Synthesis, crystal study, and anti-proliferative activity of some 2-benzimidazolylthioacetophenones towards triple-negative breast cancer MDA-MB-468 cells as apoptosis-inducing agents

Article abstract of DOI:10.3390/ijms17081221

On account of its poor prognosis and deficiency of therapeutic stratifications, triple negative breast cancer continues to form the causative platform of an incommensurate number of breast cancer deaths. Aiming at the development of potent anticancer agents as a continuum of our previous efforts, a novel series of 2-((benzimidazol-2-yl)thio)-1-arylethan-1-ones 5a–w was synthesized and evaluated for its anti-proliferative activity towards triple negative breast cancer (TNBC) MDA-MB-468 cells. Compound 5k was the most active analog against MDA-MB-468 (IC₅₀ = 19.90 ± 1.37 μM), with 2.1-fold increased activity compared to 5-fluorouracil (IC₅₀ = 41.26 ± 3.77 μM). Compound 5k was able to induce apoptosis in MDA-MB-468, as evidenced by the marked boosting in the percentage of florecsein isothiocyanate annexin V (Annexin V–FITC)-positive apoptotic cells (upper right (UR) + lower right (LR)) by 2.8-fold in comparison to control accompanied by significant increase in the proportion of cells at pre-G1 (the first gap phase) by 8.13-fold in the cell-cycle analysis. Moreover, a quantitative structure activity relationship (QSAR) model was established to investigate the structural requirements orchestrating the anti-proliferative activity. Finally, we established a theoretical kinetic study.

Full text of DOI:10.3390/ijms17081221

International Journal of
Molecular Sciences
Article
Synthesis, Crystal Study, and Anti-Proliferative
Activity of Some 2-Benzimidazolylthioacetophenones
towards Triple-Negative Breast Cancer MDA-MB-468
Cells as Apoptosis-Inducing Agents
Hatem A. Abdel-Aziz 1,*, Wagdy M. Eldehna 2, Hazem Ghabbour 3,4, Ghada H. Al-Ansary 5,
Areej M. Assaf 6 and Abdullah Al-Dhfyan 7
1
Department of Applied Organic Chemistry, National Research Center, Dokki, Giza 12622, Egypt
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City,
2
Cairo 11829, Egypt; wagdy2000@gmail.com
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457,
3
Riyadh 11451, Saudi Arabia; ghabbourh@yahoo.com
Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, Abbassia,
4
5
Cairo 11566, Egypt; ghada.mohamed@pharma.asu.edu.eg
Department of Biopharmaceutics and Clinical Pharmacy, Faculty of Pharmacy, The University of Jordan,
6
Amman 11942, Jordan; areej_assaf@ju.edu.jo
Stem Cell & Tissue Re-Engineering Program, Research Center, King Faisal Specialized Hospital & Research
7
Center, MBC-03, P.O. Box 3354, Riyadh 11211, Saudi Arabia; pharm101696@hotmail.com
*
Correspondence: hatem_741@yahoo.com; Tel.: +966-146-77341; Fax: +966-146-76220
Academic Editors: Ge Zhang and Charles J. Malemud
Received: 20 April 2016; Accepted: 11 July 2016; Published: 29 July 2016
Abstract: On account of its poor prognosis and deficiency of therapeutic stratifications, triple negative
breast cancer continues to form the causative platform of an incommensurate number of breast cancer
deaths. Aiming at the development of potent anticancer agents as a continuum of our previous
efforts, a novel series of 2-((benzimidazol-2-yl)thio)-1-arylethan-1-ones 5a–w was synthesized and
evaluated for its anti-proliferative activity towards triple negative breast cancer (TNBC) MDA-MB-468
cells. Compound 5k was the most active analog against MDA-MB-468 (IC50 = 19.90 ˘ 1.37 µM), with
2.1-fold increased activity compared to 5-fluorouracil (IC50 = 41.26
˘
3.77 µM). Compound 5k was
able to induce apoptosis in MDA-MB-468, as evidenced by the marked boosting in the percentage
of florecsein isothiocyanate annexin V (Annexin V–FITC)-positive apoptotic cells (upper right
(UR) + lower right (LR)) by 2.8-fold in comparison to control accompanied by significant increase
in the proportion of cells at pre-G1 (the first gap phase) by 8.13-fold in the cell-cycle analysis.
Moreover, a quantitative structure activity relationship (QSAR) model was established to investigate
the structural requirements orchestrating the anti-proliferative activity. Finally, we established
a theoretical kinetic study.
Keywords: synthesis; X-ray; anti-proliferative; breast cancer MDA-MB-468 cells; apoptosis
1. Introduction
Pertaining to the frequency of diagnosis worldwide, breast cancer is regarded as the second most
frequently diagnosed cancer and the most frequently diagnosed tumor among women. Also, it is
regarded as the fifth leading cause of cancer mortality [
diagnosed breast cancer cases and 522,000 breast cancer deaths occurred worldwide [
of breast cancer is still unknown, although different risk factors have been established—to name
1]. In 2012, an estimated 1.67 million newly
1
]. The etiology
Int. J. Mol. Sci. 2016, 17, 1221; doi:10.3390/ijms17081221
www.mdpi.com/journal/ijms

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just a few, first-degree relative’s breast cancer family history, mammographic density, benign breast
disease, younger age at menarche, low parity, older age at first birth, older age at menopause, high
postmenopausal body mass index, low premenopausal body mass index, and endogenous hormone
levels have been established as risk factors for breast cancer [2,3].
Breast cancer is regarded as a diverse group of diseases with multiple intrinsic tumor subtypes that
have various treatment modalities and long-term survival probabilities. The immunohistochemical
expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth
factor receptor-2 (HER-2) forms the platform of characterization of clinically defined breast cancer
subtypes [
4]. In approximately 15%–20% of globally diagnosed breast cancers, the tumors do not
express ER, PR, or HER-2. Such malignancies are designated as triple-negative breast cancer (TNBC) [
5
].
In the current medical era, TNBC, among all the breast cancer subgroups, has stood out as the
greatest clinical challenge as these tumors have no clinically validated molecularly targeted therapy,
are prevalent in younger women, associated with the worst prognosis, and often relapse rapidly.
Also, TNBC are highly proliferative, poorly differentiated, often grade III carcinomas, genetically
unstable, and preferentially metastasize to the brain and lungs [6–10]. Therefore, there is a critical need
to develop potent and effective novel therapies to improve the outcomes of TNBC treatment.
Because of their similarity to some naturally occurring nucleotides and their existence in several
naturally occurring compounds, benzimidazole derivatives possess a wide range of biological activities
and therapeutic effects [11
privileged scaffold and has been copiously explored as an anti-proliferative agent targeting different
breast cancer cells [14 21]. Surveying the literature revealed that different benzimidazole-based
–13]. In the field of medicinal chemistry, benzimidazole represents a highly
–
scaffolds were developed with significant activity toward the TNBC MDA-MB-468 cells in the
anticancer drug screening program of the U.S. National Cancer Institute (NCI), according to their
applied protocol against full NCI 60 human cell lines panel (Figure 1).
Figure 1. Structures of some reported benzimidazoles I–VIII, by other research groups, with
anti-proliferative activity against triple-negative breast cancer MDA-MB-468 cells [22–27].
Many researchers have reported the utility of 2-aryl benzimidazole derivatives as anti-proliferative
agents against TNBC. Attaching a heterocyclic moiety, 5-tert-butyl-1H-pyrazol-3-yl, in position 1
and an aryl moiety, 4-chlorophenyl, in position 2 of benzimidazole core resulted in compound
(NSC: 751047) with good anti-proliferative activity against MDA-MB-468 (IC50 = 2.4 M) [22] (Figure 1),
while substitution of position 2 of 5-flouro and 5-methoxybenzimidazole with 1,2,4-oxadiazole moiety
I
µ

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through a phenyl ring afforded compounds II and III, respectively, (NSC: 761109 and 761814) with IC50
values of 3.01 and 6.54 M, respectively, against MDA-MB-468 [23] (Figure 1). Moreover, introduction
of different aryl groups through a pyrazole linker at position 2 of the benzimidazole core, as in
compounds IV and (NSC: 768400 and 768399), achieved significant efficacy against MDA-MB-468
(IC50 values of 0.93 and 3.5 M, respectively) [24] (Figure 1). Interestingly, linking different aryl
moeities, through variable heterocyclic groups, via a three-atom linker, namely a propan-1-one group
to position 2 of the benzimidazole core led to compounds VI VIII (NSC: 761980, NSC: 759205 and
µ
V
µ
–
NSC: 7604520) with low or sub-micromolar anti-proliferative activity against MDA-MB-468 (IC50 = 1.93
,
0.79 and 0.69 µM, respectively) [25–27] (Figure 1).
In addition, we recently introduced an efficacious benzimidazole-based scaffold as for
development of potent antitumor agents that prove to have anti-proliferative activity not only toward
the cancer stem cells but also toward the bulk of tumor cells of the colon HT-29 cell line [28]. The design
of such a scaffold relies on linking different aryl or heteroaryl groups to position 2 of the benzimidazole
core through a thio ethan-2-one linker. From the findings reported above, we came to the conclusion
that linking the 2-position of benzimidazole scaffold to a terminal aryl or heteroaryl group directly or
via variable spacers—an aryl, a heteroaryl, or a propan-1-one group—affords promising molecules
that have significant anti-proliferative activity against TNBC.
Regarding these points and as a continuation of our research program on the design and synthesis
of effective antitumor candidates [29–35], it was thought worthwhile to extend our investigations
around our study [28] to probe for benzimidazole derivatives having anti-proliferative activity towards
TNBC. Our structure-based design was three-fold: (i) preserving benzimidazole structure with
subsitution at 2-position; (ii) maintaining a terminal lipophilic group; and (iii) establishing a three-atom
thio ethane-1-one linker to afford more flexibility for the designed molecules (Figure 2). Thus, the
present work reports the synthesis of benzimidazoles 5a–w and their in vitro anti-proliferative activity
against the TNBC MDA-MB-468 cell line. Moreover, the most active member in this study, 5k, was
selected to be further investigated regarding its effects on cell cycle progression and potential apoptotic
effect in the MDA-MB-468 cells, to acquire perception of the mechanism of the anti-proliferative activity
of the prepared compounds. Eventually, a theoretical kinetic study was constituted.
Figure 2. Structure-based design of target benzimidazoles 5a–w as anti-triple-negative breast cancer
(TNBC) agents.
2. Results
2.1. Synthetic Approach to Prepare the Target Derivatives
The target compounds were prepared following our recently published procedure [28] via
the reaction of compound with different aromatic ketones 3a in glacial acetic acid in the
2
–w

Int. J. Mol. Sci. 2016, 17, 1221
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presence of two equivalents conc. H2SO4 to afford a quantitative yield from the sulfate salts
4a The prepared sulfate salts 4a were subsequently neutralized to afford the target
–
w
.
–w
2-((benzimidazol-2-yl)thio)-1-arylethan-1-ones 5a–w in an excellent yield of 82%–96% (Scheme 1).
H
N
NH2
NH2
i
SH
N
1
2
ii
ii
O
HO
O
OH
O
HO
Me
OH
O
S
R
R
S
4,5
R
Ar Me
3u-w
O
O
3a-t
H
a
4-CH3
4-NH2
4-Cl
2-F
4-F
b
c
d
e
f
O
O
O
R
O
Ar
Ar
H
N
H
N
H
N
S
S
H
N
S
S
O
O
O
O
g
h
i
j
k
l
m
n
o
p
q
r
2,4-di-F
4-Br
N
H
N
N
O
O
O
O
S
N
H
H
H
S
4-NO2
2-OH
3-OH
4-OH
2-OCH3
4-OCH3
3-OH,4-OCH3
2,4-(OCH3)2
3,4-(OCH3)2
3,4-O-CH2-O
3,4,5-(OCH3)3
4-OC2H5
4u-w
4a-t
Ar
4,5
iii
iii
u
v
naphthalin-2-yl
furan-2-yl
w
thiophen-2-yl
H
H
N
N
S
O
S
O
N
N
s
t
Ar
R
5u-w
5a-t
Scheme 1. Synthesis of the target 2-((benzimidazol-2-yl)thio)-1-arylethan-1-ones 5a
corresponding sulfate salts 4a Reagents and conditions: ( ) CS2/KOH/ethanol/reflux 2 h;
(ii) Glacial acetic acid/reflux 0.5 h; (iii) Aqueous Na2CO3/r.t. (room temperature) 2 h.
–w and their
–
w.
i
Infrared (IR) spectra for compounds 5a–w displayed absorption bands attributable for the NH
group in the range 3324–3460 cm´1, also a (C=O) band in the range of 1654–1690 cm´1. Also, their
1H-nuclear magnetic resonance (NMR) spectra displayed one singlet D2O-exchangeable signal due to
the NH proton in the range of
within
5.00 ppm. Furthermore, the 13C-NMR spectra of compounds 5a
in the region 182.16–193.90 ppm due to the carbon of carbonyl group, whereas the carbons of the
δ
12.52–12.80 ppm, whereas (–CH2–) protons appeared as singlet signals
δ
–w
showed signals resonating
δ
(–CH2–) group appeared in the region of δ 38.46–43.97 ppm.
2.2. Single Crystal Analysis of Compounds 4u and 5v
Crystals of compounds 4u and 5v were selected to analyze their single-crystal X-ray
crystallographic after slow evaporation from solutions of ethanol. The instrument used
is Bruker SMART APEX II D8 Venture diffractometer (Bruker, Karlsruhe, Germany) with
graphite-monochromated Mo K radiation ( = 0.71073 Å) at 100 and 150 K, respectively. A direct
α
λ
method was applied to solve the structures that were subsequently refined with SHELXTL [36].
The positions of all the non-H-atoms were provided by E-maps. Using anisotropic temperature
factors, the full-matrix least-squares refinement was carried out on F2’s for all non-H-atoms.
Crystallographic data was deposited in the Cambridge Crystallographic Data Center and assigned the
following deposition numbers: CCDC 1058838 and 1455648 for compounds 4u and 5v, respectively.
In Figures 2 and 3, the crystallographic structures of compounds 4u and 5v are represented,
respectively. The exact structure is unambiguously defined by the single crystal X-ray study on
both derivatives. The crystal structure of 4u confirmed two crystallographically independent cation
molecules with one sulfate anion, in the presence of one molecule of ethanol in its asymmetric

Int. J. Mol. Sci. 2016, 17, 1221
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unit, as shown in Figure 3. The asymmetric unit of 5v contains one molecule only, as depicted in
Figure 4. Table 1 listed the crystallographic data and the refinement for the crystals. Tables 2 and 3
summarized some selected geometric parameters for 4u and 5v, respectively. Also, Figure S1 (in
Supplementary Materials) displayed the molecular packing of compound 5v, while Table S1 showed
˝
the hydrogen-bond geometry (Å, ) for compound 5v.
Figure 3. An ORTEP diagram of the final X-ray structure of compound 4u.
Figure 4. An ORTEP diagram of the final X-ray structure of compound 5v.
Table 1. Crystallographic data and refinements for compounds 4u and 5v.
Compound
4u
Crystal data
5v
Chemical formula
2(C19H15N2OS) C2H6O¨ SO4
780.94
C13H10N2O2S
258.29
Mr
Crystal system, space group
Orthorhombic, P212121
100
Monoclinic, P21/c
100
Temperature (K)
a, b, c (Å)
β (˝)
14.2409 (8), 15.8339 (9), 16.2043 (8) 11.4097 (7), 10.8336 (7), 10.1256 (7)
90.00
3653.9 (3)
4
114.165 (2)
1141.93 (13)
4
V (Å3)
Z
Radiation type
Mo Kα
Mo Kα
µ (mm´1
Crystal size (mm)
)
0.26
0.28
0.44 ˆ 0.26 ˆ 0.16
0.47 ˆ 0.36 ˆ 0.11

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Table 1. Cont.
Compound
4u
5v
Data collection
Bruker APEX-II D8 venture
diffractometer
Diffractometer
CCD area detector diffractometer
Absorption correction
multi-scan, SADABS
0.90, 0.92
multi-scan, SADABS
0.89, 0.93
Tmin, Tmax
Number of measured,
independent and observed
[I > 2σ(I)] reflections
11,220, 11,220, 8647
9364, 1999, 1689
0.090
Rint
Refinement
R[F2 > 2ó(F2)], wR(F2), S
Number of reflections
Number of parameters
Number of restraints
0.050, 0.121, 1.05
0.086, 0.250, 1.10
11,220
494
0
1999
163
0
by a mixture of independent and
constrained refinement
by a mixture of independent and
constrained refinement
H-atom treatment
∆ρmax, ∆ρmin (e¨ Å´3
)
0.76, ´0.37
1,058,838
1.12, ´0.87
1,455,648
CCDC number
Table 2. Selected geometric parameters (Å, ˝) for compound 4u.
Bond Distances
S1A–C7A
S1A–C8A
S1B–C7B
S1B–C8B
S2–O3
S2–O5
S2–O2
S2–O4
O1A–C9A
1.722 (3)
1.811 (3)
1.724 (3)
1.804 (3)
1.476 (3)
1.479 (3)
1.478 (3)
1.467 (2)
1.203 (6)
O6–C21
N1A–C7A
N1A–C1A
N2A–C6A
N2A–C7A
N1B–C7B
N1B–C1B
N2B–C7B
N2B–C6B
1.437 (6)
1.333 (4)
1.406 (4)
1.387 (4)
1.338 (4)
1.342 (4)
1.398 (4)
1.339 (4)
1.381 (4)
Bond Angles
C7A–S1A–C8A
C7B–S1B–C8B
O3–S2–O5
O4–S2–O5
O3–S2–O4
O2–S2–O3
O2–S2–O4
O2–S2–O5
C1A–N1A–C7A
C6A–N2A–C7A
C1B–N1B–C7B
C6B–N2B–C7B
N1A–C1A–C2A
N1A–C1A–C6A
N2A–C6A–C1A
N2A–C6A–C5A
102.76 (15)
99.02 (15)
108.55 (14)
111.31 (14)
110.49 (14)
108.54 (16)
109.46 (15)
108.43 (14)
108.2 (3)
108.7 (3)
108.0 (3)
108.7 (3)
130.9 (3)
N1A–C7A–N2A
S1A–C7A–N1A
S1A–C8A–C9A
O1A–C9A–C10A
O1A–C9A–C8A
N1B–C1B–C2B
N1B–C1B–C6B
N2B–C6B–C5B
N2B–C6B–C1B
S1B–C7B–N1B
S1B–C7B–N2B
N1B–C7B–N2B
S1B–C8B–C9B
O1B–C9B–C10B
O1B–C9B–C8B
O6–C21–C20
109.8 (3)
130.1 (2)
105.5 (2)
121.0 (4)
120.6 (3)
131.1 (3)
106.5 (3)
131.8 (3)
107.0 (3)
128.7 (3)
121.3 (2)
109.9 (3)
107.8 (2)
122.0 (3)
120.4 (3)
111.9 (4)
106.5 (3)
106.9 (3)
131.1 (3)

Int. J. Mol. Sci. 2016, 17, 1221
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Table 3. Selected geometric parameters (Å, ˝) for compound 5v.
Bond Distances
Cl1–C13
S1–C7
S1–C8
S1–C8
O1–C9
1.7361 (12)
N1–C1
N1–C7
N2–C6
N2–C6
N2–C7
1.3831 (13)
1.3686 (12)
1.3947 (13)
1.3947 (13)
1.3157 (12)
1.7521 (10)
1.7949 (10)
1.7949 (10)
1.2146 (12)
Bond Angles
C6–S1–C7
C3–O1–C4
C7–N1–C8
C7–N2–C13
O1–C3–C2
O1–C4–C5
O1–C4–C1
O2–C5–C6
102.2 (2)
106.5 (4)
103.8 (4)
106.7 (4)
111.2 (5)
116.2 (4)
109.8 (4)
123.4 (5)
S1–C6–C5
S1–C7–N2
N1–C7–N2
S1–C7–N1
N1–C8–C9
N1–C8–C13
N2–C13–C8
N2–C13–C12
113.5 (4)
117.3 (3)
114.4 (5)
128.4 (4)
130.2 (4)
109.6 (4)
105.5 (5)
132.2 (5)
2.3. Biological Evaluation of the Target Derivatives as Anti-Cancer Agents
2.3.1. In Vitro Anti-Proliferative Activity against MDA-MB-468
The WST-1 assay, as described by Ngamwongsatit et al. [37], was adopted to evaluate the
anti-proliferative activity of the synthesized 2-((benzimidazol-2-yl)thio)-1-arylethan-1-ones 5a
–
w
against human breast cancer cell line MDA-MB-468. 5-FU (florouracil) was selected as a positive
control due to its broad spectrum of anticancer activity. The anti-proliferative activity was expressed as
growth inhibitory concentration (IC50) values, which represent the compound concentrations required
to produce a 50% inhibition of cell growth after 48 hours of incubation compared to untreated controls
(Table 4).
Table 4. In vitro anti-proliferative activity of compounds 5a
–
w
against breast MDA-MB-468 cancer
cell line.
Compound
Aryl
IC50 (µM) a
5a
5b
5c
5d
C6H5
22.31 ˘ 2.04
74.25 ˘ 6.23
53.79 ˘ 5.02
31.97 ˘ 3.07
NA b
4-Me–C6H4
4-NH2–C6H4
4-Cl–C6H4
2-F–C6H4
5e
5f
5g
5h
]
4-F–C6H4
2,4-di-F–C6H3
4-Br–C6H4
24.96 ˘ 2.55
50.78 ˘ 5.11
53.83 ˘ 6.53
32.80 ˘ 3.17
4-NO2–C6H4
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
2-OH–C6H4
3-OH–C6H4
4-OH–C6H4
22.41 ˘ 2.13
19.90 ˘ 1.37
21.98 ˘ 1.91
22.32 ˘ 2.06
25.35 ˘ 2.15
22.06 ˘ 1.93
26.60 ˘ 2.24
24.30 ˘ 2.13
22.79 ˘ 1.87
22.72 ˘ 2.03
23.91 ˘ 2.19
66.45 ˘ 7.12
30.34 ˘ 3.01
28.17 ˘ 2.24
41.26 ˘ 3.77
2-OCH3–C6H4
4-OCH3–C6H4
3-OH-4-OCH3–C6H3
2,4-(OCH3)2–C6H3
3,4-(OCH3)2–C6H3
3,4-O-CH2–O–C6H3
3,4,5-(OCH3)3–C6H2
4-OC2H5–C6H4
naphthalin-2-yl
furan-2-yl
thiophen-2-yl
5-Fluorouracil
a IC50 values are the mean
˘
S.D. of three separate experiments; b NA: Compounds having IC50 value > 100
µM.

Int. J. Mol. Sci. 2016, 17, 1221
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The obtained results of the tested benzimidazole derivatives 5a
–
w
indicated that most of
the prepared compounds showed good to moderate anti-proliferative activity against the tested
MDA-MB-468 cancer cell line. Compound 5k merged as the most potent member against MDA-MB-468
(IC50 = 19.90
˘
1.37
µ
M) as it was 2.1 times more potent and efficacious than 5-fluorouracil
5f, and 5j showed superior anti-proliferative
1.91 to 26.60 2.24 M) compared to 5-fluorouracil,
M). In addition, compounds 5d 5i 5v, and 5w with
2.24 M, respectively, displayed good
activity against MDA-MB-468. On the other hand, compounds 5b 5c 5g 5h, and 5u were moderately
(IC50 = 41.26 ˘ 3.77 µM). Moreover, analogs 5a
,
–t
activity (IC50 values ranging from 21.98
the reference drug, (IC50 = 41.26 3.77
IC50 = 31.97 ˘ 3.07, 32.80 3.17, 30.34
˘
˘
µ
˘
µ
,
,
˘
˘
3.01 and 28.17
˘
µ
,
,
,
active against MDA-MB-468 with IC50 values ranging from 50.78 ˘ 5.11 to 74.25 ˘ 6.23 µM.
2.3.2. Structure Activity Relationship Study (SAR Study) of the Target Compounds
Observing the results in Table 4, valuable data could be extracted regarding the structure activity
correlation of our compounds. Foremost, the effect of grafting diverse substituents on the terminal aryl
moiety on the activities of the synthesized compounds 5a
bearing unsubstituted phenyl group showed good activity (IC50 = 22.31
–
t
was closely investigated. Compound 5a
2.04 M) in comparison to
˘
µ
5-fluorouracil (IC50 = 41.26 ˘ 3.77 µM), implying a doubling of the anti-proliferative activity.
Introduction of fluorine atom, a classical bioisostere of the hydrogen atom, at the 4-position as in
compound 5f resulted in comparable activity to the unsubstituted analogue 5a
22.31 2.04 M, respectively). Interestingly, transferring the fluorine atom from the 4-position to the
2-position, 5e, resulted in an inactive derivative. Again, di-substitution with two fluorine atoms in the
2- and 4-positions, 5g, was not favorable to the activity (IC50 = 50.78 5.11 M) compared to the mono
4-F substituted derivative 5f (IC50 = 24.96 2.55 M). Incorporation of more bulky halogens as chlorine
and bromine led to compounds 5d and 5h, respectively, with decreased activity (IC50 = 31.97 ˘ 3.07
and 53.83 6.53 M, respectively), suggesting that incorporation of a small halogen fluorine
atom only in the 4-position is markedly advantageous to the activity. The order of activities
of the halogenated derivatives 5d decreased in the order of 4-F > 4-Cl > 2,4-di-F > 4-Br > 2-F
Also, grafting an electron-withdrawing nitro group as in compound 5i resulted only in moderate
improvement of the activity (IC50 = 32.80 3.17
M) compared to 5-FU (IC50 = 41.26 ˘ 3.77 µM),
while introduction of methyl or amino groups, electron-donating groups, reduced the activity
against MDA-MB-468, as shown in 5b and 5c analogs (IC50 = 74.25 6.23 and 53.79 5.02 M,
respectively). Contrariwise, substitution with electron-donating hydroxyl, methoxy, or ethoxy groups
as in compounds 5j maintained the activity in the good range of activity regardless of their positions
(
IC50 = 24.96 ˘ 2.55 and
˘
µ
˘
µ
˘
µ
˘
µ
–
h
.
˘
µ
˘
˘
µ
–
t
or numbers (IC50 = (19.90 ˘ 1.37)–(26.60 ˘ 2.24) µM).
On the other hand, scrutinizing the anti-proliferative activity of compounds 5u–w gave us
insight about the effect of exchanging the phenyl group of 5a for other aryl or heteroaryl moieties.
Replacement of the phenyl ring of 5a with a naphthalin-2-yl group in compound 5u decreased the
activity (IC50 = 66.45
or 2-thienyl groups (compounds 5v and 5w) moderately reduced the activity (IC50 = 30.34 ˘ 3.01 and
28.17 2.24 M, respectively). In conclusion, we can assume that incorporation of an unsubstituted
˘
7.12 µM). Moreover, bioisosteric replacement of the phenyl moiety with 2-furyl
˘
µ
phenyl group or its substitution with electron-donating hydroxyl, methoxy, or ethoxy groups is
beneficial for activity against the MDA-MB-468 cell line, while introduction of heterocycles, such as
2-furyl or 2-thienyl, could not effectively replace the phenyl ring.
2.3.3. Cell-Cycle Analysis and Apoptotic Changes Investigation of Compound 5k
Cell reproduction necessitates DNA replication with a concomitant nuclear division followed by
cytoplasmic partitioning to sporulate two daughter cells. Such a successive routine is known as the
“cell cycle” and involves four distinguishable phases. The G1 phase is a gap integrated between the M
phase (nuclear division) and the S phase (DNA synthesis); another gap called G2 phase also occurs
between S and M. These gaps permit the repair of DNA damage and replication errors [38].

Int. J. Mol. Sci. 2016, 17, 1221
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To understand the mechanism behind the tumor suppression activity of the prepared compounds,
the most active member in this study, 5k, was selected to be further investigated regarding its effects on
cell cycle progression and its potential apoptotic effects in the MDA-MB-468 cell line. The MDA-MB-468
cells were treated with IC50 concentration of compound 5k for 24 h and its effect on the normal cell cycle
was detected by fluorescence-activated cell sorting (FACS) analysis (Figure 5, Figure S2). Interestingly,
exposure of MDA-MB-468 cells to 5k induced a remarkable augmentation in the proportion of cells at
pre-G1 phase by 8.13-fold. The increase was accompanied by concomitant noteworthy mitigation in
the percentage of cells at the G0/G1, S, and G2/M phases by 2.21-, 2.43-, and 11.83-fold in comparison
to the control, respectively.
Figure 5. Bar chart shows percentage of MDA-MB-468 cells at each phase of cell cycle in control cells
and cells treated with compound 5k.
2.3.4. Evaluation of the Apoptotic Effect of Compound 5k by Fluorescein Isothiocyanate
(FITC)-Labeled Annexin V (Annexin V–FITC) Assay
The apoptotic effect of 5k was further evaluated by Annexin VFITC/PI (AV/PI) dual staining
assay to examine the occurrence of phosphatidylserine externalization and also to understand whether
it is due to physiological apoptosis or nonspecific necrosis.
In this study MDA-MB-468 cells were treated with compound 5k for 48 h at 19.9 µM (IC50)
to examine the apoptotic effect. It was observed that 5k showed significant apoptosis against
MDA-MB-468 cells, as shown in Figures 6 and 7. Results indicated that 5k showed 75.78% of apoptosis
at 19.9 µM whereas 27.06% of apoptosis was observed in the control (untreated cells), comprising
a 2.8-fold improvement compared to the control. This experiment suggests that 5k significantly induces
apoptosis in MDA-MB-468 cells.
Figure 6. Fluorescein isothiocyanate (FITC)-labeled annexin V (Annexin V–FITC) staining. The cells
were treated with dimethylsulfoxide (DMSO) as a control or with compound 5k at IC50 concentration
for 24 h. The experiment was done in triplicate.

Int. J. Mol. Sci. 2016, 17, 1221
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100
80
60
40
20
0
a
Figure 7. Effect of compound 5k on the percentage of annexin V–FITC-positive staining in
MDA-MB-468 cells. Data are presented as means
p < 0.0001.
˘
S.D. a Indicates statistical difference from control at
2.4. 2 Dimensional-Quantitative Structure Activity Relationship (2D-QSAR) Analysis for the
Anti-Proliferative Activity of the Prepared Derivatives 5a–w
2.4.1. Elaboration of QSAR Model
QSAR analysis of the anti-tumor activity of the prepared derivatives 5a–w was performed to
establish a correlation between the biochemical data and the compound structures; moreover, it aids us
in identifying the positive and negative structural features within the three scaffolds. DS 2.5 software
(Discovery Studio 2.5, Accelrys, Co., Ltd., Accelrys, San Diego, CA, USA) was used to run the analysis.
A set of 21 synthesized derivatives (5a–d, 5f–I, and 5k–w) was applied as a training set with
their experimentally detected logIC50 against the MDA-MB-468 cancer cell line in the QSAR modeling.
The two remaining synthesized members were used as an external test set to assess the predictive
power and validate the established QSAR model. Various molecular descriptors for the training
set molecules were calculated using the “Calculate Molecular Properties” module. 2D Descriptors
entangled: topological descriptors, molecular properties, molecular property counts, AlogP, surface
area, and volume. As for the 3D descriptors: dipole, principal moments of inertia, jurs descriptors,
surface area, and volume, and shadow indices. To search for the best QSAR regression equation,
genetic function approximation (GFA) was utilized, i.e., multiple linear regression modeling (MLR).
2.4.2. QSAR Study Results
The best performing QSAR model is represented by Equation (1);
Potency (LogIC50) against MDA-MB-468 cell line
LogIC50 “ 4.1269 ` 0.0599 Num_ExplicitAtoms ´ 0.0139 Molecular_SAVol ` 3.0316
(1)
CHI_V_3_C ´ 0.0920 Jurs_RPCS
Adopting Equation (1), QSAR model was graphically represented. This was accomplished by
plotting the experimental values against the predicted bioactivity values logIC50 for the training set
compounds, as shown in Figure 8. Also, the estimated and experimental activities data and the
calculated descriptors of the training set compounds were summarized in Table 5. The Least-Squares
method was used to build the models, r2 = 0.842, r2 (adj) = 0.803, r2 (pred) = 0.795, Least-Squared
error = 0.004 for model 1, where r2 (adj) is r2 adjusted for the number of terms in the model; r2 (pred) is
the prediction r2, equivalent to q2 from a leave-one-out cross-validation.

Int. J. Mol. Sci. 2016, 17, 1221
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Figure 8. Predicted versus experimental logIC50 of the tested compounds against MDA-MB-468 cell
line according to Equation (1) (r2 = 0.842).
Table 5. Estimated IC50 data of the training set against MDA-MB-468 cell line and calculated descriptors
governing IC50 according to Equation (1).
Experimental
Activity
Predicted
Activity
(LogIC50)
Compound
Residual Num_ExplicitAtoms Molecular_SAVol CHI_V_3_C
Jurs_RPCS
(LogIC50
)
5a
5b
5c
5d
5f
5g
5h
5i
5k
5l
5m
5n
5o
5p
5q
5r
1.3485
1.8707
1.7307
1.5047
1.3972
1.7057
1.7310
1.5159
1.2989
1.3420
1.3487
1.4040
1.3436
1.4249
1.3856
1.3577
1.3564
1.3786
1.8225
1.4820
1.4498
1.4134
1.8742
1.6624
1.5789
1.4763
1.4841
1.7260
1.4788
1.3084
1.3186
1.3758
1.4366
1.3587
1.3990
1.4065
1.4848
1.3416
1.3311
1.8264
1.4312
1.4865
´0.0649
´0.0035
0.0683
´0.0742
´0.0791
0.2216
31
34
33
31
31
31
31
33
32
32
35
35
36
39
39
34
43
38
38
28
28
414.53
430.44
426.68
443.37
423.74
432.96
462.96
441.92
423.66
423.66
445.34
445.34
454.48
476.16
476.16
444.90
506.98
465.86
452.50
396.29
417.77
0.429
0.596
0.525
0.618
0.492
0.536
0.756
0.541
0.504
0.504
0.477
0.497
0.553
0.545
0.547
0.528
0.579
0.497
0.628
0.405
0.523
0.817
0.834
0.736
0.860
0.807
0.752
0.844
0.928
3.675
3.563
0.720
0.727
2.662
0.630
0.616
0.633
0.296
0.710
1.671
0.637
0.655
0.0050
0.0371
´0.0095
0.0234
´0.0271
´0.0326
´0.0151
0.0259
´0.0209
´0.1271
0.0148
5s
5t
5u
5v
5w
0.0475
´0.0039
0.0508
´0.0367
2.4.3. QSAR Validation
Two of the prepared compounds (5e and 5j) were utilized to carry out the external validation of
the determined QSAR equation. 5e and 5j were chosen as they exhibit mild and excellent activities.
The observed activities versus those provided by QSAR study are presented in Table 6.
Table 6. External validation of the established QSAR model.
Experimental
Activity
Predicted
Activity
(LogIC50)
Compound
Residual Num_ExplicitAtoms Molecular_SAVol CHI_V_3_C
Jurs_RPCS
(LogIC50
)
5e
5j
2.1497
1.3504
2.1497
1.3504
0.00
0.00
31
32
423.74
423.66
0.473
0.482
0.795
3.313

Int. J. Mol. Sci. 2016, 17, 1221
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2.5. Theoretical Kinetic ADME Study of the Target Derivatives 5a–w
A theoretical kinetic study carried out by Discovery Studio 2.5 software (Accelrys) was adopted
to predict the ADME of the prepared derivatives 5a , Table 7. The lipophilicity was evaluated by
–
w
calculating AlogP98, whereas the PSA_2D descriptor was adopted to estimate the polar surface area.
Moreover, solubility level was predicted where all members of this study seemed to possess low
solubility. In accordance with this anticipation, absorption levels implicate that they are well absorbed.
Also, they are predicted to be non-inhibitors of CYP2D not to mention compounds 5b, 5q, and 5s,
which are expected to inhibit CYP2D. LogP for compound 5k was determined experimentally and
found to equal 3.75. It is worth mentioning that all compounds passed Lipinski’s rule of five.
Table 7. Computer-aided ADME study of the prepared derivatives.
Solubility Absorption
CYP2D6
Compound AlogP98 a
Solubility c
PSA_2D b
CYP2D6 f
Level d
Level e
Probability g
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
3.684
4.171
2.938
4.349
3.890
3.890
4.095
4.433
3.579
3.442
3.442
3.442
3.668
3.668
3.426
3.652
3.652
3.453
3.635
4.017
3.287
3.080
3.410
43.616
43.616
70.156
43.616
43.616
43.616
43.616
43.616
86.440
64.432
64.432
64.432
52.547
52.547
73.362
61.477
61.477
61.477
70.407
52.547
28.624
56.171
43.616
b
´4.830
´5.324
´4.307
´5.560
´5.158
´5.152
´5.478
´5.636
´5.034
´4.355
´4.363
´4.366
´4.911
´4.887
´4.486
´4.959
´4.949
´5.112
´5.013
´5.102
´4.870
´4.348
´4.643
c
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0.316
0.554
0.376
0.376
0.336
0.376
0.326
0.287
0.257
0.326
0.336
0.386
0.376
0.396
0.425
0.306
0.524
0.346
0.554
0.366
0.306
0.099
0.425
5s
5t
5u
5v
5w
a
Lipophilicity descriptor; Polar surface area; Solubility parameter. (0–
´
2 = optimal,
´
2–
´
4 = good,
´4–´6 = low
,
´
6–
8 = very low); d Solubility level. (0 = extremely low, 1 = very low but possible, 2 = low,
´
3 = good, 4 = optimal); e Absorption level. (0 = good, 1 = moderate, 2 = low, 3 = very low); f CYP2D inhibition.
(0 = non inhibitor, 1 = inhibitor); g CYP2D6 Probability: 0–0.5 = non inhibitor; 0.5–1 = inhibitor.
3. Discussion
In summary, a novel series of 2-((benzimidazol-2-yl)thio)-1-arylethan-1-one derivatives 5a
been synthesized Their anti-proliferative activity against triple-negative breast cancer MDA-MB-468
cells was evaluated. Compound 5k was found to be the most active compound in this study with
IC50 value of 19.90 1.37 M as it was 2.1 times more potent and efficacious than 5-fluorouracil
IC50 = 41.26 ˘ 3.77 µM). Also, analogs 5a 5f, and 5j possessed excellent anti-proliferative activity
with IC50 values ranging from 21.98 1.91 to 26.60 2.24 M, which are better than the used
–
w has
.
˘
µ
(
,
–t
˘
˘
µ
reference drug. The preliminary SAR study showed that incorporation of unsubstituted phenyl
group or its substitution with electron-donating hydroxyl, methoxy, or ethoxy groups are essential
elements for the anti-tumor activity against MDA-MB-468, while introduction of heterocycles, such as
2-furyl or 2-thienyl, could not effectively replace the phenyl ring. In a cell-cycle analysis, compound
5k increased the percentage of MDA-MB-468 cells at pre-G1 by 8.13-fold and G2/M phase by
11.83-fold. Furthermore, treatment of MDA-MB-468 cells with 5k led to a marked increase in the
percentage of annexin V–FITC-positive apoptotic cells (UR + LR) by 2.8-fold compared to the control.

Int. J. Mol. Sci. 2016, 17, 1221
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In addition, a QSAR model was established to investigate the structural requirements controlling
activity against MDA-MB-468. Of note, the anticipated activities by the QSAR model were very near
to the experimentally determined activities. Accordingly, this model could be conveniently applied for
the prediction of more effective hits bearing the same structural framework. A theoretical kinetic study
was constituted to anticipate the ADME of the prepared benzimidazoles. Moreover, single crystal
X-ray diffraction has been included for compounds 4u and 5v.
Through this work we planned to add a scientific contribution for the treatment of the resistant
type; TNBC by exploring different 2-benzimidazole derivatives. Our design was inspired by previously
reported active scaffolds. Among the designed and synthetized molecules, many of them showed
2-fold increase in activity compared to 5-FU; this led us to develop a fruitful SAR analysis that will be
a guideline for our future work.
4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were determined using a Gallenkamp melting point apparatus (WeissTechnik,
Loughborough, UK) and are uncorrected. Infrared (IR) Spectra were recorded as KBr disks using
the Perkin Elmer FT-IR Spectrum BX apparatus (PerkinElmer, Boston, MA, USA). Mass spectra were
measured on an Agilent TripleQuadrupole 6410 QQQ LC/MS equipped with an ESI (electrospray
ionization) source (Agilent Technologies, Santa Clara, CA, USA). NMR Spectra were recorded on
1
a Bruker NMR spectrometer (Bruker, Karlsruhe, Germany). H spectrum was run at 500 MHz and
13C spectrum was run at 125 MHz in deuterated dimethyl sulfoxide (DMSO-d6). Chemical shifts are
expressed in
δ values (ppm) using the solvent peak as internal standard. All coupling constant (J)
values are given in hertz. The abbreviations used are as follows: s, singlet; d, doublet; m, multiplet.
Elemental analyses were carried out at the Regional Center for Mycology and Biotechnology, Al-Azhar
University, Cairo, Egypt. Analytical thin layer chromatography (TLC) on silica gel plates containing
UV indicator (Merck KGaA, Darmstadt, Germany) was employed routinely to follow the course of
reactions and to check the purity of products. All reagents and solvents were purified and dried
by standard techniques. Compounds
reported [28].
4 & 5a, b, f, h, 4 & 5l–o, 4 & 5q, r, t, v, and w, are previously
4.1.2. Benzoimidazole-2-Thiol 2
Prepared according to the reported procedures [39].
4.1.3. General Procedures for Synthesis of Sulfate Salts 4a–w
Prepared according to the reported procedures [28].
2-((2-(4-Aminophenyl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4c). White crystals, (yield
˝
97%), m.p. 210–213 C; IR (KBr,
ν δ ppm: 5.23
cm´1): 3450 (NH) and 1684 (C=O); 1H-NMR (DMSO-d6)
(s, 2H, CH2), 6.65 (d, 2H, H-3 and H-5 of 4-NH2C6H4, J = 8.5 Hz), 6.70 (s, 2H, NH2), 7.49–7.51 (m, 2H,
H-5 and H-6 of 2-mercaptobenzimidazole), 7.71–7.73 (m, 2H, H-4 and H-7 of 2-mercaptobenzimidazole),
7.87 (d, 2H, H-2 and H-6 of 4-NH2C6H4, J = 8.5 Hz), 10.55 (s, 1H, NH), 12.64 (s, 1H, NH); 13C-NMR
(DMSO-d6)
δ ppm: 41.55 (CH2), 113.40, 113.59, 114.66, 125.76, 130.92, 131.62, 132.74, 151.68, 189.11,
195.74 (C=O); ESI MS m/z: 665 [M + 1]+; Anal. Calcd. for C30H28N6O6S3: C, 54.20; H, 4.25; N, 12.64;
Found C, 54.28; H, 4.21; N, 12.75.
2-((2-(4-Chlorophenyl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4d). White crystals, (yield
˝
98%), m.p. 227–230 C; IR (KBr,
(s, 2H, CH2), 7.37–7.40 (m, 2H, H-5 and H-6 of 2-mercaptobenzimidazole), 7.62–7.65 (m, 2H, H-4 and
ν δ ppm: 5.29
cm´1): 3400 (NH) and 1683 (C=O); 1H-NMR (DMSO-d6)

Int. J. Mol. Sci. 2016, 17, 1221
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H-7 of 2-mercaptobenzimidazole), 7.68 (d, 2H, H-3 and H-5 of 4-ClC6H4, J = 8.5 Hz), 8.08 (d, 2H, H-2
and H-6 of 4-ClC6H4, J = 8.5 Hz), 10.55 (s, 1H, NH), 12.61 (s, 1H, NH); 13C-NMR (DMSO-d6)
ppm:
δ
56.50 (CH2), 113.84, 120.85, 122.77, 124.68, 129.53, 130.89, 134.12, 134.86, 139.48, 150.64, 192.07 (C=O);
ESI MS m/z: 703 [M + 1]+, 704 [M + 2]+; Anal. Calcd. for C30H24Cl2N4O6S3: C, 15.21; H, 3.44; N, 7.96;
Found C, 15.30; H, 3.49; N, 8.02.
2-((2-(2-Fluorophenyl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4e). White crystals, (yield
˝
97%), m.p. 219–222 C; IR (KBr,
ν δ ppm: 5.15
cm´1): 3435 (NH) and 1669 (C=O); 1H-NMR (DMSO-d6)
(s, 2H, CH2), 7.37–7.40 (m, 2H, H-5 and H-6 of 2-mercaptobenzimidazole), 7.41–7.48 (m, 2H, Ar–H),
7.60–7.62 (m, 2H, H-4 and H-7 of 2-mercaptobenzimidazole), 7.75–7.79 (m, 1H, Ar–H), 7.94 (t, 1H, H-6
of 2-FC6H4, J = 7.5 Hz), 10.64 (s, 1H, NH), 12.73 (s, 1H, NH). ESI MS m/z: 671 [M + 1]+; Anal. Calcd.
for C30H24F2N4O6S3: C, 53.72; H, 3.61; N, 8.35; Found C, 53.67; H, 3.65; N, 8.38.
2-((2-(2,4-Difluorophenyl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4g). White crystals,
˝
1
(yield 96%), m.p. 206–209 C; IR (KBr,
ppm: 5.27 (s, 2H, CH2), 7.30 (t, 1H, Ar–H, J = 8.5 Hz), 7.43–7.46 (m, 2H, H-5 and H-6 of
ν
cm´1): 3468 (NH) and 1680 (C=O); H-NMR (DMSO-d6)
δ
2-mercaptobenzimidazole), 7.52 (t, 1H, Ar–H, J = 9.0 Hz), 7.65–7.69 (m, 2H, H-4 and H-7 of
2-mercaptobenzimidazole), 8.03 (t, 1H, Ar–H, J = 9.0 Hz),10.52 (s, 1H, NH), 12.67 (s, 1H, NH); 13C-NMR
(DMSO-d6)
δ ppm: 44.06 (CH2), 105.70, 105.91, 106.12, 113.09, 113.27, 113.76, 120.81, 125.25, 133.50,
133.85, 150.71, 189.03 (C=O). ESI MS m/z: 707 [M + 1]+; Anal. Calcd. for C30H22F4N4O6S3: C, 50.99;
H, 3.14; N, 7.93; Found C, 50.91; H, 3.18; N, 8.01.
2-((2-(2-Hydroxyphenyl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4j). White crystals,
˝
(yield 98%), m.p. 238–240 C; IR (KBr, ν δ ppm:
cm´1): 3466 (NH) and 1684 (C=O); 1H-NMR (DMSO-d6)
5.30 (s, 2H, CH2), 7.02–7.11 (m, 4H, Ar–H), 7.38–7.42 (m, 2H, H-5 and H-6 of 2-mercaptobenzimidazole),
7.67–7.70 (m, 2H, H-4 and H-7 of 2-mercaptobenzimidazole), 9.59 (s, 1H, OH), 10.60 (s, 1H, NH), 12.87
(s, 1H, NH). ESI MS m/z: 667 [M + 1]+, 668 [M + 2]+; Anal. Calcd. for C30H26N4O8S3: C, 54.04; H, 3.93;
N, 8.40; Found C, 54.12; H, 3.91; N, 8.46.
2-((2-(3-Hydroxyphenyl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4k). White crystals,
˝
1
(yield 97%), m.p. 228–230 C; IR (KBr,
ppm: 5.34 (s, 2H, CH2), 7.11 (d, 1H, H-4 of 3-OHC6H4, J = 8.0 Hz), 7.40–7.44 (m, 2H, H-2 and
ν
cm´1): 3418 (NH) and 1670 (C=O); H-NMR (DMSO-d6)
δ
H-3 of 3-OHC6H4), 7.47–7.49 (m, 2H, H-5 and H-6 of 2-mercaptobenzimidazole), 7.54 (d, 1H, H-6
of 3-OHC6H4, J = 8.0 Hz), 7.70–7.72 (m, 2H, H-4 and H-7 of 2-mercaptobenzimidazole), 9.73 (s, 1H,
OH), 10.48 (s, 1H, NH), 12.61 (s, 1H, NH); 13C-NMR (DMSO-d6)
δ ppm: 41.58 (CH2), 113.69, 115.09,
120.03, 121.82, 125.59, 130.55, 133.19, 136.46, 151.08, 158.23, 192.52 (C=O). ESI MS m/z: 667 [M + 1]+;
Anal. Calcd. for C30H26N4O8S3: C, 54.04; H, 3.93; N, 8.40; Found C, 54.13; H, 4.03; N, 8.46.
2-((2-(2,4-Dimethoxyphenyl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4p). White crystals,
(yield 98%), m.p. 235–237 ˝C; IR (KBr,
ν
cm´1): 3377 (NH) and 1680 (C=O); 1H-NMR
(DMSO-d6)
δ ppm: 3.88 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 5.08 (s, 2H, CH2), 6.67 (d, 1H, H-5
of 2,4-(OCH3)2C6H3, J = 9.0 Hz), 6.74 (s, 1H, H-3 of 2,4-(OCH3)2C6H3), 7.43–7.44 (m, 2H, H-5 and H-6
of 2-mercaptobenzimidazole), 7.65–7.66 (m, 2H, H-4 and H-7 of 2-mercaptobenzimidazole), 7.79 (d, 1H,
H-6 of 2,4-(OCH3)2C6H3, J = 9.0 Hz), 10.46 (s, 1H, NH), 12.37 (s, 1H, NH); 13C-NMR (DMSO-d6)
δ ppm:
45.31 (CH2), 56.33 (OCH3), 56.73 (OCH3), 98.92, 107.24, 113.37, 117.65, 121.38, 125.12, 133.06, 151.57,
162.02, 165.95, 191.78 (C=O). ESI MS m/z: 755 [M + 1]+; Anal. Calcd. for C34H34N4O10S3: C, 54.10; H,
4.54; N, 7.42; Found C, 54.16; H, 4.59; N, 7.38.
2-((2-(3,4,5-Trimethoxyphenyl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4s). White
˝
1
crystals, (yield 98%), m.p. 213–215 C; IR (KBr,
ν
cm´1): 3427 (NH) and 1675 (C=O); H-NMR
(DMSO-d6) δ ppm: 3.77 (s, 6H, 2OCH3), 3.89 (s, 12H, 4OCH3), 5.29 (s, 4H, 2CH2), 7.40–7.67 (m, 12H,
Ar–H), 10.67 (s, 2H, 2NH), 12.49 (s, 2H, 2NH). ESI MS m/z: 815 [M + 1]+, 816 [M + 2]+; Anal. Calcd. for
C36H38N4O12S3: C, 53.06; H, 4.70; N, 6.88; Found C, 52.96; H, 4.63; N, 6.80.

Int. J. Mol. Sci. 2016, 17, 1221
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2-((2-(Naphthalen-2-yl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4u). White crystals,
(yield 96%), m.p. 246–250 ˝C; IR (KBr, cm´1): 3412 (NH) and 1670 (C=O); 1H-NMR (DMSO-d6)
ν
δ
ppm: 5.38 (s, 2H, CH2), 7.31–7.33 (m, 2H, H-5 and H-6 of 2-mercaptobenzimidazole), 7.57–7.60 (m, 2H,
H-4 and H-7 of 2-mercaptobenzimidazole), 7.66–7.74 (m, 2H, H-6 and H-7 of naphthalene), 8.04 (d, 2H,
Ar–H, J = 8.5 Hz), 8.08 (d, 1H, Ar–H, J = 8.5 Hz), 8.17 (d, 1H, Ar–H, J = 8.0 Hz), 8.85 (s, 1H, H-1 of
naphthalene), 10.54 (s, 1H, NH), 12.68 (s, 1H, NH); 13C-NMR (DMSO-d6)
δ ppm: 56.49 (CH2), 113.96,
123.93, 124.13, 127.72, 128.26, 129.01, 130.14, 131.27, 132.56, 132.83, 135.79, 136.26, 150.58, 193.14 (C=O).
ESI MS m/z: 735 [M + 1]+; Anal. Calcd. for C38H30N4O6S3: C, 62.11; H, 4.11; N, 7.62; Found C, 62.18;
H, 4.13; N, 7.57.
4.1.4. General Procedure for Preparation of the Target Derivatives 5a–w
An aqueous solution (10 mL) of sodium bicarbonate was added to a stirred suspension of the
adequate sulfate salts 4a–w (4 mmol) in water (20 mL). The mixture was stirred for 2 h at room
temperature. The obtained solid was collected by filtration, washed several times with water, then
dried and recrystallized from ethanol to furnish compounds 5a–w.
2-((1H-Benzo˝[d]imidazol-2-yl)thio)-1-(4-aminophenyl)ethan-1-one (5c). White crystals (yield 85%),
m.p. 175–178 C; IR (KBr, ν δ ppm: 4.87 (s, 2H,
cm´1): 3412 (NH) and 1680 (C=O); 1H-NMR (DMSO-d6)
CH2), 6.21 (s, 2H, NH2), 6.59 (d, 2H, H-3 and H-5 of 4-NH2C6H4, J = 9.0 Hz), 7.10–7.14 (m, 4H, H-4,
H-5, H-6 and H-7 of 2-mercaptobenzimidazole), 7.76 (d, 2H, H-2 and H-6 of 4-NH2C6H4, J = 8.5 Hz),
12.53 (s, 1H, NH); 13C-NMR (DMSO-d6)
δ ppm: 40.57 (CH2), 109.93, 113.03, 121.82, 122.78, 123.27,
131.42, 132.70, 150.41, 154.72, 190.66 (C=O); ESI MS m/z: 284 [M + 1]+; Anal. Calcd. for C15H13N3OS:
C, 63.58; H, 4.62; N, 14.83; Found C, 63.81; H, 4.60; N, 14.89.
2-((1H-Benzo[d]imidazol-2-yl)thio)-1-(4-chlorophenyl)ethan-1-one (5d). White crystals (yield 90%),
m.p. 179–181 ˝C (reported: 189–191 ˝C [40]); IR (KBr,
ν
cm´1): 3410 (NH) and 1675 (C=O); 1H-NMR
(DMSO-d6)
δ ppm: 5.04 (s, 2H, CH2), 7.09–7.13 (m, 2H, H-5 and H-6 of 2-mercaptobenzimidazole),
7.40–7.42 (m, 2H, H-4 and H-7 of 2-mercaptobenzimidazole), 7.64 (d, 2H, H-3 and H-5 of 4-ClC6H4,
J = 8.0 Hz), 8.08 (d, 2H, H-2 and H-6 of 4-ClC6H4, J = 8.5 Hz), 12.64 (s, 1H, NH); 13C-NMR (DMSO-d6)
δ
ppm: 40.49 (CH2), 112.45, 121.87, 129.41, 130.82, 134.67, 139.06, 149.82, 193.09 (C=O); ESI MS m/z:
302.9 [M]+, 304.9 [M + 2]+.
2-((1H-Benzo˝[d]imidazol-2-yl)thio)-1-(2-fluorophenyl)ethan-1-one (5e). White crystals (yield 89%),
m.p. 145–148 C; IR (KBr,
ν δ ppm: 4.93 (s, 2H,
cm´1): 3420 (NH) and 1654 (C=O); 1H-NMR (DMSO-d6)
CH2), 7.09–7.12 (m, 2H, H-5 and H-6 of 2-mercaptobenzimidazole), 7.37–7.49 (m, 4H, Ar–H), 7.70–7.74
(m, 1H, Ar–H), 8.08 (t, 1H, Ar–H, J = 7.5 Hz), 12.66 (s, 1H, NH); 13C-NMR (DMSO-d6)
δ
ppm: 43.11
(CH2), 109.23, 117.29, 118.50, 121.69, 122.18, 124.61, 125.38, 131.09, 136.01, 149.80, 157.31, 160.50, 162.52,
191.90 (C=O); ESI MS m/z: 287 [M + 1]+, 288 [M + 2]+; Anal. Calcd. for C15H11FN2OS: C, 62.92; H, 3.87;
N, 9.78; Found C, 63.09; H, 3.90; N, 9.84.
2-((1H-Benzo[d]imidazol-2-yl)thio)-1-(2,4-difluorophenyl)ethan-1-one (5g). White crystals (yield 94%),
˝
m.p. 116–120 C; IR (KBr, ν δ ppm: 4.91 (s, 2H,
cm´1): 3420 (NH) and 1675 (C=O); 1H-NMR (DMSO-d6)
CH2), 7.08–7.12 (m, 2H, H-5 and H-6 of 2-mercaptobenzimidazole), 7.26 (t, 1H, Ar–H, J = 8.5 Hz),
7.39–7.41 (m, 2H, H-4 and H-7 of 2-mercaptobenzimidazole), 7.47 (t, 1H, Ar–H, J = 9 Hz), 8.00 (q, 1H,
Ar–H, J = 8.5 Hz), 12.61 (s, 1H, NH); 13C-NMR (DMSO-d6)
δ ppm: 42.95 (CH2), 105.54, 105.75, 105.96,
112.89, 113.04, 121.88, 133.35, 149.73, 190.71 (C=O); ESI MS m/z: 304 [M]+, 305 [M + 1]+; Anal. Calcd.
for C15H10F2N2OS: C, 59.20; H, 3.31; N, 9.21; Found C, 59.46; H, 3.29; N, 9.32.
2-((1H-Benzo[d]imidazol-2-yl)thio)-1-(2-hydroxyphenyl)ethan-1-one (5j). White crystals (yield 85%),
˝
˝
1
m.p. 205–208 C (reported: 201 C [41]); IR (KBr,
(DMSO-d6) ppm: 4.91 (s, 2H, CH2), 6.99–7.15 (m, 4H, Ar–H), 7.38–7.41 (m, 2H, H-4 and H-7 of
ν
cm´1): 3408 (NH) and 1670 (C=O); H-NMR
δ
2-mercaptobenzimidazole), 7.83–7.95 (m, 2H, Ar–H), 9.37 (s, 1H, OH), 12.53 (s, 1H, NH); ESI MS m/z:
285 [M + 1]+.

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2-((1H-Benzo[d]imidazol-2-yl)thio)-1-(3-hydroxyphenyl)ethan-1-one (5k). White crystals (yield 87%),
m.p. 228–230 ˝C (reported: 224–227 ˝C [42]); IR (KBr, cm´1): 3336 (NH) and 1660 (C=O); 1H-NMR
(DMSO-d6) ppm: 5.01 (s, 2H, CH2), 7.08–7.10 (m, 3H, H-5, H-6 of 2-mercaptobenzimidazole and H-4
of 3-OHC6H4), 7.37–7.45 (m, 4H, H-4, H-7 of 2-mercaptobenzimidazole and H-5, H-6 of 3-OHC6H4),
7.54 (s, 1H, H-2 of 3-OHC6H4), 9.89 (s, 1H, OH), 12.61 (s, 1H, NH); 13C-NMR (DMSO-d6)
ppm: 40.47
ν
δ
δ
(CH2), 114.96, 119.86, 121.28, 121.61, 130.43, 137.23, 150.01, 158.14, 193.72 (C=O); ESI MS m/z: 285.
2-((1H-Benzo[d]imidazol-2-yl)thio)-1-(2,4-dimethoxyphenyl)ethan-1-one (5p). White crystals (yield
˝
92%), m.p. 208–211 C; IR (KBr,
ν δ ppm: 3.92
cm´1): 3413 (NH) and 1655 (C=O); 1H-NMR (DMSO-d6)
(s, 3H, OCH3), 3.96 (s, 3H, OCH3), 4.80 (s, 2H, CH2), 6.65 (d, 1H, H-5 of 2,4-(OCH3)2C6H3, J = 9.0 Hz),
6.71 (s, 1H, H-3 of 2,4-(OCH3)2C6H3), 7.06–7.09 (m, 2H, H-5 and H-6 of 2-mercaptobenzimidazole),
7.40–7.41 (m, 2H, H-4 and H-7 of 2-mercaptobenzimidazole), 7.73 (d, 1H, H-6 of 2,4-(OCH3)2C6H3,
J = 8.5 Hz), 12.60 (s, 1H, NH); 13C-NMR (DMSO-d6)
δ ppm: 43.97 (CH2), 56.20 (OCH3), 56.60 (OCH3),
98.88, 104.25, 106.96, 110.65, 114.19, 118.27, 121.63, 132.88, 150.15, 161.52, 165.36, 193.07 (C=O); ESI MS
m/z: 329 [M + 1]+; Anal. Calcd. for C17H16N2O3S: C, 62.18; H, 4.91; N, 8.53; Found C, 62.40; H, 4.94;
N, 8.45.
2-((1H-Benzo[d]imidazol-2-yl)thio)-1-(3,4,5-trimethoxyphenyl)ethan-1-one (5s). White crystals (yield
88%), m.p. 233–235 ˝C; IR (KBr,
ν
cm´1): 3405 (NH) and 1670 (C=O); 1H-NMR (DMSO-d6)
δ
ppm:
3.77 (s, 3H, OCH3), 3.86 (s, 6H, OCH3), 5.04 (s, 2H, CH2), 7.12–7.43 (m, 6H, Ar–H), 12.68 (s, 1H, NH);
13C-NMR (DMSO-d6)
ppm: 39.37 (CH2), 56.60 (OCH3), 60.67 (OCH3), 106.58, 121.92, 131.12, 142.73,
δ
149.87, 153.29, 192.94 (C=O); Anal. Calcd. for C18H18N2O4S: C, 60.32; H, 5.06; N, 7.82; Found C, 60.41;
H, 5.03; N, 7.75.
2-((1H-Benzo˝[d]imidazol-2-yl)thio)-1-(naphthalen-2-yl)ethan-1-one (5u). White crystals (yield 92%),
m.p. 160–162 C; IR (KBr, ν δ ppm: 5.20 (s, 2H,
cm´1): 3415 (NH) and 1673 (C=O); 1H-NMR (DMSO-d6)
C
H2), 7.10–7.13 (m, 2H, H-5 and H-6 of 2-mercaptobenzimidazole), 7.41–7.43 (m, 2H, H-4 and H-7
of 2-mercaptobenzimidazole), 7.64–7.72 (m, 2H, Ar–H), 8.02–8.07 (m, 3H, Ar–H), 8.15 (d, 1H, Ar–H,
J = 8.0 Hz), 8.85 (s, 1H, H-1 of naphthalene), 12.80 (s, 1H, NH); 13C-NMR (DMSO-d6)
ppm: 40.42
δ
(CH2), 121.92, 124.19, 127.59, 128.20, 128.91, 129.42, 130.12, 131.11, 132.59, 133.19, 135.69, 149.99, 193.90
(C=O); ESI MS m/z: 319 [M + 1]+; Anal. Calcd. for C19H14N2OS: C, 71.68; H, 4.43; N, 8.80; Found C,
71.81; H, 4.40; N, 8.73.
4.2. Biological Evaluation
4.2.1. In Vitro Evaluation of the Anti-Proliferative Activity
The synthesized derivatives 5a–w was evaluated for their anti-proliferative activity via the
Stem Cell Therapy and Tissue Reengineering Program in the King Faisal Specialized Hospital and
Research Center, Riyadh, Saudi Arabia. In vitro anti-proliferative activity was measured by the cell
growth inhibition assay. This assay was conducted using a WST-1 reagent (Sigma-Aldrich Chemie
Gmbh, Munich, Germany) for determination of the IC50 for each compound and the results are given in
Table 4. MDA-MB-468 breast cancer cell line was purchased from the American Type Culture Collection
(Manassas, Virginia, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)
(Sigma-Aldrich Chemie Gmbh), supplemented with 10% FBS (Lonza, Visp, Switzerland), 100 IU/mL
penicillin, 100 mg/mL streptomycin, and 2 mmol/L L-glutamine (Sigma). Cells were seeded into
96-well plates at 0.4
containing the desired concentrations of the test compounds. After 48 h, 10
ˆ
104/well and incubated overnight. The medium was replaced with a fresh one
L of the WST-1 reagent
µ
˝
were added to each well and the plates were re-incubated for 4 h at 37 C. The amount of formazan was
quantified using an ELISA reader (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm. The IC50
values were calculated according to the equation for Boltzmann sigmoidal concentration response curve
using the nonlinear regression models (GraphPad, Prism Version 5, San Diego, CA, USA). The results
reported are means of at least three separate experiments. Significant differences were analyzed by

Int. J. Mol. Sci. 2016, 17, 1221
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one-way analysis of variance (ANOVA) wherein the differences were considered to be significant at
p < 0.05.
4.2.2. Cell Cycle Analysis
The MDA-MB-468 cells were subjected to treatment with 19.90
µM of compound 5k for
24 h. Consequently, the cells were washed twice with ice-cold phosphate buffered saline (PBS).
The treated cells were collected by centrifugation, fixed in ice-cold 70% (v/v) ethanol, washed with PBS,
re-suspended with 0.1 mg/mL RNase, stained with 40 mg/mL PI, and analyzed by flow cytometry
using FACScalibur (Becton Dickinson, BD, San Jose, CA, USA). The cell cycle distributions were
calculated using CellQuest software (Becton Dickinson).
4.2.3. Annexin V–FITC Apoptosis Assay
The MDA-MB-468 cells were seeded as described above and then incubated with 19.90 µM of
compound 5k for 24 h. Cells were harvested, washed twice with PBS, and centrifuged. In brief, 105 of
cells were treated with annexin V–FITC and propidium iodide (PI) using the apoptosis detection kit
(BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol. Annexin V–FITC and PI
binding were analyzed by flow cytometry on FACScalibur (BD Biosciences) without gating restrictions
using 10,000 cells. Data were collected using logarithmic amplification of both the FL1 (FITC) and
the FL2 (PI) channels. Quadrant analysis of co-ordinate dot plots was performed with CellQuest
software. Unstained cells were used to adjust the photomultiplier voltage and for compensation setting
adjustment to eliminate spectral overlap between the FL1 and the FL2 signal.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/
17/8/1221/s1.
Acknowledgments: The authors would like to extend their sincere appreciation to the Deanship of Scientific
Research at King Saud University (Riyadh, Saudi Arabia) for its funding of this research through the Research
Group Project no. PRG-1436-038.
Author Contributions: Hatem A. Abdel-Aziz and Wagdy M. Eldehna conceived and designed the experiments;
Wagdy M. Eldehna, Hazem Ghabbour, Ghada H. Al-Ansary carried out the experiments; Wagdy M. Eldehna
and Ghada H. Al-Ansary analyzed and interpreted the data; Hazem Ghabbour carried out the single crystal
analysis of compounds 4u and 5v and interpreted their data; Hatem A. Abdel-Aziz, Wagdy M. Eldehna and
Ghada H. Al-Ansary prepared the manuscript; Areej M. Assaf and Abdullah Al-Dhfyan performed the biological
screening. All authors have read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
International Agency for Research on Cancer. IGlobocan: Estimated Cancer Incidence, Mortality, and Prevalence
Worldwide in 2012; IARC: Lyon, France, 2014.
Adami, H.; Hunter, D.; Trichopoulos, D. Textbook of Cancer Epidemiology; Oxford University Press:
New York, NY, USA, 2002; pp. 301–373.
Barnard, M.E.; Boeke, C.E.; Tamimi, R.M. Established breast cancer risk factors and risk of intrinsic tumor
subtypes. Biochim. Biophys. Acta 2015, 1856, 73–85. [CrossRef] [PubMed]
Atkinson, R.L.; Yang, W.T.; Rosen, D.G.; Landis, M.D.; Wong, H.; Lewis, M.T.; Creighton, C.J.; Sexton, K.R.;
Hilsenbeck, S.G.; Sahin, A.A.; et al. Cancer stem cell markers are enriched in normal tissue adjacent to triple
negative breast cancer and inversely correlated with DNA repair deficiency. Breast Cancer Res. 2013, 15, R77.
[CrossRef] [PubMed]
5.
6.
Rahman, M.; Davis, S.R.; Pumphrey, J.G.; Bao, J.; Nau, M.M.; Meltzer, P.S.; Lipkowitz, S. TRAIL induces
apoptosis in triple-negative breast cancer cells with a mesenchymal phenotype. Breast Cancer Res. Treat. 2009
,
113, 217–230. [CrossRef] [PubMed]
Rakha, E.A.; Reis-Filho, J.S.; Ellis, I.O. Basal-like breast cancer: A critical review. J. Clin. Oncol. 2008, 26,
2568–2581. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2016, 17, 1221
18 of 19
7.
8.
9.
Fadare, O.; Tavassoli, F.A. Clinical and pathologic aspects of basal-like breast cancers. Nat. Clin. Pract. Oncol.
2008, 5, 149–159. [CrossRef] [PubMed]
Smid, M.; Wang, Y.; Zhang, Y.; Sieuwerts, A.M.; Yu, J.; Klijn, J.G.; Foekens, J.A.; Martens, J.W. Subtypes of
breast cancer show preferential site of relapse. Cancer Res. 2008, 68, 3108–3114. [CrossRef] [PubMed]
Maire, V.; Baldeyron, C.; Richardson, M.; Tesson, B.; Vincent-Salomon, A.; Gravier, E.; Marty-Prouvost, B.;
De Koning, L.; Rigaill, G.; Dumont, A.; et al. TTK/hMPS1 is an attractive therapeutic target for triple-negative
breast cancer. PLoS ONE 2013, 8, e63712. [CrossRef] [PubMed]
10. Cleator, S.; Heller, W.; Coombes, R.C. Triple-negative breast cancer: therapeutic options. Lancet Oncol. 2007
8, 235–244. [CrossRef]
,
11. Bansal, Y.; Silakari, O. The therapeutic journey of benzimidazoles: A review. Bioorg. Med. Chem. 2012, 20,
6208–6236. [CrossRef] [PubMed]
12. Fei, F.; Zhou, Z. New substituted benzimidazole derivatives: A patent review (2010–2012). Expert Opin.
Ther. Pat. 2013, 23, 1157–1179. [CrossRef] [PubMed]
13. Yadav, G.; Ganguly, S. Structure activity relationship (SAR) study of benzimidazole scaffold for different
biological activities: A mini-review. Eur. J. Med. Chem. 2015, 97, 419–443. [CrossRef] [PubMed]
14. Gümüs¸, F.; Algül, Ö.; Eren, G.; Erog˘lu, H.; Diril, N.; Gür, S.; Özkul, A. Synthesis, cytotoxic activity on MCF-7
cell line and mutagenic activity of platinum (II) complexes with 2-substituted benzimidazole ligands. Eur. J.
Med. Chem. 2003, 38, 473–480. [CrossRef]
15. Bielawski, K.; Wolczynski, S.; Bielawska, A. Inhibition of DNA topoisomerase I and II, and growth inhibition
of MDA-MB-231 human breast cancer cells by bis-benzimidazole derivatives with alkylating moiety.
Pol. J. Pharmacol. 2004, 56, 373–378. [PubMed]
16. Thimmegowda, N.R.; Swamy, S.N.; Kumar, C.S.A.; Kumar, Y.C.S.; Chandrappa, S.; Yip, G.W.; Rangappa, K.S.
Synthesis, characterization and evaluation of benzimidazole derivative and its precursors as inhibitors of
MDA-MB-231 human breast cancer cell proliferation. Bioorg. Med. Chem. Lett. 2008, 18, 432–435. [CrossRef]
[PubMed]
17. Taher, A.T.; Khalil, N.A.; Ahmed, E.M. Synthesis of novel isatin-thiazoline and isatin-benzimidazole
conjugates as anti-breast cancer agents. Arch. Pharmacal Res. 2011, 34, 1615–1621. [CrossRef] [PubMed]
18. Romero-Castro, A.; León-Rivera, I.; Ávila-Rojas, L.C.; Navarrete-Vázquez, G.; Nieto-Rodríguez, A. Synthesis
and preliminary evaluation of selected 2-aryl-5(6)-nitro-1H-benzimidazole derivatives as potential anticancer
agents. Arch. Pharmacal Res. 2011, 34, 181–189. [CrossRef] [PubMed]
19. Bonham, S.; O’Donovan, L.; Carty, M.P.; Aldabbagh, F. First synthesis of an aziridinyl fused
pyrrolo[1,2-a]benzimidazole and toxicity evaluation towards normal and breast cancer cell lines.
Org. Biomol. Chem. 2011, 9, 6700–6706. [CrossRef] [PubMed]
20. Rahim, A.S.A.; Salhimi, S.M.; Arumugam, N.; Pin, L.C.; Yee, N.S.; Muttiah, N.N.; Keat, W.B.; Hamid, S.A.;
Osman, H.; Mat, I.B. Microwave-assisted synthesis of sec/tert-butyl 2-arylbenzimidazoles and their
unexpected antiproliferative activity towards ER negative breast cancer cells. J. Enzym. Inhib. Med. Chem.
2013, 28, 1255–1260. [CrossRef] [PubMed]
21. Yoon, Y.K.; Ali, M.A.; Wei, A.C.; Choon, T.S.; Osman, H.; Parang, K.; Shirazi, A.N. Synthesis and evaluation
of novel benzimidazole derivatives as sirtuin inhibitors with antitumor activities. Bioorg. Med. Chem. 2014
,
22, 703–710. [CrossRef] [PubMed]
22. Abonia, R.; Cortés, E.; Insuasty, B.; Quiroga, J.; Nogueras, M.; Cobo, J. Synthesis of novel 1,2,5-trisubstituted
benzimidazoles as potential antitumor agents. Eur. J. Med. Chem. 2011, 46, 4062–4070. [CrossRef] [PubMed]
23. Kamal, A.; Reddy, T.S.; Vishnuvardhan, M.V.P.S.; Nimbarte, V.D.; Rao, A.V.S.; Srinivasulu, V.; Shankaraiah, N.
Synthesis of 2-aryl-1,2,4-oxadiazolo-benzimidazoles: Tubulin polymerization inhibitors and apoptosis
inducing agents. Bioorg. Med. Chem. 2015, 23, 4608–4623. [CrossRef] [PubMed]
24. Kamal, A.; Shaik, A.B.; Polepalli, S.; Kumar, G.B.; Reddy, V.S.; Mahesh, R.; Garimella, S.; Jain, N. Synthesis of
arylpyrazole linked benzimidazole conjugates as potential microtubule disruptors. Bioorg. Med. Chem. 2015
,
23, 1082–1095. [CrossRef] [PubMed]
25. Husain, A.; Rashid, M.; Mishra, R.; Parveen, S.; Shin, D.-S.; Kumar, D. Benzimidazole bearing oxadiazole
and triazolo-thiadiazoles nucleus: Design and synthesis as anticancer agents. Bioorg. Med. Chem. Lett. 2012
,
22, 5438–5444. [CrossRef] [PubMed]
26. Rashid, M.; Husain, A.; Mishra, R. Synthesis of benzimidazoles bearing oxadiazole nucleus as anticancer
agents. Eur. J. Med. Chem. 2012, 54, 855–866. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2016, 17, 1221
19 of 19
27. Husain, A.; Rashid, M.; Shaharyar, M.; Siddiqui, A.A.; Mishra, R. Benzimidazole clubbed with
triazolo-thiadiazoles and triazolo-thiadiazines: New anticancer agents. Eur. J. Med. Chem. 2013, 62,
785–798. [CrossRef] [PubMed]
28. Abdel-Aziz, H.A.; Ghabbour, H.A.; Eldehna, W.M.; Al-Rashood, S.T.A.; Al-Rashood, K.A.; Fun, H.-K.;
Al-Tahhan, M.; Al-Dhfyan, A. 2-((Benzimidazol-2-yl)thio)-1-arylethan-1-ones: Synthesis, crystal study and
cancer stem cells CD133 targeting potential. Eur. J. Med. Chem. 2015, 104, 1–10. [CrossRef] [PubMed]
29. Aboul-Fadl, T.; Kadi, A.; Abdel-Aziz, H.A. Novel N,N1-Hydrazino-bis-isatin Derivatives with Selective
Activity Against Multidrug-Resistant Cancer Cells. U.S. Patent 20120252860, 4 October 2012.
30. Abdel-Aziz, H.A.; Ghabbour, H.A.; Eldehna, W.M.; Qabeel, M.M.; Fun, H.-K. Synthesis, crystal structure and
biological activity of cis/trans amide rotomers of (Z)-N1-(2-oxoindolin-3-ylidene)formohydrazide. J. Chem.
2014, 2014, 760434. [CrossRef]
31. Fares, M.; Eldehna, W.M.; Abou-Seri, S.M.; Abdel-Aziz, H.A.; Aly, M.H.; Tolba, M.F. Design, synthesis and
in vitro antiproliferative activity of novel isatin-quinazoline hybrids. Arch. Pharm. Chem. Life Sci. 2015, 348,
144–154. [CrossRef] [PubMed]
32. Eldehna, W.M.; Ibrahim, H.S.; Abdel-Aziz, H.A.; Farrag, N.N.; Youssef, M.M. Design, synthesis and in vitro
antitumor activity of novel N-substituted-4-phenyl/benzylphthalazin-1-ones. Eur. J. Med. Chem. 2015, 89,
549–560. [CrossRef] [PubMed]
33. Eldehna, W.M.; Altoukhy, A.; Mahrous, H.; Abdel-Aziz, H.A. Design, synthesis and QSAR study of certain
isatin-pyridine hybrids as potential anti-proliferative agents. Eur. J. Med. Chem. 2015, 90, 684–694. [CrossRef]
[PubMed]
34. Alafeefy, A.M.; Ahmad, R.; Abdulla, M.; Eldehna, W.M.; Al-Tamimi, A.S.; Abdel-Aziz, H.A.; Al-Obaid, O.;
Carta, F.; Al-Kahtani, A.A.; Supuran, C.T. Development of certain new 2-substituted-quinazolin-
4-yl-aminobenzenesulfonamide as potential antitumor agents. Eur. J. Med. Chem. 2016, 109, 247–253.
[CrossRef] [PubMed]
35. Eldehna, W.M.; Fares, M.; Ibrahim, H.S.; Aly, M.H.; Zada, S.; Ali, M.M.; Abou-Seri, S.M.; Abdel-Aziz, H.A.;
Abou El Ella, D.A. Indoline ureas as potential anti-hepatocellular carcinoma agents targeting VEGFR-2:
Synthesis, in vitro biological evaluation and molecular docking. Eur. J. Med. Chem. 2015, 100, 89–97.
[CrossRef] [PubMed]
36. Sheldrick, G.M. A short history of SHELX. Acta Crystallogr. Sect. A 2008, 64, 112–122. [CrossRef] [PubMed]
37. Ngamwongsatit, P.; Banada, P.P.; Panbangred, W.; Bhunia, A.K. WST-1-based cell cytotoxicity assay as
a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line.
J. Microbiol. Methods 2008, 73, 211–215. [CrossRef] [PubMed]
38. Massagué, J. G1 cell-cycle control and cancer. Nature 2004, 432, 298–306. [CrossRef] [PubMed]
39. Mavrova, A.T.; Denkova, P.; Tsenov, Y.A.; Anichina, K.K.; Vutchev, D.I. Synthesis and antitrichinellosis
activity of some bis(benzimidazol-2-yl)amines. Bioorg. Med. Chem. 2007, 15, 6291–6297. [CrossRef] [PubMed]
40. Loghmani-Khouzani, H.; Hajiheidari, D. Synthesis of difluorinated
β-ketosulfones and novel
gem-difluoromethylsulfone-containing heterocycles as fluorinated building blocks. J. Fluor. Chem. 2010, 131,
561–569. [CrossRef]
41. Turan-Zitouni, G.; Demirayak, S.; Erol, K.; Ozdemir, M. Synthesis of some 2-[(benzazole-
2-yl)thoiacetyl]phenol derivatives and preliminary investigation on their vasodilatory activity. Farmaco 1994
40, 755–757. [PubMed]
,
42. Madsen, P.; Knudsen, L.B.; Wiberg, F.C.; Carr, R.D. Discovery and structure-activity relationship of the
first non-peptide competitive human glucagon receptor antagonists. J. Med. Chem. 1998, 41, 5150–5157.
[CrossRef] [PubMed]
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