Design, synthesis and biological evaluation of 1,3-diphenyl-1H-pyrazole derivatives containing benzimidazole skeleton as potential anticancer and apoptosis inducing agents

Article abstract of DOI:10.1016/j.ejmech.2015.07.031

A series of forty different pyrazole containing benzimidazole hybrids (6-45) have been designed, synthesized and evaluated for their potential anti-proliferative activity against three human tumor cell lines - lung (A549), breast (MCF-7), and cervical (HeLa). Some of the compounds, specifically 9, 17, and 28, showed potent growth inhibition against all the cell lines tested, with IC₅₀ values in the range of 0.83-1.81 μM. Breast cancer cells were used for further detailed studies to understand the mechanism of cell growth inhibition and apoptosis inducing effect of compounds. The morphology, cell migration and long term clonogenic survival of MCF-7 breast cancer cells were severely affected by treatment with these compounds. Flow-cytometry revealed the compounds arrested MCF-7 cells in the G1 phase of the cell cycle via down regulation of cyclin D2 and CDK2. Fluorescent staining and DNA fragmentation studies showed that cell proliferation was inhibited by induction of apoptosis. Moreover, the compounds led to collapse of mitochondrial membrane potential (DΨm) and increased levels of reactive oxygen species (ROS) were noted. The ease of synthesis and the remarkable biological activities make these compounds promising new frameworks for the development of cancer therapeutics.

Full text of DOI:10.1016/j.ejmech.2015.07.031

Accepted Manuscript
Design, synthesis and biological evaluation of 1,3-diphenyl-1H-pyrazole derivatives
containing benzimidazole skeleton as potential anticancer and apoptosis inducing
agents
T.Srinivasa Reddy, Hitesh Kulhari, V.Ganga Reddy, Vipul Bansal, Dr. Ahmed Kamal,
Outstanding Scientist, Dr. Ravi Shukla
PII:
S0223-5234(15)30163-X
10.1016/j.ejmech.2015.07.031
EJMECH 8015
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 April 2015
Revised Date: 13 July 2015
Accepted Date: 16 July 2015
Please cite this article as: T.S. Reddy, H. Kulhari, V.G. Reddy, V. Bansal, A. Kamal, R. Shukla, Design,
synthesis and biological evaluation of 1,3-diphenyl-1H-pyrazole derivatives containing benzimidazole
skeleton as potential anticancer and apoptosis inducing agents, European Journal of Medicinal
Chemistry (2015), doi: 10.1016/j.ejmech.2015.07.031.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Design, synthesis and biological evaluation of 1,3-diphenyl-1H-pyrazole derivatives
containing benzimidazole skeleton as potential anticancer and apoptosis inducing agents
a,b,c,d
a,b,c,d
a
c,d
T. Srinivasa Reddy,
Hitesh Kulhari,
*
V. Ganga Reddy, Vipul Bansal, Ahmed
a,b
c,d,e
Kamal, * Ravi Shukla,
a
Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500 007, India
b
IICT - RMIT Research Centre, CSIR-Indian Institute of Chemical Technology, Hyderabad
5
00 007, India
c
Ian Potter NanoBioSensing Facility, NanoBiotechnology Research Laboratory, School of
Applied Sciences, RMIT University, Melbourne 3000, Australia
d
Health Innovations Research Institute, RMIT University, Melbourne 3083, Australia
e
Centre for Advanced Materials and Industrial Chemistry, RMIT University, Melbourne
3
000, Australia

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of 1,3-diphenyl-1H-pyrazole derivatives
containing benzimidazole skeleton as potential anticancer and apoptosis inducing agents
a,b,c,d
a,b,c,d
a
c,d
T. Srinivasa Reddy,
Hitesh Kulhari,
V. Ganga Reddy, Vipul Bansal, Ahmed
a,b
c,d,e
Kamal, * Ravi Shukla,
*
a
Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500 007, India
b
IICT - RMIT Research Centre, CSIR-Indian Institute of Chemical Technology, Hyderabad
5
00 007, India
c
Ian Potter NanoBioSensing Facility, NanoBiotechnology Research Laboratory, School of
Applied Sciences, RMIT University, Melbourne 3000, Australia
d
Health Innovations Research Institute, RMIT University, Melbourne 3083, Australia
e
Centre for Advanced Materials and Industrial Chemistry, RMIT University, Melbourne
3
000, Australia
*
Corresponding authors:
Dr. Ravi Shukla
School of Applied Sciences, RMIT University
GPO Box 2476, Melbourne 3001 VIC Australia
E-mail: ravi.shukla@rmit.edu.au
Tel:
0061 3 9925 2970
Fax: 0061 3 9925 2882
Dr. Ahmed Kamal
Outstanding Scientist,
CSIR-Indian Institute of Chemical Technology,
Hyderabad, India-500 007.
E-mail: ahmedkamal@iict.res.in
Tel: +91-040-27193157;
Fax: +91-40-27193189
1

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Abstract
A series of forty different pyrazole containing benzimidazole hybrids (6-45) have been
designed, synthesized and evaluated for their potential anti-proliferative activity against three
human tumor cell lines - lung (A549), breast (MCF-7), and cervical (HeLa). Some of the
compounds, specifically 9, 17, and 28, showed potent growth inhibition against all the cell
lines tested, with IC values in the range of 0.83-1.81 µM. Breast cancer cells were used for
5
0
further detailed studies to understand the mechanism of cell growth inhibition and apoptosis
inducing effect of compounds. The morphology, cell migration and long term clonogenic
survival of MCF-7 breast cancer cells were severely affected by treatment with these
compounds. Flow-cytometry revealed the compounds arrested MCF-7 cells in the G1 phase
of the cell cycle via down regulation of cyclin D2 and CDK2. Fluorescent staining and DNA
fragmentation studies showed that cell proliferation was inhibited by induction of apoptosis.
Moreover, the compounds led to collapse of mitochondrial membrane potential (DΨm) and
increased levels of reactive oxygen species (ROS) were noted. The ease of synthesis and the
remarkable biological activities make these compounds promising new frameworks for the
development of cancer therapeutics.
Key words
Pyrazole; benzimidazole; anti-proliferative activity; apoptosis; RT-PCR.
2

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1
.0 Introduction
Cancer is a major cause of death worldwide. Thus, the incidence of and mortality rate
from cancer has become globally important [1]. Chemotherapy using drugs that target cell
division, angiogenesis or that induce cancer cell death by various signaling pathways is one
strategy to treat cancer. However, due to side effects and the development of drug resistance
in cancer cells, there is need to design, synthesize and develop more potent and safer
chemotherapeutic agents [2].
Apoptosis is a programmed cell death that plays an important role in the maintenance of
tissue homeostasis. Generally, it is considered that apoptosis is regulated by pro and anti-
apoptotic proteins, such as the Bcl-2 and inhibitor of apoptosis protein (IAP) family
members, and executed through caspases; mainly via two major and inter-related pathways,
the mitochondrial-dependent ‘intrinsic’ pathway and the death receptor-mediated ‘extrinsic’
caspase-8 pathway [3]. Disruption or inappropriate regulation of these processes results in
several diseases including cancer [4]. Thus, targeting critical regulators of apoptosis with the
goal of inducing apoptosis in cancer cells has emerged as an attractive strategy in cancer
therapy [5].
Pyrazoles are an important class of heterocyclic compounds and are promising scaffolds
in medicinal chemistry. Much attention has been paid to pyrazole based compounds because
of their broad range of biological effects, including anti-inflammatory, anti-microbial, anti-
oxidant, anti-depressant, anti-influenza and anti-cancer activities [6-10]. Several recent
reports suggest pyrazole derivatives as promising anticancer agents, indicating their use in the
development of new anticancer agents [11-14]. Among the anticancer pyrazoles, 1,3-diphenyl
pyrazoles have been reported to be highly potent and efficient cytotoxic agents [15-17].
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On the other hand, benzimidazole ring system has also emerged as an important
heterocyclic system due to its wide range of biological activities, as well as synthetic
applications in medicinal chemistry [18-19]. Recently, various benzimidazole derivatives
have been reported to have potential anti-tumor/anti-proliferative activity against different
cancers [20-22]. Due to the structural similarity of the benzimidazole nuclei with some of the
naturally occurring moieties such as purines, they easily interact with biomolecules of living
systems. Moreover, benzimidazoles attached to other heterocyclic moieties have resulted in
compounds (hybrid molecules) with improved anti-cancer activities [23-24].
A pharmacophore hybridization approach for the synthesis of new bioactive compounds
is an effective strategy and is being used in modern medicinal chemistry. Hybridization of
two different bioactive molecules with complementary pharmacophoric functions or with
different mechanisms of action often show enhanced effects [25-26]. Prompted by these
observations, and continuing our work on benzimidazole and pyrazole derivatives with
significant anti-cancer activities [27-28], we synthesized pyrazole containing benzimidazole
derivatives (6-45) with a view to producing promising anti-cancer agents.
2
2
.0 Results and Discussion
.1 Chemistry
The synthetic route for 2-(1,3-diphenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazoles (6-45)
is outlined in Scheme 1. These analogs were synthesized by adapting a reported procedure
13,16].The key aldehyde intermediates (4a-d) were prepared in two steps. Condensation of
[
the acetophenones (1a-d) with phenyl hydrazine (2) in ethanol produced the corresponding
acetophenone phenyl hydrazones (3a-d). This was followed by cyclization of the
acetophenone phenyl hydrazones via the Vilsmeir–Haack reaction. The desired target
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compounds (6-45) were obtained in good yields by refluxing aldehyde intermediates (4a-d)
with different substituted O-phenylenediamines (5a-d) in ethanol and sodium metabisulphate.
<Scheme 1>
2
4
.2 In vitro anticancer activity
All the newly synthesized 2-(1,3-diphenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazoles (6-
5) were evaluated for their cytotoxicity against three human tumor cell lines: lung (A549),
breast (MCF-7), cervical (HeLa) and against normal keratinocyte (HaCaT) cells using the 3-
4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) growth inhibition assay.
-Fluorouracil was used as a positive control and the results are summarized in Table.1. As
(
5
shown in Table 1, compounds 9, 17 and 28 exhibited potent broad spectrum anti-proliferative
activity against the three cancer cell lines, with IC values of 0.83-1.81 µM. These were
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0
superior to 5-fluorouracil which showed IC values of 2.13-4.16 µM. The highest growth
5
0
inhibitory activity observed in the series was against the MCF-7 cancer cell line, with
compound 9 being most potent (IC -0.83 µM), followed by the compounds 17 (IC -0.93
5
0
50
µM) and 28 (IC -1.17 µM). The other two cancer cell lines, A549 and HeLa, also showed
5
0
sensitivity towards these three compounds with not more than 1.81 µM concentration. All
compounds, with the exception of 13-15, 23-25, 33-35 and 43-45, exhibited higher or
comparable cytotoxic activity against certain cell lines with respect to the standard, 5-
Fluorouracil. The intermediates 1,3- diphenyl pyrazole carboxaldehyde (4 a-d) displayed
moderate growth inhibition on the tested cancer cells with IC values of 8.66-29.5 µM. We
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0
also investigated the effect of these compounds on normal human keratinocytes (HaCaT)
proliferation. Interestingly, most of the synthesized compounds had no effect on keratinocyte
cell growth (IC >50 µM). The exceptions were compounds 12, 14, 19, 24, 31 and 43, that
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0
exhibited IC50 values in the range of 21.35-44.67 µM (Table. 1). These results indicate
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selectivity of 1,3-diphenyl-1H-pyrazole derivatives towards tumor cells compared to the
normal HaCaT cells.
By investigating the variation in selectivity of the three cell lines to the compounds, it
was revealed that structural variations and modification on the B ring of pyrazolo-
benzimidazole derivatives led to different cytotoxic activities. Structure–activity relationships
in these hybrids demonstrated that compounds 7-9, 17-19, 27-29 and 37-39, with mono-
substituted halogens (fluorine, chlorine, and bromine) on the benzimidazole B ring, exhibited
more potent activities than compounds 11, 20, 30 and 40 or 10, 21, 31 and 42 with methoxy
and methyl substitutions, respectively. An electron withdrawing trifluoromethyl (CF3)
substitution on the B-ring of compounds 12, 22, 31 and 41 resulted in moderate to lower
cytotoxic activity. In addition, the presence of dichloro groups (14, 24, 34 and 44) on the
phenyl ring and dimethyl groups (13, 23, 33 and 43) on the benzimidzole B ring also caused
lower inhibitory activities. In contast, varying the substitution groups on the A-ring did not
impact anti-proliferative activity of these hybrids.
<Table.1>
An in silico study of compounds 6-45 was performed in QikProp 3.7 (Schrödinger, 2013)
[
29] to determine the Lipinski’s parameters and the predicted human oral absorption (%
ABS) (Table. S1 Supplementary information). According to the ‘Lipinski’s rule of five’,
compounds that violate more than one of these rules (no. of violations), may face
bioavailability issues. It was observed that all the compounds followed the rule, indicative of
their ‘drug-like’ nature. The predicte percent human oral absorption (% ABS) was 100%
indicating complete absorption via the oral route. Moreover, the number of likely metabolic
reactions (#metab) was between 0-3, indicating lower propensity for metabolic degradation.
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2
.3 Morphological analysis
Based on promising cytotoxic activity, the three most potent compounds from this series,
compunds 9, 17 and 28 were used for further mechanistic studies. To determine whether
these compounds could cause loss of cell viability, MCF-7 cells were treated with the IC50
concentrations for 48 h and cells were observed under a phase contrast microscope. As shown
in Figure 1, as determined by the distinctive morphological features of cells including
detachment and cell shrinkage, numbers of viable MCF-7 cells were significantly reduced
after treatment with the compounds compared to control, untreated cells.
<Figure 1>
2
.4 Colony formation inhibition assay
In order to investigate the long term cytotoxic potential of compounds 9, 17 and 28,
clonogenic cell survival assay was performed. This assay measures the ability of a single cell
to grow into a colony. Because it reflects all modes of cell death or arrest, it is considered a
standard for measuring long term cell viability [30]. As shown in Figure 2, exposure of MCF-
7
cells to the compounds inhibited the colony formation of MCF-7 cells, with all reducing
clonogenic survival by approximately 50%. These results are in agreement with GI values,
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0
indicating the compounds can effectively suppress growth and proliferation of MCF-7 cells.
<Figure 2>
2
.5 In vitro cell migration assay
In vitro cell migration assay/wound healing assay was then performed. Migration of
cancer cells is a key step in tumor metastasis, and as such, measures of migration capacity
correlate with the metastatic potential of cancer cells [31]. Migration was assessed by
comparing the number of cells moving into the induced wound area in control and treatment
wells. Photographs, taken at different time intervals during the assay (0, 24, 48 and 72 h),
7

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showed the number of invasive breast cancer cells penetrating into the respective wound
areas were inhibited after treatment with these compounds (9, 17 and 28) when compared to
the control group (Figure 3). These results indicate that the compounds can suppress the
migration of MCF-7 cells.
<Figure 3>
2
.6 Effect on mRNA expression levels of markers of cell adhesion
Cellular migration and invasiveness are considered to be associated with decrease in the
expression of cell adhesion markers, such as E-cadherin and β-catenin [32]. E-cadherin is a
trans-membrane protein acting at cell-to-cell junctions where it influences epithelial-
mesenchymal transition. Studies have shown down-regulation of the E-cadherin expression in
tumors of many organs, including human breast [33]. β-catenin plays an important role in the
regulation of the Wnt signaling pathway that is crucial for cell-to-cell interactions in organ
development [34]. To evaluate the effect of these compounds (9, 17 and 28) on E-cadherin
and β-catenin levels, total RNA was extracted from compound treated MCF-7 cells and
relative E-cadherin, β-catenin expression levels were determined by RT-PCR. Results shown
in Figure 4 indicate the pyrazolo-benzimidazole hybrids enhanced the expression of E-
cadherin (1.6-2.2 times) and β-catenin (1.4-1.6 times) in MCF-7 cancer cells. Compound 9,
which more potently inhibits cell migration, increased expression of E-cadherin 2.2 times.
These results suggest the hybrids, at least in part, may inhibit cell migration via up-regulation
of cell adhesion markers in MCF-7 cells.
<Figure 4>
2
.7 Cell cycle analysis
We then used flow cytometry to explore the effects of the compounds 9, 17 and 28 on cell
cycle. MCF-7 cells were treated with the IC concentrations of the compounds for 24 h,
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0
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fixed with ethanol and stained with propidium iodide. As shown in Figure 5, treatment of
MCF-7 cells with compounds 9, 17 and 28 increased cell cycle arrest at the G1 phase,
resulting in cell population increase in the G1 phase (85.2%, 84.7% and 81.3%, respectively)
compared to the control cells (72.3%). In line with this finding, the cell populations in S and
G2/M phases decreased significantly after treatments. These results demonstrate that, in
MCF-7 cells, cell cycle arrest in the G0/G1 phase contributes to the cytotoxicity of the
compounds.
<Figure 5>
2
.8 Effect of compounds on expression levels of cell cycle check point markers
To study possible mechanisms responsible for arrest at G1 phase, we examined effects on
expression levels of some cell cycle specific genes such as cyclins and cyclin dependent
kinases (CDKs) by RT-PCR. As shown in Figure 6, treatment with hybrids 9, 17 and 28
resulted in significantly decreased levels of cyclin D1; a known regulator of G1/S phase
transition. There was a small increase of 1.1 to 1.3 times in levels of cyclin B1, whereas the
mRNA levels of CDK1 were not significantly changed. Thus, pyrazole-benzimidazole
hybrids may decrease levels of cell cycle regulators such as cyclin D1 and CDK2 in MCF-7
cells, leading to the cell cycle arrest and inhibition of cell growth.
<Figure 6>
2
.9 Studies of apoptosis
Apoptosis is one of the major pathways that leads to the process of cell death and is
associated with chromatin condensation, nuclear shrinking and fragmented nuclei. Tumor
cells often have irregular apoptotic pathways and induction of tumor cell apoptosis by natural
or synthetic compounds is considered an effective therapy for cancer [35-36]. Moreover,
9

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many pyrazole and benzimidazole derivatives induce apoptosis in different cancer cell lines
[
37-40]. Hence it was of interest to investigate the effects of the hybrid compounds on
apoptosis inducing effect of these hybrids in MCF-7 breast cancer cells.
2
.9.1 Hoechst staining
To examine apoptosis inducing effect, MCF-7 cells were treated with the IC50
concentration of compounds 9, 17 and 28 for 24 h and morphological changes studied
following Hoechst 33242 staining. As shown in Figure 7, control cells treated with DMSO
exhibited uniformly dispersed chromatin, whereas 25-34% of compound treated cells showed
typical apoptotic characteristics; including condensation of chromatin (brightly stained cells)
and appearance of nuclear fragmentation (arrowheads indicate an apoptotic nucleus). Among
them, compound 9 was the most potent in inducing apoptosis (42% apoptotic cells) compared
to the other two compounds; that is 17 (26% apoptotic cells) and 28 (32% apoptotic cells).
These results demonstrate the hybrids have the potential to induce cellular apoptosis in MCF-
7
cells.
<Figure 7>
2
.9.2 DNA fragmentation assay
During the apoptotic process, activated nucleases degrade the higher order chromatin
structure of DNA into mono and oligo nucleosomal DNA-fragments. Thus, fragmentation of
DNA leads to loss of DNA content which results in a characteristic DNA ladder on agarose
gel electrophoresis [41]. Thus, DNA fragmentation assay was carried out to further confirm
the apoptosis inducing effect of these hybrids. MCF-7 cells were treated with the IC50
concentration of compunds 9, 17 and 28 for 24 h and chromosomal DNA was extracted.
Apoptotic degradation of DNA was analyzed by agarose gel electrophoresis. A typical DNA
smeared ladder pattern, indicative of fragmentation, was observed in DNA from the pyrazole-
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benzimidazole compound treated cells (Figure 8). The observed smear is the result of DNA
breaks at multiple positions across the chromosomal DNA. The smear intensity was most
obvious in DNA from compound 9 and 28 treated cells, whereas compound 17 resulted in
moderate smear formation. In contrast, there was little or no degradation of DNA from
control cells. The findings support the concept that compounds 9, 17 and 28 induce apoptosis
in MCF-7 cells.
<Figure 8>
2
.9.3 Effect of compounds on mitochondrial membrane potential
The maintenance of mitochondrial membrane potential (DΨm) is important for mitochondrial
integrity and bioenergetic function. Previous reports suggest loss of mitochondrial membrane
potential leads to apoptosis [42]. To examine this possibility, MCF-7 cells were treated with
hybrid compounds 9, 17 and 28 for 24 h and incubated with rhodamine 123. Mitochondria
that maintain normal DΨm retain the dye, which gives strong green fluorescence. However,
loss of mitochondrial membrane potential leads to depolarization which results in less uptake
of rhodamine 123 [43]. As shown in Figure 9a, compared with the untreated control,
compounds 9, 17 and 28 treated cells exhibit a marked reduction in green fluorescence.
Quantitation of fluorescence intensity demonstrated the compound treated cells exhibit 44-
6
2% loss of mitochondrial membrane potential compared to the control cells (Figure 9b).
Among the hybrids, compound 9 is more potent in inducing mitochondrial depolarization
62%). The results suggest that, in MCF-7 cells, collapse of mitochondrial membrane
potential also contributes to the apoptotic effects of the compunds.
(
<Figure 9>
1
1

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2
.9.4 Effect of compounds on reactive oxygen species (ROS) levels
The increase in levels of the ROS in the mitochondrion may result in oxidative damage to the
mitochondrial membrane which leads to apoptosis [44]. Thus, we next examined the effects
of these hybrids 9, 17 and 28 on ROS generation using carboxy-2',7'-dichlorofluorescein
diacetate (Carboxy-DCFDA) dye which upon cleavage by intracellular esterases oxidizes to
highly fluorescent carboxy-2',7'-dichlorofluorescein (Carboxy-DCF). As shown in Figure
1
0a, increased ROS production was observed in compound treated cells that showed strong
green fluorescence in comparison to the control cells. Quantitative analysis by
spectrofluorometry revealed that the ROS levels increased 2-4 times higher when compared
to control cells (Figure 10b). This data suggests cytotoxicity of the hybrids is also dependent
on ROS production.
<Figure 10>
2
.9.5 Effect on the mitochondrial mediated apoptotic pathway
Mitochondria play an important role in cell death signaling pathways such as apoptosis [45].
Mitochondrial dependent apoptotic pathway is regulated by proteins of the Bcl-2 family
which are associated with both pro and anti-apoptotic effects in cancer cells. The relative
equilibrium of proapoptotic (Bax) and anti-apoptotic (Bcl-2) proteins is critical for
maintaining cellular homeostasis. A change in the Bax/Bcl-2 ratio induces the release of
cytochrome C from mitochondria into the cytosol leading to activation of caspases and
induction of apoptosis [46]. To examine the possible role of mitochondrial mediated
apoptotic pathway, the effect of these hybrids (9, 17 and 28) on the expression of levels of
Bcl-2, Bax and caspases-3 and 7 was analysed by RT-PCR. As shown in Figure 11, treatment
of MCF-7 cells with IC concentrations of 9, 17 and 28 resulted in significantly increased
5
0
levels of bax (proapoptotic) and reduced levels of Bcl-2 (anti-apoptotic) mRNAs. In addition,
as shown in Figure 11, the compounds upregulated expression of the proapoptotic, caspase-3
1
2

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and 7. Thus, these mitochondrial mediated pathways may also play a role in induction of
apoptosis by pyrazole-benzimidazole hybrids.
<Figure 11>
3
.0 Conclusion
In summary, we synthesized a series of pyrazole-benzimidazole hybrids (6-45) and
investigated their antiproliferative activities on four different human tumor cell lines. The
results reveal most of the synthesized compounds significantly inhibit cancer cell
proliferation; some with superior antiproliferative activity than 5-fluorouracil. Compounds 9,
1
7 and 28 showed potent broad spectrum cytotoxic activity against all tested cancer cell lines.
Preliminary structure-activity relationships among the series are discussed. The compounds
inhibit in vitro cell migration, possibly through restoration of E-cadherin, β-catenin
expression and also inhibit the long term colonogenic survival of MCF-7 cells. Moreover, the
compounds induces G1 phase cell cycle arrest in MCF-7 cells through down regulation of
cyclin D1 as well as CDK2. The apoptotic effects of these compounds on MCF-7 cells was
studied by Hoechst staining and the DNA ladder assay. Results suggest the compounds
induce apoptosis via increased production of ROS, elevation of Bax/Bcl-2 ratio, and
activation of caspase-3 and 7. Therefore, these results imply that the pyrazolyl-benzimidazole
hybrids respresent targets for further optimization and development of anticancer agents to
treat breast cancer.
4
4
.0 Experimental Section
.1 Chemistry
All reagents and solvents used were of commercial grade and were used without any further
purification. Progress of reactions was monitored by thin layer chromatography (TLC)
performed on silica gel glass plates containing 60 F-254 and visualized on TLC was achieved
under UV light or with iodine indicator. Melting points were measured with an Electro-
1
3

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1
13
thermal melting point apparatus, and are uncorrected. H and C NMR spectra were recorded
on INOVA (400 MHz) or Gemini Varian-VXR-unity (200 MHz) or Bruker UXNMR/XWIN-
NMR (300 MHz) instruments. Chemical shifts (d) are reported in ppm downfield from
internal TMS standards. Signal multiplicities are represented by s (singlet), d (doublet), t
(
triplet), ds (double singlet), dd (double doublet), m (multiplet) and br s (broad singlet). ESI
spectra were recorded on Micro mass, Quattro LC using ESI+ software with capillary voltage
.98 kV and ESI mode positive ion trap detector. IR spectra were recorded on KBr disc using
a FTIR bruker Vector 22 Spectrophotometer.
.1.1 General procedure for the synthesis of 1, 3 diphenyl pyrazole carboxaldehydes (4a-
3
4
d)
The 1,3-diphenyl-1H-pyrazole-4-carboxaldehydes intermediates (4a-d) were synthesized
based on a literature method as follows: para-substituted acetophenones (1a-d, 20 mmol)
interact with phenylhydrazine (2, 25 mmol)
in anhydrous ethanol to form
acetophenonephenylhydrazones (3a-d), which were added to a cold solution of DMF (25 mL)
and POCl (5 mL) and stirred at 50–60 ºC for 6 h. The resulting mixture was poured into ice-
3
cold water, a saturated solution of sodium bicarbonate was added to neutralize the mixture,
extracted with ethyl acetate and then concentrated in vacuo. The resulting residue was
purified by column chromatography to afford pure 1,3-diphenyl-1H-pyrazole-4-
carbaldehydes (4a-d) in good yields. The spectroscopic data of the obtained aldehyde
compounds was in agreement with the reported data [16,47].
4
.1.2 General procedure for the synthesis of 2-(1,3-diphenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole derivatives (6-45)
A solution of Na S O (40 mmol) in H O (1.6 mL) was added to a mixture of appropriate 1,3
2
2
5
2
diphenyl carboxaldehydes (10 mmol) and different substituted o-phenylenediamines (10
1
4

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mmol) in ethanol. After completion of the reaction, the resulting mixture was stirred at reflux
for 4 h,the mixture diluted with 50 mL of H O and then extracted with ethyl acetate (2X40
2
mL). The combined extracts were washed with brine, dried over Na SO , and evaporated in
2
4
vacuo. The crude product was purified by column chromatography using EtOAc/hexane as
the eluent to produce pure 2-(1,3-diphenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazole
derivatives in good yields.
4
.1.2.1
2-(1,3-diphenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazole (6)
1
White solid, yield 77%, Mp: 178-182 ºC.; H NMR (300 MHz, CDCl +DMSO-d6) δ 8.60 (s,
3
1
H), 7.82 – 7.73 (m, 4H), 7.55 (dd, J = 6.0, 3.1 Hz, 2H), 7.45 (t, J = 7.8 Hz, 2H), 7.39 – 7.27
1
3
(
m, 4H), 7.20 (dd, J = 6.0, 3.2 Hz, 2H). C NMR (75 MHz, CDCl +DMSO-d6) δ 150.2,
3
1
45.2, 138.7, 138.3, 131.8, 128.8, 128.7, 127.8, 127.7, 126.3, 121.5, 118.2, 114.3, 112.3. MS
+
(
ESI): m/z 337 [M+H] . HRMS (ESI) calcd for C H N [M+H]+ 337.14477; found:
2
2
16
4
3
37.14257.
4
.1.2.2
2-(1,3-diphenyl-1H-pyrazol-4-yl)-6-fluoro-1H-benzo[d]imidazole (7)
1
Light brown solid, yield 71%, Mp: 189-192 ºC.; H NMR (300 MHz, CDCl ) δ 8.63 (s, 1H),
3
7
7
1
.74 (d, J = 7.8 Hz, 2H), 7.71-7.62 (m, 2H), 7.59-7.46 (m, 6H), 7.37 (t, J = 7.36 Hz, 2H),
13
.03 – 6.93 (m, 1H). C NMR (75 MHz, CDCl ) δ 161.1, 157.9, 150.7, 146.9, 139.2, 132.3,
3
29.5, 129.2, 129.1, 128.8, 128.6, 127.4, 119.1, 112.5, 111.0, 110.7.; MS (ESI): m/z 355
+
[
M+H] . HRMS (ESI) calcd for C H N F [M+H]+ 355.13535; found: 355.13342.
2
2
16
4
4
.1.2.3
6-chloro-2-(1,3-diphenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazole (8)
1
White solid, yield 69%, Mp: 196-198 ºC.; H NMR (300 MHz, CDCl ) δ 8.67 (s, 1H), 7.77
3
(d, J = 7.8 Hz, 2H), 7.68 (dd, J = 6.4, 3.0 Hz, 2H), 7.55 – 7.42 (m, 6H), 7.35 (t, J = 7.4 Hz,
1
3
2
H), 7.20 (d, J = 7.4 Hz, 1H). C NMR (75 MHz, DMSO-d6) δ 150.1, 147.4, 138.4, 131.4,
1
5

ACCEPTED MANUSCRIPT
+
1
28.8, 128.7, 127.7, 127.6, 126.31, 124.6, 118.1, 117.8, 111.5.; MS (ESI): m/z 371 [M+H] .
HRMS (ESI) calcd for C H N Cl [M+H]+ 371.10580; found: 371.10401.
2
2
16
4
4
.1.2.4
6-bromo-2-(1,3-diphenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazole (9)
1
Light brown solid, yield 75%, Mp: 184-187 ºC.; H NMR (500 MHz, CDCl ) δ 8.67 (s, 1H),
3
7
6
1
.78 (d, J = 7.62 Hz, 2H), 7.74-7.67 (m, 2H), 7.53 – 7.45 (m, 6H), 7.34 (t, J = 7.32 Hz, 2H),
.
13
.87 (dd, J = 8.69, 2.28 Hz, 1H) C NMR (75 MHz, CDCl ) δ 150.8, 146.6, 139.1, 132.2,
3
29.5, 129.2, 129.1, 128.9, 128.6, 127.3, 125.8, 119.1, 115.6, 112.3.; MS (ESI): m/z 415
+
[
M+H] . HRMS (ESI) calcd for C H N Br [M+H]+ 415.05529; found: 415.05284.
2
2
16
4
4
.1.2.5
2-(1,3-diphenyl-1H-pyrazol-4-yl)-6-methyl-1H-benzo[d]imidazole (10)
1
Light brown solid, yield 67%, Mp: 175-1177 ºC.; H NMR (300 MHz, CDCl ) δ 8.64 (s, 1H),
3
7
2
1
1
3
.77 (d, J = 7.6 Hz, 2H), 7.69 (d, J = 3.2 Hz, 2H), 7.55 – 7.39 (m, 6H), 7.34 (d, J = 7.4 Hz,
1
3
H), 7.06 (d, J = 7.5 Hz, 1H), 2.45 (s, 3H). C NMR (126 MHz, CDCl ) δ 150.7, 145.8,
3
39.3, 132.2, 129.5, 129.2, 129.0, 128.8, 128.7, 127.4, 127.1, 126.5, 124.1, 119.3, 119.1,
+
18.8, 113.0, 21.6.; MS (ESI): m/z 351 [M+H] . HRMS (ESI) calcd for C H N [M+H]+
23
19
4
51.16042; found: 351.15831.
4
.1.2.6
2-(1,3-diphenyl-1H-pyrazol-4-yl)-6-methoxy-1H-benzo[d]imidazole (11)
1
Light yellow solid, yield 73%, Mp: 191-193 ºC.; H NMR (500 MHz, CDCl ) δ 8.67 (s, 1H),
3
7
7
1
1
.80 – 7.75 (m, 2H), 7.70 (ddd, J = 10.8, 5.8, 3.4 Hz, 2H), 7.53 – 7.44 (m, 6H), 7.34 (t, J =
1
3
.4 Hz, 2H), 6.88 (dd, J = 8.8, 2.4 Hz, 1H), 3.83 (s, 3H). C NMR (126 MHz, CDCl ) δ
3
56.5, 150.7, 145.3, 139.3, 132.6, 129.5, 129.3, 129.0, 128.7, 127.9, 127.4, 127.1, 119.3,
+
19.1, 118.8, 112.9, 112.2, 55.8.; MS (ESI): m/z 367 [M+H] . HRMS (ESI) calcd for C23
H ON [M+H]+ 367.15534; found: 367.15330.
1
9
4
1
6

ACCEPTED MANUSCRIPT
4
.1.2.7
2-(1,3-diphenyl-1H-pyrazol-4-yl)-6-(trifluoromethyl)-1H-
benzo[d]imidazole (12)
Brown solid, yield 73%, Mp: 201-203 ºC.; H NMR (500 MHz, CDCl ) δ 8.71 (s, 1H), 7.77
1
3
(d, J = 7.3 Hz, 2H), 7.70-7.64 (m, 2H), 7.55 – 7.43 (m, 7H), 7.35 (t, J = 6.9 Hz, 1H), 7.21 (s,
1
3
1
1
H). C NMR (75 MHz, CDCl ) δ 150.9, 148.0, 139.1, 132.1, 129.6, 129.3, 129.1, 128.6,
3
+
27.4, 126.5, 125.2, 124.8, 119.7, 119.2, 112.1.; MS (ESI): m/z 405 [M+H] . HRMS (ESI)
calcd for C H N F [M+H]+ 405.13216; found: 405.12978.
2
3
16
4 3
4
.1.2.8
2-(1,3-diphenyl-1H-pyrazol-4-yl)-5,6-dimethyl-1H-benzo[d]imidazole (13)
1
Light brown solid, yield 73%, Mp: 184-186 ºC.; H NMR (300 MHz, CDCl ) δ 8.55 (s, 1H),
3
7
.78 – 7.59 (m, 5H), 7.46-7.35 (m, 3H), 7.34 (s, 2H), 7.17 (t, J = 8.6 Hz, 2H), 2.35 (s, 6H).
+
MS (ESI): m/z 365 [M+H] , HRMS (ESI) calcd for C H N [M+H]+ 365.17612; found:
24
21
4
3
65.17662.
4
.1.2.9
5,6-dichloro-2-(1,3-diphenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazole (14)
1
Light brown solid, yield 66%, Mp: 213-215 ºC.; H NMR (300 MHz, DMSO-d6) δ 8.90 (s,
1
3
1
H), 7.85 – 7.72 (m, 5H), 7.68 (s, 2H), 7.46 (t, J = 7.8 Hz, 2H), 7.31-7.38 (m, 3H); C NMR
(75 MHz, DMSO) δ 150.1, 147.4, 138.4, 131.4, 128.8, 128.7, 127.7, 127.6, 126.3, 124.6,
+
1
18.1, 111.5.; MS (ESI): m/z 405 [M+H] . HRMS (ESI) calcd for C H N Cl [M+H]+
2
2
15
4
2
4
05.06683; found: 405.06473.
4
.1.2.10
2-(1,3-diphenyl-1H-pyrazol-4-yl)-1H-naphtho[2,3-d]imidazole (15)
1
Light brown solid, yield 76%, Mp: 197-199 ºC.; H NMR (300 MHz, CDCl ) δ 8.61 (s, 1H),
3
7
.92-7.83 (m, 4H), 7.81 – 7.70 (m, 4H), 7.48 (d, J = 7.7 Hz, 2H), 7.39 (d, J = 7.1 Hz, 2H),
1
3
7
.34 (s, 2H), 7.20 (t, J = 8.5 Hz, 2H). C NMR (75 MHz, CDCl ) δ 151.2, 149.7, 139.2,
3
1
7

ACCEPTED MANUSCRIPT
1
32.3, 130.6, 129.6, 129.4, 129.3, 129.1, 128.9, 127.8, 127.3, 123.9, 119.2, 112.7.; MS (ESI):
+
m/z 387 [M+H] . HRMS (ESI) calcd for C H N [M+H]+ 387.16081; found: 387.16105.
2
6
19
4
4
.1.2.11
2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazole
(16)
1
White solid, yield 73%, Mp: 179-182 ºC.; H NMR (300 MHz, CDCl ) δ 8.71 (s, 1H), 7.89-
3
1
3
7
.79 (m, 3H), 7.68–7.44 (m, 5H), 7.36 (s, 2H), 7.22-7.13 (m, 3H). C NMR (75 MHz,
CDCl +DMSO-d6) δ 163.7, 160.4, 149.2, 145.1, 138.6, 129.6, 129.5, 128.8, 128.7, 127.9,
3
+
1
27.9 126.2, 121.3, 118.1, 114.5, 114.2, 112.2.; MS (ESI): m/z 355 [M+H] . HRMS (ESI)
calcd for C H FN [M+H]+ 355.13496; found: 355.13483.
2
2
16
4
4
.1.2.12
5-fluoro-2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (17)
1
Light brown solid, yield 79%, Mp: 189-192 ºC.; H NMR (500 MHz, CDCl ) δ 8.54 (s, 1H),
3
7
7
1
1
.69 (d, J = 7.4 Hz, 2H), 7.66 – 7.60 (m, 2H), 7.52-7.41 (m, 4H), 7.33 (t, J = 7.4 Hz, 1H),
1
3
.12 (t, J = 8.6 Hz, 2H), 7.00 (td, J = 9.2, 2.4 Hz, 1H). C NMR (126 MHz, CDCl ) δ 164.4,
3
62.3, 160.6, 158.7, 149.7, 139.2, 130.5, 129.6, 128.9, 128.4, 127.4, 119.2, 116.3, 116.1,
+
12.5, 111.2, 111.0.; MS (ESI): m/z 373 [M+H] . HRMS (ESI) calcd for C H F N
2
2
15
2
4
[
M+H]+ 373.12647; found: 373.12513.
4
.1.2.13
5-chloro-2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (18)
1
White solid, yield 77%, Mp: 198-200 ºC.; H NMR (300 MHz, CDCl ) δ 8.62 (s, 1H), 7.75
3
(d, J = 7.7 Hz, 2H), 7.70 – 7.62 (m, 2H), 7.54 – 7.45 (m, 3H), 7.38 -7.31 (m, 2H), 7.23-7.19
1
3
(
m, 2H), 6.90 (dt, J = 16.4, 8.5 Hz, 1H); C NMR (75 MHz, CDCl ) δ 163.5, 160.2, 149.1,
3
1
45.4, 138.0, 137.2, 135.0, 129.4, 128.6, 127.1, 126.3, 122.3, 118.0, 114.5, 113.6, 109.7; MS
1
8

ACCEPTED MANUSCRIPT
+
(
ESI): m/z 389 [M+H] . HRMS (ESI) calcd for C H ClFN [M+H]+ 389.09655; found:
22 15 4
3
89.09677.
4
.1.2.14
5-bromo-2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (19)
1
Light brown solid, yield 71%, Mp: 207-209 ºC.; H NMR (500 MHz, CDCl ) δ 8.67 (s, 1H),
3
7
.96 – 7.86 (m, 2H), 7.82 (d, J = 7.4 Hz, 2H), 7.75 – 7.61 (m, 1H), 7.54-7.46 (m, 3H), 7.35 (t,
1
3
J = 6.5 Hz, 1H), 7.28 (dd, J = 8.3, 1.4 Hz, 1H), 7.13 – 7.00 (m, 2H). C NMR (75 MHz,
CDCl ) δ 163.5, 160.3, 149.1, 146.1, 138.4, 129.6, 129.5, 128.7, 127.7, 126.1, 124.1, 117.9,
3
+
1
14.3, 114.1, 113.9, 111.6.; MS (ESI): m/z 433 [M+H] . HRMS (ESI) calcd for C H BrFN
2
2
25
4
[
M+H]+ 433.04641; found: 433.04597.
4
.1.2.15
2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-methoxy-1H-
benzo[d]imidazole (20)
Light yellow solid, yield 77%, Mp: 213-215 ºC.; H NMR (300 MHz, CDCl ) δ 8.58 (s, 1H),
1
3
7
.78 – 7.62 (m, 4H), 7.47 (t, J = 7.8 Hz, 2H), 7.34 (t, J = 7.4 Hz, 1H), 7.28 (s, 1H), 7.16 (t, J
1
3
=
8.6 Hz, 2H), 7.01 (s, 1H), 6.91 (dd, J = 8.8, 2.4 Hz, 1H), 3.85 (s, 3H). C NMR (75 MHz,
CDCl ) δ 164.8, 161.4, 156.6, 149.6, 139.1, 130.5, 130.4, 129.5, 128.71, 128.5, 127.1, 119.0,
3
+
1
16.1, 115.8, 112.8, 112.3, 55.7.; MS (ESI): m/z 385 [M+H] . HRMS (ESI) calcd for
C H FN O [M+H]+ 385.14507; found: 385.14507.
23
18
4
4
.1.2.16
2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-methyl-1H-
benzo[d]imidazole (21)
1
Light brown solid, yield 65%, Mp: 185-187 ºC.; H NMR (500 MHz, CDCl ) δ 8.35 (s, 1H),
3
7
7
.58 – 7.49 (m, 4H), 7.40 (d, J = 7.3 Hz, 1H), 7.34 (t, J = 7.4 Hz, 2H), 7.28 – 7.22 (m, 2H),
1
3
.06 (dd, J = 8.2, 1.0 Hz, 1H), 6.98 (t, J = 8.69 Hz, 2H), 2.44 (s, 3H). C NMR (126 MHz,
1
9

ACCEPTED MANUSCRIPT
CDCl ) δ 164.1, 162.1, 149.8, 145.23, 139.2, 132.7, 130.4, 130.3, 129.5, 128.8, 128.4, 127.1,
3
+
1
24.2, 119.0, 115.9, 115.8, 112.7, 21.6.; MS (ESI): m/z 369 [M+H] . HRMS (ESI) calcd for
C H FN [M+H]+ 369.15027; found: 369.15027.
23
18
4
4
.1.2.17
2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-(trifluoromethyl)-1H-
benzo[d]imidazole (22)
1
Light brown solid, yield 70%, Mp: 217-219 ºC.; H NMR (500 MHz, CDCl ) δ 8.78 (s, 1H),
3
7
.98 – 7.91 (m, 2H), 7.88-7.82 (m, 3H), 7.72-7.67 (m, 1H), 7.49 (t, J = 7.47 Hz, 2H), 7.46
1
3
(
dd, J = 8.4, 1.2 Hz, 1H), 7.35 (t, J = 7.47 Hz, 1H), 7.10 (t, J = 8.71 Hz, 2H). C NMR (75
MHz, CDCl3) δ 163.6, 160.3, 149.3, 147.0, 138.8, 138.2, 137.1, 129.49, 128.7, 127.3, 126.3,
+
1
25.7, 123.6, 123.2, 122.1, 118.5, 114.2, 111.9, 110.3.; MS (ESI): m/z 423 [M+H] . HRMS
(
ESI) calcd for C H F N [M+H]+ 423.12182; found: 423.12187.
23 15 4 4
4
.1.2.18
2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-5,6-dimethyl-1H-
benzo[d]imidazole (23)
Light brown solid, yield 73%, Mp: 187-189 ºC.; H NMR (300 MHz, CDCl ) δ 8.56 (s, 1H),
1
3
7
.78 – 7.59 (m, 4H), 7.46 (t, J = 7.8 Hz, 2H), 7.34 (t, J = 7.4 Hz, 2H), 7.25 (s, 1H), 7.14 (t, J
1
3
=
8.6 Hz, 2H), 2.38 (s, 6H). C NMR (75 MHz, CDCl ) δ 164.7, 161.4, 149.8, 144.7, 139.1,
3
1
31.7, 130.3, 130.2, 129.4, 128.6, 128.4, 127.0, 118.9, 115.8, 115.5, 112.7, 20.3.; MS (ESI):
+
m/z 383 [M+H] . HRMS (ESI) calcd for C H FN [M+H]+ 383.16617; found: 383.16614.
2
4
20
4
4
.1.2.19
5,6-dichloro-2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (24)
1
Light brown solid, yield 79%, Mp: 203-205 ºC.; H NMR (300 MHz, CDCl ) δ 8.74 – 8.60
3
(m, 1H), 7.93-7.82 (m, 4H), 7.62 (s, 2H), 7.47 (t, J = 7.0 Hz, 2H), 7.39 – 7.27 (m, 1H), 7.06
13
(
t, J = 7.6 Hz, 2H). C NMR (126 MHz, CDCl ) δ 163.9, 161.9, 150.0, 148.0, 139.1, 130.3,
3
2
0

ACCEPTED MANUSCRIPT
1
30.2, 129.5, 129.3, 128.2, 127.1, 125.9, 118.8, 115.3, 112.0, 30.8, 29.5.; MS (ESI): m/z 423
+
[
M+H] . HRMS (ESI) calcd for C H ClFN [M+H]+ 423.05680; found: 423.05699.
2
2
14
4
4
.1.2.20
2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-naphtho[2,3-
d]imidazole (25)
1
Brown solid, yield 70%, Mp: 193-195 ºC.; H NMR (500 MHz, CDCl ) δ 8.74 (s, 1H), 7.89-
3
7
7
1
1
.84 (m, 3H), 7.78 – 7.69 (m, 4H), 7.47 (t, J = 7.8 Hz, 2H), 7.40 (dq, J = 6.2, 3.1 Hz, 2H),
13
.35 (t, J = 7.1 Hz, 1H), 7.25 (s, 1H), 7.20 (t, J = 8.5 Hz, 2H). C NMR (126 MHz, CDCl ) δ
3
63.7, 161.8, 150.2, 150.0, 139.1, 130.5, 130.4, 130.1, 129.7, 129.4, 128.4, 127.6, 127.0,
+
23.4, 118.7, 115.1, 114.9, 112.6.; MS (ESI): m/z 405 [M+H] . HRMS (ESI) calcd for
C H FN [M+H]+ 405.15063; found: 405.15071.
26
18
4
4
.1.2.21
2-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazole
(26)
1
Light brown solid, yield 73%, Mp: 177-179 ºC.; H NMR (500 MHz, CDCl ) δ 8.55 (s, 1H),
3
7
2
.66 (d, J = 7.7 Hz, 2H), 7.59 (d, J = 8.54 Hz, 2H), 7.56-7.52 (m, 2H), 7.42 (t, J = 7.9 Hz,
1
3
H), 7.36 (d, J = 8.4 Hz, 2H), 7.31 (t, J = 7.47 Hz, 1H), 7.28 – 7.23 (m, 2H). C NMR (126
MHz, CDCl ) δ 149.6, 145.3, 139.1, 135.1, 130.7, 129.8, 129.5, 129.1, 127.3, 123.0, 119.1,
3
+
1
12.3.; MS (ESI): m/z 371 [M+H] ; HRMS (ESI) calcd for C H ClN [M+H]+ 371.10567;
2
2
16
4
found: 371.10580.
4
.1.2.22
2-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-fluoro-1H-
benzo[d]imidazole (27)
1
Light brown solid, yield 69%, Mp: 188-191 ºC.; H NMR (300 MHz, CDCl ) δ 8.69 (s, 1H),
3
7
.91-7.80 (m, 4H), 7.60 (s, 1H), 7.52-7.46 (m, 3H), 7.36 (d, J = 7.55 Hz, 2H), 7.25 (d, J = 9.1
1
3
Hz, 1H), 6.97 (t, J = 9.2 Hz, 1H). C NMR (75 MHz, CDCl ) δ 149.7, 147.8, 139.08, 134.2,
3
2
1

ACCEPTED MANUSCRIPT
+
1
30.7, 129.7, 129.5, 129.4, 128.3, 127.1, 125.4, 118.8, 112.2. MS (ESI): m/z 389 [M+H] ;
HRMS (ESI) calcd for C H ClFN [M+H]+ 389.09671; found: 389.09636.
2
2
15
4
4
.1.2.23
5-chloro-2-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (28)
1
Light brown solid, yield 71%, Mp: 208-210 ºC.; H NMR (300 MHz, CDCl ) δ 8.78 (d, J =
3
8
.7 Hz, 1H), 7.90 (dd, J = 16.6, 8.0 Hz, 4H), 7.72 (d, J = 11.1 Hz, 1H), 7.65 – 7.48 (m, 4H),
+
7
.41 (d, J = 7.9 Hz, 2H), 7.21 (d, J = 8.3 Hz, 1H). MS (ESI): m/z 405 [M+H] .; HRMS (ESI)
calcd for C H Cl N [M+H]+ 405.06729; found: 405.06685.
2
2
15
2
4
4
.1.2.24
5-bromo-2-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (29)
1
Light brown solid, yield 76%, Mp: 202-204 ºC.; H NMR (300 MHz, CDCl ) δ 8.87 (s, 1H),
3
7
7
1
.96 (d, J = 8.5 Hz, 2H), 7.93 – 7.83 (m, 3H), 7.54 (t, J = 7.8 Hz, 2H), 7.44-7.34 (m, 4H),
13
.29 (d, J = 8.4 Hz, 1H). C NMR (75 MHz, CDCl ) δ 147.9, 145.2, 137.5, 132.1, 129.5,
3
+
28.5, 128.0, 126.5, 125.4, 123.2, 117.1, 111.0.; MS (ESI): m/z 449 [M+H] . HRMS (ESI)
calcd for C H BrClN [M+H]+ 449.01686; found: 449.01589.
2
2
15
4
4
.1.2.25
2-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-methoxy-1H-
benzo[d]imidazole (30)
1
Light brown solid, yield 75%, Mp: 191-193 ºC.; H NMR (500 MHz, CDCl ) δ 8.55 (s, 1H),
3
7
2
.70 (d, J = 7.7 Hz, 2H), 7.61 (d, J = 8.54 Hz, 2H), 7.48-7.43 (m, 3H), 7.41 (d, J = 8.39 Hz,
1
3
H), 7.38-7.29 (m, 2H), 6.90 (dd, J = 8.8, 2.4 Hz, 1H), 3.84 (s, 3H). C NMR (75 MHz,
CDCl ) δ 155.3, 148.7, 144.4, 138.5, 133.2, 130.3, 128.9, 128.4, 127.5, 126.2, 118.0, 112.3,
3
+
1
4
10.8, 96.5, 54.9.; MS (ESI): m/z 401 [M+H] . HRMS (ESI) calcd for C H ClN O [M+H]+
2
3
18
4
01.11680; found: 401.11694.
2
2

ACCEPTED MANUSCRIPT
4
.1.2.26
2-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-(trifluoromethyl)-1H-
benzo[d]imidazole (31)
Light brown solid, yield 71%, Mp: 216-218 ºC.; H NMR (500 MHz, CDCl ) δ 8.55 (s, 1H),
1
3
7
2
1
.71 (d, J = 8.39 Hz, 2H), 7.60 – 7.55 (m, 3H), 7.51 (d, J = 7.78 Hz, 2H), 7.45 (t, J = 8.39 Hz,
1
3
H), 7.42 – 7.37 (m, 2H), 7.34 (dt, J = 7.47, 1.52 Hz, 1H). C NMR (75 MHz, CDCl ) δ
3
49.8, 148.0, 139.0, 134.1, 130.7, 129.7, 129.6, 129.4, 128.3, 127.1, 118.8, 112.3.; MS (ESI):
+
m/z 439 [M+H] . HRMS (ESI) calcd for C H ClF N [M+H]+ 439.09211; found:
23
15
3
4
4
39.09206.
4
.1.2.27
2-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-methyl-1H-
benzo[d]imidazole (32)
1
Light brown solid, yield 67%, Mp: 176-178 ºC.; H NMR (300 MHz, CDCl ) δ 8.48 (s, 1H),
3
7
1
1
.63 (d, J = 7.8 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.45-7.28 (m, 7H), 7.08 (d, J = 8.12 Hz,
1
3
H), 2.46 (s, 3H). C NMR (75 MHz, CDCl ) δ 148.3, 144.1, 138.0, 132.6, 130.4, 130.0,
3
+
28.6, 128.3, 127.0, 125.7, 122.3, 117.5, 111.9, 20.4.; MS (ESI): m/z 385 [M+H] . HRMS
(
ESI) calcd for C H ClN [M+H]+ 385.12185; found: 385.12093.
23 17 4
4
.1.2.28
2-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-5,6-dimethyl-1H-
benzo[d]imidazole (33)
Light brown solid, yield 71%, Mp: 182-184 ºC.; H NMR (500 MHz, CDCl ) δ 8.41 (s, 1H),
1
3
7
2
1
.56 (d, J = 8.08 Hz, 2H), 7.48 (t, J = 8.39 Hz, 2H), 7.36-7.30 (m, 3H), 7.26-7.21 (m, 4H),
1
3
.34 (s, 6H). C NMR (75 MHz, CDCl ) δ 149.7, 143.3, 134.4, 133.1, 130.0, 129.5, 129.2,
3
28.7, 128.4, 127.9, 127.3, 126.9, 126.5, 119.2, 118.8, 114.3, 110.7, 109.3, 20.3.; MS (ESI):
+
m/z 399 [M+H] .; HRMS (ESI) calcd for C H ClN [M+H]+ 399.13745; found: 399.13714.
2
4
20
4
2
3

ACCEPTED MANUSCRIPT
4
.1.2.29
5,6-dichloro-2-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (34)
Brown solid, yield 72%, mp: 195-197 ºC; H NMR (300 MHz, CDCl ) δ 8.39 (s, 1H), 7.92-
1
3
7
.80 (m, 4H), 7.65 (s, 1H), 7.52 (t, J = 7.74 Hz, 2H) 7.4 – 7.32 (m, 4H). MS (ESI): m/z 439
+
[
M+H] ; HRMS (ESI) calcd for C H Cl N [M+H]+ 439.0284; found: 439.0282.
2
2
14
3
4
4
.1.2.30
2-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-naphtho
[2,3-
d]imidazole (35)
Light brown solid, yield 75%, Mp: 184-186 ºC.; H NMR (500 MHz, CDCl ) δ 8.65 (s, 1H),
1
3
7
1
1
.92-7.84 (m, 3H), 7.70 – 7.59 (m, 4H), 7.45 – 7.37 (m, 6H), 7.32 (t, J = 7.4 Hz, 1H), 7.25 (s,
1
3
H). C NMR (75 MHz, CDCl ) δ 150.0, 149.8, 139.1, 134.3, 130.8, 130.3, 129.9, 129.4,
3
+
28.4, 127.7, 127.1, 123.5, 118.8, 112.7.; MS (ESI): m/z 421 [M+H] .; HRMS (ESI) calcd for
C H ClN [M+H]+ 421.12184; found: 421.12176.
26
18
4
4
.1.2.31
2-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazole
(36)
1
Light yellow solid, yield 78%, Mp: 179-182 ºC.; H NMR (300 MHz, CDCl +DMSO-d6) δ
3
8
2
.69 (s, 1H), 7.81 (dd, J = 8.2, 3.4 Hz, 4H), 7.56 (dd, J = 5.9, 3.1 Hz, 2H), 7.48 (t, J = 7.9 Hz,
1
3
H), 7.32 (t, J = 7.4 Hz, 1H), 7.23 – 7.14 (m, 2H), 6.89 (d, J = 8.8 Hz, 2H), 3.77 (s, 3H). C
NMR (75 MHz, CDCl +DMSO-d6) δ 158.6, 149.5, 145.0, 138.2, 128.5, 128.3, 128.2, 125.6,
3
+
1
23.8, 120.8, 117.5, 112.5, 111.5, 54.0.; MS (ESI): m/z 367 [M+H] . HRMS (ESI) calcd for
C H ON [M+H]+ 367.15534; found: 367.15330.
23
19
4
4
.1.2.32
6-fluoro-2-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (37)
2
4

ACCEPTED MANUSCRIPT
1
Light brown solid, yield 71%, Mp: 176-179 ºC.; H NMR (500 MHz, CDCl ) δ 8.64 (s, 1H),
3
7
.73 (d, J = 7.9 Hz, 2H), 7.63 (d, J = 8.54 Hz, 2H), 7.47-7.43 (m, 4H), 7.32 (t, J = 7.4 Hz,
1
3
1
H), 7.02-6.95 (m, 3H), 3.85 (s, 3H); C NMR (126 MHz, CDCl ) δ 160.2, 159.9, 158.3,
3
1
50.7, 147.2, 139.3, 129.6, 129.4, 128.9, 126.8, 124.6, 118.8, 113.9, 112.3, 110.4, 110.2,
+
5
5.1.; MS (ESI): m/z 385 [M+H] ; HRMS (ESI) calcd for C H ON F [M+H]+ 385.1464;
2
3
18
4
found: 385.1506.
4
.1.2.33
6-chloro-2-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (38)
1
Light brown solid, yield 75%, Mp: 188-190 ºC; H NMR (300 MHz, CDCl ) δ 8.47 (s, 1H),
3
7
6
1
.62 (d, J = 7.74 Hz, 2H), 7.50-7.39 (m, 4H), 7.36-7.24 (m, 3H), 7.18 (d, J = 7.6 Hz, 1H),
1
3
.89 (d, J = 8.5 Hz, 2H), 3.78 (s, 3H). C NMR (75 MHz, CDCl3) δ 160.1, 150.7, 147.0,
39.0, 129.7, 129.4, 128.7, 128.1, 127.1, 124.2, 123.1, 118.9, 114.2, 112.0, 55.2.; MS (ESI):
+
m/z 401 [M+H] . HRMS (ESI) calcd for C H ON Cl [M+H]+ 401.11637; found:
23
18
4
4
01.11412.
4
.1.2.34
6-bromo-2-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (39)
Light brown solid, yield 78%, Mp: 194-196 ºC.; H NMR (300 MHz, CDCl ) δ 8.67 (s, 1H),
1
3
7
7
1
.81 (d, J = 8.1 Hz, 2H), 7.68 (d, J = 8.3 Hz, 2H), 7.57-7.42 (m, 4H), 7.36 (t, J = 7.2 Hz, 1H),
13
.11-7.02 (m, 3H), 3.87 (s, 3H); C NMR (75 MHz, CDCl ) δ 160.2, 151.1, 146.8, 139.5,
3
29.8, 129.5, 128.8, 127.1, 125.6, 124.3, 119.0, 115.5, 114.3, 112.0, 55.2.; MS (ESI): m/z 445
+
[
M+H] .; HRMS (ESI) calcd for C H BrN O [M+H]+ 445.06593; found: 445.06581.
2
3
18
4
4
.1.2.35
6-methoxy-2-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (40)
2
5

ACCEPTED MANUSCRIPT
1
Light yellow solid, yield 74%, Mp: 177-179 ºC.; H NMR (300 MHz, CDCl ) δ 8.69 (s, 1H),
3
7
6
1
1
3
.90 – 7.66 (m, 4H), 7.59-7.48 (m, 3H), 7.45 (t, J = 7.6 Hz, 2H), 7.33 (d, J = 7.2 Hz, 1H),
1
3
.89 (d, J = 8.5 Hz, 2H), 3.82 (s, 3H), 3.78 (s, 3H). C NMR (75 MHz, CDCl ) δ 162.0,
3
60.0, 158.7, 138.3, 136.1, 130.3, 129.1, 128.6, 125.7, 123.8, 118.1, 115.9, 112.5, 105.8,
+
01.6, 55.4, 55.3.; MS (ESI): m/z 397 [M+H] . HRMS (ESI) calcd for C H O N [M+H]+
2
4
21
2
4
97.16590; found: 397.16368.
4
.1.2.36
2-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-5-(trifluoromethyl)-1H-
benzo[d]imidazole (41)
1
Brown solid, yield 71%, Mp: 190-192 ºC.; H NMR (300 MHz, CDCl ) δ 8.63 (s, 1H), 7.72
3
(d, J = 7.7 Hz, 2H), 7.57 (d, J = 8.6 Hz, 2H), 7.55-7.46 (m, 4H), 7.33 (t, J = 7.3 Hz, 1H), 7.11
1
3
(
d, J = 7.2 Hz, 1H), 6.98 (d, J = 8.6 Hz, 2H), 3.84 (s, 3H). C NMR (75 MHz, CDCl ) δ
3
1
60.0, 150.8, 147.9, 138.8, 129.4, 127.2, 123.8, 119.9, 118.9, 114.1, 110.6, 54.9. MS (ESI):
+
m/z 435 [M+H] . HRMS (ESI) calcd for C H ON F [M+H]+ 435.14272; found:
24
18
4 3
4
35.13945.
4
.1.2.37
2-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-6-methyl-1H-
benzo[d]imidazole (42)
Light brown solid, yield 68%, Mp: 175-177 ºC.; H NMR (500 MHz, CDCl ) δ 8.53 (s, 1H),
1
3
7
5
1
1
.66 (d, J = 7.8 Hz, 2H), 7.55 (d, J = 8.7 Hz, 2H), 7.40 (t, J = 7.9 Hz, 2H), 7.30 (dd, J = 12.4,
13
.0 Hz, 4H), 6.94 – 6.90 (m, 2H), 3.80 (s, 3H), 2.33 (s, 3H). C NMR (75 MHz, CDCl ) δ
3
60.2, 150.6, 145.5, 139.3, 132.5, 130.0, 129.5, 129.2, 128.8, 128.6, 127.0, 126.4, 124.7,
+
24.1, 119.0, 118.7, 114.4, 114.0, 112.8, 110.1, 55.3, 21.6.; MS (ESI): m/z 381 [M+H] .
HRMS (ESI) calcd for C H ON [M+H]+ 381.17099; found: 381.16916.
2
4
21
4
2
6

ACCEPTED MANUSCRIPT
4
.1.2.38
2-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-5,6-dimethyl-1H-
benzo[d]imidazole (43)
Light brown solid, yield 67%, Mp: 187-189 ºC.; H NMR (300 MHz, CDCl ) δ 8.93 (s, 1H),
1
3
7
6
1
1
.65 (d, J = 7.5 Hz, 2H), 7.45 – 7.30 (m, 4H), 7.26 – 7.11 (m, 2H), 6.78 (d, J = 8.6 Hz, 2H),
1
3
.68-6.73 (m, 1H), 3.71 (s, 3H), 2.33 (s, 6H); C NMR (75 MHz, CDCl ) δ 160.2, 150.5,
3
45.0, 139.4, 131.5, 130.0, 129.5, 129.2, 128.7, 128.5, 126.9, 126.4, 124.7, 119.8, 119.1,
+
18.7, 114.3, 112.9, 110.8, 55.3, 20.3.; MS (ESI): m/z 395 [M+H] . HRMS (ESI) calcd for
C H ON [M+H]+ 395.18664; found: 395.18477.
25
23
4
4
.1.2.39
5,6-dichloro-2-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (44)
1
Light brown solid, yield 79%, Mp: 208-210 ºC.; H NMR (500 MHz, CDCl ) δ 8.73 (s, 1H),
3
7
2
1
.82 (d, J = 7.7 Hz, 2H), 7.78 (d, J = 8.6 Hz, 2H), 7.66 (s, 2H), 7.49 (dd, J = 10.7, 5.1 Hz,
1
3
H), 7.33 (t, J = 7.4 Hz, 1H), 6.95 – 6.86 (m, 2H), 3.81 (s, 3H). C NMR (75 MHz, CDCl ) δ
3
59.3, 150.3, 148.0, 138.8, 129.2, 129.1, 126.5, 124.7, 124.2, 118.2, 113.2, 111.5, 54.7.; MS
+
(
ESI): m/z 435 [M+H] . HRMS (ESI) calcd for C H ON Cl [M+H]+ 435.07739; found:
23
17
4
2
4
35.07502.
4
.1.2.40
2-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-naphtho[2,3-
d]imidazole (45)
Light brown solid, yield 76%, Mp: 211-213 ºC.; H NMR (500 MHz, CDCl ) δ 8.75 (s, 1H),
1
3
7
.89-7.81 (s, 4H), 7.74 (d, J = 7.8 Hz, 2H), 7.64 (d, J = 8.6 Hz, 2H), 7.45 (t, J = 7.9 Hz, 2H),
.39 (dq, J = 6.5, 3.3 Hz, 2H), 7.32 (t, J = 7.4 Hz, 1H), 7.01 (d, J = 8.6 Hz, 2H), 3.83 (s, 3H).
7
1
3
C NMR (75 MHz, CDCl ) δ 149.2, 147.2, 142.9, 139.0, 138.4, 130.9, 127.2, 123.5, 121.2,
3
2
7

ACCEPTED MANUSCRIPT
+
1
14.1, 110.3, 108.5, 105.7, 101.8, 101.6, 55.4.; MS (ESI): m/z 417 [M+H] .; HRMS (ESI)
calcd for C H N O [M+H]+ 417.17142; found: 417.17126.
2
7
21
4
4
.2 Cell Culture
MCF-7 (breast), HeLa (cervical) and A549 (lung) cancer cells were purchased from
ATCC (Manassas, VA). The A549 cells were grown in RPMI 1640 medium (GIBCO-
Invitrogen, NY) with 10% fetal bovine serum (FBS) and supplemented with glutamine (2
mmol/L), penicillin G (100 µg/mL), and streptomycin (100 µg/mL) at 37 ºC under 5% CO2.
MCF-7 and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)
o
containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin at 37 C under 5%
CO The culture medium was replaced with fresh medium every two days. After reaching 80-
2
.
9
0% confluence, cells were treated with 0.25% trypsin-EDTA for further passages. Cells were
used at passages 4-8 for all the experiments.
4
.2.1 MTT assay
The in vitro cytotoxicity of test compounds on MCF-7, HeLa and A549 cells was
performed using the MTT assay. Cells under study were seeded into 96 well plates at a
density of 3000-5000 cells per well depending on their doubling times and allowed to adhere
for 24 h at 37 ˚C and 5% CO . After 24 h incubation, the medium was replaced with culture
2
medium containing test compounds dissolved in dimethyl sulphoxide (DMSO) and culture
medium (DMSO) only was included as a control. After 48 h incubation, the medium
containing test compounds was aspirated and 100 µL of culture media containing 5 mg/mL
MTT (Thiazoyl blue tetrazolium bromide) was added to each well and cells were further
incubated for 4 hours in dark at 37 ºC. After 4 hours incubation, the media containing MTT
was removed and 100 µL DMSO was added to each well to solubilize the crystallized
formazan product. The plates were read on a micro-plate reader at 570 nm and a reference
2
8