Conventional and microwave-assisted synthesis of new 1H-benzimidazole-thiazolidinedione derivatives: A potential anticancer scaffold

Article abstract of DOI:10.1016/j.ejmech.2017.06.035

A series of new benzimidazole bearing thiazolidinedione derivatives has been designed, synthesized by using conventional as well as microwave-assisted methods. Microwave-assisted synthesis caused a significant reduction in the reaction times and improvement in the yields of all the derivatives. All the new synthesized compounds were evaluated for their in vitro cytotoxic potential against selected human cancer cell lines of breast (MDAMB-231), prostate (PC-3), cervical (HeLa), lung (A549) and bone (HT1080) along with a normal kidney cells (HeK-293T). The compounds 17n, 17p and 17q were found to be potent cytotoxic with IC₅₀ values in the range of 0.096–0.63 μM on PC-3, HeLa, A549 and HT1080 cancer cells. Most of the compounds have found to be safe on normal HeK-293T kidney cells in comparison to cancer cells. The treatment of cells with 17p and 17q showed the typical apoptotic morphological features like fragmentation and shrinkage of nuclei. Further, test compounds resulted in inhibition of cell migration through disruption of F-actin protein assembly. Hoechst, DCFH-DA staining, mitochondrial membrane and annexin binding assays revealed that the cancer cell proliferation was inhibited through induction of apoptosis in A549 cells.

Full text of DOI:10.1016/j.ejmech.2017.06.035

Accepted Manuscript
Conventional and microwave-assisted synthesis of new 1H-benzimidazole-
thiazolidinedione derivatives: A potential anticancer scaffold
Pankaj Sharma, T. Srinivasa Reddy, Niggula Praveen Kumar, Kishna Ram Senwar,
Suresh K. Bhargava, Nagula Shankaraiah
PII:
S0223-5234(17)30481-6
10.1016/j.ejmech.2017.06.035
EJMECH 9530
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 May 2017
Revised Date: 20 June 2017
Accepted Date: 21 June 2017
Please cite this article as: P. Sharma, T.S. Reddy, N.P. Kumar, K.R. Senwar, S.K. Bhargava,
N. Shankaraiah, Conventional and microwave-assisted synthesis of new 1H-benzimidazole-
thiazolidinedione derivatives: A potential anticancer scaffold, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.06.035.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Conventional and microwave-assisted synthesis of new 1H-benzimidazole-
thiazolidinedione derivatives: A potential anticancer scaffold
a
b
a
a
Pankaj Sharma, T. Srinivasa Reddy, Niggula Praveen Kumar, Kishna Ram Senwar, Suresh
b
a
K. Bhargava, * Nagula Shankaraiah *
a
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
Centre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science, RMIT
b
University, GPO BOX 2476, Melbourne-3001, Australia

ACCEPTED MANUSCRIPT
Conventional and microwave-assisted synthesis of new 1H-benzimidazole-
thiazolidinedione derivatives: A potential anticancer scaffold
a
b
a
a
Pankaj Sharma, T. Srinivasa Reddy, Niggula Praveen Kumar, Kishna Ram Senwar,
b
a
Suresh K. Bhargava, * 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
Abstract
A series of new benzimidazole bearing thiazolidinedione derivatives has been designed,
synthesized by using conventional as well as microwave-assisted methods. Microwave-
assisted synthesis caused a significant reduction in the reaction times and improvement in
the yields of all the derivatives. All the new synthesized compounds were evaluated for their
in vitro cytotoxic potential against selected human cancer cell lines of breast (MDAMB-
231), prostate (PC-3), cervical (HeLa), lung (A549) and bone (HT1080) along with a
normal kidney cells (HeK-293T). The compounds 17n, 17p and 17q were found to be
potent cytotoxic with IC values in the range of 0.096 to 0.63 µM on PC-3, HeLa, A549
5
0
and HT1080 cancer cells. Most of the compounds have found to be safe on normal HeK-
93T kidney cells in comparison to cancer cells. The treatment of cells with 17p and 17q
2
showed the typical apoptotic morphological features like fragmentation and shrinkage of
nuclei. Further, test compounds resulted in inhibition of cell migration through disruption of
F-actin protein assembly. Hoechst, DCFH-DA staining, mitochondrial membrane and
annexin binding assays revealed that the cancer cell proliferation was inhibited through
induction of apoptosis in A549 cells.
Keywords: Benzimidazole, thiazolidinedione, cytotoxicity, apoptosis, microwave.
1

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1. Introduction
In the drug discovery of potential antitumor molecules, significant efforts have been made
for the synthesis of new heterocyclic motifs as the main structural design. Such a
heterocyclic scaffolds ‘benzimidazole’ has been recognised as a ‘Master Scaffold’
considering for their broad spectrum of biological profiles and affinities towards different
targets [1,2]. The molecules based on benzimidazole derivatives been reported to have
potential anticancer activities against different types of cancers [3,4,5]. Bendamustine [6]
and Veliparib [7] are benzimidazole based drugs approved for the cancers treatment. The
different substitutions on benzimidazole and its derivatives have been luring researchers
throughout the world to investigate their therapeutic potential [8,9,10,11].
On the other hands, thiazolidinedione templates are privileged structural fragments in
modern medicinal chemistry and reported to have a diverse range of biological activities
[
12,13]. The molecules based on this skeleton act as antidiabetic agents via peroxisome
proliferator-activated receptor-γ (PPAR-γ, pioglitazone and its analogues) [14]. Apart from
antidiabetic activity, these scaffolds have shown a broad spectrum of activities such as anti-
inflammatory [15], antimicrobial [16], wound-healing [17] etc. GSK1059615 (Figure 1) is a
thiazolidinedione derivative known to exert its anticancer effect through the inhibition of
PI3K-α [18]. Liu et al. [19] and Jung et al. [20] reported thiazolidinediones as potential
anticancer agents through inhibition of Raf/MEK/ERK and PI3K/Akt signaling pathways.
The research on thiazolidinedione for their anticancer activity has recently gained interest
and proven promising for cancer treatment. Furthermore, the different heterocyclic ring
systems like pyrrolidine, morpholine, piperidine etc. have been recognized as the
fundamental building blocks of the most drugs in today's market [21]. These nitrogen-
containing heterocycles have the capability to form hydrogen-bonding for selective protein
binding and enhanced drug-likeness properties [22]. The significance of these rings are well
understood in modern medicinal chemistry and drug discovery, since they are known to
play a noteworthy role in molecular properties like three-dimensionality, lipophilicity or
polarity, metabolic stability, and toxicity.
<
insert figure 1>
In the recent years, microwave assisted synthesis have become a well established tool for
the fast assembly of novel chemical entities [23]. The rate of many chemical reactions are
increased because of selective absorption of MW energy. The application of microwave
2

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irradiation is the use of catalysts or mineral supported reagents which enables various
organic reactions to happen expeditiously at ambient pressure, thus providing unique
chemical processes with extraordinary features such as enhanced reaction rates and higher
yields [24].
From the past few decades, the amalgamation of two dissimilar chemical
entities/pharmacophores with two (or more than two) structural domains having
same/different biological functions is being under constant escalation for the exploration of
novel and highly active compounds [25,26]. The advantage of using hybridization approach
is to turn on different targets by a single molecular entity, thereby increasing therapeutic
efficacy and exerting synergistic action with reduced side effects. Hence, in continuation of
our research interest [27,28,29] in discovering and developing new potential anticancer
agents, we herein designed and synthesized new benzimidazole bearing thiazolidinedione
derivatives as potential cytotoxic agents. We have successfully synthesized a series of new
derivatives under microwave-assisted technique as well as conventional method using
toluene as a solvent with catalytic amounts of piperidine. We have appended the structural
features of 2-phenyl-1H-benzimidazole to the head-part and various heterocyclic as well as
benzyl ring systems like morpholine, piperidine, pyrrolidine, benzyl, 3,4,5-
trimethoxybenzyl etc. to the tail-part of thiazolidinedione (Figure 2). We hypothesized that
the synthesized New Chemical Entities (NCEs) could act as potential cytotoxic agents that
might act through the induction of apoptosis. In this context, herein we present the synthesis
and biological evaluation of new benzimidazole bearing thiazolidinedione derivatives as
potential cytotoxic agents and also compared their cytotoxicity profile with a normal kidney
cell line.
<
insert figure 2>
2
. Results and discussion
.1. Chemistry
2
The synthetic strategy for thiazolidinediones 2, 6a–d, 8a–c, 10 and new benzimidazole-
thiazolidinediones 17a–t are outlined in Scheme 1 and Scheme 2. The final products 17a–t
were synthesized in a convergent approach by employing Knoevenagel condensation
reaction. The reaction of chloroacetic acid (1) with thiourea afforded thiazolidinedione 2
(TZD) ring which was subsequently converted into potassium salt of TZD (3). On the other
3

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hand, different heterocyclic amines 4a–d were reacted with chloroacetyl chloride to obtain
the intermediates 5a–d. The chloroacetylated intermediates 5a–d, benzyl bromides 7a–c
and phenacyl bromide 9 were reacted with potassium salt of TZD 3 to obtain different N-
substituted thiazolidinediones 6a–d, 8a–c and 10, respectively. Thus, the N-substitutions of
thiazolidinedione with a variety of heterocyclic amines, benzyl and phenacyl moieties were
introduced with different functionalities as well as the scope of diversity.
<
insert Scheme 1>
Alternatively, 3,4-diaminobenzoic acid (11) was converted to its methyl ester 12, then
reacted with different substituted benzaldehydes 13a–d in the presence of sodium
metabisulfite to afford 1H-benzo[d]imidazole-5-carboxylates 14a–d. The ester
functionalities in 14a–d were reduced with lithium aluminium hydride to obtain alcohols
15a–d which subsequently were oxidised to their corresponding aldehydes 16a–d. The title
compounds 17a–t were accomplished by reacting different thiazolidinediones 2, 6a–d, 8a–c
and 10 with 2-phenyl-1H-benzimidazole-5-carbaldehydes 16a–d under Knoevenagel
reaction using piperidine in dry toluene. The Knoevenagel condensation reaction for
derivatives 17a–t was performed under both conventional as well as microwave-assisted
synthesis. Comparative study results obtained by microwave assisted synthesis; versus
conventional heating method showed that the conventional heating required more reaction
time i.e. 5–11 h, however all the reations were completed within 5–10 min. under
microwave irradiation technique which caused significant reduction in the reaction times
and the yields have been improved from 57–79% (conventional heating) to 74–95%
(
microwave-assisted reaction) (Table 1).
insert Scheme 2>
insert table 1>
All the synthesized derivatives were characterized by melting point, HRMS (ESI), FT-IR,
<
<
1
13
H and C NMR techniques. The target model compound 17a could be easily confirmed by
1
H NMR as the aldehydic -CH proton at 10.04 ppm disappeared and a sharp singlet peak of
newly formed methine -CH- proton appeared at 7.84 ppm by condensation reaction. All the
other aromatic protons of 17a were found in the range of 7.77–7.36 ppm and methoxy
1
3
groups appeared as two separate singlets at 4.01 and 3.80 ppm. The examination of C
NMR spectrum of compound 17a showed two carbonyl signals at 167.6 and 167.1 ppm. All
4

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other aromatic carbons of 17a appeared in the range of 153.5–103.9 ppm and aliphatic
methoxy carbons at 66.0 and 55.7 ppm. FT-IR spectrum of 17a showed the carbonyl
-1
-1
1
13
stretching at 1686 cm and -NH- stretching at 3240 cm . The H and C NMR spectra of
all other derivatives 17b–t were found almost in a similar pattern like 17a for their
respective aliphatic and aromatic peaks. The HRMS (ESI) of all the final products showed
+
the characteristic [M + H] molecular ion peaks to their corresponding molecular formula.
2.2. Pharmacology
2.2.1. In vitro cytotoxic activity
All the new derivatives of benzimidazole bearing thiazolidinediones 17a–t were evaluated
for their in vitro cytotoxic potential against selected human cancer cell lines viz. breast
(MDAMB-231), prostate (PC-3), cervical (HeLa), lung (A549), bone (HT1080) and a
normal kidney cells (HeK-293T) using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) reduction assay [10]. The IC values of 17a–t and reference
5
0
standard nocodazole have been listed in Table 2. It is apparently observed from the primary
screening results in Table 2 that few of the synthesized compounds exhibited potent
cytotoxicities below 1.0 µM on tested cancer cell lines. From the close analysis of Table 2,
compound 17o was found to be potent cytotoxic with IC value of 0.27 µM on MDAMB-
5
0
231 cancer cells. The derivatives 17n, 17q and 17t were found to be active on PC-3 cancer
cells with IC values of 0.63 µM, 0.11 µM and 0.43 µM respectively. Moreover, 17g, 17l,
5
0
17n, 17p, and 17q displayed the potential cytotoxicities on cervical (HeLa) cancer cell lines
with IC s of 0.51 µM, 0.95 µM, 0.18 µM, 0.32 µM and 0.25 µM respectively. The
5
0
compounds 17b, 17g, 17j, 17l, 17m, 17n, 17o, 17p, 17q, 17r and 17t were found to be
active in the range of 0.096 µM–0.98 µM on lung cancer (A549) cell lines which showed
the selective cytotoxicity against this cancer cells. Interestingly, 17q is the highest cytotoxic
compound in this series which exhibited IC value of 0.096 µM, whereas the reference
5
0
standard displayed IC of 1.87 µM. Furthermore, 17l, 17n, 17o, 17p, 17q, 17r and 17t
5
0
were found to be active on HT1080 (bone cancer) cell lines in the range of 0.16 µM–0.98
µM. It could be noticed from Table 2 that 17l, 17m, 17n and 17q showed a broad spectrum
of cytotoxic activity on all the examined cancer cells in the range of 0.096 to 4.58 µM.
<
insert table 2>
5

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From Table 2, it could be observed that compounds 17a–d having free -NH functionality
produced albeit less active compounds (above 1.0 µM) when compared to N-substituted
ones (except 17b on A549 cancer cells). The presence of oxo-ethyl linked heterocyclic rings
like morpholine, piperidine, and pyrrolidine produced compounds >1.0 µM with exception
of 17g, 17h and 17j which were fairly selective against A549 cancer cells with below IC50
of 1.0 µM. Changing the oxo-ethyl linked heterocyclic ring system to benzyl group (17l–q)
produced more active compounds below 1.0 µM on HeLa, A549, and HT1080 cancer cells.
One of the compound 17n containing 3-methyl benzyl moiety at the tail-part of
thiazolidinedione was found to be active on all the tested cancer cell lines below 1.78 µM,
however, compounds 17o–q having 3,4,5-trimethoxy benzyl substitution at tail-part
produced bioactive compounds below 1.0 µM on most of the tested cancer cell lines. On the
other hand, substitutions on head-part of thiazolidinedione with benzimidazole containing
3,4,5-trimethoxyphenyl groups generally provided more number of bioactive compounds,
however, the most active compound 17q contained 4-isobutoxy-3-methoxy group on
benzimidazole.
These compounds were further evaluated for their in vitro cytotoxicity on normal kidney
cells (HeK-293T), to find out safety and selectivity against non-cancer cells. It was quite
interesting to note that most of the compounds displayed potent cytotoxicity towards cancer
cells in comparison to normal HeK-293T kidney cells. It was our keen observation that
three of the most active compounds i.e. 17n, 17p and 17q were found to be moderately to
high selective towards all the tested cancer cell line when compared to normal kidney cells.
Compound 17n displayed IC50 values almost 1.9, 5.3, 18.6, 25.8 and 17.6 folds
more in MDAMB-231, PC-3, HeLa, A549 and HT1080 respectively when compared to
normal HeK-293T cells. Compound 17p displayed 21.1, 52 and 25.3 times more selectivity
in HeLa, A549 and HT1080 cancer cell respectively, however, compound 17q showed 1.5,
60.5, 26.6, 69.3 and 41.6 times more selectivity in MDAMB-231, PC-3, HeLa, A549 and
HT1080 respectively in comparison to normal HeK-293T kidney cells. Based on the
cytotoxicity and selectivity of 17p and 17q against A549 cells, these were selected for
further mechanistic studies of cell growth inhibition.
2.2.2. In vitro cell migration assay/Wound healing assay
Migration is a key feature of practically each biological process for the development and
maintenance of cells, however it is known to play a major role in tumor progression and
6

ACCEPTED MANUSCRIPT
metastatic cascade in cancer cells [30]. Since, cell migration is linked with the metastatic
activity of cancer cells, therefore we studied the effect of compounds 17p and 17q on A549
cells using in vitro cell migration assay/wound healing assay [30]. This assay is based on
the observation that, upon creating a new artificial wound, so called “scratch” 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 of cells
with anticancer molecules restricts the migration of cells to close the wound. Henceforth, to
test the effect of compounds on cell migration, wounds were created on A549 cell’s
monolayer by using a 200 µL sterile pipette tip and treated cells with the IC50
concentrations of 17p and 17q were allowed to migrate into the wounded area. The images
of cells to fill-up the wounds were captured at 0, 24 and 48 h after the treatment with the
compounds by using phase contrast microscopy. It can be inferred from Figure 3 that the
compounds 17p and 17q effectively suppressed the migration of cells into the wounded
area, however almost complete healing was observed in DMSO treated control cells after 48
h.
<
insert figure 3>
2.2.3. Effect on F-actin polymerisation
Previous report suggested that the migration or motility of cancer cells is an integrated sum
of multiple processes triggered by the formation of membrane protrusions or remodelling of
cytoskeleton [31]. The cytoskeleton is made-up of various filamentous structures like actin
filaments and microtubules. The polymerisation of these actin filaments is considered to be
the driving force for the formation of membrane protrusions which help in cancer cell
migration as well as stress fibre assembly [32]. It was seen in Figure 3 that these conjugates
inhibited the migration of A549 cancer cells, henceforth it was considered of our interest to
examine the effect of these compounds on actin polymerisation and stress fibre formation.
The formation of actin cytoskeleton in A549 cells was investigated by using rhodamine
phalloidin [33], a red fluorescent dye which binds specifically to F-actin proteins. A549
cancer cells were treated with IC concentrations of 17p and 17q followed by staining with
5
0
rhodamine-phalloidin. Results from the Figure 4 clearly demonstrated that the DMSO-
treated control A549 cancer cells have a more F-actin extensions however 17p and 17q
treatment caused the decreased F-actin extensions at the periphery. These results
7

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collectively revealed the ability of these compounds to inhibit the migration potential of
A549 cells through disruption of F-actin assembly.
<
insert figure 4>
2.2.4. Hoechst staining
Chromatin condensation, DNA fragmentation and nuclear shrinkage are some of the
characteristic morphological features of apoptotic cell death. Therefore, we examined the
apoptosis-inducing effect of compounds 17p and 17q on A549 cancer cells using the
Hoechst 33242 nuclear staining method [29]. A549 cancer cells were treated with IC50
concentrations of 17p and 17q, stained with Hoechst 33242 and observed for nuclear
morphological changes under fluorescence microscope. The results from Figure 5 clearly
demonstrated that the nuclear structure of control cells was intact, however 17p and 17q
treated cells showed the characteristic changes in the morphological features like
fragmented and shrinked nuclei (indicated with white arrows) which are typical apoptotic
feature.
<
insert figure 5>
2.2.5. Effect on intracellular reactive oxygen species (ROS)
Earlier reports suggested that increase in the levels of intracellular reactive oxygen species
ROS) triggers apoptosis induction by altering membrane potential [34,35,36]. We were
(
intrigued by the fact that apoptotic induction in A549 cancer cells could be due to the
generation of intracellular ROS, henceforth we tested this possibility by using DCFH-DA
(2'-7'-dichlorodihydrofluorescein diacetate) fluorescent dye [37]. DCFH-DA is a non-polar
and non-fluorescent dye which gets converted into the highly fluorescent polar derivative
DCF when oxidized by intracellular ROS. It could be easily inferred from the Figure 6 that
the DMSO-treated control A549 cells showed less fluorescence, however treatment with
IC concentrations of 17p and 17q showed a significant increase in the green fluorescence.
5
0
This test evidenced the production intracellular ROS, suggesting clearly that these
compounds induced apoptosis in A549 cancer cells.
<
insert figure 6>
2.2.6. Effect on mitochondrial membrane potential (DΨm)
8

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Mitochondria play an important role in energy metabolism and can also be the source of
signals that initiate apoptotic cell death [38]. It has been reported in literature that the loss of
mitochondrial membrane potential and increase of intracellular ROS levels are closely
connected processes that occur during apoptosis [39]. Henceforth, we examined the effect
of 17p and 17q on DΨm of A549 cancer cell line by using rhodamine-123 dye [40].
Mitochondria having normal DΨm give a strong green fluorescence; however mitochondrial
energization induces the quenching of fluorescence due to less uptake of rhodamine-123,
thus causing depolarisation. The change in DΨm can be monitored by shift in the
fluorescence intensity by the use of spectrofluorometer. From the Figure 7a and 7b it can
be seen the loss of mitochondrial membrane potential of A549 cells. The peak changes from
control (DMSO treated) to left (after treatment with IC concentrations of 17p and 17q) as
5
0
shown in Figure 7a indicated depolarisation by the loss of DΨm. The loss in DΨm dropped
to 31.6% (17p) and 37.3% (17q) in comparison to the control. The results from loss of
DΨm clearly indicate the induction of apoptosis through the collapse of mitochondrial
membrane potential on A549 cells.
<
insert figure 7>
2.2.7. Annexin V-FITC/Propidium iodide staining assay
The apoptosis inducing effect of compounds 17p and 17q on A549 cancer cells was further
investigated using annexin V-FITC/propidium iodide staining assay [41]. This assay
facilitate the detection of 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+).
A549 cells were treated with IC concentrations of 17p and 17q and stained with annexin
5
0
V-FITC/propidium iodide. Results from Figure 8 showed that the percentage of total
apoptotic cells (early and late apoptotic cells) increased from 11.4% (control) to 33.8% and
30.2% after treatment with 17p and 17q respectively. Therefore, the percentage increase in
early and late apoptotic cells clearly indicated that the compound 17p and 17q induced
apoptosis on A549 cancer cells.
<
insert figure 8>
3
. Experimental
.1. Synthesis
3
9

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3.1.1. Materials and methods
All the reagents, chemicals, starting materials and solvents were procured from commercial
sources and were used as such without further purifications. The reactions were monitored
by TLC (thin layer chromatography-MERCK silica gel 60-F254 pre-coated aluminium
plates) under ultra-violet (UV) light. All melting points are uncorrected and were obtained
by using Stuart® SMP30 apparatus. All the microwave reactions were performed using
1
13
monowave 300 instrument (Anton Paar). H and C NMR were taken on Bruker Avance
500 MHz spectrometer using tetramethylsilane (TMS) as the internal standard and chemical
1
shifts are reported in ppm. Chemical shifts are referenced to TMS (δ 0.00 for H NMR and
1
3
1
13
1
C NMR), DMSO˗d (δ 2.50 for H NMR and 39.5 for C NMR) or CDCl (δ 7.26 for H
6
3
1
3
NMR and 77.15 for C NMR). 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.
Wherever required, column chromatography was performed using silica gel (100–200
mesh).
3.1.2. Conventional procedure for the synthesis of benzimidazole-thiazolidinedione (17a–t).
To the suspension of benzimidazole aldehydes (16a–d, 0.32 mmol), thiazolidinedione (2,
a–d, 8a–c and 10, 0.35 mmol), in dry toluene (5 mL) was added catalytic amount of
6
piperidine. The reaction mixture was refluxed until complete disappearance of starting
materials (5–11 h) was observed by TLC. After cooling, the formed precipitates were
filtered, washed with hexane and recrystallized using ethanol to obtain final products 17a–t
as yellow solids in 57–79% yields (table 1).
3.1.3. Microwave assisted procedure for the synthesis benzimidazole-thiazolidinedione
(17a–t).
A suspension of benzimidazole aldehydes (16a–d, 0.32 mmol), thiazolidinedione (2, 6a–d,
8a–c and 10, 0.35 mmol), in dry toluene (5 mL) was added catalytic amount of piperidine in
o
a microwave vial (10 mL). The reaction mixture was irradiated at 150 C for 5–10 min.
After cooling, the formed precipitates were filtered, washed with hexane and recrystallized
using ethanol to obtain final products 17a–t as yellow solids in 74–95% yields (table 1).
1
0

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3.1.3.1.
(Z)-5-((2-(3,4,5-Trimethoxyphenyl)-1H-benzo[d]imidazol-5-yl)methylene)
thiazolidine-2,4-dione (17a)
-1
Yellow solid; mp: 295–297 °C; FT-IR (cm ): 3240, 3007, 1735, 1686, 1590, 1291, 1131,
1
8
08; H NMR (500 MHz, CDCl +DMSO-d ): δ 7.83 (s, 1H), 7.69 (s, 1H), 7.61 (d, J = 8.4
3
6
1
3
Hz, 1H), 7.49–7.47 (m, 2H), 7.34–7.32 (m, 1H), 3.90 (s, 6H), 3.77 (s, 3H); C NMR (125
MHz, CDCl +DMSO-d ): δ 167.6, 167.1, 153.5, 152.9, 139.2, 133.1, 130.8, 127.6, 126.8,
3
6
1
24.4, 120.4, 122.9, 103.9, 60.0, 55.7; HRMS (ESI): m/z calcd for C H N O S 412.0967,
20 18 3 5
+
found 412.0950 [M + H] .
3.1.3.2. (Z)-5-((2-(3,4-Dimethoxyphenyl)-1H-benzo[d]imidazol-5-yl)methylene)thiazolidine-
2,4-dione (17b)
-1
Yellow solid; mp: 279–281 °C; FT-IR (cm ): 3251, 3001, 1728, 1683, 1591, 1285, 1134,
1
8
2
3
1
10;; H NMR (500 MHz, CDCl +DMSO-d ): δ 7.81 (d, J = 14.0 Hz, 1H), 7.73–7.70 (m,
3
6
H), 7.66 (s, 1H), 7.58–7.56 (m, 1H), 7.30 (d, J = 8.4 Hz, 1H), 6.96 (d, J = 8.2 Hz, 1H),
.90 (s, 3H), 3.85 (s, 3H); (125 MHz, CDCl +DMSO-d ): δ 167.7, 167.2, 153.7, 150.3,
3
6
48.6, 133.2, 126.5, 124.6, 121.9, 120.1, 119.5, 110.8, 109.6, 55.4, 55.3; HRMS (ESI): m/z
+
calcd for C H N O S 382.0862, found 382.0863 [M + H] .
1
9
16
3
4
3.1.3.3.
(Z)-5-((2-(2,4,5-Trimethoxyphenyl)-1H-benzo[d]imidazol-5-yl)methylene)
thiazolidine-2,4-dione (17c)
-1
Yellow solid; mp: 278–280 °C; FT-IR (cm ): 3297, 3145, 2970, 1730, 1685, 1477, 1276,
1
1
1
3
1
4
035, 798; H NMR (500 MHz, CDCl +DMSO-d ): δ 7.92 (s, 1H), 7.88 (s, 1H), 7.85 (s,
3
6
H), 7.72 (d, J = 8.4 Hz, 1H), 7.45–7.43 (m, 1H), 6.66 (s, 1H), 4.06 (s, 3H), 3.91 (s, 3H),
13
.82 (s, 3H); C NMR (125 MHz, DMSO-d ): δ 168.1, 167.5, 152.2, 151.8, 142.9, 133.5,
6
26.4, 120.1, 111.9, 108.2, 97.7, 56.3, 55.9, 55.8; HRMS (ESI): m/z calcd for C H N O S
2
0
18
3
5
+
12.0967, found 412.0969 [M + H] .
.1.3.4. (Z)-5-((2-(4-Isobutoxy-3-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)methylene)
3
thiazolidine-2,4-dione (17d)
-1
Yellow solid; mp: 290–292 °C; FT-IR (cm ): 3306, 3160, 2958, 1728, 1683, 1467, 1269,
1
1
8
1
022, 796; H NMR (500 MHz, DMSO-d ): δ 7.94 (s, 1H), 7.78–7.69 (m, 4H), 7.44 (d, J =
6
.2 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 3.89 (s, 3H), 3.82 (d, J = 6.7 Hz, 2H), 2.09–2.02 (m,
13
H), 1.00 (d, J = 6.7 Hz, 6H); C NMR (125 MHz, DMSO-d ): δ 168.1, 167.6, 153.9,
6
1
1

ACCEPTED MANUSCRIPT
150.2, 149.1, 133.4, 126.8, 124.6, 121.8, 120.4, 119.7, 112.9, 110.1, 74.4, 55.7, 27.7, 19.0;
+
HRMS (ESI): m/z calcd for C H N O S 424.1331, found 424.1333 [M + H] .
2
2
22
3
4
3.1.3.5.
(Z)-3-(2-Morpholino-2-oxoethyl)-5-((2-(3,4,5-trimethoxyphenyl)-1H-
benzo[d]imidazol-5-yl)methylene)thiazolidine-2,4-dione (17e)
-1
Yellow solid; mp: 198–200 °C; FT-IR (cm ): 3315, 2991, 1739, 1688, 1649, 1470, 1285,
1
1
8
3
1
4
022, 819; H NMR (500 MHz, CDCl +DMSO-d ): δ 8.00 (s, 1H), 7.76 (s, 1H), 7.65 (d, J =
3
6
.4 Hz, 1H), 7.49 (s, 2H), 7.40 (d, J = 8.5 Hz, 1H), 4.55 (s, 2H), 3.90 (s, 6H), 3.77 (s, 3H),
1
3
.66–3.48 (m, 8H); C NMR (125 MHz, CDCl +DMSO-d ): δ 166.9, 165.1, 163.0, 153.6,
3
6
52.4, 139.2, 134.7, 131.6, 128.5, 126.7, 124.8, 124.4, 117.9, 103.9, 65.8, 65.7, 60.0, 55.7,
4.4, 42.0, 41.8; HRMS (ESI): m/z calcd for C H N O S 539.1600, found 539.1594 [M +
2
6
27
4
7
+
H] .
3.1.3.6.
(Z)-5-((2-(3,4-Dimethoxyphenyl)-1H-benzo[d]imidazol-5-yl)methylene)-3-(2-
morpholino-2-oxoethyl)thiazolidine-2,4-dione (17f)
-1
Yellow solid; mp: 268–270 °C; FT-IR (cm ): 3210, 2987, 1736, 1682, 1600, 1440, 1266,
1
8
07; H NMR (500 MHz, CDCl +DMSO-d ): δ 8.19 (s, 1H), 7.93–7.72 (m, 4H), 7.50 (d, J
3
6
=
8.2 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H), 4.61 (s, 2H), 3.89 (s, 3H), 3.84 (s, 3H), 3.64–3.44
1
3
(
m, 8H); C NMR (125 MHz, CDCl +DMSO-d ): δ 166.9, 165.2, 163.1, 153.7, 150.6,
3 6
148.7, 134.8, 126.5, 124.7, 121.6, 191.7, 117.7, 11.3, 109.8, 65.8, 55.4, 55.3, 44.4, 42.1,
+
4
1.8; HRMS (ESI): m/z calcd for C H N O S 509.1495, found 509.1497 [M + H] .
2
5
25
4
6
3.1.3.7.
(Z)-3-(2-Morpholino-2-oxoethyl)-5-((2-(2,4,5-trimethoxyphenyl)-1H-
benzo[d]imidazol-5-yl)methylene)thiazolidine-2,4-dione (17g)
-1
Yellow solid; mp: 303–305 °C; FT-IR (cm ): 3312, 2980, 1741, 1685, 1651, 1453, 1282,
1
1
023, 805; H NMR (500 MHz, CDCl +DMSO-d ): δ 8.03 (s, 1H), 7.91–7.89 (m, 2H), 7.76
3
6
(d, J = 8.4 Hz, 1H), 7.46–7.44 (m, 1H), 6.80 (s, 1H), 4.62 (s, 2H), 4.10 (s, 3H), 3.96 (s, 3H),
1
3
3
.90 (s, 3H), 3.70–3.52 (m, 8H, merged with DMSO-moisture); C NMR (125 MHz,
CDCl +DMSO-d ): δ 167.0, 165.2, 163.1, 152.2, 151.8, 151.5, 142.8, 134.9, 126.2, 125.0,
3
6
117.4, 112.0, 108.0, 97.2, 65.8, 56.1, 55.8, 55.7, 44.4, 42.1, 41.8; HRMS (ESI): m/z calcd
+
for C H N O S 539.1600, found 539.1606 [M + H] .
2
6
27
4
7
3.1.3.8.
(Z)-3-(2-Oxo-2-(piperidin-1-yl)ethyl)-5-((2-(3,4,5-trimethoxyphenyl)-1H-
benzo[d]imidazol-5-yl)methylene)thiazolidine-2,4-dione (17h)
1
2

ACCEPTED MANUSCRIPT
-1
Yellow solid; mp: 180–182 °C; FT-IR (cm ): 3250, 2955, 1730, 1677, 1630, 1285, 1230,
1
8
13; H NMR (500 MHz, DMSO-d ): δ 7.69 (s, 1H), 7.49 (s, 1H), 7.40–7.38 (m, 3H), 7.05
6
(d, J = 8.1 Hz, 1H), 4.57 (s, 2H), 3.90 (d, J = 8.5 Hz, 9H), 3.63 (t, J = 5.3 Hz, 2H), 3.52–
1
3
3
.48 (m, 2H), 1.73–1.69 (m, 4H), 1.65–1.55 (m, 2H); C NMR (125 MHz, CDCl +DMSO-
3
d6): δ 167.4, 165.5, 162.1, 152.9, 153.6, 139.4, 134.9, 126.9, 125.1, 124.0, 118.0, 103.8,
60.3, 55.7, 45.2, 42.8, 41.7, 25.5, 24.6, 23.6; HRMS (ESI): m/z calcd for C H N O S
27 29 4 6
+
5
37.1808, found 537.1808 [M + H] .
3.1.3.9. (Z)-3-(2-Oxo-2-(piperidin-1-yl)ethyl)-5-((2-(2,4,5-trimethoxyphenyl)-1H-
benzo[d]imidazol-5-yl)methylene)thiazolidine-2,4-dione (17i)
-1
Yellow solid; mp: 265–267 °C; FT-IR (cm ): 3249, 2967, 1732, 1677, 1626, 1406, 1287,
1
1
245, 998, 815; H NMR (500 MHz, CDCl +DMSO-d ): δ 7.94 (s, 1H), 7.89–7.86 (m, 1H),
3
6
7.78 (s, 1H), 7.66–7.63 (m, 1H), 7.33, (d, J = 8.5 Hz, 1H), 6.68 (s, 1H), 4.48 (s, 2H), 4.03
13
(
s, 3H), 3.90 (s, 3H), 3.85 (s, 3H), 3.45–3.42 (m, 4H), 1.60–1.48 (m, 6H); C NMR (125
MHz, CDCl +DMSO-d ): δ 167.0, 165.2, 162.2, 152.1, 151.7, 151.5, 142.8, 134.7, 128.4,
3
6
127.6, 126.2, 124.9, 117.5, 115.5, 111.9, 108.0, 96.7, 55.9, 55.8, 55.6, 45.1, 42.6, 42.0, 25.6,
+
2
4.8, 23.6; HRMS (ESI): m/z calcd for C H N O S 537.1808, found 537.1798 [M + H] .
2
7
29
4
6
3.1.3.10.
(Z)-3-(2-Oxo-2-(pyrrolidin-1-yl)ethyl)-5-((2-(3,4,5-trimethoxyphenyl)-1H-
benzo[d]imidazol-5-yl)methylene)thiazolidine-2,4-dione (17j)
-1
Yellow solid; mp: 269–271 °C; FT-IR (cm ): 3241, 2988, 1735, 1670, 1632, 1290, 1255,
1
8
13; H NMR (500 MHz, CDCl +DMSO-d ): δ 8.12 (s, 1H), 7.88–7.49 (m, 2H), 7.55–7.50
3
6
(m, 3H), 4.48 (s, 2H), 3.91 (s, 6H), 3.74 (s, 3H), 3.53 (t, J = 6.7 Hz, 2H), 3.30 (t, J = 6.9 Hz,
1
3
2
1
1
5
H), 1.95–1.89 (m, 2H), 1.82–1.77 (m, 2H); C NMR (125 MHz, CDCl +DMSO-d ): δ
3 6
67.1, 165.3, 162.7, 153.7, 153.2, 139.3, 140.0, 128.8, 128.1, 126.8, 125.2, 124.7, 118.0,
04.0, 60.1, 56.0, 45.8, 45.0, 43.1, 25.2, 23.6; HRMS (ESI): m/z calcd for C H N O S
26
27
4
6
+
23.1651, found 523.1662 [M + H] .
.1.3.11. (Z)-3-(2-(2,6-Dimethylmorpholino)-2-oxoethyl)-5-((2-(4-isobutoxy-3-methoxy
3
phenyl)-1H-benzo[d]imidazol-5-yl)methylene)thiazolidine-2,4-dione (17k)
-1
Yellow solid; mp: 302–304 °C; FT-IR (cm ): 3234, 2978, 1737, 1683, 1631, 1442, 1259,
1
1
082, 1019, 809; H NMR (500 MHz, DMSO-d ): δ 8.11 (s, 1H), 7.93–7.71 (m, 4H), 7.50
6
(d, J = 8.2 Hz, 1H), 7.14 (d, J = 8.2 Hz, 1H), 4.79 (d, J = 16.6 Hz, 1H), 4.50 (d, J = 16.6 Hz,
1H), 4.15 (d, J = 12.7 Hz, 1H), 3.89–3.81 (m, 6H), 3.58–3.44 (m, 2H), 2.79 (t, J = 11.4 Hz,
1
3

ACCEPTED MANUSCRIPT
1
6
1
1
H), 2.33 (t, J = 11.7 Hz, 1H), 2.08–2.03 (m, 1H), 1.13–1.08 (m, 6H), 1.00 (d, J = 6.4 Hz,
1
3
H); C NMR (125 MHz, DMSO-d ): δ 167.2, 165.4, 163.1, 150.3, 149.1, 135.1, 126.6,
6
24.7, 121.8, 119.8, 117.7, 112.9, 110.1, 74.5, 71.3, 71.0, 55.7, 49.3, 46.8, 42.6, 27.7, 19.0,
+
8.6, 18.3; HRMS (ESI): m/z calcd for C H N O S 579.2277, found 579.2284 [M + H] .
3
0
35
4
6
3.1.3.12. (Z)-3-Benzyl-5-((2-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazol-5-yl)methylene)
thiazolidine-2,4-dione (17l)
-1
Yellow solid; mp: 263–265 °C; FT-IR (cm ): 3428, 3010, 2967, 1735, 1670, 1357, 1275,
1
1
2
4
1
1
211, 1021, 777; H NMR (500 MHz, CDCl +DMSO-d ): δ 8.06 (s, 1H), 7.84–7.81 (m,
3
6
H), 7.73 (d, J = 8.4 Hz, 1H), 7.59 (s, 2H), 7.47 (d, J = 8.5 Hz, 1H), 7.39–7.28 (m, 4H),
1
3
.90 (s, 2H), 3.98 (s, 6H), 3.87 (s, 3H); C NMR (125 MHz, CDCl +DMSO-d ): δ 167.1,
3
6
65.4, 153.8, 153.0, 139.3, 135.3, 135.1, 134.9, 128.3, 127.6, 124.6, 117.8, 113.2, 104.0,
03.9, 59.9, 55.8, 44.4; HRMS (ESI): m/z calcd for C H N O S 502.1437, found 502.1435
27
24
3
5
+
[
M + H] .
3.1.3.13.
(Z)-3-(3-Methylbenzyl)-5-((2-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazol-5-
yl)methylene)thiazolidine-2,4-dione (17m)
-1
Yellow solid; mp: 235–237 °C; FT-IR (cm ): 3349, 2967, 1733, 1655, 1352, 1129, 1001,
1
7
44; H NMR (500 MHz, CDCl +DMSO-d ): δ 8.13 (s, 1H), 7.86 (s, 1H,), 7.74 (d, J = 8.4
3
6
Hz, 1H), 7.55 (s, 2H,), 7.49 (d, J = 9.5 Hz, 1H), 7.24 (t, J = 7.6 Hz, 1H), 7.13–7.09 (m, 3H),
1
3
4
1
1
.80 (s, 2H), 3.90 (s, 6H), 3.74 (s, 3H), 2.28 (s, 3H); C NMR (125 MHz, DMSO-d ): δ
6
67.3, 165.5, 153.7, 153.2, 139.3, 137.8, 135.4, 135.0, 128.8, 128.5, 128.3, 128.0, 126.8,
24.8, 124.7, 124.6, 118.0, 104.0, 60.1, 56.0, 44.5, 20.9; HRMS (ESI): m/z calcd for
+
C H N O S 516.1593, found 516.1598 [M + H] .
28
26
3
5
3.1.3.14.
(Z)-3-(3-Methylbenzyl)-5-((2-(2,4,5-trimethoxyphenyl)-1H-benzo[d]imidazol-5-
yl)methylene)thiazolidine-2,4-dione (17n)
-1
Yellow solid; mp: 197–199 °C; FT-IR (cm ): 3422, 3002, 2942, 1734, 1678, 1375, 1278,
1
1
206, 1027, 778; H NMR (500 MHz, CDCl +DMSO-d ): δ 7.96 (s, 1H), 7.92 (s, 1H), 7.78
3
6
(s, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.16-7.10 (m, 3H), 7.03 (d, J =
7.0 Hz, 1H), 6.63 (s, 1H), 4.78 (s, 2H), 4.03 (s, 3H), 3.90 (s, 3H), 3.89 (s, 3H), 2,26 (s, 3H);
1
3
C NMR (125 MHz, CDCl +DMSO-d ): δ 167.1, 165.4, 152.2, 151.8, 151.1, 142.8, 137.5,
3
6
134.8, 134.7, 128.3, 128.2, 128.0, 126.4, 125.0, 124.7, 117.6, 115.4, 111.8, 107.6, 96.6,
1
4

ACCEPTED MANUSCRIPT
5
5.9, 55.6, 44.3, 20.7; HRMS (ESI): m/z calcd for C H N O S 516.1593, found 516.1591
2
8
26
3
5
+
[
M + H] .
3.1.3.15.
(Z)-3-(3,4,5-Trimethoxybenzyl)-5-((2-(3,4,5-trimethoxyphenyl)-1H-benzo[d]
imidazol-5-yl)methylene)thiazolidine-2,4-dione (17o)
-1
Yellow solid; mp: 134–136 °C; FT-IR (cm ): 3351, 2997, 1727, 1662, 1580, 1120, 815,
1
7
95, 617; H NMR (500 MHz, CDCl ): δ 8.01 (s, 1H), 7.86–7.52 (m, 2H), 7.41–7.36 (m,
3
13
3
H), 6.71 (s, 2H), 4.82 (s, 2H), 3.91 (s, 3H), 3.85–3.83 (m, 15H); C NMR (125 MHz,
CDCl ): δ 168.3, 166.4, 154.0, 153.4, 140.8, 138.0, 135.3, 131.1, 128.3, 126.4, 124.6, 119.5,
3
106.4, 104.3, 61.2, 61.0, 56.4, 56.3, 45.7; HRMS (ESI): m/z calcd for C H N O S
30 30 3 8
+
5
92.1754, found 592.1757 [M + H] .
3.1.3.16. (Z)-5-((2-(3,4-Dimethoxyphenyl)-1H-benzo[d]imidazol-5-yl)methylene)-3-(3,4,5-
trimethoxybenzyl)thiazolidine-2,4-dione (17p)
-1
Yellow solid; mp: 246–248 °C; FT-IR (cm ): 3334, 3001, 1729, 1663, 1586, 1126,798,
1
6
15; H NMR (500 MHz, DMSO-d ): δ 8.12 (s, 1H), 7.90–7.64 (m, 4H), 7.48 (d. J = 8.2
6
Hz, 1H), 7.15 (d, J = 8.9 Hz, 1H), 6.62 (s, 2H), 4.77 (s, 2H), 3.88 (s, 3H), 3.84 (s, 3H), 3.74
13
(
s, 6H), 3.62 (s, 3H); C NMR (125 MHz, DMSO-d ): δ 167.5, 165.7, 152.9, 150.7, 148.9,
6
137.1, 135.0, 131.1, 128.8, 128.2, 126.7, 121.9, 119.7, 117.9, 111.8, 109.8, 105.2, 59.9,
5
6.0, 55.8, 55.5, 44.8; HRMS (ESI): m/z calcd for C H N O S 562.1648, found 562.1635
2
9
28
3
7
+
[
M + H] .
3.1.3.17. (Z)-5-((2-(4-Isobutoxy-3-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)methylene)-3-
(3,4,5-trimethoxybenzyl)thiazolidine-2,4-dione (17q)
-1
Yellow solid; mp: 298–300 °C; FT-IR (cm ): 3342, 2960, 1670, 1594, 1462, 1220, 1126,
1
8
00; H NMR (500 MHz, CDCl ): δ 7.97 (s, 1H), 7.75 (s, 1H), 7.70–7.68 (m, 1H), 7.61 (d, J
3
=
8.1 Hz, 1H), 7.48 (s, 1H), 7.33 (d, J = 8.1 Hz, 1H), 6.91 (d, J = 8.3 Hz, 1H), 6.61–6.56
(m, 2H), 4.74 (s, 2H), 3.90 (s, 3H), 3.77–3.70 (m, 11H), 2.14–2.06 (m, 1H), 0.98 (d, J = 6.3
1
3
Hz, 6H); C NMR (125 MHz, CDCl ): δ 168.3, 166.4, 153.4, 151.4, 150.1, 137.9, 135.5,
3
131.1, 127.9, 126.2, 121.7, 119.8, 119.0, 112.9, 110.5, 106.4, 75.6, 61.0, 56.3, 56.2, 45.6,
+
2
8.2, 19.4; HRMS (ESI): m/z calcd for C H N O S 604.2117, found 604.2121 [M + H] .
3
2
34
3
7
3.1.3.18. (Z)-3-(2-Oxo-2-phenylethyl)-5-((2-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazol-
5-yl)methylene)thiazolidine-2,4-dione (17r)
1
5

ACCEPTED MANUSCRIPT
-1
Yellow solid; mp: 183–185 °C; FT-IR (cm ): 3331, 2967, 1745, 1670, 1576, 1456, 1240,
1
1
2
7
016, 814; H NMR (500 MHz, CDCl +DMSO-d ): δ 8.01 (s, 1H), 7.97 (d, J = 8.4 Hz,
3
6
H,), 7.78 (s, 1H), 7.70 (d, J = 8.4 Hz, 1H,), 7.61 (t, J = 7.5 Hz, 1H,), 7.55 (s, 2H), 7.50–
13
.47 (m, 2H), 7.42–7.40 (m, 1H), 5.16 (s, 2H), 3.94 (s, 6H), 3.84 (s, 3H); C NMR (125
MHz, CDCl +DMSO-d ): δ 190.0, 166.7, 165.0, 153.5, 152.9, 143.0, 139.3, 134.9, 133.8,
3
6
133.5, 128.5, 127.7, 126.7, 125.0, 124.0, 117.8, 113.9, 103.9, 60.0, 55.7, 47.2; HRMS
+
(
ESI): m/z calcd for C H N O S 530.1386, found 530.1384 [M + H] .
2
8
24
3
6
3.1.3.19. (Z)-3-(2-Oxo-2-phenylethyl)-5-((2-(2,4,5-trimethoxyphenyl)-1H-benzo[d]imidazol-
5-yl)methylene)thiazolidine-2,4-dione (17s)
-1
Yellow solid; mp: 264–266 °C; FT-IR (cm ): 3346, 2942, 1732, 1678, 1585, 1450, 1226,
1
1
027, 813; H NMR (500 MHz, CDCl +DMSO-d ): δ 7.98–7.97 (m, 3H), 7.90 (s, 1H), 7.80
3
6
(s, 1H), 7.66–7.61 (m, 2H), 7.49 (d, J = 7.6 Hz, 2H), 7.36 (d, J = 8.2 Hz, 1H), 6.67 (s, 1H),
1
3
5
.16 (s, 2H), 4.04 (s, 3H), 3.90 (s, 3H), 3.86 (s, 3H); C NMR (125 MHz, CDCl +DMSO-
3
d6): δ 189.9, 166.9, 165.1, 152.1, 151.7, 151.6, 142.8, 135.2, 133.7, 133.5, 128.4, 127.6,
126.1, 125.0, 117.3, 115.9, 111.9, 107.9, 96.6, 55.9, 55.6, 47.0; HRMS (ESI): m/z calcd for
+
C H N O S 530.1386, found 530.1391[M + H] .
28
24
3
6
3.1.3.20. (Z)-5-((2-(3,4-Dimethoxyphenyl)-1H-benzo[d]imidazol-5-yl)methylene)-3-(2-oxo-
2-phenylethyl)thiazolidine-2,4-dione (17t)
-1
Yellow solid; mp: 237–239 °C; FT-IR (cm ): 3350, 2957, 1735, 1667, 1578, 1470, 1220,
1
1
7
7
007, 815; H NMR (500 MHz, CDCl +DMSO-d ): δ 8.11 (s, 1H), 8.08 (d, J = 7.2 Hz, 2H),
3
6
.84 (s, 1H), 7.80–7.78 (m, 2H), 7.73–7.68 (m, 2H), 7.59–7.56 (m, 2H), 7.48–7.46 (m, 1H),
1
3
.11 (d, J = 8.4 Hz, 1H), 5.28 (s, 2H), 3.92 (s, 3H), 3.87 (s, 3H); C NMR (125 MHz,
CDCl +DMSO-d ): δ 190.7, 166.9, 165.1, 153.8, 150.7, 148.7, 135.2, 134.0, 133.6, 128.7,
3
6
128.0, 126.5, 124.8, 121.5, 119.8, 117.6, 111.4, 109.8, 55.4, 47.5; HRMS (ESI): m/z calcd
+
for C H N O S 500.1280, found 500.1280 [M + H] .
2
7
22
3
5
3
.2. Biological assays
.2.1. Cell cultures
3
Breast (MDAMB-231), prostate (PC-3), cervical (HeLa), lung (A549), bone (HT1080) and
normal kidney cells (HeK-293T) were procured from ATCC (American Type Cell Culture
Collection), Maryland, USA and were grown in suitable MEM (Minimum Essential
1
6

ACCEPTED MANUSCRIPT
Medium, Sigma) or DMEM (Dulbecco Modified Eagle medium, Sigma) with supplement
of 10% fetal bovine serum stabilized with 1X antibiotic-antimycotic solution (Sigma) in a
CO incubator with 5% CO and 90% relative humidity at 37 °C. When the confluency of
2
2
cells reached upto 80-90%, were treated with 0.25 % trypsin/1 mM EDTA solution for
further passage.
3.2.2. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay
The cytotoxic potential of compounds 17a–t was examined using MTT reduction assay. 1 ×
4
1
0 cells/well were seeded in 96-well plates in 100 µL DMEM, supplemented with 10%
FBS in each well and incubated in CO incubator for 24 h at 37 °C. The medium was
2
replaced with culture medium containing test compounds dissolved in DMSO (Dimethyl
sulfoxide). Wells having only DMSO were used as the control. After incubating for 48 h, 10
µL of MTT was added to each well and further incubated for 4 hour in dark at 37 °C. After
4
h of incubation period, the MTT media was removed and DMSO (100 µL) was put into
each well to dissolve the crystallized formazan product. The absorbance was recorded on a
micro-plate reader 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 performed at least three
independent experiments and IC values were computed by using Probit Software.
5
0
3.2.3. In vitro cell migration assay/Wound healing assay
5
A549 cells (5 × 10 cells/well) were grown as confluent monolayers for 24 h in six well
plate. Then artificial wound/scratch were created on cell monolayers using 200 µL sterile tip
of pipette and non-adherent cells were removed by washing twice with PBS (Phosphate-
buffered saline). The media containing IC concentrations of compounds 17p and 17q were
5
0
added to each well and the wells without compound were shown as Control. The
movement/migration of cells across the wounded/scratched area was photographed by using
Nikon phase contrast microscope immediately at 0 h, 24 h and 48 h of time interval
subsequent to treatment in three or more randomly selected fields.
3.2.4. F-actin staining
6
A549 cancer cells (1 × 10 cells/well) were grown on cover slips for 24 h in 6 well plate and
incubated with IC concentrations of compound 17p and 17q for 24 h. After compounds
5
0
treatment, the cells were washing with PBS and fixed with 4% para-formaldehyde solution.
1
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Cells were incubated with rhodamine-phalloidin (red fluorescent dye) for staining of F-actin
and with Hoechst 33242 for nuclear staining. Then cells were washed thrice with PBS and
mounted with ProLong Gold anti-fade reagent (Molecular Probes, Eugene, OR) on
microscopic slide and visualized by microscopy (Nikon). The fluorescent images were
captured by using 20X magnification lenses.
3.2.5. Hoechst nuclear staining
4
A549 lung cancer cells (5 × 10 cells/well) were cultured on coverslips in a 6 wells plate
and permitted to adhere for 24 h. The culture medium containing IC concentrations of
5
0
compound 17p and 17q were added to them. After incubation for 24 h, culture medium was
separated; cells were washed and fixed with 4% para-formaldehyde solution for 10 min at 4
°
C. Cells were washed two times with PBS and stained with Hoechst 33242 (5 µg/
mL) at room temperature for 30 min. The excess dye was separated by washing cells two
times with PBS and suspension of cells was mounted on slides and examined for change in
the morphology under fluorescence microscopy at an absorbance of 350 nm for excitation
and 460 nm for emission (Nikon, magnification 40x).
3.2.6. Measurement of reactive oxygen species (ROS) levels
Intracellular ROS levels in A549 cancer cells were assessed by using DCFH-DA (2'-7'-
5
dichlorodihydrofluorescein diacetate). A549 cells (5 × 10 cells/mL) were plated in 6 wells
plate and were allowed to grow overnight. The cells were incubated for 24 h with IC50
concentrations of 17p and 17q. After treatment with compounds, the media was replaced
with DCFH-DA (10 µM) containing culture medium followed by incubation for 30 minutes
4
at room temperature in dark. Cells (5 × 10 cells/mL) were washed with PBS, collected
from well-plate and suspended again in PBS. The fluorescence intensity of the DCF (2', 7'-
dichlorofluorescein produced by the hydrolysis of DCFH-DA from each sample was
analyzed by spectrofluorometer at an excitation of 488 nm and emission wavelength 525
nm. Fluorescent images were captured by using Nikon ECLIPSETE2000-S fluorescence
microscope.
3.2.7. Measurement of Mitochondrial Membrane Potential (DΨm)
5
A549 cells (5 × 10 cells/mL) were cultured in 6 well plates and allowed to adhere
overnight. The cells were treated with IC concentrations of 17p and 17q for 24 h. After
5
0
24 h of treatment, the adherent cells were collected by trypsinisation, washed with PBS (500
1
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µL) and suspended in a solution of PBS containing rhodamine-123 (10 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. The samples were analyzed for
fluorescence using spectrofluorometer.
3.2.8. Annexin V-FITC/propidium iodide dual staining assay
To quantify the percentage of apoptotic cells, annexin V-FITC/propidium iodide dual
6
staining assay was performed using A549 cells. Cells (10 /mL per well) were seeded in six-
well plates and allowed to grow for 24 h. After treatment with IC concentrations of 17p
5
0
and 17q for 24 h, cells were collected by trypsinisation. The collected cells were then
washed two times with ice-cold PBS, then incubated with 200 µL1 × binding buffer
containing 5 µL Annexin V-FITC, and then in 300 µL1 × binding buffer containing 5 µL
Propidium iodide (PI) at room temperature in the dark for 5 min and incubate for 15 min.
After 15 min of the incubation period, cells were analysed for a percentage of apoptosis
using BD-C6 accuri flow-cytometer.
4. Conclusion
In conclusion, a series of new benzimidazole-containing thiazolidinedione derivatives was
synthesized by using conventional as well as microwave-assisted technique and evaluated
for their in vitro cytotoxic activity against different human cancer cell lines viz. MDAMB-
231, PC-3, HeLa, A549, HT1080 and a normal kidney cell (HeK-293T). Some of the
synthesized compounds were found to be more potent with IC values ranging from 0.096
5
0
to 0.98 µM. Apart from this, a number of compounds were found to be active against A549
cancer cells below 1.0 µM in comparison to others tested cancer cells. The cytotoxic
profiles on normal HeK-293T cells revealed that most of these compounds were more
selective towards the cancer cells when compared to normal cells. The exposure of A549
lung cancer cells to 17p and 17q resulted in remarkable inhibition of cell migration through
disruption of F-actin assembly. The apoptosis inducing effect of the compounds were
studied by different techniques like Hoechst 33242 nuclear staining, DCFH-DA,
rhodamine-123, and annexin V-FITC/propidium iodide staining. Moreover, 17p and 17q
treatment led to the collapse of the mitochondrial membrane potential (DΨm) and increased
levels of reactive oxygen species (ROS) in A549 cells. Overall, the different studies
suggested that these new compounds have the potential to be developed as cytotoxic agents
1
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and their structural modifications could lead to the generation of promising anticancer
agents.
Acknowledgements
P.S. is thankful to DoP, Ministry of Chemicals & Fertilizers, Govt. of India, for the award
of NIPER fellowship. Dr. N. Shankaraiah gratefully acknowledges SERB, DST, Govt. of
India for research grant (YSS-2015-001709).
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Table 1. Time and yield data of newly synthesized compounds 17a–t using conventional
and microwave irradiation method.
a
b
Compound
Conventional Method
time (h)
Microwave Method
time (min)
c
c
% yield
61
63
61
71
67
57
64
65
59
61
65
64
66
58
67
62
70
63
79
66
% yield
79
85
82
86
79
74
76
78
76
78
85
80
80
78
84
88
91
95
92
88
1
1
1
1
1
1
1
7a
7b
7c
7d
7e
7f
7g
8
7
8
10
5
5
6
5
5
8
8
8
7
7
7
7
10
7
7
8
8
8
10
5
5
5
17h
5
17i
5
17j
5
1
1
1
1
7k
7l
7m
7n
9
6
7
8
1
1
1
1
7o
7p
7q
7r
7s
6
6
11
5
5
1
1
7t
5
5
a
Conventional Method: Toluene, piperidine (cat.), reflux, 5–11 h.
Microwave-assisted synthesis: Toluene, piperidine (cat.), 150 C, 5–10 min.
b
o
c
Isolated yields
2
3

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Table 2. IC values (in µM) of benzimidazole-thiazolidinediones 17a–t on selected human
5
0
a
cancer cell lines and a normal kidney cell (HeK-293T)
b
c
d
e
f
g
Compound MDAMB-231
PC-3
HeLa
A549 HT1080
HeK-293T
1
1
1
1
1
1
1
7a
7b
7c
7d
7e
7f
7g
6.83
13.6
24.53
7.78
14.90
>50
21.3
34.92
>50
11.97
8.72
6.32
2.98
5.94
>50
11.7
14.66
>50
8.37
NT
2.50
4.37
0.63
10.24
>50
0.11
>50
NT
1.67
7.83
15.4
5.22
16.3
7.51
0.51
23.27
6.89
4.78
NT
0.95
1.44
0.18
2.24
0.32
0.25
26.26
NT
3.57
0.57
1.84
5.11
2.93
8.93
0.66
0.98
14.5
0.91
NT
0.67
0.93
0.13
0.53
0.13
0.096
0.88
NT
3.12
2.43
6.35
4.45
1.68
3.32
1.12
3.65
8.76
2.47
NT
0.66
4.58
0.19
0.98
0.27
0.16
0.57
NT
13.6
21.7
15.3
14.1
18.79
>50
35.7
21.5
>50
12.7
NT
8.21
18.9
3.35
16.6
6.76
6.65
13.57
NT
1
7h
1
1
7i
7j
2.88
NT
1
7k
7l
7m
7n
1
1
1
2.29
3.26
1.78
0.27
29.10
4.37
>50
1
1
1
7o
7p
7q
1
1
7r
7s
NT
1
N
7t
2.04
-
0.43
-
7.48
2.83
0.74
1.87
0.59
-
27.8
-
h
a
c
b
5
0% inhibitory concentration after 48 h of compound treatment; breast cancer cells;
d
e
f
g
prostate cancer cells; cervical cancer cells; lung cancer cells; bone cancer cells; Normal
h
Kidney cells; N: Nocodazole as standard; NT - not tested.
Figure captions
Figure 1. The structure of some bioactive thiazolidinediones and benzimidazoles.
Figure 2. The design of small molecules bearing benzimidazole-thiazolidinedione as
anticancer agents.
Figure 3. Effect of compounds 17p and 17q on migration potential of A549 cells. Cells
were exposed to IC concentrations of 17p and 17q and scratched with sterile 200 µL
5
0
pipette tip. The images were captured at 0, 24 and 48 h using phase contrast microscope.
Figure 4. Effects of compounds 17p and 17q on F-actin polymerisation in A549 cancer
cells. The cells were treated with IC50 concentrations of 17p and 17q and stained with
2
4

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Rhodamine-phalloidin (red fluorescent dye: actin filament) and Hoechst 33242 (nucleus:
blue).
Figure 5. Effect of compounds 17p and 17q on nuclear morphology of A549. Cells were
treated with IC concentrations of 17p and 17q and stained with Hoechst 33242 for 24 h.
5
0
Images were captured by a fluorescence microscope (Nikon).
Figure 6. Effect of compounds 17p and 17q on intracellular reactive oxygen species (ROS)
production. A549 Cells were treated with IC concentrations of 17p and 17q for 24 h and
5
0
stained with DCFH-DA and photographed using fluorescence microscope (Nikon).
Figure 7. Compound 17p and 17q triggered change in mitochondrial membrane potential
(
DΨm). (a) A549 cells were treated with Control-DMSO and IC concentrations 17p and
50
17q and processed by rhodamine-123 staining followed by flow cytometry analysis. The
peak change to the left indicate depolarization. (b) Data shown are mean ± SD from three
independent experiments.
Figure 8. Annexin V- FITC/propidium iodide dual staining assay. A549 cells were treated
with IC concentrations of 17p and 17q and stained with Annexin V-FITC/PI and analysed
5
0
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 captions
Scheme 1. Synthesis of thiazolidinedione 2 and N-substituted thiazolidinediones 6a–d, 8a–c
and 10.
Scheme 2. Synthesis of benzimidazole-thiazolidinedione derivatives 17a–t.
2
5

ACCEPTED MANUSCRIPT
N
N
H
Br
N
O
S
Cl
R
O
N
O
O
O
S
N
O
HN
S
O
O
Benzsuberone-thiazolidinedione
GSK1059615
α-bromoacryloylamido-thiazolidinedione
COOH
N
H N
O
2
O
O
NH
S
N
O
N
Cl
N
N
N
H
N
H
N
H
Cl
Veliparib
Bendamustine
Nocodazole
Figure 1. The structure of some bioactive thiazolidinediones and benzimidazoles.
R6
H
N
O
N
Different
Substitutuions at Tail part
Nitrogen
R5
S
Head part
N
R4
R3
O
Figure 2. The design of small molecules bearing benzimidazole-thiazolidinedione as
anticancer agents.
Figure 3. Effect of compounds 17p and 17q on migration potential of A549 cells. Cells
were exposed to IC concentrations of 17p and 17q and scratched with sterile 200 µL
5
0
pipette tip. The images were captured at 0, 24 and 48 h using phase contrast microscope.
2
6

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Figure 4. Effects of compounds 17p and 17q on F-actin polymerisation in A549 cancer
cells. The cells were treated with IC50 concentrations of 17p and 17q and stained with
Rhodamine-phalloidin (red fluorescent dye: actin filament) and Hoechst 33242 (nucleus:
blue).
Figure 5. Effect of compounds 17p and 17q on nuclear morphology of A549. Cells were
treated with IC concentrations of 17p and 17q and stained with Hoechst 33242 for 24 h.
5
0
Images were captured by a fluorescence microscope (Nikon).
2
7

ACCEPTED MANUSCRIPT
Figure 6. Effect of compounds 17p and 17q on intracellular reactive oxygen species (ROS)
production. A549 Cells were treated with IC concentrations of 17p and 17q for 24 h and
5
0
stained with DCFH-DA and photographed using fluorescence microscope (Nikon).
Figure 7. Compound 17p and 17q triggered change in mitochondrial membrane potential
(
DΨm). (a) A549 cells were treated with Control-DMSO and IC concentrations 17p and
50
17q and processed by rhodamine-123 staining followed by flow cytometry analysis. The
peak change to the left indicate depolarization. (b) Data shown are mean ± SD from three
independent experiments.
2
8