Synthesis and biological evaluation of new benzimidazole-thiazolidinedione hybrids as potential cytotoxic and apoptosis inducing agents

Article abstract of DOI:10.1016/j.ejmech.2016.08.029

A series of new benzimidazole-thiazolidinedione hybrids has been synthesized and evaluated for their cytotoxic potential against a selected human cancer cell lines of prostate (PC-3 and DU-145), breast (MDA-MB-231), lung (A549) and a normal breast epithelial cells (MCF10A). Among the tested compounds, 11p exhibited promising cytotoxicity with IC₅₀value of 11.46?±?1.46?μM on A549 lung cancer cell line and did not show significant toxicity on normal MCF10A cells. Lung cancer cells (A549) have been used to know the mechanism of cell growth inhibition and apoptosis inducing effect with compound 11p. The treatment of A549?cells with 11p showed typical apoptotic morphology like cell shrinkage, chromatin condensation and horseshoe shaped nuclei formation. Flow-cytometry analysis revealed the G2/M phase of cell cycle arrest in a dose dependent manner. Preliminary mechanistic studies suggested that the cell migration was inhibited through the disruption of F-actin protein. Acridine orange-ethidium bromide (AO-EB), DAPI, annexin V-FITC/propidium iodide, rhodamine-123 and MitoSOX assays suggested the induction of apoptosis in A549 cells by compound 11p.

Full text of DOI:10.1016/j.ejmech.2016.08.029

Accepted Manuscript
Synthesis and biological evaluation of new benzimidazole-thiazolidinedione hybrids
as potential cytotoxic and apoptosis inducing agents
Pankaj Sharma, T. Srinivasa Reddy, Dinesh Thummuri, Kishna Ram Senwar, Niggula
Praveen Kumar, V.G.M. Naidu, Suresh K. Bharghava, Nagula Shankaraiah
PII:
S0223-5234(16)30672-9
10.1016/j.ejmech.2016.08.029
EJMECH 8825
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 June 2016
Revised Date: 1 August 2016
Accepted Date: 14 August 2016
Please cite this article as: P. Sharma, T. Srinivasa Reddy, D. Thummuri, K.R. Senwar, N. Praveen
Kumar, V.G.M. Naidu, S.K. Bharghava, N. Shankaraiah, Synthesis and biological evaluation of new
benzimidazole-thiazolidinedione hybrids as potential cytotoxic and apoptosis inducing agents, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.08.029.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis and biological evaluation of new benzimidazole-thiazolidinedione hybrids as
potential cytotoxic and apoptosis inducing agent
a
b
c
a
Pankaj Sharma, T. Srinivasa Reddy, Dinesh Thummuri, Kishna Ram Senwar, Niggula
a
c
b
a
Praveen Kumar, V. G. M. Naidu, Suresh K. Bharghava, Nagula Shankaraiah *
a
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
b
Centre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science, RMIT
University, GPO BOX 2476, Melbourne-3001, Australia
c
Department of Pharmacology and Toxicology, National Institute of Pharmaceutical
Education and Research (NIPER), Hyderabad 500 037, India

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of new benzimidazole-thiazolidinedione hybrids as
potential cytotoxic and apoptosis inducing agents
a
b
c
a
Pankaj Sharma, T. Srinivasa Reddy, Dinesh Thummuri, Kishna Ram Senwar, Niggula
a
c
b
a
Praveen Kumar, V. G. M. Naidu, Suresh K. Bharghava, Nagula Shankaraiah *
a
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
b
Centre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science, RMIT
University, GPO BOX 2476, Melbourne-3001, Australia
c
Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education
and Research (NIPER), Hyderabad 500 037, India
Abstract:- A series of new benzimidazole-thiazolidinedione hybrids has been synthesized and
evaluated for their cytotoxic potential against a selected human cancer cell lines of prostate (PC-
3
and DU-145), breast (MDA-MB-231), lung (A549) and a normal breast epithelial cells
(
MCF10A). Among the tested compounds, 11p exhibited promising cytotoxicity with IC value
5
0
of 11.46 ± 1.46 µM on A549 lung cancer cell line and did not show significant toxicity on
normal MCF10A cells. Lung cancer cells (A549) have been used to know the mechanism of cell
growth inhibition and apoptosis inducing effect with compound 11p. The treatment of A549
cells with 11p showed typical apoptotic morphology like cell shrinkage, chromatin condensation
and horseshoe shaped nuclei formation. Flow-cytometry analysis revealed the G2/M phase of
cell cycle arrest in a dose dependent manner. Preliminary mechanistic studies suggested that the
cell migration was inhibited through the disruption of F-actin protein. Acridine orange-ethidium
bromide (AO-EB), DAPI, annexin V-FITC/propidium iodide, rhodamine-123 and MitoSOX
assays suggested the induction of apoptosis in A549 cells by compound 11p.
Keywords: Benzimidazole, thiazolidinedione, apoptosis, MitoSOX, annexin, cell migration.
∗
Corresponding author. Tel.: (+91) 040 23073740; Fax: (+91) 040 23073751; E-mail:
shankar@niperhyd.ac.in; shankarnbs@gmail.com
1

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1.0. Introduction
Cancer is one the leading causes of death throughout the world. Despite availability of several
chemotherapeutic agents, no effective treatments have been presented that can deal with the
sequelae of cancer and still the disease remains tenacious and deadly [1]. Apoptosis is a critical
cellular process which plays a central role in the maintenance of tissue growth and homeostasis
[
2]. The inappropriate regulation of apoptotic pathway has been indicated in many diseases
including cancer [3]. Therefore, the design and synthesis of novel chemotherapeutic agents that
can induce apoptosis has emerged as an attractive approach in cancer drug discovery [4].
In the search for potential anticancer agents, significant efforts have been focused on the
development of scaffolds containing heterocyclic structures as their key structural design. Such
heterocyclics, thiazolidinedione is a privileged fragment in modern medicinal chemistry
considering its broad spectrum and affinity towards various biological targets [5]. The drugs
containing thiazolidinedione as a core skeleton act as hypoglycaemic agents through insulin
sensitization (pioglitazone and its analogues) [6]. Apart from their hypoglycaemic activity, the
molecules based on this scaffold have emerged as a broad spectrum of activities such as
antimicrobial [7], anti-inflammatory agents [8] etc. The research on thiazolidinediones has
recently gained huge interest and proven promising for cancer treatment [9]. These molecules act
as the agonists of peroxisome proliferator-activated receptor-γ (PPAR-γ), which is expressed in
many human tumours, including lung, breast, colon, prostate and bladder [10] and they modulate
the apoptosis and proliferation of several cancer cell lines. GSK1059615 is a thiazolidinedione
derivative known to exert its anticancer effects through the inhibition of PI3K-α [11]. Liu et al.
[
12] and Fletcher et al. [13] reported a thiazolidinedione with potential anticancer activity
through the blockade of the Raf/MEK/ERK and PI3K/Akt signaling pathways. Very recently,
Lesyk et al. reported 5-ene-4-thiazolidinones as potential antileukemic agents [14].
<Insert Figure 1 here>
On the other hand, benzimidazole scaffold (Figure 1) can also be considered as "Master Key"
due to its wide applications in medicinal chemistry [15] and has been luring researchers all over
the world to explore their therapeutic potential [16]. The structural resemblance of
benzimidazole with naturally occurring moieties like purines, makes them to interact with
biomolecules of living system. Furthermore, the incorporation of medicinally active moieties
like morpholine, quinoline, piperidine, thiazole onto a core of bioactive scaffold provides the
means for accessing wider range of pharmacological profiles. The significance of these
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fundamental building blocks is well understood by modern medicinal chemists, because they
play a key role in molecular properties such as the three dimensionality, electronic distribution,
scaffold rigidity, lipophilicity, polarity, metabolic stability, molecular reactivity and toxicity
[
17].
The amalgamation of two dissimilar bioactive pharmacophores made into a single molecule is
successful and frequently used approach in modern medicinal chemistry for the exploration of
novel and highly active compounds which may have synergistic effect on biological properties
[
18]. Therefore, owing to our interest in the synthesis of biologically active scaffolds [19], herein
we have designed and synthesized novel benzimidazole-thiazolidinedione derivatives by aiming
at the identification of new chemical entities (NCEs) as potent cytotoxic agents. We have
attached the structural features of benzimidazole to the head part and various fundamental
building blocks like morpholine, isoxazole, piperidine, thiazole and tetrahydroisoquinoline to the
tail part of thiazolidinedione (Figure 2). We hypothesized that the newly synthesized molecules
could enhance the cytotoxic activity that might be through induction of apoptosis and cell cycle
arrest. In this context, herein we present the synthesis of new benzimidazole-thiazolidinedione
hybrids and tested them on selected human cancer cell lines and also compared their toxicity
profile with a normal cell line. One of the compounds that displayed promising cytotoxic activity
against A549 cancer cells was further evaluated for mechanistic studies accountable for cell
death.
<Insert Figure 2 here>
2.0. Chemistry
The protocol for the synthesis of benzimidazole-thiazolidinedione hybrids 11a–t was carried out
in a convergent approach by employing Knoevenagel condensation reaction between N-
substituted thiazolidinedione 6a–g and 1-alkyl-1H-benzo[d]imidazole-2-carbaldehydes 10a–c as
outlined in Scheme 1. This reaction permitted to accomplish the products in a ‘greener’
condition by reacting an activated methylene group with appropriate aldehydes. The
thiazolidinedione (TZD) ring 2 was synthesized by reacting chloroacetic acid (1) with thiourea
under reflux in conc. HCl [20]. TZD 2 was converted to its potassium salt 3 using potassium
hydroxide. Later, the chloroacetylated key intermediates 5a–g were synthesized from precursors
4a–g by reacting with appropriate heterocyclic primary and secondary amines with chloroacetyl
chloride and achieved in almost quantitative yields. Next, potassium salt of TZD 3 was reacted
with different chloroacetylated amine intermediates 5a–g in DMF at 60 °C to obtain N-
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substituted thiazolidinediones 6a–g in good yields. N-Alkylation of the thiazolidinedione with a
range of heterocyclic amines introduced the basic functionalities and scope of diversity.
Alternatively, 1-alkyl-1H-benzo[d]imidazole-2-carbaldehydes 10a–c were prepared according to
the earlier reports [21]. o-Phenylenediamine 7 was reacted with glycolic acid under the reflux
conditions in HCl (4N) to obtain 1H-benzo[d]imidazole-2-yl)methanol (8). The obtained
intermediate 8 was converted to 1-alkyl-1H-benzo[d]imidazole-2-yl)methanols 9a–c by the
reaction with alkyl iodide in the presence of potassium carbonate. Further, 1-alkyl-1H-
benzo[d]imidazole-2-carbaldehydes 10a–c were obtained from the oxidation of primary alcohols
9a–c by using Dess-Martin periodinane reagent. Finally, the designed compounds 11a–t were
accomplished by reacting N-substituted thiazolidinediones 6a–g and 1-alkyl-1H-
benzo[d]imidazole-2-carbaldehydes 10a–c under Knoevenagel condensation using piperidine as
1
13
base. All the synthesized compounds 11a–t were confirmed by using FT-IR, H NMR, C NMR
and HRMS (ESI) spectroscopic techniques. The products 11a could be easily deduced since
there was a clear loss of aldehydic -CH proton at 10.13 ppm and showed a sharp signal of newly
generated methine -CH double bond at 7.99 ppm by condensation reaction. The close
13
examination of C NMR of compound 11a revealed three carbonyl groups at 169.6, 164.8 and
162.6 ppm. All the other aromatic and aliphatic carbon of 11a was found in the range of 147.3–
1
13
1
11.0 ppm and 45.7–23.6 ppm respectively. The H NMR and C NMR spectra of all the
synthesized compounds 11b–t were found almost in a similar pattern like 11a. The HRMS (ESI)
+
of 11a–t showed the characteristic [M+H] corresponding peaks to their molecular formula.
<Insert Scheme 1 here>
3
3
.0. Pharmacology
.1. In vitro cytotoxic activity
The newly synthesized benzimidazole-thiazolidinedione hybrids 11a–t were screened for their
cytotoxicity against selected human cancer cell lines viz. prostate (PC-3 and DU-145), breast
(MDA-MB-231), lung (A549) and one normal breast epithelial cell (MCF10A) using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [22]. The concentrations
required to cause 50% inhibition of cancer cell growth are expressed as IC values. The IC
50
5
0
(µM) values of test compounds 11a–t and reference standard 5-FU are listed in the Table 1. It is
evident from the initial results that some of the synthesized compounds possessed moderate to
potent growth inhibition against the tested cancer cells and showed selective potency against
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A549 cell line. From the close examination of IC (µM), it can be inferred that most of the
50
compounds were inactive against PC-3 cancer cells. The IC values for compounds 11b, 11f,
5
0
11g, 11j, 11l, 11p and 11r were found to be in the range of 29.74 ± 2.08 to 45.11 ± 2.51 µM on
DU-145 cancer cells. Furthermore, the compounds 11j, 11l, 11p, and 11r showed cytotoxic
activity in the range between 29.18 ± 0.93 µM to 36.03 ± 1.53 µM on MDA-MB-231 cells,
however rest of the compounds were found to be inactive (IC >50 µM). Compounds 11j, 11l,
5
0
1
1p, and 11r were found selectively cytotoxic against A549 cancer cells with IC values range
50
from 11.46 ± 1.46 µM to 20.63 ± 1 .72 µM. The compound 11p was found to be highest
cytotoxic on A549 cancer cell line with IC of 11.46 ± 1.46 µM. From the IC values, it was
5
0
50
noted in general that the N-substitution on the tail position of thiazolidinedione with oxoethyl
linked morpholine, piperidine, pyrrolidine and 2,6-dimethylmorpholine did not produce the
bioactive compounds. However, most of the compounds containing 6,7-dimethoxy-
tetrahydroisoquinoline at the tail side of thiazolidinedione were found to be active (below 50
µM). On the other hand, substitution on head position of TZD with 1-methyl-benzimidazole
produced more active compounds compared to ethyl and isopropyl groups.
From the cytotoxicity data in Table 1, most active compounds 11j, 11l, 11p and 11r displaying
IC50 ≤ 20 µM were tested on non-tumorigenic breast epithelial cell line (MCF10A) to find out
the selectivity toward cancer cells. Interestingly, compound 11j, 11l and 11p were found to be
selective towards cancer cells since they did not exhibit cytotoxicity (IC50 > 100 µM) on
MCF10A cells. However, compound 11r showed almost 2.5 times more selectivity for A549
cancer cells in comparison to normal MCF10A cells. The promising cytotoxic activity of
compound 11p on A549 cancer cells encouraged us to study its effects at cellular level.
<Insert Table 1 here>
3.2. In vitro cell migration assay/Wound healing assay
Migration of cells is a key attribute of practically each biological process, particularly cancer
cells where it is known to play a major role in tumor progression and metastatic cascade [23a].
As cell migration and motility are connected with the metastatic activity of cancer cells,
therefore we have investigated the effect of compound 11p on A549 cells using wound healing
assay [23b]. This assay is based on the observation that, upon making of a new artificial wound
on a confluent cell monolayer, the cells on the edge of the wound will move toward the opening
to close the wound until new cell–cell interactions are established. The treatment with anticancer
agent restricts the migration of cells to fill up the wound. Wounds were created on a confluent
5

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cell monolayer culture of A549 cells by using a sterile 200 µL pipette tip and treated with 5 µM,
10 µM and 15 µM of compound 11p. The migration of A549 cells was recorded by microscopic
observations at 0 h, 24 h and 48 h. Results from the Figure 3a clearly showed that there was
almost complete healing of wound in control after 48 h, however healing was strongly
suppressed in the cells treated with 11p. Quantification of cell in Figure 3b showed 77.8% (5
µM), 74.3% (10 µM) and 60.4% (15 µM) of migrated A549 cells in relative comparison to
control after 24 h. The effect was more prominent with 15 µM, wherein only 29.6% of cells have
migrated after 48 h.
<Insert Figure 3 here>
3.3. Effect on F-actin polymerisation
Previous reports suggest that migration of cells is an integrated sum of multi-step processes
triggered by the formation of membrane protrusions, remodelling of cytoskeleton, regulation of
microtubules and actin [24a]. As an important part of cytoskeleton, F-actin is reported to be a
potential target in cancer drug discovery. The polymerisation of F-actin filaments is the driving
force for cell migration as well as motility. As evidenced by the Figure 3 that compound 11p
inhibited the cell migration of A549 lung cancer cells, henceforth it was considered of interest to
examine its effect on actin polymerisation. The formation of the actin cytoskeleton in A549 cells
was studied by staining the cells with rhodamine-phalloidin [24b], a red fluorescent stain that
specifically binds to F-actin protein. Results from the Figure 4 demonstrated that 5 µM, 10 µM
and 15 µM of compound 11p disrupted the actin fibre assembly and led to the decrease in F-
actin extensions. Thus, it is likely that the loss of F-actin extensions at the periphery of the cell
membrane resulted in inhibition of migration ability of the A549 cells.
<Insert Figure 4 here>
3.4. Cell cycle analysis
Most of the chemotherapeutic compounds exert their cytotoxic effect either by induction of
apoptosis or by arresting cell cycle at a specific checkpoint [25]. The inappropriate regulation of
cell cycle by anticancer agent has been considered to be an effective strategy in the development
of new cancer therapeutics. Therefore, it was our interest to examine the effect of compound 11p
on cell cycle progression of A549 cells by using flow cytometry analysis method [22]. The A549
cells were treated with 5 µM, 10 µM and 15 µM of compound 11p for 24 h and stained with
propidium iodide. Results from the Figure 5 clearly demonstrated the noteworthy increase in the
6

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ratio of cells in G2/M phase from 23.3% (control) to 41.4% (5 µM), 49.3% (10 µM) and 57.8%
(15 µM) respectively. Concomitantly, there was a decrease in the ratio of cells in G0/G1 phase
in a dose dependent manner. These results clearly indicated that the treatment of 11p led to
G2/M phase of cell cycle arrest.
<Insert Figure 5 here>
3.5. Morphological observations using phase contrast microscopy
The induction of apoptosis by chemotherapeutic agents has always been an ideal choice in
developing new anticancer therapeutics. Henceforth, to examine the effect on apoptosis, A549
cells were treated with one of the most active compounds 11p and were observed under phase
contrast microscopy. It can be inferred from the Figure 6 that the different concentrations i.e. 5
µM, 10 µM and 15 µM of compound 11p treated A549 cells showed characteristic apoptotic
morphological features like cell shrinkage, cell wall deformation and reduced number of viable
cells, whereas these distinctive morphological feature were absent in the control cells.
<Insert Figure 6 here>
3.6 Acridine orange-ethidium bromide (AO-EB) staining
The morphological changes caused by the most potent compound 11p in A549 cells were further
studied by using acridine orange-ethidium bromide (AO-EB) staining to find out whether the
inhibition is due to apoptosis or nonspecific necrosis [26]. AO-EB staining method distinguishes
the live cells from dead cells, because AO permeates the cells with intact membrane and stain
the nuclei green, however ethidium bromide is taken-up by the cells only when there is a loss of
membrane integrity, and stains the nucleus orange. The compound 11p untreated and treated
A549 cells were stained with AO-EB and analysed under fluorescence microscope. It can be
seen from Figure 7 that the control A549 cells have a normal morphology, whereas the early
signs of apoptosis were characterized by condensed chromatin and cell membrane blebbing at
concentration of 5 µM of compound 11p. Apoptotic body formation, irregular distribution of
chromatin and destructive fragmentation of nuclei which marginated into horse-shoe shaped
structure was observed at 10 µM and 15 µM respectively, whereas these distinctive
morphological features were absent in the control A549 lung cancer cells.
<Insert Figure 7 here>
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3.7. DAPI staining
4
′,6-Diamidino-2-phenylindole (DAPI) is a nuclear stain which strongly binds to adenine-
thymine clusters of the minor groove of double-stranded DNA and detect the chromatin
condensation or nuclear damage. Binding of DAPI to DNA produces a ~20-fold enhanced
fluorescence, apparently due to the displacement of water molecules from both DAPI and the
minor groove [27]. DAPI stains the apoptotic cells as bright coloured due to the condensed
nucleus which is a typical apoptotic feature. Henceforth, it was considered of interest to detect
chromatin condensation and nuclear damage induced by the compound 11p in A549 cells. DAPI
staining was performed according to the known literature procedure [26]. The results from the
Figure 8 clearly demonstrate that the nuclear structure of untreated cells was intact, however
compound 11p treated A549 cells exhibited condensed, pyknotic, fragmented and horse-shoe
shaped nuclei.
<Insert Figure 8 here>
3.8. Effect on mitochondrial membrane potential (DΨm)
Previous reports suggest that the damage of mitochondrial membrane integrity and loss or
collapse of mitochondrial membrane potential are early events the process of apoptotic induction
[
23b, 24b]. Therefore, we tested this possibility by examining the effect of compound 11p on
DΨm of A549 cancer cell line by using rhodamine-123 staining [28], a cationic dye that emits
green fluorescence. Mitochondrion with normal DΨm retain rhodamine, however the disruption
of DΨm result in loss of rhodamine from the mitochondria and a decrease in intracellular green
fluorescence. The dissipation in DΨm can be observed by the shift in the green fluorescence
intensity by using flow-cytometer. Result from the Figure 9 showed the peak shift from control
(Figure 9A-a) to left (Figure 9A-b to d and e is overlay of a-d) which indicates depolarisation
by the loss of DΨm. The relative loss of DΨm was found to be 24.6% at 5 µM compared to
control which further dropped to 30.6% at 10 µM and 45.8% at 15 µM respectively (Figure 9B).
These results collectively suggest the induction of apoptosis in A549 cells through collapse of
DΨm.
<Insert Figure 9 here>
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3.9 Measurement of Superoxide Production (Mitosox red assay)
The production of high levels of ROS has been implicated in apoptotic induction by triggering
oxidative damage to the mitochondrial membrane potential and permeability [29a]. Henceforth,
we were intrigued by the fact that apoptosis induction in A549 cells could be due to the
production of ROS, henceforth we tested this possibility using MitoSOX red [29b], a chemical
probe which selectively detects superoxide ROS in the mitochondria of live cells. Oxidation of
MitoSOX by superoxide ROS produces red fluorescence which can be visualized by
fluorescence microscopy. A549 cells were treated with different concentrations of compound
11p and stained with MitoSOX red. Results from Figure 10 indicate the dose dependent
increase in the red fluorescence when compared to control. It can be seen in Figure 10 that
DMSO-treated control A549 cells exhibited weak and diffuse MitoSOX red fluorescence,
however cells treated with 5 µM, 10 µM and 15 µM of compound 11p were brightly stained
signifying superoxide generation.
<Insert Figure 10 here>
3.10. Annexin V-FITC/Propidium iodide staining assay
To quantify the percentage of cells undergoing apoptosis by compound 11p, Annexin V-
FITC/Propidium iodide staining assay was performed according to known procedures [19b].
This assay facilitate the detection of live cells (Q1-LL; AV-/PI-), early apoptotic cells (Q1-LR;
AV+/PI-), late apoptotic cells (Q1-UR; AV+/PI+) and necrotic cells (Q1-UL; AV-/PI+). A549
cells were treated with 5 µM, 10 µM and 15 µM of compound 11p and stained with Annexin V-
FITC/propidium iodide. Results from the Figure 11 showed that the percentage of total
apoptotic cells (early and late apoptotic cells) increased from 3.4% (control) to 19.0% (5 µM),
23.7% (10 µM) and 31.3% (15 µM) respectively. Therefore, the percentage increase in early and
late apoptotic cells from 3.4% to 31.3% indicate that the compound 11p induced apoptosis in
A549 cancer cells in dose dependent manner.
<Insert Figure 11 here>
4.0. Conclusion
In conclusion, a series of new benzimidazole bearing thiazolidinedione hybrids has been
synthesized and evaluated for their in vitro cytotoxic activity against selected human cancer cell
lines viz. PC-3, DU-145, MDA-MB-231, A549 and one normal cell line (MCF10A). Some of the
9

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compounds like 11j, 11p and 11r exhibited potent cytotoxic activity with IC ≤ 15 µM against
5
0
A549 cell line and were non-toxic to normal breast epithelial cells (MCF10A). The exposure of
compound 11p to A549 cells resulted in G2/M phase of cell cycle arrest, inhibition of in vitro
cell migration and disruption of F-actin filaments in a dose dependent manner. The apoptosis
inducing effect of 11p was further analysed by acridine orange/ethidium bromide (AO-EB)
staining, DAPI staining and annexin binding assay. Moreover, the compound 11p could induce
apoptosis through the collapse of mitochondrial membrane potential (DΨm) and increased levels
of superoxide ROS. Overall, the ease in synthetic preparation and significant cytotoxic
properties makes these compounds a promising starting point for the development of potent
cancer chemotherapeutic agents in the drug discovery process.
5.0. Materials & Methods
All the chemicals, reagents and starting materials were procured from commercial available
providers and were used as such. The monitoring of reactions were performed by thin layer
chromatography (TLC-MERCK pre-coated silica gel 60-F254 aluminium plates) under UV
1
light. Melting points were checked using Stuart® SMP30 apparatus and are uncorrected. H and
1
3
C NMR were taken on Bruker Avance 500 MHz spectrometer using tetramethylsilane (TMS)
as the internal standard and chemical shifts are reported in ppm. Chemical shifts are referenced
1
13
1
13
to TMS (δ 0.00 for H NMR and C NMR), DMSO˗d (δ 2.50 for H NMR and 39.5 for C
6
1
13
NMR) or CDCl (δ 7.26 for H NMR and C NMR). Spin multiplicities are reported as s
3
(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.
5.1. General reaction procedure for the synthesis of (Z)-5-((1-alkyl-1H-benzo[d]imidazol-2-
yl)methylene)-3-(2-oxoethyl)thiazolidine-2,4-dione derivatives 11a–t.
To a mixture of thiazolidinedione (6a, 0.5 mmol), 1-methyl-1H-benzo[d]imidazole-2-
carbaldehydes (10a, 0.55 mmol) in ethanol (4 mL) was added catalytic amount of piperidine.
The reaction mixture was refluxed until complete conversion as observed by TLC. After cooling,
the precipitates were filtered, washed and recrystalized with ethanol, to obtain pure (Z)-5-((1-
methyl-1H-benzo[d]imidazol-2-yl)methylene)-3-(2-oxo-2-(pyrrolidin-1-yl)ethyl)thiazolidine-
2,4-dione derivative 11a as yellow solids. All the other compounds 11b–t were obtained in a
similar reaction procedure of 11a in moderate to high yields.
1
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5.1.1.
(Z)-5-((1-Methyl-1H-benzo[d]imidazol-2-yl)methylene)-3-(2-oxo-2-(pyrrolidin-1-
-1
yl)ethyl)thiazolidine-2,4-dione (11a): Yellow solid, Yield 82%; mp: 264–266 °C; FT–IR: (cm ):
1
2
7
1
2
1
985, 2964, 1740, 1679, 1659, 1385, 1220, 750; H NMR (500 MHz, DMSO–d ): δ 7.99 (s, 1H),
6
.78 (d, J = 8.1 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.39 (t, J = 7.9 Hz, 1H), 7.32 (t, J = 7.5 Hz,
H), 4.50 (s, 2H), 4.02 (s, 3H), 3.55 (t, J = 6.6 Hz, 2H), 3.31 (t, J = 7.3 Hz, 2H), 1.97–1.89 (m,
1
3
H), 1.84–1.77 (m, 2H); C NMR (125 MHz, DMSO–d ): δ 169.6, 164.8, 162.6, 147.3, 142.6,
6
35.8, 128.1, 124.2, 123.1, 119.6, 116.7, 111.0, 45.7, 45.0, 42.7, 29.8, 25.8, 23.6; HRMS (ESI):
+
m/z calcd for [M+H] C H N O S 371.1178; found 371.1171.
1
8
19
4
3
5.1.2.
(Z)-5-((1-Ethyl-1H-benzo[d]imidazol-2-yl)methylene)-3-(2-oxo-2-(pyrrolidin-1-
-1
yl)ethyl)thiazolidine-2,4-dione (11b): Yellow solid, Yield 81%; mp: 229–231 °C; FT–IR: (cm ):
1
2
7
1
1
1
4
3
998, 2970, 1744, 1667, 1654, 1376, 1225, 754; H NMR (500 MHz, DMSO–d ): δ 7.99 (s, 1H),
6
.78 (d, J = 8.1 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.39 (t, J = 7.9 Hz, 1H), 7.32 (t, J = 7.2 Hz,
H), 4.60–4.54 (m, 2H), 4.50 (s, 2H), 3.55 (t, J = 6.7 Hz, 2H), 3.31 (t, J = 7.3 Hz, 2H), 1.97–
13
.89 (m, 2H), 1.84–1.77 (m, 2H), 1.35 (t, J = 7.3 Hz, 3H); C NMR (125 MHz, CDCl ): δ
3
70.4, 165.8, 162.7, 146.5, 143.7, 134.8, 129.9, 124.5, 123.3, 120.9, 115.5, 109.8, 46.2, 45.7,
+
2.7, 38.6, 26.1, 24.0, 15.8; HRMS (ESI): m/z calcd for [M+H] C H N O S 385.1334; found
19
21
4
3
85.1332.
5.1.3.
(Z)-5-((1-Isopropyl-1H-benzo[d]imidazol-2-yl)methylene)-3-(2-oxo-2-(pyrrolidin-1-
-1
yl)ethyl)thiazolidine-2,4-dione (11c): Yellow solid, Yield 76%; mp: 259–261 °C; FT–IR: (cm ):
1
2
7
981, 2930, 1735, 1670, 1662, 1383, 1208, 751; H NMR (500 MHz, DMSO–d ): δ 8.06 (s, 1H),
6
.87–7.84 (m, 1H), 7.80–7.77 (m, 1H), 7.36–7.29 (m, 2H), 5.29–5.19 (m, 1H), 4.50 (s, 2H), 3.54
(t, J = 6.7 Hz, 2H), 3.31 (t, J = 7.9 Hz, 2H), 1.97–1.89 (m, 2H), 1.84–1.76 (m, 2H), 1.63 (d, J =
1
3
6
1
.9 Hz, 6H); C NMR (125 MHz, CDCl ): δ 170.7, 165.9, 162.8, 146.4, 144.1, 133.8, 130.0,
3
24.1, 123.0, 121.1, 116.4, 111.9, 48.2, 46.2, 45.7, 42.7, 26.1, 24.0, 21.9; HRMS (ESI): m/z
+
calcd for [M+H] C H N O S 399.1491; found 399.1482.
2
0
23
4
3
5.1.4.
(Z)-5-((1-Methyl-1H-benzo[d]imidazol-2-yl)methylene)-3-(2-oxo-2-(piperidin-1-
-1
yl)ethyl)thiazolidine-2,4-dione (11d): Yellow solid, Yield 74%; mp: 255–257 °C; FT–IR: (cm ):
1
3
002, 1739, 1661, 1612, 1383, 1226, 1103, 753; H NMR (300 MHz, CDCl +DMSO–d ): δ 7.89
3
6
(s, 1H), 7.82 (d, J = 7.4 Hz, 1H), 7.48–7.27 (m, 3H), 4.55 (s, 2H), 3.97 (s, 3H), 3.58–3.40 (m,
1
3
4
1
H), 1.74–1.39 (m, 6H); C NMR (125 MHz, DMSO–d ): δ 169.6, 164.9, 162.6, 147.3, 142.6,
6
35.8, 128.1, 124.2, 123.1, 119.6, 116.7, 111.0, 45.0, 42.5, 42.2, 29.8, 25.8, 25.1, 23.7; HRMS
+
(
ESI): m/z calcd for [M+H] C H N O S 385.1334; found 385.1332.
1
9
21
4
3
1
1

ACCEPTED MANUSCRIPT
5.1.5.
(Z)-5-((1-Ethyl-1H-benzo[d]imidazol-2-yl)methylene)-3-(2-oxo-2-(piperidin-1-
-1
yl)ethyl)thiazolidine-2,4-dione (11e): Yellow solid, Yield 72%; mp: 228–230 °C; FT–IR: (cm ):
1
2
1
1
1
2
980, 1749, 1680, 1620, 1383, 1230, 750; H NMR (300 MHz, CDCl ): δ 7.87 (d, J = 7.9 Hz,
3
H), 7.80 (s, 1H), 7.46–7.29 (m, 3H), 4.55 (s, 2H), 4.40–4.43 (m, 2H), 3.60–3.39 (m, 4H), 1.75–
1
3
.52 (m, 6H), 1.48 (t, J = 7.2 Hz, 3H); C NMR (125 MHz, CDCl ): δ 170.5, 166.0, 162.6,
3
46.6, 143.7, 134.9, 130.3, 124.7, 123.6, 121.0, 115.5, 109.9, 46.0, 43.6, 42.3, 38.8, 26.3, 25.4,
+
4.4, 15.9; HRMS (ESI): m/z calcd for [M+H] C H N O S 399.1491; found 399.1489.
20
23
4
3
5.1.6.
(Z)-5-((1-Isopropyl-1H-benzo[d]imidazol-2-yl)methylene)-3-(2-oxo-2-(piperidin-1-
-1
yl)ethyl)thiazolidine-2,4-dione (11f): Yellow solid, Yield 79%; mp: 219–221 °C; FT–IR: (cm ):
1
2
983, 2939, 1745, 1680, 1659, 1380, 1225, 754; H NMR (300 MHz, CDCl +DMSO–d ): δ 7.92
3
6
(s, 1H), 7.85–7.80 (m, 1H), 7.63–7.57 (m, 1H), 7.37–7.27 (m, 2H), 5.05–4.93 (m, 1H), 4.56 (s,
1
3
2
1
2
H), 3.60–3.43 (m, 4H), 1.76–1.51 (m, 12H); C NMR (125 MHz, DMSO–d ): δ 169.9, 165.0,
6
62.6, 146.3, 143.2, 133.5, 128.8, 124.0, 122.9, 120.1, 116.7, 112.9, 47.6, 45.0, 42.5, 42.1, 25.8,
+
5.0, 23.7, 21.3; HRMS (ESI): m/z calcd for [M+H] C H N O S 413.1647; found 413.1642.
2
1
25
4
3
5.1.7.
(Z)-5-((1-Methyl-1H-benzo[d]imidazol-2-yl)methylene)-3-(2-morpholino-2-
-1
oxoethyl)thiazolidine-2,4-dione (11g): Yellow solid, Yield 85%; mp: >300 °C; FT–IR: (cm ):
1
3
045, 1733, 1668, 1617, 1389, 1230, 1110, 1034, 751; H NMR (500 MHz, DMSO–d ): δ 8.00
6
(s, 1H), 7.78 (d, J = 7.9 Hz, 1H), 7.69 (d, J = 7.9 Hz, 1H), 7.39 (t, J = 7.3 Hz, 1H), 7.32 (t, J =
7
2
1
.8 Hz, 1H), 4.64 (s, 2H) 4.02 (s, 3H), 3.68–3.62 (m, 2H), 3.61–3.52 (m, 4H), 3.47–3.41 (m,
1
3
H); C NMR (125 MHz, DMSO–d ): δ 169.6, 164.9, 163.3, 147.3, 142.6, 135.8, 128.0, 124.2,
6
+
23.1, 119.6, 116.8, 111.0, 65.8, 44.5, 42.1, 41.9, 29.8; HRMS (ESI): m/z calcd for [M+H]
C H N O S 387.1127; found 387.1120.
18
19
4
4
5.1.8.
(Z)-5-((1-Ethyl-1H-benzo[d]imidazol-2-yl)methylene)-3-(2-morpholino-2-
-
oxoethyl)thiazolidine-2,4-dione (11h): Yellow solid, Yield 89%; mp: 288–290 °C; FT–IR: (cm
1
1
)
: 3047, 1739, 1669, 1614, 1382, 1236, 1113, 1019, 754; H NMR (500 MHz, DMSO–d ): δ
6
7.99 (s, 1H), 7.78 (d, J = 7.9 Hz, 1H), 7.72 (d, J = 7.9 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.32 (t, J
=
7.3 Hz, 1H), 4.63 (s, 2H) 4.63–3.52 (m, 2H), 3.69–3.62 (m, 2H), 3.61–3.53 (m, 4H), 3.48–3.42
1
3
(
m, 2H), 1.35 (t, J = 7.0 Hz, 3H); C NMR (125 MHz, DMSO–d ): δ 169.6, 164.9, 163.3, 146.4,
6
142.8, 134.7, 128.5, 124.3, 123.2, 119.8, 116.3, 111.0, 65.8, 44.5, 42.1, 41.9, 38.0, 15.6; HRMS
+
(
ESI): m/z calcd for [M+H] C H N O S 401.1283; found 401.1278.
1
9
21
4
4
5.1.9.
(Z)-5-((1-Isopropyl-1H-benzo[d]imidazol-2-yl)methylene)-3-(2-morpholino-2-
-
oxoethyl)thiazolidine-2,4-dione (11i): Yellow solid, Yield 82%; mp: 270–272 °C; FT–IR: (cm
1
2

ACCEPTED MANUSCRIPT
1
1
)
: 3046, 1745, 1662, 1624, 1381, 1235, 1129, 1019, 751; H NMR (300 MHz, CDCl +DMSO–
3
d6): δ 7.93 (s, 1H), 7.86–7.80 (m, 1H), 7.63–7.57 (m, 1H), 7.38–7.27 (m, 2H), 5.06–4.92 (m,
13
1
H), 4.58 (s, 2H), 3.81–3.51 (m, 8H), 1.72 (d, J = 7.0 Hz, 6H); C NMR (125 MHz, DMSO–
d6): δ 169.9, 165.0, 163.4, 146.3, 143.2, 133.5, 128.8, 124.1, 123.0, 120.1, 116.8, 112.9, 65.9,
+
4
7.6, 44.5, 42.1, 41.9, 21.3; HRMS (ESI): m/z calcd for [M+H] C H N O S 415.1440; found
20
23
4
4
4
15.1440.
.1.10. (Z)-3-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-2-oxoethyl)-5-((1-methyl-1H-
5
benzo[d]imidazol-2-yl)methylene)thiazolidine-2,4-dione (11j): Yellow solid, Yield 84%; mp:
-1
1
2
70–272 °C; FT–IR: (cm ): 3031, 2944, 1741, 1683, 1650, 1376, 1217, 1119, 1021, 750; H
NMR (500 MHz, CDCl ): δ 7.89 (d, J = 7.9 Hz, 1H), 7.85 (s, 1H), 7.43–7.32 (m, 3H), 6.68–6.57
3
(m, 2H), 4.67–4.61 (m, 4H), 3.93 (s, 3H), 3.90–3.80 (m, 7H), 3.73 (t, J = 6.4 Hz 1H), 2.93 (t, J =
1
3
5
1
1
4
4
.5 Hz, 1H) 2.80 (t, J = 5.5 Hz, 1H); C NMR (125 MHz, DMSO–d ): δ 169.7, 164.9, 164.4,
6
64.3, 147.5, 147.4, 147.3, 147.2, 147.1, 142.6, 135.8, 128.1, 128.0, 126.2, 125.8, 124.6, 124.2,
24.1, 123.1, 119.6, 116.8, 116.7, 111.8, 111.7, 111.0, 109.9, 109.8, 55.4, 45.0, 43.6, 42.5, 42.3,
+
2.0, 29.8, 27.9, 27.1; HRMS (ESI): m/z calcd for [M+H] C H N O S 493.1546; found
25
25
4
5
93.1542.
.1.11. (Z)-3-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-2-oxoethyl)-5-((1-ethyl-1H-
5
benzo[d]imidazol-2-yl)methylene)thiazolidine-2,4-dione (11k): Yellow solid, Yield 87%; mp:
-1
1
2
69–271 °C; FT–IR: (cm ): 3023, 2945, 1745, 1682, 1653, 1383, 1215, 1123, 1019, 751; H
NMR (500 MHz, CDCl ): δ 7.88 (d, J = 7.9 Hz, 1H), 7.82 (s, 1H), 7.43–7.32 (m, 3H), 6.66–6.60
3
(m, 2H), 4.69–4.60 (m, 4H), 4.41–4.34 (m, 2H), 3.89–3.81 (m, 7H), 3.73 (t, J = 5.8 Hz, 1H),
1
3
2
.93 (t, J = 5.8 Hz, 1H), 2.80 (t, J = 5.8 Hz, 1H), 1.47 (t, J = 7.3 Hz, 3H); C NMR (125 MHz,
CDCl ): δ 170.6, 166.0, 163.4, 148.4, 148.3, 148.1, 148.0, 147.9, 146.6, 143.9, 134.0, 130.0,
3
1
5
26.9, 125.6, 124.8, 124.7, 123.6, 123.0, 121.0, 115.8, 111.8, 111.3, 109.9, 109.5, 109.0, 56.2,
6.1, 56.0, 46.2, 44.5, 42.8, 42.6, 42.4, 40.5, 38.7, 28.9, 28.0, 16.0; HRMS (ESI): m/z calcd for
+
[
M+H] C H N O S 507.1702; found 507.1701.
26
27
4
5
5
1
2
.1.12. (Z)-3-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-2-oxoethyl)-5-((1-isopropyl-
H-benzo[d]imidazol-2-yl)methylene)thiazolidine-2,4-dione (11l): Yellow solid, Yield 88%; mp:
-1
1
42–244 °C; FT–IR: (cm ): 3047, 1745, 1680, 1652, 1375, 1220, 1128, 1023, 750; H NMR
(
500 MHz, CDCl ): δ 7.91 (s, 1H), 7.88–7.83 (m, 1H), 7.60–7.53 (m, 1H), 7.34–7.28 (m, 2H),
3
6.66–6.57 (m, 2H), 5.00–4.91 (m, 1H), 4.68–4.60 (m, 4H), 3.92–3.79 (m, 7H), 3.72 (t, J = 6.3
1
3
Hz, 1H), 2.93 (t, J = 5.8 Hz, 1H), 2.79 (t, J = 5.7 Hz, 1H), 1.70 (d, J = 6.9 Hz, 6H); C NMR
1
3

ACCEPTED MANUSCRIPT
(
125 MHz, CDCl ): δ 170.8, 166.1, 163.4, 148.3, 148.2, 148.1, 148.0, 147.9, 146.5, 144.2, 134.0,
3
130.0, 126.9, 125.5, 124.8, 124.3, 123.2, 123.0, 121.2, 116.6, 112.1, 112.7, 111.3, 109.5, 108.9,
56.2, 56.1, 56.0, 48.3, 46.2, 44.5, 42.8, 42.5, 42.3, 40.5, 28.9, 28.0, 22.1; HRMS (ESI): m/z calcd
+
for [M+H] C H N O S 521.1859; found 521.1860.
2
7
29
4
5
5.1.13.
(Z)-3-(2-(2,6-Dimethylmorpholino)-2-oxoethyl)-5-((1-methyl-1H-benzo[d]imidazol-2-
yl)methylene)thiazolidine-2,4-dione (11m): Yellow solid, Yield 90%; mp: 293–295 °C; FT–IR:
-1
1
(
cm ): 2977, 1740, 1669, 1624, 1384, 1234, 1082, 749; H NMR (500 MHz, DMSO–d ): δ 7.98
6
(s, 1H), 7.77 (d, J = 8.1 Hz, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.32 (t, J =
7.3 Hz, 1H), 4.78 (d, J = 16.8 Hz, 1H), 4.49 (d, J = 16.4 Hz, 1H), 4.14 (d, J = 12.8 Hz, 1H), 4.01
(s, 3H), 3.87 (d, J = 13.0 Hz, 1H), 3.63–3.41 (m, 2H), 2.80 (t, J = 11.0 Hz 1H), 2.33 (t, J = 11.3
1
3
δ
Hz, 1H), 1.15–1.06 (m, 6H); C NMR (125 MHz, DMSO–d ): 169.6, 164.9, 163.0, 147.3,
6
1
1
42.6, 135.8, 128.0, 124.2, 123.1, 119.6, 116.8, 111.0, 71.3, 71.0, 49.3, 46.8, 42.2, 29.8, 18.5,
+
8.3; HRMS (ESI): m/z calcd for [M+H] C H N O S 415.1440; found 415.1435.
20
23
4
4
5.1.14.
(Z)-3-(2-(2,6-Dimethylmorpholino)-2-oxoethyl)-5-((1-ethyl-1H-benzo[d]imidazol-2-
yl)methylene)thiazolidine-2,4-dione (11n): Yellow solid, Yield 89%; mp: 279–281 °C; FT–IR:
-1
1
(
cm ): 2978, 1742, 1671, 1620, 1383, 1246, 1080, 751; H NMR (500 MHz, DMSO–d ): δ 7.99
6
(s, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.38 (t, J = 7.9 Hz, 1H), 7.32 (t, J =
7.6 Hz, 1H,), 4.78 (d, J = 16.8 Hz, 1H), 4.60–4.53 (m, 2H), 4.49 (d, J = 16.6 Hz, 1H), 4.14 (d, J
=
12.8 Hz, 1H), 3.87 (d, J = 13.0 Hz, 1H), 3.62–3.41 (m, 2H), 2.80 (t, J = 11.0 Hz, 1H), 2.34 (t,
1
3
J = 11.1 Hz, 1H), 1.34 (t, J = 7.2 Hz, 3H) 1.16–1.07 (m, 6H); C NMR (125 MHz, DMSO–d ):
6
δ 169.7, 164.9, 163.0, 146.4, 142.8, 134.7, 128.5, 124.3, 123.2, 119.8, 116.3, 111.0, 71.3, 71.0,
+
4
9.3, 46.8, 42.2, 38.0, 18.5, 18.3, 15.7; HRMS (ESI): m/z calcd for [M+H] C H N O S
2
1
25
4
4
4
29.1597; found 429.1607.
.1.15. (Z)-3-(2-(2,6-Dimethylmorpholino)-2-oxoethyl)-5-((1-isopropyl-1H-benzo[d]imidazol-2-
5
yl)methylene)thiazolidine-2,4-dione (11o): Yellow solid, Yield 85%; mp: 259–261 °C; FT–IR:
-1
1
(
cm ): 2983, 1739, 1661, 1615, 1384, 1235, 1080, 754; H NMR (500 MHz, DMSO–d ): δ 8.06
6
(s, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.78 (d, J = 7.6 Hz, 1H), 7.37–7.27 (m, 2H), 5.28–5.19 (m,
1
1
7
1
1
H), 4.78 (d, J = 16.8 Hz, 1H), 4.49 (d, J = 16.6 Hz, 1H), 4.14 (d, J = 12.8 Hz, 1H), 3.87 (d, J =
2.8 Hz, 1H), 3.63–3.41 (m, 2H), 2.80 (t, J = 11.3 Hz, 1H), 2.33 (t, J = 11.7 Hz,1H), 1.62 (d, J =
1
3
.0 Hz, 6H) 1.16–1.06 (m, 6H); C NMR (125 MHz, DMSO–d ): δ 169.9, 165.0, 163.1, 146.3,
6
43.2, 133.5, 128.7, 124.0, 122.9, 120.1, 116.8, 112.9, 71.3, 71.0, 49.3, 47.6, 46.8, 42.1, 21.3,
+
8.5, 18.3; HRMS (ESI): m/z calcd for [M+H] C H N O S 443.1753; found 443.1760.
22
27
4
4
1
4

ACCEPTED MANUSCRIPT
5.1.16. (Z)-2-(5-((1-Methyl-1H-benzo[d]imidazol-2-yl)methylene)-2,4-dioxothiazolidin-3-yl)-N-
-
(4-phenylthiazol-2-yl)acetamide (11p): Yellow solid, Yield 62%; mp: 280–282 °C; FT–IR: (cm
1
1
)
: 3302, 3028, 1734, 1677, 1542, 1411, 1383, 1003, 740; H NMR (500 MHz, DMSO–d ): δ
6
1
2
1
1
4
2.8 (brs, 1H), 8.04 (s, 1H), 7.91 (d, J = 7.5 Hz, 2H), 7.79 (d, J = 8.1 Hz, 1H), 7.71–7.67 (m,
1
3
H), 7.47–7.29 (m, 5H), 4.68 (s, 2H), 4.02 (s, 3H); C NMR (125 MHz, DMSO–d ): δ 169.7,
6
64.8, 164.7, 157.2, 148.9, 147.3, 142.6, 135.8, 134.0, 128.7, 128.0, 127.8, 125.6, 124.2, 123.2,
+
19.6, 117.0, 111.1, 108.5, 43.0, 29.8; HRMS (ESI): m/z calcd for [M+H] C H N O S
2
3
18
5
3
2
76.0851; found 476.0844.
5.1.17. (Z)-2-(5-((1-Ethyl-1H-benzo[d]imidazol-2-yl)methylene)-2,4-dioxothiazolidin-3-yl)-N-
-
(4-phenylthiazol-2-yl)acetamide (11q): Yellow solid, Yield 55%; mp: 249–251 °C; FT–IR: (cm
1
1
)
: 3314, 3024, 1742, 1670, 1535, 1390, 1383, 1015, 745; H NMR (500 MHz, DMSO–d ): δ
6
12.8 (brs, 1H), 8.04 (s, 1H), 7.91 (d, J = 7.5 Hz, 2H), 7.80 (d, J = 8.1 Hz, 1H), 7.73 (d, J = 8.1
Hz, 1H), 7.68 (s, 1H), 7.47–7.30 (m, 5H), 4.68 (s, 2H), 6.62–4.55 (m, 2H), 1.35 (t, J = 7.3 Hz,
3
1
H); 13C NMR (125 MHz, DMSO–d ): δ 169.7, 164.8, 164.7, 157.3, 148.9, 146.4, 142.8, 134.7,
6
34.0, 128.7, 128.5, 127.8, 125.6, 124.3, 123.2, 119.8, 116.5, 111.0, 108.5, 43.0, 38.0, 15.7;
+
HRMS (ESI): m/z calcd for [M+H] C H N O S 490.1008; found 490.1000.
24
20
5
3 2
5.1.18.
(Z)-N-(5-(tert-Butyl)isoxazol-3-yl)-2-(5-((1-methyl-1H-benzo[d]imidazol-2-
yl)methylene)-2,4-dioxothiazolidin-3-yl)acetamide (11r): Yellow solid, Yield 81%; mp: 259–261
-1
1
°
C; FT–IR: (cm ): 3222, 3066, 1748, 1688, 1578, 1406, 1217, 961, 751; H NMR (500 MHz,
DMSO–d ): δ 11.5 (brs, 1H), 8.01 (s, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H),
6
7
9
1
.39 (t, J = 7.6 Hz, 1H), 7.32 (t, J = 7.3 Hz, 1H), 6.56 (s, 1H), 4.55 (s, 2H), 4.02 (s, 3H), 1.28 (s,
1
3
H); C NMR (125 MHz, DMSO–d ): δ 180.7, 169.6, 164.8, 164.5, 157.3, 147.3, 142.6, 135.8,
6
28.0, 124.2, 123.2, 119.6, 116.9, 111.0, 93.0, 43.3, 32.4, 29.8, 28.2; HRMS (ESI): m/z calcd for
+
[
M+H] C H N O S 440.1392; found 440.1389.
21
22
5
4
5
2
.1.19. (Z)-N-(5-(tert-Butyl)isoxazol-3-yl)-2-(5-((1-ethyl-1H-benzo[d]imidazol-2-yl)methylene)-
,4-dioxothiazolidin-3-yl)acetamide (11s): Yellow solid, Yield 78%; mp: 254–257 °C; FT–IR:
-1
1
(
cm ): 3240, 3028, 1744, 1667, 1570, 1406, 1235, 981, 757; H NMR (500 MHz, DMSO–d ): δ
6
1
2
1.5 (brs, 1H), 8.02 (s, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.41–7.30 (m,
1
3
H), 6.56 (s, 1H), 4.62–4.53 (m, 4H), 1.34 (t, J = 7.3 Hz, 3H), 1.28 (s, 9H); C NMR (125
MHz, DMSO–d ): δ 180.7, 169.6, 164.8, 164.5, 157.3, 146.4, 142.8, 134.7, 128.5, 124.0, 123.2,
6
+
119.8, 116.4, 111.0, 93.0, 43.3, 38.0, 32.4, 28.2, 15.7; HRMS (ESI): m/z calcd for [M+H]
C H N O S 454.1549; found 454.1535.
22
24
5
4
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5.1.20.
(Z)-N-(5-(tert-Butyl)isoxazol-3-yl)-2-(5-((1-isopropyl-1H-benzo[d]imidazol-2-
yl)methylene)-2,4-dioxothiazolidin-3-yl)acetamide (11t): Yellow solid, Yield 78%; mp: 265–267
-1
1
°
C; FT–IR: (cm ): 3260, 3021, 1745, 1645, 1555, 1419, 1220, 980, 749; H NMR (500 MHz,
DMSO–d ): δ 11.5 (brs, 1H), 8.08 (s, 1H), 7.86 (d, J = 7.9 Hz, 1H), 7.78 (d, J = 7.7 Hz, 1H),
6
7.38–7.29 (m, 2H), 6.56 (s, 1H), 5.30–5.21 (m, 1H), 4.55 (s, 2H), 1.62 (d, J = 6.9 Hz, 6H), 1.28
1
3
(
s, 9H); C NMR (125 MHz, DMSO–d ): δ 180.7, 169.9, 164.8, 164.5, 157.3, 146.2, 143.2,
6
133.5, 128.7, 124.1, 122.9, 120.1, 116.9, 112.9, 93.0, 47.6, 43.2, 32.4, 28.2, 21.3; HRMS (ESI):
+
m/z calcd for [M+H] C H N O S 468.1706; found 468.1708.
2
3
26
5
4
6
6
.0. Pharmacology
.1. Cell cultures
Breast (MDA-MB-231), Prostate (PC-3 and DU-145) and normal breast epithelial cells
(
MCF10A) [American Type Cell Culture Collection (ATCC), Maryland, USA] and lung cancer
A549) cells were obtained from National Centre for Cell Science (NCCS) Pune, India and were
(
grown in appropriate DMEM (Dulbecco Modified Eagle medium, Sigma) or MEM (Minimum
Essential Medium, Sigma) supplemented with 10% fetal bovine serum stabilized with 1X
antibiotic-antimycotic solution (Sigma) in a CO incubator with 5% CO and 90% relative
2
2
humidity at 37 °C in incubator. When the cells reached upto 80-90% of confluency, were treated
with 0.25 % trypsin/1 mM EDTA solution for further passage.
6.2. MTT assay
4
The cytotoxic evaluation of 11a–t was performed by using MTT assay. 1 × 10 cells per well
were seeded in well of 96-well microculture plates in 100 µL DMEM, supplemented with 10%
FBS in each and incubated for 24 h at 37 °C in a CO incubator. The culture media was removed
2
after 24 h and 100 µL of test compounds in DMSO. Wells without test compound were used as
the control. After incubating for 48 h, 10 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) was added to each well and further incubated for 4 hour in dark at
37 °C. Then, the supernatant from each well was cautiously removed, formazon crystals were
dissolved in 100 µL of DMSO and absorbance was recorded at 570 nm wavelength and at a
reference wavelength of 630 nm. The percentage inhibition was calculated as 100–[(Mean OD of
treated cell × 100)/Mean OD of vehicle treated cells (DMSO)]. All the experiments were
repeated at least three independent experiments.
1
6

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6.3. In vitro cell migration assay/Wound healing assay
5
A549 cells (5 × 10 cells/well) were cultured in 6 well plate as confluent monolayers for 24 h.
Then artificial scratch on the monolayers were created with 200 µL sterile pipette tip and washed
twice with PBS to remove non-adherent cells. The media containing 5µM, 10µM and 15 µM of
compound 11p or without compound 11p (Control–DMSO) were added to each well. The
migration of cells across the scratched area were photographed by using phase contrast
microscope (Nikon) at 0 h (immediately), 24 h and 48 h time interval after treatment in three or
more randomly selected fields.
6.4. F-actin staining
6
A549 cells (1 × 10 cells/well) were grown on cover slips in 6 well plates for 24 h and then
incubated with 5 µM, 10 µM and 15 µM of compound 11p for 24 h. After the compound
treatment, cells were washed with PBS and fixed with 4% para-formaldehyde in PBS. Cells were
incubated with rhodamine-phalloidin 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 visualized by flourescent
microscopy (Nikon). Images were captured using 20X magnification objective lenses.
6.5. Cell cycle analysis
6
A549 cells (1 × 10 cells/well) in 6 well plate were treated with 5 µM, 10 µM and 15 µM of
compound 11p for 24 h. Cells were collected by trypsinisation, washed with 150 mM PBS and
fixed with 70% ethanol for 30 min at 4 °C. After fixing, cells were again 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 of PBS] for 15 min in dark at room temperature. The
samples were then analyzed for propidium iodide fluorescence from 15,000 events by flow
cytometry using BD Accuri C6 flow-cytometer.
6.6. Acridine orange–ethidium bromide (AO–EB) staining
6
A549 cells (1 × 10 cell/ml) were treated with 5 µM, 10 µM and 15 µM of compound 11p.
Plates were incubated in an atmosphere of 5% CO at 37 °C for 24 h, followed by addition of
2
fluorescent dyes containing acridine orange-ethidium bromide (AO-EB; 10 µL) into each well in
equal volumes (10 µg/mL) and within 10 minutes cells were visualized under fluorescence
1
7

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microscope (Nikon, Inc. Japan) with excitation (488 nm) and emission (550 nm) at 200×
magnification.
6.7. DAPI staining
6
A549 cells (1 × 10 cells/well) were cultured in six well plates and treated with 5 µM, 10 µM
and 15 µM of compound 11p or without compound 11p (Control–DMSO). The untreated and
treated cells were washed two times with PBS, followed by fixation with 4% paraformaldehyde
and staining with 10 µg/mL of DAPI. The cells were observed for apoptotic characteristics under
fluorescence microscope (Nikon, Inc. Japan) with excitation at 359 nm and emission at 461 nm
using DAPI filter at 200 × magnification.
6.8. Measurement of mitochondrial membrane potential (DΨm)
5
A549 cells were cultured in 6 well plates at a density of 5 × 10 cells/mL and allowed to adhere
overnight. The cells were treated with 5 µM, 10 µM and 15 µM of compound 11p for 24 h.
After 24 h of 11p treatment, the adherent cells were collected by trypsinisation, washed with
PBS and resuspended in solution of PBS containing rhodamine-123 (5 µg/mL) and additionally
incubated at room temperature for 30 minutes. Cells were washed two times with PBS to remove
excess dye and resuspended in PBS. 3000 cells from each sample were analyzed by flow
cytometer (BD-c6 accuri) forrhodamine-123 fluorescence.
6.9. Mitosox red assay
5
A549 cells were seeded at a density of 1×10 cells/well in cell culture medium in a 24-well plate
and allowed to adhere overnight. Cells were treated with 5 µM, 10 µM and 15 µM of compound
11p, 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 (Nikon).
6.10. AnnexinV-FITC/Propidium iodide dual staining assay
The Annexin V-FITC/propidium iodide dual staining assay was carried out using A549 cells, to
6
quantify the percentage of apoptotic cells. Cells (1 × 10 /mL per well) were plated in 6 well
culture plates and allowed to grow for 24 h. After treatment with 5 µM, 10 µM and 15 µM of
compound 11p 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
1
8

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Annexin V-FITC, and then in 300 ꢀL1 × binding buffer containing 5 ꢀL Propidium iodide (PI)
in the dark for 5 minutes at room temperature and incubate for 15 minutes. After 15 minutes of
incubation, cells were analysed for percentage of apoptosis using BD-C6 accuri flow-cytometer.
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. IC values (µM) of the tested compounds 11a–t against selected human cancer cell
5
0
a
lines and normal cell line (MCF10A)
Figure 1. Structure of some reported bioactive thiazolidinedione and benzimidazoles as
anticancer agents.
Figure 2. Design of small molecules containing benzimidazole–thiazolidinedione as anticancer
agents.
Figure 3. In vitro cell migration assay. (a) A549 cells were treated with compound 11p (5 µM,
10 µM and 15 µM) and artificial scratches were done with sterile 200 mL pipette. The images
were captured by using phase contrast microscopy at 0 h, 24 h and 48 h. (b) The relative
percentage of cells migrated were quantified using Nikon NIS elements software. Data presented
are mean±SD of three similar independent experiments.
Figure 4. Effects of compound 11p on polymerisation of F-actin filaments in A549 cancer cells.
The A549 cells were treated with 5 µM, 10 µM and 15 µM of compound 11p and stained with
Rhodamine-phalloidin (red fluorescent dye: actin filament) and Hoechst 33242 (nucleus: blue).
Figure 5. Cell cycle distribution of A549 lung cancer cells treated with compound 11p for 24 h.
(a) Cells were treated with 11p, fixed in ethanol, stained with propidium iodide and analysed by
flow-cytometry. (b) Data are mean ± SD from three independent experiments.
Figure 6. Morphological changes observed in A549 lung cancer cells after treatment with
compound 11p for 24 h.
Figure 7. Morphological changes observed by AO–EB staining of compound 11p in A549 cells.
DMSO treatment (control); treatment with 5 µM, 10 µM and 15 µM respectively for 24 h.
Figure 8. Nuclear morphology of A549 cells after DAPI staining. Cells were treated with
different concentrations of compound 11p for 24 h and stained with DAPI. The control
represents DAPI stain of A549 cells without 11p treatment. The images were captured with
fluorescence microscope.
Figure 9. Compound 11p triggered change in mitochondrial membrane potential (DΨm). (A)
A549 cells were treated with [(a) Control-DMSO, (b) 5 µM, (c) 10µM, (d) 15 µM and (e)
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overlay of a-d] and processed by rhodamine-123 staining followed by flow cytometry analysis.
The peak change to the left indicate depolarisation. (B) Data represented as mean ± SD from
three independent experiments.
Figure 10. Effect of compound 11p on production of superoxide in A549 lung cancer cells.
Fluorescent microscopic images of A549 cells stained with MitoSOX after 48 h of treatment
with different concentrations of compound 11p.
Figure 11. Annexin V-FITC/propidium iodide staining assay. A549 cells were treated with
compound 11p and stained with Annexin V-FITC/PI and analysed for apoptosis using flow
cytometer. The percentage of Annexin V-FITC and/or Propidium iodide positive cells is
reported inside the quadrants. Cells in the lower left quadrant (Q1-LL: AV-/PI-): live cells;
lower right quadrant (Q1-LR: AV+/PI-): early apoptotic cells; upper right quadrant (Q1-UR:
AV+/PI +): late apoptotic cells and upper left quadrant (Q1-UL: AV-/PI+): necrotic cells.
Scheme
1.
Synthesis
of
(Z)-5-((1-alkyl-1H-benzo[d]imidazol-2-yl)methylene)-3-(2-
oxoethyl)thiazolidine-2,4-dione derivatives 11a–t.
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Table 1. IC values (µM) of the tested compounds 11a–t against selected human cancer cell
5
0
a
lines and normal cell line (MCF10A)
b
c
MDA-MB-
e
f
Compound
1a
1b
1c
1d
1e
1f
1g
PC-3
DU-145
>50
d
A549
>50
MCF10A
231
1
>50
>50
>50
>50
-
1
45.11 ± 2.51
>50
31.16 ± 1.94
>50
-
1
>50
>50
-
1
>50
>50
>50
>50
>50
>50
-
1
>50
>50
-
1
>50
42.19 ± 2.61
41.06 ± 2.61
>50
>50
28.15 ± 3.09
38.45 ± 2.91
>50
-
1
>50
>50
-
1
1h
>50
>50
-
1
1i
1j
>50
>50
>50
>50
-
1
41.10 ± 3.08
33.54 ± 3.15
>50
37.52 ± 1.45
>50
29.74 ± 2.08
>50
36.03 ± 1.53
>50
13.35 ± 1.26
26.21 ± 1.54
20.63 ± 1.72
29.40 ± 1.69
>50
>100
1
1k
1l
1m
1n
-
1
30.63 ± 1.24
>50
>50
>50
29.18 ± 0.93
>50
33.82 ± 1.37
>50
>50
35.98 ± 1.52
>100
1
>50
-
-
1
1
>50
>50
>50
1o
1p
1q
>50
>50
-
>100
-
1
39.87 ± 3.16
>50
31.41 ± 1.52
>50
31.36 ± 1.12
>50
11.46 ± 1.46
>50
1
1
1r
1s
>50
15.30 ± 0.92
44.83 ± 1.90
>50
38.71 ± 4.03
1
>50
-
-
-
1
1t
>50
>50
g
5
-FU
45.32 ± 2.08
40.58 ± 1.83
30.47 ± 1.90
a
IC50 values are the concentrations that cause 50% inhibition of cancer cell growth. Data represent the mean values
b
c
±
standard deviation of three independent experiments performed in triplicate; prostate cancer (PC-3); prostate
d
e
f
cancer (DU-145); breast cancer (MDA-MB-231); lung cancer (A549); normal breast epithelial cell (MCF10A);
g
5
-Fluorouracil (5-FU) as reference standard.
H
N
Br
O
Cl
N
R
O
O
S
O
S
O
N
O
Benzsuberone-thiazolidinedione
α-bromoacryloylamido-thiazolidinedione
H
2
N
O
COOH
N
N
N
N
N
H
N
H
Veliparib
Bendamustine
Figure 1. Structure of some reported bioactive thiazolidinedione and benzimidazoles as
anticancer agents.
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O
S
N
N
Head part
R' Tail part
N
R
O
O
Figure 2. Design of small molecules containing benzimidazole–thiazolidinedione as anticancer
agents.
Figure 3. In vitro cell migration assay. (a) A549 cells were treated with compound 11p (5 µM,
10 µM and 15 µM) and artificial scratches were done with sterile 200 µL pipette. The images
were captured by using phase contrast microscopy at 0 h, 24 h and 48 h. (b) The relative
percentage of cells migrated were quantified using Nikon NIS elements software. Data presented
are mean±SD of three similar independent experiments.
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Figure 4. Effects of compound 11p on polymerisation of F-actin filaments in A549 cancer cells.
The A549 cells were treated with 5 µM, 10 µM and 15 µM of compound 11p and stained with
Rhodamine-phalloidin (red fluorescent dye: actin filament) and Hoechst 33242 (nucleus: blue).
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