Design, synthesis and in vitro antitumor evaluation of novel pyrazole-benzimidazole derivatives

Article abstract of DOI:10.1016/j.bmcl.2021.128097

A series of novel pyrazole-benzimidazole derivatives (6–42) have been designed, synthesized and evaluated for their in vitro antiproliferative activity against the HCT116, MCF-7 and Huh-7 cell lines. Among them, compounds 17, 26 and 35 showed significant

Full text of DOI:10.1016/j.bmcl.2021.128097

Bioorg. Med. Chem. Lett. 43 (2021) 128097
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and in vitro antitumor evaluation of novel
pyrazole-benzimidazole derivatives
Bo Ren, Rong-Chun Liu, Kegong Ji, Jiang-Jiang Tang*, Jin-Ming Gao
Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling, 712100 Shaanxi, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of novel pyrazole-benzimidazole derivatives (6–42) have been designed, synthesized and evaluated for
their in vitro antiproliferative activity against the HCT116, MCF-7 and Huh-7 cell lines. Among them, compounds
17, 26 and 35 showed significant antiproliferative activity against HCT116 cell lines with the IC50 values of 4.33,
Benzimidazole-pyrazole
Antiproliferative activity
Cells apoptosis
5.15 and 4.84 μM, respectively. Moreover, fluorescent staining studies showed compound 17 could induce cancer
Cell cycle
cells apoptosis. The flow cytometry assay revealed that compound 17 could induce cell cycle arrest at G0/G1
phase. All in all, these consequences suggest that pyrazole-benzimidazole derivatives could serve as promising
compounds for further research to develop novel and highly potent cancer therapy agents.
Introduction
Recent years there have been reported an increasing number of molec-
ulars containing 1,3-diphenylpyrazole moieties showed various bio-
Cancer, described as the uncontrolled proliferation and spread of
abnormal cells, is one of the most siginificant health problems today.1
Chemotherapy drugs are widely used in the clinical treatment of cancer.
However chemotherapeutic drugs such as cisplatin, thiotepa, paclitaxel
and chlorambucil failed to control the growth of cancer cells completely.
The traditional chemotherapy is facing the challenge of lack selectivity
towards tumor cells.2 Therefore, the development of the new antitumor
agents has been getting more and more attention.3
activities such as antitumor, immunosuppressive, antiotubercular and
antibacterial (some moleculars are showed in Fig. 2).16–19
Inspired by the combination principle of some molecules encom-
passing benzimidazole moiety and 1,3-diphenylpyrazole moiety for the
synthesis of new bioactive compounds20–23 (Fig. 3), we designed and
synthesized a new series of heterocyclic antitumor molecules by the
joining of two important pharmacophores: 1,3-diphenylpyrazole and
benzimidazole. Different substituents on benzimidazole ring and ben-
zene ring were introduced to investgate their anti-proliferative activity
against cancer cells in vitro.
Heterocyclic compounds are well-documented pharmaceutically
active products, and have been found to play a significant role in the
drug design and discovery. Hence, heterocyclic moieties are often used
in the design and synthesis of efficient and pharmacological agents for
the cure of various diseases, such as antioxidant, anticancer agent,
antibacterial agent. Among the N-heterocyclic pharmacophores,
benzimidazole4–8 and pyrazole9–12 moieties are of interest because of
their wide range of clinical applications.
The synthesis of the target compounds was described in Scheme 1. 4-
Substituted acetophenone (1a and 1b) was condensed with phenyl-
hydrazine (2) in reflux ethanol containing 5% glacial acetic acid.24,25
The ethylidene hydrazine (3a and 3b) was formed as precipitate at room
temperature and filtered. The next step was Vilsmeier-Haack-Arnold
ring closing formylation,26 by treating the ethylidene hydrazine (3a
and 3b) with POCl3/DMF.27 The compounds (5a and 5b) were obtained
in good yields by heating aldehyde intermediates (4a and 4b) with O-
phenylenediamine in DMF and sodium metabisulfite.28 Then respec-
tively reacted with various alkyl bromide in the presence of cesium
carbonate in acetonitrile at room temperature or in the presence of so-
dium hydride in tetrahydrofuran at 0 ◦C to yield the desired compounds
pyrazol-benzimidazole derivatives (6–42). 37 novel derivatives were
characterized by 1H NMR, 13C NMR, 19F NMR and (HR)ESI-MS. The
Benzimidazole nucleus is used as privileged structural motif in the
development of a wide range of drugs with interest in numerous me-
dicinal areas, such as anti-inflammation, anti-microbial, and anti-can-
cer.13–15 The heterocycle has been applied into various marketed
anticancer drugs such as bendamustine, veliparib, pracinostat and
galeterone (Fig. 1).
1,3-Diphenylpyrazole moieties are highly important pharmacologi-
cally active organic compounds found in medicinally active substances.
* Corresponding author.
E-mail address: tangjiang11@nwafu.edu.cn (J.-J. Tang).
https://doi.org/10.1016/j.bmcl.2021.128097
Received 27 January 2021; Received in revised form 26 April 2021; Accepted 5 May 2021
Available online 9 May 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

B. Ren et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128097
Fig. 1. Drugs possessing benzimidazole motifs.
Fig. 2. Examples of molecules with 1,3-diphenylpyrazole.
Fig. 3. Examples of pyrazole-benzimidazole derivatives.
purity of all derivatives was greater than 97% by HPLC detection.
All the synthesized benzimidazole-pyrazoles derivatives (6–42) were
screened for their antiproliferative activity against three human tumor
cell lines: HCT116, MCF-7 and Huh-7 cancer cells lines by using sulfo-
rhodamine B (SRB) growth inhibition assay.29 The derivatives were
dissolved in DMSO (less than 0.1%) and etoposide (VP-16) was used as
positive control. The IC50 values of the derivatives are listed in Table 1.
The derivatives (except compound 6 and 38), possessing different
substititued aryl or alkyl group on benzimidazole, showed higher anti-
control etoposide (VP-16, IC50 = 15.6
Furthermore, most derivatives showed moderate anti-prolifeative ac-
tivities against MCF-7 cells with IC50 values ranging from 10 to 16.4 M,
which is better than VP-16 (IC50 = 18.9 M). However, the derivatives
μM) against HCT116 cells.
μ
μ
showed poor activities (IC50 > 20 μM) against Huh-7 cells. In general,
the compounds showed more selectively significant anti-proliferative
activities against HTC116 than MCF-7 and Huh-7 cells.
In comparison with compound 6 and 7–25, when R was substituted
with Br, derivatives with single or multiple substitution on the benzyl
group exhibited considerable antiproliferative activities against HCT116
proliferative activity (IC50 ranges from 4.33 to 10.0 μM) than positive
2

B. Ren et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128097
Scheme 1. Reagents and conditions: (i) HOAc, EtOH, reflux, yield: for 3a: 95%; for 3b: 90%; (ii) POCl3, DMF; NaHCO3, reflux, yield: for 4a: 90%; for 4b: 86% (iii) o-
diaminobenzene, sodium metabisulfite, DMF, 110 ◦C, 4 h, reflux, yield: for 5a: 80%; for 5b: 74% (iv) alkyl bromide, Cs2CO3, MeCN, rt, yield: 60–85%; for compound
26: propyl bromide, NaH, THF, 0 ◦C, yield: 70%.
(colon cancer), but did not significantly affect the inhibitory activity
against MCF-7 (breast cancer) and Huh-7 (liver cancer), which indicated
that the introduction of substituted benzyl groups can increase the
selectivity index of the derivative against HCT116 (breast cancer) when
R was substituted with Br. Derivatives with same substituents in
different positions will also exhibit different inhibitory activities. For
instance, for derivatives 7, 8 and 9, the order of their antiproliferative
activities against HCT116 cells is meta > ortho > para, and derivative 8
(with the substitution of 3-fluorobenzyl group) exhibited higher inhib-
through membranes, and thus enables estimation of transport properties
in the intestine and blood brain barrier.30 TPSA values of more than half
of derivatives were 35.65 Å, while the TPSA value of VP-16 is 160.83 Å.
The absorption (%ABS) was determine using the equation %ABS = 109
ꢀ 0.345 × TPSA following the method of Zhao et al.31 Compounds 6–29
derivatives showed %ABS values more than 95%, indicating potential
for good oral bioavailability. To find out whether the calculated clogP
(the logarithm of partition coefficient of a compound between n-octanol
and water) and antiproliferative activity are related, the clogP values of
these derivatives were calculated via Molinspiration and the results
were showed in Table 2. It showed that there was no obvious connection
between clogP values and their antiproliferative activities, while intro-
ducing substituents on benzimidazole core affected the hydrophobicity
to a certain extent.
itory activity against HCT116 cells (IC50 = 5.43 μM) and selectivity
index (3.1). For derivatives 12–15, the inhibitory activity and selectivity
of meta-substituted derivatives are also higher than other positions.
Derivative 17 bearing 4-(trifluoromethyl)benzyl group showed better
activities than derivative 16 bearing 2-(trifluoromethyl)benzyl group,
17 also exhibited the highest antitumor activity against HCT116 with
The morphological characteristics displayed by cells would exhibits
some changes like cell shrinkage and chromatin accumulation when cell
apoptosis occurs. In order to understand whether compound 17 can
induce apoptosis, HCT116 cells were treated with different concentra-
IC50 value of 4.33 μM and with high selectity index value of 3.4. Then we
introduced the hydrocarbon group in the benzimidazole ring to inves-
tigate its performance. For derivatives 26–28, increasing its hydropho-
bic properties can also increase the inhibitory activity against HCT116
and selectivity index. Interestingly, derivative 29 exhibited lower
cytotoxic effect against two cell lines (HCT116, MCF-7) and selectivity
index probably because of the bulkiness of the substituted alkyl group.
Derivatives with R substituted with OMe showed higher activities
against MCF-7 cells than R substituted with Br (for example, 31 vs 9, 33
vs 12, 34 vs 13, 35 vs 15), but it has weak or poor influence on the
inhibitory activity against HCT116, this leads to the decrease of the
selectity index.
tions of compound 17 (1, 2.5 or 5 μM) for 48 h and then stained with
Hoechst 33258. The morphological changes were observed by using
fluorescence microscopy (Fig. 4). The control (DMSO) exhibited slight
blue fluorescence, while HCT116 cells treated with different concen-
trations of compound 17 exhibited bright blue fluorescence due to
staining of the condensed chromatin in apoptotic cells (Fig. 5). The re-
sults showed that compound 17 was significant in inducing apoptosis in
HCT116 cells. We speculate that compound 17 might exert an effect on
inhibitory effects on tubulin polymerization.
The values of topological polar surface area (TPSA) and percent
absorption (%ABS) could be used to predict absorption of the pyrazole-
benzimidazole derivatives. TPSA is the surface belonging to polar atoms
and is described as a value connected with passive molecular transport
To determine whether the derivatives could induce alterations in the
cell cycle progression, we evaluated the effect of the compound 17 on
the cell cycle progression by using flow cytometric analysis in HCT116
cells after treatment with 1 or 5 µM of the compound for 24 h. As shown
3

B. Ren et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128097
Table 1
The in vitro anti-proliferative evaluation of target compounds 6–42 against selected cell lines.
No.
R
R′
IC50
,
μ
Ma
HCT116
MCF-7
Huh-7
SIb
6
7
Br
Br
19.1 ± 1.1
15.5 ± 2.1
19.0 ± 0.3
0.8
6.53 ± 0.14
15.9 ± 0.3
>20
2.4
8
Br
5.43 ± 0.36
16.7 ± 1.3
>20
3.1
9
Br
Br
8.01 ± 0.09
9.65 ± 0.07
15.2 ± 0.3
14.2 ± 0.2
>20
>20
1.9
1.5
10
11
12
Br
Br
7.26 ± 0.04
7.49 ± 0.27
15.5 ± 0.2
15.2 ± 0.3
>20
>20
2.1
2.0
13
14
15
16
Br
Br
Br
Br
6.67 ± 0.36
6.63 ± 0.36
5.40 ± 0.15
8.80 ± 0.08
17.1 ± 0.6
15.4 ± 1.9
19.5 ± 0.9
16.4 ± 0.3
>20
2.6
2.3
3.6
1.9
>20
>20
18.8 ± 0.3
17
18
Br
Br
4.33 ± 0.26
9.79 ± 0.13
14.7 ± 2.3
13.4 ± 0.6
>20
3.4
1.4
19.3 ± 1.0
19
20
Br
Br
6.35 ± 0.15
8.97 ± 0.44
14.0 ± 0.4
14.7 ± 0.6
>20
>20
2.2
1.6
21
Br
7.83 ± 0.03
19.4 ± 0.90
>20
2.5
22
23
24
Br
Br
Br
9.67 ± 0.18
8.07 ± 0.50
5.96 ± 0.15
16.0 ± 0.4
14.8 ± 0.5
15.2 ± 0.4
>20
>20
>20
1.7
1.8
2.6
25
Br
9.83 ± 0.14
12.7 ± 0.3
>20
1.3
26
Br
5.15 ± 0.10
16.4 ± 1.1
>20
3.2
27
28
Br
Br
5.19 ± 0.38
5.22 ± 0.09
16.3 ± 0.9
14.7 ± 0.7
>20
>20
3.1
2.8
29
Br
7.49 ± 0.35
16.8 ± 0.5
>20
2.2
30
Br
7.26 ± 0.22
15.3 ± 0.3
>20
2.1
31
32
33
Br
6.17 ± 0.24
6.72 ± 0.26
7.19 ± 0.73
12.3 ± 1.6
11.9 ± 1.5
12.5 ± 1.1
>20
2.0
1.8
1.7
OMe
OMe
>20
18.4 ± 0.5
34
OMe
5.78 ± 0.03
11.1 ± 0.9
>20
1.9
(continued on next page)
4

B. Ren et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128097
Table 1 (continued)
No.
R
R′
IC50
,
μ
Ma
HCT116
MCF-7
Huh-7
SIb
35
OMe
4.84 ± 0.14
12.8 ± 1.1
>20
2.6
36
37
OMe
OMe
5.58 ± 0.22
5.99 ± 0.14
11.0 ± 1.4
11.3 ± 1.3
>20
>20
2.0
1.9
38
OMe
16.5 ± 0.20
11.5 ± 0.4
>20
0.7
39
40
OMe
OMe
10.0 ± 0.51
6.27 ± 0.14
12.5 ± 2.6
13.9 ± 1.1
>20
>20
1.3
2.2
41
42
Ec
OMe
OMe
6.03 ± 0.24
10.6 ± 1.3
>20
1.8
7.98 ± 0.42
15.6 ± 0.21
11.7 ± 1.6
18.9 ± 0.8
>20
1.5
1.2
17.3 ± 0.6
a
b
c
Values are means from three independent experiments. Compounds were measured at different concentrations for 48 h.
Selective Index (SI): [IC50 MCF-7]/[IC50HCT116].
Positive control etoposide (VP-16).
Table 2
clogP, TPSA and %ABS values of the derivatives 6–42.a
Compound
clogP
TPSA
%ABS
Compound
clogP
TPSA
%ABS
6
7.69
7.83
7.83
7.83
7.83
8.40
8.55
8.55
8.51
8.81
8.57
8.57
7.98
8.55
8.70
9.27
8.61
8.70
8.96
35.65
35.65
35.65
35.65
35.65
35.65
35.65
35.65
35.65
35.65
35.65
35.65
35.65
35.65
35.65
35.65
44.89
35.65
35.65
96.70
96.70
96.70
96.70
96.70
96.70
96.70
96.70
96.70
96.70
96.70
96.70
96.70
96.70
96.70
96.70
93.51
96.70
96.70
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
VP-16
8.62
7.23
6.95
6.27
7.26
6.47
6.93
7.49
7.64
7.64
7.90
6.21
7.07
8.36
7.70
7.78
7.97
6.35
ꢀ 0.11
35.65
35.65
35.65
35.65
35.65
61.96
44.89
44.89
44.89
44.89
44.89
68.68
44.89
44.89
44.89
44.89
44.89
44.89
160.83
96.70
96.70
96.70
96.70
96.70
87.62
93.51
93.51
93.51
93.51
93.51
85.31
93.51
93.51
93.51
93.51
93.51
93.51
53.51
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
aTPSA calculations were done by using Molinspiration. ClogP values were calculated by using Molinspiration. %ABS: Percentage of absorption. %ABS = 109 ꢀ 0.345 *
TPSA31.
in Fig. 4, with the concentrations of compound 17 increased, the per-
centage of HCT116 cells in the G0/G1 phase increased from 44.1% to
67.1%, while the percentage of HCT116 cells in the S phase decreased
from 45.0% to 22.7%. These results showed that compound 17 induced
cell cycle arrest at the G0/G1 phase in a concentration-dependent
manner. Its’ mechanism is significantly different from the positive
control VP-16.
cells with IC50 value and selective index of 4.33 μM and 3.4, respec-
tively. In addition, mechanism of action research shown that compound
17 could significantly induce cell cycle arrest and apoptosis in HCT116
cells. Furthermore, all the consequences showed that pyrazole-
benzimidazole derivatives will serve as antiproliferative leads for
further chemical optimization.
In conclusion, we designed and synthesized 37 novel pyrazole-
benzimidazole derivatives and evaluated their anti-proliferative activ-
ities against the tested cells. Moreover, a majority of derivatives
exhibited potent antiproliferative activities against HCT116 with IC50
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
values ranging from 4.33 μM to 9.83 μM. Among them, compound 17
exhibited the most potent anti-proliferative activity against the HCT116
5

B. Ren et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128097
Fig. 4. Hoechst staining of compound 17 in HCT116 cells. After the treatment with compound 17 (1, 2.5 or 5 μM) for 48 h, HCT116 cells were stained by Hoechst
33258 solution, then detected by fluorescent microscope (20×). Error bar (white in DMSO): 20 μm.
Fig. 5. Cell cycle analysis of different concentrations of compound 17 and VP-16 in HCT116 cells. HCT116 cells were treated with compound 17 (1 or 5
16 (10 M) for 24 h, then stained with PI. A flow cytometer was used to analyze.
μM) and VP-
μ
8 Khalil MAA. J Heterocycl Chem. 2012;49:806–813.
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Appendix A. Supplementary data
Supplementary data (experimental section and spectral data con-
taining m.p., 1H NMR, 13C NMR, 19F NMR, (HR)ESI-MS and HPLC) to
this article can be found online at https://doi.org/10.1016/j.bmcl.20
21.128097.
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