Synthesis, anticancer activity and molecular docking studies on 1,2-diarylbenzimidazole analogues as anti-tubulin agents

Article abstract of DOI:10.1016/j.bioorg.2019.103219

Twenty-four 1,2-diarylbenzimidazole derivatives were designed, synthesized and biologically evaluated. It turned out that most of them were potential anticancer drugs. Among them, compound c24 showed the highest anti-tumor activity (GI₅₀ = 0.71–2.41 μM against HeLa, HepG2, A549 and MCF-7 cells), and low toxicity to normal cells (CC₅₀ > 100 μM against L02 cells). In the microtubule binding assay, c24 showed the most potent inhibition of microtubule polymerization (IC₅₀ = 8.47 μM). The binding ability of compound c24 to tubulin crystal was verified by molecular docking simulation experiment. Further studies on HepG2 and HeLa cells showed that compound c24 could cause mitotic arrest of tumor cells to G2/M phase then inducing apoptosis. To sum up, compound c24 is a promising microtubule assembly inhibitor.

Full text of DOI:10.1016/j.bioorg.2019.103219

BioorganicChemistry92(2019)103219
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, anticancer activity and molecular docking studies on 1,2-
diarylbenzimidazole analogues as anti-tubulin agents
⁎
Ya-Liang Zhanga,1, Rong Yanga,1, Lin-Ying Xiaa, Ruo-Jun Mana,b, Yi-Chun Chua, Ai-Qin Jiangc,
,
⁎
⁎
Zhong-Chang Wanga, , Hai-Liang Zhua,
a State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210023, People’s Republic of China
b Guangxi Biological Polysaccharide Separation, Purification and Modification Research Platform, Guangxi University for Nationalities, Nanning 530006, People’s Republic
of China
c Medical School, Nanjing University, Nanjing 210093, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Twenty-four 1,2-diarylbenzimidazole derivatives were designed, synthesized and biologically evaluated. It
turned out that most of them were potential anticancer drugs. Among them, compound c24 showed the highest
anti-tumor activity (GI50 = 0.71–2.41 μM against HeLa, HepG2, A549 and MCF-7 cells), and low toxicity to
normal cells (CC50 > 100 μM against L02 cells). In the microtubule binding assay, c24 showed the most potent
inhibition of microtubule polymerization (IC50 = 8.47 μM). The binding ability of compound c24 to tubulin
crystal was verified by molecular docking simulation experiment. Further studies on HepG2 and HeLa cells
showed that compound c24 could cause mitotic arrest of tumor cells to G2/M phase then inducing apoptosis. To
sum up, compound c24 is a promising microtubule assembly inhibitor.
Tubulin inhibitor
Benzimidazole
Cell cycle arrest
Apoptosis
Molecular docking
1. Introduction
research on its inhibitor has attracted much attention in recent years
[11,12].
The incidence of cancer in China is on the rise year by year, so it is
urgent to develop more effective cancer prevention and treatment
programs [1]. Malignant tumor is a kind of disease with unlimited cell
proliferation and abnormal differentiation, while chemotherapy drugs
have the effect of “cytotoxicity” and promoting differentiation, which
can kill cancer cells [2]. Chemotherapy, as a main treatment method,
plays an increasingly important role in the overall comprehensive
treatment. It is not only suitable for advanced [3] and recurrent cases
[4], but also plays a role in the treatment of early patients.
Combretastatin A-4 (CA-4, Fig. 1) was a product originally isolated
from the Combretum caffrum in South Africa [13]. The structure-activity
relationship showed that the cis-configuration of two aromatic rings
and the three methoxy groups of A-ring were important pharmacores
for the strong anti-cancer activity of CA-4 [14]. However, cis-double
bonds in CA-4 structure were easy to be isomerized into more stable
trans-double bonds in thermokinetics during the process of preservation
and use, thus reducing the activity [15,16]. Therefore, many researches
have been devoted to replacing the cis-double bond of CA-4 with dif-
ferent rigid linkers including heterocyclic ring systems to avoid the
problem of structural isomerization of CA-4 [17,18]. Meanwhile, the B-
ring of CA-4 was changed to obtain CA-4 analogues with better phar-
macodynamics and pharmacokinetics characteristics. The structure of
the designed compound was modified to replace the double bond of CA-
4 with heterocyclic ring, which not only stabilized the cis-configuration
but also improved the stability of the compound.
Microtubules are composed of tubulin and microtubule binding
protein, which are the main components of cytoskeleton [5]. Micro-
tubules have the kinetic properties of polymerization and depolymer-
ization, whose key functions are maintaining cell morphology, cell di-
vision and proliferation, organelle composition and transport, and
signaling material transmission [6,7]. Anti-cancer drugs targeting mi-
crotubules use their kinetic properties to either promote or inhibit their
polymerization [8]. Studies have shown that there are three mainly
different inhibitory binding sites on tubulin: paclitaxel site, vincristine
site and colchicine site [9,10]. Due to the small volume of the colchicine
site cavity and the simple structure of the corresponding inhibitor, the
Benzimidazole was one of the earliest nitrogen-containing hetero-
cyclic compounds [19], whose structure as a kind of dominant frame-
work had been widely studied in the field of pharmacy. Compounds
containing benzimidazole had a wide range of biological activities, such
⁎ Corresponding authors.
E-mail addresses: jianaq@nju.edu.cn (A.-Q. Jiang), wangzhongchang2006@163.com (Z.-C. Wang), zhuhl@nju.edu.cn (H.-L. Zhu).
1 These authors equally contributed to this paper.
https://doi.org/10.1016/j.bioorg.2019.103219
Received 27 January 2019; Received in revised form 11 July 2019; Accepted 24 August 2019
Availableonline26August2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

Y.-L. Zhang, et al.
BioorganicChemistry92(2019)103219
Fig. 1. Structure of reference and target compounds.
as anti-inflammatory, antihypertensive, anticoagulant, and especially
had a good application prospect as anti-cancer drugs [20,21]. Lu et al.
[22] designed and synthesized a novel compound containing benzimi-
dazole structure that could inhibit tubulin. Studies have shown that
compound 1 (Fig. 1) has a strong inhibitory effect on microtubule, with
GI50 value of 30 nM and stable metabolism in human liver microsome
with a half-life of 51 min. Wang’s team [23] synthesized a series of 1-
benzene acyl-2-(1-methylindol-3-yl)-benzimidazole derivatives. Com-
pound 2 (Fig. 1) showed high activity and GI50 values were 2.4, 3.8 and
5.1 μM against A549, HepG2 and MCF-7 cell. Ahmed Kamal et al. [24]
synthesized 20 arylpyrazole linked benzimidazole compounds. As a
known pro-tubulin depolymerizer, Nocodazole [25] (Fig. 1) bound to
the microtubule at the colchicine site although it had no structural si-
milarity with colchicine. Benzimidazole was important in the structure
of Nocodazole. On the basis of our previous research, we designed and
synthesized a series of 1,2-diarylbenzimidazole derivatives (Fig. 1). The
addition of benzimidazole structure to the microtubule inhibitor
structure could increase both the structural stability and the bioactivity
of the compound. The structure of the positive drug CA-4 was modified.
The same 3,4,5-trimethoxy group as the ring A in the structure of CA-4
was designed, and the ring B was replaced differently. Only benzimi-
dazole was used as the bridge to connect the ring A and ring B, and the
two benzene rings were substituted at the 1 and 2 positions of benzi-
midazole, respectively. The synthetic methods and biological activities
of these compounds were described in this paper. The binding modes of
these compounds to tubulin were studied by in vitro cell experiment and
computer simulation.
corresponding a1-a5 were obtained and purified by silica gel column
chromatography. Compounds a1-a5 were dissolved in mixture solution
(tetrahydrofuran and ethanol), and sodium dithionite solution was
added for reduction reaction. After the alkalization by NaHCO3, com-
pounds b1-b5 were obtained and purified by silica gel column chro-
matography. Compounds b1-b5 were dissolved in anhydrous ethanol
and then refluxed with substituted benzaldehyde to form imidazole
ring. Recrystallized in ethanol system, c1-c24 were obtained. All syn-
thesized compounds c1-c24 were determined by melting test, NMR,
ESI-MS and elemental analysis, which were consistent with the depicted
structures.
2.2. In vitro bioactivity assay
2.2.1. Inhibition of tubulin polymerization
The results for the in vitro tubulin polymerization inhibition of
compounds c1-c24 showed that compound c24 exhibited the highest
activity with IC50 (8.47 μM, Table 2). In the molecular structure of CA-
4, the 3,4,5-trimethoxyl structure on A-ring was an essential condition
for good activity. Eight compounds, c1-c8, were synthesized pre-
liminarily to figure out which one of these R1, R2 rings functions better
as A-ring. The substituents of R2 ring in c1-c4 were 3,4,5-trimethoxy,
and R1 ring mimicking the B-ring of CA-4 were non-substituent, 3-OH-
4-OCH3, 4-OCH3 and 3-OH, respectively. Compounds c5-c8 inter-
changed R1 ring with R2 ring of c1-c4. The IC50 value of c1-c4 is bigger
than that of c5-c8. Therefore, it is more appropriate to confirm that R1
ring was A-ring, and the substituent group is 3,4,5-trimethoxy. Ac-
cording to the IC50 value, the activity was compared as follows: 3-
Br < 2-Br < 4-Br (c10 < c11 < c9), 2-OCH3 < 3-OCH3 < 4-
OCH3 (c13 < c12 < c7). Two more groups of compounds were syn-
thesized, including meta- and para-substitutions. Results of bioactivity
comparison were as follows: 3-OH < 4-OH (c8 < c14), 3-CH3 < 4-
CH3 (c16 < c15). Therefore, the para-substitutions could improve the
activity of the compound more than that of the ortho-substitution and
the meta-substitution. Four more para-substituted compounds were
2. Results and discussion
2.1. Chemistry
The synthesis of compounds c1-c24 (Table 1) followed the general
method depicted in Scheme 1. o-Fluoronitrobenzene was refluxed with
substituted aniline in water, neutralized with NaHCO3, and then
2

Y.-L. Zhang, et al.
BioorganicChemistry92(2019)103219
Table 1
Structures of compounds c1–c24.
.
Compounds
R1
R2
Compounds
R1
R2
c1
H
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
H
c13
c14
c15
c16
c17
c18
c19
c20
c21
c22
c23
c24
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
2-OCH3
4-OH
c2
3-OH-4-OCH3
4-OCH3
c3
4-CH3
c4
3-OH
3-CH3
c5
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
4-SCH3
4-NO2
c6
3-OH-4-OCH3
4-OCH3
c7
4-F
c8
3-OH
4-Cl
c9
4-Br
2,4-(OH)2
3,4-(OH)2
2,4-(OCH3)2
3,4-(OCH3)2
c10
c11
c12
3-Br
2-Br
3-OCH3
synthesized in the hope of finding the most active substituents. In
conclusion, the activity sequencing of different substituents was as
Table 2
In vitro tubulin inhibitory activity of compound c1-c24.
follows:
4-SCH3 < 4-NO2 < 4-Cl < 4-Br < 4-F < 4-CH3 < 4-
Compounds
IC50
SD (μM)
Compounds
IC50
SD (μM)
OH < 4-OCH3 (c17 < c18 < c20 < c8 < c19 < c15 < c14 <
c7). The effect of double substitution on activity was further con-
sidered. The substitutions of -OCH3 and -OH with good activity were
selected as substituents to synthesize ortho-para disubstituted com-
pounds and meta-para disubstituted compounds respectively. 2,4-
(OH)2 < 3,4-(OH)2 (c21 < c22) and 2,4-(OCH3)2 < 3,4-(OCH3)2
(c23 < c24). Therefore, the activity of meta-para disubstitution was
superior to that of ortho-para disubstitution, in which 3,4-(OCH3)2 had
the optimal activity.
c1
109.73
128.12
87.92
117.96
145.77
50.22
9.35
3.05
3.46
2.11
1.59
2.36
0.78
0.13
0.44
0.57
0.62
0.55
0.42
0.07
c13
c14
c15
c16
c17
c18
c19
c20
c21
c22
c23
c24
39.34
17.16
19.60
21.30
140.00
125.66
19.75
29.43
16.36
14.45
11.77
8.47
0.53
0.41
0.29
0.37
c2
c3
c4
c5
2.08
c6
2.01
c7
0.64
c8
25.22
28.60
37.62
35.44
28.21
5.95
0.71
0.52
0.39
0.28
c9
c10
c11
c12
CA-4
0.11
2.2.2. Anti-proliferative activity
Anti-proliferative activity of compounds c1-c24 on human cervical
carcinoma (HeLa), human liver cancer cell (HepG2), human non-small
cell lung carcinoma (A549), human breast cancer cell (MCF-7) was
studied. The results were shown in Table 3. Among the 24 compounds,
the anti-proliferative activity of compound c24 was high, which was
1.71 μM, 0.71 μM, 2.41 μM and 1.94 μM against HeLa, HepG2, A549
and MCF-7 cells, respectively. R1 ring with 3,4,5-trimethoxy was more
active than R2 ring with 3,4,5-trimethoxy. Ortho-substitution activity
was poorer, followed by meta-substitution, and para-substitution ac-
tivity was the highest. The activity of compounds with double sub-
stitution was better than that of a single substitution. In different cell
lines, the structure-activity relationship was basically consistent with
the description in 2.2.1, but there were still some differences, which
may be due to different absorption capacity of the compounds. The
MTT assay was performed on human normal liver cells (L02) to test the
toxicity of the compounds. CC50 values were all above 100 μM, so the
toxicity of the compounds was relatively weak.
Scheme 1. General synthesis of compound c1-c24. Reagents and conditions: (i) substituted aniline, H2O, reflux, 6 h; (ii) Na2S2O4, THF/EtOH, 0 °C~r.t., 3 h; (iii)
substituted benzaldehyde, EtOH, reflux, 3 h.
3

Y.-L. Zhang, et al.
BioorganicChemistry92(2019)103219
Table 3
In vitro antiproliferative activity and cytotoxicity of compound c1-c24.
Compounds
GI50
SD (μM)
CC50
L02
SD (μM)
HeLa
HepG2
A549
MCF-7
c1
12.35
11.43
15.42
12.32
6.59
10.75
1.74
3.37
7.75
12.96
11.82
2.78
2.66
3.11
2.34
1.69
9.77
13.42
7.52
6.85
6.33
4.23
2.06
1.71
1.57
1.22
0.98
0.99
1.01
0.59
0.97
0.15
0.21
0.76
1.31
1.15
0.17
0.13
0.33
0.13
0.08
0.96
1.51
0.67
0.72
0.71
0.41
0.17
0.14
0.52
11.72
10.18
10.44
11.63
9.48
4.31
0.93
2.46
2.08
3.18
4.22
2.45
3.35
1.87
1.96
2.49
8.87
7.41
2.32
2.17
1.69
1.07
0.84
0.71
0.65
1.05
1.02
1.01
1.09
15.26
14.63
13.55
16.41
2.99
13.39
1.11
3.38
2.78
10.04
15.31
2.74
4.45
4.78
2.44
1.75
2.03
12.71
3.53
9.04
2.37
3.97
5.56
2.41
2.12
1.41
1.35
1.13
1.41
13.94
12.58
12.16
17.55
15.49
12.42
1.37
1.01
1.11
1.21
1.67
1.52
1.15
> 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
c2
c3
c4
c5
1.05
0.28
c6
0.31
0.09
0.20
0.16
0.30
0.37
0.13
0.21
0.16
0.20
0.16
0.27
0.71
0.13
0.19
0.18
0.11
0.11
0.07
0.09
1.21
c7
0.08
0.12
c8
0.34
0.21
5.48
0.47
0.52
c9
5.11
c10
c11
c12
c13
c14
c15
c16
c17
c18
c19
c20
c21
c22
c23
c24
CA-4
0.91
13.22
13.19
5.04
1.08
1.36
1.19
0.18
0.61
0.19
0.41
0.22
0.13
0.18
5.39
0.58
0.72
0.35
0.54
6.81
4.27
6.37
10.41
10.89
6.58
0.89
1.22
0.95
0.27
0.96
0.21
0.32
0.53
0.31
0.61
0.61
8.33
0.91
0.43
0.51
0.26
0.08
0.33
4.02
4.39
3.29
1.94
1.21
2.2.3. Cell cycle arrest
2.2.5. Cell apoptosis
Tubulin inhibitors could inhibit the polymerization of tubulin and
thus interfere with mitosis. Cell cycle assay was performed by PI
staining to verify whether compound c24 could prevent mitosis. HeLa
and HepG2 cells were treated with 0.4, 0.8, 1.6, 2.4 and 3.2 μM c24 for
24 h, respectively, forming a concentration gradient. Meanwhile, HeLa
and HepG2 cells were treated with c24 (0.8 μM) for gradient time (12 h,
24 h, 36 h and 48 h). It showed in Fig. 2 that compound c24 caused cell
cycle arrest into G2/M phase in concentration- and time-dependent
manners. With the increase of concentration of c24, the percentage of
cells in G2/M phase increased 2.5-fold as that of untreated cells. The
rates of HepG2 cells in G2/M were 19.4%, 25.8%, 32.2%, 37.2%, and
49.2%, respectively, after 24 h of treatment with 0.4, 0.8, 1.6, 2.4 and
3.2 μM c24. When HepG2 cells were treated with 0.8 μM c24 for 48 h,
the percentage of cells in G2/M phase increased from 14.9% to 34.2%,
compared with that untreated cells. The number of HeLa cells in G2/M
phase also increased with the increase of time and concentration.
Compared with untreated cells, the maximum number of HeLa cells in
G2/M phase increased by 2–3 folds. As the concentration and time
increased, the number of S-phase cells also increased, which may be
due to gradual apoptosis after long-term or high-concentration. Com-
pound c24 had a similar effect on HeLa to HepG2. Therefore, com-
pound c24 could block the cell in G2/M phase and affect the normal
mitosis of the cell.
Annexin V-FITC/PI assay was used to verify whether compound c24
could induce apoptosis of cancer cells. HeLa and HepG2 cells were
treated with 0.4, 0.8, 1.6, 2.4 and 3.2 μM c24 for 24 h, respectively, to
form a concentration gradient. Meanwhile, HeLa and HepG2 cells were
treated with 0.8 μM c24 for 12 h, 24 h, 36 h and 48 h, forming a time
gradient. The percentage of FITC positive cells, namely the sum of early
and late apoptotic cells, was used to represent the apoptotic rate. As
shown in Fig. 4, the apoptotic rate of HepG2 cells was concentration-
and time-dependent. With the increase of compound c24 concentration,
the apoptotic rate increased gradually up to 30%. Apoptotic cells were
20.85%, 22.87%, 26.66%, 28.16%, and 28.77%, respectively, after 24 h
of treatment with 0.4, 0.8, 1.6, 2.4 and 3.2 μM c24. When cells were
treated with the same concentration, apoptotic rate increased with the
increase in treatment time. After 48 h of treatment, the apoptotic rate
reached 25.88%, twice that of normal cells. Apoptotic rates of HepG2
cells treated by 0.8 μM c24 at 12 h, 24 h, 36 h and 48 h were 14.32%,
15.92%, 16.82%, 20.09 and 25.88%, respectively. Compared with
HepG2 cells, the apoptotic rate of HeLa cells treated by compound c24
was similar. After treatment with 0.8 μM c24 for 48 h and 3.2 μM c24
for 24 h, the number of apoptotic cells was approximately twice that of
untreated cells. Compound c24 could also induce dose-dependent and
time-dependent apoptosis of HeLa cells. In addition, an analysis of data
from both cell lines showed that the treatment with the compound for a
short period of time did not result in apoptosis or mild apoptosis. This
indicated that after compound treatment, cancer cells firstly underwent
mitotic arrest, which may lead to apoptosis. Therefore, compound c24
could induce apoptosis of tumor cells, similar to other tubulin depoly-
merization agents.
2.2.4. Cellular reactive oxygen species accumulation
Acute high concentrations of reactive oxygen species (ROS) in cells
could damage lipids, DNA and proteins in cells and disrupt normal cell
function. Cancer cells had low clearance efficiency and higher sensi-
tivity to ROS than normal cells. ROS was selectively harmful to cancer
cells, which could be used in treatment. To verify whether c24 could
lead to the increase of intracellular ROS content, flow cytometry assay
was performed with DCFH-DA staining. After the treatment of c24,
there was a right-forward peak shift in the FL1-H, indicating that the
content of intracellular ROS was increased in a time-dependent and
concentration-dependent manner (Fig. 3). ROS increased in HeLa cells
with the same trend. In conclusion, c24 could lead to ROS accumulation
in HeLa and HepG2 cells.
2.2.6. Morphological effect of microtubule
The effect of the compound on microtubule was observed by im-
munofluorescence staining. Colchicine (0.5 μM), paclitaxel (0.5 μM)
and compound c24 (1.6 and 3.2 μM) were used to treat HepG2 and
HeLa cells for 24 h, respectively. Microtubules were labeled with FITC,
showing green under confocal microscopy, and nuclei were labeled
with DAPI, showing blue. As shown in Fig. 5, microtubule in normal
cultured cells was fibrous and clearly defined, and normal spindle shape
4

Y.-L. Zhang, et al.
BioorganicChemistry92(2019)103219
Fig. 2. Compound c24 induced the cell cycle of HepG2 and HeLa arrest in the G2/M phase. Normal cultured cells were used as negative control (NC). (A and B)
HepG2 cells were exposed to 0.4, 0.8, 1.6, 2.4 and 3.2 μM c24 for 24 h. (C) HepG2 exposed to 0.8 μM c24 for 12 h, 24 h, 36 h and 48 h. (D and E) HeLa cells were
exposed to 0.4, 0.8, 1.6, 2.4 and 3.2 μM c24 for 24 h. (F) HeLa were exposed to 0.8 μM c24 for 12 h, 24 h, 36 h and 48 h. Cells were collected, stained with PI, and
analyzed by flow cytometry. Data shown are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.
could be observed. Colchicine and paclitaxel could cause morphological
changes and abnormalities of tubulin. Colchicine was a microtubule
depolymerization agent, which could inhibit the polymerization of
microtubule. Under confocal microscope, microtubule fibers were ob-
served to become short and dispersed from slender fibrous shape. Pa-
clitaxel was a promoter of tubulin polymerization, significantly en-
hancing the ability of tubulin polymerization, and then microtubule
fibers became long and thick. After treatment with compound c24, the
cells were normally elongated and rounded. After HepG2 and HeLa cells
were treated by compound c24, the changes of microtubule fibers were
similar to colchicine but different from paclitaxel. With the increase of
compound c24 concentration, the number of morphological changed or
apoptotic cells increased. At the same time, some features of mitotic
mutations, such as polykaryocyte and abnormal mitotic spindles, could
be observed. The results showed that compound c24 promoted the
depolymerization of tubulin and affected the normal mitosis of cells.
5

Y.-L. Zhang, et al.
BioorganicChemistry92(2019)103219
Fig. 3. Reactive oxygen species analysis of HepG2 and HeLa cells after compound c24 treatment. Normal cultured cells were used as negative control (NC). CA-4 was
treated as a positive agent. (A) HepG2 cells were exposed to 0.4, 0.8, 1.6, 2.4 and 3.2 μM c24 for 24 h. (B) HepG2 cells exposed to 0.8 μM c24 for 12 h, 24 h, 36 h and
48 h (C) HeLa cells were exposed to 0.4, 0.8, 1.6, 2.4 and 3.2 μM c24 for 24 h. (D) HeLa cells were exposed to 0.8 μM c24 for 12 h, 24 h, 36 h and 48 h. Cells were
collected, stained with DCFH-DA, and analyzed by flow cytometry. Data shown are representative of three independent experiments.
2.3. Computational simulation
positive drug, especially the alignment of 3,4,5-trimethoxy. One of the
3,4,5-trimethoxy groups in colchicine was linked to the protein residues
of tubulin (CYS B: 241) by a hydrogen bonding (distance = 1.98 Å)
(Fig. 6A and B). Similarly, in CA-4, the 3,4,5-trimethoxy group was
connected to the protein residue (CYS B: 241) by a hydrogen bond
(distance = 2.31 Å) (Fig. 6C and D). This hydrogen bond was one of
important interactions. The 3,4,5-trimethoxy group of c24 was also
linked to the protein residue (CYS B: 241) by hydrogen bonds (dis-
tance = 2.38 Å, Fig. 6E and F). Other functional groups of c24 were
connected to the microtubule protein in other ways, such as π-cation
interactions. These bonds could ensure that c24 bound tightly to tu-
bulin. Meanwhile, 3,4,5-trimethoxyphenyl of c24 was surrounded by
hydrophobic residues such as leucine, isoleucine and valine.
2.3.1. Molecular docking
Discovery Studio (DS, version 3.5) software was performed for
molecular docking to simulate the binding ability and mode of com-
pound c1-c24 and tubulin. On the basis of the crystal complex of col-
chicine and microtubule protein (PDB ID: 1SA0 [26]), colchicine at the
binding site of colchicine was replaced with compound c1-c24. Among
them, the most effective compound c24 showed the lowest interaction
energy −50.72 kcal/mol, while the CDocker energy of positive drug
CA-4 was −50.63 kcal/mol. The CDocker energy of compound c24 and
CA-4 are basically the same. Therefore, the binding ability of compound
c24 to colchicine binding sites is similar to that of positive drug CA-4.
Fig. 6 showed the modes of colchicine, CA-4 and c24 bound to tubulin.
There were many similarities between the binding of compound c24
to tubulin and the positive drug. 3,4,5-Trimethoxybenzene was an im-
portant functional group of colchicine and CA-4 as microtubule in-
hibitors. The docking direction of c24 was the same as that of the
2.3.2. 3D-QSAR model
Synthetic benzimidazole derivatives were analyzed by using the
QSAR program built into the DS software to obtain the systematic SAR
spectra as an anti-tubulin polymeric agent, so as to explore more
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Fig. 4. Compound c24 induced apoptosis of HepG2 and HeLa. Normal cultured cells were used as negative control (NC). (A and B) HepG2 cells were exposed to 0.4,
0.8, 1.6, 2.4 and 3.2 μM c24 for 24 h. (C) HepG2 exposed to 0.8 μM c24 for 12 h, 24 h, 36 h and 48 h. (D and E) HeLa cells were exposed to 0.4, 0.8, 1.6, 2.4 and
3.2 μM c24 for 24 h. (F) HeLa were exposed to 0.8 μM c24 for 12 h, 24 h, 36 h and 48 h. Cells were collected, stained with PI and FITC-Annexin V, and analyzed by
flow cytometry. Data shown are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.
effective and more selective anti-tumor drugs. The IC50 value was
converted into pIC50 value, and the training set and test set were ran-
domly screened by the random multi-molecular method. The training
set accounted for 83% of all compounds and the test set was the re-
maining four compounds. The difference between the converted pIC50
value and the predicted value by computer simulation was shown in
Fig. 7. The R2 value was 0.839, indicating a high degree of fitting be-
tween the calculated value and the predicted value. The 3D-QSAR
model could also be used to predict the biological activity of benzimi-
dazole derivatives.
was expected to decrease activity, and the blue regions were regions of
low electron density (positive charge), which was expected to increase
activity. In the van der Waals force grid, the green area refers to the
area where the expected spatial volume will increase activity, and the
yellow area refers to the area where the expected spatial volume will
decrease activity. Good anti-tubulin polymerization agents should have
a strong van der Waals force (green area) and strong polar groups (blue
area). Based on the 3D-QSAR model, further structural modification of
the compound could be carried out, laying a foundation for future re-
search.
All the molecules in the 3D-QSAR model are arranged on the elec-
trostatic potential grid (Fig. 8A) and van der Waals force grid (Fig. 8B)
in an isosurface manner. In the electrostatic potential grid, the red re-
gions were regions of high electron density (negative charge), which
3. Conclusion
1,2-Diarylbenzimidazole derivatives were synthesized and their
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BioorganicChemistry92(2019)103219
Fig. 5. Effects of colchicine (0.5 μM), paclitaxel (0.5 μM) and c24 (1.6 μM and 3.2 μM) on microtubules in HepG2 and HeLa cells (tubulins, green; nuclei, blue.)
activities were evaluated. The compound showed good proliferation
activity of anticancer cells in vitro in MTT assay. Among all compounds,
c24 showed the highest anti-proliferative activity, corresponding to
HeLa, HepG2, A549 and MCF-7, and the IC50 values were 1.71, 0.71,
2.41 and 0.94 μM, respectively. The activity of anti-tubulin poly-
merization was well demonstrated in tubulin inhibition experiments.
The microtubule inhibition value of c24 was 8.47 μM. MTT assay of
normal liver cells (L02) showed that the compounds were relatively less
cytotoxic. Cell cycle tests showed that, like most tubulin inhibitors [27],
compound c24 could block tumor cells in G2/M phase, then affecting
mitosis. In addition, confocal assay showed that c24 promoted the
depolymerization of tubulin, mitotic arrest, and formation of poly-
karyotic cells. According to the results of reactive oxygen species de-
tection, it was observed that c24 could effectively increase the content
of reactive oxygen species in cells. Combined with the results of
apoptosis assay, it was speculated that c24 could promote apoptosis
and might be related to the increase in the content of reactive oxygen
species [28]. In addition, the possibility of c24 binding to colchicine
binding sites of tubulin was studied by computer simulation of mole-
cular docking and the construction of 3D-QSAR model. Through the
analysis of the binding model of compound c24 to tubulin, it was ob-
served that it had some strong interactions with protein, which might
be helpful for its anti-microtubule polymerization. The study of the
binding relationship between the compounds and tubulin was helpful
for the further structural modification of the compounds and the de-
velopment of more effective tubulin polymerization inhibitors.
mixture was reflux for 6 h under stirring. Then the reaction complex
was cooled to room temperature, neutralized with NaHCO3, extracted
with ethyl acetate, dried with anhydrous sodium sulfate, and vacuum
evaporated to obtain the crude product of compound a1. Compound a1
was purified by silica gel column chromatography. Compounds a2-a5
were prepared by performing the method above.
4.1.2. General procedure for synthesis of b1-b5
In the ice bath conditions, a mixture of compound a1 (6.09 g,
20.0 mmol) and anhydrous ethanol (50 mL) in tetrahydrofuran
(100 mL) was added into sodium dithionite aqueous solution (55.71 g,
320 mmol). The mixture was stirred at room temperature for 3 h, then
saturated NaHCO3 solution and extracted with ethyl acetate. Collect the
organic layer, wash with saturated NaHCO3 solution, dry with anhy-
drous sodium sulfate and concentrate to obtain crude compound b1.
Compounds b2-b5 were prepared by performing the method above.
4.1.3. General procedure for synthesis of c1-c24
Compound b5 (0.09 g, 0.5 mmol) was dissolved in anhydrous
ethanol (5 mL), and 3,4,5-trimethoxybenzaldehyde (0.10 g, 0.5 mmol)
was added. After stirring and refluxing for 3 h, solid crude products of
compound c1 were filtered and recrystallized in the ethanol system.
Compounds c2-c24 were prepared by performing the method above.
4.1.4. Structural characterization of compounds
4.1.4.1. 3,4,5-Trimethoxy-N-(2-nitrophenyl)aniline (a1). Orange powder
(12.17 g, 40% yield), m.p. 182–183 °C. 1H NMR (600 MHz, DMSO‑d6) δ
9.31 (s, 1H, -NH-), 8.11 (dd, J = 8.5, 1.5 Hz, 1H, Ar-H), 7.51 (t,
J = 7.1 Hz, 1H, Ar-H), 7.24 (d, J = 8.1 Hz, 1H, Ar-H), 6.86 (t,
J = 7.2 Hz, 1H, Ar-H), 6.68 (s, 2H, Ar-H), 3.76 (s, 6H, -OCH3), 3.67 (s,
3H, -OCH3). MS (ESI): 305.11 [M + H]+; Anal. Calcd for C15H16N2O5: C,
59.21; H, 5.30; N, 9.21. Found: C, 59.07; H, 5.28; N, 9.23.
4. Experimental
4.1. Chemistry general
All chemicals used were purchased from Aladdin (Shanghai, China).
All reagents and solvents were of analytical or synthetic grade. Melting
points were taken on an X4 MP apparatus (Jingsong Corp, Shanghai,
China). All the 1H NMR spectra were registered in DMSO‑d6 on Bruker
AM 600 model spectrometer (Rhenistetten-Forchheim, Germany), op-
erating at 600 MHz and using tetramethylsilane (TMS) as internal
standard and DMSO as solvents. The progress of the reaction and purity
of the products were checked on TLC plates coated with Merck silica gel
60 GF254, and the spots were visualized in UV light (254 nm and
365 nm). 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.
4.1.4.2. N1-(3,4,5-trimethoxyphenyl)benzene-1,2-diamine (b1). Brown
powder (3.29 g, 60% yield), m.p. 189–190 °C. 1H NMR (600 MHz,
DMSO‑d6) δ 7.01 (d, J = 7.0 Hz, 1H, Ar-H), 6.97 (s, 1H, -NH-), 6.81
(t, J = 7.0 Hz, 1H, Ar-H), 6.72 (d, J = 7.9 Hz, 1H, Ar-H), 6.54 (t,
J = 7.5 Hz, 1H, Ar-H), 6.05 (s, 2H, Ar-H), 4.72 (s, 2H, -NH2), 3.65 (s,
6H, -OCH3), 3.56 (s, 3H, -OCH3). MS (ESI): 275.13 [M + H]+; Anal.
Calcd for C15H18N2O3: C, 65.68; H, 6.61; N, 10.21. Found: C, 65.51; H,
6.59; N, 10.23.
4.1.4.3. 1-Phenyl-2-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole
(c1). Yellow powder (0.14 g, 78% yield), m.p. 165–167 °C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.70 – 7.40 (m, 5H, Ar-H), 7.10 – 6.93 (m, 4H,
Ar-H), 6.77 (s, 1H, Ar-H), 6.50 (t, J = 7.2 Hz, 1H, Ar-H), 3.87 (s, 3H,
-OCH3), 3.71 (s, 3H, -OCH3), 3.58 (s, 3H, -OCH3). MS (ESI): 361.15
4.1.1. General procedure for synthesis of a1-a5
o-Fluoronitrobenzene (14.10 g, 100 mmol) and 3,4,5-trimethox-
yaniline (21.98 g, 120 mmol) were added into water (150 mL). The
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Fig. 6. The binding mode compound c24 is in the colchicine binding site (CBS) of tubulin (PDB ID: 1SA0). In CBS and colchicine (A) or CA-4 (C) or compound c24 (E)
(The interactive 3D plots are shown separately between. Only the interacting residues are shown. Hydrogen bonds are shown as green dashed lines and π-cations are
purple lines. A 2D plot of the interaction between CBS and colchicine (B) or CA-4 (D) or compound c24 (F) is shown.
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H), 6.72 (s, 1H, Ar-H), 6.65 (s, 1H, Ar-H), 6.63 (s, 1H, Ar-H), 6.60 (t,
J = 2.4 Hz, 3H, Ar-H), 3.82 (s, 3H, -OCH3), 3.69 (s, 3H, -OCH3), 3.60 (s,
3H, -OCH3). MS (ESI): 377.14 [M + H]+; Anal. Calcd for C22H20N2O4:
C, 70.20; H, 5.36; N, 7.44. Found: C, 70.15; H, 5.37; N, 7.45.
4.1.4.7. 2-Phenyl-1-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole
(c5). White powder (0.15 g, 81% yield), m.p. 171–173 °C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.23 – 7.15 (m, 6H, Ar-H), 6.95 (s, 2H, Ar-H),
6.68 (s, 1H, Ar-H), 6.58 (s, 1H, Ar-H), 6.27 (s, 1H, Ar-H), 3.85 (s, 3H,
-OCH3), 3.71 (s, 3H, -OCH3), 3.60 (s, 3H, -OCH3). MS (ESI): 361.15
[M + H]+; Anal. Calcd for C22H20N2O3: C, 73.32; H, 5.59; N, 7.77.
Found: C, 73.18; H, 5.57; N, 7.74.
4.1.4.8. 2-Methoxy-5-(1-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazol-
2-yl) phenol (c6). White powder (0.14 g, 67% yield), m.p. 191–193 °C.
1H NMR (600 MHz, DMSO‑d6) δ 8.57 (s, 1H, -OH), 7.94 (s, 1H, Ar-H),
6.67 – 6.37 (m, 6H, Ar-H), 6.05 (d, J = 6.0 Hz, 1H, Ar-H), 5.62 (d,
J = 6.0 Hz, 1H, Ar-H), 3.77 (s, 3H, -OCH3), 3.66 (s, 3H, -OCH3), 3.63 (s,
3H, -OCH3), 3.55 (s, 3H, -OCH3). 13C NMR (151 MHz, DMSO‑d6) δ:
157.92, 153.96, 148.73, 142.89, 138.02, 132.49, 131.55, 128.76,
123.65, 120.31, 118.62, 117.07, 116.58, 111.22, 105.91, 60.68,
56.72. MS (ESI): 407.15 [M + H]+; Anal. Calcd for C23H22N2O5: C,
67.97; H, 5.46; N, 6.89. Found: C, 68.13; H, 5.44; N, 6.87.
Fig. 7. Correlation between predicted pIC50 values of tubulin inhibitory activity
and experimental values, with 4 test compounds (orange) and 20 training
compounds (blue).
[M + H]+; Anal. Calcd for C22H20N2O3: C, 73.32; H, 5.59; N, 7.77.
Found: C, 73.57; H, 5.57; N, 7.75.
4.1.4.4. 2-Methoxy-5-(2-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazol-
1-yl) phenol (c2). White powder (0.13 g, 65% yield), m.p. 194–196 °C.
1H NMR (600 MHz, DMSO‑d6) δ 8.56 (s, 1H, -OH), 7.96 (s, 1H, Ar-H),
6.69– 6.51 (m, 4H, Ar-H), 6.47 (t, J = 7.8 Hz, 2H, Ar-H), 6.05 (s, 1H,
Ar-H), 5.69 (d, J = 1.2 Hz, 1H, Ar-H), 3.80 (s, 3H, -OCH3), 3.66 (s, 3H,
-OCH3), 3.53 (s, 3H, -OCH3), 3.36 (s, 3H, -OCH3). 13C NMR (151 MHz,
DMSO‑d6) δ: 152.35, 149.01, 146.42, 140.07, 135.30, 133.58, 129.92,
126.24, 125.97, 121.11, 119.62, 116.13, 112.36, 110.09, 61.51, 57.05.
MS (ESI): 407.15 [M + H]+; Anal. Calcd for C23H22N2O5: C, 67.97; H,
5.46; N, 6.89. Found: C, 67.72; H, 5.48; N, 6.91.
4.1.4.9. 2-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-benzo[d]
imidazole (c7). White powder (0.14 g, 71% yield), m.p. 178–180 °C. 1
H
NMR (600 MHz, DMSO‑d6) δ 7.13 – 7.01 (m, 5H, Ar-H), 6.73 – 6.70 (m,
3H, Ar-H), 6.61 (t, J = 7.0 Hz, 1H, Ar-H), 6.29 (s, 1H, Ar-H), 3.84 (s,
3H, -OCH3), 3.69 (s, 3H, -OCH3), 3.63 (s, 3H, -OCH3), 3.57 (s, 3H,
-OCH3). 13C NMR (151 MHz, DMSO‑d6) δ: 153.28, 149.69, 146.71,
142.07, 138.85, 131.96, 130.82, 126.91, 122.51, 119.13, 114.06,
108.58, 102.35, 61.02, 58.04. MS (ESI): 391.16 [M + H]+; Anal.
Calcd for C23H22N2O4: C, 70.75; H, 5.68; N, 7.18. Found: C, 70.92; H,
5.66; N, 7.16.
4.1.4.5. 1-(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-1H-benzo[d]
imidazole (c3). White powder (0.15 g, 77% yield), m.p. 171–173 °C. 1
H
4.1.4.10. 3-(1-(3,4,5-Trimethoxyphenyl)-1H-benzo[d]imidazol-2-yl)
phenol (c8). White powder (0.15 g, 80% yield), m.p. 172–174 °C. 1H
NMR (600 MHz, DMSO‑d6) δ 9.62 (s, 1H, -OH), 7.75 (t, J = 4.5 Hz, 1H,
Ar-H), 7.31 – 6.80 (m, 7H, Ar-H), 6.75 (s, 2H, Ar-H), 3.75 (s, 3H,
-OCH3), 3.69 (s, 6H, -OCH3). MS (ESI): 377.14 [M + H]+; Anal. Calcd
for C22H20N2O4: C, 70.20; H, 5.36; N, 7.44. Found: C, 70.41; H, 5.34; N,
7.45.
NMR (600 MHz, DMSO‑d6) δ 7.21 – 7.14 (m, 4H, Ar-H), 6.95 (s, 2H, Ar-
H), 6.71 (s, 2H, Ar-H), 6.48 (s, 2H, Ar-H), 3.85 (s, 3H, -OCH3), 3.76 (s,
3H, -OCH3), 3.60 (s, 3H, -OCH3), 3.51 (s, 3H, -OCH3). MS (ESI): 391.16
[M + H]+; Anal. Calcd for C23H22N2O4: C, 70.75; H, 5.68; N, 7.18.
Found: C, 70.89; H, 5.67; N, 7.19.
4.1.4.6. 3-(2-(3,4,5-Trimethoxyphenyl)-1H-benzo[d]imidazol-1-yl)
phenol (c4). White powder (0.14 g, 71% yield), m.p. 163–165 °C. 1H
NMR (600 MHz, DMSO‑d6) δ 9.13 (s, 1H, -OH), 7.09 – 6.98 (m, 4H, Ar-
4.1.4.11. 2-(4-Bromophenyl)-1-(3,4,5-trimethoxyphenyl)-1H-benzo[d]
imidazole (c9). White powder (0.16 g, 72% yield), m.p. 176–177 °C. 1
H
Fig. 8. (A) The isosurface of the coefficients on the electrostatic potential grid in the 3D-QSAR model. (B) The isosurface of the coefficients on the van der Waals grid
in the 3D-QSAR model.
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BioorganicChemistry92(2019)103219
NMR (600 MHz, DMSO‑d6) δ 7.41 (d, J = 8.5 Hz, 2H, Ar-H), 7.18 (d,
J = 8.4 Hz, 2H, Ar-H), 7.13 (s, 1H, Ar-H), 7.01 (s, 2H, Ar-H), 6.73 (s,
1H, Ar-H), 6.62 (t, J = 6.6 Hz, 1H, Ar-H), 6.28 (s, 1H, Ar-H), 3.84 (s,
3H, -OCH3), 3.69 (s, 3H, -OCH3), 3.60 (s, 3H, -OCH3). MS (ESI): 439.06
[M + H]+; Anal. Calcd for C22H19BrN2O3: C, 65.15; H, 4.36; N, 6.38.
Found: C, 64.89; H, 4.35; N, 6.36.
6.57 (s, 1H, Ar-H), 3.82 (s, 3H, -OCH3), 3.68 (s, 3H, -OCH3), 3.60 (s, 3H,
-OCH3), 2.14 (s, 3H, -CH3). MS (ESI): 375.16 [M + H]+; Anal. Calcd for
C
23H22N2O3: C, 73.78; H, 5.92; N, 7.48. Found: C, 73.79; H, 5.94; N,
7.47.
4.1.4.19. 2-(4-(Methylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)-1H-benzo
[d]imidazole (c17). White powder (0.16 g, 81% yield), m.p.
187–189 °C. 1H NMR (600 MHz, DMSO‑d6) δ 7.15 – 7.02 (m, 6H, Ar-
H), 6.73 (s, 1H, Ar-H), 6.62 (t, J = 7.2 Hz, 2H, Ar-H), 6.30 (s, 1H, Ar-
H), 3.84 (s, 3H, -OCH3), 3.70 (s, 3H, -OCH3), 3.59 (s, 3H, -OCH3), 2.36
4.1.4.12. 2-(3-Bromophenyl)-1-(3,4,5-trimethoxyphenyl)-1H-benzo[d]
imidazole (c10). White powder (0.19 g, 85% yield), m.p. 176–178 °C.
1H NMR (600 MHz, DMSO‑d6) δ 7.23 – 7.10 (m, 5H, Ar-H), 6.96 (s, 2H,
Ar-H), 6.51 (s, 1H, Ar-H), 6.40 (s, 1H, Ar-H), 6.22 (s, 1H, Ar-H), 3.81 (s,
3H, -OCH3), 3.69 (s, 3H, -OCH3), 3.60 (s, 3H, -OCH3). MS (ESI): 439.06
[M + H]+; Anal. Calcd for C22H19BrN2O3: C, 65.15; H, 4.36; N, 6.38.
Found: C, 64.98; H, 4.37; N, 6.39.
(s, 3H, -SCH3). MS (ESI): 407.14 [M + H]+
; Anal. Calcd for
C23H22N2O3S: C, 67.96; H, 5.46; N, 6.89. Found: C, 67.69; H, 5.44; N,
6.87.
4.1.4.20. 2-(4-Nitrophenyl)-1-(3,4,5-trimethoxyphenyl)-1H-benzo[d]
imidazole (c18). White powder (0.17 g, 83% yield), m.p. 170–172 °C.
1H NMR (600 MHz, DMSO‑d6) δ 8.07 (d, J = 8.8 Hz, 2H, Ar-H), 7.40 (d,
J = 4.7 Hz, 2H, Ar-H), 6.89 (s, 1H, Ar-H), 6.77 (s, 1H, Ar-H), 6.70 (s,
1H, Ar-H), 6.58 (s, 1H, Ar-H), 6.49 (s, 1H, Ar-H), 6.03 (s, 1H, Ar-H),
3.82 (s, 3H, -OCH3), 3.68 (s, 3H, -OCH3), 3.61 (s, 3H, -OCH3). MS (ESI):
406.13 [M + H]+; Anal. Calcd for C22H19N3O5: C, 65.18; H, 4.72; N,
10.37. Found: C, 65.39; H, 4.71; N, 10.33.
4.1.4.13. 2-(2-Bromophenyl)-1-(3,4,5-trimethoxyphenyl)-1H-benzo[d]
imidazole (c11). White powder (0.19 g, 86% yield), m.p. 177–179 °C.
1H NMR (600 MHz, DMSO‑d6) δ 7.52 (s, 1H, Ar-H), 7.41 (s, 3H, Ar-H),
7.18 (s, 2H, Ar-H), 7.13 (s, 1H, Ar-H), 6.91 (s, 1H, Ar-H), 6.73 (s, 1H,
Ar-H), 6.61 (d, J = 6.0 Hz, 1H, Ar-H), 3.83 (s, 3H, -OCH3), 3.70 (s, 3H,
-OCH3), 3.60 (s, 3H, -OCH3). MS (ESI): 439.06 [M + H]+; Anal. Calcd
for C22H19BrN2O3: C, 65.15; H, 4.36; N, 6.38. Found: C, 65.38; H, 4.35;
N, 6.36.
4.1.4.21. 2-(4-Fluorophenyl)-1-(3,4,5-trimethoxyphenyl)-1H-benzo[d]
imidazole (c19). White powder (0.14 g, 75% yield), m.p. 167–169 °C.
1H NMR (600 MHz, DMSO‑d6) δ 7.23 (dd, J = 7.9, 5.6 Hz, 2H, Ar-H),
7.03 – 6.97 (m, 4H, Ar-H), 6.69 (s, 1H, Ar-H), 6.59 (t, J = 7.0 Hz, 2H,
Ar-H), 6.28 (s, 1H, Ar-H), 3.82 (s, 3H, -OCH3), 3.68 (s, 3H, -OCH3), 3.58
(s, 3H, -OCH3). 13C NMR (151 MHz, DMSO‑d6) δ: 153.90, 149.13,
141.85, 138.16, 132.27, 126.27, 123.08, 118.37, 114.25, 106.44,
4.1.4.14. 2-(3-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-benzo[d]
imidazole (c12). White powder (0.14 g, 72% yield), m.p. 179–181 °C.
1H NMR (600 MHz, DMSO‑d6) δ 7.15 (s, 1H, Ar-H), 7.08 – 7.00 (m, 4H,
Ar-H), 6.71 (t, J = 7.2 Hz, 2H, Ar-H), 6.64 – 6.59 (m, 2H, Ar-H), 6.30 (s,
1H, Ar-H), 3.84 (s, 3H, -OCH3), 3.70 (s, 3H, -OCH3), 3.64 (s, 3H,
-OCH3), 3.61 (s, 3H, OCH3). MS (ESI): 391.16 [M + H]+; Anal. Calcd
for C23H22N2O4: C, 70.75; H, 5.68; N, 7.18. Found: C, 70.48; H, 5.67; N,
7.20.
60.80, 57.10. MS (ESI): 379.14 [M + H]+
; Anal. Calcd for
C
22H19FN2O3: C, 69.83; H, 5.06; N, 7.40. Found: C, 69.62; H, 5.08; N,
7.09.
4.1.4.15. 2-(2-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-benzo[d]
imidazole (c13). White powder (0.14 g, 70% yield), m.p. 175–177 °C.
1H NMR (600 MHz, DMSO‑d6) δ 7.62 (s, 2H, Ar-H), 7.08 – 7.00 (m, 3H,
Ar-H), 6.71 (t, J = 7.2 Hz, 2H, Ar-H), 6.64 – 6.59 (m, 2H, Ar-H), 6.15 (s,
1H, Ar-H), 3.84 (s, 3H, -OCH3), 3.70 (s, 3H, -OCH3), 3.65 (s, 3H,
-OCH3), 3.59 (s, 3H, -OCH3). MS (ESI): 391.16 [M + H]+; Anal. Calcd
for C23H22N2O4: C, 70.75; H, 5.68; N, 7.18. Found: C, 71.00; H, 5.69; N,
7.17.
4.1.4.22. 2-(4-Chlorophenyl)-1-(3,4,5-trimethoxyphenyl)-1H-benzo[d]
imidazole (c20). White powder (0.14 g, 73% yield), m.p. 173–175 °C.
1H NMR (600 MHz, DMSO‑d6) δ 7.26 – 7.23 (m, 4H, Ar-H), 7.10 (s, 1H,
Ar-H), 7.00 (s, 2H, Ar-H), 6.71 (s, 1H, Ar-H), 6.61 (t, J = 6.6 Hz, 1H, Ar-
H), 6.28 (s, 1H, Ar-H), 3.83 (s, 3H, -OCH3), 3.69 (s, 3H, -OCH3), 3.60 (s,
3H, -OCH3). MS (ESI): 395.11 [M + H]+
;
Anal. Calcd for
C22H19ClN2O3: C, 66.92; H, 4.85; N, 7.09. Found: C, 67.15; H, 4.84;
N, 7.08.
4.1.4.16. 4-(1-(3,4,5-Trimethoxyphenyl)-1H-benzo[d]imidazol-2-yl)
phenol (c14). White powder (0.13 g, 70% yield), m.p. 164–166 °C. 1H
NMR (600 MHz, DMSO‑d6) δ 9.49 (s, 1H, -OH), 9.16 (s, 1H, Ar-H), 7.08
– 6.98 (m, 4H, Ar-H), 6.71 (s, 1H, Ar-H), 6.62 – 6.53 (m, 3H, Ar-H),
6.27 (s, 1H, Ar-H), 3.83 (s, 3H, -OCH3), 3.68 (s, 3H, -OCH3), 3.56 (s, 3H,
-OCH3). MS (ESI): 377.14 [M + H]+; Anal. Calcd for C22H20N2O4: C,
70.20; H, 5.36; N, 7.44. Found: C, 70.41; H, 5.34; N, 7.42.
4.1.4.23. 4-(1-(3,4,5-Trimethoxyphenyl)-1H-benzo[d]imidazol-2-yl)
benzene-1,3-diol (c21). White powder (0.14 g, 73% yield), m.p.
183–185 °C. 1H NMR (600 MHz, DMSO‑d6) δ 8.80 (s, 1H, -OH), 8.53
(s, 1H, -OH), 7.08 – 7.03 (m, 3H, Ar-H), 6.84 (s, 1H, Ar-H), 6.81 (s, 1H,
Ar-H), 6.80 (s, 1H, Ar-H), 6.72 (s, 1H, Ar-H), 6.46 (s, 1H, Ar-H), 6.39 (s,
1H, Ar-H), 3.83 (s, 3H, -OCH3), 3.71 (s, 3H, -OCH3), 3.59 (s, 3H,
-OCH3). MS (ESI): 393.14 [M + H]+; Anal. Calcd for C22H20N2O5: C,
67.34; H, 5.14; N, 7.14. Found: C, 67.26; H, 5.16; N, 7.12.
4.1.4.17. 2-(p-Tolyl)-1-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole
(c15). White powder (0.14 g, 77% yield), m.p. 184–186 °C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.07 – 7.04 (m, 3H, Ar-H), 6.95 (d, J = 7.8 Hz,
1H, Ar-H), 6.90 (d, J = 7.8 Hz, 1H, Ar-H), 6.66 (s, 2H, Ar-H), 6.57 (s,
2H, Ar-H), 6.22 (s, 1H, Ar-H), 3.83 (s, 3H, -OCH3), 3.69 (s, 3H, -OCH3),
3.58 (s, 3H, -OCH3), 2.15 (s, 3H, -CH3). 13C NMR (151 MHz, DMSO‑d6)
δ: 152.33, 149.52, 146.41, 140.29, 137.54, 136.11, 134.96, 133.87,
124.01, 123.71, 120.86, 119.25, 115.61, 113.03, 111.55, 108.09,
61.51, 54.12, 21.49. MS (ESI): 375.16 [M + H]+; Anal. Calcd for
4.1.4.24. 4-(1-(3,4,5-Trimethoxyphenyl)-1H-benzo[d]imidazol-2-yl)
benzene-1,2-diol (c22). White powder (0.16 g, 81% yield), m.p.
189–191 °C. 1H NMR (600 MHz, DMSO‑d6) δ 8.70 (s, 1H, -OH), 8.69
(s, 1H, -OH), 7.89 – 7.79 (m, 2H, Ar-H), 7.71 (d, J = 6.6 Hz, 1H, Ar-H),
7.64 – 7.41 (m, 3H, Ar-H), 7.10 (s, 1H, Ar-H), 6.94 (d, J = 8.6 Hz, 1H,
Ar-H), 6.72 (t, J = 12.5 Hz, 1H, Ar-H), 3.76 (s, 3H, -OCH3), 3.71 (s, 3H,
-OCH3), 3.59 (s, 3H, -OCH3). MS (ESI): 393.14 [M + H]+; Anal. Calcd
for C22H20N2O5: C, 67.34; H, 5.14; N, 7.14. Found: C, 67.42; H, 5.12; N,
7.16.
C
23H22N2O3: C, 73.78; H, 5.92; N, 7.48. Found: C, 73.92; H, 5.90; N,
7.46.
4.1.4.18. 2-(m-Tolyl)-1-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole
(c16). White powder (0.14 g, 76% yield), m.p. 167–169 °C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.27 – 7.03 (m, 2H, Ar-H), 7.00 (d, J = 1.2 Hz,
2H, Ar-H), 6.91 (s, 2H, Ar-H), 6.90 (s, 2H, Ar-H), 6.76 (s, 1H, Ar-H),
4.1.4.25. 2-(2,4-Dimethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-benzo
[d]imidazole (c23). White powder (0.14 g, 68% yield), m.p.
191–193 °C. 1H NMR (600 MHz, DMSO‑d6) δ 7.41 (s, 1H, Ar-H), 7.00
(s, 1H, Ar-H), 6.94 (s, 1H, Ar-H), 6.74 (s, 1H, Ar-H), 6.61 (d, J = 2.3 Hz,
11

Y.-L. Zhang, et al.
BioorganicChemistry92(2019)103219
1H, Ar-H), 6.48 (d, J = 2.3 Hz, 1H, Ar-H), 6.32 – 6.29 (m, 1H, Ar-H),
6.27 (dd, J = 8.6, 2.3 Hz, 2H, Ar-H), 3.91 (s, 3H, -OCH3), 3.80 (s, 3H,
-OCH3), 3.70 (s, 3H, -OCH3), 3.61 (s 3H, -OCH3), 3.59 (s 3H, -OCH3).
13C NMR (151 MHz, DMSO‑d6) δ: 152.49, 150.91, 146.24, 146.05,
140.55, 137.47, 136.67, 134.26, 133.62, 120.49, 120.12, 119.11,
118.73, 117.45, 115.62, 113.79, 111.77, 97.70, 61.24, 55.78. MS
(ESI): 421.17 [M + H]+; Anal. Calcd for C24H24N2O5: C, 68.56; H,
5.75; N, 6.66. Found: C, 68.81; H, 5.74; N, 6.65.
and stained with 1 mL of DCFH-DA solution (in DMEM) for 20 min in
the cell incubator at 37 °C. Then cells were collected and washed twice
with fresh DMEM. The samples were tested on BD Accuri C6 flow cy-
tometry (BD, USA) and the data was analyzed with Flowjo 7.6.1
(FlowJo, USA).
4.2.5. Cell apoptosis assay
Cells were seeded into the 6-well plate (500,000 cells/mL) and
treated with compound c24 under various conditions (concentration
and time) respectively. Cells were collected, washed twice on ice and
resuspended in 500 μL staining buffer. Then it was stained with 5 μL
AnnexinV-FITC and 5 μL PI in dark for 10 min. The samples were tested
on BD Accuri C6 flow cytometry (BD, USA) and the data was analyzed
with Flowjo 7.6.1 (FlowJo, USA).
4.1.4.26. 2-(3,4-Dimethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-benzo
[d]imidazole (c24). White powder (0.14 g, 67% yield), m.p.
190–192 °C. 1H NMR (600 MHz, DMSO‑d6) δ 7.36 (d, J = 6.5 Hz, 1H,
Ar-H), 7.23 – 7.17 (m, 1H, Ar-H), 7.08 – 7.01 (m, 2H, Ar-H), 6.72 – 6.62
(m, 3H, Ar-H), 6.33 (dd, J = 8.3, 1.9 Hz, 2H, Ar-H), 3.84 (d, J = 6.5 Hz,
3H, -OCH3), 3.70 (s, 3H, -OCH3), 3.67 (s, 3H, -OCH3), 3.61 (d,
J = 3.2 Hz, 6H, -OCH3). 13C NMR (151 MHz, DMSO‑d6) δ 157.57,
153.97, 152.33, 142.84, 137.88, 137.63, 132.59, 131.57, 129.76,
123.64, 123.05, 120.21, 119.66, 117.03, 116.45, 111.31, 105.74,
4.2.6. Immunofluorescence
Cells were seeded into the 6-well plate (500,000 cells/mL) in which
a glass cover was pre-positioned, and treated with compounds under
various conditions (concentration and time) respectively. Cells were
uniformly distributed in six-well plates with glass covers. After treat-
ment, cells were fixed with fixed with 4% paraformaldehyde for
0.5–1 h, washed with PBS. After permeated with 1% Triton X-100 so-
lution for 30 min, cells were incubated with immune dyeing sealing for
1 h. Then cells were incubated with Alexa Fluor 488 labeled tubulin
antibody (1:500) at 4 °C overnight in the dark. Then cells were washed
and stained by DAPI for 10 min, avoiding light. The cover glasses were
fixed with anti-fluorescence quenching agent on the glass slide. Images
were collected using the confocal laser scanning microscope (Olympus,
Japan) and processed in Photoshop (Adobe, USA).
60.69, 56.62. MS (ESI): 421.17 [M + H]+
;
Anal. Calcd for
C24H24N2O5: C, 68.56; H, 5.75; N, 6.66. Found: C, 68.31; H, 5.73; N,
6.67.
4.2. Biological assays
4.2.1. MTT assay
Human cervical carcinoma (HeLa), human liver cancer cell
(HepG2), human non-small cell lung carcinoma (A549), human breast
cancer cell (MCF-7) and human normal liver cells (L02) were cultured
in DMEM containing 10% fetal bovine serum (FBS) and 1% Penicillin-
Streptomycin in 5% CO2 atmosphere at 37 °C. Seed the cells in the
logarithmic phase into 96-well plates (5000–10,000 cells per well).
After 8–12 h, the compounds of various concentration (0.01, 0.1, 1, 10
and 100 μM) were added into the 96-wells plates. After 48 h, 10 μL MTT
(5 mg mL−1) were added to each well to incubate 4 h. Then discard the
media and add DMSO (150 μL) to dissolve formazan completely on a
shaker for 10 min. The absorbance was measured at 570 nm wavelength
by microplate reader (Tecan, Switzerland). Three replicate wells were
used for each concentration and each independent experiment was
conducted at least three times. The GI50 was the compound con-
centration required to inhibit cell proliferation by 50% and the CC50
was the compound concentration to cause cytotoxic by 50%.
4.3. Docking simulations
The software Discovery Studio (version 3.5) was used to optimize
the molecular structure and microtubule protein, and DS-CDOCKER
was selected for simulated docking. Compound c24 and positive drug
CA-4 molecules were simulated in the three-dimensional X-ray struc-
ture (PDB ID: 1SA0) of the microtubule protein. The whole tubulin
complex was defined as the receptor, and the interaction site was se-
lected according to the binding position of colchicine ligand (CN2700).
During the molecular docking process, the CN2700 molecule was re-
moved and replaced by c24. All binding water and ligands in the
docking results were removed from the protein and labeled with polar
hydrogen. The types of interactions between proteins and ligands were
analyzed, and the different binding bonds were labeled.
4.2.2. Tubulin polymerization assay
Microtubule was purified from fresh pig brain according to the
previous method [29] with modification. Compounds of various con-
centrations (from 0.1 to 50 μM) were plated into 96-well plates (10 μL/
well). Purified tubulin in G-PEM buffer was prepared freshly on ice and
added into plates. Then the mixture was monitored by microplate
reader at 350 nm at 37 °C. IC50 value was the concentration of the
compound when the inhibitory rate of microtubule assembly by 50%
after incubation for 20 min.
4.4. 3D-QSAR modeling
Using the software Discovery Studio (version 3.5), the ligand-based
3D-QSAR method was used for processing. The IC50 value of the in-
hibitory protein was converted to obtain the corresponding pIC50 value.
All definitions of descriptors could be observed with the help of DS 3.5
software and they were calculated by QSAR protocol of DS 3.5. The
coordination conformation of each molecule selects the CDOCKER re-
sult with the lowest interaction energy. The results of 3D-QSAR mod-
eling can be evaluated according to the correlation coefficient of cross-
validation. The random correlation risk was tested by scrambling (Y
scrambling). The ability of the compound to inhibit tubulin was ran-
domly reordered 20 times, and leave-one-out verification test was
conducted respectively. The 3D-QSAR model was also verified by the
test set, in which the compounds were not included in the training set.
4.2.3. Cell cycle analysis
Cells were seeded into the 6-well plate (500,000 cells/mL) and
treated with compound c24 under various conditions (concentration
and time) respectively. Then cells were collected, washed twice on ice
and fixed with 70% ethanol at 4 °C overnight. Centrifugate and discard
the supernatant. Cells were treated with RNAse (100 μL) for 30 min and
stained with 400 μL PI in dark. The samples were tested on BD Accuri
C6 flow cytometry (BD, USA) and the data was analyzed with Flowjo
7.6.1 (FlowJo, USA).
Acknowledgement
4.2.4. Reactive oxygen detection
Cells were seeded into the 6-well plate (500,000 cells/mL) and
treated with compound c24 under various conditions (concentration
and time) respectively. Then cells were collected, washed twice on ice
This work was supported by the Public Science and Technology
Research Funds Projects of Ocean (State Oceanic Administration,
People’s Republic of China, No. 201505023).
12

Y.-L. Zhang, et al.
BioorganicChemistry92(2019)103219
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