New (E)-1-alkyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-ones: Synthesis, in vitro cytotoxicity evaluation and apoptosis inducing studies

Article abstract of DOI:10.1016/j.ejmech.2016.07.019

A new series of (E)-benzo[d]imidazol-2-yl)methylene)indolin-2-one derivatives has been synthesized and evaluated for their in vitro cytotoxic activity against a panel of selected human cancer cell lines of prostate (PC-3 and DU-145) and breast (BT-549, MDA-MB-231, MCF-7, 4T1), non-small lung (A549) and gastric (HGC) cancer cells along with normal breast epithelial cells (MCF10A). Among the tested compounds, 8l showed significant cytotoxic activity against MDA-MB-231 and 4T1 cancer cells with IC₅₀values of 3.26 ± 0.24 μM and 5.96 ± 0.67 μM respectively. The compounds 8f, 8i, 8l and 8o were also screened on normal human breast epithelial cells (MCF10A) and found to be safer with lesser cytotoxicity. The treatment of MDA-MB-231 cells with 8l led to inhibition of cell migration ability through disruption of F-actin protein assembly. The flow-cytometry analysis reveals that the cells arrested in G0/G1 phase of the cell cycle. Further, the compound 8l induced apoptosis of MDA-MB-231 cells was characterized by different staining techniques such as Acridine Orange/Ethidium Bromide (AO/EB), DAPI, annexin V-FITC/PI, Rhodamine-123 and MitoSOX red assay. Western blot studies demonstrated that the compound 8l treatment led to activation of caspase-3, increased expression of cleaved PARP, increased expression of pro-apoptotic Bax and decreased expression of anti-apoptotic Bcl-2 in MDA-MB-231 cancer cells.

Full text of DOI:10.1016/j.ejmech.2016.07.019

Accepted Manuscript
New (E)-1-alkyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-ones: Synthesis, in
vitro cytotoxicity evaluation and apoptosis inducing studies
Pankaj Sharma, Dinesh Thummuri, T. Srinivasa Reddy, Kishna Ram Senwar, V.G.M.
Naidu, Gannoju Srinivasulu, Suresh K. Bharghava, Nagula Shankaraiah
PII:
S0223-5234(16)30567-0
10.1016/j.ejmech.2016.07.019
EJMECH 8737
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 April 2016
Revised Date: 27 June 2016
Accepted Date: 10 July 2016
Please cite this article as: P. Sharma, D. Thummuri, T.S. Reddy, K.R. Senwar, V.G.M. Naidu,
G. Srinivasulu, S.K. Bharghava, N. Shankaraiah, New (E)-1-alkyl-1H-benzo[d]imidazol-2-
yl)methylene)indolin-2-ones: Synthesis, in vitro cytotoxicity evaluation and apoptosis inducing studies,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.019.
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ACCEPTED MANUSCRIPT
Graphical Abstract
New (E)-1-alkyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-ones: Synthesis, in vitro
cytotoxicity evaluation and apoptosis inducing studies
Pankaj Sharma,a Dinesh Thummuri,b T. Srinivasa Reddy,c Kishna Ram Senwar,a V. G. M.
Naidu,b Gannoju Srinivasulu,a Suresh K. Bharghava,c Nagula Shankaraiaha*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER) Hyderabad 500 037, India
bDepartment of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and
Research (NIPER) Hyderabad 500 037, India
cCentre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science, RMIT
University, GPO BOX 2476, Melbourne–3001, Australia

ACCEPTED MANUSCRIPT
New (E)-1-alkyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-ones: Synthesis, in vitro
cytotoxicity evaluation and apoptosis inducing studies
Pankaj Sharma,a Dinesh Thummuri,b T. Srinivasa Reddy,c Kishna Ram Senwar,a V. G. M.
Naidu,b Gannoju Srinivasulu,a Suresh K. Bharghava,c Nagula Shankaraiaha*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
bDepartment of Pharmacology and Toxicology, National Institute of Pharmaceutical Education
and Research (NIPER), Hyderabad 500 037, India
cCentre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science, RMIT
University, GPO BOX 2476, Melbourne-3001, Australia
List of abbreviations
AO–EB: Acridine Orange–Ethidium Bromide
DAPI: 4',6-Diamidino-2-phenylindole
PI: Propidium iodide
VEGFR: Vascular endothelial growth factor receptor
PDGFR: Platelet-derived growth factor receptors
PARP: Poly(ADP-ribose) polymerases
Bcl2: B-cell lymphoma 2 Protein
Bax: Bcl2-Associated X Protein
CDK: Cyclic-dependent kinase
DΨm: Mitochondrial membrane potential
ROS: Reactive oxygen species
RNS: Reactive nitrogen species
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide
NMR: Nuclear magnetic resonance
DMSO: Dimethyl sulfoxide
DMEM: Dulbecco modified eagle medium
MEM: Minimum essential medium
PBS: Phosphate-buffered saline
Corresponding author. Tel.: (+91) 040 23073740; Fax: (+91) 040 23073751; E-mail:
shankar@niperhyd.ac.in; shankarnbs@gmail.com
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Abstract
A new series of (E)-benzo[d]imidazol-2-yl)methylene)indolin-2-one derivatives has been
synthesized and evaluated for their in vitro cytotoxic activity against a panel of selected human
cancer cell lines of prostate (PC-3 and DU-145) and breast (BT-549, MDA-MB-231, MCF-7,
4T1), non-small lung (A549) and gastric (HGC) cancer cells along with normal breast epithelial
cells (MCF10A). Among the tested compounds, 8l showed significant cytotoxic activity against
MDA-MB-231 and 4T1 cancer cells with IC50 values of 3.26 ± 0.24 µM and 5.96 ± 0.67 µM
respectively. The compounds 8f, 8i, 8l and 8o were also screened on normal human breast
epithelial cells (MCF10A) and found to be safer with lesser cytotoxicity. The treatment of
MDA-MB-231 cells with 8l led to inhibition of cell migration ability through disruption of F-
actin protein assembly. The flow-cytometry analysis reveals that the cells arrested in G0/G1
phase of the cell cycle. Further, the compound 8l induced apoptosis of MDA-MB-231 cells was
characterized by different staining techniques such as Acridine Orange/Ethidium Bromide
(AO/EB), DAPI, annexin V-FITC/PI, Rhodamine-123 and MitoSOX red assay. Western blot
studies demonstrated that the compound 8l treatment led to activation of caspase-3, increased
expression of cleaved PARP, increased expression of pro-apoptotic Bax and decreased
expression of anti-apoptotic Bcl-2 in MDA-MB-231 cancer cells.
Keywords: 3-Alkenyl-indolin-2-one, apoptosis, anticancer, benzimidazole, Knoevenagel
condensation.
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1.0. Introduction
Cancer is one of the life threatening diseases characterized by uncontrolled growth of cells,
leading to invasion of surrounding tissue and often spreading to other parts of the body. Despite
of various chemotherapeutic strategies, no effective treatments are available that can tackle the
sequelae of metastasis and still the disease remains tenacious and deadly [1]. The toxicity as well
as resistance to chemotherapeutics makes it crucial to discover novel drugs and new targets that
can overcome the side-effects of existing anticancer drugs. Chemotherapeutic agents act by
activating the cell death signalling pathways and apoptosis [2]. The inappropriate regulation of
apoptosis signalling pathways has been indicated in many diseases including cancer [3].
Henceforth, the development of new chemotherapeutic agents that can induce apoptosis in
cancer cell has emerged as an attractive approach in cancer drug discovery [2].
In the search for prospective anticancer agents, considerable effort has been made on the
development of heterocyclic motifs based on their structural design. It is worth noting that isatin
(indolin-2,3-dione) a “privileged heterocyclic scaffold” has been found to be an attractive
pharmacophore in modern medicinal chemistry for the development of new antitumor agents [4].
The antineoplastic and cytotoxic activities of isatin/oxindole based molecules are related with its
affinity to inhibit tyrosine kinases like c-Kit, VEGFR-1, VEGFR-2, PDGFRb [5] and cyclic-
dependent kinases (CDKs) [6]. Recently, USFDA approved drug Sunitinib (Figure 1) is the first
multikinase inhibitor containing 5-fluoro-3-alkenyl-oxindole, used for the treatment of
gastrointestinal stromal tumor and renal cell carcinoma [7]. Moreover, Semaxanib [8a], SU5402
[8b], SU6668 [8c] and SU14813 [8d] are some clinical drug candidates of this class of molecules
known to exhibit anticancer potential [9,10]. Henceforth, a plethora of biologically active C3-
substituted oxindoles have generated a huge interest in medicinal chemistry to develop novel
molecules of this class for the treatment of cancer.
On the other hand, benzimidazole scaffold can be considered as “Master Key” as it is an
important core in numerous compounds acting at different targets to elicit diverse range of
medicinal properties [11]. The different substitutions on benzimidazole nucleus have been luring
researchers all over the world to assess their therapeutic potential. The structural similarity of
benzimidazole with naturally occurring moieties like purines, makes them to interact with
biomolecules of living systems. In the recent years, it has been reported that the hybrid
molecules of benzimidazole with various heterocyclic moieties resulted in the discovery of
potential anticancer molecules against different cancers cells [12].
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<Insert Figure 1 here>
The development of novel bio-active scaffolds and efficient synthetic routes has always been
fascinating medicinal chemists in the process of drug discovery. Hence, in the pursuit of
developing a prospective anticancer agent, we incorporated the structural features of 3-alkenyl-
oxindoles and benzimidazole by aiming at the identification of new small molecules with potent
anticancer effects on selected human cancer cells. The synthesis of aforesaid conjugates could be
possible by a pharmacophore hybrid approach of modern medicinal chemistry. Hybridization of
two dissimilar bioactive molecules with different mechanisms or complementary
pharmacophoric functions often lead to synergistic effects [13]. Henceforth, owing to the
therapeutic significance of 3-alkenyl-indolin-2-ones and our interest in the synthesis of
biologically active framework with medicinal potential [14], the present work explicates the
synthesis of a new series of (E)-benzo[d]imidazol-2-yl)methylene)indolin-2-ones by combining
the two pharmacophores; 3-alkenyl-oxindole and benzimidazole with a view to create promising
cytotoxic agents (Figure 2). The amalgamation of a benzimidazole moiety onto oxindole
scaffold might discover the potential to access a new aspect of structural diversity to the
molecules via Knoevenagel condensation reaction.
<Insert Figure 2 here>
2.0. Results and discussion
2.1. Chemistry
The 1-alkyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-ones 8a–z were synthesized in a
convergent approach by employing the versatile Knoevenagel condensation reaction between
oxindoles 3a–f and 1-alkyl-1H-benzo[d]imidazole-2-carbaldehydes 7a–e as shown in Scheme 1.
The anilines 1a–f were converted into isonitrosoacetanilides via reaction with chloral hydrate
and hydroxylamine hydrochloride followed by cyclization under acidic conditions to give 3-
iminoindolin-2-ones, and then hydrolysed to furnish isatins 2a–f in 30–80% yields [15]. Next,
the isatins 2a–f were converted in to their corresponding oxindoles 3a–f under reflux conditions
in hydrazine-hydrate [16].
<Insert Scheme 1 here>
Next, the 1-alkyl-1H-benzo[d]imidazole-2-carbaldehydes 7a–e were synthesized according to
the previous reports [17]. o-Phenylenediamines 4a–b were reacted with glycolic acid in 4N HCl
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under reflux conditions formed benzimidazolyl methanols 5a–b. The obtained intermediates
5a–b, reacted subsequently with alkyl iodide in the presence of potassium carbonate to give 1-
alkylated benzimidazolyl methanols 6a–e. 1-Alkyl-1H-benzo[d]imidazole-2-carbaldehydes 7a–e
were obtained from the oxidation of 6a–e by using Dess-Martin reagent. Finally, the title
compounds 1-alkyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-ones 8a–z were obtained by
reacting oxindoles 3a–f and 1-alkyl-1H-benzo[d]imidazole-2-carbaldehydes 7a–e under
Knoevenagel condensation using piperidine as base. It is worth to note that all the compounds
8a–z were obtained as single isomers by this condensation reaction. To determine the
configuration at double, one of the representative compound 8l proton chemical shifts was
assigned by using gDQFCOSY experiment (see ESI) and the geometry around double of
compound 8l was confirmed by the NOE experiments. The presence of NOEs between H4-H12
and H5-H6 was suggested E-configuration (Figure 3). The intermediate 1-alkyl-1H-
benzo[d]imidazole-2-carbaldehydes 7d and 7e obtained as a inseparable mixture of C-5 and C-6
tautomers from unsymmetrical o-phenylenediamines 4b and were reacted to give a C-5 and C-6
tautomeric mixture of final compounds 8r–z. All the synthesized compounds 8a–z were
carefully characterized by FT–IR, HRMS, 1H and 13C NMR spectroscopy techniques.
<Insert Figure 3 here>
2.2. Pharmacology
2.2.1. In vitro cytotoxic activity
The newly synthesized 1-alkyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one derivatives
8a–z were tested for their in vitro cytotoxic activity against prostate (PC-3 and DU-145), breast
(BT-549, MDA MB-231, MCF-7 and 4T1), non-small lung (A549), gastric (HGC) cancer cell
lines and normal breast epithelial cell (MCF10A) by using 3-(4,5-dimethylthiazol-2-yl)-2,5
diphenyltetrazolium bromide (MTT) assay [18]. The IC50 (µM) values (concentration required to
inhibit 50% of cancer cells growth) of tested compounds 8a–z and reference standard (Sunitinib)
has been listed in the Table 1. Results from the Table 1 indicated that some of the synthesized
compounds exhibited potential cytotoxicity against DU-145, 4T1, MDA-MB-231 and A549
cancer cell lines and were found to be active in the range of 3.26±0.24 to 9.36±0.76 µM. From
the close examination of IC50 values, it is observed that 8c, 8d, 8f, 8g, 8i, 8n, and 8x were active
and less than 50 µM on most of the tested cell lines. It is quite interesting to note that compound
8l displayed a cytotoxicity of IC50<15 µM in all the tested cancer cell lines which are
comparable to that of standard Sunitinib. Furthermore, compound 8o was found to be selectively
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cytotoxic toward MDA-MB-231 and 4T1 cancer cell lines with IC50 of 7.23±0.83 and 9.36±0.76
µM respectively. It could be easily inferred from IC50 values in Table 1 that halogenated
analogues at C5-position of oxindole increased biological response with the exception of
compound 8o. On the other hand, N-methyl and N-ethyl substituted benzimidazole did not have
much impact on the cytotoxic activity. However, among the all compounds 8c, 8f, 8i, 8l and 8o
containing N-isopropyl substitution on benzimidazole showed potent cytotoxic activity.
From the cytotoxicity data, the compounds 8f, 8i, 8l and 8o showed IC50 value ≤ 25 µM on
MDA-MB-231 cell line, were further checked for in vitro cytotoxicity on normal breast
epithelial cell line (MCF10A) to find out the specificity toward cancer cells. It was quite
interesting to note that compound 8l was found to be moderately selective towards the DU-145,
4T1, MDA-MB-231 and A549 cancer cell where IC50 was found to be almost 8, 9.5, 17 and 6.5
fold higher in MCF10A respectively. Compound 8o displayed almost 10.6 and 14 fold more
selectivity towards 4T1 and MDA-MB-231 cancer cell lines when compared to normal breast
epithelial cells. Compound 8i was almost 2 to 3 fold more selective towards all the cancer cells
compared to normal breast epithelial cell (IC50 in MCF10A was 68.83±4.42 µM), however, 8f
showed IC50 >100 µM on MCF10A. The remarkable biological activity of compound 8l on
MDA-MB-231 cancer cell line prompted us to investigate its effects at cellular level,
particularly, the mechanisms responsible for cell growth inhibition.
<Insert Table 1 here>
2.2.2. In vitro cell migration assay/Wound healing assay
Migration of cells is crucial to a broad variety of biological processes, particularly cancer cells in
which migration is a key step in metastatic cascade [19a]. In vitro cell migration assay/wound
healing assay is based on the observation that, upon formation of a new artificial wound on a
confluent cell monolayer, the cells on the edge of the newly created wound will move toward the
opening to close the wound until new establishment of cell–cell interactions. The treatment of
cells with anticancer agent restricts the migration of cells to fill up the wound. Therefore, we
have investigated the effect of the most active compound 8l on migration potential of metastatic
MDA-MB-231 cells by using in vitro cell migration assay [19b]. Wounds were created on a
confluent cell monolayer culture of MDA-MB-231 cells and were independently treated with 1
µM, 2 µM and 4 µM of compound 8l. The migration of MDA-MB-231 cancer cells to fill up the
wound was recorded by microscopic observations and images of the wounded areas were
photographed at 0 h and 24 h of compound treatment. The results from the Figure 4 clearly
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showed that wound gap in control has decreased considerably by migration of cells in control
after 24 h, whereas the treatment with 2 µM and 4 µM of compound 8l significantly inhibited the
migration of cell towards the wounded area. These results undoubtedly point towards that the
migration of MDA-MB-231 cancer cells was appreciably suppressed by these benzimidazole-
indolin-2-one hybrids.
<Insert Figure 4 here>
2.2.3. Effect on F-actin polymerisation
Motility of cancer cells is a integrated sum of multi-step processes triggered by the formation of
membrane protrusions in response to chemo-attractants [20a]. It is reported that the actin
polymerisation is the driving force for the formation of membrane protrusions which help in
cancer cell motility and stress fibre assembly [20b]. As seen in Figure 4 that compound 8l
inhibited the migration of MDA-MB-231 cancer cells, it was considered of interest to examine
its effect on actin polymerisation and stress fibre formation. The formation of actin cytoskeleton
in MDA-MB-231 cancer cells was studied by staining the cells using rhodamine-phalloidin (red
fluorescent dye) [20c], which specifically binds to F-actin. Results from the Figure 5 showed
that the control cells have a more number of F-actin extensions as well as stress fibre formation
at the periphery, however treatment of compound 8l led to disruption of stress fibres around the
nucleus and F-actin extensions were decreased. These results together disclose that the treatment
of compound 8l inhibit the migration potential of MDA-MB-231 cancer cells, at least in parts
through disruption of F-actin assembly.
<Insert Figure 5 here>
2.2.4. Cell cycle analysis
Many of the cytotoxic compounds exert their growth inhibitory effect by arresting the cell cycle
at a specific checkpoint [21]. Thus, the blockade of cell cycle progression by chemotherapeutic
agents always has been an ideal choice for developing anti-cancer therapeutics. In vitro
screening results revealed that the compound 8l showed significant cytotoxic activity against
MDA-MB-231 cells. Therefore, we examined the effect of compound 8l on distributions of cell
in different phases of cell cycle using flow cytometry analysis method [19b]. MDA-MB-231
cancer cells were treated with 1 µM, 2 µM and 4 µM of compound 8l for 24 h, cells were fixed
in ethanol and stained with propidium iodide which were further analysed by flow cytometry.
The results from the Figure 6 showed that the ratio of MDA-MB-231 cells in G0/G1 phase from
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50.4% in control (DMSO) increased to 57.2% at 1 µM, 64.3% at 2 µM and 74.7% at 4 µM
respectively. Concomitantly, there was a decrease in the number of cells in both S and G2/M
phase in a dose dependent manner. Similar observation was seen with sunitinib (positive control)
treated MDA-MB-231 cells wherein the ratio of cells in G0/G1 phase increased from 50.4% in
control to 67.6% at 4 µM. Henceforth, result from the flow cytometry analysis clearly indicated
that the treatment of MDA-MB-231 cells with compound 8l led to G0/G1 cell cycle arrest
(Figure 6B).
<Insert Figure 6 here>
2.2.5. Morphological observations using phase contrast microscopy
The induction of apoptosis by chemotherapeutic agents has always been a superlative choice in
developing anti-cancer therapeutics. Therefore, to examine the loss of cell viability as well as
induction of apoptosis, MDA-MB-231 cancer cells were treated with different concentrations of
compound 8l. Cell morphology was observed and photographs were taken under the phase
contrast microscope. It can be seen from the Figure 7 that the different concentrations i.e 1 µM,
2 µM and 4 µM of compound 8l treated MDA-MB-231 cells showed the characteristic apoptotic
features like cell shrinkage, cell wall deformation and reduced number of viable cells in
comparison to control cell, where these distinctive morphological features were absent in control
cells.
<Insert Figure 7 here>
2.2.6. Acridine orange–ethidium bromide (AO–EB) staining
The morphological changes induced by the most active compound 8l in MDA-MB-231 cells
were further studied by using acridine orange–ethidium bromide (AO–EB) staining to identify
whether the inhibition is due to apoptosis or nonspecific necrosis [22]. AO–EB staining
technique discriminate the live cells from dead cells, since AO permeates the live as well as dead
cells with intact membrane and stain the cells green, however EB can stain only dead cells with
loss in membrane integrity and stain the nucleus orange. The compound 8l treated and untreated
MDA-MB-231 cells were stained with AO–EB dye and analysed under fluorescence
microscope. It can be inferred from the Figure 8 that the control cells have a normal
morphology as well green in colour, however at 1 µM, early signs of apoptosis were
characterized by condensed chromatin and cell membrane blebbing. Irregular distribution of
chromatin which marginated into horse-shoe shaped nuclei and destructive fragmentation of
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chromatin was observed at 2 µM and 4 µM respectively, whereas these distinctive
morphological features were absent in the control MDA-MB-231 cancer cells.
<Insert Figure 8 here>
2.2.7. DAPI staining
DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye that binds strongly to A-T rich
regions in DNA and detects the chromatin condensation or nuclear damage. DAPI passes
through the membrane of live cells less efficiently; therefore the effectiveness of the stain in live
cells is lower. DAPI stains the apoptotic cells as bright coloured due to the condensed nucleus
which is a typical apoptotic characteristic. Therefore, it was considered of interest to detect the
effect of compound 8l in MDA-MB-231 cells by using DAPI staining technique [23], which
distinguishes live cells from apoptotic cells based on nuclear morphology. DAPI forms
fluorescent complexes with chromatin and stain nuclei bright blue fluorescent with a DAPI filter.
As shown in the Figure 9, the nuclear structure of control cells was intact, however compound
8l treated MDA-MB-231 cells exhibited condensed and horse-shoe shaped nuclei, which are the
typical characteristic features of apoptotic induction.
<Insert Figure 9 here>
2.2.8. Effect on mitochondrial membrane potential (DΨm)
Mitochondria play a key role in energy metabolism, as they build their membrane potential from
respiratory substrates derived from the electron transport chain. Literature reports show that the
loss or collapse of mitochondrial membrane potential may be an early event in the process of
apoptosis [24a]. Therefore, we examined the effect of compound 8l on mitochondrial membrane
potential of MDA-MB-231 cancer cell line by using rhodamine-123 [24b]. Mitochondria having
normal DΨm produce a strong green fluorescence, however mitochondrial energization induces
quenching of fluorescence due to less uptake of rhodamine-123 which leads to
depolarisation. The loss of DΨm can be monitored by the shift in the green fluorescence
intensity by the use of spectrofluorometer. From the Figure 10A and 10B it can be seen the dose
dependent loss of mitochondrial membrane potential of MDA-MB-231 cells. Results from the
Figure 10A shows that the peak changes from control (Figure 10A-a) to the left (Figure 10A-b
to d and e is overlay of a-d) indicates depolarisation by the loss of DΨm. At concentration of 1
µM, the loss in DΨm was 25.2% compared to control which further dropped to 32.5% at 2 µM
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and 38.5% at 4 µM respectively. The results from loss of DΨm clearly indicate the induction of
apoptosis through the collapse of mitochondrial membrane potential in MDA-MB-231 cells.
<Insert Figure 10 here>
2.2.9. Effect on superoxide production (MitoSOX™ Red assay)
The production of ROS has been implicated in apoptotic induction by triggering oxidative
damage to the mitochondrial membrane potential and permeability [25a]. We were intrigued by
the fact that the apoptosis induction could be due to the ROS generation. Henceforth, we tested
this possibility using MitoSOX™ red, a chemical probe which react with mitochondrial
generated superoxide and accumulate in mitochondria [25b]. MitoSOX™ Red is a novel
fluorogenic dye which selectively detects superoxide in the mitochondria of live cells.
MitoSOX™ red is quickly oxidized by superoxide species but not by other reactive oxygen
species (ROS) and reactive nitrogen species (RNS). The oxidized product is highly fluorescent
upon binding to nucleic acid and produces red fluorescence. Fluorescent microscopic images of
MDA-MB-231 cancer cells stained with MitoSOX™ Red indicator were taken after 48 h
treatment with different concentrations of the compound 8l. It can be easily inferred from the
Figure 11 that the treatment of compound 8l caused a dose-dependent increase in MitoSOX™
Red fluorescence in MDA-MB-231 cancer cell. As shown in Figure 11, DMSO-treated control
MDA-MB-231 cancer cells exhibited weak and diffuse MitoSOX™ Red fluorescence, however
the cells treated with 1 µM, 2 µM and 4 µM of compound 8l were brightly stained suggesting
superoxide generation.
<Insert Figure 11 here>
2.2.10. Western blotting analysis
Caspases, Bax, Bcl2 and PARP are some of the proteins whose expression plays a critical role in
the apoptotic process. Particularly, caspase-3 is a member of cysteine-aspartic acid
protease family which is known for catalyzing specific cleavage of many key cellular proteins
[26a]. Herein, to investigate the molecular mechanisms of compound 8l on apoptosis, we have
checked the expression of Bcl2, Bax, PARP and caspase-3 by using western blot method [26b,
26c]. Results from the Figure 12 indicated that compound 8l treatment led to the dose dependent
increased expression of cleaved PARP and caspase-3 in MDA-MB-231 cells, which is a
hallmark feature of apoptosis. Moreover, the compound 8l treatment resulted in decreased
expression of anti-apoptotic Bcl2 and increased expression of proapoptotic Bax proteins in a
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dose dependent manner. Collectively, these results illustrate that compound 8l induced apoptosis
through apoptosis-related protein expression.
<Insert Figure 12 here>
2.2.11. Annexin V-FITC/Propidium iodide dual staining assay
Further, to quantify the number of apoptotic cells after the treatment with different
concentrations of compound 8l, annexin V-FITC/propidium iodide dual staining assay has been
carried out [27]. The Annexin V-FITC/PI binding assay detects live cells (Q2-LL; AV-/PI-),
early apoptotic cells (Q2-LR; AV+/PI-), late apoptotic cells (Q2-UR; AV+/PI+) and necrotic
cells (Q2-UL; AV-/PI+). MDA-MB-231 cells were treated with 1 µM, 2 µM and 4 µM of
compound 8l for 24 h and stained with annexin V-FITC and propidium iodide. Results from the
Figure 13 indicated that the percentage of total apoptotic cells (early and late apoptotic cells)
from 7.7% (control) increased to 13.3% (1 µM), 23.1% (2 µM) and 30.5% (4 µM) respectively.
The significant increase in early and late apoptotic from 7.7% to 30.5% in a dose dependent
manner clearly indicates that the compound 8l induced apoptosis in MDA-MB-231 cells.
<Insert Figure 13 here>
3.0. Conclusion
In conclusion, a new series of (E)-benzo[d]imidazol-2-yl)methylene)indolin-2-ones hybrids 8a–z
has been synthesized and evaluated for their in vitro cytotoxic potential against different human
cancer cell lines including normal breast epithelial cells. The preliminary studies have given
away that a few of the synthesized hybrids were active on all the tested cancer cell lines with less
than 50 µM (IC50 value). The cytotoxicity profile revealed that compound 8l displayed broad
spectrum of cytotoxic activity against all the tested cancer cell lines. Further, compounds 8f, 8i,
8l and 8o were found to be safer with lesser cytotoxicity on normal breast epithelial cells
(MCF10A). The exposure of MDA-MB-231 cancer cells to compound 8l inhibited the in vitro
cellular migration through the disruption of cytoskeleton and arrested the cells in G0/G1 phase
of the cell cycle. The detailed studies like AO/EB staining, DAPI staining and Annexin V-
FITC/Propidium iodide assays suggested the compound 8l induced apoptosis in MDA-MB-231
cancer cells. Moreover, the compound 8l treatment resulted in the collapse of mitochondrial
membrane potential and elevated intracellular generation of superoxide ROS. Western blotting
analysis demonstrated that compound 8l induces apoptotic cell death, accompanied by the
increased expression of cleaved PARP and caspase-3. Additionally, exposure of MDA-MB-231
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cancer cells to 8l decreased the expression of anti-apoptotic Bcl2 and increased the expression of
proapoptotic Bax proteins. Overall, these findings propose that (E)-benzo[d]imidazol-2-
yl)methylene)indolin-2-one hybrids have the potential to be developed as lead molecule and
further their structural modification may create promising new anticancer agents.
4.0 Materials & Methods
4.1 Chemistry
All the reagents and starting materials were obtained from commercially available suppliers and
were used without further purification; substituted anilines, piperidine, NH2OH.HCl
NH2NH2.H2O, glycolic acid, methyl iodide, ethyl iodide, isopropyl iodide, Dess-Martin
periodinane (Spectrochem); chloral hydrate (Aldrich) and phenylenediamines (alfa-aeser). The
reactions were monitored by thin layer chromatography (TLC), using MERCK pre-coated silica
gel 60-F254 aluminum plates. Visualization of spots on TLC plates were done by UV light.
Percentage purity of compounds was determined by using waters Acquity UPLC instrument.
Melting points were checked using Stuart® SMP30 melting point apparatus and are uncorrected.
1H and 13C NMR spectra were recorded on Bruker Avance 500 MHz spectrometer using
tetramethyl silane (TMS) as the internal standard and are reported in parts per million (ppm)
1
downfield from TMS. Chemical shifts are referenced to DMSO˗d6 (δ 2.50) (for H spectra) or
13
DMSO˗d6 (δ 39.5) (for C spectra). Spin multiplicities are reported as s (singlet), brs (broad
singlet), d (doublet), dd (double doublet), t (triplet) and m (multiplet). Coupling constant (J)
values are reported in hertz (Hz). HRMS were determined with Agilent QTOF mass
spectrometer 6540 series instrument and were performed in the ESI techniques at 70 eV.
Column chromatography was performed using silica gel 60–120 or 100–200 mesh.
4.1.1 General procedure for the synthesis of (E)-benzo[d]imidazol-2-yl)methylene)indolin-2-
ones (8a–z)
To a mixture of oxindole (3a–f, 0.3 mmol), 1-alkyl-1H benzo[d]imidazole-2-carbaldehydes (7a–
e, 0.33 mmol) in ethanol (2 mL), was added catalytic amount of piperidine. The reaction mixture
was stirred at reflux until complete consumption of the oxindole observed by TLC. After
cooling, the precipitate was filtered, washed with cold ethanol, and dried in air to furnish pure
(E)-benzo[d]imidazol-2-yl)methylene)indolin-2-ones 8a–z orange/yellow solids in moderate to
good yields. Compounds 8c, 8f, 8i, 8l and 8o did not precipitated out from ethanol, were purified
by column chromatography with silica gel (60–120) by using ethyl acetate:hexane (2:8 to 3:7).
12

ACCEPTED MANUSCRIPT
4.1.1.1 (E)-3-((1-Methyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one (8a). Orange solid,
1
Yield 87%; mp: 255–257 °C; FT–IR: (cm-1): 3186, 3151, 3063, 1705, 1605, 1329, 781, 763; H
NMR (500 MHz, DMSO–d6): δ 10.71 (brs, 1H, NH), 9.49 (d, J = 7.6 Hz, 1H, Ar–H), 7.84 (d, J
= 7.9 Hz, 1H, Ar–H), 7.69 (d, J = 8.1 Hz, 1H, Ar–H), 7.58 (s, 1H, C=CH), 7.42–7.29 (m, 3H,
13
Ar–H), 7.07 (t, J = 7.8 Hz, 1H, Ar–H), 6.90 (d, J = 7.8 Hz, 1H, Ar–H), 4.02 (s, 3H, CH3); C
NMR (75 MHz, DMSO–d6): δ 168.4, 147.2, 143.1, 142.3, 135.1, 130.9, 130.2, 127.5, 123.6,
122.5, 120.9, 120.6, 119.2, 117.3, 110.3, 109.1, 29.8; HRMS (ESI): m/z calcd for C17H14N3O
276.1137, found 276.1125 [M+H]+; Purity: 98.5%.
4.1.1.2 (E)-3-((1-Ethyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one (8b). Orange solid,
1
Yield 79%; mp: 266–268 °C; FT–IR: (cm-1): 3176, 3133, 3033, 1703, 1615, 1339, 791, 759; H
NMR (500 MHz, DMSO–d6): δ 10.67 (brs, 1H, NH), 9.48 (d, J = 7.6 Hz, 1H, Ar–H), 7.85 (d, J
= 7.9 Hz, 1H, Ar–H), 7.79 (d, J = 7.9 Hz, 1H, Ar–H) 7.55 (s, 1H, C=CH), 7.41–7.30 (m, 3H,
Ar–H), 7.07 (t, J = 7.6 Hz, 1H, Ar–H), 6.90 (d, J = 7.6 Hz, 1H, Ar–H), 4.58–4.50 (m, 2H, CH2),
13
1.38 (t, J = 7.3 Hz, 3H, CH3); C NMR (75 MHz, DMSO–d6): δ 168.7, 146.5, 143.3, 142.7,
134.3, 131.2, 130.7, 127.8, 124.0, 122.8, 121.2, 120.8, 119.7, 117.2, 110.4, 109.5, 38.2, 15.4;
HRMS (ESI): m/z calcd for C18H16N3O 290.1293, found 290.1282 [M+H]+; Purity: 99.0%.
4.1.1.3 (E)-3-((1-Isopropyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one (8c). Yellow
solid, Yield 83%; mp: 184–186 °C; FT–IR: (cm-1): 3152, 3074, 2981, 1705, 1611, 1335, 767,
1
739; H NMR (500 MHz, DMSO–d6): δ 10.74 (brs, 1H. NH), 9.28 (d, J = 7.8, 1H, Ar–H), 7.84
(d, J = 7.7 Hz, 2H, Ar–H), 7.63 (s, 1H, C=CH), 7.37–7.28 (m, 3H, Ar–H), 7.03 (t, J = 6.9 Hz,
1H, Ar–H), 6.90 (d, J = 7.6 Hz, 1H, Ar–H), 5.18–5.09 (m, 1H, CH), 1.65 (d, J = 7.0 Hz, 6H,
13
2CH3); C NMR (75 MHz, CDCl3+DMSO–d6): δ 169.4, 146.5, 143.5, 143.1, 133.1, 131.7,
130.7, 127.3, 123.6, 122.1, 121.4, 120.9, 120.1, 118.1, 111.4, 109.4, 47.9, 21.3; HRMS (ESI):
m/z calcd for C19H18N3O 304.1450, found 304.1443 [M+H]+; Purity: 98.1%.
4.1.1.4 (E)-5-Fluoro-3-((1-methyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one (8d).
Yellow solid, Yield 90%; mp: 277–279 °C; FT–IR: (cm-1): 3163, 3068, 1703, 1620, 1301, 808,
1
730;; H NMR (500 MHz, DMSO–d6): δ 10.72 (brs, 1H, NH), 9.45 (dd, J = 2.7, 10.1 Hz, 1H,
Ar–H), 7.82 (d, J = 8.1 Hz, 1H, Ar–H), 7.67 (d, J = 8.2 Hz, 1H, Ar–H), 7.59 (s, 1H, C=CH),
7.38 (t, J = 7.3 Hz, 1H, Ar–H), 7.32 (t, J = 7.8 Hz, 1H, Ar–H), 7.19–7.13 (m, 1H, Ar–H), 6.89–
6.84 (m, 1H, Ar–H), 4.01 (s, 3H, CH3); 13C NMR (125 MHz, DMSO–d6): δ 168.8, 157.3 (d, JCF
= 234.3 Hz), 147.4, 142.8, 139.9, 135.6, 130.1 (d, JCF = 2.8 Hz), 124.3, 123.1, 122.1 (d, JCF
9.9 Hz), 119.8, 119.1, 117.4 (d, JCF = 23.6 Hz), 115.0 (d, JCF = 27.2 Hz) 110.9, 110.1 (d, JCF
=
=
13

ACCEPTED MANUSCRIPT
8.2 Hz), 30.3; HRMS (ESI): m/z calcd for C17H13FN3O 294.1043, found 294.1036 [M+H]+;
Purity: 99.5%.
4.1.1.5
(E)-3-((1-Ethyl-1H-benzo[d]imidazol-2-yl)methylene)-5-fluoroindolin-2-one
(8e).
Yellow solid, Yield 89%; mp: 276–278 °C; FT–IR: (cm-1): 3166, 3071, 2975, 1702, 1613, 1320,
1
812, 734; H NMR (500 MHz, DMSO–d6): δ 10.76 (brs, 1H, NH), 9.45 (dd, J = 2.7, 10.2 Hz,
1H, Ar–H), 7.85 (d, J = 8.1 Hz, 1H, Ar–H), 7.73 (d, J = 8.1 Hz, 1H, Ar–H), 7.60 (s, 1H, C=CH),
7.42–7.32 (m, 2H, Ar–H), 7.22–7.17 (m, 1H, Ar–H), 6.91–6.86 (m, 1H, Ar–H), 4.59–4.52 (m,
2H, CH2), 1.38 (t, J = 7.3 Hz, 3H, CH3); 13C NMR (75 MHz, DMSO–d6): δ 168.7, 157.5 (d, JCF
= 234.4 Hz), 146.3, 142.7, 139.7, 134.4, 130.2, 124.3, 123.1, 121.9 (d, JCF = 9.9 Hz), 119.8,
118.6, 117.4 (d, JCF = 24.2 Hz), 114.7 (d, JCF = 27.5 Hz) 110.7, 110.1 (d, JCF = 7.7 Hz), 38.3,
15.5; HRMS (ESI): m/z calcd for C18H15FN3O 308.1199, found 308.1198 [M+H]+; Purity:
99.5%.
4.1.1.6 (E)-5-Fluoro-3-((1-Isopropyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one (8f).
Yellow solid, Yield 84%; mp: 218–220 °C; FT–IR: (cm-1): 3163, 3069, 1705, 1616, 1320, 812,
1
739; H NMR (500 MHz, DMSO–d6): δ 10.76 (brs, 1H, NH), 9.30 (dd, J = 2.7, 10.1 Hz, 1H,
Ar–H), 7.88–7.83 (m, 2H, Ar–H), 7.70 (s, 1H, C=CH), 7.39–7.31 (m, 2H, Ar–H), 7.22–7.16 (m,
1H, Ar–H) 6.91–6.86 (m, 1H, Ar–H), 5.21–5.12 (m, 1H, CH), 1.66 (d, J = 7.0 Hz, 6H, 2CH3);
13C NMR (75 MHz, CDCl3+DMSO–d6): δ 169.5, 158.0 (d, JCF = 234.4 Hz), 146.1, 143.4, 139.2,
133.1, 131.2, 123.6, 122.4, 121.9 (d, JCF = 9.9 Hz), 120.3, 119.2, 116.8 (d, JCF = 24.2 Hz), 114.8
(d, JCF = 27.4 Hz), 111.5, 109.7 (d, JCF = 8.2 Hz), 48.0, 21.4; HRMS (ESI): m/z calcd for
C19H17FN3O 322.1356, found 322.1345 [M+H]+; Purity: 99.5%.
4.1.1.7 (E)-5-Chloro-3-((1-methyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one (8g).
Orange solid, Yield 81%; mp: 279–281 °C; FT–IR: (cm-1): 3157, 3065, 1703, 1608, 1317, 807,
1
724; H NMR (500 MHz, DMSO–d6): δ 10.85 (brs, 1H, NH), 9.68 (d, J = 2.3 Hz, 1H, Ar–H),
7.80 (d, J = 7.9 Hz, 1H, Ar–H), 7.71 (d, J = 8.1 Hz, 1H, Ar–H), 7.63 (s, 1H, C=CH), 7.43–7.31
(m, 3H, Ar–H), 6.91 (d, J = 7.8 Hz, 1H, Ar–H), 4.04 (s, 3H, CH3); 13C NMR (75 MHz, DMSO–
d6): δ 168.5, 147.1, 142.5, 142.1, 135.4, 130.5, 129.3, 127.4, 125.2, 124.3, 123.1, 122.4, 119.5,
119.2, 110.8, 110.6, 30.1; HRMS (ESI): m/z calcd for C17H13ClN3O 310.0747, found 310.0743
[M+H]+; Purity: 99.5%.
4.1.1.8
(E)-5-Chloro-3-((1-ethyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one
(8h).
Orange solid, Yield 74%; mp: 286–288 °C; FT–IR: (cm-1): 3160, 3054, 1703, 1605, 1320, 812,
738;1H NMR (500 MHz, DMSO–d6): δ 10.89 (brs, 1H, NH), 9.66 (d, J = 2.1 Hz, 1H, Ar–H),
14

ACCEPTED MANUSCRIPT
7.81 (d, J = 7.6 Hz, 1H, Ar–H), 7.74 (d, J = 7.7 Hz, 1H, Ar–H) 7.60 (s, 1H, C=CH), 7.45–7.31
(m, 3H, Ar–H), 6.91 (d, J = 8.3 Hz, 1H, Ar–H), 4.62–4.49 (m, 2H, CH2), 1.38 (t, J = 7.3 Hz, 3H,
CH3); 13C NMR (75 MHz, DMSO–d6): δ 168.6, 146.4, 142.7, 142.3, 134.6, 130.7, 129.8, 127.6,
125.3, 124.5, 123.2, 122.5, 119.8, 118.9, 110.9, 110.8, 38.5, 15.7; HRMS (ESI): m/z calcd for
C18H15ClN3O 324.0904, found 324.0894 [M+H]+; Purity: 99.5%.
4.1.1.9 (E)-5-Chloro-3-((1-isopropyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one (8i).
Yellow solid, Yield 82%; mp: 243–245 °C; FT–IR: (cm-1): 3171, 3079, 2974, 1698, 1602, 1308,
813, 738; 1H NMR (500 MHz, DMSO–d6): δ 10.86 (brs, 1H,NH), 9.50 (d, J = 2.1 Hz, 1H, Ar–
H), 7.86 (d, J = 7.9 Hz, 1H, Ar–H), 7.80 (d, J = 7.8 Hz, 1H, Ar–H), 7.70 (s, 1H, C=CH), 7.41–
7.30 (m, 3H, Ar–H), 6.91 (d, J = 8.2 Hz, 1H, Ar–H), 5.21–5.10 (m, 1H, CH), 1.66 (d, J = 6.9 Hz,
6H, 2CH3); 13C NMR (75 MHz, CDCl3+DMSO–d6): δ 169.2, 146.1, 143.2, 141.6, 133.1, 130.6,
130.1, 127.4, 126.2, 123.7, 122.4, 122.3, 120.2, 119.3, 111.5, 110.3, 47.9, 21.6; HRMS (ESI):
m/z calcd for C19H17ClN3O 338.1060, found 338.1053 [M+H]+; Purity: 99.5%.
4.1.1.10 (E)-5-Bromo-3-((1-methyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one (8j).
Orange solid, Yield 75%; mp: 243–245 °C; FT–IR: (cm-1): 3160, 3012, 2841, 1697, 1604, 1318,
789, 736; 1H NMR (500 MHz, DMSO–d6): δ 10.85 (brs, 1H, NH), 9.81 (d, J = 2.3 Hz, 1H, Ar–
H), 7.78 (d, J = 7.8 Hz, 1H, Ar–H), 7.69 (d, J = 8.0 Hz, 1H, Ar–H), 7.61 (s, 1H, C=CH), 7.50
(dd, J = 2.0, 8.2 Hz, 1H, Ar–H), 7.42–7.32 (m, 2H, Ar–H), 6.86 (d, J = 7.8 Hz, 1H, Ar–H), 4.03
(s, 3H, CH3); 13C NMR (75 MHz, DMSO–d6): δ 168.4, 147.3, 142.6, 142.5, 135.5, 133.3, 130.3,
129.3, 124.3, 123.1, 122.9, 119.6, 119.3, 113.0, 111.4, 110.9, 30.2; HRMS (ESI): m/z calcd for
C17H13BrN3O 354.0242, found 354.0241 [M+H]+; Purity: 97.0%.
4.1.1.11 (E)-5-Bromo-3-((1-ethyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one (8k).
Orange solid, Yield 76%; mp: 273–275 °C; FT–IR: (cm-1): 3164, 3052, 1704, 1605, 1321, 811,
1
739; H NMR (300 MHz, DMSO–d6): δ 10.38 (brs, 1H, NH), 9.77 (d, J = 1.7 Hz, 1H, Ar–H),
7.90 (d, J = 7.2 Hz, 1H, Ar–H), 7.63 (s, 1H, C=CH), 7.54–7.30 (m, 4H, Ar–H), 6.81 (d, J = 8.1
13
Hz, 1H, Ar–H), 4.54–4.43 (m, 2H, CH2), 1.51 (t, J = 7.3 Hz, 3H, CH3); C NMR (125 MHz,
DMSO–d6): δ 168.4, 146.4, 142.8, 142.7, 134.6, 133.5, 130.3, 129.7, 124.4, 123.3, 123.0, 119.8,
118.9, 113.1, 111.4, 110.8, 38.5, 15.7; HRMS (ESI): m/z calcd for C18H15BrN3O 368.0398,
found 368.0388 [M+H]+; Purity: 98.1%.
4.1.1.12 (E)-5-Bromo-3-((1-isopropyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one (8l).
Yellow solid, Yield 88%; mp: 141–143 °C; FT–IR: (cm-1): 3171, 2986, 1707, 1607, 1316, 1004,
1
811, 739; H NMR (500 MHz, DMSO–d6, 27 °C): δ 10.85 (brs, 1H, NH), 9.63 (d, J = 2.0 Hz,
15

ACCEPTED MANUSCRIPT
1H, Ar–H), 7.85 (dd, J = 7.6, 1.5 Hz, 1H, Ar–H), 7.77 (dd, J = 7.6, 1.5 Hz, 1H, Ar–H), 7.68 (s,
1H, C=CH), 7.50 (dd, J = 8.2, 2.1 Hz, 1H, Ar–H), 7.36 (dt, J = 7.6, 7.6, 1.5 Hz, 1H, Ar–H), 7.33
(dt, J = 7.6, 7.6, 1.5 Hz, 1H, Ar–H), 6.85 (d, J = 8.2 Hz, 1H, Ar–H), 5.21–5.10 (m, 1H, CH),
13
1.65 (d, J = 7.0 Hz, 6H, 2CH3); C NMR (75 MHz, CDCl3+DMSO–d6): δ 169.0, 146.0, 143.3,
142.1, 133.1, 132.9, 130.4, 130.2, 123.7, 122.7, 122.4, 120.2, 119.3, 113.7, 111.5, 110.8, 47.9,
21.4; HRMS (ESI): m/z calcd for C19H17BrN3O 382.0555, found 382.0553 [M+H]+; Purity:
98.8%.
4.1.1.13 (E)-5-Methyl-3-((1-methyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one (8m).
Orange solid, Yield 79%; mp: 266–268 °C; FT–IR: (cm-1): 3183, 3056, 2916, 1702, 1612, 1321,
815, 737; 1H NMR (500 MHz, DMSO–d6): δ 10.59 (brs, 1H, NH), 9.35 (s, 1H, Ar–H), 7.84 (d, J
= 8.1 Hz, 1H, Ar–H), 7.69 (d, J = 8.1 Hz, 1H, Ar–H), 7.55 (s, 1H, C=CH), 7.41–7.31 (m, 2H,
Ar–H), 7.15 (d, J = 7.8 Hz, 1H, Ar–H), 6.79 (d, J = 7.8 Hz, 1H, Ar–H), 4.02 (s, 3H, CH3), 2.35
(s, 3H, CH3); 13C NMR (75 MHz, CDCl3+DMSO–d6): δ 168.9, 147.5, 142.6, 141.0, 135.4,
131.4, 130.9, 129.9, 128.4, 123.9, 122.7, 120.9, 119.5, 117.3, 110.4, 109.1, 30.0, 20.8; HRMS
(ESI): m/z calcd for C18H16N3O 290.1293, found 290.1279 [M+H]+; Purity: 99.2%.
4.1.1.14 (E)-3-((1-Ethyl-1H-benzo[d]imidazol-2-yl)methylene)-5-methylindolin-2-one (8n).
Orange solid, Yield 84%; mp: 274–276 °C; FT–IR: (cm-1): 3166, 3067, 2980, 1704, 1615, 1323,
813, 742;1H NMR (300 MHz, DMSO–d6): δ 10.20 (brs, 1H, NH), 9.22 (s, 1H, Ar–H), 7.87 (d, J
= 7.2 Hz, 1H, Ar–H), 7.65–7.58 (m, 1H, Ar–H), 7.56 (s, 1H, C=CH), 7.51–7.28 (m, 2H, Ar–H),
7.09 (d, J = 7.9 Hz, 1H, Ar–H), 6.78 (d, J = 7.9 Hz, 1H, Ar–H), 4.54–4.41 (m, 2H, CH2), 2.38 (s,
13
3H, CH3), 1.50 (t, J = 7.3 Hz, 3H, CH3); C NMR (125 MHz, DMSO–d6): δ 168.9, 146.8,
142.9, 141.3, 134.6, 131.7, 131.2, 129.9, 128.6, 124.1, 122.9, 121.1, 119.8, 117.1, 110.6, 109.3,
38.4, 21.0, 15.7; HRMS (ESI): m/z calcd for C19H18N3O 304.1450, found 304.1438 [M+H]+;
Purity: 98.7%.
4.1.1.15 (E)-3-((1-Isopropyl-1H-benzo[d]imidazol-2-yl)methylene)-5-methylindolin-2-one (8o).
Yellow solid, Yield 79%; mp: 223–225 °C; FT–IR: (cm-1): 3185, 3055, 1701, 1606, 1314, 813,
735; 1H NMR (500 MHz, DMSO–d6): δ 10.61 (brs, 1H, NH), 9.13 (s, 1H, Ar–H), 7.86–7.81 (m,
2H, Ar–H), 7.60 (s, 1H, C=CH), 7.37–7.28 (m, 2H, Ar–H), 7.14 (d, J = 7.8 Hz, 1H, Ar–H), 6.78
(d, J = 7.8 Hz, 1H, Ar–H), 5.17–5.06 (m, 1H, CH), 2.31 (s, 3H, CH3), 1.65 (d, J = 6.9 Hz, 6H,
13
2CH3); C NMR (75 MHz, CDCl3+DMSO–d6): δ 169.5, 146.6, 143.4, 140.8, 133.1, 132.1,
131.2, 130.5, 127.7, 123.3, 122.1, 120.9, 120.1, 117.9, 111.5, 109.2, 47.9, 21.6, 20.8; HRMS
(ESI): m/z calcd for C20H20N3O 318.1606, found 318.1604 [M+H]+; Purity: 99.5%.
16

ACCEPTED MANUSCRIPT
4.1.1.16 (E)-3-((1-Methyl-1H-benzo[d]imidazol-2-yl)methylene)-5-nitroindolin-2-one (8p).
Yellow solid, Yield 84%; FT–IR: (cm-1): 3156, 3121, 2963, 1705, 1615, 1329, 821, 733; mp:
>300 °C; 1H NMR (500 MHz, DMSO–d6): δ 11.27 (brs, 1H, NH), 10.63 (d, J = 2.4 Hz, 1H, Ar–
H), 8.24 (dd, J = 2.4, 8.5 Hz, 1H, Ar–H), 7.77 (d, J = 7.9 Hz, 1H, Ar–H), 7.69 (d, J = 7.9 Hz,
1H, Ar–H), 7.66 (s, 1H, C=CH), 7.44–7.34 (m, 2H, Ar–H), 7.03 (d, J = 8.7 Hz, 1H, Ar–H), 4.03
13
(s, 3H, CH3); C NMR (125 MHz, DMSO–d6): δ 169.1, 149.0, 147.1, 142.7, 142.0, 136.6,
128.4, 127.4, 124.7, 123.7, 123.4, 121.3, 120.6, 119.7, 111.1, 109.6, 30.3; HRMS (ESI): m/z
calcd for C17H13N4O3 321.0988, found 321.0986 [M+H]+; Purity: 98.0%.
4.1.1.17
(E)-3-((1-Ethyl-1H-benzo[d]imidazol-2-yl)methylene)-5-nitroindolin-2-one
(8q).
Yellow solid, Yield 82%; mp: 285–287 °C; FT–IR: (cm-1): 3143, 3111, 2963, 1701, 1603, 1332,
1
780, 737; H NMR (500 MHz, DMSO–d6): δ 11.39 (brs, 1H, NH), 10.62 (d, J = 2.4 Hz, 1H,
Ar–H), 8.24 (dd, J = 2.4, 8.5 Hz, 1H, Ar–H), 7.78 (d, J = 7.9 Hz, 1H, Ar–H), 7.73 (d, J = 8.0 Hz,
1H, Ar–H), 7.64 (s, 1H, C=CH), 7.45–7.34 (m, 2H, Ar–H), 7.03 (d, J = 8.7 Hz, 1H, Ar–H),
13
4.60–4.52 (m, 2H, CH2), 1.38 (t, J = 7.3 Hz, 3H, CH3); C NMR (125 MHz, DMSO–d6): δ
169.1, 149.0, 146.2, 142.9, 142.0, 134.6, 128.7, 127.4, 124.7, 123.7, 123.4, 121.2, 120.2, 119.9,
111.0, 109.6, 38.5, 15.8; HRMS (ESI): m/z calcd for C18H15N4O3 335.1144, found 335.1142
[M+H]+; Purity: 98.2%.
4.1.1.18
(E)-3-((1,5-Dimethyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one
(1:1
inseparable mixture of C-5 and C-6 tautomers of benzimidazole, 8r). Orange solid, Yield 71%;
1
mp: 217–219 °C; FT–IR: (cm-1): 3175, 3071, 1705, 1610, 1333, 781; H NMR (500 MHz,
DMSO–d6): δ 10.69 (brs, 2H, NH), 9.52–9.48 (m, 2H, Ar–H), 7.71 (d, J = 8.4 Hz, 1H, Ar–H),
7.63 (s, 1H, C=CH), 7.56–7.53 (m, 3H, Ar–H), 7.46 (s, 1H, C=CH), 7.53–7.29 (m, 2H, Ar–H),
7.21 (d, J = 8.4 Hz, 1H, Ar–H), 7.15 (d, J = 8.3 Hz, 1H, Ar–H), 7.06 (t, J = 7.6 Hz, 2H, Ar–H),
6.89 (d, J = 7.6 Hz, 1H, Ar–H), 3.99 (s, 3H, CH3), 3.97 (s, 3H, CH3), 2.49 (s, 3H, CH3), 2.46 (s,
13
3H, CH3); C NMR (125 MHz, DMSO–d6): δ 168.9, 168.8, 147.5, 147.2, 143.5, 143.4, 143.2,
141.2, 135.9, 133.9, 133.8, 132.2, 131.2, 131.1, 130.2, 130.0, 128.1, 128.0, 125.8, 124.7, 121.3,
121.2, 121.1, 121.0, 119.3, 119.2, 117.9, 117.8, 110.3, 110.2, 109.5, 109.4, 30.2, 30.1, 21.5,
21.1; HRMS (ESI): m/z calcd for C18H16N3O 290.1293, found 290.1279 [M+H]+; Purity: 98.4%.
4.1.1.19
(E)-3-((1-Ethyl-5-methyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one
(8:2
inseparable mixture of C-5 and C-6 tautomers of benzimidazole, 8s). Data for major tautomer:
Yield 74%; mp: 239–241 °C; FT–IR: (cm-1): 3143, 3066, 1698, 1605, 1332, 807, 713; 1H NMR
(500 MHz, DMSO–d6): δ 10.71 (brs, 1H, NH), 9.49 (d, J = 7.6 Hz, 1H, Ar–H), 7.72 (d, J = 8.2
17

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Hz, 1H, Ar–H), 7.52 (s, 1H, C=CH), 7.49 (s, 1H, Ar–H), 7.33 (t, J = 7.5 Hz, 1H, Ar–H), 7.15 (d,
J = 8.4 Hz, 1H, Ar–H), 7.06 (t, J = 7.3 Hz, 1H, Ar–H), 6.90 (d, J = 7.6 Hz, 1H, Ar–H), 4.51–
13
4.46 (m, 2H, CH2), 2.48, (s, 3H, CH3), 1.37 (t, J = 7.2 Hz, 3H, CH3); C NMR (125 MHz,
DMSO–d6): δ 168.9, 146.2, 143.5, 141.3, 134.8, 134.4, 131.2, 130.3, 128.0, 124.8, 121.3, 121.1,
119.5, 117.5, 110.2, 109.5, 38.3, 21.5 15.6; HRMS (ESI): m/z calcd for C19H18N3O 304.1450,
found 304.1441 [M+H]+; Purity: 98.8%.
4.1.1.20 (E)-3-((1-Ethyl-5-methyl-1H-benzo[d]imidazol-2-yl)methylene)-5-fluoroindolin-2-one
(7:3 inseparable mixture of C-5 and C-6 tautomers of benzimidazole, 8t). Data for major
tautomer: Yield 76%; mp: 270–272 °C; FT–IR: (cm-1): 3153, 3072, 1704, 1616, 1323, 811, 793;
1H NMR (500 MHz, DMSO–d6): δ 10.62 (brs, 1H, NH), 9.45–9.41 (m, 1H, Ar–H), 7.60 (s, 1H,
C=CH), 7.50 (s, 1H, Ar–H), 7.48 (d, J = 8.4 Hz, 1H, Ar–H), 7.17 (d, J = 8.1 Hz, 1H, Ar–H),
7.10–7.13 (m, 1H, Ar–H), 6.85–6.80 (m, 1H, Ar–H), 4.51–4.44 (m, 2H, CH2), 2.46 (s, 3H, CH3),
1.39 (t, J = 7.4 Hz, 3H, CH3); 13C NMR (125 MHz, CDCl3+DMSO–d6): δ 168.8, 157.2 (d, JCF
=
234.4 Hz), 146.1, 143.2, 139.7, 132.7, 132.3, 130.1 (d, JCF = 2.7 Hz), 125.9, 122.1 (d, JCF = 10.8
Hz), 119.3, 118.3, 117.0 (d, JCF = 23.6 Hz), 114.9 (d, JCF = 27.4 Hz) 109.9, 109.8 (d, JCF = 8.2
Hz), 38.4, 21.0, 15.6; HRMS (ESI): m/z calcd for C19H17FN3O 322.1356, found 322.1349
[M+H]+; Purity: 98.8%.
4.1.1.21 (E)-5-Chloro-3-((1,5-dimethyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one (1:1
inseparable mixture of C-5 and C-6 tautomers of benzimidazole, 8u). Yellow solid, Yield 75%;
1
mp: 141–143 °C; FT–IR: (cm-1): 3157, 3024, 1702, 1607, 1217, 798, 776; H NMR (500 MHz,
DMSO–d6): δ 10.82 (brs, 2H, NH), 9.69–9.66 (m, 2H, Ar–H), 7.67 (d, J = 8.2 Hz, 1H, Ar–H),
7.60–7.54 (m, 4H, Ar–H), 7.46 (s, 1H, C=CH), 7.36 (d, J = 8.2 Hz, 2H, Ar–H), 7.22 (d, J = 8.4
Hz, 1H, Ar–H), 7.16 (d, J = 8.3 Hz, 1H, Ar–H), 6.89 (d, J = 7.6 Hz, 2H, Ar–H), 4.00 (s, 3H,
CH3), 3.98 (s, 3H, CH3), 2.48 (s, 3H, CH3), 2.46 (s, 3H, CH3); 13C NMR (125 MHz, DMSO–d6):
δ 168.6, 168.5, 147.2, 146.9, 143.1, 142.3, 142.2, 142.1, 141.1, 135.8, 134.3, 133.9, 132.5,
130.5, 130.4, 129.0, 128.8, 127.6, 127.5, 126.2, 125.2, 125.0, 122.7, 119.4, 119.3, 119.2, 119.1,
119.0, 110.8, 110.7, 110.5, 110.4, 30.3, 30.1, 21.5, 21.1; HRMS (ESI): m/z calcd for
C18H15ClN3O 324.0904, found 324.0898 [M+H]+; Purity: 98.3%.
4.1.1.22 (E)-5-Chloro-3-((1-ethyl-5-methyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one
(8:2 inseparable mixture of C-5 and C-6 tautomers of benzimidazole, 8v). Data for major
tautomer (ethanol extra peaks): Yield 80%; mp: 261–263 °C; FT–IR: (cm-1): 3160, 2979, 1698,
1
1605, 1324, 801, 788; H NMR (500 MHz, DMSO–d6): δ 10.84 (brs, 1H, NH), 9.67 (d, J = 2.1
18

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Hz, 1H, Ar–H), 7.68 (d, J = 8.2 Hz, 1H, Ar–H), 7.56 (s, 1H, C=CH), 7.50 (s, 1H, C=CH), 7.37
(dd, J = 2.1, 8.2 Hz, 1H, Ar–H), 7.17 (d, J = 8.4 Hz, 1H, Ar–H), 6.90 (d, J = 8.2 Hz, 1H, Ar–H),
4.53–4.46 (m, 2H, CH2), 2.48, (s, 3H, CH3), 1.37 (t, J = 7.3 Hz, 3H, CH3); 13C NMR (125 MHz,
DMSO–d6): δ 168.6, 146.0, 142.2, 141.2, 134.8, 134.4, 130.5, 129.1, 127.5, 125.3, 125.1, 122.6,
119.5, 119.0, 110.8, 110.3, 38.4, 21.5 15.7; HRMS (ESI): m/z calcd for C19H17ClN3O 338.1060,
found 338.1046 [M+H]+; Purity: 98.4%.
4.1.1.23 (E)-5-Bromo-3-((1,5-dimethyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one (1:1
inseparable mixture of C-5 and C-6 tautomers of benzimidazole, 8w). Yellow solid, Yield 71%;
1
mp: 293–295 °C; FT–IR: (cm-1): 3168, 3072, 1692, 1608, 1304, 1218, 799, 777; H NMR (500
MHz, DMSO–d6): δ 10.83 (brs, 2H, NH), 9.83–9.79 (m, 2H, Ar–H), 7.64 (d, J = 8.2 Hz, 1H,
Ar–H), 7.60–7.53 (m, 4H, Ar–H), 7.51–7.44 (m, 3H, Ar–H), 7.22 (d, J = 8.2 Hz, 2H, Ar–H),
7.17 (d, J = 8.3 Hz, 1H, Ar–H), 6.85 (d, J = 7.6 Hz, 2H, Ar–H), 3.99 (s, 3H, CH3), 3.98 (s, 3H,
13
CH3), 2.48 (s, 3H, CH3), 2.46 (s, 3H, CH3); C NMR (125 MHz, DMSO–d6): δ 168.5, 168.4,
147.2, 146.9, 143.1, 142.6, 142.5, 142.4, 141.0, 135.9, 134.3, 133.9, 133.3, 133.2, 132.6, 130.4,
130.3, 128.9, 128.7, 126.2, 126.1, 125.1, 123.2, 123.1, 119.4, 119.3, 119.1, 113.0, 11.4, 111.3,
110.5, 110.4, 30.3, 30.2, 21.6, 21.1; HRMS (ESI): m/z calcd for C18H15BrN3O 368.0398, found
368.405 [M+H]+; Purity: 98.1%.
4.1.1.24 (E)-5-Bromo-3-((1-ethyl-5-methyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-one
(7:3 inseparable mixture of C-5 and C-6 tautomers of benzimidazole, 8x). Data for major
tautomer: Yield 70%; mp: 264–266 °C; FT–IR: (cm-1): 3160, 2980, 1703, 1605, 1322, 801,
792;1H NMR (500 MHz, DMSO–d6): δ 10.85 (brs, 1H, NH), 9.81 (d, J = 2.0 Hz, 1H, Ar–H),
7.65 (d, J = 8.4 Hz, 1H, Ar–H), 7.55 (s, 1H, Ar–H), 7.49 (s, 1H, C=CH), 7.16 (d, J = 8.2 Hz, 1H,
Ar–H), 6.85 (d, J = 8.2 Hz, 1H, Ar–H), 4.53–4.46 (m, 2H, CH2), 2.48, (s, 3H, CH3), 1.36 (t, J =
7.3 Hz, 3H, CH3); 13C NMR (125 MHz, DMSO–d6): δ 168.5, 145.9, 142.5, 141.2, 134.8, 134.4,
133.3, 130.3, 129.0, 125.1, 123.1, 119.4, 118.9, 113.0, 111.3, 110.3, 38.3, 21.5 15.7; HRMS
(ESI): m/z calcd for C19H17BrN3O 382.0555, found 382.0548 [M+H]+; Purity: 98.0%.
4.1.1.25 (E)-3-((1,5-Dimethyl-1H-benzo[d]imidazol-2-yl)methylene)-5-methylindolin-2-one (1:1
inseparable mixture of C-5 and C-6 tautomers of benzimidazole, 8y) Yellow solid, Yield 73%;
1
mp: 260–262 °C; FT–IR: (cm-1): 3171, 3051, 2963, 1705, 1607, 1329, 781, 763; H NMR (300
MHz, DMSO–d6): δ 10.02 (brs, 2H, NH), 9.24 (s, 2H, Ar–H), 7.75 (d, J = 8.2 Hz, 1H, Ar–H),
7.65 (s, 1H, C=CH), 7.68–7.40 (m, 3H, Ar–H), 7.32 (d, J = 8.3 Hz, 2H, Ar–H), 7.24–7.14 (m,
3H, Ar–H), 7.08 (d, J = 7.7 Hz, 2H, Ar–H), 6.78 (d, J = 7.7 Hz, 2H, Ar–H), 3.97 (s, 3H, CH3),
19

ACCEPTED MANUSCRIPT
3.96 (s, 3H, CH3), 2.54 (s, 3H, CH3), 2.53 (s, 3H, CH3), 2.39 (s, 3H, CH3), 2.38 (s, 3H, CH3); 13C
NMR (125 MHz, DMSO–d6): δ 169.0, 168.9, 147.3, 147.0, 142.9, 141.0, 140.9, 135.6, 135.5,
133.7, 133.6, 132.0, 131.9, 131.3, 131.2, 131.1, 130.3, 130.1, 129.7, 128.4, 128.3, 125.6, 124.5,
121.0, 119.1, 119.0, 117.4, 117.3, 110.0, 109.0, 30.0, 29.9, 21.3, 20.9, 20.8, 20.7; HRMS (ESI):
m/z calcd for C19H18N3O 304.1450, found 304.1435 [M+H]+; Purity: 98.0%.
4.1.1.26 (E)-3-((1-Ethyl-5-methyl-1H-benzo[d]imidazol-2-yl)methylene)-5-methylindolin-2-one
(8:2 inseparable mixture of C-5 and C-6 tautomers of benzimidazole, 8z). Data for major
tautomer: Yield 75%; mp: > 300 °C; FT–IR: (cm-1): 3177, 2982, 1704, 1609, 1321, 1229, 824,
1
729; H NMR (500 MHz, DMSO–d6): δ 10.60 (brs, 1H, NH), 9.33 (d, J = 2.0 Hz, 1H, Ar–H),
7.71 (d, J = 7.9 Hz, 1H, Ar–H), 7.48–7.43 (m, 2H, Ar–H), 7.17–7.08 (m, 2H, Ar–H), 6.77 (d, J =
7.3 Hz, 1H, Ar–H), 4.55–4.51 (m, 2H, CH2), 2.49, (s, 3H, CH3), 2.33 (s, 3H, CH3), 1.35 (t, J =
7.4 Hz, 3H, CH3); 13C NMR (125 MHz, DMSO–d6): δ 168.9, 146.3, 141.3, 141.2, 134.8, 134.0,
131.5, 130.6, 129.9, 128.5, 124.7, 121.2, 119.5, 117.2, 110.2, 109.2, 38.3, 21.5, 21.0, 15.6;
HRMS (ESI): m/z calcd for C20H20N3O 318.1606, found 318.1608 [M+H]+; Purity: 98.5%.
4.2 Pharmacology
4.2.1 Cell Cultures
Prostate (PC-3 and DU-145), Breast (BT549, MDA-MB-231, MCF-7 and 4T1), lung cancer
(A549) and gastric cancer (HGC) cancer cells were obtained from American Type Cell Culture
Collection (ATCC), Maryland, USA and were cultured in appropriate DMEM (Dulbecco
modified Eagle medium, Sigma) or MEM (Minimum Essential Medium, Sigma) supplemented
with 10% fetal bovine serum with 1X stabilized antibiotic-antimycotic solution (Sigma) in a
CO2 incubator at 37 C with 5% CO2 and 90% relative humidity.
4.2.2 MTT Assay
The cytotoxic activity of the compounds 8a–z was determined using MTT assay. 1 × 104 cells
per well were seeded in 100 µL DMEM, supplemented with 10% FBS in each well of 96-well
microculture plates and incubated for 48 h at 37 °C in a CO2 incubator. Compounds 8a–z,
diluted to the desired concentrations in culture medium, were added to the wells with respective
vehicle control. After 48 h of incubation, 10 µL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) (5 mg/mL) was added to each well and the plates were further incubated at
37 °C for 4 h. The supernatant from each well was carefully removed, formazan crystals were
dissolved in 100 µL of DMSO and absorbance was recorded at 570 nm wavelength on a
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spectrophotometer (SpectraMax, Molecular devices). The compounds which exhibited >50%
inhibition of cell viability at 50 µM in preliminary screening; were further selected to generate
drug response curve (DRC). IC50 values were determined from DRC plot by linear regression
method: % cell inhibition (from control OD) versus different concentrations (µM). All the values
were expressed as Mean ± SEM of three independent experiments in which each treatment was
done in triplicate wells.
4.2.3 Acridine orange–ethidium bromide (AO–EB) staining
MDA-MB-231 cells were plated at a concentration of 1 × 106 cell/ml and treated with different
concentration of compound 8l. Plates were incubated in an atmosphere of 5% CO2 at 37 °C for
48 h. 10 µL of fluorescent dyes containing Acridine Orange (AO) and Ethidium Bromide (EB)
added into each well in equal volumes (10 µg/mL) respectively and within 10 minutes the cells
were visualized under fluorescence microscope (Nikon, Inc. Japan) with excitation (488 nm) and
emission (550 nm) at 200× magnification.
4.2.4 DAPI nuclear staining
MDA-MB-231 cells (1 × 106 cells/well) were grown in 6-well plates and treated with or without
compound 8l at concentrations ranging from 1.0 µM, 2.0 µM and 4.0 µM for 24 h. The treated
and untreated cells were washed twice with PBS, fixed with 4% paraformaldehyde, and stained
with 10 µg/mL of DAPI and the cells were observed for apoptotic characteristics like nuclear
fragmentation and chromatin condensation under fluorescence microscope (Nikon, Inc. Japan)
with excitation at 359 nm and emission at 461 nm using DAPI filter at 200× magnification.
4.2.5 In vitro cell migration assay/Wound healing assay
MDA-MB-231 cells (5 × 105 cells/well) were cultured in 6 well plates for 24 h. The confluent
monolayers were then scratched with 200 µL pipette tip. The wounded monolayers were washed
twice with PBS to remove non-adherent cells. Then, media containing the different
concentrations (1, 2, and 4 µM) of the compound 8l were added to each well. Cells which
migrated across the scratched wound were photographed under the phase contrast microscope
(Nikon) at 0 and 24 h time interval after treatment.
4.2.6 F-actin staining
MDA-MB-231 cells (1 × 106 cells/well) were grown on coverslips in 6 well plates for 24 h and
then incubated with 1, 2, and 4 µM concentrations of compound 8l for 12 h. After the compound
21

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treatment, cells were washed with PBS and fixed with 4% para-formaldehyde in PBS. Cells were
incubated with rhodamine phalloidin (red fluorescent dye) for actin staining and Hoechst 33242
for nucleus staining. After washing thrice with PBS, cells were mounted with ProLong Gold
anti-fade reagent (Molecular Probes, Eugene, OR) on microscopic slide and were observed by
confocal microscopy (Nikon). Images were captured using 20× objective lenses.
4.2.7 Cell cycle analysis
MDA-MB-231 cells (1 × 106 cells /well) in 6 well plate were treated with different 1, 2, and 4
µM concentrations of the compound 8l for 24 h. Cells were collected by trypsininsation, washed
with 150 mM PBS and were fixed with 70% ethanol for 30 min at 4 ºC. After fixing, cells were
washed with PBS and stained with 400 µL of Propidium Iodide staining buffer [PI (200 µg),
Triton X (100 µL), DNAse-free RNAse A (2 mg) in 10 mL PBS] for 15 min at room temp in
dark. The samples were then analyzed for propidium iodide fluorescence from 15,000 events by
flow cytometry using BD Accuri C6 flow-cytometer.
4.2.8 MitoSOX™ Red assay
Mitochondrial superoxide (O2*-) production were determined by using MitoSOX™ Red. MDA-
MB-231 cells were seeded at a density of 1x105 cells/ per well in cell culture medium in a 24-
well plate and allowed to adhere overnight. Cells were treated with different concentrations of
8l, the medium was removed after 24 h and washed with PBS. A solution of MitoSOX™ Red
mitochondrial superoxide indicator in HBSS (Hank's balanced salt solution) was added and
incubated at 37 ºC for 15 min. Cells were then washed three times with PBS to remove the
excess dye. Images were captured using the fluorescent microscope.
4.2.9 Measurement of mitochondrial membrane potential (DΨm)
In this assay, MDA-MB-231 cells were cultured in 6 well plates at a density of 5 × 105 cells/mL
and allowed to adhere overnight. The cells were treated with the 1, 2, and 4 µM concentrations
of the compound 8l for 24 h. After 24 h of 8l treatment, the adherent cells were collected by
trypsinsation, washed with PBS and resuspended in solution of PBS containing rhodamine-123
(10 µg/mL) and further incubated at room temperature for 30 minutes. Cells were washed two
times with PBS to remove excess dye and resuspended in PBS. The samples were analyzed for
rhodamine-123 fluorescence using spectrofluorometer with an excitation and emission
wavelengths of 480 and 530 nm.
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4.2.10 Annexin V-FITC/Propidium iodide dual staining assay
The Annexin V-FITC/propidium iodide dual staining assay was carried out using MDA-MB-231
cells, to quantify the percentage of apoptotic cells. MDA-MB-231 cells (1 × 106/mL per well)
were plated in six-well culture plates and allowed to grow for 24 h. After treatment with
increasing concentrations of compound 8l (1, 2 and 4 ꢀM) for 24 h, cells were collected by
trypsinisation. The collected cells were washed two times with ice-cold PBS, then incubated
with 200 ꢀL1 × binding buffer containing 5 ꢀL Annexin V-FITC, and then in 300 ꢀL1 × binding
buffer containing 5 ꢀL Propidium iodide (PI) for 5 min at room temperature in the dark. After 15
minutes of incubation, cells were analysed for apoptosis using BD-c6 accuri flow-cytometer.
4.2.11 Western blotting analysis
For western blotting, MDA-MB-231 cells were treated with compound 8l for 24 h. Whole cell
extracts were prepared using RIPA (Sigma-Aldrich, St. Louis, MO, USA). Western blotting
analysis was performed using anti-Caspase-3 (rabbit, 1:1000, Santa Cruz, CA), anti-Bcl2 (rabbit,
1:1000, Santa Cruz, CA), anti-Bax (rabbit, 1:500, Santa Cruz, CA), anti-β-actin (rabbit, 1:1000,
Santa Cruz, CA) and HRP-conjugated secondary antibodies (Santa Cruz, CA). The antigen-
antibody complex was visualized with an ECL detection kit (Amersham Bioscience). For
subsequent antibody treatment, membranes were stripped in stripping buffer and reprobed with
another antibody [28]. The immune blots were quantified by densitometry scanning with NIH
ImageJ software.
Acknowledgements
P.S. and K.R.S. are thankful to DoP, Ministry of Chemicals & Fertilizers, Govt. of India, for the
award of NIPER fellowship. NS gratefully acknowledges SERB, DST, Govt. of India for
research grant (YSS-2015-001709).
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Table, Figures captions, Figures and Scheme
Table 1. In-vitro cytotoxic activity (IC50 in µM)a of benzo[d]imidazol-2-yl)methylene)indolin-2-
ones 8a–z.
Figure 1. Representative examples of 3-alkenyl-indolin-2-ones and benzimidazoles as
anticancer agents.
Figure 2. Molecular hybridization approach for the design of small molecules as anticancer
agents.
Figure 3. Determination of compound 8l configuration by NOE analysis.
Figure 4. Effect of compound 8l on in vitro migration potential of MDA-MB-231 breast cancer
cells. Scratches were created with sterile 200 µL pipette and images were captured using phase
contrast microscopy at 0 h and 24 h after treatment with 1 µM, 2 µM and 4 µM of compound 8l.
Figure 5. Effects of compound 8l on the structures F-actin in MDA-MB-231 cells. The MDA-
MB-231 cells were treated with 1 µM, 2 µM and 4 µM of compound 8l. Compound treatment
led to disrupted stress fibre network. F-actin was stained with Rhodamine-phalloidin (red
fluorescent dye). The blue colour indicates Hoechst 33242 staining for nucleus.
Figure 6. Cell cycle analysis of MDA-MB-231 cancer cells treated with compound 8l (1 µM, 2
µM and 4 µM) and sunitinib (4 µM) for 24 h. (A) The cell cycle distribution was performed by
using propidium iodide staining method and analysed by flow cytometry. (B) Data from 10,000
cells was collected for each data file. The percentage of cells in G2/M, S and G0/G1 phase were
quantified by means of Flowjo software.
Figure 7. Morphological changes observed in MDA-MB-231 cells by the treatment of
compound 8l for 24 h.
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Figure 8. AO–EB staining of compound 8l in MDA-MB-231 cells. (a) DMSO treatment
(control); (b, c and d) treatment with 1 µM, 2 µM and 4 µM respectively for 24 h.
Figure 9. Nuclear morphology in MDA-MB-231 cells stained with DAPI. MDA-MB-231 cells
treated with compound 8l for 24 h were stained with DAPI. The images were captured with
fluorescence microscope with a DAPI filter.
Figure 10. Effect of compound 8l on mitochondrial membrane potential. (A) MDA-MB-231
cells were treated with compound 8l with different concentrations [(a) Control, (b) 1 µM, (c) 2
µM, (d) 4 µM and (e) overlay of a-d] and stained with rhodamine 123. The Peak changes to the
left indicate depolarisation. (B) The intensity of rhodamine 123 fluorescence was determined by
flow cytometer. Data are mean ± SD from three independent experiments, each with triplicates.
Figure 11. Effect of compound 8l on mitochondrial superoxide production in MDA-MB-231
cancer cells. Fluorescent microscopic images of MDA-MB-231 cells stained with MitoSOX™
Red indicator after 48 h treatment with different concentrations of compound 8l.
Figure 12. MDA-MB-231 cells were treated with compound 8l at 1, 2 and 4 µM concentration
for 24 h and then total protein extracts were analysed for Bcl2, Bax, caspase-3 and PARP. (A)
Western blotting analysis of Bcl2, Bax, PARP and caspase-3. (B) Bar graph representing the
fold changes in the protein expression compared to control. Data expressed as mean ± SEM
(n=3). **P<0.01 and ***P < 0.001 significant vs. control.
Figure 13. Annexin V- FITC/propidium iodide dual staining assay. MDA-MB-231cells were
treated with compound 8l and stained with Annexin V-FITC/PI and analysed for apoptosis using
flow cytometer. The 10,000 cells from each sample were analysed by flow cytometry. The
percentage of cells positive for Annexin V-FITC and/or Propidium iodide is reported inside the
quadrants. Cells in the lower left quadrant (Q2-LL: AV-/PI-): live cells; lower right quadrant
(Q2-LR: AV+/PI-): early apoptotic cells; upper right quadrant (Q2-UR: AV+/PI +): late
apoptotic cells and upper left quadrant (Q2-UL: AV-/PI+): necrotic cells.
Scheme 1. Synthesis of (E)-1-alkyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-ones 8a–z.
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