Design, synthesis and anti-tumor activity of novel benzimidazole-chalcone hybrids as non-intercalative topoisomerase II catalytic inhibitors

Article abstract of DOI:10.3390/molecules25143180

Chemical diversification of type II topoisomerase (Topo II) inhibitors remains indispensable to extend their anti-tumor therapeutic values which are limited by their side effects. Herein, we designed and synthesized a novel series of benzimidazole-chalcone hybrids (BCHs). These BCHs showed good inhibitory effect in the Topo II mediated DNA relaxation assay and anti-proliferative effect in 4 tumor cell lines. 4d and 4n were the most potent, with IC₅₀values less than 5 μM, superior to etoposide. Mechanistic studies indicated that the BCHs functioned as non-intercalative Topo II catalytic inhibitors. Moreover, 4d and 4n demonstrated versatile properties against tumors, including inhibition on the colony formation and cell migration, and promotion of apoptosis of A549 cells. The structure-activity relationship and molecular docking analysis suggested possible contribution of the chalcone motif to the Topo II inhibitory and anti-proliferative potency. These results indicated that 4d and 4n could be promising lead compounds for further anti-tumor drug research.

Full text of DOI:10.3390/molecules25143180

molecules
Article
Design, Synthesis and Anti-Tumor Activity of Novel
Benzimidazole-Chalcone Hybrids as
Non-Intercalative Topoisomerase II
Catalytic Inhibitors
1
,
1
1
2
1
1,
Wei Zhou * , Wenjin Zhang , Yi Peng , Zhi-Hong Jiang
, Lanyue Zhang and Zhiyun Du *
1
School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology,
Guangzhou 510006, China; wenjinzhang8@163.com (W.Z.); doris_py@163.com (Y.P.);
zhanglanyue@gdut.edu.cn (L.Z.)
State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in
Medicine and Health, Macau University of Science and Technology, Avenida Wai Long, Taipa,
Macau 999078, China; zhjiang@must.edu.mo
2
*
Correspondence: zhou_wei@gdut.edu.cn (W.Z.); zhiyundu@foxmail.com (Z.D.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 17 April 2020; Accepted: 9 July 2020; Published: 12 July 2020
Abstract: Chemical diversification of type II topoisomerase (Topo II) inhibitors remains indispensable
to extend their anti-tumor therapeutic values which are limited by their side effects. Herein,
we designed and synthesized a novel series of benzimidazole-chalcone hybrids (BCHs). These BCHs
showed good inhibitory effect in the Topo II mediated DNA relaxation assay and anti-proliferative
effect in 4 tumor cell lines. 4d and 4n were the most potent, with IC50 values less than 5 µM, superior
to etoposide. Mechanistic studies indicated that the BCHs functioned as non-intercalative Topo II
catalytic inhibitors. Moreover, 4d and 4n demonstrated versatile properties against tumors, including
inhibition on the colony formation and cell migration, and promotion of apoptosis of A549 cells.
The structure-activity relationship and molecular docking analysis suggested possible contribution of
the chalcone motif to the Topo II inhibitory and anti-proliferative potency. These results indicated
that 4d and 4n could be promising lead compounds for further anti-tumor drug research.
Keywords: type II topoisomerase; benzimidazole; chalcone; anti-tumor
1
. Introduction
Type II topoisomerase (Topo II) is a predominant enzyme involved in DNA replication,
recombination, and repair [
a prominent target for anticancer drug research [
such as etoposide and doxorubicin, have been clinically applied as anti-cancer drugs. However,
possible serious risk of drug resistance and side effects limits their wide therapeutic use [ ]. Topo II
inhibitors can be classified into Topo II poisons and Topo II catalytic inhibitors according to their
different mechanism of action [ ]. The former type stabilizes the covalent DNA-Topo II cleavable
complexes while the latter type inhibits any other step in the catalytic cycle. Accumulating evidences
have suggested that Topo II catalytic inhibitors may be more potent and less toxic than poisons [ ].
10]. Therefore, the research
1,
2]. Due to its important roles in cell life cycle, Topo II has become
3,4]. In the last decades, typical Topo II inhibitors,
5,6
7,8
2
Moreover, catalytic inhibitors could resist possible side effects of poisons [9,
for novel Topo II catalytic inhibitor is of great significance.
Benzimidazoles have various of biological activities, such as anti-bacterial, anti-inflammatory,
anti-tumor, and so on [11 15]. Studies have shown that the benzimidazole scaffold rings play a critical
role in the antitumor activity [16 17]. In recent years, the modification of benzimidazole rings has
–
,
Molecules 2020, 25, 3180; doi:10.3390/molecules25143180
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3180
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become one of the hottest topics in the development of anti-cancer agents. Several benzimidazole
derivatives have been reported as new Topo II inhibitors ( and 3, Figure 1) [18 19]. Mechanistic
and molecular docking studies have revealed that benzimidazole-containing derivatives act as Topo II
1,
2
,
catalytic inhibitors by blocking the adenosine triphosphate (ATP) binding site of the enzyme [16,18,20].
Figure 1. Design of benzimidazole-chalcone hybrids as topoisomerase II inhibitors.
Chalcone derivatives have also been extensively investigated for their various physiological
activities, such as anti-cancer, anti-oxidant, anti-inflammatory, anti-infective activities [21 22]. Naturally
occurring or synthetic chalcone derivatives ( and , Figure 1) have attracted much attention not only
,
4,
5
6
for their good tumor inhibitory activity, but also for their relatively safety for healthy tissues [23–27].
Based on the facts above, we focused on Topo II inhibitors, and designed a series of benzimidazole
derivatives cooperated with the chalcone fragment using a classic pharmacophore hybridization strategy
(
Figures 1 and 2) [28,29]. Compounds were synthesized and evaluated for Topo II inhibitory activity,
and their mechanism of action were also explored. The results indicated that benzimidazole-chalcone
hybrids (BCHs) were a class of non-embedded Topo II catalytic inhibitors. Moreover, most of the
BCHs were found to not only effectively inhibit catalytic function of Topo II, but also suppress cell
proliferation (HepG2, A549, LNCaP and MG63) and induce cell apoptosis.
Figure 2. Chemical structures of designed Topo II inhibitors 4a–4u.

Molecules 2020, 25, 3180
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2
. Results and Discussion
2
.1. Chemistry
A series of benzimidazole-chalcone hybrids 4a
–
4u was synthesized as described in Scheme 1.
(
1H-Benzo[d]imidazol-2-yl) methanol (1a) was obtained by refluxing o-phenylenediamine with glycolic
acid in hydrochloric acid. The substituted-(1-benzyl-1H-benzo[d]imidazol-2-yl) methanols 2a 2b were
synthesized with 47-88% yields using 1a and appropriate benzyl bromides in the presence of K CO .
,
2
3
Subsequently, substituted-1-benzyl-1H-benzo[d]imidazole-2-carbaldehydes 3a
,
3b were obtained in
5
6–78% yields by oxidation of the corresponding primary alcohols with Dess–Martin reagent. The target
compounds 4a–4u were finally prepared in 70–86% yields from 3a or 3b by using the Claisen-Schmidt
reaction in the presence of appropriate acetophenones. Chemical structures of the target compounds
4
a–4u are presented in Figure 2.
◦
a) glycolic acid, 4N HCl, 100 C, 6 h;
) Dess-Martin periodinane, DCM, 0 C, 1 h; (d) ArCOCH3,
Scheme 1. The synthetic route of 4a
–
4u. Reagents and conditions: (
◦
(b
) benzyl bromides, K CO , DMF, rt, 24 h; (
c
2
3
EtOH, 10% NaOH, rt, 1 h.
2
.2. BCHs as Potent Topo II Inhibitors
The Topo II-mediated DNA relaxation assay was performed to evaluate the inhibitory effect of
BCHs against the catalytic activity of Topo II. Etoposide was used as the comparative control and
pBR322 DNA plasmid as the substrate [30]. BCHs were supposed to inhibit DNA relaxation catalyzed
by Topo II. The decrease of pBR322 and the increase of relaxed DNA were indicators of catalytic
activity of Topo II. According to the western blot and semi-quantification results (Figure 3), it was
found that most BCHs showed strong Topo II inhibition activity at 50
, and 4q showed relatively stronger inhibitory potency at 10 L (Figure 3) than the others. Generally,
compounds with an electron-donating group such as (-C(CH ) , -CH CH , -CH , -OCH ) at R position
µL (Figure 3), while 4d, 4n, 4o,
4
p
µ
3
2
2
3
3
3
2
showed stronger inhibitory activity on Topo II than that of compounds with an electron-withdrawing
group (-NO , -CN). Substitutions at R with electron-withdrawing groups, such as-NO (4k) and -CN
2
2
2
(4u) groups, significantly impaired the inhibitory potency, while a bromine (compounds 4c
, 4d) and
chlorine (compounds 4i 4j) atom at the position were basically tolerated. The impact of diversification
,
at R position to the inhibitory activity were found to be inconclusive, as a methyl group or a fluorine
1
atom were moderately tolerated. Based on 4g, more molecules with electron-donating groups at R2
were prepared and screened. Among all compounds of the series, 4n was identified as the most potent
one, which inhibited 94.9% at 20
µM and 93.1% at 10 µM of the Topo II’s catalytic activity in DNA
relaxation, compared with the positive control group without Topo II.
2
.3. Anti-Proliferative Activity of BCHs on Tumor Cells
Given that etoposide, a typical Topo II inhibitor, can be served as an effective anti-cancer drug,
we moved on to evaluate the anti-tumor activity of our compounds on tumor cells. The cytotoxicity
of BCHs was evaluated using the MTT assay against four human tumor cell lines, including HepG2
(human hepatoma cell line), A549 (human lung cancer cell line), LNCaP (human prostate cancer cell
line), MG63 (human osteosarcoma cell line). Etoposide was included as the comparative control.
The inhibitory activities are expressed as IC50 and listed in Table 1.

Molecules 2020, 25, 3180
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Figure 3. Topo II inhibiting activity of BDHs in Topo II-mediated DNA relaxation assay. lane D:
pBR322 DNA; lane T: pBR322 DNA + Topo II; lane E: pBR322 DNA + Topo II + etoposide (100 M);
other lanes: pBR322 DNA + Topo II + compounds. ( ) BCHs at concentration of 50 M; ( ) BCHs
at concentration of 20 M; ( ) BCHs at concentration of 10 M; ( ) semi-quantification of Topo II
inhibiting activity of BCHs at 50, 20, 10 M, respectively, with positive control (lane D: pBR322 DNA) as
00%, negative control (lane T: pBR322 DNA + Topo II) as 0% and comparative control (lane E: pBR322
DNA + Topo II + etoposide 100 µM).
µ
A,
B
µ
C,D
µ
E
µ
F–H
µ
1
Table 1. Inhibition of cancer cells proliferation for compounds 4a–4u.
IC50 (Mean ± SD) (µM) a
Topo II Inhibition
Compound
Inhibition Rate (%) b
A549
HePG2
MG63
LNCaP
Indicator
4
a
2.8 ± 0.2
3.6 ± 0.6
8.3 ± 2.8
3.6 ± 0.7
5.5 ± 1.5
6.1 ± 4.9
3.3 ± 0.6
2.9 ± 0.2
5.6 ± 2.7
3.1 ± 0.1
8.8 ± 1.0
3.7 ± 1.1
3.1 ± 0.7
3.8 ± 1.8
3.4 ± 0.4
5.7 ± 0.8
4.3 ± 1.0
5.8 ± 0.7
4.0 ± 0.6
4.6 ± 0.9
6.9 ± 1.1
4.5 ± 1.0
5.5 ± 1.7
10.8 ± 5.7
4.4 ± 1.3
4.6 ± 0.8
4.9 ± 1.5
4.4 ± 0.9
7.0 ± 0.5
5.1 ± 0.3
3.9 ± 0.3
4.6 ± 0.2
4.5 ± 0.2
6.5 ± 0.3
5.5 ± 0.3
6.5 ± 0.2
4.0 ± 0.5
5.5 ± 0.8
6.7 ± 0.6
4.7 ± 0.8
6.7 ± 0.6
5.5 ± 0.5
4.7 ± 0.5
4.4 ± 0.1
5.7 ± 0.6
5.7 ± 0.8
3.5 ± 0.1
4.2 ± 0.2
7.7 ± 0.4
5.4 ± 0.1
8.6 ± 0.5
8.5 ± 1.5
3.6 ± 0.2
3.6 ± 0.1
6.1 ± 0.2
6.1 ± 0.1
15.7 ± 2.7
3.4 ± 0.2
3.5 ± 0.4
3.6 ± 0.3
3.4 ± 0.5
5.7 ± 0.5
4.3 ± 1.4
5.8 ± 0.3
28.3 ± 0.7
67.1 ± 2.1
75.5 ± 2.6
86.9 ± 2.7
54.8 ± 1.0
44.2 ± 0.3
90.2 ± 2.1
−0.9 ± 0.2
93.9 ± 1.4
38.5 ± 1.0
59.2 ± 0.9
65.8 ± 1.3
n.d. [
94.9 ± 2.9
97.4 ± 1.6
108.6 ± 1.0
105.2 ± 0.6
106.4 ± 0.6
+
+
++
++
+
+
++
-
4
b
4
c
4
d
4
e
4
f
4
g
4
h
4
4
i
j
++
+
+
4
k
10.7
10.3
±
±
0.6
0.6
4
l
+
c]
n.d. [c]
++
++
++
++
++
4
m
5.0 ± 0.4
4.1 ± 0.6
6.2 ± 0.6
3.7 ± 0.6
4.1 ± 0.9
4.3 ± 1.3
4
n
4
4
4
o
p
q
4
r

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Table 1. Cont.
IC50 (Mean ± SD) (µM) a
Topo II Inhibition
Inhibition Rate (%) b
Indicator
Compound
A549
5.5 ± 1.0
12.1 1.1
HePG2
MG63
LNCaP
4
s
5.6 ± 0.2
22.4 ± 1.8
8.1 ± 0.4
>100
3.8 ± 0.8
4.9 ± 0.3
20.1 ± 2.4
18.0 ± 0.5
86.4 ± 2.4
52.4 ± 1.1
13.3 ± 1.6
103.1 ± 1.2
41.2 ± 1.5
70.9 ± 0.7
23.2 ± 1.6
12.5 ± 2.5
73.6 ± 0.6
++
+
+
-
-
+
4
t
±
47.7
18.5
±
±
1.6
3.0
4
u
8.2 ± 1.6
2
a
74.3
67.3
11.8
±
±
±
2.7
1.3
1.2
>100
>100
chalcone
etoposide
86.4 ± 0.9
5.2 ± 1.3
12.1
±
1.1
a, Each assay was performed in quadruplicate with a number of determinations n > 2, and the results are expressed
as mean
±
SD, whereas the IC50 means the concentration of drug needed to reduce the cell number to 50% of vehicle.
b
,
Topo II inhibition potency of compounds at 20 µM was semi-quantified in the DNA relaxation assay, normalized
by positive control (lane D) as 100%, negative control (lane T) as 0%, comparative control (lane E). Indicators were
set as follows: -, no obvious inhibition (<25%); +, weak inhibition ( 25%, <75%); ++, strong inhibition ( 75%).
≥
≥
[c]
Not determined.
Most of the tested compounds displayed strong antiproliferative activity in the four tested
cancer cell lines. In particular, for BCHs with the best inhibitory activity against A549 cells, the IC50
values of 4a and 4h reached 2.75
µM and 2.86
µM, and the IC50 values of 4d and 4n with better
inhibitory activity against Topo II also reached 3.55
µ
M and 3.81 M, respectively. Notably, 4a showed
µ
approximately 4-fold increase of inhibition potency, compared with that of etoposide on A549 cells
and LNCaP cells. Cellular permeability and metabolic stability of compounds in different tumor
cell lines could contribute to the gap between molecular and cellular pharmacological activity.
The anti-proliferative effect of the synthesized compounds was largely consistent with their Topo
II catalytic inhibition activity, except for compound 4h, which was possibly due to an off-target
mechanism. The anti-proliferative activity of these series of compounds was generally better than
the Topo II inhibitors we previously reported [31]. Compared with the benzimidazole or chalcone
pharmacophore separately, which represented by 2a and chalcone, respectively, this series of hybrids
showed remarked improvement in anti-proliferative activity against tumor cell lines. This work
was good practice of the classic pharmacophore hybridization strategy, which was also employed in
another series of Topo II poisons containing both the pharmacophores of merbarone and etoposide,
with similar anti-proliferative activity in cancer cell lines [28]. After balanced consideration of both
inhibitory activities against Topo II catalytic activity and cell proliferation, 4d and 4n were chosen for
further studies.
2
.4. BCHs as Non-Intercalative Topo II Catalytic Inhibitors
Since two typical mechanisms-of-action of Topo II inhibitors exist, assays for a mechanistic study
were performed. One mode of mechanism-of-action of Topo II poisons is to stabilize the Topo II-DNA
complex and promote the formation of linear DNA, while the other mode is to interfere the binding of
ATP to the enzyme and block the catalytic activity of the ATPase. The Topo II-mediated DNA cleavage
assay was employed to determine the mode of action, with etoposide, a typical Topo II poison, as the
comparative control. In the assay, proteinase K was added to cleavage Topo II-DNA complex stabilized
by the Topo II poison etoposide, which eventually produced linear DNA. According to the results,
the stains in the gel which represented linear DNA was observed in the etoposide group. In contrast,
no obvious linear form of DNA was noticed in neither 4d nor 4n group (Figure 4A,B), indicating that
4
d
and 4n were not a Topo II poison at concentration of 50
µM. More importantly, pretreatment of 4d
or 4n (group E + 4d, E + 4n) significantly decreased the amount of linear DNA compared with group E.
It implied that etoposide could be antagonized by 4d or 4n, suggesting that 4d, 4n and the other BCHs
of the series were Topo II catalytic inhibitors.

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Figure 4.
pBR322 DNA without or with Topo II; Lane 3-5: addition of etoposide (100
Topo II with supercoiled pBR322 DNA; Lane 6-7: pretreatment of pBR322 DNA and Topo II with 4d
or 4n (50 M) followed by addition of etoposide. In each lane, the positions of supercoiled DNA (S),
relaxed DNA (R), linear DNA (L) and nicked DNA (N) are indicated. ( ) Semi-quantification of the
western blot gel in panel A. Data were presented as Mean SD (n = 3). * p < 0.05 indicates statistically
significant difference of relaxed DNA compared with group E (Student’s t-test). ( ) The unwinding
(A) Western blot gel of the Topo II-DNA cleavage assay. Lane 1-2: control group of supercoiled
µ
M), 4d or 4n (50 M) on
µ
µ
B
±
C
assay of compounds. Lane D: pBR322 DNA; lane T: pBR322 DNA + Topo I; other lanes: pBR322
DNA + Topo I + 4d, 4n or EB of different concentrations.
In order to verify whether our Topo II catalytic inhibitors function as DNA intercalators or
non-intercalators, a Topo I-mediated DNA unwinding assay was carried out with 4d and 4n. In this
assay, the supercoiled DNA could be completely relaxed by excessive Topo I (Figure 4C). However,
addition of DNA intercalators, such as ethidium bromide (EB), could block the relaxation. As shown
in Figure 4C, the relaxation of DNA was partially inhibited by different concentrations of EB, while no
inhibiting effect was observed in groups adding 4d or 4n, even at as high as 50 µM.
In view of these results above, this series of BCHs, exemplified by 4d and 4n, inhibited Topo II
enzyme activity and suppress tumor cell proliferation. But it did not stabilize Topo II-DNA complex as
Topo II poisons nor intercalate with DNA. Thus, it could be agreeably inferred that this series of BCHs
acted as non-intercalative Topo II catalytic inhibitors.
2
.5. Inhibition of Clonogenic Survival of A549 Cells by 4d and 4n
The clonogenic cell viability test is a long-term cell viability assay, which determines whether the
cells can proliferate infinitely, thus maintaining their reproductive capacity to form colonies. This assay
was used to examine the anti-colony forming ability of 4d and 4n in A549 cells. A549 cells were
incubated for 24 h with different concentrations of 4d and 4n (0–3
according to previously described protocols [32 33]. The survival of A549 cells decreased to 10% after
M treatment of 4d or 4n, even the survival of A549 cells was almost 0% in the 2 M treatments of
or 4n (Figure 5). Etoposide also showed good anti-colony forming ability in A549 cells. The IC50
M and 0.47
M. According to these results, we concluded that 4d and 4n
had strong inhibition of A549 cell colony formation after a short exposure.
µM), and the assay was performed
,
1
µ
µ
4
d
values for inhibition of A549 cells clonogenic by compound 4d and 4n were 0.54
respectively, while etoposide was 0.70
µ
µM
µ

Molecules 2020, 25, 3180
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Figure 5. Compound 4d and 4n inhibited the clonogenic survival of A549 cells. (
colonies were counted and compared with the untreated control as survival percentage.
) Dose-dependent curves of 4n 4n and etoposide. Data represented here are the mean of three
A) The developed
(
B
,
independent experiments performed in triplicates.
2
.6. Inhibition of Wound Healing in A549 Cell by 4d and 4n
The wound healing assay is a classic and common method used to investigate the effect of
antitumor drugs on tumor migration. According to previous studies, scratch-wound healing assay
was used to assess the effect of compounds on the migration and interaction capacity of A549 cells.
A sterile tip scratched the monolayer of cells and a wound was created. Then it was inspected after
the treatments of 1
time. Obviously, the migration ability of treated cells was time dependent. The scratch gaps in 4d- and
-treated A549 cells were wider than control with increased treating time (Figure 6). Combining the
µM and 2 µM 4d or 4n. The untreated control cells migrated across the well with
4
n
results of cytotoxicity, clonogenic survival and scratch-wound assay, we concluded that compound 4d
and 4n were able to inhibit tumor viability, colony formation, and migration of A549 cells.
2
.7. Cell Apoptosis of A549 Cells by 4d and 4n
We also investigated the effects of compounds in inducing A549 cells apoptosis using fluorescence
microscope by staining with DAPI [34,35]. The results in Figure 7A indicate that the nuclear structure
of the control cells was intact with round shape while the A549 cells treated with the 4d and 4n showed
nuclear concentration and micronuclei formation. Morphological alterations were semi-quantified
with Nuclear area factor (NAF), which was regarded as an early indicator of cell morphological changes
occurring during apoptosis (Figure 7B). It was calculated by the product of average nuclear area and
circularity. According to the figure, the BCHs could probably induce apoptosis of A549 cells compared
with the control group.

Molecules 2020, 25, 3180
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Figure 6. Compound 4d and 4n inhibited wound healing of A549 cells. (
by JuLI Intelligent fluorescent cell monitor at different time points (0, 24, 48 and 72 h). Scale bar: 500
) Quantification of the cell scrape results using ImageJ software. Values are means SD, **** indicates
A
) The wound was captured
µm.
(
B
±
p < 0.0001 (Student’s t-test).
Figure 7. Morphological observation on A549 cells by DAPI staining. (
treated with 4d and 4n (1 µM, 2 µM), with etoposide (2 µM) as a comparative control. (B
A
) DAPI staining of A549 cells
) Morphological
alterations were semi-quantification by NAF calculated by the product of nuclear area and circularity.
Values (% of control) were presented as means SD (n = 3), labeled with * p < 0.05 or with *** p < 0.001
±
compared with the control group (Student’s t-test). Scale bar: 100 µm.
2
.8. Molecular Docking
To further rationalize the inhibitory activity and SAR of BCHs, a flexible ligand receptor docking
investigation of 4d and 4n was carried out on SYBYL platform with a Surflex-Dock module. The crystal
structure data of Topo II protein was originated from the protein data bank (PDB code: 1ZXM). Various
binding pockets had been searched for and finally the ATP-binding pocket was identified as the
optimal binding site represented by high total scores. 4d formed two hydrogen bounds with residues
SER149 via the fluorine atom and the carbonyl group, while 4n interacted with the enzyme via two
hydrogen bounds with ASN150 and ALA167 through the carbonyl group and methoxy group of the
chalcone motif. Both compounds were evaluated with higher total score (6.53 and 9.62 of 4d and 4n
,

Molecules 2020, 25, 3180
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respectively) than that of etoposide (total score 6.52) according to the docking results (Supplementary
Materials Table S1). As we can see in Figure 8, both hybrid 4d and 4n were found to locate in the cavity
which were supposed to accommodate ATP, which was demonstrated in the cocrystal structure of
adenosine diphosphate (ADP) and the N-terminal ATPase domain of Topo II (Supplementary Materials
Figures S1–S3). Given that, it can be inferred that inhibition mode of hybrid 4d and 4n against Topo II
was catalytic inhibition through occupying ATP binding pocket of N-terminal ATPase domain of Topo
II, and modification of may furtherly promote the inhibitory activity.
Figure 8. Schematic illustration of proposed binding mode of 4d and 4n with the ATP binding pocket
of Topo II (PDB code: 1ZXM) (A), compound 4d; (B), compound 4n.
3
3
3
. Materials and Methods
.1. Chemistry
.1.1. Synthesis and Characterization
1H- and 13C-NMR spectra were recorded using TMS as the internal standard in CDCl with a
3
1
Bruker BioSpin GmbH (Bruker, Billerica, MA, USA) spectrometer at 400 MHz for H and 101 MHz for
C. Mass spectra (ESI-MS) were recorded on a 4000 QTRAP type LC/MS (SCIEX, Framingham, MA,
13
USA). The purities of final compounds were higher than 95%, confirmed by an analytical HPLC on a
dual pump LC-20AB system (Shimadzu, Tokyo, Japan). HPLC conditions: methanol/H O = 80/20 (v/v);
2
flow rate, 0.5 mL/min; column, Ultimate XB-C18.
3
.1.2. Procedure for the Synthesis of Intermediate 1a
A solution of o-phenylenediamine (10.8 g, 10 mmol, 1 equiv) and glycolic acid (22.8 g, 30 mmol,
◦
3
equiv) in 4N HCl (70 mL) was stirred at 100 C for 6 h. After the reaction was complete, the reaction
mixture was cooled to room temperature, and an appropriate amount of NaHCO was added to
3
neutralize hydrochloric acid, and an off-white solid precipitate was formed. The precipitated solid was
filtered, washed with water and dried in a vacuum oven to get the crude product, which then was
purified by column chromatography (200 mesh silica gel, ethyl acetate/ petroleum ether = 8:1) to yield
the title compound 1a.
3
.1.3. General Procedure for the Synthesis of Intermediates 2a and 2b
To a solution of 1a (2.96 g, 20 mmol, 1 equiv) in DMF (30 mL) was added the appropriate benzyl
bromide (40 mmol, 2 equiv) and K CO (100 mmol, 5 equiv), and the reaction mixture was stirred at
2
3
room temperature overnight. After removal of the solvent under vacuum the residue was partitioned
between DCM (70 mL) and H O (70 mL). The organic layer was separated, and the aqueous layer
2
was extracted with DCM (70 mL
×
2). The combined organic layers were concentrated in vacuo to

Molecules 2020, 25, 3180
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provide a crude product, which was purified by column chromatography (200 mesh silica gel, ethyl
acetate/petroleum ether = 8:1) to yield the title compounds 2a and 2b.
(
1-(2-Fluorobenzyl)-1H-benzo[d]imidazol-2-yl)methanol (2a). Light yellow thick liquid, yield: 47%.
1
H- NMR (CDCl3)
.14–7.07 (m, 2H), 5.57 (d, J = 1.1 Hz, 2H), 4.70 (d, J = 5.5 Hz, 2H), 3.92 (t, J = 5.5 Hz, 1H). 13C-NMR
CDCl3) 160.6 (d, J = 203.5 Hz), 155.3, 141.1, 136.4, 130.1 (d, J = 72.7 Hz), 129.4 (d, J = 6.5 Hz),
δ 7.57–7.52 (m, 1H), 7.43–7.36 (m, 1H), 7.34–7.26 (m, 1H), 7.26–7.17 (m, 3H),
7
(
δ
1
24.7 (d, J = 16.2 Hz), 124.2, 123.1, 119.4, 115.6 (d, J = 16.2 Hz), 111.2, 57.9, 45.2(d, J = 4.0 Hz). MS (ESI):
+
calcd for C H FN O [M + H] 257.1, found 257.1. HPLC purity: 97.5%.
15
13
2
(
1-(4-Methylbenzyl)-1H-benzo[d]imidazol-2-yl)methanol (2b). Light yellow thick liquid, yield: 88%.
1
H- NMR (CDCl3)
.54 (t, J = 0.9 Hz, 2H), 4.70 (d, J = 5.5 Hz, 2H), 3.92 (t, J = 5.5 Hz, 1H). 13C-NMR (CDCl3)
36.8, 136.5, 134.0, 129.2, 127.3, 124.2, 123.1, 119.4, 111.2, 57.9, 47.4, 21.0. MS (ESI): calcd for C H N O
δ
7.57–7.52 (m, 1H), 7.43–7.37 (m, 1H), 7.22 (pd, J = 7.1, 1.5 Hz, 2H), 7.09–7.00 (m, 4H),
5
1
[
δ
155.2, 141.1,
16
16
2
+
M + H] 253.1, found 253.1. HPLC purity: 95.9%.
3
.1.4. General Procedure for the Synthesis of Intermediates 3a and 3b
◦
A solution of 2a or 2b (10 mmol, 1 equiv) in DCM (20 mL) was stirred at 0 C for 15 min, then added
◦
Dess-Martin periodinane (11 mmol, 1.1 equiv) was added and the mixture stirred at 0 C for another
1
h. After the reaction was complete, it was returned to room temperature and an appropriate amount
of a saturated Na S O solution was added to remove the excess Dess-Martin periodinane. Next the
2
2
3
mixture was diluted with DCM (70 mL) and H O (70 mL). The organic layer was separated, and the
2
aqueous layer was extracted with DCM (70 mL
in vacuo to provide a crude product, which was purified by column chromatography (200 mesh silica
×
2). The combined organic layers were concentrated
gel, ethyl acetate/petroleum ether = 10:1) to yield the title compounds 3a and 3b.
1
-(2-Fluorobenzyl)-1H-benzo[d]imidazole-2-carbaldehyde (3b). Light yellow semisolid, yield: 78%.
1
H- NMR (CDCl3)
δ
9.78 (1H, s), 7.69 (dd, J = 6.5, 1.4 Hz, 1H), 7.44 (dd, J = 7.2, 1.4 Hz, 1H), 7.34–7.20
182.0, 160.6 (d, J = 203.4 Hz),
(
m, 4H), 7.14–7.07 (m, 2H), 5.59 (d, J = 1.1 Hz, 2H). 13C-NMR (CDCl3)
δ
1
1
2
51.1, 139.2, 137.4, 130.1 (d, J = 65.7 Hz), 129.4 (d, J = 6.6 Hz), 124.7 (d, J = 5.7 Hz), 124.2 (d, J = 16.2 Hz),
+
23.3, 120.1, 115.6 (d, J = 16.2 Hz), 113.1, 44.5 (d, J = 4.0 Hz). MS (ESI): calcd for C H FN O [M + H]
15
11
2
55.1, found 255.1. HPLC purity: 96.5%.
1
-(4-Methylbenzyl)-1H-benzo[d]imidazole-2-carbaldehyde (4b). Light yellow semisolid, yield: 56%.
1
H-NMR (CDCl3)
.32–7.20 (m, 2H), 7.09–7.00 (m, 4H), 5.54 (t, J = 1.0 Hz, 2H), 2.34 (s, 2H). 13C-NMR (CDCl3)
50.9, 139.2, 137.6, 136.8, 133.7, 129.2, 127.3, 124.6, 123.3, 120.1, 113.1, 46.5, 21.0. MS (ESI): calcd for
δ
9.61 (s, 1H), 7.69 (dd, J = 6.5, 1.4 Hz, 1H), 7.44 (dd, J = 7.2, 1.4 Hz, 1H),
7
1
δ
182.2,
+
C H N O [M + H] 251.1, found 251.1. HPLC purity: 95.7%.
16
14
2
3
.1.5. General Procedure for the Synthesis of Compounds 4a–4u
Compounds 3a or 3b (2 mmol, 1 equiv) and an appropriate acetophenone (2 mmol, 1 equiv)
were dissolved in EtOH (10 mL) and 10% NaOH (0.5 mL) was added slowly. Then, the mixture was
stirred at room temperature for 4 h. The solid was filtered off and recrystallized from ethanol to give a
light-yellow solid.
0
(
E)-3-(1-(2-Fluorobenzyl)-1H-benzo[d]imidazol-2-yl)-1-(p-tolyl)prop-2-en-1-one
(
4a).
4 -Methyl-
◦
acetophenone and 3a were used as reactants to give 4a
.
Yield: 73%, mp: 167.3–168.5 C.
δ 8.40 (d, J = 15.0 Hz, 1 H), 8.04 (d, J = 8.2 Hz, 2 H), 7.90–7.85 (m, 2 H), 7.39–7.27
1
H-NMR (CDCl3)
(
m, 6 H), 7.15–7.10 (m, 1 H), 7.01 (t, J = 7.1 Hz, 1 H), 6.80 (t, J = 7.0 Hz, 1 H), 5.60 (s, 2 H), 2.44 (s, 3 H).
1
3
C-NMR (CDCl3)
δ 188.5, 160.1 (d, J = 247.0 Hz), 148.8, 144.5, 143.3, 135.8, 134.9, 130.0 (d), 129.5, 129.0,
1
28.7, 128.0 (d), 127.4, 124.8 (d, J = 3.6 Hz), 124.4, 123.7, 122.8 (d, J = 14.3 Hz), 120.4, 115.9 (d, J = 21.0 Hz),

Molecules 2020, 25, 3180
11 of 18
+
1
10.2, 41.1 (d, J = 5.2 Hz), 21.7. HRMS (ESI): calcd for C H FN O [M + H] 371.1554, found 371.1571.
24 19 2
HPLC purity: 95.8%.
0
(
E)-3-(1-(4-Methylbenzyl)-1H-benzo[d]imidazol-2-yl)-1-(p-tolyl)prop-2-en-1-one
(
4b).
4 -Methyl-
◦
1
acetophenone and 3b were used as reactants to give 4b. Yield: 82%, mp: 207.6–209.2 C. H-NMR
(
7
CDCl3)
δ
8.37 (d, J = 15.0 Hz, 1 H), 8.03 (d, J = 8.2 Hz, 2 H), 7.88–7.83 (m, 2 H), 7.37–7.28 (m, 5 H),
.10 (d, J = 8.0 Hz, 2 H), 7.01 (d, J = 8.0 Hz, 2 H), 5.51 (s, 2 H), 2.44 (s, 3 H), 2.30 (s, 3 H). 13C-NMR
(
1
CDCl3)
δ
188.6, 148.7, 144.5, 143.3, 138.0, 136.0, 134.9, 132.7, 129.8, 129.5, 129.0, 128.5, 127.7, 126.3,
+
24.2, 123.6, 120.3, 110.3, 47.0, 21.7, 21.1. HRMS (ESI): calcd for C H N O [M + H] 367.1805, found
25
22
2
3
67.1809. HPLC purity: 96.2%.
0
E)-1-(4-Bromophenyl)-3-(1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)prop-2-en-1-one (4c). 4 -Bromo-
acetophenone and 3b were used as reactants to give 4c. Yield: 81%, mp: 194.5–196.3 C. H-NMR
(
◦
1
(
7
CDCl3)
δ
8.31 (d, J = 15.0 Hz, 1 H), 7.97 (d, J = 8.5 Hz, 2 H), 7.89–7.84 (m, 2 H), 7.65 (d, J = 8.5 Hz, 2 H),
.37–7.30 (m, 3 H), 7.11 (d, J = 8.1 Hz, 2 H), 7.00 (d, J = 8.0 Hz, 2 H), 5.51 (s, 2 H), 2.30 (s, 3 H). 13C-NMR
(
1
CDCl3)
δ
188.0, 148.4, 143.3, 138.1, 136.1, 136.1, 132.6, 132.1, 130.3, 129.8, 128.6, 127.6, 126.3, 124.5, 123.7,
+
20.4, 110.4, 47.0, 21.1. HRMS (ESI): calcd for C H BrN O [M + H] 431.0754, found 431.0751. HPLC
24
19
2
purity: 95.9%.
0
E)-1-(4-Bromophenyl)-3-(1-(2-fluorobenzyl)-1H-benzo[d]imidazol-2-yl) prop -2-en-1-one (4d). 4 -Bromo-
(
◦
1
acetophenone and 3a were used as reactants to give 4d. Yield: 78%, mp: 156.7–157.5 C. H-NMR
CDCl3) 8.34 (d, J = 14.9 Hz, 1 H), 7.99 (d, J = 8.6 Hz, 2 H), 7.92–7.84 (m, 2 H), 7.66 (d, J = 8.5 Hz, 2 H),
.40–7.28 (m, 4 H), 7.16–7.10 (m, 1 H), 7.02 (t, J = 7.5 Hz, 1 H), 6.80 (t, J = 7.6 Hz, 1 H), 5.60 (s, 2 H).
(
δ
7
13
C-NMR (CDCl3)
δ 187.9, 160.1 (d, J = 246.9 Hz), 148.4, 143.3, 136.1, 135.9, 132.1, 130.3, 130.1 (d), 128.8,
1
28.3, 128.1 (d), 127.9, 124.8 (d), 124.6, 123.9, 122.7 (d), 120.5, 115.8 (d), 110.2, 41.1 (d). HRMS (ESI):
+
calcd for C H BrFN O [M + H] 435.0503, found 435.0508. HPLC purity: 98.4%.
23
16
2
0
E)-1-(4-Fluorophenyl)-3-(1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)prop-2-en-1-one (4e). 4 -Fluoro-
(
◦
1
acetophenone and 3b were used as reactants to give 4e. Yield: 79%, mp: 206.7–207.4 C. H-NMR
CDCl3) 8.34 (d, J = 15.0 Hz, 1 H), 8.15 (dd, J = 8.8, 5.4 Hz, 2 H), 7.88–7.83 (m, 2 H), 7.38–7.29 (m, 3 H),
.18 (t, J = 8.6 Hz, 2 H), 7.11 (d, J = 7.9 Hz, 2 H), 7.01 (d, J = 8.0 Hz, 2 H), 5.51 (s, 2 H), 2.30 (s, 3 H).
(
δ
7
1
3
C-NMR (CDCl3) δ 187.5, 166.0 (d), 148.5, 143.3, 138.0, 136.0, 133.8 (d), 132.6, 131.5 (d), 129.8, 128.3,
+
1
27.9, 126.3, 124.4, 123.7, 120.4, 115.9 (d), 110.4, 47.0, 21.1. HRMS (ESI): calcd for C H FN O [M + H]
24
19
2
3
71.1554, found 371.1558. HPLC purity: 98.6%.
0
E)-3-(1-(2-Fluorobenzyl)-1H-benzo[d]imidazol-2-yl)-1-(4-fluorophenyl)prop-2-en-1-one (4f). 4 -Fluoro-
acetophenone and 3a were used as reactants to give 4f. Yield: 70%, mp: 165.9–167.2 C. H-NMR
(
◦
1
(
7
CDCl3)
δ
8.37 (d, J = 14.9 Hz, 1 H), 8.19–8.14 (m, 2 H), 7.88 (t, J = 11.7 Hz, 2 H), 7.39–7.27 (m, 4 H),
.16 (dt, J = 18.5, 8.6 Hz, 3 H), 7.02 (t, J = 7.5 Hz, 1 H), 6.80 (t, J = 7.2 Hz, 1 H), 5.60 (s, 2 H). 13C-NMR
(CDCl3)
δ
187.4, 166.0 (d), 160.1 (d), 148.5, 143.3, 135.8, 133.8 (d), 131.5 (d), 130.1 (d), 128.1, 128.0 (d),
1
28.0, 124.8 (d), 124.5, 123.8, 122.8 (d), 120.4, 116.0 (d), 115.8 (t), 110.2, 41.1 (d). HRMS (ESI): calcd for
+
C H F N O [M + H] 375.1303, found 375.1302. HPLC purity: 99.3%.
23
16
2
2
(
E)-1-(4-Methoxyphenyl)-3-(1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)prop-2-en-1-one
-Methoxyacetophenone and 3b were used as reactants to give 4g. Yield: 80%, mp: 196.2–197.6 C.
(
4g).
0
◦
4
1
H-NMR (CDCl3)
δ 8.37 (d, J = 15.0 Hz, 1 H), 8.13 (d, J = 8.9 Hz, 2 H), 7.87–7.82 (m, 2 H), 7.37–7.27
13
(
(
1
m, 3 H), 7.10 (d, J = 7.9 Hz, 2 H), 7.03–6.97 (m, 4 H), 5.51 (s, 2 H), 3.90 (s, 3 H), 2.29 (s, 3 H). C-NMR
CDCl3) 187.3, 164.0, 148.8, 143.3, 138.0, 136.0, 132.7, 131.2, 130.5, 129.8, 128.5, 127.4, 126.3, 124.2,
δ
+
23.5, 120.3, 114.0, 110.3, 55.6, 47.0, 21.1. HRMS (ESI): calcd for C H N O [M + H] 383.1754, found
25 22 2 2
3
83.1760. HPLC purity: 96.7%.

Molecules 2020, 25, 3180
12 of 18
(
E)-3-(1-(2-Fluorobenzyl)-1H-benzo[d]imidazol-2-yl)-1-(4-methoxyphenyl)prop-2-en-1-one
-Methoxyacetophenone and 3a were used as reactants to give 4h. Yield: 73%, mp: 155.8–157.3 C.
(
4h).
0
◦
4
1
H-NMR (CDCl3)
δ
8.40 (d, J = 14.9 Hz, 1 H), 8.14 (d, J = 8.8 Hz, 2 H), 7.89–7.84 (m, 2 H), 7.34 (dt, J = 26.0,
.6 Hz, 4 H), 7.15–7.10 (m, 1 H), 7.00 (dd, J = 11.3, 8.3 Hz, 3 H), 6.80 (t, J = 7.6 Hz, 1 H), 5.60 (s, 2 H),
.90 (s, 3 H). 13C-NMR (CDCl3)
187.3, 164.0, 160.1 (d, J = 247.0 Hz), 148.9, 143.3, 135.8, 131.3, 130.4,
30.0 (d), 128.7, 128.0 (d), 127.0, 124.8 (d), 124.3, 123.7, 122.8 (d), 120.3, 115.8 (d), 114.0, 110.2, 55.6,
8
3
1
δ
+
4
1.1 (d). HRMS (ESI): calcd for C H FN O [M + H] 387.1503, found 387.1504. HPLC purity: 99.8%.
24 19 2 2
0
E)-1-(4-Chlorophenyl)-3-(1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)prop-2-en-1-one (4i). 4 -Chloro-
acetophenone and 3b were used as reactants to give 4i. Yield: 84%, mp: 206.4–208.1 C. H-NMR
(
◦
1
(
7
CDCl3)
δ
8.32 (d, J = 15.0 Hz, 1 H), 8.05 (d, J = 8.5 Hz, 2 H), 7.89–7.83 (m, 2 H), 7.48 (d, J = 8.5 Hz, 2 H),
.38–7.29 (m, 3 H), 7.11 (d, J = 7.9 Hz, 2 H), 7.00 (d, J = 8.0 Hz, 2 H), 5.51 (s, 2 H), 2.30 (s, 3 H). 13C-NMR
(
1
CDCl3)
δ
187.8, 148.4, 143.3, 140.0, 138.1, 136.1, 135.7, 132.6, 130.2, 129.8, 129.1, 128.5, 127.7, 126.3, 124.5,
+
23.7, 120.4, 110.4, 47.0, 21.1. HRMS (ESI): calcd for C H ClN O [M + H] 387.1259, found 387.1273.
24
19
2
HPLC purity: 98.2%.
0
E)-1-(4-Chlorophenyl)-3-(1-(2-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)prop-2-en-1-one (4j). 4 -Chloro-
(
◦
1
acetophenone and 3a were used as reactants to give 4j. Yield: 78%, mp: 166.2–167.3 C. H-NMR
CDCl3) 8.35 (d, J = 14.9 Hz, 1 H), 8.07 (d, J = 8.5 Hz, 2 H), 7.92–7.84 (m, 2 H), 7.49 (d, J = 8.5 Hz, 2 H),
.33 (ddd, J = 19.5, 12.8, 7.8 Hz, 4 H), 7.16–7.10 (m, 1 H), 7.01 (t, J = 7.5 Hz, 1 H), 6.80 (t, J = 7.7 Hz, 1 H),
.60 (s, 2 H). 13C-NMR (CDCl3)
187.7, 160.1 (d), 148.5, 143.3, 140.0, 135.9, 135.7, 130.2, 130.1 (d), 129.1,
28.2, 128.1 (d), 127.9, 124.8 (d), 124.6, 123.9, 122.8 (d), 120.5, 115.8 (d), 110.2, 41.1 (d). HRMS (ESI):
(
δ
7
5
δ
1
+
calcd for C H ClFN O [M + H] 391.1008, found 391.1004. HPLC purity: 97.9%.
23
16
2
0
4 -Nitro-
(
E)-3-(1-(4-Methylbenzyl)-1H-benzo[d]imidazol-2-yl)-1-(4-nitrophenyl)prop-2-en-1-one (4k).
◦
1
acetophenone and 3b were used as reactants to give 4k. Yield: 76%, mp: 209.8–211.5 C. H-NMR
(
7
1
1
CDCl3)
.12 (d, J = 8.0 Hz, 2 H), 7.00 (d, J = 8.1 Hz, 2 H), 5.52 (s, 2 H), 2.31 (s, 3 H). 13C-NMR (CDCl3)
50.4, 148.0, 143.3, 142.0, 138.2, 136.1, 132.6, 129.8, 129.7, 129.7, 127.0, 126.2, 124.8, 123.9, 123.9, 120.5,
δ
8.33 (dd, J = 15.0, 8.8 Hz, 3 H), 8.24 (d, J = 8.8 Hz, 2 H), 7.92–7.85 (m, 2 H), 7.39–7.32 (m, 3 H),
δ
187.6,
+
10.4, 47.0, 21.1. HRMS (ESI): calcd for C H N O [M + H] 398.1499, found 398.1492. HPLC
24 19 3 3
purity: 98.0%.
(
E)-1-(3,4-Dimethoxyphenyl)-3-(1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)prop-2-en-1-one
,4 -Dimethoxyacetophenone and 3b were used as reactants to give 4l. Yield: 82%, mp: 193.8–195.4 C.
(
4l).
0
0
◦
3
1
H-NMR (CDCl3)
δ 8.39 (d, J = 14.9 Hz, 1 H), 7.88–7.82 (m, 2 H), 7.66 (s, 1 H), 7.33 (dd, J = 14.4, 6.5 Hz,
13
3
H), 7.15–6.98 (m, 5 H), 6.91 (dd, J = 16.3, 8.3 Hz, 1 H), 5.52 (s, 2 H), 3.97 (s, 6 H), 2.30 (s, 3 H). C-NMR
(
1
CDCl3)
δ
187.2, 153.8, 149.3, 148.8, 143.3, 138.0, 136.0, 132.7, 130.6, 129.8, 128.2, 127.3, 126.3, 124.2, 123.9,
+
23.6, 120.2, 110.7, 110.3, 110.1, 56.1, 47.0, 21.1. HRMS (ESI): calcd for C H N O [M + H] 413.1867,
26
24
2
3
found 413.1866. HPLC purity: 95.0%.
(
3
1
7
1
1
1
4
E)-1-(3,4-Dimethoxyphenyl)-3-(1-(2-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)prop-2-en-1-one
(
4m).
Yield: 76%, mp:
8.33 (d, J = 14.9 Hz, 1 H), 7.83–7.75 (m, 3 H), 7.59 (d, J = 2.0 Hz, 1 H),
.33–7.20 (m, 4 H), 7.08–7.03 (m, 1 H), 6.94 (t, J = 7.6 Hz, 1 H), 6.87 (d, J = 8.5 Hz, 1H), 6.74 (t, J = 6.9 Hz,
H), 5.53 (s, 2 H), 3.91 (d, J = 2.8 Hz, 6 H). 13C-NMR (CDCl3)
187.2, 160.1 (d), 153.9, 149.4, 148.9, 143.3,
35.8, 130.6, 130.1 (d), 128.5, 128.1 (d), 127.1, 124.8 (d), 124.3, 123.9, 123.7, 122.8 (d), 120.3, 115.8 (d),
0
0
,4 -Dimethoxyacetophenone and 3a were used as reactants to give 4m.
◦
54.4–155.2 C. H-NMR (CDCl ) δ
3
1
δ
+
10.7, 110.2, 110.1, 56.2, 56.1, 41.1 (d). HRMS (ESI): calcd for C H FN O [M + H] 417.1609, found
25
21
2
3
17.1607. HPLC purity: 99.2%.
0
0
0
(
E)-3-(1-(4-Methylbenzyl)-1H-benzo[d]imidazol-2-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4n). 3 ,4 ,5 -
◦
Trimethoxyacetophenone and 3b were used as reactants to give 4n. Yield: 86%, mp: 190.7–191.8 C.

Molecules 2020, 25, 3180
13 of 18
1H-NMR (CDCl3)
2
δ
8.33 (d, J = 15.0 Hz, 1 H), 7.86 (t, J = 10.7 Hz, 2 H), 7.39–7.29 (m, 5 H), 7.11 (d, J = 7.9 Hz,
H), 7.02 (d, J = 7.9 Hz, 2 H), 5.52 (s, 2 H), 3.95 (s, 9 H), 2.30 (s, 3 H). 1 C-NMR (CDCl ) δ 187.7, 153.3,
3
3
1
4
48.6, 143.3, 143.1, 138.0, 136.0, 132.7, 129.8, 128.1, 127.9, 126.3, 124.4, 123.7, 120.2, 110.4, 106.4, 61.0, 56.5,
+
7.0, 21.1. HRMS (ESI): calcd for C H N O [M + H] 443.1965, found 443.1961. HPLC purity: 99.6%.
27
26
2
4
0
0
0
(
E)-3-(1-(2-Fluorobenzyl)-1H-benzo[d]imidazol-2-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4o). 3 ,4 ,5 -
◦
Trimethoxyacetophenone and 3a were used as reactants to give 4o. Yield: 84%, mp: 152.7–153.5 C.
1
H-NMR (CDCl3)
δ
8.36 (d, J = 14.9 Hz, 1 H), 7.93–7.84 (m, 2 H), 7.40–7.30 (m, 5 H), 7.14 (dd, J = 16.9,
.9 Hz, 1 H), 7.02 (t, J = 8.1 Hz, 1 H), 6.83 (t, J = 7.6 Hz, 1 H), 5.61 (s, 2 H), 3.96 (d, J = 3.1 Hz, 9 H),
.89 (d, J = 7.4 Hz, 1 H). 13C-NMR (CDCl3)
187.6, 160.1 (d), 153.3, 148.7, 143.2 (d), 135.8, 132.6, 130.1 (d),
28.1, 128.1, 127.8, 124.8 (d), 124.5, 123.8, 122.8 (d), 120.3, 115.8 (d), 110.3, 106.3, 61.0, 56.5, 41.1 (d).
7
3
1
δ
+
HRMS (ESI): calcd for C H FN O [M + H] 447.1715, found 447.1719. HPLC purity: 99.5%.
26
23
2
4
(
E)-1-(4-Ethylphenyl)-3-(1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)prop-2-en-1-one (4p).
4-Ethyl-
◦
1
acetophenone and 3b were used as reactants to give 4p. Yield: 84%, mp: 203.6–205.3 C. H-NMR
(
7
1
1
CDCl3)
.10 (d, J = 7.7 Hz, 2 H), 7.01 (d, J = 7.8 Hz, 2 H), 5.50 (s, 2 H), 2.73 (q, J = 7.5 Hz, 2 H), 2.30 (s, 3 H),
.28 (t, J = 7.6 Hz, 3 H). 13C-NMR (CDCl3)
188.6, 150.6, 148.7, 143.3, 138.0, 136.0, 135.1, 132.7, 129.8,
29.1, 128.5, 128.3, 127.7, 126.3, 124.2, 123.6, 120.3, 110.3, 47.0, 29.0, 21.1, 15.2. HRMS (ESI): calcd for
δ 8.37 (d, J = 15.0 Hz, 1 H), 8.05 (d, J = 8.0 Hz, 2 H), 7.90–7.80 (m, 2 H), 7.39–7.27 (m, 5 H),
δ
+
C H N O [M + H] 381.1961, found 381.1968. HPLC purity: 97.1%.
26
24
2
(
E)-1-(4-Ethylphenyl)-3-(1-(2-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)prop-2-en-1-one (4q).
4-Ethyl-
◦
1
acetophenone and 3a were used as reactants to give 4q. Yield: 78%, mp: 165.1–166.3 C. H-NMR
CDCl3) 8.35 (d, J = 15.0 Hz, 1 H), 8.00 (d, J = 8.3 Hz, 2 H), 7.83–7.78 (m, 2 H), 7.33–7.20 (m, 6 H),
.08–7.03 (m, 1 H), 6.94 (t, J = 7.2 Hz, 1 H), 6.73 (t, J = 7.6 Hz, 1 H), 5.53 (s, 2 H), 2.67 (q, J = 7.6 Hz, 2 H),
.21 (t, J = 7.6 Hz, 3 H). 13C-NMR (CDCl3)
188.5, 160.1 (d), 150.7, 148.8, 143.3, 135.8, 135.1, 130.1 (d),
(
7
1
1
1
δ
δ
29.1, 128.7, 128.3, 128.0 (d), 127.4, 124.8 (d), 124.4, 123.7, 122.8 (d), 120.4, 115.8 (d), 110.2, 41.1 (d), 29.1,
+
5.2. HRMS (ESI): calcd for C H FN O [M + H] 385.1771, found 385.1779. HPLC purity: 95.1%.
25
21
2
(
E)-1-(4-Isopropylphenyl)-3-(1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)prop-2-en-1-one (4r). 1-[4-(1-
◦
methylethyl)phenyl]ethanone and 3b were used as reactants to give 4r. Yield: 83%, mp: 204.7–206.2 C.
1
H-NMR (CDCl3)
δ 8.37 (d, J = 15.0 Hz, 1 H), 8.06 (d, J = 8.3 Hz, 2 H), 7.88–7.82 (m, 2 H), 7.37–7.27
(
1
1
m, 5 H), 7.10 (d, J = 8.0 Hz, 2 H), 7.01 (d, J = 8.0 Hz, 2 H), 5.50 (s, 2 H), 3.04–2.94 (m, 1 H), 2.30 (s, 3 H),
.29 (d, J = 6.9 Hz, 6 H). 13C-NMR (101 MHz, CDCl3)
δ 188.5, 155.2, 148.8, 143.3, 138.0, 136.0, 135.2,
32.7, 129.8, 129.1, 128.5, 127.7, 126.9, 126.3, 124.2, 123.6, 120.3, 110.3, 47.0, 34.4, 23.7, 21.1. HRMS (ESI):
+
calcd for C H N O [M + H] 395.2118, found 395.2115. HPLC purity: 99.2%.
27
26
2
(
E)-3-(1-(2-Fluorobenzyl)-1H-benzo[d]imidazol-2-yl)-1-(4-isopropylphenyl)prop-2-en-1-one (4s). 1-[4-(1-
◦
methylethyl)phenyl]ethanone and 3a were used as reactants to give 4s. Yield: 72%, mp: 166.5–167.8 C.
1
H-NMR (CDCl3)
δ 8.41 (d, J = 15.0 Hz, 1 H), 8.08 (d, J = 8.4 Hz, 2 H), 7.90–7.85 (m, 2 H), 7.39–7.27
(
(
1
4
m, 6 H), 7.15–7.10 (m, 1 H), 7.01 (t, J = 7.6 Hz, 1 H), 6.80 (t, J = 8.3 Hz, 1 H), 5.60 (s, 2 H), 3.04–2.96
m, 1 H), 1.29 (d, J = 6.9 Hz, 6 H). 13C-NMR (CDCl3)
188.5, 160.1 (d), 155.2, 148.8, 143.3, 135.8, 135.2,
30.0 (d), 129.2, 128.7, 128.0 (d), 127.4, 126.9, 124.8 (d), 124.4, 123.7, 122.8 (d), 120.4, 115.8 (d), 110.2,
δ
+
1.1 (d), 34.5, 23.7. HRMS (ESI): calcd for C H FN O [M + H] 399.1867, found 399.1862. HPLC
26 23 2
purity: 97.8%.
(
3
E)-4-(3-(1-(4-Methylbenzyl)-1H-benzo[d]imidazol-2-yl)acryloyl)benzonitrile (4t). 4-Acetylbenzonitrile and
◦
b were used as reactants to give 4t. Yield: 82%, mp: 208.6–209.7 C. H-NMR (CDCl ) δ 8.33
3
1
(
7
1
d, J = 15.0 Hz, 1 H), 8.18 (d, J = 8.2 Hz, 2 H), 7.91–7.79 (m, 4 H), 7.36 (d, J = 7.2 Hz, 3 H),
.12 (d, J = 7.9 Hz, 2 H), 7.00 (d, J = 7.9 Hz, 2 H), 5.52 (s, 2 H), 2.30 (s, 3 H). 13C-NMR (CDCl3)
187.7,
48.0, 143.2, 140.5, 138.2, 136.1, 132.6, 132.5, 129.8, 129.5, 129.1, 127.1, 126.2, 124.8, 123.9, 120.5, 117.9,
δ

Molecules 2020, 25, 3180
14 of 18
+
1
16.6, 110.5, 47.0, 21.1. HRMS (ESI): calcd for C H N O [M + H] 378.1601, found 378.1606. HPLC
25 19 3
purity: 97.6%.
(
3
E)-4-(3-(1-(2-Fluorobenzyl)-1H-benzo[d]imidazol-2-yl)acryloyl) benzonitrile (4u). 4-Acetylbenzonitrile and
◦
a were used as reactants to give 4u. Yield: 79%, mp: 167.5–169.4 C. H-NMR (CDCl ) δ 8.34
3
1
(d, J = 14.9 Hz, 1 H), 8.20 (d, J = 8.4 Hz, 2 H), 7.93 (d, J = 14.9 Hz, 1 H), 7.86 (d, J = 6.7 Hz, 1 H),
7
1
1
.82 (d, J = 8.4 Hz, 2 H), 7.41–7.30 (m, 4 H), 7.16–7.11 (m, 1 H), 7.03 (t, J = 7.4 Hz, 1 H), 6.82 (t, J = 7.5 Hz,
H), 5.61 (s, 2 H). 13C-NMR (CDCl3)
187.7, 160.1 (d), 148.1, 143.3, 140.4, 135.9, 132.6, 130.2 (d), 129.3,
29.1, 128.1 (d), 127.2, 124.9, 124.8 (d), 124.0, 122.7 (d), 120.6, 117.9, 116.6, 115.9 (d), 110.3, 41.2 (d).
δ
+
HRMS (ESI): calcd for C H FN O [M + H] 382.1350, found 382.1352. HPLC purity: 95.4%.
24
16
3
3
.2. Biology
3
.2.1. Topo II Mediated DNA Relaxation Assay In Vitro
We used Topo II assay kit (TopoGEN, Buena Vista, CO, USA) to study the effects of compounds
on Topo II-catalyzed DNA relaxation according to the kit instructions with minor modifications.
Relaxation assays were performed in a final volume of 20 L in Topo II reaction buffer (50 mM Tris-HCl,
pH 8.0, 150 mM NaCl, 10 mM MgCl , 2 mM ATP, 0.5 mM DTT, and 30 g/mL BSA) with 0.2
of pBR322 DNA (TaKaRa, Shiga, Japan). Then compounds and 1 U human Topo II were added to
µ
µ
µg
2
the reaction at a constant solvent volume. Etoposide (100
The reaction was incubated for 30 min at 37 C. The reaction was stopped with 5
µ
M) was used as a comparative control.
◦
×
stop buffer (5
µL
per 20
glycerol. The mixtures were applied to 1% agarose gel and subjected to electrophoresis for 1.5 h at
5 V, in TAE buffer (40 mM Tris-acetate, 2 mM EDTA). Gels were stained for 30 min in an aqueous
solution of Ged Red (0.5 g/mL). DNA bands were visualized by UV light transillumination and then
µ
L reaction volume). 5 stop buffer contained 5% sarcosyl, 0.02% bromophenol blue, and 25%
×
7
µ
photographed with an Alpha Innotech digital imaging system (Boston Microscopes, Boston, MA, USA).
Semi-quantification was performed on western blot gel photographs by using software ImageJ 1.53a,
with positive control (lane D: pBR322 DNA) as 100%, negative control (lane T: pBR322 DNA + Topo II)
as 0% and comparative control (lane E: pBR322 DNA + Topo II + etoposide 100 µM).
3
.2.2. Topo II Mediated DNA Cleavage Assay
The mixture of Topo II (6 units), 0.1 g of supercoiled pBR322 DNA, and 50
or 100 M etoposide) were performed in a final volume of 20 L in Topo II reaction buffer (50 mM
µ
µM compounds
(
µ
µ
Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM MgCl , 2 mM ATP, 0.5 mM DTT, and 30
After incubation at 37 C for 6 min to achieve cleavage relegation equilibrium, the cleavage intermediates
µ
g/mL BSA).
2
◦
were captured by the addition of 2
at pH 8.0. Proteinase K (2
µ
L of 1% SDS followed by the addition of 2
µ
L of 250 mM Na EDTA
4
◦
µ
L, 0.8 mg/mL) was added and the reactions were incubated at 45 C for
3
0
2
0 min to digest Topo II. The sample was mixed with 5
.5% bromophenol blue and 0.5% xylene cyano FF in 10 mM Tris-HCl, pH 7.9) and heated at 72 C for
min. Electrophoresis was carried out in a 1% agarose gel in TAE buffer (40 mM Tris-acetate and 2 mM
µ
L of agarose gel loading buffer (30% sucrose,
◦
EDTA) at 75 V for 1 h. The gel was stained in Ged Red aqueous solution (0.5 µg/mL) for 30 min and
kept on electrophoresis at 75 V for 30–45 min. Cleavage was monitored by the conversion of negatively
supercoiled plasmid to nicked DNA. DNA bands were visualized by UV light and photographed with
an Alpha Innotech digital imaging system. Semi-quantification was performed on western blot gel
photographs by using software ImageJ 1.53a.
3
.2.3. DNA Unwinding Assay
The relaxed pBR322 DNA used for this unwinding assay was previously relaxed with Topo I
in the reaction buffer (50 mM Tris-HCl, pH = 7.5, 50 mM KCl, 10 mM MgCl , 0.5 mM DTT, 0.1 mM
2
EDTA, and 30 µg/mL BSA). Then other components were added. Assay mixtures contained 0.1 µg

Molecules 2020, 25, 3180
15 of 18
of relaxed pBR322 plasmid DNA, Topo I (1 units), and compounds in 20
µL of Topo I reaction buffer.
After incubating the DNA and drug for 10 min at room temperature, Topo I was added, and the
◦
reaction was incubated at 37 C for 30 min. The sample was mixed with 5
µL of agarose gel loading
buffer (30% sucrose, 0.5% bromophenol blue and 0.5% xylene cyano FF in 10 mM Tris-HCl, pH 7.9).
Samples were subjected to electrophoresis in 1 TAE buffer (40 mM Tris-acetate, 2 mM EDTA). DNA
×
bands were visualized by transillumination with UV light and then photographed with an Alpha
Innotech digital imaging system (Boston Microscopes).
3
.2.4. In Vitro Anti-Proliferation Assay
MTT assay was used to measure the growth inhibitory effects of 4a–4u in various cancer cell
lines. HepG2, A549, LNCaP, MG63 cells obtained from National Infrastructure of Cell Line Resource
Beijing, China). HepG2, LNCaP and MG63 cells were cultured with DMEM medium supplemented
(
with 10% FBS, while A549 were with RPMI-1640 medium. Each cell line was plated at a density of
◦
5
2
000 per well in 96-well microplates, incubated at 37 C in a humidified incubator with 5% CO for
2
4 h. Compounds 4a
–
4u were added to the wells at increasing concentrations (0–50
µM). After 48 h,
the medium was replaced with new medium (100
were further incubated at 37 C for 4 h. At the end of the incubation, the untransformed MTT was
µ
L, containing 50
µ
g MTT) in each well. The cells
◦
removed, and 100 µL of DMSO was added. The microplates were well shaken to dissolve the formazan
dye, and the absorbance at 570 nm was measured using a microplate reader (Bio-Tek). The amount of
absorbance (A570) is proportional to the cell numbers. The inhibition rates of different concentrations of
test compounds were calculated with the following equation. The IC50 values of test compounds were
calculated after nonlinear regression of logarithmic concentrations and cell proliferation inhibition rates:
ꢀ
ꢁ
A570control − A570sample × 100
Cell proliferation inhibition rate % =
%
A570
control
3
.2.5. Clonogenic Assay
To determine the population dependence and proliferative capacity of A549 cells after treatment
of compounds, the clonogenic assay was implemented on the basis of the method previously reported
with minor changes. A549 cells were plated at a density of 700 per well in six-well plates and grown at
◦
3
7 C for 24 h and then treated with increasing concentrations of compound (0–3
µM) for another 24 h,
subsequently, medium was replaced with fresh medium and incubated for two weeks. The colonies
developed were fixed with methanol and stained using 0.4% trypan blue. The number of colonies in
each plate was photographed with a digital camera and then counted with ImageJ 1.53a.
3
.2.6. Cell Migration Assay
5
Approximately 3
Wound generation was performed using a 10
was washed twice with PBS to remove the floating/detached cells. After, the cells were exposed to each
of 4d and 4n (1 and 2 M) for 24 h and were allowed to migrate in the medium (RPMI-1640 with 2%
×
10 A549 cells per well were cultured in a six-well plate to 80% confluence.
µL pipet tip across the center of the well and each well
µ
FBS). The wound was assessed by a JuLI Intelligent fluorescent cell monitor (NanoEnTek, Seoul, South
Korea) at different time points (0, 24, 48 and 72 h).
3
.2.7. DAPI Staining
Apoptotic cell death induced by 4d or 4n was examined by fluorescence microscopy using
4
DAPI staining. Briefly, A549 cells were seeded to six-well plate in a density of 8
×
10 per well and
incubated with or without the indicated concentration of 4d or 4n for 48 h. Then DAPI (1 µg/mL final
concentration) was added and incubated for 10 min. After one wash with PBS, cells were photo-graphed
using a Olympus CX31 Fluorescence Microscope (Olympus, Shinjuku, Japan). Apoptotic cells were

Molecules 2020, 25, 3180
16 of 18
identified by morphologic alterations (nuclear condensation and cell fragmentation). The photographic
analyses and quantification of apoptotic cells were performed by using software ImageJ 1.53a. NAF
was calculated by the product of average nuclear area and circularity.
4
. Conclusions
In conclusion, a novel series of benzimidazole-chalcone hybrids were designed and synthesized as
potential Topo II-targeting anticancer agents. Most of the tested compounds displayed strong inhibitory
activity against the catalytic function of Topo II. Mechanistic studies indicated that 4d and 4n act as
non-intercalative Topo II catalytic inhibitors. The BCHs displayed favorable antiproliferative activities
with low micromolar IC50 values toward the four tested cancer cell lines, especially on A549 cells.
Hybrid 4d and 4H not only showed excellent anti-proliferative activity with respective IC50 values
of 3.6, 3.8 µM on A549 cells, but also inhibited A549 cell migration and probably induced cancer cell
apoptosis. The mode of action postulated by the molecular docking suggests a possible occupation of
the ATP binding pocket of Topo II by 4d and 4n. This research identified novel benzimidazole-chalcone
hybrids and diversified the scope of Topo II inhibitors, which would provide valuable foundation for
the research on anticancer drugs. Ideally further research on pharmaceutical kinetics and toxicity of the
optimized compounds may forward one step further to the discovery of a novel chemotherapeutic drug.
Supplementary Materials: The following are available online, H- and 13C-NMR spectra of final compounds,
1
detailed docking results of 4d, 4n and original ligand with Topo II (1ZXM), Table S1: Total scores of 4n, 4d and
original ligand of Topo II (1ZXM), Figure S1: Docking model of 4n with Topo II protein (1ZXM), Figure S2:
Docking model of 4d with Topo II protein (1ZXM), Figure S3: Docking model of Topo II protein (1ZXM)with its
original ligand, Figure S4: Docking results of Topo II protein (1ZXM)with its original ligand.
Author Contributions: Conceptualization, W.Z. (Wei Zhou); Formal analysis, Y.P.; Investigation,
W.Z. (Wenjin Zhang); Methodology, W.Z. (Wenjin Zhang); Project administration, Z.D.; Resources, L.Z.; Supervision,
Z.-H.J.; Validation, L.Z.; Writing–original draft, W.Z. (Wei Zhou); Writing–review & editing, W.Z. (Wei Zhou) and
Z.D. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by National Science Foundation of China (grant number: 81502911) and
Municipal Program for Science and Technology of Guangzhou (grant number: 202002030212).
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
Topo II type II topoisomerase
BCHs benzimidazole-chalcone hybrids
IC50
ATP
EB
DAPI
ADP
half maximal inhibitory concentration
adenosine triphosphate
ethidium bromide
0
4 ,6-diamidino-2-phenylindole
adenosine diphosphate
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Sample Availability: Samples of the compounds are available from the authors.
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