Discovery of novel indole-1,2,4-triazole derivatives as tubulin polymerization inhibitors

Article abstract of DOI:10.1002/ddr.21805

A series of novel indole-1,2,4-triazole derivatives have been designed, synthesized, and evaluated as potential tubulin polymerization inhibitors. The top hit 12, bearing the 3,4,5-trimethoxyphenyl moiety, exhibited substantial anti-proliferative activity against HepG2, HeLa, MCF-7, and A549 cells in vitro with IC₅₀ values of 0.23 ± 0.08 μM, 0.15 ± 0.18 μM, 0.38 ± 0.12 μM, and 0.30 ± 0.13 μM, respectively. It also inhibited tubulin polymerization with the IC₅₀ value of 2.1 ± 0.12 μM, which was comparable with that of the positive controls. Furthermore, compound 12 regulated the expression of cell cycle-related proteins (Cyclin B1, Cdc25c, and Cdc2) and apoptosis-related proteins (Bcl-2, Bcl-x, and Mcl-1). Mechanistically, compound 12 could arrest cell cycle at the G2/M phase, thus induce an increase of apoptotic cell death. In addition, molecular docking hinted the possible interaction mode of compound 12 into the colchicine binding site of tubulin heterodimers. According to the applications of microtubule-targeting agents in both direct and synergistic cancer therapies, we hope this work might be of significance for future researches.

Full text of DOI:10.1002/ddr.21805

Received: 9 January 2021
DOI: 10.1002/ddr.21805
Revised: 1 February 2021
Accepted: 5 February 2021
R E S E A R C H A R T I C L E
Discovery of novel indole-1,2,4-triazole derivatives as tubulin
polymerization inhibitors
Meng-Ke Wu1 | Ruo-Jun Man1
Zhu-Gui Zhou3
| Yan-Juan Liao1 | Hai-Liang Zhu2
|
1Guangxi Biological Polysaccharide Separation, Purification and Modification Research Platform, Guangxi University for Nationalities, Nanning, China
2State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, China
3College of Chemistry and Chemical Engineering, Guangxi Key Laboratory of Polysaccharide Materials and Modifications, Guangxi University for Nationalities,
Nanning, China
Correspondence
Abstract
Ruo-Jun Man, Guangxi Biological
Polysaccharide Separation, Purification and
A series of novel indole-1,2,4-triazole derivatives have been designed, synthesized,
Modification Research Platform, Guangxi
and evaluated as potential tubulin polymerization inhibitors. The top hit 12, bearing
the 3,4,5-trimethoxyphenyl moiety, exhibited substantial anti-proliferative activity
against HepG2, HeLa, MCF-7, and A549 cells in vitro with IC50 values of 0.23
0.08 μM, 0.15 0.18 μM, 0.38 0.12 μM, and 0.30 0.13 μM, respectively. It also
inhibited tubulin polymerization with the IC50 value of 2.1 0.12 μM, which was
comparable with that of the positive controls. Furthermore, compound 12 regulated
the expression of cell cycle-related proteins (Cyclin B1, Cdc25c, and Cdc2) and
apoptosis-related proteins (Bcl-2, Bcl-x, and Mcl-1). Mechanistically, compound 12
could arrest cell cycle at the G2/M phase, thus induce an increase of apoptotic cell
death. In addition, molecular docking hinted the possible interaction mode of com-
pound 12 into the colchicine binding site of tubulin heterodimers. According to the
applications of microtubule-targeting agents in both direct and synergistic cancer
therapies, we hope this work might be of significance for future researches.
University for Nationalities, Nanning 530006,
China.
Email: manruojun@163.com
Hai-Liang Zhu, State Key Laboratory of
Pharmaceutical Biotechnology, Nanjing
University, Nanjing 210023, China.
Email: zhuhl@nju.edu.cn
Zhu-Gui Zhou, College of Chemistry and
Chemical Engineering, Guangxi Key Laboratory
of Polysaccharide Materials and Modifications,
Guangxi University for Nationalities, Nanning,
530006, China.
Email: zhouzhugui@126.com
Funding information
Guangxi Biological Polysaccharide Separation,
Purification and Modification Research
Platform, Grant/Award Number:
GKZY18076005; Guangxi Natural Science
Foundation, Grant/Award Number:
2019AC20294; Guangxi University for
Nationalities Research Funded Project, Grant/
Award Number: 2018KJQD13; Lianyungang
Talent Project
K E Y W O R D S
colchicine-binding site, indole-1,2,4-triazole, microtubules, molecular docking
1
|
INTRODUCTION
toxicity, are preferred over traditional cytotoxic treatment. Among
them, microtubule-targeting agents (MTAs) are a major advancement
In the past decades, the reported natural and synthetic compounds
targeting the mitotic spindle apparatus have attracted wide attention,
and microtubules (MTs) have been recognized as a crucial target for
the development of potential novel anticancer agents (Mahindroo
et al., 2006). Currently, the drugs, which can bind to specific target or
multitargets with the characteristics of high-efficiency and low
in the treatment of a considerable fraction of cancers in the pharmacy
field (Meng et al., 2009). MTs are cytoskeleton protein polymers com-
posed of α,β-tubulin heterodimers that are vital components in
eukaryotic cells (Amos, 2004; Dumontet & Jordan, 2010; Jordan &
Wilson, 2004; Kavallaris et al., 2001; Risinger et al., 2009). They play
an essential role in the process of cells division, cell shape formation,
maintenance, cell signaling, cell transport of organelles, as well as the
secretion, regulation of motility, and segregation of chromosomes
Meng-Ke Wu and Ruo-Jun Man contributed equally to the work
Drug Dev Res. 2021;1–13.
wileyonlinelibrary.com/journal/ddr
© 2021 Wiley Periodicals, LLC.
1

2
WU ET AL.
(McIntosh et al., 2002; Walczak, 2000). Upon breaking down the bal-
ance between tubulin polymerization and depolymerization, MTs
function will be interfered, interrupting the cell division and encourag-
ing the apoptosis (Honore et al., 2005; Teicher, 2008; Nakagawa-Goto
et al., 2011). Therefore, MTs have become an attractive target for the
exploitation of novel chemotherapeutic drugs and therapies (Brennan
et al., 2013; Gan et al., 2013).
synergistic therapies for treating cancers, developing anti-tubulin
agents remains a challenge.
Previous studies have yielded numerous small molecule com-
pounds with different heterocycles such as piperazine, imidazoles, thi-
azole, indoles, pyrazoline, and oxadiazole (Kamal, Kumar, et al., 2014a;
Kamal, Reddy, et al., 2014b; Szczepankiewicz et al., 2001). These syn-
thetic compounds were potent in the inhibition of tubulin polymeriza-
tion. In particular, indole analogues (3 and 4, Figure 1) have been
identified to possess potent antitumor and tubulin depolymerization
activity (Tanitame et al., 2004). Moreover, 1,2,4-triazole analogues
have also been a common choice in potent tubulin polymerization
inhibitors (5, Figure 1) (Alhamadsheh et al., 2007; Campbell &
Cronan, 2001; Li, Li, et al., 2011b; Li, Luo, & Zhu, 2011a).
The design of targeting drugs is a popular field. A lot of modified
compounds which can bind to tubulin have been reported. Because
of the effective interference with microtubule polymerization, colchi-
cine (compound 1, Figure 1) has been identified a potentially useful
anticancer drug, and its binding site on tubulin has been character-
ized already (Nakagawa-Goto et al., 2011; Ravelli et al., 2004). How-
ever, due to the high adverse effect, the clinical development of
Structure activity relationship (SAR) studies of colchicine and
CA-4 (CA-4P) revealed that the 3,4,5-trimethoxy phenyl ring was
essential for activity (Pettit et al., 2000; Pettit & Rhodes, 1998; Solum
et al., 2014). Similar compounds were designed and synthesized. Due
to their performances at the colchicine binding site on tubulin, these
compounds with identical skeleton could inhibit tubulin polymeriza-
tion well and exhibit potent antitumor activity in vivo against various
tumors. According to the molecular modeling studies and the results
of previous researches, we attempted to integrate these two groups
to screen new basic scaffolds for the design of a series of novel tubu-
lin assembling inhibitors (7–24, Figure 1). As the designing strategy,
we started the investigation by using the indole-containing backbone,
and set the 3,4,5-trimethoxypheny moiety as one choice of the
substitutes.
colchicine for cancer treatment was not successful (Zhou
&
Giannakakou, 2005). Among the natural compounds, combretastatins
could affect microtubule dynamics by binding to the colchicine site
(Sharma et al., 2010). Combretastatin A-4 (CA-4, 2a, Figure 1)
exhibited strong growth inhibition at nanomolar concentrations
against a wide variety of human cancers including multidrug resistant
(MDR) types (Flynn et al., 2011). Low water solubility of CA-4 limited
its efficacy in vivo, hence, water soluble combretastatin analogs have
been further developed (Yu et al., 2015). Combretastatin A-4P (CA-
4P, 2b, Figure 1), a water-soluble disodium phosphate derivative, has
shown promising results in human clinical trials. CA-4P is under eval-
uation in phase III trials for the treatment of anaplastic thyroid cancer
and in phase II trials for non-small-cell lung cancer and platinum-
resistant ovarian cancer (Rustin et al., 2010; Zweifel et al., 2011).
With no commercialized cases but great potential in direct or
Here, we searched more novel agents to extend our research and
gain further insight into the ensuing structure–activity relationships
MeO
OMe
OMe
MeO
MeO
MeO
OMe
MeO
MeO
O
OH
MeO
MeO
OMe
O
R
NH
N
MeO
H
O
2
3
1
2a
2b
R = OH, Combertastatin A-4 (CA-4)
R = OPO3Na2, Combertastatin A-4P (CA-4P)
OXi8006
Colchicine
R2
R1
R3
R4
N
S
O
H3C
N
NH2
N
O
N
N
N
R5
N
N
N
H
Ph
N
H
CH3
OMe
OMe
MeO
4
5
7-24
CA224
FIGURE 1 Chemical structures of known tubulin inhibitors and designed structures

WU ET AL.
3
(SARs). Our screening strategy was to perform modifications on the
aromatic ring to find the one with the best inhibitory activity. In
this work, we synthesized a series of tubulin inhibitors bearing
indole-1,2,4-triazole scaffold and emphasized the rationale behind the
discovery of inhibitors, the discussion of synthetic method, SAR,
bioactivities, and the effect of the inhibitors on the viability of tumor
cells.
iodomethane to yield compound 6d, with sodium hydride as catalyst.
The hydrazides (compound 6e) were obtained from the reaction of
esters 6d and 85% hydrazine hydrate in ethanediol. Treatment of the
hydrazide with phenyl isothiocyanate under refluxing gave the key
intermediate 6. After dissolving in acetonitrile, compound 6 was
reacted with different substituted benzyl bromide to afford the target
compounds 7–24. All of the target compounds (Table 1) gave satisfac-
tory analytical and spectroscopic data, which were in full accordance
with their assigned structures.
2
|
RESULTS AND DISCUSSION
Chemistry
2.1
|
2.2
|
Anti-proliferation assay
As illustrated in Scheme 1, 19 novel indole-1,2,4-triazole derivatives
were synthesized via the general pathway. They were obtained in six
steps and started from indole-3-carbxaldehyde. In the stirred solution
of indole-3-carbxaldehyde (6a) in acetone, KMnO4 was added and the
reaction was heated for 5–6 h. The obtained compound 6b was dis-
solved in MeOH, then H2SO4 was added softly, and the reaction was
refluxed to gain compound 6c. Compound 6c was further treated with
All the synthesized compounds 6–24 were evaluated for their in vitro
anti-proliferation activity against HepG2 (human hepatoma cells),
HeLa (human cervix cell), MCF-7 (human breast cancer cell), and A549
(human lung cancer cell). All of the chosen tumor cell lines were typi-
cal ones associated with the polymerization of tubulin. As reported in
Table 1, the results indicated that the top hit 12 exhibited appreciable
antiproliferative effect, especially for HeLa cells. Compared with
Compounds
R1
H
F
R2
H
R3
H
R4
H
R5
H
F
7
8
H
H
H
9
H
H
H
H
F
Cl
Br
H
H
H
H
H
H
H
H
H
Cl
H
OCH3
OCH3
H
H
H
F
Cl
Br
H
H
H
H
H
H
F
H
H
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
OCH3
H
OCH3
H
H
H
H
H
H
F
Cl
Br
I
CH3
CF3
SCHEME 1 General synthesis
and structures of compounds
(7–24). Reagents and conditions:
(a) KMnO4, acetone, reflux, 5–6 h;
(b) H2SO4, methanol, 80ꢀC,
8–11 h; (c) CH3I, NaH, THF,
35ꢀC; (d) hydrazine hydrate (85%),
ethanediol, 180ꢀC, 6 h; (e) phenyl
isothiocyanate, ethyl alcohol,
80ꢀC, 4–6 h; (f) substituted
benzyl bromide, acetonitrile,
90ꢀC, 12 h

4
WU ET AL.
TABLE 1 The substitutes of the synthesized compounds.
was a typical epithelial cell line for evaluation of cytotoxicity. The
results shown in Table 2 indicated that most of the obtained com-
pounds had low toxicity upon 293T cells.
Compounds
R1
H
F
R2
H
R3
H
R4
H
R5
H
F
7
8
H
H
H
9
H
H
H
H
F
Cl
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2.5
|
Structure–activity relationship analysis
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
H
OCH3
H
H
OCH3
OCH3
H
OCH3
OCH3
H
Subsequently, SAR were derived from analysis of antiproliferative and
tubulin inhibitory activity data. Some general hints could be inferred
based on the data. Initially, the phenyl group seemed necessary
because compound 6 with a short chain was less potent in all the
compounds. If there was no substitution (compound 7), the phenyl
group also showed limited improvement on the activity. Second, the
electron-donating groups (–OMe, −Me; compounds 10–12, 23) were
more favorable than the electron-withdrawing ones (–F, –Cl, –Br, –I,
–CF3; compounds 8, 9, 13–22, 24). In the electron-donating situation,
introducing more such substitutes might enhance the potency; while
in the electron-withdrawing situation, especially compounds 13–22,
reducing the size of such substitutes might be beneficial whereas the
position seemed a less important parameter. That was to say, although
we introduced a variety of substitutes to enrich the diversity by
changing the position of the halogen atom from the ortho-position to
the meta-position and para-position, the fluoro-substituted ones were
more beneficial. Actually, for fluoro-substituted ones, the ortho-
position was better. Additionally, when the substitutes were electron-
withdrawing, introducing multisubstitution (compounds 8, 9) could
also improve the activity. In brief, the new class of compounds raised
several potent tubulin inhibitors, which have the potential to further
explore the values of development.
OCH3
H
Cl
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
Cl
H
H
Br
H
H
H
F
H
H
Cl
H
H
Br
I
H
H
H
H
CH3
CF3
H
H
H
colchicine and ABT-751 as the positive controls, IC50 values of com-
pound 12 were found to be favorable against the four carcinoma cell
lines (IC50 = 0.23 0.08 μM, 0.15 0.18 μM, 0.38 0.12 μM, and
0.30 0.13 μM, respectively).
2.3
|
Tubulin polymerization inhibition
Since compounds 6–24 were designed for inhibiting tubulin polymeriza-
tion, we evaluated them on the inhibition of tubulin polymerization
in vitro. The results were also shown in Table 1. Accordingly, these
agents showed inhibitory activities of tubulin and the displayed IC50
values ranged from 2.1 0.12 to 35.79 1.05 μM, as compared with
2.52 0.23 μM of colchicine. Among all the compounds, compound 12
showed the most potent anti-tubulin (IC50 = 2.1 0.12 μM) activity,
which was even better than that of the positive controls. Besides, 10
and 11 also appreciably inhibited tubulin polymerization (IC50 = 5.08
0.16, 3.4 0.43 μM), respectively. The correlation coefficient (R2)
between the tubulin inhibition and the anti-proliferation potency was
calculated as 0.7833 (larger than a general 0.4 for checking the positive-
correlation). Therefore we could conclude that the synthesized com-
pounds were potential inhibitors of tubulin assembly and the inhibitory
effect was roughly proportional with the antiproliferative potency.
2.6
|
Cell apoptosis
To investigate the ability of compound 12 to induce cell death, flow
cytometry of cells double stained with Annexin V-FITC/propidium
iodide (PI) was conducted. In this study, HeLa cells were treated with
compound 12 for 24 h at the concentrations of 0, 0.1, 0.15, and
0.2 μM to examine the apoptotic effect. As shown in Figure 2, the
results displayed that the early apoptotic cells were increased from
2.99% to 4.98%. Moreover, the percentage of apoptotic cell signifi-
cantly increased from 14.0% to 35.3% along with the increasing doses
of compound 12. Thus, compound 12 was efficiently in the induction
of apoptosis in a dose-dependent manner.
To further check the compound 12-induced cell apoptosis, we
performed the western blot analysis. It is well-known that regulation
of Bcl-2 proteins shares the signaling pathways induced by anti-
microtubule compounds. Whereas antiapoptotic members (Bcl-2, Bcl-
x1, and Mcl-1) are capable of antagonizing the proapoptotic proteins.
In agreement with these observations, we found that the levels of
Bcl-2 and Bcl-x1 after 24 h treatment with compound 12 were gradu-
ally decreased, while the expression of Mcl-1 (another antiapoptotic
member of the Bcl-2 family) was also down-regulated. Altogether, our
findings indicated that compound 12 was able to downregulate the
2.4
|
Cytotoxicity test
All the compounds were evaluated for their toxicity against the non-
cancer cell line (human kidney epithelial cell 293T) with the median
cytotoxic concentration (CC50) data via the MTT assay. This cell line

WU ET AL.
5
TABLE 2 IC50 values (μM) of the synthesized compounds 6–24 and the positive controls in human tumor cell lines and normal cell line
IC50 SD (μM)
CC50 (μM)
Compounds
HepG2a
HeLaa
MCF-7a
A549a
Tubulinb
293Tc
105
167
98.7
107
115
136
126
108
142
118
88.9
147
97.6
189
113
157
124
99.6
121
93.5
102
6
11.57 0.24
5.58 0.12
1.49 0.06
1.56 0.14
0.95 0.09
0.37 0.56
0.23 0.08
1.33 0.05
2.44 0.09
2.84 0.17
2.63 0.14
3.04 0.36
2.95 0.45
3.58 0.39
2.42 0.04
2.67 0.55
3.76 0.20
0.99 0.35
2.08 0.39
0.35 0.20
3.82 0.36
10.34 0.14
3.14 0.31
2.27 0.17
1.88 0.08
0.54 0.87
0.38 0.25
0.15 0.18
1.15 0.34
2.63 0.66
1.86 0.32
3.12 0.23
2.88 0.14
2.34 0.67
2.14 0.76
1.23 0.35
2.12 0.16
2.67 0.17
1.07 0.76
1.96 0.22
0.28 0.11
0.95 0.08
13.13 0.17
3.56 0.74
1.46 0.86
2.74 0.11
0.78 0.39
0.45 0.18
0.38 0.12
1.48 0.07
2.97 0.12
2.46 0.29
2.73 0.67
2.47 0.10
3.78 0.09
2.56 0.47
2.46 0.77
2.19 0.29
3.67 0.43
0.86 0.26
1.93 0.76
0.44 0.09
4.12 0.36
8.55 0.42
4.14 0.32
1.24 0.37
2.86 0.15
0.58 0.31
0.34 0.32
0.30 0.13
1.30 0.11
3.25 0.76
3.87 1.10
2.66 0.56
3.38 0.19
2.58 0.23
2.14 0.21
2.76 0.81
3.97 0.54
3.26 0.05
0.95 0.28
2.87 0.54
0.37 0.14
6.10 0.54
35.79 1.05
23.77 0.64
7.09 0.32
8.87 0.98
6.08 0.16
4.40 0.43
2.10 0.12
9.64 0.78
13.45 0.12
15.58 0.34
10.99 0.76
14.79 0.22
13.34 0.57
11.56 0.30
12.82 0.39
13.67 0.24
19.78 0.67
6.92 0.21
12.44 0.76
2.52 0.23
8.24 0.69
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Colchicined
ABT-751d
aInhibition of the growth of tumor cell lines.
bInhibition of tubulin polymerization.
cInhibition of the growth of normal cell line.
dUsed as a positive control.
FIGURE 2 Annexin V-FITC/PI dual-immuno-fluorescence staining after treatment with different concentrations of compound 12 on HeLa
cells revealed significantly increased percentage of apoptotic cells. (a) The apoptosis rate of HeLa cells treated with 0, 0.1, 0.15, and 0.2 μM for
24 h. (b) The percentages of apoptotic cells were calculated after the treatment of compound 12. Images were representative of three
independent experiments. (c) Western blot analysis of Bcl-2, Bcl-x1, and Mcl-1 after treatment of HeLa cells with compound 12 at the
concentrations of 0, 0.1, 0.15, and 0.2 μM for 24 h

6
WU ET AL.
FIGURE 3 (a) Effect of
compound 12 on the cell cycle
distribution of HeLa cells in a
dose-dependent manner (0, 0.1,
0.15, and 0.2 μM). Images were
representative of three
independent experiments.
(G1 phase, green; S phase,
yellow; and G2/M phase, blue).
(b) Western blot analysis of the
levels of the G2/M-related
proteins Cdc2, Cyclin B1, and
Cdc25c after treating HeLa cells
with compound 12 at the
mentioned concentrations
for 24 h
expression of antiapoptotic proteins in line, thus effectively to
increase the percentage of apoptotic cells.
et al., 2014]). All docking runs were applied under Discovery Studio
3.5. The interaction energy as the docking calculation of the synthe-
sized compounds ranged from −64.22 to −36.21 kcal/mol. It was
clear that compound 12 showed the lowest interaction energy
(−64.22 kcal/mol) among all the synthesized compounds. In order to
give a better understanding of the tested activity, we examined the
interaction mode of the most potent inhibitor 12 within tubulin. For
the control colchicine, its trimethoxyphenyl was buried deeply into
the β-subunit of tubulin, surrounded by several hydrophobic amino
acids, such as Ala, Val, Leu, and Ile (Figure 4(a)). The H-bond of colchi-
cine with Val181 from α-subunit strengthened their interaction with
each other. Of course, other kinds of interactions, such as π-alkyl
bond, were also helpful. As shown in Figure 4(c) and (d), compound
12 maintained the orientation, especially that of the trimethoxyphenyl
motif which mimicked that of colchicine (LOC503). As for the
3,4,5-trimethoxypheny moiety, both compound 12 and LOC503
might form one H-bond with Ala250, whereas compound 12 might
introduce more H-bonds with Gly237 and Val 238 according to the
binding patterns. Moreover, the direction of phenyl from 12 was also
similar to the seven-membered ring of LOC503, heading to the gap
between tubulin heterodimers. However, π-cation bond between β
chain Lys352 and indole ring of 12 was new and different from the H-
bond of colchicine. Meanwhile, the numbers of ring contributed more
π-interactions, compared with LOC503 (Figure 4(a) and (c)). The 3D
interaction mode in Figure 4 revealed that compound 12 was well
embedded into the colchicine binding pocket of tubulin dimers and
exhibited the most potent affinity for tubulin.
2.7
|
Cell cycle arrest
To further characterize the cell growth inhibitory properties of the new
compound 12 on the cell cycle, another flow cytometry assay was car-
ried out. We attempted to confirm whether HeLa cells were blocked in
mitosis with G2/M cell cycle arrest. Compound 12 with the various con-
centrations (0, 0.1, 0.15, and 0.2 μM) was used on HeLa cells for 24 h.
The cells were stained with PI for 30 min. The results of cell cycle profile
were subsequently analyzed by flow cytometry. A concomitant decrease
in cell population (G1 and S) was detected. As shown in Figure 3, com-
pound 12 caused a rise in G2/M arrest with increased drug concentra-
tion. The percentage of cells arrested at the G2/M phases was observed
increasing from 18.19% to 37.19%, 46.12%, and 79.6%, respectively.
Then we analyzed the proteins that controlled the cell cycle path-
way. Generally speaking, entry into mitosis of eukaryotic cell was reg-
ulated by activation of Cdc2 kinase, Cdc25c phosphorylation, and
Cyclin B1 binding. As shown in Figure 3, compound 12 caused a dose-
dependent decrease in Cdc2, Cdc25c, and Cyclin B1. This result indi-
cated that the arrest at G2/M induced by the compound 12 caused
significant variations in Cyclin B1 expression, followed by a remark-
able decrease in the level of the phosphorylated forms of both Cdc2
and Cdc25c, especially with the added concentration of 0.2 μM. The
decline in the level of Cdc2 after 24 h was highly significant. The
results, which were consistent with the cell cycle analysis, further
illustrated the mechanism of the cell cycle arrest effect.
3
|
CONCLUSION
2.8
|
Molecular docking
A comprehensive series of indole derivatives was developed as a
potent cell growth inhibitor, and initial experiments demonstrated that
these compounds exhibited significant anticancer activity and tubulin
polymerization inhibition activity. We also elucidated SARs between
tubulin and the synthesized indole derivatives at colchicine binding
site. In summary, compound 12 showed the most potent anticancer
We also visualized the possible binding mode of the colchicine binding
site on tubulin from a molecular modeling study for compounds 6–24.
The protein crystal structure of tubulin–colchicine complex was
obtained from the Protein Data Bank (PDB code: 4O2B [Prota

WU ET AL.
7
FIGURE 4 Binding mode of compound 12 with the target protein tubulin (PDB code: 4O2B). (a) 2D and (b) 3D diagram of the interaction mode
that colchicine with the colchicine binding site. (c) 2D and (d) 3D diagram of the interaction mode that compound 12 with the colchicine binding site
activity against HepG2, HeLa, MCF-7, A549 with IC50 values 0.23
0.08 μM, 0.15 0.18 μM, 0.38 0.12 μM, 0.30 0.13 μM, and per-
formed impressed tubulin polymerization inhibitory activity with an
IC50 value of 2.1 0.12 μM, which was comparable to that of positive
controls. Importantly, the flow cytometry studies demonstrated that
compound 12 induced apoptosis in HeLa cells and arrested cells in the
G2/M phase of the cell cycle. In order to further study the mecha-
nism, we performed the western blot analysis consulting most micro-
tubule targeting agents. At 0.2 μM in HeLa cells, compound 12
induced downregulation of the anti-apoptotic proteins Bcl-2, Bcl-x1,
and particularly Mcl-1, which was a key factor governing cell survival.
Moreover, compound 12 induced cell cycle arrest at the G2/M phase
accompanied with a decrease in mitotic cells. Several key regulators
proteins (Cyclin B1, Cdc2, and Cdc25c) coordinated the progress of
cells from G2 to M phase. Compound 12 treatment led to a decrease
in the level of Cyclin B1 with a concomitant decrease in Cdc2 and
Cdc25c activity. Finally, Molecular docking manifested the inhibitor-
tubulin protein interaction and revealed that compound 12 exten-
sively bound to the tubulin at the colchicine binding site. Based on
these interesting results of the novel compounds, our work might be
served as the starting point as well as provide a reliable approach for
reasonable design of tubulin polymerization inhibitors in future.
4
|
EXPERIMENTAL SECTION
| General
4.1
All chemicals and reagents used in the current study were of analytical
grade. The synthesized compounds were chemically characterized by
thin layer chromatography (TLC) on Merck precoated silica GF254
plates and visualized in UV light (254 nm). Melting points (uncorrected)

8
WU ET AL.
were determined on a XT4MP apparatus (Taike Corp., Beijing, China). All
the 1H NMR spectra were recorded on a Bruker DPX 400 model
Spectrometer in DMSO-d6 and chemical shifts were reported in ppm (δ).
ESI-MS spectra were recorded on a Mariner System 5304 Mass
spectrometer. Elemental analyses were performed on a CHN-O-Rapid
instrument and were within 0.4% of the theoretical values.
stirred at 90ꢀC until TLC analysis indicated the completion of organic
reaction. Then, cool the reaction system to room temperature. The
resulting solid was obtained and washed with cold anhydrous ethanol,
dried through anhydrous MgSO4, and crystallized from anhydrous
ethanol to get the pure products.
4.2.5
|
5-(1-Methyl-1H-indol-3-yl)-4-phenyl-4H-
4.2
|
Chemistry
1,2,4-triazole-3-thiol (6)
4.2.1
compounds 6b–6d
|
General procedure for the preparation of
White powder, yield: 82%, m.p. 147–148ꢀC; 1H NMR (400 MHz, DMSO-
d6) δ: 13.96 (s, 1H), 8.04 (d, J = 7.9 Hz, 1H), 7.62 (p, J = 3.5, 3.0 Hz, 3H),
7.52–7.41 (m, 3H), 7.27 (dd, J = 15.3, 1.2 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H),
6.36 (s, 1H), 3.61 (s, 3H); MS(ESI): 307.1 [M+ H]+; Anal. Calcd for
To a stirred solution of indole-3-carboxaldehyde 6a (50 mmol) in ace-
tone (100 ml) was added KMnO4 (10 mmol). The mixture was refluxed
under stirring for 5–6 h. The obtained compounds 6b (40 mmol) in
MeOH (100 mL) was added H2SO4 (2.9 ml, 40 mmol) dropwise at 0ꢀC.
The mixture was further stirred 8–11 h at 80ꢀC. MeOH was evaporated
and the pH value was adjusted to 5–6 with aqueous NaHCO3. Then the
mixture was extracted three times, then organic layer was dried through
MgSO4 and concentrated under reduced pressure. Methyl indole-
3-carboxylate 6c was treated with CH3I in the presence of NaH in THF
at 35ꢀC for 5 h to get products 6d, respectively. All the reactions were
monitored by the thin layer chromatography (TLC).
C17H14N4S: C 66.64, H 4.61, N 18.29, found: C 66.63, H 4.62, N 18.30.
4.2.6
|
3-(5-[Benzylthio]-4-phenyl-4H-
1,2,4-triazol-3-yl)-1-methyl-1H-indole (7)
White powder, yield: 47.7%, m.p. 150–151ꢀC; 1H NMR (400 MHz,
DMSO-d6) δ: 8.16 (d, J = 7.8 Hz, 1H), 7.67–7.53 (m, 4H), 7.46 (d,
J = 8.2 Hz, 1H), 7.38–7.23 (m, 7H), 7.26–7.15 (m, 1H), 6.49 (s, 1H), 4.37
(s, 2H). 3.62 (m, 3H). MS(ESI): 397.1 [M + H]+; Anal. Calcd for
C24H20N4S: C 72.70, H 5.08, N 14.13, found: C 72.71, H 5.09, N 14.11.
4.2.2
|
General procedure for the preparation of
compounds 6e
4.2.7
|
3-(5-([2,6-Difluorobenzyl]thio)-4-phenyl-
4H-1,2,4-triazol-3-yl)-1-methyl-1H-indole (8)
For the synthesis of substituted compounds 6e
a mixture of
corresponding esters 6d and 85% hydrazine hydrate (1:2) in
ethanediol (30 ml) was heated (180ꢀC) to reflux for 6 h. After that, the
solution was completely removed under reduced pressure, and the
residue crystallized from ethanol.
Light yellow powder, yield: 38.4%, m.p. 177–179ꢀC; 1H NMR (400 MHz,
DMSO-d6) δ: 8.16 (d, J = 8.0 Hz, 1H), 7.68–7.55 (m, 4H), 7.51–7.34 (m,
3H), 7.26 (t, J = 7.3 Hz, 1H), 7.14 (dt, J = 31.8, 7.9 Hz, 3H), 6.51 (s, 1H),
4.27 (s, 2H). 3.61 (s, 3H). MS(ESI): 433.1 [M + H]+; Anal. Calcd for
C24H18F2N4S: C 66.65, H 4.20, N 12.95, found: C 66.66, H 4.19, N 12.93.
4.2.3
|
General procedure for the preparation of
compounds 6
4.2.8
|
3-(5-([3-Chloro-4-fluorobenzyl]thio)-
4-phenyl-4H-1,2,4-triazol-3-yl)-1-methyl-1H-indole (9)
To a solution of the hydrazides 6e (10 mmol) in ethanol (50 ml) at 80ꢀC,
phenyl isothiocyanate (15 mmol) were added, and the reaction mixture
was refluxed for 4–6 h. Excess solvent was evaporated under reduced
pressure and the residue was dissolved in sodium hydroxide aqueous
solution (0.5 h) and then acidified with dilute hydrochloric acid (10%) to
pH 5. The precipitate was filtered off, dried, and crystallized from etha-
nol. In general, compounds 6 was prepared with this method.
Yellow powder, yield: 52.3%, m.p. 183–185ꢀC; 1H NMR (400 MHz,
DMSO-d6) δ: 8.15 (d, J = 7.9 Hz, 1H), 7.66–7.55 (m, 5H), 7.46 (d,
J = 8.2 Hz, 1H), 7.42–7.33 (m, 3H), 7.25 (t, J = 7.1 Hz, 1H), 7.17 (t,
J = 7.9 Hz, 1H), 6.51 (s, 1H), 4.37 (s, 2H). 3.63 (s, 3H). MS(ESI): 449.1
[M + H]+; Anal. Calcd for C24H18ClFN4S: C 64.21, H 4.04, N 12.48,
found: C 64.20, H 4.03, N 12.49.
4.2.4
|
General synthesis method of the target
4.2.9
|
3-(5-([4-Methoxybenzyl]thio)-4-phenyl-4H-
compounds 7–24
1,2,4-triazol-3-yl)-1-methyl-1H-indole (10)
Acetonitrile (10 ml) containing compounds 6 (1 mmol) and a variety of
Light yellow powder, yield: 69.1%, m.p. 155–157ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ: 8.20 (d, J = 8.0 Hz, 1H), 7.87–7.70 (m, 2H),
benzyl bromide (1.5 mmol) was added to the reaction mixture and

WU ET AL.
9
7.50 (d, J = 8.2 Hz, 2H), 7.35 (dd, J = 8.0, 1.3 Hz, 2H), 7.24 (dd,
J = 17.1, 7.6 Hz, 4H), 7.22–7.08 (m, 2H), 6.50 (s, 1H), 4.33 (s, 2H),
3.87 (s, 3H) 3.73 (s, 3H),. MS(ESI): 427.2 [M + H]+; Anal. Calcd for
4.2.14
|
3-(5-([2-Bromobenzyl]thio)-4-phenyl-4H-
1,2,4-triazol-3-yl)-1-methyl-1H-indole (15)
C
25H22N4OS: C 70.40, H 5.20, N 13.14, found: C 70.41, H 5.22,
Light yellow powder, yield: 41.8%, m.p. 160–161ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ: 8.17 (d, J = 7.9 Hz, 1H), 7.65–7.54 (m, 4H),
7.50–7.38 (m, 2H), 7.34 (d, J = 6.8 Hz, 3H), 7.25 (t, J = 7.6 Hz, 1H),
7.24–7.10 (m, 2H), 6.50 (s, 1H), 4.38 (s, 2H),3.62 (s, 3H). MS(ESI):
477.1 [M + H]+; Anal. Calcd for C24H19BrN4S: C 60.64, H 4.03, N
11.79, found: C 60.63, H 4.02, N 11.80.
N 13.13.
4.2.10
|
3-(5-([3,5-Dimethoxybenzyl]thio)-
4-phenyl-4H-1,2,4-triazol-3-yl)-1-methyl-1H-
indole (11)
White powder, yield: 55.6%, m.p, 175–177ꢀC; 1H NMR (400 MHz,
DMSO-d6) δ: 7.95 (dt, J = 7.9, 1.0 Hz, 1H), 7.64–7.54 (m, 5H),
7.58–7.43 (m, 4H), 7.24 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.12 (ddd,
J = 8.0, 7.0, 1.0 Hz, 1H), 6.85 (s, 1H), 4.45 (s, 2H) 3.70 (s, 6H), 3.63 (s,
3H). MS(ESI): 457.2 [M + H]+; Anal. Calcd for C26H24N4O2S: C 68.40,
H 5.30, N 12.27, found: C 68.38, H 5.30, N 12.28.
4.2.15
|
3-(5-([3-Fluorobenzyl]thio)-4-phenyl-4H-
1,2,4-triazol-3-yl)-1-methyl-1H-indole (16)
Yellow powder, yield: 40.1%, m.p. 171–173ꢀC; 1H NMR (400 MHz,
DMSO-d6) δ: 8.16 (d, J = 7.9 Hz, 1H), 7.68–7.52 (m, 3H), 7.47 (d,
J = 8.1 Hz, 1H), 7.46–7.30 (m, 3H), 7.34–7.06 (m, 5H), 6.51 (s, 1H),
4.39 (s, 2H), 3.63 (s, 3H). MS(ESI): 415.1 [M + H]+; Anal. Calcd for
C24H19FN4S: C 69.54, H 4.62, N 13.52, found: C 69.53, H 4.63,
4.2.11
|
1-Methyl-3-(4-phenyl-
N 13.51.
5-([3,4,5-trimethoxybenzyl]thio)-4H-1,2,4-triazol-3-yl)-
1H-indole (12)
4.2.16
|
3-(5-([3-Chlorobenzyl]thio)-4-phenyl-4H-
Light yellow powder, yield: 66.3%, m.p. 189–190ꢀC; 1H NMR
(600 MHz, DMSO-d6): δ: 8.14 (d, J = 8.0 Hz, 1H), 7.61 (t, J = 7.4 Hz,
1H), 7.56 (t, J = 7.6 Hz, 2H), 7.46 (d, J = 8.2 Hz, 1H), 7.25 (dd, J = 12.8,
7.3 Hz, 3H), 7.17 (t, J = 7.5 Hz, 1H), 6.57 (s, 2H), 6.50 (s, 1H), 4.26 (s,
2H), 3.69 (s, 6H), 3.63 (d, J = 3.8 Hz, 6H). MS(ESI): 487.2 [M + H]+;
Anal. Calcd for C27H26N4O3S: C 66.65, H 5.39, N 11.51, found: C
66.64, H 5.38, N 11.50.
1,2,4-triazol-3-yl)-1-methyl-1H-indole (17)
Light yellow powder, yield: 56.4%, m.p. 166–167ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ: 8.15 (d, J = 8.0 Hz, 1H), 7.68–7.54 (m, 3H),
7.50–7.41 (m, 2H), 7.40–7.29 (m, 5H), 7.25 (s, 1H), 7.21 (dt, J = 32.1,
7.2 Hz, 1H), 6.50 (d, J = 1.9 Hz, 1H), 4.37 (d, J = 1.9 Hz, 2H), 3.62 (m,
3H). MS(ESI): 431.1 [M + H]+; Anal. Calcd for C24H19ClN4S: C 66.89,
H 4.44, N 13.00, found: C 66.88, H 4.43, N 13.01.
4.2.12
|
3-(5-([2-Fluorobenzyl]thio)-4-phenyl-4H-
1,2,4-triazol-3-yl)-1-methyl-1H-indole (13)
4.2.17
|
3-(5-([3-Bromobenzyl]thio)-4-phenyl-4H-
1,2,4-triazol-3-yl)-1-methyl-1H-indole (18)
Yellow powder, yield 53.3%, m.p. 170–171ꢀC; 1H NMR (400 MHz,
DMSO-d6) δ: 8.17 (d, J = 7.9 Hz, 1H), 7.60 (dt, J = 14.4, 6.9 Hz, 3H),
7.50–7.38 (m, 2H), 7.34 (d, J = 6.9 Hz, 3H), 7.28–7.12 (m, 4H). 6.49 (s,
1H), 4.37 (s, 2H), 3.67 (s, 3H). MS(ESI): 415.1 [M + H]+; Anal. Calcd for
White powder, yield: 47.7%, m.p. 164–166ꢀC; 1H NMR (400 MHz,
DMSO-d6) δ: 8.76 (s, 1H), 7.95 (dd, J = 8.0, 1.1 Hz, 1H), 7.66–7.54 (m,
3H), 7.58–7.41 (m, 3H), 7.24 (d, J = 7.0 Hz, 2H), 7.12 (dt, J = 7.5, 7.0,
1.1 Hz, 3H), 6.85 (s, 1H), 4.46 (s, 2H), 3.70 (s, 3H). MS(ESI): 477.1 [M
+ H]+; Anal. Calcd for C24H19BrN4S: C 60.64, H 4.03, N 11.79, found:
C 60.65, H 4.04, N 11.81.
C
24H19FN4S: C 69.54, H 4.62, N 13.52, found: C 69.53, H 4.63,
N 13.54.
4.2.13
|
3-(5-([2-Chlorobenzyl]thio)-4-phenyl-4H-
1,2,4-triazol-3-yl)-1-methyl-1H-indole (14)
4.2.18
|
3-(5-([4-Fluorobenzyl]thio)-4-phenyl-4H-
1,2,4-triazol-3-yl)-1-methyl-1H-indole (19)
Light yellow powder, yield: 36.4%, m.p. 162–164ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ: 8.16 (d, J = 7.9 Hz, 1H), 7.59 (dt, J = 14.4,
6.9 Hz, 5H), 7.48 (dd, J = 13.7, 7.6 Hz, 4H), 7.36–7.21 (m, 2H), 7.17 (t,
J = 7.5 Hz, 1H), 6.50 (s, 1H), 4.45 (s, 2H). 3.63 (s, 3H). MS(ESI): 431.1
[M + H]+; Anal. Calcd for C24H19ClN4S: C 66.89, H 4.44, N 13.00,
found: C 66.90, H 4.43, N 13.01.
Yellow powder, yield: 46.7%, m.p. 169–170ꢀC; 1H NMR (400 MHz,
DMSO-d6) δ: 8.18 (d, J = 7.9 Hz, 1H), 7.61–7.55 (m, 3H), 7.50–7.38 (m,
2H), 7.34 (d, J = 6.9 Hz, 3H), 7.23–7.09 (m, 4H). 6.52 (s, 1H), 4.40 (s, 2H),
3.61 (s, 3H). MS(ESI): 415.1 [M + H]+; Anal. Calcd for C24H19FN4S: C
69.54, H 4.62, N 13.52, found: C 69.53, H 4.63, N 13.51.

10
WU ET AL.
4.2.19
|
3-(5-([4-Chlorobenzyl]thio)-4-phenyl-4H-
Calcd for C25H19F3N4S: C 60.64, H 4.12, N 12.06, found: C 60.65, H
4.13, N 12.04.
1,2,4-triazol-3-yl)-1-methyl-1H-indole (20)
Light yellow powder, yield: 36.4%, m.p. 165–167ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ: 8.16 (d, J = 8.0 Hz, 1H), 7.68–7.55 (m, 3H),
7.49 (dd, J = 18.3, 8.3 Hz, 3H), 7.35 (dd, J = 13.1, 7.6 Hz, 4H), 7.25 (t,
J = 7.0 Hz, 1H), 7.17 (t, J = 7.5 Hz, 1H), 6.50 (s, 1H), 4.35 (s, 2H),3.63
(s, 3H). MS(ESI): 449.1 [M + H]+; Anal. Calcd for C24H19ClN4S: C
66.89, H 4.44, N 13.00, found: C 66.87, H 4.45, N 12.99.
4.3
|
Cell culture
Human hepatoma cell (HepG2), carcinoma of cervix cell (HeLa), human
breast cancer cells (MCF-7), human lung adenocarcinoma cells (A549),
and human renal epithelial cells (293T) were maintained in DMEM
(Dulbecco's modified Eagle's medium) with 1% antibiotic (100 U/ml
penicillin and 100 μg/ml streptomycin) and 10% FBS (fetal bovine
serum) in a humidified atmosphere in 5% CO2 at 37ꢀC.
4.2.20
|
3-(5-([4-Bromobenzyl]thio)-4-phenyl-4H-
1,2,4-triazol-3-yl)-1-methyl-1H-indole (21)
White powder, yield: 55.1%, m.p. 161–162ꢀC; 1H NMR (400 MHz,
DMSO-d6) δ: 8.15 (d, J = 7.8 Hz, 1H), 7.68–7.55 (m, 4H), 7.49 (dd,
J = 18.1, 8.3 Hz, 3H), 7.35 (dd, J = 13.0, 7.6 Hz, 3H), 7.21 (dt, J = 32.7,
7.0 Hz, 2H),), 6.55 (s, 1H), 4.46 4.35 (s, 2H), 3.63 (s, 3H). MS(ESI):
477.1 [M + H]+; Anal. Calcd for C24H19BrN4S: C 60.64, H 4.03, N
11.79, found: C 60.63, H 4.04, N 11.80.
4.4
|
Antiproliferation assay
According to the previous reports (Tsyganov et al., 2013; Zheng
et al., 2014), target tumor cells were grown to log phase in DMEM
medium supplemented with 10% FBS. After reaching a dilution of 105
cells ml−1 with the medium, 100 μl of the obtained cell suspension
was added to each well of 96-well culture plates and then incubation
was performed at 37ꢀC in 5% CO2 atmosphere for 12 h before being
subjected to antiproliferation assessment. Tested samples at preset
concentrations (0.1, 1, 10, and 100 μM) were added to 96 wells with
colchicine as positive reference. After a 24 h exposure period, 25 ml
of PBS containing 2.5 mg/ml of MTT was added to each well, and the
cells were incubated at 37ꢀC for 4 h. Then, the solution was poured
out and 150 μl DMSO was added to dissolve the purple formazan
crystals produced. The optical absorbance was measured at 570 nm.
Each assay was carried out for at least three times.
4.2.21
|
3-(5-([4-Iodobenzyl]thio)-4-phenyl-4H-
1,2,4-triazol-3-yl)-1-methyl-1H-indole (22)
Yellow powder, yield: 40.7%, m.p. 197–199ꢀC; 1H NMR (400 MHz,
DMSO-d6) δ: 8.15 (d, J = 7.9 Hz, 1H), 7.71–7.55 (m, 6H), 7.46 (d,
J = 8.1 Hz, 1H), 7.36 (d, J = 6.8 Hz, 2H), 7.25 (t, J = 7.0 Hz, 1H), 7.18
(d, J = 8.3 Hz, 2H), 6.50 (s, 1H), 4.33 (s, 2H), 3.63 (s, 3H). MS(ESI):
523.0 [M + H]+; Anal. Calcd for C24H19IN4S: C 55.18, H 3.67, N
10.72, found: C 55.19, H 3.66, N 10.71.
4.5
|
Tubulin polymerization assay
4.2.22
|
1-Methyl-3-(5-([4-methylbenzyl]thio)-
4-phenyl-4H-1,2,4-triazol-3-yl)-1H-indole (23)
According to the previous reports (Yang et al., 2019; Yang
et al., 2020), bovine brain tubulin was purified as described previously.
To evaluate the effect on tubulin assembly in vitro, various concentra-
tions of the compounds (6–24) were preincubated with 10 μM tubulin
in glutamate buffer at 30ꢀC to 0ꢀC. And then the addition of GTP, the
mixtures were transferred to cuvettes at 0ꢀC in a recording spectro-
photometer and warmed up to 30ꢀC. The IC50 was defined as the
compound concentration that inhibited the extent of assembly by
50% after 20 min incubation. In all experiments three replicate wells
were used for each drug concentration.
Light yellow powder, yield: 69.1%, m.p. 155–157ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ: 8.16 (d, J = 7.9 Hz, 1H), 7.67–7.54 (m, 3H),
7.46 (d, J = 8.2 Hz, 1H), 7.33 (dd, J = 8.0, 1.3 Hz, 2H), 7.24 (dd,
J = 17.1, 7.6 Hz, 3H), 7.22–7.08 (m, 3H), 6.50 (s, 1H), 4.33 (s, 2H),
3.63 (s, 3H), 2.27 (s, 3H). MS(ESI): 411.2 [M + H]+; Anal. Calcd for
C
25H22N4S: C 73.14, H 5.40, N 13.65, found: C 73.13, H 5.42,
N 13.66.
4.2.23
|
1-Methyl-3-(4-phenyl-
5-((4-[trifluoromethyl]benzyl)thio)-4H-1,2,4-triazol-
3-yl)-1H-indole (24)
4.6
|
In vitro cytotoxicity test of 293T
In this study, the cytotoxic activity in vitro was measured using the
MTT assay. It is generally known that the human renal epithelial cells
(293T) was used to test the toxicity of compounds. The prepared sus-
pension was added into the 96-well plates to be incubated for about
12 h. Subsequently various concentrations of the test compounds
Light yellow powder, yield: 47.3%, m.p. 178–179ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ: 7.73 (s, 1H), 7.60 (dd, J = 23.2, 9.2 Hz, 7H),
7.46 (d, J = 8.1 Hz, 1H), 7.31 (d, J = 7.6 Hz, 2H), 7.29–7.14 (m, 2H),
6.49 (s, 1H), 4.46 (s, 2H), 3.36 (s, 3H). MS(ESI): 465.1 [M + H]+; Anal.

WU ET AL.
11
(0.1, 1, 10, and 100 μM) were added to the per well and grown at
37ꢀC for 24 h. And then 5 mg/ml of MTT (20 μl) was added each well
4 h before the termination of the incubation. After the supernatant
was removed, 150 ml DMSO was added to dissolve the formazan
crystals. The absorbance was read at a wavelength of 570 nm. Each
assay was replicated for three times.
After being washed three times in TBST, the membranes were incu-
bated with the suitable HRP-conjugated secondary antibodies for 2 h
at room temperature. Follow by three additional washes with TBST,
the membranes were detected with the SuperSignal West Pico chemi-
luminescence substrate (Abcam) and imaged by darkroom exposure.
4.10
|
Docking simulations
4.7
|
Cell apoptosis assay
The 3D X-ray structure of tubulin (PDB code: 4O2B) was chosen as
the template for the modeling study of compound 12. The crystal
structures of tubulin domain bound to colchicine were retrieved from
the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.
do). Molecular docking was carried out using the Discovery Studio
(version 3.5) as implemented through the graphical user interface DS-
CDOCKER protocol consulting the references (Li, Pan, et al., 2020a;
Li, Wu, et al., 2020b; Pan et al., 2020). The synthesized compounds
6–24 were constructed using Chem. 3D ultra 14.0 software. All bound
water and ligands were eliminated from the protein and the polar
hydrogen was added. The whole tubulin complex was defined as a
receptor and the site sphere was selected based on the ligand binding
location of colchicine (LOC503), then the LOC503 molecule was
removed and replaced by compound 12 during the molecular docking
procedure. All the types of the interactions of the docked protein with
ligand were analyzed at the end of molecular docking.
Approximately 105 cells/well were seeded in a 6-well plate and
allowed to adhere. The medium was replaced with fresh culture
medium containing compound 12 at final concentrations of 0, 0.1,
0.15, and 0.2 μM. After 24 h, cells were trypsinized, washed twice
with PBS, subsequently centrifuged (1500 rpm at 4ꢀC for 5 min) and
stained with 5 μl of Annexin V-FITC and 5 μl of PI in binding buffer
for 15 min at room temperature (25ꢀC) in the dark. In the test, about
10,000 cells were counted. Apoptosis analysis was performed using a
FACScaliber (FACScan; BD Biosciences) equipped with a CellQuest
software (BD Biosciences).
4.8
|
Cell cycle analysis
About 5 × 105 cells/well were plated in 6-well plates and incubated at
37ꢀC for 24 h to adhere. The medium was then washed off and fresh
culture medium containing 0, 0.1, 0.15, and 0.2 μM compound 12 was
added. After the incubation, the cells were collected, centrifuged,
fixed overnight at 4ꢀC with ice-cold 70% ethanol. The next day etha-
nol was removed, and the cells were in PBS (phosphate buffered
saline) containing stained with 0.1 mg/ml of RNase, 5 μg/ml PI and
stained at 37ꢀC for 30 min, and then subjected to flow cytometric
analysis. The percentage of cells in the G1, S, and G2/M phases of the
cell cycle were determined with Flowjo 7.6.1 software.
ACKNOWLEDGMENTS
This research was funded by Natural Science Foundation of Guangxi
Province under Grant No.2019AC20294, Guangxi University for
Nationalities Research Funded Project (No. 2018KJQD13) and
Guangxi biological polysaccharide separation, purification and modifi-
cation research platform (GKZY18076005). Dr. H.L. Zhu thanks for
the support of Lianyungang talent project.
CONFLICT OF INTEREST
The authors declare no conflicts of interest.
4.9
|
Western blot analysis
DATA AVAILABILITY STATEMENT
HeLa cells were incubated in the presence of compound 12. After
24 h, the cells were collected, centrifuged, and washed twice with ice-
cold PBS (phosphate buffered saline) and the pellet was lysed in RIPA
lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 0.1% SDS, 1% NP-40,
0.25% Sodium deoxycholate, and 1 mM EDTA, pH 8.0) with freshly
added protease inhibitor cocktail (Roche) on ice for 30 min, and then
centrifuged at 15,000g at 4ꢀC for 10 min. The supernatant was col-
lected, and the protein concentration was calculated with a BCA pro-
tein assay kit (Thermo Scientific, Rockford, Illinois). The proteins were
separated via SDS-PAGE (Bio-Rad), and boiled at 100ꢀC for 5 min.
After electrophoresis, the proteins were electrotransferred to PVDF
membranes (Bio-Rad) and then blocked with 5% skim milk at room
temperature for 2 h. Membranes were incubated with primary anti-
bodies against Cyclin B1 (Cell Signaling), p-cdc2Tyr15 (Cell Signaling),
Bcl-2, Bcl-x1 (Millipore), and β-actin (Sigma-Aldrich) overnight at 4ꢀC.
The data that support the findings of this study are available on
request from the corresponding author. The data are not publicly
available due to privacy or ethical restrictions.
ORCID
Ruo-Jun Man
https://orcid.org/0000-0001-5434-4229
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of this article.
How to cite this article: Wu M-K, Man R-J, Liao Y-J, Zhu H-L,
Zhou Z-G. Discovery of novel indole-1,2,4-triazole derivatives
as tubulin polymerization inhibitors. Drug Dev Res. 2021;1–13.
https://doi.org/10.1002/ddr.21805