hr staining with DHE. (b)
Flow cytometry analysis of HeLa cells. (c) The statistical graph of DHE positive ratio
of action and pharmacological mechanism. The excessive ROS
3.2.6
Effects on mitochondrial
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production could induce G2/M phase cell cycle arrest by mod-
ulating the expression of p21 (He et al., 2011). Cells treated
with compound 12 resulted in a significant up-regulation of
p21 in a time-dependent manner. Chk1, upon activation, phos-
phorylates Cdc25C at Ser-216 leading to inactivation of cy-
clin B1/cdc2 (CDK1) complex, which leads to G2/M arrest.
As shown in Figure 9, compound 12 induced an up-regulation
of Chk1 and affected the expression levels of related proteins.
membrane potential
Mitochondria play a core role in the mitochondrial-induced ap-
optosis pathway via changes of the mitochondrial membrane
potential. The detection of mitochondrial membrane potential
was performed to study whether HeLa cells apoptosis is due to
mitochondria malfunction with JC-1 dye being used to detect
changes in mitochondrial membrane potential. At high concen-
tration, the dye aggregates which gives a red to orange fluores-
cence due to high membrane potential. At low concentration,
the dye is predominantly a monomer that yields a green fluo-
rescence due to low membrane potential. As shown in Figure 7,
a dose-dependent decrease in red fluorescence and an increase
in green fluorescence were observed. These results demonstrate
that compound 12 induced mitochondrial membrane potential
collapse which leads to mitochondrial dysfunction.
3.3
Molecular modeling
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To further elucidate the mode of action and to aid in further
optimization of the compound, binding mode of compound
12 was computationally evaluated based on the previous work
(Qi et al., 2018). To investigate potential binding mode, com-
pound 12 was docked into the colchicine binding site of tu-
bulin (PDB ID: 1SA0) based on our previous study. Some of
3.2.7
Cellular ROS generation
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ROS generation is known to be associated with mitochon-
drial membrane potential (ΔΨm) changes. The effect of 12
on intracellular ROS level was probed using dihydroethid-
ium (DHE) since it played an important role in anticancer
activity. As shown in Figure 8, a significant increase of red
fluorescence emission was observed when HeLa cells were
treated with compound 12 compared with the control. ROS
production induced by compound 12 was dose-dependent
which was quantified by flow cytometry. These results indi-
cated that compound 12 increased intracellular ROS levels.
3.2.8
Effects on expression of G2/M cell
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cycle regulatory proteins
The expression of ROS producing-related and cell cycle-
related proteins was detected to preliminarily investigate mode
FIGURE 9 Western blot analysis of cell cycle-related protein

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FIGURE 10 (a) Comparison between
pose of cocrystallized DAMA-colchicine
(red) with tubulin and docking poses of 12.
(b) Proposed binding mode of 12. Residues
within 4 Å of 12 are shown
the higher docking poses ranked by total scores were shown
in Figure 10a. The binding orientation of compound 12 was
very similar to that of the cocrystallized DAMA-colchicine.
As shown in Figure 10b, a hydrogen bond is present between
compound 12 and VaL 181 in the loop of α-tubulin. The tri-
methoxyphenyl moiety made nonpolar interaction with Cys
241, Leu 242, Leu248, Val 318, and Ile 378 of β-tubulin.
Institutions of Henan Province (Grant No. 15A350005), and
Research Fund of Henan University (2015YBZR043).
CONFLICT OF INTEREST
The authors declare that there are no conflicts of interests.
ORCID
4
CONCLUSIONS
Jianguo Qi
https://orcid.org/0000-0002-8087-1169
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We have synthesized a series of quinoxaline derivatives that
were evaluated for antiproliferative activity against human
cancer cell lines (Hela, SMMC-7721, HT29). Among these
compounds, Compound 12 exhibited potent cytotoxicity
with IC50 ranging from 0.19 to 0.51 μM. Compound 12 in-
hibited the tubulin polymerization and induced cell cycle
arrest at G2/M phase. Further, compound 12 induced an
increase of intracellular ROS levels. Compound 12 also
led to cancer cells apoptosis via mitochondrial pathway.
The Western blot analysis showed that compound 12 in-
duced up-regulation of p21 and affected the expression of
cell cycle-related proteins. This work provided new agents
for the development of the promising anticancer drugs.
Optimization and structure–activity relationship (SAR)
studies are currently underway and will be reported in
due course. Furthermore, we are actively investigating the
mechanism of action, which will give more insight into the
biological activity observed.
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ACKNOWLEDGMENTS
TheauthorsaregratefulforthefinancialsupportfromProgram
for Science Technology Innovation Talents in Universities of
Henan Province (16HASTIT029) and the National Natural
Science Foundation of China (No. 21272056). In addition,
this work was also supported by National Natural Science
Foundation of China (No. U1704176, No. U1404819),
Key Scientific Research Program of the Higher Education

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How to cite this article: Qi J, Huang J, Zhou X,
et al. Synthesis and biological evaluation of
quinoxaline derivatives as tubulin polymerization
inhibitors that elevate intracellular ROS and triggers
apoptosis via mitochondrial pathway. Chem Biol
Drug Des. 2019;00:1–11. https://doi.org/10.1111/
cbdd.13459
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