Synthesis of imidazothiadiazole-benzimidazole conjugates as mitochondrial apoptosis inducers

Article abstract of DOI:10.1039/c4md00219a

A series of imidazothiadiazole-benzimidazole conjugates (3a-z) were synthesized and evaluated for their cytotoxic activity against a set of four selected human cancer cell lines. Amongst them, compounds 3b and 3y exhibited significant antiproliferative activity in ME-180 (cervical) cell line. Flow cytometric analysis showed that these two compounds arrested the cell cycle in the G₀/G₁ phase leading to the loss of mitochondrial membrane potential followed by apoptotic cell death. Further, Hoechst 33258 staining, DNA fragmentation assay, Annexin V staining assay and caspase-3 also suggested that 3b and 3y induced cell death by apoptosis. Docking studies revealed that compound 3b binds to the Gly142, Phe101, Asn140 and Arg143 on B-cell lymphoma 2 (Bcl-2) proteins and inhibition of Bcl-2 protein could be the possible mechanism of action for these compounds.

Full text of DOI:10.1039/c4md00219a

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Synthesis of imidazothiadiazole–benzimidazole
conjugates as mitochondrial apoptosis inducers†
Cite this: DOI: 10.1039/c4md00219a
a
Ahmed Kamal, Swapna Ponnampalli,a M. V. P. S. Vishnuvardhan,a
*
M. P. Narasimha Rao,a Kishore Mullagiri,a V Lakshma Nayaka and Bagul Chandrakantb
A series of imidazothiadiazole–benzimidazole conjugates (3a–z) were synthesized and evaluated for their
cytotoxic activity against a set of four selected human cancer cell lines. Amongst them, compounds 3b and
3y exhibited significant antiproliferative activity in ME-180 (cervical) cell line. Flow cytometric analysis
showed that these two compounds arrested the cell cycle in the G0/G1 phase leading to the loss of
mitochondrial membrane potential followed by apoptotic cell death. Further, Hoechst 33258 staining,
DNA fragmentation assay, Annexin V staining assay and caspase-3 also suggested that 3b and 3y induced
cell death by apoptosis. Docking studies revealed that compound 3b binds to the Gly142, Phe101,
Asn140 and Arg143 on B-cell lymphoma 2 (Bcl-2) proteins and inhibition of Bcl-2 protein could be the
possible mechanism of action for these compounds.
Received 22nd May 2014
Accepted 4th August 2014
DOI: 10.1039/c4md00219a
www.rsc.org/medchemcomm
analogues reports the activity of these compounds against
numerous transplanted animal tumours.18 Terzioglu and co-
Introduction
workers reported the hydrazone derivatives of 2,6-dimethyli-
midazo[2,1-b][1,3,4]-thiadiazole-5-carbohydrazide as promising
anticancer agents against ovarian cancer cell line (OVCAR),13
this nding prompting further investigation of the antitumor
effects of imidazo[2,1-b][1,3,4]-thiadiazoles.14–17
The benzimidazole moiety is structurally related to purine
bases and is extensively found in a variety of natural products.
Several promising anticancer agents are known to contain this
heterocycle. The ring system of benzimidazole can be consid-
ered as a new device for the target-specic transcription aspect
at binding sites related to biological systems.19,20 The cytotoxic
benzimidazoles act mainly as topoisomerase inhibitors, alky-
lating agents, DNA binding agents and also as poly(ADP-ribose)
polymerase-1 (PARP-1) inhibitors.21–28 Some representative imi-
dazo[2,1-b][1,3,4]thiadiazoles and benzimidazole derivatives are
shown in Fig. 1.
Cancer is one of the major global health problems, in which a
cell or a group of cells exhibit uncontrolled growth and inva-
sion, and is the second-leading cause of death in the Western
world.1 According to the World Health Organization (WHO), it is
estimated that 12 million deaths could occur due to cancer in
2030. The discovery and development of new, effective and
more selective compounds is the most important task for the
treatment of cancer. It is credible that the future of tumor
therapy is in the development of molecularly targeted drugs that
particularly block key mechanisms that are involved in devel-
opment and progression of specic types of tumor.2 The search
for such potential anticancer agents has been directed to the
discovery of small synthetic molecules with promising anti-
carcinogenic activity. Nitrogen-bridgehead fused heterocyclic
containing an imidazole ring is a very common structural
moiety in many pharmacologically active molecules that exhibit
a wide range of activities for a diverse number of targets.3–8
One of the most widely used heterocyclic systems is imidazo
[2,1-b][1,3,4]thiadiazole. Imidazothiadiazoles show
a wide
spectrum of biological properties like antibacterial,9 anti-
fungal,10 antitubercular11 and anticancer.12–16 Imidazothiadia-
zoles display different types of molecular mechanisms in cancer
chemotherapy.13–18 Previous work on 2-amino-1,3,4-thiadiazole
aMedicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical
Technology, Hyderabad-500007, India. E-mail: ahmedkamal@iict.res.in; Fax: +91-
40-27193189; Tel: +91-40-27193157
bDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education
and Research, Hyderabad 500037, India
† Electronic supplementary information (ESI) available: Experimental procedures,
spectral data of all the nal compounds. See DOI: 10.1039/c4md00219a
Fig. 1 Structures of some representative imidazo[2,1-b][1,3,4]thia-
diazole and benzimidazole derivatives.
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Our earlier efforts toward the discovery of new synthetic
small molecules led to the development of a number of hybrids/
conjugates as potent cytotoxic agents.29–32 In continuation of
these efforts, an attempt has been made, considering the
signicant bioactivities of conjugates with an imidazo[2,1-b]-
[1,3,4]thiadiazole as the core moiety. As reported in the litera-
ture, imidazothiadiazoles exhibit tubulin polymerization inhi-
bition and show G2/M cell cycle arrest17 whereas benzimidazoles
mostly act as topoisomerase I and DNA binding agents and
show G1/G0 cell cycle arrest.25 We designed imidazothiadiazole–
benzimidazole conjugates with a view to develop effective
cytotoxic agents and to investigate their cytotoxic pathway for
mammalian cells. Therefore, in the present study we synthe-
sized some imidazothiadiazoles that are linked to a benzimid-
azole scaffold. The promising activity exhibited by some of these
conjugates prompted us to examine their role in the cell
proliferation and apoptosis of the human cervical cancer cell
line (ME-180). The biological assays of these imidazothiadia-
zole–benzimidazole conjugates show G0/G1 cell cycle arrest
leading to mitochondrial apoptotic cell death.
Results and discussion
Chemistry
The synthesis of imidazothiadiazole–benzimidazole conjugates
(3a–z) was carried out with commercially available 5-methyl-
1,3,4-thiadiazol-2-amine (4) and bromoketones (5a–d) as the
starting materials as shown in Scheme 1. The reaction between
4 and appropriate bromoketones (5a–d) in acetone produced
the intermediate compounds 6a–d, which were isolated and
used in the succeeding step without any further purication to
generate imidazo[2,1-b][1,3,4]thiadiazoles 7a–d. The imidazo
[2,1-b][1,3,4]thiadiazole aldehydes 8a–d were obtained by
employing the Vilsmeier reaction with the corresponding 7a–
d.33 The oxidative cyclization of the appropriately substituted
o-phenylenediamine and 8a–d in the presence of Na2S2O5 in
ethanol afforded the desired imidazothiadiazole–benzimid-
azole conjugates (3a–z) with good yields.34
Scheme 1 Reagents and conditions: (i) acetone, reflux, 6–8 h; (ii) 2 N
HCl, reflux, 1 h, 79–85%; (iii) POCl3, DMF, reflux, 1 h, 70–76%; (iv)
substituted o-phenylenediamine, Na2S2O5, EtOH, reflux, 4 h, 65–79%.
well as electron-withdrawing substituents while keeping the core
imidazothiadiazole–benzimidazole moiety unchanged. Some
interesting trends have been observed in the SAR studies of these
newly synthesized compounds. The substitution pattern of R1 on
the phenyl ring of the imidazothiadiazole unit shows some
impact on the antiproliferative activity of these compounds.
From the cell line assays, it appears that a number of compounds
with electron-donating substituents like H, methyl and methoxy
at R1 position (3b, 3c, 3i, 3q, 3s, 3u and 3y) exhibited considerable
cytotoxic activity (IC50 values of 0.5 to 5.8 mM and 1.04 to 7.7 mM
Biological evaluation
Anticancer activity. The conjugates 3a–z were screened for against ME-180 and A375, respectively). Whereas in the case of
their anticancer activity against a set of selected human cancer electron-withdrawing substituents like uoro at R1 position (3d,
cell lines of ME-180 (cervical), A375 (melanoma), DU145 (pros- 3j, 3o, 3r, 3t and 3w) on the phenyl ring of the imidazothiadiazole
tate) and A549 (lung) by using MTT assay35 and the results unit, there was comparatively less cytotoxicity (IC50 values of 5.8
obtained are summarized in Table 1. All these conjugates to 13.9 mM and 7.3 to 22.2 mM against ME-180 and A375,
exhibited cytotoxic activity with IC50 values ranging from 0.5 to respectively). The substitution pattern (R2, R3 and R4) on the
>30 mM, while doxorubicin and nocodazole were used as the fused phenyl ring of the benzimidazole subunit considerably
positive controls. Amongst these conjugates, two of them, 3b affects the cytotoxic activity of these conjugates. Amongst the
and 3y, showed signicant cytotoxic activity against ME-180 synthesized compounds, most of the electron-withdrawing halo
(IC50 values of 0.5 and 0.7 mM) and A375 (IC50 values of 2.8 and substituted compounds such as 3b, 3c, 3i, 3q, 3s and 3y displayed
1.04 mM) cell lines, whereas the other conjugates showed higher potency than compounds with other substitution patterns
promising to moderate activity against different cancer cell on the phenyl ring of the benzimidazole subunit. Especially,
lines.
chloro substitution at R3 and R4 played an important role in the
With a view to studying the structure–activity relationship overall activity prole of these compounds (3b, 3p, 3q and 3y)
(SAR), we varied the substitution pattern of the phenyl rings on with IC50 values in the range of 0.5 to 5.8 mM and 1.04 to 4.8 mM
imidazothiadiazole and benzimidazole with electron-donating as against ME-180 and A375, respectively.
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Table
1
Cytotoxic activitya of imidazothiadiazole–benzimidazole Table 2 Cell cycle phase distribution of ME-180 cells following
treatment with compounds 3b, 3y and doxorubicin
derivatives (3a–z) expressed in mM
Compound
ME-180b
A375c
DU145d
A549e Sample
Sub-G1
G1
S
G2/M
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
3x
19.9
0.5
5.4
13.9
15.8
3.9
16.9
3.7
22.5
2.8
7.7
17.7
22.9
5.2
20.8
5.8
19.8
19.1
25.1
>30
>30
19.6
>30
14.2
>30
15.8
23.3
>30
25.1
16.9
19.9
14.4
15.8
>30
7.3
>30
10.7
12.5
>30
14.4
11.8
20.6
2.4
>30
13.6
16.9
>30
18.4
13.6
16.4
16.2
17.9
12.7
16.5
16.6
18.4
Control
3b; 0.5 mM
3b; 1 mM
3y; 0.5 mM
3y; 1 mM
DOXO; 1 mM
7
5.4
16.3
14.1
11.6
7.7
59.9
68.1
72.4
66.6
75.7
64.8
19.3
15.7
9.3
12.3
10.1
16.1
13.2
10.4
1.6
6.7
2.3
10.9
4.6
5.8
6.8
7.3
phase and was comparable to doxorubicin at 1 mM. Doxorubicin
at lower concentration arrests G2/M phase (data not shown);
however upon increasing the concentration to 1 mM, it shows
G0/G1 arrest. Interestingly, this was also accompanied by
compensatory decrease in G2 phase of cells. Conjugates 3b and
3y caused a G0/G1 phase arrest, as the majority of accumulation
effect was detected by exposure for 48 h. These results suggested
that both the conjugates (3b and 3y) inhibited the cellular
proliferation via G0/G1 phase arrest in a dose-dependent
manner.
Hoechst staining. Apoptosis is one of the major pathways
that lead to the process of cell death. The classic characteristics
of apoptosis are chromatin condensation and fragmented
nuclei. Therefore it was considered of interest to investigate the
apoptosis-inducing effect of these two potential conjugates (3b
and 3y) by Hoechst staining (H 33258) on the cervical cancer cell
line (ME-180). In this assay, cells were treated with 3b and 3y at
1 mM for 24 h. Manual eld quantication of apoptotic cells
based on cytoplasmic condensation, presence of apoptotic
bodies, nuclear fragmentation and relative uorescence of the
test compounds (3b and 3y) was used as a measure of how well
these compounds induced cell death by apoptosis (Fig. 2).
DNA fragmentation assay. DNA fragmentation is well known
and a typical biochemical hallmark of apoptotic cell death.
From the cytotoxicity studies, it was observed that compounds
12.3
16.9
8.7
15.7
6.6
1.9
2.6
10
3.6
18.1
4.5
9.7
6.1
15.8
0.7
16.4
0.5
1.7
12.5
19.9
10.9
18.1
7.7
>30
11.7
10.2
7.9
>30
4.4
>30
9.8
10.3
>30
12.5
10.3
19.4
1.1
4.8
4.3
14.2
5.01
22.2
6.1
11.4
7.6
20.4
1.04
19.07
1.2
3y
3z
Doxorubicin
Nocodazole
0.2
2.1
2.5
a
IC50 or 50% inhibitory concentration, each value being the average of
three individual experiments. b Cervical cancer. c Melanoma. d Prostate
cancer. e Lung cancer.
These observations reveal that halo substitution on the fused
phenyl ring of the benzimidazole subunit and electron-
donating groups on the phenyl ring of the imidazothiadiazole
unit are important for maintaining antiproliferative activity.
Therefore, the conjugates 3b and 3y with 6-chloro and 5-chloro/
uoro on the phenyl ring of the benzimidazole subunit and
unsubstituted/methyl-substituted phenyl ring of the imidazo-
thiadiazole unit exhibited higher cytotoxicity against ME-180
(IC50 values of 0.5 and 0.7 mM) and A375 (IC50 values of 2.8 and
1.04 mM) cell lines as compared with other substitution patterns
of these conjugates. Hence, the SAR studies based on the cell
viability values obtained for ME-180 cells encouraged us to
investigate the inuence of conjugates 3b and 3y on the cell
cycle.
Cell cycle analysis. To study the mechanism underlying the
cytotoxic activity of 3b and 3y, cell cycle disruption and
apoptotic ability of these conjugates were investigated with
ME-180 cells. The effect on cell cycle distribution was examined
by uorescence activated cell sorting (FACS) analysis.36 ME-180
cells were exposed to 0.5 and 1 mM of 3b and 3y for 48 h.
Compound 3b showed 74% G0/G1 at 0.5 mM, which, upon
increasing the concentration to 1 mM was 88.7%, whereas 3y
showed 80.7% at 0.5 mM and 87.3% at 1 mM (Table 2). These
Fig. 2 Hoechst staining of the conjugates (3b and 3y) at 1 mM in
results showed that the accumulation of cells was in the G0/G1 cervical cancer cells (ME-180).
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The DNA fragmentation analysis37 revealed that compounds
3b and 3y induced a discrete ladder pattern in ME-180 cell line
at 1 mM aer 48 h of incubation thereby showing signicant
fragmentation in a similar pattern to that of doxorubicin, which
was used as a positive control; however, no such effect was
observed in untreated cells (Fig. 3).
Annexin V FITC dual staining assay. The apoptotic effect of
these conjugates (3b and 3y) was further evaluated by Annexin V
FITC/PI (AV/PI) dual staining assay38–41 to examine the occur-
rence of phosphatidylserine externalization and also to under-
stand whether it is due to physiological apoptosis or nonspecic
necrosis. In this assay, ME-180 cells were treated with 3b and 3y
for 48 h at 2 mM concentration to study the apoptotic effect. It
was observed that these compounds showed promising
apoptotic effect against ME-180 cells as shown in Fig. 4, and the
results indicated that 3b and 3y showed 22.6% and 16.5%
apoptosis, respectively. In the untreated cells (control) 5%
apoptosis was observed and the standard doxorubicin showed
15.1% apoptosis at 2 mM concentration. These results suggest
that the two conjugates induce apoptosis in ME-180 cells.
Moreover these studies clearly indicated that the cell cycle arrest
at G0/G1 phase takes place by the induction of cellular
apoptosis.
Fig. 3 DNA fragmentation of the conjugates (3b and 3y) in ME-180
cancer cells.
3b and 3y considerably inhibit the growth of human cervical
cancer cell line ME-180. Therefore it was of interest to deter-
mine the mechanism of cell death in the same cell line.
Fig. 4 Annexin V FITC: control cells of ME-180, ME-180 cells treated with 3b, 3y and doxorubicin for 24 h at 1 mM. Q2 (late apoptotic); Q4 (early
apoptotic).
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Fig. 5 The loss of mitochondrial membrane potential was monitored by JC-1 stain. High DJm in control mitochondria causes association with
the dye in an aggregated state leading to red-orange fluorescence emission. Compounds 3b and 3y lead to dissipation of DJm, and the dye is
now partitioned into the cytosol, resulting in green fluorescence.
cytotoxic effect of 3b and 3y by virtue of apoptotic cell death.
This prompted us to treat ME-180 cells with compounds 3b and
3y to examine the activation of caspase-3,45 with doxorubicin as
a positive control. The results indicate about 3- to 5-fold
increase in caspase-3 activity in cells treated with a 1 mM
concentration of these compounds (Fig. 6).
However, under identical conditions, doxorubicin showed
about 3- to 4-fold increase in caspase-3 activity as compared to
the control. These results are in line with the prediction of
apoptosis induction by the activation of caspase-3 in ME-180
cells treated with 3b and 3y.
Loss of mitochondrial membrane potential using JC-1 dye.
The loss of mitochondrial membrane potential (DJm) is a
major characteristic of apoptosis.42 Measurement of DJm
serves as a marker to estimate the overall function of mito-
chondria during the induction of apoptosis. Therefore, in this
study we examined the effect of 3b and 3y on DJm. ME-180 cells
were treated with compounds 3b and 3y at 1 mM concentration
for 24 h and stained with JC-1 dye. In the control cells, the dye
was concentrated in the mitochondrial matrix where it forms
red uorescent aggregates (J-aggregates) because of the elec-
trochemical potential gradient. Whereas in the cells treated
with 3b and 3y, apoptosis was induced and the mitochondrial
membrane was depolarized thus preventing the accumulation
of the JC-1 dye in the mitochondria. Therefore, the dye in the
monomeric form is dispersed throughout the entire cell leading
to a shi in the colour from orange to green uorescence (JC-1
monomers). The apoptotic cells with green uorescence are
easily differentiated from the healthy cells with orange uo-
rescence. Therefore it was quite evident that these conjugates
(3b and 3y) signicantly depolarize the mitochondrial
membrane potential (Fig. 5) thereby inducing cell death in ME-
180 cells.
Molecular docking studies. Biological studies have shown
that these molecules induce cell death by a mitochondrial
apoptotic pathway. Aer the treatment of cancer cells with these
molecules, a change in membrane potential was observed. To
Activation of caspase induction. Caspase-3 is activated in the
apoptotic cell, both by extrinsic (death ligand) and intrinsic
(mitochondrial) pathways.43 In intrinsic activation, cytochrome-
c from the mitochondria works in combination with caspase-9,
apoptosis-activating factor 1 (Apaf-1), and ATP to process pro-
caspase-3.44 These molecules are sufficient to activate caspase-3
in vitro, but other regulatory proteins are necessary for in vivo
Fig. 6 Effect of the compounds 3b, 3y and doxorubicin on caspase-3
activity. ME-180 cells were treated for 48 h with 1 mM concentration.
Doxorubicin is used as a positive control. Values indicated are the
activation. Hence it was considered worthwhile to examine the mean Æ SD of two different experiments performed in triplicate.
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assay proved that these conjugates induce cell death by mito-
chondrial apoptosis. Flow cytometric analysis revealed that the
cell cycle arrest is in the G0/G1 phase. Further, Hoechst 33258
staining, DNA fragmentation assay, Annexin staining and acti-
vation of caspase-3 also provided evidence that they caused cell
death by apoptosis. Docking studies of 3b and 3y on Bcl-2
protein suggested that Bcl-2 inhibition could be the possible
mechanism for the cytotoxic activity of these compounds.
Moreover, the overall results indicate that these conjugates
based on the imidazothiadiazole–benzimidazole scaffold have
the potential to be exploited for their anticancer property and
could be considered as an interesting lead for the efficient
treatment against cervical cancer.
Acknowledgements
The authors P. S., M. P. N. R., M. V. P. S. V., and M. K. are
thankful to CSIR, New Delhi and UGC, New Delhi for the award
of research fellowships. We also acknowledge CSIR for nancial
support under the 12th Five Year Plan Project “Affordable
Cancer Therapeutics (ACT)” (CSC0301).
Fig. 7 Docking pose for compound 3b. The compound is shown in
yellow and the protein is shown in green. Hydrogen bond is shown as a
red dotted line. Residues forming hydrogen bonding interactions and
p–p interactions are highlighted in different colors.
investigate the possible mechanism, docking studies were
carried out with compounds 3b and 3y on B-cell lymphoma 2
(Bcl-2) protein. Coordinates of protein structure were obtained
from the Protein Data Bank (PDB ID 2O22).46 Geometry of the
molecules was optimized by Gaussian 09 using PM3 semi-
empirical method.47 Docking studies were performed using
AutoDock 4.2 docking soware.48 For the purpose of docking
protocol validation, docking of reference ligand was performed.
Docking results showed a similar docking pose to the pose of
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