2-(4-Chlorobenzyl)-6-arylimidazo[2,1-b][1,3,4]thiadiazoles: Synthesis, cytotoxic activity and mechanism of action

Article abstract of DOI:10.1016/j.ejmech.2014.07.054

The cytotoxic activity of a new series of 2-(4′-chlorobenzyl)-5,6- disubstituted imidazo[2,1-b][1,3,4]thiadiazoles against different human and murine cancer cell lines is reported. Among the tested compounds, two derivatives namely 2-(4-chlorobenzyl)-6-(2

Full text of DOI:10.1016/j.ejmech.2014.07.054

European Journal of Medicinal Chemistry 84 (2014) 687e697
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
2-(4-Chlorobenzyl)-6-arylimidazo[2,1-b][1,3,4]thiadiazoles: Synthesis,
cytotoxic activity and mechanism of action
,
,
Sujeet Kumar a 1, Mahesh Hegde b 1, Vidya Gopalakrishnan b, Vinaya Kumar Renuka a,
Sureshbabu A. Ramareddy a, Erik De Clercq d, Dominique Schols d,
Anil Kumar Gudibabande Narasimhamurthy c, Sathees C. Raghavan b, Subhas S. Karki a
,
*
a Department of Pharmaceutical Chemistry, KLE University's College of Pharmacy, Bangalore 560010, India
b Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
c Dept. of Physics, M. S. Ramaiah Institute of Technology, Bangalore 560054, India
d Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
The cytotoxic activity of a new series of 2-(40-chlorobenzyl)-5,6-disubstituted imidazo[2,1-b][1,3,4]
thiadiazoles against different human and murine cancer cell lines is reported. Among the tested com-
pounds, two derivatives namely 2-(4-chlorobenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]
thiadiazole-5-carbaldehyde 4i and 2-(4-chlorobenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]
thiadiazol-5-yl thiocyanate 5i emerged as the most potent against all the cell lines. To investigate the
mechanism of action, we selected compounds 4i for cell cycle study, analysis of mitochondrial membrane
potential and Annexin V-FITC flow cytometric analysis and DNA fragmentation assay. Results showed
that 4i induced cytotoxicity by inducing apoptosis without arresting the cell cycle.
Article history:
Received 11 June 2013
Received in revised form
15 July 2014
Accepted 16 July 2014
Available online 17 July 2014
Keywords:
Anti-cancer drugs
Cancer therapeutics
Cytotoxicity
© 2014 Elsevier Masson SAS. All rights reserved.
Apoptosis
Cell death
1. Introduction
new compounds with improved activity against several tumor cell
lines, which was superior to standard antitumorals such as
Cancer is a class of diseases in which cells, or a group of cells
display uncontrolled growth, invasion, and sometimes metastasis.
It affects people at all ages with the risk of most types increasing
with age. Earlier we reported on the antitumor activity of 2-aralkyl-
5-substituted-6-(4-fluorophenyl)imidazo[2,1-b][1,3,4]thiadiazoles
[1] which were mainly synthesized according to Scheme 1. Thus,
the 2-amino-5-(4-chlorobenzyl)-1,3,4-thiadiazole 1 was treated
with 4-fluorophenacyl bromide to obtain compound 2a (R ¼ C6H4-
4-F) [1]. This compound served as a precursor of the bromo-,
formyl- and thiocyanate derivatives (3a), (4a) and (5a) (R ¼ C6H4-4-
F), respectively [1]. The promising results obtained in the cytotox-
icity assays prompted us to prepare second-generation derivatives
where the 4-fluorophenyl group was replaced by other substituted
phenyl rings or by a coumarin-3-yl group [2e4]. It is known that the
substitution on C-5 is important for cytotoxic activity. As will be
seen in the Results and discussion section, this approach yielded
melphalan or parental compound Levamisole.
2. Chemistry
In order to determine structureeactivity (SAR) relationships, 36
compounds were prepared according to the strategy outlined in
Scheme 1. Different 2-bromoketones were used in the first step, to
afford compounds 2aei (Table 1). The bromination step was carried
out with bromine in glacial acetic acid, yielding compounds 3aei,
while the formylation used standard Vilsmeier conditions to give
compounds 4aei. The thiocyanation was performed by treatment
with bromine and potassium thiocyanate, affording products 5aei
(Table 1).
3. Results and discussion
3.1. Chemistry
* Corresponding author.
All the synthesized compounds showed absorption bands
E-mail address: subhasskarki@gmail.com (S.S. Karki).
Equal first authors.
ranging from 3185 to 3004 cmꢀ1 for CeH aromatic stretching and
1
http://dx.doi.org/10.1016/j.ejmech.2014.07.054
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

688
S. Kumar et al. / European Journal of Medicinal Chemistry 84 (2014) 687e697
Scheme 1. Synthesis of 2,5,6-substituted imidazo[2,1-b][1,3,4]thiadiazoles.
2980e2824 cmꢀ1 for CeH aliphatic stretching. Compounds 4aei
showed vibration bands between 1672 and 1653 cmꢀ1 for C]O
stretching. Compounds 5a to 5i showed vibration bands at
2163e2157 cmꢀ1 for SCN in their respective IR spectra.
There was not much improvement in cytotoxic activity of
compounds 2aei with respect to standard. When a bromine group
was introduced at the 5th position of the imidazo[2,1-b][1,3,4]
thiadiazole moiety resulting in the formation of 5-bromo de-
rivatives (3aei), the IC50 value of the coumarin analog (compound
In 1H NMR, the presence of singlet between
d
8.64 and 7.88 ppm
for the imidazole proton (C5eH) confirmed the cyclization of 2-
amino-5-(4-chlorobenzyl)-1,3,4-thiadiazole with respective
3i, IC50 3.7e5.2 mM) towards all cell lines was, at average, two times
1
less potent, than the reference drug melphalan (IC50 1.4e2.1).
However, when the eCHO group was introduced at the C-5
position of the imidazo[2,1-b][1,3,4]thiadiazole moiety, which
resulted in the formation of the 5-formyl derivatives (4aei), there
was much improvement in cytotoxicity towards all three-cell lines
phenacyl bromide. All the electrophilic substitution reactions car-
ried out on imidazo[2,1-b][1,3,4]thiadiazole derivatives 2aei,
afforded the expected 5-substituted derivatives (3aei, 4aei, 5aei).
All these 5-substituted derivatives showed the absence of C5eH in
their respective spectra and confirmed the substitution at C-5 po-
by compounds 4c, 4d and 4i (IC50 0.7e6.0 mM). Compound 4i was
sition. Derivatives 4aei showed a singlet between
9.94 ppm for the CHO proton. All the compounds showed promi-
nent signals for aromatic protons around 8.91e6.92 ppm. Bridge
headed methylene proton at C2 appeared between 4.60 to
4.26 ppm for all derivatives. The compounds 2d, 3d, 4d, and 5d
showed OCH3 proton between 3.84e3.78 ppm. Compounds 2e,
3e, 4e and 5e showed a methyl proton between 2.37e2.32 ppm as
d
10.08 to
three times more cytostatic against L1210, CEM and HeLa tumor
cells than melphalan.
In separate experiments, when the eSCN group was introduced
at the C-5 position of the imidazo[2,1-b][1,3,4]thiadiazole moiety,
which resulted in the formation of 5-thiocyanate derivatives
d
d
d
(5aei), the cytotoxicity of compound 5i (IC50 1.0e1.4 mM) was two
times more potent against all cell lines tested.
d
singlet. The structures of all the compounds were finally ascer-
tained by mass spectra. In the case of a representative compound
(3b), the structure was also determined by X-Ray analysis, as
commented upon in the Supporting information section.
The structureeactivity relationship has been studied at the C-5
and C-6 position in the skeleton of imidazo[2,1-b][1,3,4]thiadiazole.
Substitution of bromine at C-5 position did not produce any
improvement in cytotoxicity activity, except for compound 3i (IC50
3.7e5.2 mM) with the coumarin moiety at the C-6 position. Intro-
duction of a formyl group at the C-5 position by Vilsmeier reaction
resulted in improvement of cytotoxic activity (see compounds 4c
3.2. Bioevaluations
IC50 4.0e4.6
Replacement of the phenyl group at the C-6 by a coumarin-3-yl
group gave compound 4i with an IC50 of 0.89 M, 0.75 M and
0.90 M for L1210, CEM & HeLa, respectively. In the next step of
modification, eSCN group was introduced at the C-5 position and
produced an improvement in cytotoxic activity (compounds 5a, 5d,
mM, 4d IC50 3.8e6.0 mM and 4i IC50 0.7e0.9 mM).
The cytotoxic activities of the 36 compounds were evaluated
against human HeLa cervical carcinoma, CEM T-lymphocytes and
murine L1210 leukemia cells, and the most potent compounds were
studied for their cytotoxicity against REH and K562 cells. The mode
of cytotoxic action was also studied using REH and K562 cells, as
described below.
m
m
m
5g & 5i) in the range of 1.0 mMe24 mM for all three cell lines.
The compounds 4i and 5i exhibited highest levels of cytotox-
icity, based on above studies and were tested further in two leu-
kemia cell lines to evaluate their mechanism of action (Table 3).
3.2.1. Cytotoxicity in human & murine tumor cell lines
The evaluation of the compounds in Scheme 1 towards human
cervix carcinoma HeLa and T-lymphocyte CEM cells as well as
murine leukemia L1210 cells was undertaken in order to determine
whether the compounds were cytotoxic to human transformed
cells. The data are presented in Table 2 in comparison with
melphalan and levamisole.
3.2.2. 4i and 5i induce cytotoxicity in leukemic cells
In the present study we have used two leukemic cell lines, REH
and K562 to evaluate the cytotoxic effects of 4i and 5i (Fig. 1).

S. Kumar et al. / European Journal of Medicinal Chemistry 84 (2014) 687e697
689
Table 1
Structure and molecular weight of imidazo[2,1-b][1,3,4]thiadiazoles (2aei, 3aei, 4aei, and 5aei).
Structure
R
Code
Mol formula
C17H11ClFN3S
Mol wt
MP (ꢁC)
4-F-ph
4-Cl-ph
4-Br-ph
4-OCH3-ph
4-CH3-ph
2,4-di-Cl-ph
Ph
4-NO2-ph
Coumarin-3-yl
a
b
c
d
e
f
g
h
i
343.81
360.26
404.71
355.84
339.84
394.71
325.82
370.81
393.85
167e170
178e179
164e165
174e176
178e181
144e147
193e195
195e198
202e205
C17H11Cl2N3S
C17H11BrClN3S
C18H14ClN3OS
C18H14ClN3S
C
C
17H10Cl3N3S
17H12ClN3S
C17H11ClN4O2S
20H12ClN3O2S
C
4-F-ph
4-Cl-ph
4-Br-ph
4-OCH3-ph
4-CH3-ph
2,4-di-Cl-ph
Ph
4-NO2-ph
Coumarin-3-yl
a
b
c
d
e
f
g
h
i
C17H10BrClFN3S
422.70
439.16
483.61
434.74
418.74
473.60
404.71
449.71
472.74
155e157
143e145
134e137
126e128
131e133
105e107
149e152
158e160
208e210
C
C
17H10BrCl2N3S
17H10Br2ClN3S
C18H13BrClN3OS
C18H13BrClN3S
C
C
17H9BrCl3N3S
17H11BrClN3S
C17H10BrClN4O2S
20H11BrClN3O2S
C
4-F-ph
4-Cl-ph
4-Br-ph
4-OCH3-ph
4-CH3-ph
2,4-di-Cl-ph
Ph
4-NO2-ph
Coumarin-3-yl
a
b
c
d
e
f
g
h
i
C18H11ClFN3OS
371.82
388.27
432.72
383.85
367.85
422.72
353.83
398.82
421.86
138e140
118e120
129e131
118e121
83e85
127e129
119e121
157e159
130e133
C
C
18H11Cl2N3OS
18H11BrClN3OS
C19H14ClN3O2S
C19H14ClN3OS
C
C
18H10Cl3N3OS
18H12ClN3OS
C18H11ClN4O3S
21H12ClN3O3S
C
4-F-ph
4-Cl-p
4-Br-ph
4-OCH3-ph
4-CH3-ph
2,4-di-Cl-ph
Ph
4-NO2-ph
Coumarin-3-yl
a
b
c
d
e
f
g
h
i
C18H10ClFN4S2
400.88
417.33
461.79
412.92
396.92
451.78
382.89
427.89
450.92
148e150
181e183
181e183
151e153
155e157
130e132
121e123
167e169
196e198
C
C
18H10Cl2N4S2
18H10BrClN4S2
C19H13ClN4OS2
C19H13ClN4S2
C
C
18H9Cl3N4S2
18H11ClN4S2
C18H10ClN5O2S2
21H11ClN4O2S2
C
Results showed that although both molecules induced cytotoxicity
in the leukemic cells, 4i was more potent. 5i exhibited cytotoxic
effects on both REH and K562 cells, when analyzed using trypan
blue and MTT assays (Fig. 1AeD), with an IC50 value of ~6 and
~3 mM. In contrast, 4i showed maximum cytotoxic effect on REH
cells with an IC50 value of ~700 nM (Fig. 1E and F), while in
K562 cells, it showed an IC50 of ~900 nM (Fig. 1G and H).
found that 4i is more potent than 5i, we have used 4i to test its
ability to induce reactive oxygen species (ROS). Results showed no
ROS generation after treatment of 4i (1 mM), when analyzed after 5,
15, 30 and 60 min, while a positive control showed robust ROS
production (Fig. 3). Hence, our data suggested production of ROS is
not involved during 4i-mediated activation of cell death (Fig. 3).
3.2.5. DNA fragmentation assay
3.2.3. 4i and 5i induce cell death without affecting cell cycle
progression
Internucleosomal cleavage of DNA is one of the hallmarks of
apoptosis and cell death. Hence, we wondered whether 4i treat-
ment can induce DNA fragmentation, as described earlier [1]. The
Flow cytometric analysis of the effects of 4i and 5i (0.1, 0.5 and
1
mM) on REH cells did not show any cell cycle arrest. Besides, in the
data showed formation of fragmented DNA ladders from 0.5 mM
case of 5i, the concentrations used did not exhibit any effect on cell
cycle progression (Fig. 2A and B). In contrast, 4i showed maximum
effect resulting in the accumulation of dead cells in the sub G1
phase (Fig. 2C and D). These results thus suggested that 4i and 5i
induced cell death, without causing cell cycle arrest.
onwards of 4i treatment (Fig. 4). This suggested that 4i treatment
resulted in DNA fragmentation indicating apoptosis.
3.2.6. 4i treatment results in activation of apoptosis
To check whether treatment with 4i induces apoptosis or ne-
crosis, we have stained REH cells treated with 4i (1 mM) with pro-
3.2.4. Effect of 4i on intracellular ROS production
Production of reactive oxygen species after treatment with
drugs is one of the important mediators of cell death [5]. Since we
pidium iodide and Annexin V-FITC antibody, and subjected it to
flow cytometric analysis. Results showed a higher amount of
annexin-positive cells in the case of 4i treated REH cells compared

690
S. Kumar et al. / European Journal of Medicinal Chemistry 84 (2014) 687e697
Table 2
pathway of apoptosis could be responsible during 4i-mediated
apoptosis.
Inhibitory effects of compounds on the proliferation of murine leukemia cells
(L1210) and human T-lymphocyte cells (CEM) and human cervix carcinoma cells
(HeLa).
3.2.8. Effect of 4i treatment is robust in cancerous cells compared to
normal cells
a
Compound
IC50
(m
M)
L1210
CEM
HeLa
To investigate the effect of 4i on cancerous versus normal cells,
we treated REH and 293T cells with 0.5 mM of 4i and performed live
dead assay using ethidium bromide stain at 48 h time point. Results
showed that 4i is more cytotoxic against REH cells (~6 fold)
compared to 293T normal cells (Suppl. Fig. 4A and B). This suggests
that 4i could effectively kill the cells with high proliferation rate
which is a characteristic feature of cancerous cells.
2a
2b
2c
2d
2e
2f
2g
>250
>250
>250
>250
>250
>250
>250
>250
ꢂ250
>250
>250
>10
>250
>250
238 18
58 14
>250
106
>250
73
3.7 0.9
17
5.8 0.4
4.0 0.5
3.8 0.1
17
79
22
13
0.75 0.10
24
30 12
61 18
10
22
46 19
14
20
1.4 0.4
1.4 0.4
>250
>250
95 12
>250
>250
>250
38
5
>250
247
>250
214 23
>10
>250
4
109
8
>250
>250
>10
>250
>250
188 11
66 12
>250
118 43
>250
2h
2ib
4. Conclusions
3a
3b
3c
3d
3e
3f
3g
3h
3i
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
>250
In summary, a series of 36 derivatives with various structural
features on imidazo[2,1-b][1,3,4]thiadiazoles were synthesized and
their cytotoxicity evaluation was performed against human cervix
carcinoma HeLa, T-lymphocyte CEM, as well as murine leukemia
L1210 cells. Our structure activity relationship studies indicated
that, formyl (4c, 4d & 4i) and thiocyanate (5a, 5d, 5g & 5i) sub-
stituents are required for good cytotoxicity activity. Replacements
of the phenyl group at the 6th position by coumarin produced more
cytotoxic derivatives (3i, 4i & 5i). Therefore derivative 4i was
selected for further study to understand the mechanism of action
against REH and K562 cells. The cell cycle study, analysis of mito-
chondrial membrane potential, DNA fragmentation and Annexin V-
FITC staining suggested that 4i was able to induce apoptosis
without arresting cell cycle.
ꢂ250
63
1
ꢂ250
31
2
5
ꢂ250
94 24
5.2 0.2
4
ꢂ250
4.5 0.6
20
15
2
5
2
20
16
4.2 0.6
6.0 1.0
21
64
45 16
17
0.90 0.08
20
20
22
0
2
4.6 0.2
5.3 0.3
23
82
20
15
2
7
2
2
1
7
6
2
1
2
0
0.89 0.29
23
4
3
0
2
3
49 25
81 13
5. Experimental section
16
32
4
2
2
2
9.0 5.0
22
18
12
16
1.0 0.0
NT
0
1
8
1
5.1. Chemicals and reagents
49 12
24
5g
5h
5i
6
4
6
The melting points are uncorrected. Silica gel plates were used
for TLC by using CHCl3/MeOH in various proportions. The IR spectra
were recorded in KBr on a Jasco 430þ; the1H NMR spectra were
recorded in CDCl3/DMSO on a Bruker (400 MHz), and J values
were reported in hertz (Hz). Mass spectra were recorded in
triple quadrapole LCMS-6410 from Agilent technologies.
Phenacyl bromide [7], 4-chlorophenacyl bromide [8], 4-
bromophenacyl bromide [9], 4-methoxyphenacyl bromide [10], 4-
methylphenacyl bromide [11], 2,4-dichlorophenacyl bromide
[12], 4-fluorophenacyl bromide [13], 4-nitrophenacyl bromide
[14], 3-(bromoacetyl)-2H-chromen-2-one [15], 2-amino-5-(4-
chlorobenzyl)-1,3,4-thiadiazole 1, 2a, 3a, 4a and 5a were prepared
according to the literature [1]. Compounds 2b, 2c, 2e and 2h have
been previously reported [16].
50 12
1.3 0.0
2.13 0.02
Melphalan
Levamisole
206
6
ꢂ250
a
50% inhibitory concentration.
No doseeresponse at higher concentration.
b
Table 3
Inhibitory effects of compounds on the proliferation of REH and K562 cell lines.
a
Compound
IC50
REH
(mM)
K562
4i
5i
0.67
6.20
0.9
3.1
a
50% inhibitory concentration.
5.2. General procedure for the synthesis of the imidazo[2,1-b][1,3,4]
thiadiazoles (2d, 2f, 2g and 2i)
to the control (Fig. 5). A significant increase in the number of
apoptotic cells was seen (Fig. 5). These results suggest that 4i may
induce apoptosis as a major cause of cell death in REH cells.
The 2-amino-5-(4-chlorobenzyl)-1,3,4-thiadiazole 1 (30 mmol)
was treated with the respective phenacyl bromide (30 mmol), in
ethanol (150 mL). The mixture was refluxed for 10e12 h. Excess of
solvent was removed under reduced pressure and the solid
hydrobromide was separated by filtration, washed with cold
ethanol and dried. Neutralization of hydrobromide salts with cold
aqueous solution of sodium carbonate yielded the corresponding
free base which was filtered with a yield of 50e65%.
3.2.7. 4i treatment results in the loss of the mitochondrial
membrane potential
Mitochondria play a major role in the activation of apoptosis and
loss of mitochondrial membrane potential is one of the well-
studied aspects during induction of apoptosis [6]. Thus, we
checked for the changes in mitochondrial membrane potential after
treatment of REH cells with 4i (Fig. 6A and B). Results showed a
significant reduction in mitochondrial membrane potential at the
tested concentrations of 4i, which suggests that the loss of mito-
chondrial membrane potential is one of the key events leading to
cell death by 4i. This further indicates that activation of an intrinsic
5.2.1. 2-(4-Chlorobenzyl)-6-(4-methoxyphenyl)imidazo[2,1-b]
[1,3,4]thiadiazole (2d)
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-Yield 6.405 g, 60%, FT-IR (KBr,

S. Kumar et al. / European Journal of Medicinal Chemistry 84 (2014) 687e697
691
Fig. 1. Cytotoxic effect of 4i and 5i on REH and K562 cells. REH and K562 cells were treated with increasing concentration of 4i and 5i (0e10
mM). Trypan blue assay and MTT
assays were performed at 48 and 72 h of treatment with compound. A. Trypan blue assay for 5i-treated REH cells. B. MTT assay for 5i-treated REH cells. C. Trypan blue assay for 5i-
treated K562 cells. D. MTT assay for 5i-treated K562 cells. E. Trypan blue assay for 4i-treated REH cells. F. MTT assay for 4i-treated REH cells. G. Trypan blue assay for 4i-treated
K562 cells. H. MTT assay for 4i-treated K562 cells. The cells treated with highest concentration of DMSO used in the study served as vehicle control. In all the panels ‘C’ indicates,
DMSO treated control and error bar was plotted based on three independent experiments.
cmꢀ1): 3146, 3041, 2939, 2842, 1537, 1481, 1251, 1181, 1093, 1025,
837. NMR (400 MHz, DMSO-d6) 3.84 (s, 3H, eOCH3), 4.26 (s, 2H),
together at room temperature was added dropwise bromine
(6 mmol). After the addition, stirring was continued for 30 min. The
mixture was poured into 100 mL of water from which a solid
separated. The solid was collected by filtration and washed with
water, dried and crystallized from EtOHeCHCl3 with a yield of
60e85%.
d
6.92 (d, J ¼ 8 Hz, 2H), 7.27 (d, J ¼ 8 Hz, 2H), 7.36 (d, J ¼ 8 Hz, 2H), 7.73
(d, J ¼ 8 Hz, 2H), 7.88 (s, 1H). MS (ESI) m/z: 356.0 (355.8).
5.2.2. (2-(4-Chlorobenzyl)-6-(2,4-dichlorophenyl)imidazo[2,1-b]
[1,3,4] thiadiazole (2f)
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-Yield 5.920 g, 50%, FT-IR (KBr,
cmꢀ1): 3185, 3072, 2924, 1584, 1483, 1379, 1286, 1099, 873. NMR
5.3.1. 5-Bromo-2-(4-chlorobenzyl)-6-(4-chlorophenyl)imidazo[2,1-
b][1,3,4] thiadiazole (3b)
Obtained according to the general procedure for the synthesis of
the 5-bromo-imidazo[2,1-b][1,3,4]thiadiazole-Yield 1.865 g, 85%,
FT-IR (KBr, cmꢀ1): 3044, 2953, 2925, 1484, 1401, 1322, 1090, 824.
(400 MHz, DMSO-d6)
(m, 1H), 7.37 (d, J ¼ 8 Hz, 2H), 7.46 (m, 1H), 8.09 (d, J ¼ 8.8 Hz, 1H),
8.38 (s, 1H). MS (ESI) m/z: 395.9 (394.7).
d
4.28 (s, 2H), 7.28 (d, J ¼ 8 Hz, 2H), 7.30e7.33
NMR (400 MHz, DMSO-d6)
7.38 (d, J ¼ 8.8 Hz, 2H), 7.41 (d, J ¼ 8.8 Hz, 2H),7.95 (d, J ¼ 8.8 Hz, 2H).
MS (ESI) m/z: 439.9 (439.2).
d
4.32 (s, 2H), 7.28 (d, J ¼ 8.8 Hz, 2H),
5.2.3. 2-(4-Chlorobenzyl)-6-phenylimidazo[2,1-b][1,3,4]thiadiazole
(2g)
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-Yield 6.353 g, 65%, FT-IR (KBr,
cmꢀ1): 3101, 3068, 2941, 2910, 1528, 1483, 1435, 1193, 1020. NMR
5.3.2. 5-Bromo-6-(4-bromophenyl)-2-(4-chlorobenzyl)imidazo
[2,1-b][1,3,4] thiadiazole (3c)
Obtained according to the general procedure for the synthesis of
the 5-bromo-imidazo[2,1-b][1,3,4]thiadiazole-Yield 1.934 g, 80%,
FT-IR (KBr, cmꢀ1): 3085, 2925, 1586, 1483, 1399, 1320, 1092, 832.
(400 MHz, DMSO-d6) d 4.27 (s, 2H), 7.25e7.31 (m, 3H, ar), 7.34e7.42
(m, 4H, ar), 7.80 (d, J ¼ 8 Hz, 2H), 7.97 (s, 1H). MS (ESI) m/z: 326.40
(325.82).
NMR (400 MHz, DMSO-d6) d 4.50 (s, 2H), 7.44 (s, 4H), 7.67 (d,
J ¼ 8 Hz, 2H), 7.90 (d, J ¼ 8 Hz, 2H). MS (ESI) m/z: 481.8 (483.6).
5.2.4. 3-[2-(4-Chlorobenzyl)imidazo[2,1-b][1,3,4]thiadiazol-6-yl]-
2H-chromen-2-one (2i)
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-Yield 5.907 g, 50%, FT-IR (KBr,
cmꢀ1): 3145, 3052, 2973, 1729, 1609, 1484, 1102, 1004, 925. NMR
5.3.3. 5-Bromo-2-(4-chlorobenzyl)-6-(4-methoxyphenyl)imidazo
[2,1-b][1,3,4] thiadiazole (3d)
Obtained according to the general procedure for the synthesis of
the 5-bromo-imidazo[2,1-b][1,3,4]thiadiazole-Yield 1.738 g, 80%,
FT-IR (KBr, cmꢀ1): 3075, 3004, 2937, 2840, 1608, 1531, 1479, 1254,
(400 MHz, DMSO-d6)
(m, 5H), 7.61 (t, J ¼ 16 Hz,1H), 7.87 (d, J ¼ 8 Hz,1H), 8.56 (s,1H), 8.66
(s, 1H). MS (ESI) m/z: 392.0 (393.9).
d
4.48 (s, 2H), 7.38 (t, J ¼ 16 Hz, 1H), 7.45e7.46
1177, 1026, 826. NMR (400 MHz, DMSO-d6) d 3.78 (s, 3H, eOCH3),
4.49 (s, 2H, eCH2e), 7.03 (d, J ¼ 8 Hz, 2H), 7.44 (s, 4H), 7.87 (d,
J ¼ 8 Hz, 2H). MS (ESI) m/z: 433.90 (434.74).
5.3. General procedure for the preparation of the 5-bromo-imidazo
[2,1-b][1,3,4]thiadiazole derivatives (3bei)
5.3.4. 5-Bromo-2-(4-chlorobenzyl)-6-(4-methylphenyl)imidazo
[2,1-b][1,3,4] thiadiazole (3e)
To the respective imidazo[2,1-b][1,3,4]thiadiazole (5 mmol) 2,
sodium acetate (10 mmol) and 10 mL of glacial acetic acid stirred
Obtained according to the general procedure for the synthesis of
the 5-bromo-imidazo[2,1-b][1,3,4]thiadiazole-Yield 1.570 g, 75%,

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Fig. 2. Effect of 4i and 5i on cell cycle progression. REH cells were treated with 4i and 5i (0, 0.1, 0.5 and 1 mM). After 48 h of treatment, cells were harvested, fixed, stained with
propidium iodide and analyzed using a flow cytometer. A. Histogram representing effect of 5i on cell cycle progression. B. Bar diagram representing distribution of cells at different
phases of cell cycle following treatment with 5i. C. Histogram representing effect of 4i on cell cycle in REH cells. D. Bar diagram representing distribution of cells at different phases
of cell cycle following treatment with 4i. In both panels, B and D error bars shown were based on two independent experiments.
FT-IR (KBr, cmꢀ1): 3027, 2919, 1471, 1333, 1097, 1022, 823. NMR
(400 MHz, DMSO-d6) 2.32 (s, 3H, CH3), 4.49 (s, 2H), 7.27 (d,
5.3.6. 5-Bromo-2-(4-chlorobenzyl)-6-phenylimidazo[2,1-b][1,3,4]
thiadiazole (3g)
d
J ¼ 8 Hz, 2H), 7.44 (s, 4H), 7.84 (d, J ¼ 8 Hz, 2H). MS (ESI) m/z : 419.9
Obtained according to the general procedure for the synthesis of
the 5-bromo-imidazo[2,1-b][1,3,4]thiadiazole-Yield 1.659 g, 82%,
FT-IR (KBr, cmꢀ1): 3060, 3026, 2927, 1598, 1481, 1433, 1323, 1084,
(418.7).
1010. NMR (400 MHz, DMSO-d6)
d 4.50 (s, 2H), 7.33e7.37 (m, 1H),
7.44e7.48 (m, 6H), 7.93 (d, J¼ 7.2Hz, 2H). MS(ESI) m/z:405.9 (404.7).
5.3.5. 5-Bromo-2-(4-chlorobenzyl)-6-(2,4-dichlorophenyl)imidazo
[2,1-b][1,3,4] thiadiazole (3f)
Obtained according to the general procedure for the synthesis of
the 5-bromo-imidazo[2,1-b][1,3,4]thiadiazole-Yield 1.657 g,
70%,FT-IR (KBr, cmꢀ1): 3086, 3029, 2920, 1529, 1467, 1416, 1100,
5.3.7. 5-Bromo-2-(4-chlorobenzyl)-6-(4-nitrophenyl)imidazo[2,1-
b][1,3,4] thiadiazole (3h)
Obtained according to the general procedure for the synthesis of
the 5-bromo-imidazo[2,1-b][1,3,4]thiadiazole-Yield 1.753 g, 78%,
FT-IR (KBr, cmꢀ1): 3081, 2924, 2824, 1598, 1520, 1343, 1100, 857.
820. NMR (400 MHz, DMSO-d6)
d 4.52 (s, 2H), 7.45 (s, 4H), 7.53 (m,
2H), 7.76 (s, 1H). MS (ESI) m/z : 471.8 (473.6).

S. Kumar et al. / European Journal of Medicinal Chemistry 84 (2014) 687e697
693
Fig. 3. Detection of intracellular ROS production in REH cells following treatment with 4i. REH cells treated with 1
mM of 4i for different time periods (5, 15, 30 and 60 min),
cells were harvested, washed and stained with H2DCFDA (0.5
m
M) for 15 min at 37 ꢁC. ROS production was analyzed using flow cytometry. H2O2 treated cells were used as a positive
control, while untreated cells served as a negative control. Cells treated with H2DCFDA served as control for the study. The percent of cells which were positive for fluorescent ROS
were indicated.
NMR (400 MHz, DMSO-d6)
J ¼ 8 Hz, 2H), 8.34 (d, J ¼ 8 Hz, 2H). MS (ESI) m/z: 448.8 (449.7).
d
4.52 (s, 2H), 7.45 (s, 4H), 8.24 (d,
(KBr, cmꢀ1): 3095, 2935, 2829, 1591, 1522, 1334, 1101, 862. NMR
(400 MHz, DMSO-d6) 4.54 (s, 2H), 7.43e7.39 (m, 1H), 7.48e7.46 (m,
5H), 7.70e7.65 (m, 1H), 7.87e7.83 (m, 1H), 8.33 (s, 1H). MS (ESI) m/z:
d
471.9 (472.7).
5.3.8. 3-[5-Bromo-2-(4-chlorobenzyl)imidazo[2,1-b][1,3,4]
thiadiazol-6-yl]-2H-chromen-2-one (3i)
Obtained according to the general procedure for the synthesis of
the 5-bromoimidazo[2,1-b][1,3,4]thiadiazole-Yield 1.607 g, 68%, FT-IR
5.4. General procedure for the preparation of the imidazo[2,1-b]
[1,3,4]thiadiazole-5-carbaldehydes (4bei)
The Vilsmeier reagent was prepared at 0e5 ꢁC by dropping
POCl3 (2.3 g, 15 mmol) into a stirred solution of DMF (10 mL). The
respective imidazo[2,1-b][1,3,4]thiadiazole
2 (4 mmol) was
added slowly to the Vilsmeier reagent while maintaining stirring
and cooling for 2 h. Further stirring was continued for 6 h at
80e90 ꢁC. The resulting reaction mixture was poured into
100 mL of water; the precipitate was filtered, pressed, suspended
in water and neutralized to pH 7 with cold aqueous solution of
sodium carbonate. The solid was separated by filtration, washed
with water, dried and crystallized from EtOH with a yield of
45e58%.
5.4.1. 2-(4-Chlorobenzyl)-6-(4-chlorophenyl)imidazo[2,1-b][1,3,4]
thiadiazole-5-carbaldehyde (4b)
Obtained according to the general procedure for the synthesis
of the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde-Yield
0.854 g, 55%, FT-IR (KBr, cmꢀ1): 3071, 3028, 2954, 2914, 1670, 1477,
1312, 1193, 1027, 824. NMR (400 MHz, DMSO-d6)
d 4.42 (s, 2H),
7.30 (d, J ¼ 8 Hz, 2H), 7.39 (d, J ¼ 8 Hz, 2H), 7.49 (d, J ¼ 8 Hz, 2H),
7.84 (d, J ¼ 8 Hz, 2H), 10.08 (s, 1H, eCHO). MS (ESI) m/z: 386.0
(388.3).
Fig. 4. Evaluation of effect of 4i by DNA fragmentation assay. REH cells were treated
5.4.2. 6-(4-Bromophenyl)-2-(4-chlorobenzyl)imidazo[2,1-b][1,3,4]
thiadiazole-5-carbaldehyde (4c)
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde-Yield 0.865 g,
with 0, 0.1, 0.5,1 and 5
mM of 4i for 48 h, genomic DNA was isolated using non-
denaturing protocol and analyzed by agarose gel electrophoresis (2%). Lane 1 repre-
sents the DMSO control, Lanes 2, 3, 4 and 5 represents cells treated with 0.1, 0.5, 1 and
5 mM of 4i, respectively. ‘M’ represents the marker lane.

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Fig. 5. Annexin V-FITC flow cytometry. The mobilization of phosphatidylserine from inner membrane to outer membrane was detected using Annexin V-FITC staining. After 48 h
of treatment with 4i (1 M) cells were harvested, and stained with Annexin V-FITC and propidium iodide, and analyzed in a flow cytometer. DMSO treated cells were used as vehicle
m
control. Density plot represent control and treated cells.
50%, FT-IR (KBr, cmꢀ1): 3092, 3043, 2928, 2859, 1671, 1484, 1308,
2H, eCH2e), 7.07 (d, J ¼ 8 Hz, 2H), 7.45 (s, 4H), 7.92 (d, J ¼ 8 Hz, 2H),
9.95 (s, 1H, eCHO). MS (ESI) m/z: 383.85 (382.30).
1095, 1014. NMR (400 MHz, DMSO-d6)
d 4.55 (s, 2H), 7.44 (s, 4H),
7.69 (d, J ¼ 8 Hz, 2H), 7.91 (d, J ¼ 8 Hz, 2H), 9.97 (s, 1H, eCHO). MS
(ESI) m/z: 431.9 (432.7).
5.4.4. 2-(4-Chlorobenzyl)-6-(4-methylphenyl)imidazo[2,1-b][1,3,4]
thiadiazole-5-carbaldehyde (4e)
5.4.3. 2-(4-Chlorobenzyl)-6-(4-methoxyphenyl)imidazo[2,1-b]
[1,3,4]thiadiazole-5-carbaldehyde (4d)
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde-Yield 0.767 g,
50%, FT-IR (KBr, cmꢀ1): 3079, 3034, 2929, 2861, 1675, 1489, 1311,
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde-Yield 0.765 g,
52%, FT-IR (KBr, cmꢀ1): 3033, 2922, 2852, 1664, 1486, 1390, 1326,
1096, 1019, 829. NMR (400 MHz, DMSO-d6)
d 2.34 (s, 3H, eCH3),
4.55 (s, 2H), 7.32 (d, J ¼ 8 Hz, 2H), 7.46 (s, 4H), 7.82 (d, J ¼ 8 Hz, 2H),
1099,1010. NMR (400 MHz, DMSO-d6) d 3.82 (s, 3H, eOCH3), 4.55 (s,
9.94 (s, 1H, eCHO). MS (ESI) m/z: 366.0 (367.9).
Fig. 6. Measurement of mitochondrial membrane potential. Total mitochondrial membrane potential was measured after treatment of 4i with REH cells (0.5 and 1 mM for 48 h).
Following harvesting, cells were treated with JC-1 at 37 ꢁC for 20 min and analyzed in a flow cytometer. 2,4-DNP was used as positive control, negative control indicates DMSO-
treated cells without JC-1 staining and Control indicates JC-1 stained DMSO treated cells. A. Density plot showing JC1 staining of control and treated cells. B. Bar diagram showing
red and green fluorescence ratio. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

S. Kumar et al. / European Journal of Medicinal Chemistry 84 (2014) 687e697
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5.4.5. 2-(4-Chlorobenzyl)-6-(2,4-dichlorophenyl)imidazo[2,1-b]
[1,3,4] thiadiazole-5-carbaldehyde (4f)
4H), 7.76 (d, J ¼ 8 Hz, 2H), 7.92 (d, J ¼ 8 Hz, 2H). MS (ESI) m/z: 460.9
(461.8).
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde-Yield 0.913 g,
54%, FT-IR (KBr, cmꢀ1): 3085, 3041, 2928, 2865, 1669, 1489, 1315,
5.5.3. 2-(4-Chlorobenzyl)-6-(4-methoxyphenyl)imidazo[2,1-b]
[1,3,4]thiadiazol-5-yl thiocyanate (5d)
Obtained according to the general procedure for the synthesis
of the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde-Yield
0.218 g, 53%, FT-IR (KBr, cmꢀ1): 3007, 2965, 2842, 2154, 1610,
1101, 1015. NMR 1H (400 MHz, DMSO-d6)
d
4.44 (s, 2H), 7.31e7.29
(m, 2H), 7.40e7.37 (m, 2H), 7.50e7.48 (d, J ¼ 8 Hz, 2H), 7.57 (m, 1H,
ar), 9.77 (s, 1H, CHO). MS (ESI) m/z: 422.40 (422.72). 13C 176.92 (s),
166.93 (s), 134.47 (s), 133.25 (s), 133.18 (s), 130.42 (s), 130.22 (s),
129.60 (s), 127.44 (s), 37.67 (s). MS (ESI) m/z: 422.40 (422.72).
1527, 1480, 1099, 1035, 831. NMR (400 MHz, DMSO-d6)
d 3.81 (s,
3H, eOCH3), 4.56 (s, 2H), 7.11 (d, J ¼ 8.8 Hz, 2H), 7.46 (s, 4H), 7.92
(d, J ¼ 8.8 Hz, 2H). MS (ESI) m/z: 413.0 (412.9).
5.4.6. 2-(4-Chlorobenzyl)-6-phenylimidazo[2,1-b][1,3,4]
thiadiazole-5-carbalde hyde (4g)
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde-Yield 0.820 g,
58%, FT-IR (KBr, cmꢀ1): 3070, 2970, 2927, 2859, 1672, 1484, 1443,
1337, 1185, 1086, 846. NMR (400 MHz, DMSO-d6) d 4.56 (s, 2H), 7.48
(s, 4H), 7.49e7.53 (m, 3H), 7.91e7.93 (m, 2H), 9.96 (s,1H, eCHO). MS
(ESI) m/z: 352.0 (353.8).
5.5.4. 2-(4-Chlorobenzyl)-6-(4-methylphenyl)imidazo[2,1-b][1,3,4]
thiadiazol-5-yl thiocyanate (5e)
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde-Yield 0.214 g,
54%, FT-IR (KBr, cmꢀ1): 3029, 2915, 2157,1605,1479,1325,1123, 827.
NMR (400 MHz, DMSO-d6) d 2.36 (s, 3H, eCH3), 4.57 (s, 2H), 7.34 (d,
J ¼ 8 Hz, 2H), 7.46 (s, 4H), 7.86 (d, J ¼ 8 Hz, 2H). MS (ESI) m/z: 396.30
(396.92).
5.4.7. 2-(4-Chlorobenzyl)-6-(4-nitrophenyl)imidazo[2,1-b][1,3,4]
thiadiazole-5-carbaldehyde (4h)
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde-Yield 0.925 g,
58%, FT-IR (KBr, cmꢀ1): 3092, 2935, 2849,1670,1599,1520,1343,1102,
5.5.5. 2-(4-Chlorobenzyl)-6-(2,4-dichlorophenyl)imidazo[2,1-b]
[1,3,4]thiadiazol-5-yl thiocyanate (5f)
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde-Yield 0.230 g,
51%, FT-IR (KBr, cmꢀ1): 3085, 2920, 2854, 2163, 1586, 1462, 1099,
856. NMR (400 MHz, DMSO-d6)
d 4.57 (s, 2H), 7.45 (s, 4H), 8.26 (d,
822. NMR (400 MHz, DMSO-d6)
d 4.28 (s, 2H), 7.31e7.28 (m, 2H),
J ¼ 8 Hz, 2H), 8.34 (d, J ¼ 8 Hz, 2H), 10.06 (s, 1H, eCHO). MS (ESI) m/z:
7.37e7.35 (m, 2H), 7.46 (m, 1H), 8.09e8.07 (d, J ¼ 8 Hz, 1H), (8.39 s,
397.0 (398.8).
1H). MS (ESI) m/z: 450.8 (451.8).
5.4.8. 2-(4-Chlorobenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-
b][1,3,4] thiadiazole-5-carbaldehyde (4i)
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde-Yield 0.843 g,
50%, FT-IR (KBr, cmꢀ1): 3040, 2914, 1722, 1668, 1611, 1483, 1368,
1315, 1052. NMR (400 MHz, DMSO-d6) d 4.57 (s, 2H), 7.39e7.50 (m,
6H), 7.67e7.71 (m, 1H), 7.87e7.89 (m, 1H), 8.51 (s, 1H), 9.99 (s, 1H,
eCHO). MS (ESI) m/z: 419.9 (421.9).
5.5.6. 2-(4-Chlorobenzyl)-6-phenylimidazo[2,1-b][1,3,4]thiadiazol-
5-yl thiocyanate (5g)
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde-Yield 0.218 g,
57%, FT-IR (KBr, cmꢀ1): 3056, 2923, 2157,1481,1441,1325,1092, 840.
NMR (400 MHz, DMSO-d6) d 4.58 (s, 2H), 7.45e7.47 (m, 5H), 7.53 (t,
J ¼ 16 Hz, 2H), 7.97 (d, J ¼ 8 Hz, 2H). MS (ESI) m/z: 383.0 (382.9).
5.5.7. 2-(4-Chlorobenzyl)-6-(4-nitrophenyl)imidazo[2,1-b][1,3,4]
thiadiazol-5-yl thiocyanate (5h)
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde-Yield 0.222 g,
52%, FT-IR (KBr, cmꢀ1): 3082, 3036, 2926, 2841, 2160, 1598, 1510,
5.5. General procedure for the preparation of the imidazo[2,1-b]
[1,3,4]thiadiazol-5-yl-thiocyanates (5bei)
To a mixture of imidazo[2,1-b][1,3,4]thiadiazole 2 (10 mmol) and
potassium thiocyanate (1.56 g,16 mmol) in glacial acetic acid (50 mL)
was added bromine (1.6 g, 10 mmol) in glacial acetic acid (20 mL) at
0e5 ꢁC dropwise with stirring. Stirring was continued for 30 min at
15e20 ꢁC and then at room temperature for 1 h. The reaction mixture
was poured into ice water, filtered, washed with water and crystal-
lized from EtOHeCHCl3 with a yield of 50e57%.
1407, 1340, 1108, 1012, 854. NMR (400 MHz, DMSO-d6)
d 4.60 (s,
2H), 7.45e7.47 (s, 4H), 8.26 (d, J ¼ 8 Hz, 2H), 8.40 (d, J ¼ 8 Hz, 2H).
MS (ESI) m/z: 425.9 (427.9).
5.5.8. 2-(4-Chlorobenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-
b][1,3,4] thiadiazol-5-yl thiocyanate (5i)
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde-Yield 0.207 g,
46%, FT-IR (KBr, cmꢀ1): 3029, 2941, 1720, 1615, 1479, 1373, 1316,
5.5.1. 2-(4-Chlorobenzyl)-6-(4-chlorophenyl)imidazo[2,1-b][1,3,4]
thiadiazol-5-yl thiocyanate (5b)
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde-Yield 0.225 g,
54%, FT-IR (KBr, cmꢀ1): 3065, 2921, 2850, 2161, 1462, 1096, 832.
1063. NMR (400 MHz, DMSO-d6)
d 4.63 (s, 2H), 7.54e7.42 (m, 6H),
7.75e7.69 (m, 1H), 7.92e7.89 (m, 1H), 8.49 (s, 1H). 13C 166.08 (s),
159.80 (s), 153.93 (s), 148.58 (s), 144.40 (s), 142.95 (s), 134.44 (s),
133.04 (s), 132.63 (s), 130.39 (s), 129.59 (s), 128.66 (s), 125.03 (s),
120.45 (s), 119.78 (s), 108.67 (s), 37.71 (s). MS (ESI) m/z: 451.00
(450.92).
NMR (400 MHz, DMSO-d6)
7.40 (d, J ¼ 8.8 Hz, 2H), 7.48 (d, J ¼ 8.8 Hz, 2H), 7.92 (d, J ¼ 8.4 Hz,
2H). MS (ESI) m/z: 416.8 (417.3).
d
4.38 (s, 2H), 7.31 (d, J ¼ 8.4 Hz, 2H),
5.5.2. 6-(4-Bromophenyl)-2-(4-chlorobenzyl)imidazo[2,1-b][1,3,4]
thiadiazol-5-yl thiocyanate (5c)
6. Bioevaluations
Obtained according to the general procedure for the synthesis of
the imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde-Yield 0.240 g,
52%, FT-IR (KBr, cmꢀ1): 3063, 2924, 2852, 2161,1673,1585,1462,1398,
6.1. Cell culture
Human cell lines REH (B-cell leukemia), K562 (Chronic mye-
logenous leukemia), were cultured in RPMI1640 (Sera Lab, UK) and
1097,1009,1271, 831. NMR (400 MHz, DMSO-d6) d 4.58 (s, 2H), 7.46 (s,

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S. Kumar et al. / European Journal of Medicinal Chemistry 84 (2014) 687e697
293T (Human Embryonic Kidney cells) were cultured in DMEM
containing 10% FBS (Gibco BRL, USA) along with 100 U of Penicillin
diacetate (H2DCFDA) in REH cells as described previously [21]. REH
cells were treated with 1 M of 4i for different time points (5, 15, 30
and 60 min), cells were harvested, washed with 1ꢃ phosphate-
buffered saline and incubated with 0.5
M H2DCFDA at 37ꢁ C for
m
G/ml and 100 m
g of streptomycin/ml (SigmaeAldrich, USA) at 37 ꢁC
in a humidified atmosphere containing 5% CO2 [17].
m
15 min and fluorescence intensity was analyzed by flow cytometry
(FACS Calibur, BD Biosciences, USA). Cells treated with H2O2 were
served as the positive control.
6.1.1. Cytotoxicity in human and murine tumor cell lines
All the compounds in Scheme 1 were evaluated for their cyto-
static activity against human HeLa cervix carcinoma cells, human
CEM CD4þ T-lymphocytes as well as murine L1210 cells. All assays
were performed in 96-well microtiter plates. To each well were
added (5e7.5) ꢃ 104 tumor cells and a given amount of the test
compound. The cells were allowed to proliferate for 48 h (murine
leukemia L1210 cells) or 72 h (human lymphocytic CEM and human
cervix carcinoma HeLa cells) at 37 ꢁC in a humidified CO2-
controlled atmosphere. At the end of the incubation period, the
cells were counted in a Coulter counter. The IC50 (50% inhibitory
concentration) was defined as the concentration of the compound
that inhibited cell proliferation by 50%. These data are presented in
Tables 2 and 3 In addition, in order to assess the possible mecha-
nism of action, compounds 4i and 5i have also been evaluated
against REH and K562 leukemia cells.
6.1.7. DNA fragmentation assay
Fragmentation of genomic DNA due to apoptosis was detected
by agarose gel electrophoresis [18]. Briefly, REH cells were treated
with increasing concentrations (0, 0.1, 0.5,1 and 5 mM) of 4i for 48 h.
DMSO treated cells were used as a vehicle control. Total genomic
DNA was isolated using a standard protocol [22]. After 48 h treat-
ment, cells were harvested and washed with 1X PBS, lysed in lysis
buffer (100 mM NaCl, 10 mM Tris pH 8.0, 0.25%Triton X-100, 1 mM
EDTA and 200
treatment was applied (50
m
g/ml Proteinase K), incubated at 55 ꢁC for 5 h. RNase
m
g/ml) for 1 h at 37 ꢁC, samples were
deproteinized using phenol:chloroform and DNA content was
precipitated using 1/10th volume of 3 M sodium acetate (pH 5.2)
and an equal volume of isopropanol. The pellets were washed with
70% ethanol, dried, dissolved in TE (10:1), loaded on 2% agarose gel
and run at 50 V for 3 h.
6.1.2. Trypan blue dye exclusion assay
The viability of the leukemic cells (REH, K562) was determined
by Trypan blue dye exclusion assay [18] after treatment with 4i and
5i. Cells (0.5 ꢃ 105 cells/ml) were cultured for 24 h, following which
6.1.8. Annexin V-FITC flow cytometric analysis
Phosphatidylserine exclusion on apoptotic cells was detected by
Annexin V-FITC staining (Santa Cruz, USA). Briefly, REH cells were
increasing concentrations of the compounds (0e10
mM) were
added and incubated. Cells treated with DMSO served as the vehicle
control. Treated cells were collected after 48 and 72 h and viability
was determined by trypan blue staining. Each experiment was
repeated a minimum of three times and error bars were calculated
and plotted.
treated with 4i (1 mM) and equivalent DMSO-treated control cells
were harvested, washed with phosphate-buffered saline and
resuspended in binding buffer (HEPES-10 mM pH 7.4, 144 mM NaCl
and 25 mM CaCl2), stained with Annexin V-FITC (0.2 mg/ml) and
propidium iodide (0.05 mg/ml) for 20 min on ice and analyzed in a
flow cytometer [18].
6.1.3. MTT assay
To check the proliferation rate of the cells, following treatment
6.1.9. Measurement of mitochondrial membrane potential
with increasing concentrations of 4i and 5i (0e10 mM), REH and
Mitochondrial transmembrane potential after treatment with 4i
was measured using JC-1 (5, 50, 6, 60-tetrachloro-1, 1, 3, 30-tetrae-
thylbenzimidazolcarbocyanamide iodided; Calbiochem, USA) dye
as described earlier [18]. Briefly, after 48 h of treatment with 4i (0.5
K562 cells (0.5 ꢃ 105 cells/ml) were harvested after 48 and 72 h and
used for the MTT assay [19,20]. Cells treated with equal amount of
DMSO served as vehicle control. Experiments were repeated three
independent times, each in duplicate, and bar diagrams were
plotted with error bars.
and 1
with JC-1 (0.5
m
M), cells were harvested and incubated at 37 ꢁC for 20 min
mM), washed and analyzed by flow cytometry. 2,4-
DNP was used as a positive control. Typical green versus red ratio
of JC-1 florescence was represented with bar diagrams with error
bars using two independent experiments.
6.1.4. Live dead assay
4i mediated cell death in REH and 293T cells were studied using
live dead assay. Ethidium bromide is excluded by live cells with
intact membrane, but it can enter dead cells with broken membrane
and stains the DNA. The cells were seeded (1 ꢃ 105/ml) in 6 well
6.1.10. Statistical analysis
The values were expressed as mean
SEM for control and
plates and 4i (0.5 mM) was added to both Reh and 293T cells. DMSO
experimental samples and statistical analysis was performed using
One-way ANOVA followed by Dunnett test and significance was
calculated after comparing each value with control, for the analysis,
using GraphPad software prism 5.1. The values were considered as
statistically significant, if the p-value was equal to or less than 0.05.
served as vehicle control. Cells were harvested after 48 h, stained
with ethidium bromide (5 mg/ml) and subjected to flow cytometry.
6.1.5. Cell cycle analysis
REH cells (0.5 ꢃ 105 cells/ml) were cultured for 24 h and com-
pounds 4i or 5i, were added (0, 0.1, 0.5 and 1 mM) to the culture and
incubated. Cells were harvested after 48 h, washed, fixed with 70%
chilled ethanol and analyzed using flow cytometry following
staining with propidium iodide, as described previously [20]. A
minimum of 10,000 cells were acquired per sample and histograms
were analyzed using WinMDI 2.8 software. Experiments were
repeated two independent times and cell cycle phase was plotted in
bar diagram with error bars.
Acknowledgments
We gratefully acknowledge the support for this research effort
from All India Council for Technical Education (AICTE), New Delhi
(Ref. No. 8023/BOR/RID/RPS-169/2008-09) for SSK. The cytotoxic
activities were determined by financial support of the KU Leuven
(GOA 10/014). We thank Ms. Mrinal Srivastava. We would also like
to thank NMR facility, Indian Institute of Science, Bangalore for
characterisation of all compounds. We also thank FACS facility, IISc
for flow cytometric studies. We thank Lizette van Berckelaer for the
cytostatic determinations.
6.1.6. Detection of intracellular ROS production by flow cytometre
The total intracellular ROS production was measured using cell
permeable fluorescent probe 2,7-dichlorodihydro fluorescein

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