Synthesis, biological evaluation and molecular docking of benzimidazole grafted benzsulfamide-containing pyrazole ring derivatives as novel tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.12.031

Tubulin-targeting drugs have increasingly become the focus of anticancer drugs research. Twenty-five novel benzimidazole grafted benzsulfamide-containing pyrazole ring derivatives were synthesized and evaluated for bioactivity as potential tubulin polymerization inhibitors. Among them, compound 30 showed the most excellent inhibition against tubulin assembly (IC₅₀ = 1.52 μM) and in vitro growth inhibitory activity against a panel of four human cancer cell lines (IC₅₀ = 0.15, 0.21, 0.33 and 0.17 μM, respectively for A549, Hela, HepG2 and MCF-7). It could also validly induce A549 cell apoptosis, cause cell cycle arrest in G2/M phase and disrupt the cellular microtubule network. These results, along with molecular docking data, provided an important basis for further optimization of compound 30 as a potential anticancer agent.

Full text of DOI:10.1016/j.bmc.2018.12.031

Accepted Manuscript
Synthesis, biological evaluation and molecular docking of benzimidazole graf-
ted benzsulfamide -containing pyrazole ring derivatives as novel tubulin poly-
merization inhibitors
Yan-Ting Wang, Tian-Qi Shi, Hai-Liang Zhu, Chang-Hong Liu
PII:
S0968-0896(18)31676-6
https://doi.org/10.1016/j.bmc.2018.12.031
BMC 14680
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
26 September 2018
14 December 2018
21 December 2018
Please cite this article as: Wang, Y-T., Shi, T-Q., Zhu, H-L., Liu, C-H., Synthesis, biological evaluation and
molecular docking of benzimidazole grafted benzsulfamide -containing pyrazole ring derivatives as novel tubulin
polymerization inhibitors, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.
2
018.12.031
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Synthesis, biological evaluation and molecular docking of benzimidazole grafted
benzsulfamide -containing pyrazole ring derivatives as novel tubulin
polymerization inhibitors
a,b,
a
a,
a,
Yan-Ting Wang *, Tian-Qi Shi , Hai-Liang Zhu *, Chang-Hong Liu *
a
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210093, P. R. China
b
Department of Environmental Science & Engineering, Fudan University, Shanghai
2
00433, P. R. China
*Corresponding authors. Tel. & fax: +86-25-89682572; e-mail: zhuhl@nju.edu.cn,
chliu@nju.edu.cn
Effect of compound 30 on cell cycle progression of A549 cells and G2/M-related
proteins; 3D diagram of the interaction between compound 30 and the colchicine
binding site.
A series of novel benzimidazole grafted benzsulfamide -containing pyrazole ring
derivatives were designed, synthesized and evaluated for the inhibitory activity
against tubulin polymerization and cancer cells. Also the docking simulation of these
compounds was conducted.
1

Abstract: Tubulin-targeting drugs have increasingly become the focus of anticancer
drugs research. Twenty-five novel benzimidazole grafted benzsulfamide -containing
pyrazole ring derivatives were synthesized and evaluated for bioactivity as potential
tubulin polymerization inhibitors. Among them, compound 30 showed the most
excellent inhibition against tubulin assembly (IC50 = 1.52 μM) and in vitro growth
inhibitory activity against a panel of four human cancer cell lines (IC50 = 0.15, 0.21,
0
.33 and 0.17 μM, respectively for A549, Hela, HepG2 and MCF-7). It could also
validly induce A549 cell apoptosis, cause cell cycle arrest in G2/M phase and disrupt
the cellular microtubule network. These results, along with molecular docking data,
provided an important basis for further optimization of compound 30 as a potential
anticancer agent.
Keywords: benzimidazole; benzsulfamide; pyrazole; tubulin; inhibition; apoptosis;
molecular docking
2

1
. Introduction
Microtubules are polar cytoskeletons, which are assembled by the aggregation of
α- and β- tubulin heterodimers in a head-to-tail manner to form hollow cylindrical
filaments. [1] They are structural components of the mitotic spindle, and dynamic
microtubules play an important role in several cellular processes including
intracellular trafficking, cell migration, cell skeleton development and cell division. [2]
These functions are probably correlated with post-translational modifications such as
acetylation, polyglycylation, polyglutamylation, and tyrosination of tubulin. [3] In the
M-phase of cell cycle, microtubules assembled as a spindle direct the chromosomes
into individual cells; they therefore are crucial targets for the development of
anticancer agents. Compounds that interfere with the microtubulin equilibria in cells
are very effective in the treatment of human cancer [4, 5]. They interfere with this
dynamic equilibrium by binding to tubulins and induce cell cycle arrest, resulting in
cell death [6, 7].
Natural products such as paclitaxel, vinca alkaloids, and colchicine a (Fig. 1)
were found to bind to microtubules at specific binding sites and thus display
remarkable microtubule-targeting activity. These microtubule-targeting compounds
are functionally categorized into microtubule-stabilizers and microtubule-destabilizers
[
8]. The taxanes such as paclitaxel and docetaxel stabilize microtubules by preventing
depolymerization of tubulin. The vinca alkaloids (eg., vincristine, vinblastine, and
vinorelbine) and colchicine inhibit the polymerization of tubulin [6, 9]. Disruption of
tubulin dynamics leads to cell cycle arrest in the G2/M phase and induction of
apoptosis [10, 11].
Many molecular modeling studies and experimental data have demonstrated how
these inhibitors bind to the active site of tubulin. [12-15]. Colchicine binding is
considered a universal property of higher eukaryotic tubulin [16]. Compounds that
bind to the colchicine site have been the subject of researchers’ efforts to find new
agents that can address the limitations of existing tubulin-targeted drugs. [17, 18]
Combretastatin A-4 (CA-4) b (Fig. 1) is the most active member of the combretastatin
family, isolated from the African tree Combretum caffrum. CA-4 exhibits strong
3

antitubulin activity by binding to the colchicine-site and underwent phase II and phase
III studies in clinical. [19]
Over the past decade, a growing number of synthetic compounds related to
benzenesulfonamide have been reported to inhibit tubulin polymerization, such as
ABT-751 (Fig. 1) [20], indisulam (Fig. 1), HMN-214 (Fig. 1) [21] and
1
-phenylsulfonyl-2-(1-methylindol-3-yl)-benzimidazole f (Fig. 1) [22]. Mei-Jung
Lai’s earlier work on the modification of ABT-751 revealed that the
benzenesulfonamide moiety is crucial for the compound’s anticancer activity. [22-24]
In addition, biaryl moieties have been frequently observed in the development of
antitubulin agents. [25, 26] Compound g (Fig. 1), with both benzenesulfonamide and
biaryl moieties, was found in previous study to have remarkable antiproliferative
activity. [24] Literature surveys indicated that the N-sulfonyl-aminobiaryl moiety has
been comprehensively included in various heterocycles such as carbazole, [27]
phenanthridinones, and phenanthridines, benzimidazole. [28, 29]
In order to search for novel antitubulin agents, we designed a class of
N-sulfonyl-aminobiaryl moiety analogs based on benzimidazole grafted pyrazole
scaffold. We chose benzimidazole and pyrazole moieties because they showed widely
biological activities. Benzimidazole is a heterocyclic compound known for its
antibacterial, antifungal, antiparasitic, anti-inflammatory and antiviral activities. [30]
Some studies have also shown that benzimidazole-containing drugs have good
antitumor activity [31-35], such as Nocodazole h and compound i (Fig. 1). In addition,
pyrazoline derivatives have been reported to possess anticancer, antimicrobial and
anti-inflammatory activities. [36-39], such as compound j (Fig. 1).
To our knowledge, no reports have been dedicated to the synthesis of
benzimidazole grafted benzsulfamide -containing pyrazole ring derivatives. This
combination was suggested in an attempt to investigate the inhibitory activity against
tubulin. Through a combined virtual and experimental screening approach, we
identified several compounds as potent tubulin inhibitors, especially compound 30.
4

Fig. 1. Potential inhibitors and new inhibitor of tubulin polymerization.
. Results
.1. Chemistry
The synthesis of compounds 10, 12-35 followed the general pathway was
2
2
outlined in Scheme 1. The target compounds were obtained in four steps as described
in experimental section. All of the synthetic compounds are being reported for the
1
first time (Table 1) and gave satisfactory analysis and spectroscopic data. H NMR,
13C NMR, melting test, mass spectroscopy and elemental analysis, and analysis results
were in full accordance with their depicted structures.
5

Scheme 1. General synthesis of novel compounds (10, 12-35). I: benzene sulfonyl
chloride; II: p-toluenesulfonyl chloride; III: 4-methoxy benzene sulfonyl chloride; IV:
2
4
4
,5-dimethoxy sulfonyl chloride; V: 4-nitrobenzenesulfonyl chloride; VI:
-fluorobenzenesulfonyl; VII: 4-chlorobenzene sulfonyl chloride; VIII:
-bromobenzene sulfonyl chloride; IX: 4-trifluoromethyl sulfonyl chloride; 1, 4, 7, 10:
R
1
= CH
3
; 2, 5, 8, 11: R
1
= F; 3, 6, 9, 12: R
1
= CF
3
; 13-35: (Table 1). Reagents and
, 0℃, 15min, rt, 3 h; (c)
conditions: (a) CH
3
CH ONa, H
2
2
O, rt, 4 h; (b) DMF, POCl
3
Sodium metabisulfite, DMF, 110 ℃, 4 h; (d) EDC·HCl, triethylamine, CH
2
Cl , room
2
temperature, 12 h.
6

Table 1. Structures of compounds 10, 12-35
Compounds
R1
R2
Compounds
R1
-F
R2
4-F
1
1
1
1
1
1
1
1
1
2
2
2
2
0
2
3
4
5
6
7
8
9
0
1
2
3
-CH
3
–
24
25
26
27
28
29
30
31
32
33
34
35
-CF
-CH
-CH
-CH
-CH
-CH
-CH
-CH
3
–
H
-F
-F
-F
4-Cl
3
3
3
3
3
3
3
4- Br
4-CH
3
4-CF
3
2,5-(CH
4-F
3
)
2
-CF
-CF
-CF
-CF
-CF
-CF
-CF
-CF
3
H
3
3
3
3
3
3
3
4-OCH
3
4-Cl
2,5-(CH
)
3 2
4-Br
4-NO
2
4-CF
3
4-F
-F
-F
-F
-F
H
4-Cl
4-Br
4-OCH
2,5-(CH
4-NO
3
3
)
2
4-CF
3
2
2
.2. Crystal structure determination
The crystal structures of compounds 20 and 24 were determined by X-ray
diffraction analysis. The crystal data presented in Table 2 and Fig. 2 gave the
perspective views of 20 and 24 with the atomic labeling system. The crystallographic
data have been deposited at the Cambridge Crystallographic Data Centre (CCDC) and
the deposition numbers of 20 and 24 are 1514296 and 1514297, respectively.
7

Table 2. Crystallographic and Experimental Data for compounds 20 and 24
Compounds
Formula
Formula weight
Crystal system
Space group
a (Å)
20
24
C
28
H
19FN
4
O
2
S
C
30
H
24
F
2
N
4
3
O S
494.53
558.59
Monoclinic
C2/c
Monoclinic
P-1
29.137(9)
8.721(3)
21.941(7)
90.00
8.2599(3)
9.0030(3)
19.0369(6)
77.3790(10)
79.4050(10)
82.735(2)
1352.39(8)
2
b (Å)
c (Å)
α(o)
β(o)
113.919(8)
90.00
γ(o)
V(Å3)
5097(3)
8
Z
-3
Dc(g•cm )
1.289
1.372
-1
μ(mm )
F(000)
0.166
0.173
2048
580
θrang(o)
2.03-28.82
69038
2.22-28.30
29434
Reflns collected
Reflns unique
Goodness-of-fit on F2
6485
6551
1.080
1.000
R1, wR
R1, wR
Max, minΔρ(e Å )
2
[I>2σ(I)]
0.2394, 0.5698
0.2937, 0.6669
1.320, -1.093
0.1356, 0.3521
0.1700, 0.4243
1.202, -0.517
2
[all data]
-3
8

Fig. 2. (A) Crystal structure diagram of compound 20. (B) Crystal structure diagram
of compound 24. H atoms are shown as small spheres of arbitrary radii.
2
2
.3. Biological evaluation
.3.1. In vitro antiproliferative activities (cell viability was assessed by MTT
assay [40])
All novel derivatives 10, 12-35 were evaluated for their inhibitory effect on
growth of four cancer cell lines [human lung adenocarcinoma epithelial cell line
A549), carcinoma of cervix cell line (Hela), human hepatoma cell line (HepG2) and
(
human breast cancer cell (MCF-7)] with positive contrast drugs colchicine and CA-4.
Cells were treated with different concentrations of the given compounds, and the
viabilities were measured by MTT assay. Results (depicted in Table 3) showed that
most of the target compounds can effectively inhibit the proliferation of the four
tumor cells, especially for A549 cells. Compounds 22, 29, 30 showed better
antiproliferative activities than colchicine (IC50 = 0.22 μM for A549, IC50 = 0.36 μM
9

for Hela, IC50 = 0.42 μM for HepG2, IC50 = 0.44 μM for MCF-7), and compound 30
is better than CA-4 (IC50 = 0.16 μM for A549, IC50 = 0.24 μM for Hela, IC50 = 0.33
μM for HepG2, IC50 = 0.18 μM for MCF-7). The IC50 values of these compounds
against the four cell lines were between 0.15 and 7.26 μM.
According to the data in Table 3, we predicted that the difference of biological
activities among the tested compounds might be correlated to the variation and
modifications of the structure. For example, compounds 10 and 12 showed lower
bioactivities than any other novel compounds, which demonstrated that
benzenesulfonyl was an important structure contributing to the bioactivities of these
compounds.
1
Moreover, various substituents of R in the para-position of A-ring led to
different bioactivities, if the substituents of B-ring kept immutability. Basically, the
bioactivities of the compounds were increased in the following order: CF
3
>F>CH ,
3
which could be observed from the comparison: 30>22>15, 29>21, 28>20>13, 31>23,
3
5>27>19, 34>26>18, 33>25>17, 12>10.
Compounds with different groups in the B ring were compared in a similar way,
which showed electron-donating groups (such as OCH
preferable to electron-withdrawing groups (such as NO
gradients for compounds were
3
, CH ) in the B ring were
3
2
, F). For instance, the active
29>28>31>35>34>32>33,
2
2>21>20>23>27>26>24>25, 15>14>13>19>18>16>17. In addition, the more
electron-donating substituted groups in the B-ring, the better the antiproliferative
activity it was, and this could be demonstrated from the comparison: 15>14.
From the analysis mentioned above, some structure-activity relationship (SAR)
could be concluded: the presence of benzenesulfonyl was crucial in the cancer cell
inhibition; compounds with trifluoromethyl substituted para-position of A-ring and
methoxy and methyl substituted B-ring were found to be the most favorable for the
anticancer activity. Based on structure, activity, and structure-activity analysis, we can
conclude that compound 30 is the most bioactive compound against proliferation of
cancer cell lines A549, Hela, HepG2 and MCF-7 with IC50 values of 0.15, 0.21, 0.33
and 0.17 μM, respectively.
10

Table 3. In vitro Inhibition of Tubulin Polymerization and growth inhibition of A549,
Hela, HepG2 and MCF-7 cells by compounds 10, 12-35, combretastatin A-4 and
colchicine.
a
b
Compounds
IC50 ± SD (μM)
Colchicine binding
Vinblastine binding
d
e
inhibition %± SD
inhibition %± SD
A549
Hela
HepG2
MCF-7
Tubulinc
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
0
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
4.01 ± 0.43
3.62 ± 0.21
0.76 ± 0.20
0.46 ± 0.35
0.27 ± 0.36
3.24 ± 0.43
3.55 ± 0.52
1.86 ± 0.27
1.46 ± 0.02
0.55 ± 0.16
0.35 ± 0.04
0.20 ± 0.14
1.03 ± 0.35
2.37 ± 0.07
3.17 ± 0.25
1.72 ± 0.31
1.28 ± 0.42
0.48 ± 0.06
0.17 ± 0.51
0.15 ± 0.05
0.79 ± 0.01
1.74 ± 0.71
2.56 ± 0.06
1.69 ± 0.27
0.86 ± 0.04
0.22 ± 0.12
0.16 ± 0.04
4.95 ± 0.04
4.67 ± 0.32
0.82 ± 0.09
0.48 ± 0.21
0.37 ± 0.05
3.67 ± 0.17
4.06 ± 1.04
2.34 ± 0.06
1.52 ± 0.12
0.68 ± 0.42
0.39 ± 0.37
0.32 ± 0.08
1.17 ± 0.29
2.44 ± 0.02
3.45 ± 0.14
2.04 ± 0.29
1.43 ± 0.33
0.56 ± 0.46
0.25 ± 1.22
0.21 ± 0.67
0.95 ± 2.01
2.19 ± 0.35
3.06 ± 0.48
1.86 ± 0.22
0.97 ± 0.07
0.36 ± 0.07
0.24 ± 0.08
6.26 ± 0.08
6.03 ± 0.23
0.96 ± 0.31
0.52 ± 0.02
0.46 ± 1.03
5.06 ± 0.35
5.52 ± 0.16
3.04 ± 0.27
1.93 ± 0.09
0.72 ± 1.24
0.47 ± 0.63
0.41 ± 0.45
1.34 ± 0.05
3.52 ± 1.07
4.23 ± 0.38
2.55 ± 0.25
1.68 ± 0.81
0.59 ± 0.15
0.37 ± 0.72
0.33 ± 0.06
1.07 ± 0.90
2.73 ± 1.24
3.93 ± 0.28
2.16 ± 0.56
1.12 ± 0.73
0.42 ± 0.12
0.33 ± 0.12
7.26 ± 0.47
6.33 ± 0.38
0.83 ± 0.62
0.57 ± 0.29
0.51 ± 0.74
5.27 ± 0.65
5.93 ± 0.86
3.16 ± 0.09
2.34 ± 1.07
0.69 ± 0.48
0.53 ± 0.62
0.43 ± 0.73
1.37 ± 0.46
3.67 ± 1.24
4.96 ± 1.08
2.63 ± 1.46
1.58 ± 0.49
0.63 ± 0.72
0.42 ± 0.06
0.17 ± 0.23
1.13 ± 0.17
3.12 ± 0.04
4.32 ± 0.23
2.47 ± 0.26
1.24 ± 0.18
0.44 ± 0.18
0.18 ± 0.17
45.73 ± 1.21
44.66 ± 0.49
3.84 ± 0.74
2.64 ± 0.81
1.97 ± 0.33
29.13 ± 0.16
32.04 ± 0.28
17.69 ± 0.05
11.32 ± 0.19
3.49 ± 0.21
2.31 ± 0.44
1.82 ± 1.32
7.27 ± 0.79
20.33 ± 1.28
25.67 ± 0.56
14.08 ± 1.33
8.06 ± 0.05
2.96 ± 0.39
1.63 ± 0.06
1.52 ± 1.07
4.56 ± 0.73
16.73 ± 0.58
21.36 ± 1.05
12.67 ± 1.32
5.78 ± 0.66
2.26 ± 0.25
1.61 ± 0.31
40 ± 1.4
44 ± 2.9
74 ± 0.9
66 ± 0.2
82 ± 1.6
53 ± 0.1
38 ± 0.7
48 ± 0.9
55 ± 0.1
62 ± 1.6
77 ± 0.8
88 ± 0.4
51 ± 4.2
48 ± 0.5
48 ± 0.4
51 ± 1.3
50 ± 0.7
61 ± 2.4
85 ± 1.3
91 ± 4.1
63 ± 0.6
54 ± 0.6
40 ± 0.6
52 ± 0.3
54 ± 3.3
—
0.05 ± 1.5
-0.03 ± 2.1
0.21 ± 1.4
0.18 ± 2.8
0.07 ± 1.5
0.07 ± 0.3
0.24 ± 0.4
-0.38 ± 1.4
0.21 ± 2.6
0.05 ± 1.4
2.98 ± 1.2
0.62 ± 0.8
-0.04 ± 0.3
1.32 ± 1.1
-2.09 ± 2.7
3.02 ± 2.2
0.51 ± 2.7
0.52 ± 3.6
0.03 ± 2.3
0.06 ± 1.7
0.14 ± 4.2
0.65 ± 4.4
0.73 ± 1.7
-0.07 ± 2.1
0.05 ± 2.4
—
Colchicinef
CA-4f
89 ± 1.4
—
11

All experiments were independently performed at least three times.
aIC50 values, which include those of the parent compounds colchicine and CA-4, are
expressed as the compound concentration (μmol/L or μM) that causes 50% inhibition
of cell growth. The values are the average (±SD) of three different measurements
performed as described in the Experimental Section.
bSD: standard deviation.
cInhibition of tubulin polymerization.
d
3
Inhibition of [ H]colchicine binding.
eInhibition of vinblastine binding.
fUsed as positive controls.
2
.3.2. Tubulin polymerization inhibition assay [41]
To investigate whether the antiproliferative activities of compounds 10, 12-35
derived from an interaction with tubulin or not, these agents were evaluated for their
inhibition of tubulin polymerization.
Before the inhibition assay, tubulin was extracted from pig brain and the purified
protein was identified using SDS-PAGE gel electrophoresis (Fig. 3A). Tubulin
inhibition results (Table 3) showed that these compounds exhibited different
inhibitory activities against tubulin polymerization with IC50 values ranging from 1.52
to 45.73 μM. Compound 30 showed similar or even better inhibitory activities than
the positive control agent CA-4 (IC50 = 1.61 μM) on tubulin polymerization. While
compounds 15, 22, 29 and 30 were better than colchicine (IC50 = 2.26 μM).
Compound 30 was the most potent compound as a tubulin polymerization inhibitor
with an IC50 of 1.52 μM, which was similar to that of antiproliferative activities
against the cancer cell lines. Moreover, most of the bioactive compounds showed not
only tubulin polymerization inhibition, but also growth inhibition, which indicated the
two bioactivities might be positively correlated.
To test the inhibitory effect of compound 30 on microtubule assembly in vitro,
depolymerization activity was measured by the method described by Bonne et al. with
some modification. [42] In the control samples, which were not treated with drugs, the
12

fluorescence intensity at 350 nm increased with time. Treating with colchicine, CA-4
or compound 30 resulted in various degrees of tubulin depolymerization (Fig. 3B).
Compared to the control, compound 30 appeared to be slightly inhibiting tubulin
polymerization at 1.0 μM. However, when concentration of compound 30 was
increased to 5.0 μM, tubulin polymerization was nearly completely blocked just as 8.0
μM CA-4 did, suggesting that tubulin polymerization was inhibited by compound 30
in a concentration-dependent manner. The inhibitory mechanism of compound 30 on
microtubule morphology was similar to that of CA-4.
Fig. 3. (A) SDS-PAGE gel electrophoresis of purified tubulin extracted from pig brain.
1
: Maker, 2 and 3: purified tubulin; (B) Effects assay of compound 30 on microtubule
morphology. The evaluation of tubulin polymerization is in the presence of compound
0, colchicine and CA-4 at 37 ℃.
3
13

In addition, the new benzimidazole grafted benzsulfamide -containing pyrazole
ring derivatives were examined for potential inhibition of the binding of
[3
H]Colchicine to tubulin in parallel with CA-4 (Table 3). All except 10, 12, 17 and
3
3 were strong inhibitors of the binding reaction (48 - 91% inhibition ratio). What’s
more, compound 30 were stronger than CA-4 (89% inhibition ratio). In this assay, it
was observed that compound 30 was the strongest inhibitor, which also proved that
these compounds target tubulin at colchicine domain target. Furthermore, the
synthesized compounds were also detected the binding site-specific competition with
another type of tubulin polymerization inhibitor vinblastine, finally no compounds
exerted significant competitive binding inhibition for vinblastine (Table 3). These
outcomes validly illustrated that the binding site of the new compounds was
colchicine binding site, not vinblastine binding site.
2
.3.3. Cytotoxicity test
All the target compounds were evaluated for their toxicity against human kidney
epithelial cell 293T and mouse primary hepatocytes with the median cytotoxic
concentration (CC50) data of tested compounds by the MTT assay. As shown in Table
4
, the minimum CC50 of the 25 test compounds against 293T cells and mouse primary
hepatocytes were 37.26 μM and 55.33 μM respectively, which indicated that these
compounds were free cytotoxic activities in vitro to the given cells.
Tabel 4. The median cytotoxic concentration (CC50) data of all compounds against
2
93T cell and mouse primary hepatocytes
a
a
CC50 (μM)
CC50 (μM)
Compounds
Compounds
Primary
hepatocytes
Primary
2
93T
293T
hepatocytes
231.28
>300
1
1
1
1
1
0
2
3
4
5
187.33
>300
25
26
27
28
29
93.12
238.93
241.32
128.38
>300
1
1
66.74
37.87
158.66
188.79
102.44
231.34
55.33
7
8
9.34
2.18
186.34
87.36
14

1
1
1
1
28.32
67.43
58.66
82.19
167.45
215.63
>300
223.10
163.21
156.53
75.32
132.48
178.43
83.28
1
1
1
1
2
2
2
2
2
6
7
8
9
0
1
2
3
4
30
31
32
89.32
173.24
167.49
137.34
152.78
178.32
33
9
3
5
6.32
7.26
4.20
231.16
105.34
121.67
>300
184.56
63.23
34
35
129.32
162.35
Colchicine
CA-4
1
2
89.32
18.32
174.37
aThe cytotoxicity of each compound was expressed as the concentration of compound
that reduced cell viability to 50% (CC50).
2
.3.4. Analysis of apoptosis by fluorescence-activated cell sorting [43, 44]
Based on the antiproliferative and antitubulin activities, compound 30 with the
most potent activity was chosen for deeper mechanism researches. In the apoptosis
experiment, A549 cells treated with different concentrations of compound 30 were
stained with annexin V-FITC/PI, and analyzed by flow cytometry. As depicted in Fig.
4
, it was clear that annexin-V was accumulated in the A549 cells after treated with
compound 30 at any given concentrations. Increase of the concentration of compound
3
0 could enhance the cell apoptosis by the ratio up to 7.98%, 12.64% and 18.84% at
.1, 0.3 and 0.5 μM respectively.
0
In addition, Apoptosis-related proteins were analyzed by western blot. These
proteins were Bax, PARP, Bcl-2 and Caspase 3. As shown in Fig. 4C, the level of
Bax was efficiently up-regulated and the level of anti-apoptotic proteins Bcl-2 was
down-regulated. Caspases, witch act as the central executioners of apoptosis, and their
activation are mediated by various inducers. Synthesized as proenzymes, caspases are
themselves activated by specific proteolytic cleavage reactions. Caspase-2, -8, -9, and
-
10 are termed apical caspases, which are usually the first to be activated in the
apoptotic process. Following their activation, they in turn activate effector caspases,
in particular caspase-3. Following treatment of A549 cells with compound 30 for 24 h
15

resulted in an up regulation of cleaved caspase-3 and PARP, which may be partially
responsible for the apoptotic tendency of the A549 cells.
Fig. 4. (A) Annexin V/PI dual-immuno-fluorescence staining after treatment
with different concentrations of compound 30 revealed significantly increased number
of apoptotic and necrotic cells. Cells treated with 0, 0.1, 0.3 and 0.5 μM compound 30
for 24 h were collected and processed for analysis. (B) Apoptosis cells percentage
showed in bar diagram. The alphabets a, b, c and d represent the existence of
significant difference (P < 0.05). (C) Western blot analysis of the levels of the
apoptosis-related proteins Bax, PARP, Bcl-2 and Caspase 3 after A549 cells were
treated with 0, 0.1, 0.3, 0.5 μM compound 30 for 24 h.
2
.3.5. Cell cycle analysis [45]
Here, we assessed the cell cycle distribution of A549 cells by flow cytometry
after treating with different concentrations (0, 0.1, 0.3, 0.5 μM) of compound 30 for
4 h. Result indicated that compound 30 arrested cells in the G2/M phase and
2
decreased cells in other phases of the cell cycle (Fig. 5A and 5B). In the vehicle
treated group, about 17.62% of A549 cells were distributed in the G2/M phase.
Compound 30 increased the proportion of cells in G2/M phase in a
concentration-dependent manner (Fig. 5A and 5B). About 22.63%, 28.13%, 40.26%
16

of cells were distributed in the G2/M phase when treated with 0.1, 0.3, 0.5 μM
compound 30 for 24 h respectively.
To explore the further underlying mechanisms of G2/M phase arrest induced by
compound 30, we examined the regulated effect on the expression of several related
proteins: cyclin B1, cdc2, cdc25c and P21, which could tightly control the cell cycle
progression. Cell arrest at the prometaphase/metaphase to anaphase transition is
normally regulated by the mitotic checkpoint. M phase promoting factor (MPF) is the
major regulator of the G2 to M transition, which is a complex made up of the catalytic
subunit of cdc2 and the regulatory subunit of cyclin B1. They are held in an inactive
state by phosphorylation of cdc2 at one negative regulatory site (Tyr15). This and
other regulatory complexes are activated at different points during the cell cycle and
can also be regulated by several exogenous factors. As shown in Fig. 5C, cyclinB1
was slightly up-reguated and cdc25c was down-regulated in a dose-dependent manner.
Also, we observed there is a reduction of cdc2 in the dose-dependent manner.
Meanwhile, p21 expression increased with compound 30 in a dose-dependent manner.
Fig. 5. (A) Effect of compound 30 on cell cycle progression of A549 cells was
determined by flow cytometry analysis. A549 cells were treated with different
concentrations (0.1, 0.3, 0.5 μM) of compound 30 for 24 h. (B) The percentage of
17

cells in each cycle phase was indicated. The alphabets a, b, c and d represent the
existence of significant difference (P < 0.05). (C) Western blot analysis of the levels
of the G2/M-related proteins Cyclin B1, cdc25c, cdc2 and P21. A549 cells were
treated with 0.1, 0.3, 0.5 μM compound 30 for 24 h.
2
.3.6. Microtubule disruption and morphological aberrations
Given the importance of the microtubule system in the maintenance of cellular
morphology, an assay that involved the disruption of microtubule morphology was
performed to reveal whether compound 30 could affect microtubule morphology in
living cells. Fig. 6 clearly showed that regular morphologies of microtubule were
turned into a dose-dependent fashion when incubated with compound 30. The
interphase microtubules exhibited fibrous network-like structures in the untreated
A549 cells. With the increasing of compound concentration, more cells were found to
be contracted and rounded at the dose of 0.2 μM. Besides, when the cells were treated
with 0.5 μM compound 30, spindle microtubules were disorganized and accompanied
by misaligned chromosomes from the metaphase plate. These morphological changes
of microtubules indicated that compound 30 dramatically disrupts the microtubule
morphology, which eventually leads to cell death.
18

Fig. 6. Effects of different concentration of compound 30 (0, 0.2 μM, 0.5 μM),
colchicine (0.2 μM) and CA-4 (0.2 μM) on interphase microtubules of A549 cells.
Microtubules tagged with rhodamine (red) and nuclei tagged with DAPI (blue) were
observed under a confocal microscope. The photomicrographs shown are
representative of at least three independent experiments performed.
2
.3.7. Effects of compounds on tumor volume after treating mouse models of
cancer
Based on in-vitro experiments, we detect in-vivo activity on mouse models of
cancer treated with compound 30, with CA-4 as a contrast. The mouse model was
established by intraperitoneal inoculation of rat hepatoma H22 cells in mice. Then the
mice model was administered intraperitoneally and the dosage was 3 mg/kg. The
results were depicted in Fig. 7. As shown in Fig. 7(a), the tumor volumes of the
mouse model treated with CA-4 and compound 30 were much smaller than that of the
control group; and from eighth to twenty-eighth days, the volume of the tumor after
compound 30 treatment was smaller than that of CA-4 treatment. As shown in Figure
7
(b), the mouse model treated with compound 30 had no significant weight loss,
while the CA-4 treated mouse model decreased more than that of the control group.
Therefore, it can be concluded that compound 30 has better anticancer activity than
CA-4.
Fig. 7. (a) Effects of compound 30 and CA-4 on tumor volume after treating mouse
models of cancer. (b) Effects of compound 30 and CA-4 on body weight of mice after
treating mouse models of cancer.
19

2
.4. Docking simulations
To gain better understanding on the potency of the synthesized compounds and
guide further SAR studies, we have proceeded to examine the interaction of the novel
-(1,3-diphenyl-1H-pyrazol-4-yl)-1-(phenylsulfonyl)-1H-benzo[d]imidazole
2
derivatives with tubulin crystal structure (PDB code: 1SA0).
Data of molecular docking simulations for the synthesized compounds 10, 12-35
were described in Fig. 8A, in which the predicted binding interaction energy was used
as the criterion for ranking. As depicted in Fig. 8A, compounds 15 (－56.09 kcal/mol),
2
2 (－56.15 kcal/mol), 29 (－56.5 kcal/mol) and 30 (－56.72 kcal/mol) showed lower
interaction energy than colchicine (－56.03 kcal/mol), and compound 30 showed the
lowest, which means that compound 30 exhibited the most potent affinity for tubulin
in prediction.
Then 2D, 3D maps and surface model structure of the most potent compound 30
with 1SA0 were depicted in Fig. 8 and Fig. 9 with 2D maps of three comparisons 29,
2
2 and 15. We could see in the figures, all the compounds 30, 29, 22 and 15 formed Pi
bonds with LYS 254 and LYS 352, and are only different in the interaction position.
Compounds 30 and 22 are same in the interaction positions that Pi bonds were formed
between ring E and LYS 254 (distance: 3.7 Å, 3.8 Å), and between ring B and LYS
3
52 (distance: 6.2 Å, 6.2 Å). For compound 29, one Pi bond was formed between ring
B and LYS 254 (distance: 4.7 Å), and the other bond was formed between ring D and
LYS 352 (distance: 6.6 Å). While compound 15 formed one Pi bond between ring A
and LYS 254 (distance: 5.9 Å), and formed the other Pi bond between ring C and
LYS 352 (distance: 6.9 Å). Thus, we could see that compound 30 formed the shortest
distance either with LYS 254, or with LYS 352. From the similar interaction we could
conclude that these two Pi bonds formed with LYS 254 and LYS 352 are important
for the affinity of compound and tubulin. In addition, compound 30 formed a
Pi-Sigma bonds between ring C and ASN 258 (distance: 2.8 Å) and two H bonds
between two fluorine atoms of ring A and CYS 241 (distance: 4.8 Å, 5.2 Å).
Moreover, Compound 22 also formed a Pi-Sigma bonds between ring C and ASN 258
20

(
distance: 2.9 Å), together with a H bond formed between a fluorine atom of ring A
and CYS 241 (distance: 4.8 Å).
In addition, we compared the tubulin-docking mode of compound 30 with
another compound reported by Romagnoli et al. 2012 [46]. There were similarities
between the two combination models. Both formed two hydrogen bonds with LYS
2
41 and ASN 258, witch proved again compound 30 a potent tubulin inhibitor.
21

Fig. 8. (A) The Cdocker-interaction energy (-kcal/mol) obtained from the docking
study of compounds 10, 12-35 and Colchicine by the CDOCKER protocol. (B-F) The
binding mode between the active conformation of compound 30 and the target protein
tubulin (PDB code: 1SA0) provided by the CDOCKER protocol (Discovery Studio
3
1
.5, Accelrys, Co. Ltd). (B) 2D molecular docking modeling of compound 30 with
SA0. (C) 3D model of the interaction between compound 30 and 1SA0 binding site.
(
D) 2D molecular docking modeling of compound 29 with 1SA0. (E) 2D molecular
docking modeling of compound 22 with 1SA0. (F) 2D molecular docking modeling
of compound 15 with 1SA0. The hydrogen bonds are displayed as dotted blue arrows
and dotted green line, and the Pi-cation bonds are shown as orange lines.
Fig. 9. The surface model structure to display the interaction between compound 30
and the targeted protein tubulin.
3
. Conclusion
twenty-five novel benzimidazole grafted benzsulfamide -containing pyrazole
ring derivatives were designed and synthesized in this study. Molecular docking
simulations were performed by inserting the compounds into the colchicine binding
site of tubulin. Compound 30 showed the lowest interaction energy ( －56.72
kcal/mol), which means that compound 30 exhibited the most potent affinity for
tubulin in prediction. Moreover, these novel compounds showed potent
22

antiproliferative activities and tubulin polymerization inhibition activities by inserting
into colchicine binding site of tubulin; arresting cells in G2/M phase and inducing cell
apoptosis. Among these compounds, 30 also showed the most excellent bioactivity.
These results strongly suggested that novel benzimidazole grafted benzsulfamide
-
containing pyrazole ring derivatives can be further developed as a promising
antitumor agent for the more efficacious treatment of advanced cancers.
4
4
. Experimental section
.1. Materials and measurements
All chemicals and reagents used in current study were analytical grade. Thin
1
layer chromatography (TLC), proton nuclear magnetic resonance ( H NMR) and
elemental microanalyses (CHN) were usually used. Analytical thin-layer
chromatography (TLC) was performed on the glass-backed silica gel sheets (silica gel
6
0 Å GF254). All compounds were detected using UV light (254 nm or 365 nm).
Separation of the compounds by column chromatography was carried out with silica
gel 60 (200 - 300 mesh ASTM, E. Merck). The quantity of silica gel used was 50-100
times the weight charged on the column. Melting points were determined on a XT4
1
MP apparatus (Taike Corp., Beijing, China). H NMR spectra were measured on a
Bruker AV-300 or AV-500 spectrometer at 25 ℃ and referenced to Me Si. Chemical
4
shifts are reported in ppm (δ) using the residual solvent line as internal standard.
Splitting patterns are designed as s, singlet; d, doublet; t, triplet; m, multiplet. 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
.2. General procedure for preparation of 1-phenyl-2-(1-(p-tolyl)ethylidene)
hydrazine (4)
P-methylacetophenone (compound 1, 6.71 g, 50 mmol) was added dropwise to a
solution of phenylhydrazine hydrochloride (8.68 g, 60 mmol) coupling with sodium
acetate (10.21 g, 75 mmol) in water (120 mL). After stirring at room temperature for
3
.5 h, a large amount of solid emerged. The yellow precipitate was filtered, washed
with water, and dried to give compound 4 (yields 95 %). The corresponding
23

substituted 1-phenyl-2-(1-phenylethylidene) hydrazines were synthesized following
this way to get pure compounds 5 and 6 with a yield of 91% and 96%.
4
.3.
-phenyl-3-(p-tolyl)-1H-pyrazole-4-carbaldehyde (7)
Compound 4 (8.97 g, 40 mmol) was dissolved in DMF (10 mL), and then was
added dropwise to a cold mixed solution of DMF (20 mL) and POCl (10 mL). After
General
procedure
for
preparation
of
1
3
stirring at 55 °C for 6 h, the mixture was poured into ice-cold water, with a saturated
solution of sodium hydroxide being added to neutralize the mixture. Then a large
amount of solid emerged, which was filtered, washed with water, and dried to give
compound
7
(yields
91
%).
The
corresponding
substituted
1
,3-diphenyl-1H-pyrazole-4-carbaldehydes were synthesized following this way to
receive pure compounds 8 and 9 with a yield of 88% and 93%.
4
.4.
-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazole (10)
A solution of o-phenylenediamine (3.24 g, 30 mmol) in DMF (20 mL) was
added to a suspension of compound 7 (10.51 g, 30 mmol) and sodium pyrosulfite
11.41 g, 60 mmol) in DMF (50 mL). The heterogeneous mixture was stirred at
10 ℃ for 4 h, then cooled and poured onto a lot of ice. A large amount of solid
General
procedure
for
preparation
of
2
(
1
would
emerge,
and
was
filtered
to
give
2
-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazole (10),
a
yellow
powder,
yields
83%.
The
corresponding
substituted
2
-(1,3-diphenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazoles were synthesized following
this way, and we got pure compounds 11 and 12 with a yield of 82% and 85%.
4
2
.5.
General
synthesis
method
of
-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1-(phenylsulfonyl)-1H-benzo[d]imidazol
e (13)
Compound 13 was synthesized from a stirring mixture of the material compound
0 (0.35 g, 1mmol) and compound I (benzenesulfonyl chloride, 0.26 g, 1.5 mmol)
1
with the help of 2 - 3 drops of triethylamine and EDC·HCl (0.21 g, 1.1mmol) in 30
mL dichloromethane at room temperature for 12 h. The reaction mixture was then
24

extracted with dichloromethane (15 mL × 3) and distilled water (15 mL), and the
organic layer was concentrated under vacuum to get the crude product. The crude
product was purified by silica gel column chromatography (200 - 300 mesh) eluted
with a mixture of ethyl acetate and petroleum ether (v/v = 1:8) to afford pure product
(
compound
13,
yields
71
%).
The
corresponding
substituted
2
-(1,3-diphenyl-1H-pyrazol-4-yl)-1-(phenylsulfonyl)-1H-benzo[d]imidazoles
compounds were synthesized following this way, and we got pure compounds 14 - 35
with yields of 69 - 87%.
4
.5.1 2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1-(phenylsulfonyl)-1H-benzo[d]
imidazole (13)
Yellow powder, yield: 80%. M. p: 235 - 236 ℃, H NMR (DMSO-d
1
6
, 400 MHz)
δ: 2.38 (s, 3H); 7.31 - 7.32 (m, 5H); 7.48 - 7.51 (m, 1H); 7.57 - 7.66 (m, 8H); 7.81 -
1
3
7
2
1
.83 (m, 2H); 7.96 - 7.98 (m, 2H); 9.30 (s, 1H). C NMR (DMSO-d , 100 MHz) δ:
6
1.38, 114.54, 119.54, 125.91, 126.31, 128.15, 128.25, 128.37, 128.99, 130.09,
30.44, 132.32, 133.32, 138.98, 139.46, 143.97, 151.56. ESI-MS: 490.58
+
(
C
29
H
22
N
4
O
2
S, [M+H] ). Anal. Calcd for C29
H N
22 4
2
O S: C, 71.00; H, 4.52; N, 11.42;
O, 6.52; S, 6.54; Found: C, 71.02; H, 4.54; N, 11.45.
4
.5.2 2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1-tosyl-1H-benzo[d]imidazole (14)
1
White powder, yield: 88%. M. p: 242 - 244 ℃, H NMR (DMSO-d
6
, 400 MHz)
δ: 2.27 (s, 3H); 2.37 (s, 3H); 7.11 (d, J = 7.6, 2H); 7.3 (d, J = 8, 2H); 7.46 - 7.50 (m,
3
2
1
1
H); 7.56 - 7.59 (m, 4H); 7.61 - 7.65 (m, 2H); 7.79 - 7.81 (m, 2H); 7.96 (d, J = 0.8,
13
H); 9.25 (s, 1H). C NMR (DMSO-d , 100 MHz) δ: 21.21, 21.35, 105.83, 114.52,
6
19.58, 125.91, 126.24, 128.16, 128.20, 128.40, 128.67, 130.08, 130.43, 132.46,
33.07, 138.62, 138.94, 139.50, 143.98, 145.33, 151.60. ESI-MS: 504.60
+
(
C
30
H
24
N
4
O
2
S, [M+H] ). Anal. Calcd for C30
H N
24 4
2
O S: C, 71.41; H, 4.79; N, 11.10;
O, 6.34; S, 6.35; Found: C, 71.41; H, 4.73; N, 11.10.
4
1
.5.3 1-((2,5-dimethylphenyl)sulfonyl)-2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-
H-benzo[d]imidazole (15)
1
White powder, yield: 76%. M. p: 227 - 228 ℃, H NMR (DMSO-d
6
, 400 MHz)
δ: 2.23 (s, 3H); 2.38 (s, 3H); 2.46 (s, 3H); 7.00 (s, 2H); 7.30 - 7.32 (m, 2H); 7.46 -
25

7
1
1
1
.50 (m, 1H); 7.55 - 7.66 (m, 7H); 7.80 - 7.82 (m, 2H); 7.96 - 7.98 (m, 2H); 9.28 (s,
1
3
H). C NMR (DMSO-d , 100 MHz) δ: 20.09, 21.01, 21.38, 105.75, 114.54, 119.54,
6
26.27, 127.53, 128.24, 128.37, 129.54, 130.08, 130.43, 131.06, 132.40, 132.75,
33.25, 134.05, 138.97, 139.46, 143.98, 146.20, 151.55. ESI-MS: 518.63
+
(
C
31
H
26
N
4
O
2
S, [M+H] ). Anal. Calcd for C31
H N
26 4
2
O S: C, 71.79; H, 5.05; N, 10.80;
O, 6.17; S, 6.18; Found: C, 71.77; H, 5.04; N, 10.80.
.5.4 1-((4-fluorophenyl)sulfonyl)-2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (16)
White powder, yield: 77%. M. p: 244 - 245 ℃, H NMR (DMSO-d
δ: 2.25 (s, 3H); 7.03 - 7.18 (m, 2H); 7.20 - 7.43 (m, 4H); 7.45 - 7.65 (m, 7H); 7.81 -
.83 (m, 1H); 7.98 - 8.00 (m, 2H); 8.13 - 8.15 (m, 1H); 8.97 (s, 1H). 13C NMR
DMSO-d , 100 MHz) δ: 21.37, 105.90, 114.56, 114.79, 114.94, 115.71, 119.54,
26.22, 128.24, 128.28, 128.34, 128.37, 130.07, 130.45, 132.53, 133.19, 138.99,
39.45, 144.04, 145.14, 145.16, 151.55. ESI-MS: 508.57 (C29
S, [M+H]+).
S: C, 68.49; H, 4.16; N, 11.02; O, 6.29; S, 6.30; Found:
4
1
6
, 400 MHz)
7
(
6
1
1
H21FN
4
O
2
Anal. Calcd for C29
C, 68.45; H, 4.15; N, 11.02.
.5.5 1-((4-chlorophenyl)sulfonyl)-2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (17)
White powder, yield: 81%. M. p: 254 - 256 ℃, H NMR (DMSO-d
δ: 2.32 (s, 3H); 7.26 - 7.29 (m, 2H); 7.31 - 7.35 (m, 2H); 7.51 - 7.55 (m, 2H); 7.63 -
H21FN
4
O
2
4
1
6
, 400 MHz)
7
.65 (m, 6H); 7.83 - 7.85 (m, 5H); 8.36 (s, 1H). ESI-MS: 525.02 (C29
H
21
C N
l 4
2
O S,
+
[
M+H] ). Anal. Calcd for C29
H
21
C N
l 4
2
O S: C, 66.34; H, 4.03; N, 10.67; O, 6.09; S,
6
4
.11; Found: C, 66.34; H, 4.053; N, 10.65.
.5.6 1-((4-bromophenyl)sulfonyl)-2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (18)
Yellow powder, yield: 82%. M. p: 235 - 236 ℃, H NMR (DMSO-d
δ: 2.38 (s, 3H); 7.31 (d, J = 8, 2H); 7.47 - 7.55 (m, 5H); 7.58 - 7.66 (m, 6H); 7.82 -
1
6
, 400 MHz)
1
3
7
2
1
.84 (m, 2H); 7.95 - 7.98 (m, 2H); 9.28 (s, 1H). C NMR (DMSO-d , 100 MHz) δ:
6
1.37, 105.40, 114.49, 119.11, 119.60, 122.30, 126.48, 127.26, 128.11, 128.44,
29.59, 130.13, 130.45, 131.16, 132.06, 133.30, 138.94, 139.55, 143.88, 147.68,
26

+
1
51.62. ESI-MS: 569.47 (C29
C, 61.16; H, 3.72; N, 9.84; O, 5.62; S, 5.63; Found: C, 61.16; H, 3.75; N, 9.83.
.5.7 2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1-((4-(trifluoromethyl)phenyl)
sulfonyl)-1H-benzo[d]imidazole (19)
White powder, yield: 85%. M. p: 247 - 248 ℃, H NMR (DMSO-d
δ: 2.38 (s, 3H); 7.31 (d, J = 8, 2H); 7.46 - 7.50 (m, 1H); 7.56 - 7.66 (m, 6H); 7.69 -
.71 (m, 2H); 7.80 - 7.83 (m, 4H); 7.96 - 7.98 (m, 2H); 9.26 (s, 1H). 13C NMR
DMSO-d , 100 MHz) δ: 21.36, 105.85, 114.55, 119.55, 125.39, 125.42, 125.44,
26.24, 126.80, 128.23, 128.38, 129.24, 129.45, 130.08, 130.45, 132.49, 133.18,
38.98, 139.46, 144.03, 151.56, 152.37. ESI-MS: 558.57 (C30
S, [M+H]+).
S: C, 64.51; H, 3.79; N, 10.03; O, 5.73; S, 5.74; Found:
H
21BrN
4
O
2
S, [M+H] ). Anal. Calcd for C29
H
21BrN
4
O S:
2
4
1
6
, 400 MHz)
7
(
6
1
1
H
21
F
3
N
4
O
2
Anal. Calcd for C30
C, 64.55; H, 3.79; N, 10.03.
.5.8 2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1-(phenylsulfonyl)-1H-
benzo[d]imidazole (20)
Yellow powder, yield: 68%. M. p: 227 - 228 ℃, H NMR (DMSO-d
δ: 7.30 - 7.33 (m, 5H); 7.35 - 7.37 (m, 2H); 7.60 - 7.64 (m, 5H); 7.78 - 7.84 (m, 4H);
H
21
F
3
N
4
O
2
4
1
6
, 400 MHz)
1
3
7
1
1
1
.96 - 7.99 (m, 2H); 9.32 (s, 1H). C NMR (DMSO-d , 100 MHz) δ: 19.00, 56.51,
6
14.51, 116.48, 116.62, 119.62, 125.91, 126.44, 128.17, 128.51, 128.83, 129.05,
29.85, 130.22, 130.46, 130.72, 130.78, 132.13, 133.44, 138.89, 143.68, 148.41,
+
50.69, 162.53, 164.16. ESI-MS: 494.54 (C28
H19FN
4
O
2
S, [M+H] ). Anal. Calcd for
C
28
H19FN
4
2
O S: C, 68.00; H, 3.87; N, 11.33; O, 6.47; S, 6.48; Found: C, 68.00; H,
3
.85; N, 11.37.
4
.5.9 2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1-((4-methoxyphenyl)
sulfonyl)-1H-benzo[d]imidazole (21)
White powder, yield: 82%. M. p: 231 - 232 ℃, H NMR (DMSO-d
δ: 3.74 (s, 3H); 6.83 - 6.85 (m, 2H); 7.32 - 7.36 (m, 2H); 7.47 - 7.50 (m, 1H); 7.52 -
1
6
, 400 MHz)
7
7
1
1
.55 (m, 2H); 7.56 - 7.58 (m, 2H); 7.62 - 7.66 (m, 2H); 7.77 - 7.82 (m, 4H); 7.97 -
1
3
.98 (m, 2H); 9.29 (s, 1H). C NMR (DMSO-d , 100 MHz) δ: 18.98, 55.60, 56.52,
6
06.14, 113.27, 114.55, 116.41, 116.56, 119.59, 126.18, 127.51, 127.62, 127.64,
28.45, 130.44, 130.72, 130.78, 132.59, 133.24, 138.92, 140.90, 143.83, 150.64,
27

+
1
59.82, 162.48, 164.11. ESI-MS: 524.57 (C29
H21FN
4
O S, [M+H] ). Anal. Calcd for
3
C
29
H21FN
4
O
3
S: C, 66.40; H, 4.04; N, 10.68; O, 9.15; S, 6.11; Found: C, 66.40; H,
4
.05; N, 10.63.
4
.5.10 1-((2,5-dimethylphenyl)sulfonyl)-2-(3-(4-fluorophenyl)-1-phenyl-1H-
pyrazol-4-yl)-1H-benzo[d]imidazole (22)
White powder, yield: 74%. M. p: 242 - 243 ℃, H NMR (DMSO-d
δ: 2.22 (s, 3H); 2.45 (s, 3H); 7.00 (s, 2H); 7.31 - 7.36 (s, 2H); 7.47 - 7.50 (s, 1H); 7.54
1
6
, 400 MHz)
-
7.58 (m, 3H); 7.61 - 7.65 (m, 2H); 7.75 - 7.80 (m, 4H); 7.95 - 7.97 (m, 2H); 9.24 (s,
1
3
1
1
1
H). C NMR (DMSO-d , 100 MHz) δ: 20.06, 20.99, 106.14, 113.27, 114.52, 116.38,
6
16.59, 119.62, 126.18, 127.51, 128.50, 129.66, 130.45, 130.70, 130.79, 131.13,
32.55, 132.77, 133.12, 134.13, 138.89, 143.70, 145.94, 150.66. ESI-MS: 522.59
+
(
C
30
H23FN
4
O
2
S, [M+H] ). Anal. Calcd for C30
H
23FN
4
2
O S: C, 68.95; H, 4.44; N,
1
4
0.72; O, 6.12; S, 6.14; Found: C, 68.95; H, 4.43; N, 10.76.
.5.11 4-((2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazole
-
1-yl)sulfonyl)phenyl nitrate (23)
Yellow powder, yield: 89%. M. p: 252 - 253 ℃, H NMR (DMSO-d
δ: 7.32 - 7.37 (m, 2H); 7.47 - 7.51 (m, 1H); 7.56 - 7.58 (m, 2H); 7.62 - 7.66 (m, 2H);
1
6
, 400 MHz)
13
7
.77 - 7.85 (m, 6H); 7.96 - 7.98 (m, 2H); 8.19 (d, J = 8.8, 2H); 9.27 (s, 1H). C NMR
(
DMSO-d , 100 MHz) δ: 18.97, 56.52, 106.19, 114.55, 116.42, 116.56, 119.60,
6
1
1
23.83, 126.18, 127.38, 127.62, 127.63, 128.48, 130.46, 130.71, 130.77, 132.64,
33.18, 138.92, 143.85, 147.78, 150.64, 154.43, 162.48, 164.11. ESI-MS: 555.54
+
(
C
28
H18FN
5
O
5
S, [M+H] ). Anal. Calcd for C28
H
18FN
5
5
O S: C, 60.54; H, 3.27; N,
1
4
2.61; O, 14.40; S, 5.77; Found: C, 60.56; H, 3.25; N, 12.63.
.5.12 2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1-((4-fluorophenyl)
sulfonyl)-1H-benzo[d]imidazole (24)
Yellow powder, yield: 77%. M. p: 228 - 229 ℃, H NMR (DMSO-d
δ: 7.10 - 7.15 (m, 2H); 7.31 - 7.49 (m, 2H); 7.49 - 7.58 (m, 1H); 7.59 - 7.62 (m, 2H);
1
6
, 400 MHz)
13
7
.63 - 7.66 (m, 4H); 7.75 - 7.82 (m, 4H); 7.95 - 7.97 (m, 2H); 9.24 (s, 1H). C NMR
DMSO-d , 100 MHz) δ: 18.87, 56.47, 105.71, 114.47, 115.03, 116.46, 116.60,
19.65, 126.41, 127.48, 128.31, 130.45, 132.13, 133.28, 138.87, 143.56, 144.64,
(
6
1
28

1
44.66, 150.71, 161.72, 162.50, 163.35. ESI-MS: 512.53 (C28
Anal. Calcd for C28 S: C, 65.62; H, 3.54; N, 10.93; O, 6.24; S, 6.26; Found:
C, 65.64; H, 3.56; N, 10.92.
.5.13 1-((4-chlorophenyl)sulfonyl)-2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-
yl)-1H-benzo[d]imidazole (25)
Yellow powder, yield: 82%. M. p: 257 - 258 ℃, H NMR (DMSO-d
δ: 7.28 - 7.36 (m, 4H); 7.52 - 7.56 (m, 2H); 7.66 - 7.68 (m, 4H);7.77 - 7.81 (m, 2H);
H
18
F
2
N
4
O
2
S, [M+H]+).
H
18
F
2
N
4
O
2
4
1
6
, 400 MHz)
1
3
7
1
1
.84 - 7.86 (m, 5H); 8.42 (s, 1H). C NMR (DMSO-d , 100 MHz) δ: 18.92, 56.53,
6
15.88, 116.01, 116.04, 116.19, 118.51, 119.72, 128.99, 129.77, 129.83, 130.05,
30.15, 130.20, 130.25, 130.75, 130.88, 131.32, 131.37, 137.12, 140.56, 147.14.
+
ESI-MS: 528.98 (C28
H18ClFN
4
O
2
S, [M+H] ). Anal. Calcd for C28
H18ClFN
4
2
O S: C,
6
4
4
3.57; H, 3.43; N, 10.59; O, 6.05; S, 6.06; Found: C, 63.54; H, 3.43; N, 10.56.
.5.14 1-((4-bromophenyl)sulfonyl)-2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-
-yl)-1H-benzo[d]imidazole (26)
1
Yellow powder, yield: 83%. M. p: 261 - 262 ℃, H NMR (DMSO-d
6
, 400 MHz)
δ: 7.32 - 7.37 (m, 2H); 7.51 - 7.58 (m, 4H); 7.58 - 7.60 (m, 2H); 7.62 - 7.66 (m, 3H);
13
7
.76 - 7.80 (m, 2H); 7.81 - 7.83 (m, 2H); 7.96 - 9.98 (m, 2H); 9.31 (s, 1H). C NMR
(
DMSO-d , 100 MHz) δ: 19.00, 56.51, 105.73, 114.52, 116.46, 116.61, 119.61,
6
1
1
1
22.27, 126.40, 127.54, 127.56, 128.18, 128.49, 130.29, 130.33, 130.34, 130.45,
30.71, 130.77, 131.14, 132.20, 133.40, 138.90, 143.71, 147.76, 150.68, 162.52,
64.15. ESI-MS: 573.44 (C28
H
18BrFN
4
O
2
S, [M+H]+). Anal. Calcd for
C
28
H18BrFN
4
O
2
S: C, 58.65; H, 3.16; N, 9.77; O, 5.58; S, 5.59; Found: C, 58.64; H,
3
.13; N, 9.76.
.5.15 2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1-((4-(trifluoromethyl)
4
phenyl)sulfonyl)-1H-benzo[d]imidazole (27)
White powder, yield: 84%. M. p: 243 - 244 ℃, H NMR (DMSO-d
δ: 7.32 - 7.37 (m, 2H); 7.47 - 7.51 (m, 1H); 7.55 - 7.57 (m, 2H); 7.62 - 7.66 (m, 2H);
1
6
, 400 MHz)
13
7
.69 - 7.71 (m, 2H); 7.78 - 7.82 (m, 6H); 7.96 - 7.98 (m, 2H); 9.28 (s, 1H). C NMR
DMSO-d , 100 MHz) δ: 19.00, 56.50, 114.57, 116.39, 116.60, 116.58, 125.38,
25.42, 126.17, 126.79, 128.47, 130.47, 130.71, 130.79, 133.24, 138.92, 150.62.
(
6
1
29