Synthesis and biological evaluation of novel 2-aralkyl-5-substituted-6- (4′-fluorophenyl)-imidazo[2,1-b][1,3,4]thiadiazole derivatives as potent anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2011.02.064

Levamisole, the imidazo[2,1-b]thiazole derivative has been reported as a potential antitumor agent. In the present study, we synthesized, characterized and evaluated biological activity of its novel analogues with substitution in the aralkyl group and on

Full text of DOI:10.1016/j.ejmech.2011.02.064

European Journal of Medicinal Chemistry 46 (2011) 2109e2116
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel 2-aralkyl-5-substituted-6-(40-
fluorophenyl)-imidazo[2,1-b][1,3,4]thiadiazole derivatives as potent anticancer
agents
,
b
, ,
,
Subhas S. Karki a 1, Kuppusamy Panjamurthy a 1, Sujeet Kumar b, Mridula Nambiar a,
*
Sureshbabu A. Ramareddy b, Kishore K. Chiruvella a, Sathees C. Raghavan a
,
**
a Department of Biochemistry, Indian Institute of Science, Malleswaram, Bangalore 560 012, Karnataka, India
b Department of Pharmaceutical Chemistry, KLE University’s College of Pharmacy, Bangalore 560 010, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Levamisole, the imidazo[2,1-b]thiazole derivative has been reported as a potential antitumor agent. In
the present study, we synthesized, characterized and evaluated biological activity of its novel analogues
with substitution in the aralkyl group and on imidazothiadiazole molecules with same chemical back-
bone but different side chains namely 2-aralkyl-6-(40-fluorophenyl)-imidazo[2,1-b][1,3,4]thiadiazoles
(SCR1), 2-aralkyl-5-bromo-6-(40-fluorophenyl)-imidazo[2,1-b][1,3,4]-thiadiazoles (SCR2), 2-aralkyl-5-
formyl-6-(40-fluorophenyl)-imidazo[2,1-b][1,3,4]-thiadiazoles (SCR3) and 2-aralkyl-5-thiocyanato-6-(40-
fluorophenyl)-imidazo[2,1-b][1,3,4]-thiadiazoles (SCR4) on leukemia cells. The cytotoxic studies showed
that 3a, 4a, and 4c exhibited strong cytotoxicity while others had moderate cytotoxicity. Among these we
Received 6 January 2011
Received in revised form
13 February 2011
Accepted 24 February 2011
Available online 4 March 2011
Keywords:
Anticancer drugs
Cancer therapeutics
Cytotoxicity
Double-strand breaks
Apoptosis
chose 4a (IC50, 8 mM) for understanding its mechanism of cytotoxicity. FACS analysis in conjunction with
mitochondrial membrane potential and DNA fragmentation studies indicated that 4a induced apoptosis
without cell cycle arrest suggesting that it could be used as a potential chemotherapeutic agent.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Cell death
1. Introduction
b]thiazole derivatives of Levamisole have been reported as potential
antitumor agents (II) [2]. Later, antitumor activity of 5-formyl-6-
Development of anticancer drugs with fewer or no side effects is
important for the treatment of cancer. The search for such potential
anticancer drugs have led to the discovery of synthetic small
molecules with anti-carcinogenic activity and limited harmful side
effects particularly with respect to the immune system. Alterna-
tively, stimulation of the body’s immune system could provide
a valuable support in cancer treatment, since it is capable of erad-
icating the neoplastic cells completely. Research in this area is
expanding rapidly and some promising leads have emerged.
Levamisole (I) appears to be the most effective in patients with
small tumor burdens and it acts by stimulating the responsiveness
of lymphocytes to tumor antigens [1]. In addition, the imidazo[2,1-
arylimidazo-[2,1-b][1,3,4]thiadiazole sulfonamides (III) were also
reported [3]. The promising results obtained in that study prompted
us to prepare a new series of analogues including fluorine at posi-
tion 4 of 6-phenyl in imidazo-[2,1-b]-1,3,4-thiadiazole (IV).
R'
N
N
N
N
I
S
R
S
II
Levamisole
2,5-disubstituted-6-phenyl
imidazo[2,1-b]thiazole
F
OHC
R'
* Corresponding author. Department of Biochemistry, Indian Institute of Science,
Malleswaram, Bangalore 560 012, Karnataka, India. Tel.: þ91 80 2332 5611;
fax: þ91 80 2342 5373.
N
N
N
N
N
N
III
RHNO2S
S
IV
** Corresponding author. Tel.: þ91 80 2293 2674; fax: þ91 80 2360 0814.
E-mail addresses: subhasskarki@gmail.com (S.S. Karki), sathees@biochem.iisc.
ernet.in (S.C. Raghavan).
S
R
5-formyl-6-aryl-imidazo[2,1-b]
[1,3,4]thiadiazole sulfonamide
6-(4'-fluorophenyl) imidazo[2,1-b][1,3,4]
thiadiazoles (SCR1-SCR4)
1
These authors contributed equally.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.064

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S.S. Karki et al. / European Journal of Medicinal Chemistry 46 (2011) 2109e2116
Previously, we have reported purification and synthesis of many
fluoro phenacyl bromide. All the electrophilic substitution reactions
carried out on imidazo[2,1-b][1,3,4]thiadiazole derivatives (SCR1)
and afforded the expected 5-substituted derivatives (SCR2, SCR3,
andSCR4). Allthese 5-substituted derivatives showed the absence of
C5eH in their respective spectra, confirming the substitution at 5th
natural and synthetic compounds, respectively. These were also
studied for their biological activity and were shown to possess
different levels of cytotoxicity on cancer cells [4e17]. In the present
study, we synthesized, characterized and evaluated biological activity
of new analogues of levamisole (I) with substitution in the aralkyl
group and on fused imidazo[2,1-b][1,3,4]thiadiazole ring with same
chemical backbone but different side chains namely 2-aralkyl-6-(40-
fluorophenyl)-imidazo[2,1-b][1,3,4]thiadiazoles (SCR1), 2-aralkyl-5-
bromo-6-(40-fluorophenyl)-imidazo[2,1-b][1,3,4]-thiadiazoles (SCR2),
2-aralkyl-5-formyl-6-(40-fluorophenyl)-imidazo[2,1-b][1,3,4]-thiadi-
azoles (SCR3) and 2-aralkyl-5-thiocyanato-6-(40-fluorophenyl)-imi-
dazo[2,1-b][1,3,4]-thiadiazoles (SCR4). Based on our studies one of the
molecules, 4a was identified as the most promising lead compound.
position. Compounds 3a and 3b showed a singlet between
9.96 ppm for CHO proton respectively. All the compounds showed
prominent signals for aromatic protons around 7.00e8.05 ppm.
Methylene proton at C2 appeared between 4.21e4.58 ppm for all
synthesized derivatives. Compounds 1e, 2d and 4c showed a singlet
at 2.29 ppm for presence of methyl proton on phenyl ring. The
d 9.98 to
d
d
d
structures of all the compounds were finally ascertained by mass
spectra analysis (Table 1).
3. Pharmacology
2. Chemistry
The human T-cell leukemia cells, CEM were used for the study of
preliminary anti-cancer screening of newly synthesized compounds.
To assess the cytotoxicity, we used trypan blue dye exclusion and MTT
assays. To test this, cells growing in log phase were treated with
different concentrations of SCR1 (1ae1e), SCR2 (2ae2d), SCR3 (3a
and 3b) and SCR4 (4ae4c)). Further studies on lead cytotoxic
compound, 4a, in CEM cells were assessed by tritiated thymidine
incorporation, cell cycle analysis, measurement of mitochondrial
transmembrane potential (Djm) and DNA fragmentation assays.
We have synthesized series of 14 derivatives of imidazo[2,1-b]
[1,3,4]thiadiazoles containing aralkyl group at 2nd position by react-
ing 2-amino-5-aralkyl-1,3,4-thiadiazoles X with 4-fluoro phenacyl
bromide as depicted in Scheme 1. FurthercompoundsSCR2, SCR3 and
SCR4 were obtained by bromination, formylation and thiocyanation
respectively (Table 1). Structures of the synthesized compounds were
established on the basis of IR, 1H NMR and mass spectra analysis
(Supplementary Figs. 1e3). All synthesized compounds showed
absorption bands ranging from 3288 to 3025 cmꢀ1 for CeH aromatic
stretching, 2980 to 2840 cmꢀ1 for CeH aliphatic stretching.
Compounds 3a and 3b showed vibration bands at 1653e1676 cmꢀ1
for C]O stretching. Compounds 4ae4c showed vibration bands at
2163e2159 cmꢀ1 for SCN in their respective IR spectra. Rf values of all
the compounds were determined (Supplementary Table 1)
4. Results and discussion
In the present study we have investigated the cytotoxic effect of
1a, 1b,1c, 1d, 1e (1ae1e), 2a, 2b, 2c, 2d (2ae2d), 3a, 3b (3a and 3b),
4a, 4b, 4c (4ae4c) on T-cell leukemic cell line, CEM (Table 1). In order
to estimate the effect of compounds, CEM cells were treated with
In 1H NMR, the presence of singlet between
for imidazole proton (C5eH) confirmed the cyclization of X with 4-
d 7.83 and 8.65 ppm
increasing concentrations of compounds (10, 50, 100 and 250 mM)
Scheme 1. Reagents and conditions: (a) (i) FeC6H4eCOCH2Br, EtOH, (ii) Na2CO3; (b) Br2, gl. acetic acid; (c) (i) POCl3, DMF, 80e90 ꢁC, (ii) Na2CO3; (d) KSCN, gl. acetic acid, Br2 in gl.
acetic acid, 0e5 ꢁC.

S.S. Karki et al. / European Journal of Medicinal Chemistry 46 (2011) 2109e2116
2111
Table 1
Structure and molecular weight of novel imidazo[2,1-b]thiazole derivatives of Levamisole, 2-aralkyl-6-(40-flurophenyl)-imidazo[2,1-b][1,3,4]thiadiazoles (SCR1), bromo
(SCR2), formyl (SCR3) and thiocynate (SCR4) derivatives.
Structure
R
Code no.
Molecular formula
C17H12FN3S
Molecular weight
Melting point (ꢁC)
H
Cl
F
Br
CH3
1a
1b
1c
1d
1e
309.40
343.81
327.35
388.26
323.39
152e155
167e170
149e152
118e120
149e150
C17H11ClFN3S
C17H11F2N3S
C17H11BrFN3S
C18H14FN3S
H
Cl
F
2a
2b
2c
2d
C18H11BrFN3S
C17H10BrClFN3S
C17H10BrFN3S
C18H13BrFN3S
338.30
422.70
406.25
402.28
122e125
155e157
89e90
CH3
92e95
H
Cl
3a
3b
C18H12FN3OS
C18H11ClFN3OS
337.40
371.82
112e114
138e140
H
Cl
CH3
4a
4b
4c
C18H11FN4S2
C18H10ClFN4S2
C19H13FN4S2
366.40
371.82
380.46
133e135
148e150
100e102
and cell viability was determined by trypan blue assay (Fig. 1,
Supplementary Fig. 8). The 5-fluorouracil treated cells were used as
positive control. Since the compounds were dissolved in DMSO, cells
treated with DMSO alone served as vehicle control. The cells were
counted at intervals of every 24 h, till they attained stationary phase
and the data was plotted. Results showed that addition of compound
to the CEM cells affected viability of cells in a dose-dependent
manner, in the case of almost every compound (Fig. 1,
Supplementary Fig. 8). However, the levels of sensitivity differed
drastically among compounds. 3a, 4a and 4c induced maximum
toxicity on leukemic cells (Fig. 1), while the effect was moderate in
the case of 1a, 1c, 2c, 3b and 4b (Table 2, Supplementary Fig. 8).
However, the compounds 1b, 1d, 1e, 2a and 2b were least sensitive
(Table 2, Supplementary Fig. 8). IC50 value of each compound was
calculated for 48 and 72 h based on trypan blue assay (Table 2).
The effect of 1ae1e, 2ae2d, 3a and 3b, and 4ae4c on cell
proliferation was further tested using MTT assay. CEM cells treated
with 10, 50,100 and 250 mM of compounds were harvested after 24,
48 and 72 h and subjected to MTT assay. Results suggested that 4a
treatment lead to the reduction in cell proliferation in a dose-
dependent manner (Fig. 2, Supplementary Fig. 9). Data from MTT
assay in conjunction with trypan blue assay suggest that 4a showed
maximum cytotoxicity with an IC50 value of w8 mM and hence it
was selected as the lead compound (Table 2).
Fig. 1. Dose- and time-dependent effects of 3a, 4a and 4c on cell viability in T-cell leukemic cells. CEM cells were treated with the compounds (10, 50, 100 and 250 mM) and cell
viability was determined by Trypan blue assay. 5-flurouracil was used as the positive control. DMSO treated cells were used as vehicle control. The data shown is derived from three
independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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S.S. Karki et al. / European Journal of Medicinal Chemistry 46 (2011) 2109e2116
Table 2
IC50 value of 1ae4a and 5-FU was calculated based on Trypan blue and MTT assay at
48 h and 72 h in CEM cell lines.
Compounds
IC50 value (mM)
Trypan blue assay
MTT assay
48 h
48 h
72 h
72 h
1a
1b
1c
1d
1e
2a
2b
2c
75
220
50
100
160
200
240
58
60
60
50
100
100
100
100
100
120
40
>250
110
220
>250
>250
>250
80
>250
120
>250
>250
>250
>250
60
Fig. 3. Tritiated thymidine assay to determine the effect of 4a on cell proliferation. 4a
treated (10 M, for 12, 24 and 48 h) CEM cells were subjected to tritiated thymidine
assay. DMSO treated cells were used as control. Radioactive counts in each sample was
determined and presented as bar diagram.
2d
3a
3b
4a
4b
4c
40
20
90
10
75
18
15
52
16
80
10
40
14
8
90
35
105
8
50
80
26
110
8
40
m
10
40
10
30
The mitochondrial transmembrane potential is an important
step in the induction of cellular apoptosis. During apoptosis, the
electrochemical gradient (DJ) across the mitochondrial membrane
collapses. To test whether 4a affected the mitochondrial membrane
5-FU
Based on above results, we were interested in determining
whether the observed cytotoxicity by 4a was due to effect of 4a on
cell division. In order to test this, CEM cells were cultured in
potential, CEM cells treated with the compound (1, 5, 10 and 20 mM)
for 48 h, were subjected to JC-1 dye staining and FACS analysis. In
healthy non-apoptotic cells, the dye stains the mitochondria and
gives bright red fluorescence. However, in apoptotic cells, the
mitochondrial membrane potential collapses and the JC-1 cannot
accumulate within the mitochondria. In these cells JC-1 remains in
the cytoplasm resulting in a green fluorescent monomeric form.
The aggregate red form has absorption/emission maxima of 585/
590 nm and green monomeric form has absorption/emission
maxima of 510/527 nm. Results showed that mostly DMSO treated
cells showed red fluorescence while 4a treated cells showed green
fluorescence in a concentration dependent manner (Fig. 5) similar
to positive control, 2,4-DNP. Thus, our results indicate that 4a
altered mitochondrial membrane potential in dose-dependent
manner resulting in cytosolic accumulation of monomeric JC-1,
which further suggests the activation of apoptosis.
presence of 4a (10 m
M) and [3H] thymidine. Incorporation of triti-
ated thymidine to the DNA was determined following 12, 24 and
48 h. Data showed reduction in the incorporation in a dose-
dependent manner (Fig. 3). These results suggest that the 4a
reduced incorporation of [3H] thymidine into DNA of dividing cells
at both 24 and 48 h treatment, which indicates that 4a inhibits DNA
replication and can also induce apoptosis.
Based on the above studies, we wondered whether the effect of
4a on cell division could lead to cell cycle arrest. In order to test this,
CEM cells cultured with 4a (1, 5,10 and 20 mM) for 24 and 48 h were
stained with propidium iodide and subjected to flow cytometry.
Results showed no arrest in cell cycle progression during the time
period analyzed (Fig. 4). However, we could observe the accumu-
lation of cells at sub G1 phase in a concentration- and time-depen-
dent manner (Fig. 4). Thus, the observed accumulation of cells in sub
G1 peak indicated cell death mediated by apoptosis. Therefore, our
results suggest that 4a induces apoptosis, but not cell cycle arrest.
Since 4a could induce apoptosis, DNA fragmentation assay was
performed to detect the DNA double-strand breaks. Multiple DNA
breaks on genomic DNA can lead to smearing on an agarose gel.
Fig. 2. Determination of effect of 3a, 4a and 4c on proliferation of CEM cells. Cells treated with the compounds (10, 50, 100 and 250 mM for 24, 48 and 72 h) were subjected to MTT
assay and results were expressed as percentage of cell proliferation.

S.S. Karki et al. / European Journal of Medicinal Chemistry 46 (2011) 2109e2116
2113
Fig. 4. Analysis of cell cycle progression followed by 4a treatment. CEM cells treated with 4a (1, 5, 10 and 20
mM) for 24 and 48 h, were harvested, stained with propidium iodide and
DNA content of 10, 000 cells were analyzed by flow cytometry. Histogram representing cell population of different phases of cell cycle (sub G1, G1, S and G0/M) is represented.
CEM cells treated with different concentrations of 4a (1, 5, 10 and
20 M) were harvested using nondenaturing method for 48 h and
5. Conclusion
m
subjected to gel electrophoresis. Results showed an increase in DNA
fragmentation with increase in concentration of the compound
(Fig. 6). Therefore, different lines of evidences suggest that 4a
induces cytotoxicity by activating apoptosis.
Based on the IC50 values of the molecules studied (Table 2), it
appears that when the substituents at the 5th position of imidazo
[2,1-b][1,3,4]thiadiazole group are thiocyanate (4a and 4c) and
formyl (3a) groups, the compounds exhibit maximum cytotoxicity.
However, the introduction of bromine or hydrogen substituents
results in lesser cytotoxicity.
In the present study we have synthesized 14 novel 2-aralkyl-5-
substituted-6-(40-fluorophenyl)-imidazo[2,1-b][1,3,4]thiadiazole
derivatives, which showed strong and moderate cytotoxicity on
leukemia cell line, CEM. The derivates 3a, and 4a with formyl
(eCHO) and thiocyanate (eSCN) substitutions at 5th position
showed highest cytotoxicity. Another derivative, 4c with thiocya-
nate (eSCN) and methyl (eCH3) substitution on imidazo[2,1-b]
[1,3,4]thiadiazole at 5th and 4th positions of benzyl group,
respectively also showed higher effect on cell viability. Based on
IC50 values, 4a (8 mM) was selected as the lead compound and its
Fig. 5. Detection of loss of mitochondrial membrane potential followed by 4a treatment. CEM cells were treated with 4a (1, 5, 10 and 20 mM) for 48 h and analyzed by flow
cytometry following staining with JC-1 dye. 2,4-DNP treated CEM cells were used as positive control.

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S.S. Karki et al. / European Journal of Medicinal Chemistry 46 (2011) 2109e2116
6.2.1. General procedure for preparation of X
The mixture of 4-substituted/phenyl acetic acid (0.1 M) and
thiosemicarbazide (0.15 M) was added slowly to the round bottom
flask containing concentrated H2SO4 (30 ml) with constant stirring
in ice bath. After complete addition, ice bath was replaced by water
bath and slowly heated to 70e80 ꢁC and maintained at that
temperature for 7 h. After cooling to room temperature, the
contents of the reaction were poured into ice water and made basic
with ammonia and the precipitate was filtered, washed with water
and recrystallized from ethanol.
6.2.2. 2-Amino-5-(4-chlorobenzyl)-1,3,4-thiadiazole (XCl)
Yield 50%. M P.181e182 ꢁC FT IR ( cmꢀ1) 3262, 3100, 2971, 2915,
l
1698, 1519, 1491, 1332, 1091, 1016. 1H NMR
d: 4.30 (s, 2H. eCH2e),
7.06 (s, 2H, eNH2), 7.55 (d, 2H, J ¼ 8 Hz), 8.20 (d, 2H, J ¼ 8 Hz).
6.2.3. 2-Amino-5-(4-fluorobenzyl)-1,3,4-thiadiazole (XF)
Yield 47%. M P. 205e207 ꢁC. FT IR (
l
cmꢀ1) 3265, 3107, 2950,1685,
: 4.14 (s, 2H. eCH2e),
1515, 1334, 1232, 1148, 1092, 1052. 1H NMR
d
Fig. 6. Detection of DNA breaks induced by 4a in CEM cells. The genomic DNA was
extracted from CEM cells following treatment with 4a (1, 5, 10 and 20 mM) for 48 h and
7.04 (s, 2H, eNH2), 7.13e7.18 (m, 2H, ar), 7.30e7.34 (m, 2H, ar).
resolved on an agarose gel. DNA from DMSO treated cells served as vehicle control.
6.2.4. 2-Amino-5-(4-bromobenzyl)-1,3,4-thiadiazole (XBr)
Yield 55%. M P. 200e202ꢁC. FTIR (
l
cmꢀ1) 3395, 3275, 2986, 2948,
: 4.14 (s, 2H. eCH2e), 7.05
1598,1511,1349,1305,1047,1012.1H NMR
d
biological activity was tested. Initial studies indicate that 4a
induced cytotoxicity is mediated through apoptosis; however, it
does not affect cell cycle progression.
(s, 2H, eNH2), 7.25 (d, 2H, J ¼ 8 Hz), 7.53 (d, 2H, J ¼ 8 Hz).
6.2.5. 2-Amino-5-(4-methylbenzyl)-1,3,4-thiadiazole (XCH3)
Yield 52%. M P. 208e212 ꢁC. FT IR (
l
cmꢀ1) 3396, 3270, 3126,
: 2.27 (s, 3H,
2922, 1605, 1516, 1430, 1342, 1147, 1048, 906. 1H NMR
d
6. Experimental protocols
CH3), 4.08 (s, 2H. eCH2e), 7.03 (s, 2H, eNH2), 7.14 (s, 4H, ar).
6.1. Chemicals and reagents
6.2.6. Synthesis of the 2-aralkyl-6-(40-fluorophenyl)-imidazo[2,1-b]
[1,3,4]thiadiazole derivatives (SCR1)
All the chemicals used in the present study were analytical grade
and purchased from SigmaeAldrich, USA. Tritiated thymidine ([3H])
was purchased from BARC, India and antibodies were purchased
from Santa Cruz Biotechnology, USA. The IR spectrawere recorded in
KBr on a Jasco 430þ; the 1H NMR spectra were recorded in CDCl3 on
a Bruker (400 Mz), and J values are reported in hertz (Hz), 2-amino-
5-benzyl-1,3,4-thiadiazole (XH) was prepared according to litera-
ture [18]. The melting points are uncorrected. Silica gel plates were
used for the TLC by using CHCl3: MeOH.
The appropriate 1,3,4-thiadiazole X (30 m mol) was treated with
the 4-fluoro phenacyl bromide (32 m mol) in ethanol (150 ml). The
mixture was refluxed for 10e12 h. Excess of solvent was removed
under reduced pressure and the solid hydrobromide separated was
filtered, 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 50e60%.
6.2. Chemistry
6.2.6.1. 2-Benzyl-6-(40-fluorophenyl)-imidazo[2,1-b][1,3,4]thiadia-
zole (1a). Yield 52%. FTIR (
l
cmꢀ1): 3136, 3065, 2923, 1598, 1470,
2-amino-5-aralkyl-1,3,4-thiadiazole X was prepared from aryl
acetic acid and thiosemicarbazide in presence of H2SO4 at 70 ꢁC for
7 h. The 2-aralkyl-6-(40-fluorophenyl)-imidazo[2,1-b][1,3,4]thia-
diazoles SCR1 reported in Scheme 1 was prepared by reaction of 2-
amino-5-aralkyl-1,3,4-thiadiazole X with the 4-fluoro phenacyl
bromide, and neutralization with cold aqueous sodium carbonate
to get the free base in 50e60% yield. The 4-fluoro phenacyl bromide
was prepared by bromination of the 4-fluoro acetophenone using
the FriedeleCrafts reaction [19]. The 2-aralkyl-6-(40-fluorophenyl)-
imidazo[2,1-b][1,3,4]thiadiazole derivative, SCR1, thus obtained
was subjected to electrophilic substitution reaction [20] at the 5th
position with bromine in the presence of CH3COONa in acetic acid
to obtain the 5-bromo derivative, SCR2 in good yield. Appropriate
aldehyde, SCR3 were obtained in turn by means of the Vilsmeier
reaction on the corresponding 2-aralkyl-6-(40-fluorophenyl)-imi-
dazo[2,1-b][1,3,4]thiadiazole, SCR1. Introduction of thiocyanate
functional group at the 5th position was carried out by reaction
between 2-phenyl-6-(40-fluorophenyl)-imidazo[2,1-b][1,3,4]thia-
diazole, SCR1 and potassium thiocyanate in glacial acetic acid by
drop wise addition of bromine in glacial acetic acid to get SCR4 in
good yield.
1422, 1222. 1H NMR
d
: 4.21 (2H, s, CH2), 7.00 (2H, t, J ¼ 16),
7.23e7.32 (5H, m, ar), 7.68 (2H, q, J ¼ 12), 7.83 (IH, s, im). þESI: 310
(309.36).
6.2.6.2. 2-(4-Chlorobenzyl)-6-(40-fluorophenyl)-imidazo[2,1-b][1,3,4]
thiadiazole (1b). Yield 55%. FTIR (
l
cmꢀ1): 3115, 3042, 2917, 2882,
1537, 1483, 1413, 1227, 1092. 1H NMR
d
: 4.27 (2H, s, CH2), 7.07e7.12
(2H, m, ar), 7.25e7.27 (2H, m, ar), 7.34e7.37 (2H, m, ar), 7.73e7.78
(2H, m, ar) 7.91 (1H, s, im). þESI: 344 (343.81).
6.2.6.3. 2-(4-Fluorobenzyl)-6-(40-fluorophenyl)-imidazo[2,1-b][1,3,4]
thiadiazole (1c). Yield 50%. FTIR (
l
cmꢀ1): 3126, 3047, 2916, 1601,
1515, 1478, 1229, 1150. 1H NMR
d: 4.45 (2H, s, CH2), 7.20e7.26 (4H,
m, ar), 7.45e7.48 (2H, m, ar), 7.86e7.89 (2H, m, ar), 8.65 (1H, s,
im). þESI: 328 (327.35).
6.2.6.4. 2-(4-Bromobenzyl)-6-(4’-fluorophenyl)-imidazo[2,1-b][1,3,4]
thiadiazole (1d). Yield 57%. FTIR (
l
cmꢀ1): 3141, 3066, 2943, 2879,
1535, 1479, 1227, 1011. 1H NMR
d
: 4.43 (2H, s, CH2), 7.20e7.25 (2H,
m, ar), 7.36 (2H, d, J ¼ 8), 7.56 (2H, d, J ¼ 8), 7.84e7.88 (2H, m, ar),
8.61 (1H, s, im). þESI: 389.9 (388.26).

S.S. Karki et al. / European Journal of Medicinal Chemistry 46 (2011) 2109e2116
2115
6.2.6.5. 2-(4-Methylbenzyl)-6-(40-fluorophenyl)-imidazo[2,1-b]
[1,3,4]thiadiazole (1e). Yield 60%. FTIR (
cmꢀ1): 3110, 3025, 2909,
1522,1478,1228,1055. 1H NMR
: 2.29 (3H, s, CH3), 4.37 (2H, s, CH2),
6.2.9. Synthesis of the 2-aralkyl-5-thiocyanato-6-(40-
l
fluorophenyl)-imidazo[2,1-b][1,3,4] thiadiazole derivatives (SCR4)
To a mixture of 2-aralkyl-6-(40-fluorophenyl)-imidazo[2,1-b]
[1,3,4]thiadiazoles SCR1 (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 drop wise
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 recrystallized from
EtOHeCHCl3 with a yield of 55e65%.
d
7.17e7.24 (4H, m, ar), 7.26 (2H, d, J ¼ 8), 7.84e7.88 (2H, m, ar), 8.60
(1H, s, im). þESI: 324 (323.39).
6.2.7. Synthesis of the 2-aralkyl-5-bromo-6-(40-fluorophenyl)-
imidazo[2,1-b][1,3,4]thiadiazole derivatives (SCR2)
2-aralkyl-6-(40-fluorophenyl)-imidazo[2,1-b][1,3,4]thiadiazole
SCR1 (5 mmol), sodium acetate (10 mmol) and 10 ml of glacial
acetic acid (stirred together at room temperature) was added drop
wise 0.98 g of bromine (6 mmol). After the addition, stirring was
continued for 30 min. The mixture was poured into 100 ml water
from which a solid separated. The solid was collected by filtration
and washed with water, dried and recrystallized from EtOHeCHCl3
with a yield of 65e75%.
6.2.9.1. 2-Benzyl-5-thiocyanato-6-(40-fluorophenyl)-imidazo[2,1-b]
[1,3,4]thiadiazole (4a). Yield 62%. FTIR (
l
cmꢀ1): 3089, 3068, 2917,
2159, 1604, 1475 and 1216. 1H NMR
d: 4.41 (2H, s, CH2), 7.19 (2H, t,
J ¼ 16), 7.35e7.41 (5H, m, ar), 7.95 (2H, dd, J ¼ 8, 8). þESI: 367 (366.44)
6.2.9.2. 2-(4-Chlorobenzyl)-5-thiocyanato-6-(40-fluorophenyl)-imi-
6.2.7.1. 2-Benzyl-5-bromo-6-(40-fluorophenyl)-imidazo[2,1-b][1,3,4]
dazo[2,1-b][1,3,4]thiadiazole(4b). Yiled 65%. FTIR (
l
cmꢀ1): 3072,
: 4.58 (2H,
thiadiazole (2a). Yield 68%. FTIR (
l
cmꢀ1): 3140, 3055, 2933, 1600,
2921, 2854, 2163, 1602, 1527, 1479, 1227, 1095. 1H NMR
d
1468, 1412 and 1224. 1H NMR
7.32e7.41 (5H, m, ar), 7.96 (2H, m, ar).þESI: 389 (388.26).
d
: 4.35 (2H, s, CH2), 7.12 (2H, t, J ¼ 16),
s, CH2), 7.36e7.40 (2H, m, ar), 7.47 (4H, m, ar), 7.98e8.08 (2H, m, ar).
-ESI: 398.9 (400.88).
6.2.7.2. 2-(4-Chlorobenzyl)-5-bromo-6-(40-fluorophenyl)-imidazo[2,1-
6.2.9.3. 2-(4-Methylbenzyl)-5-thiocyanato-6-(40-fluorophenyl)-imi-
b][1,3,4]thiadiazole (2b). Yield 75%. FTIR (
l
cmꢀ1): 3063, 2923,
dazo[2,1-b][1,3,4]thiadiazole(4c). Yield 55%. FTIR (
l
cmꢀ1): 3059,
: 2.29 (3H,
2853, 1601, 1528, 1481, 1226, 1092. 1H NMR
d
: 4.50 (2H, s, CH2),
3032, 2920, 2869, 2163, 1603, 1520, 1468, 1228. 1H NMR
d
7.28e7.33 (2H, m, ar), 7.45 (4H, s, ar), 7.95e7.98 (2H, m, ar). þESI:
s, CH3), 4.50 (2H, s, CH2), 7.19 (2H, d, J ¼ 8), 7.30 (2H, d, J ¼ 8),
423.9 (422.7).
7.36e7.40 (2H, m, ar), 7.97e8.01 (2H, m, ar). þESI: 381 (380.46).
6.2.7.3. 2-(4-Fluorobenzyl)-5-bromo-6-(40-fluorophenyl)-imidazo
[2,1-b][1,3,4]thiadiazole (2c). Yield 65%. FTIR (
l
cmꢀ1): 3066, 2938,
d: 4.49 (2H, s, CH2),
6.3. Biology
1602, 1527, 1488, 1224, 1100, 1018. 1H NMR
7.19e7.23 (2H, m, ar), 7.28e7.33 (2H, m, ar), 7.45e7.48 (2H, m, ar),
7.95e7.98 (2H, m, ar). þESI: 407.9 (406.25).
6.3.1. Cell lines and culture conditions
Human T-cell leukemia cell line, CEM was used for the present
study. CEM cell line was purchased from National Center for Cell
Science, Pune, India. Cells were cultured in RPMI1640 (SERA LAB,
UK) containing 10% FBS (GIBCO BRL, USA), 100 U of Penicillin G/ml
6.2.7.4. 2-(4-Methylbenzyl)-5-bromo-6-(40-fluorophenyl)-imidazo
[2,1-b][1,3,4]thiadiazole (2d). Yield 67%. FTIR (
l
cmꢀ1): 3288, 3034,
2922,1525, 1477,1227. 1H NMR
d: 2.29 (3H, s, CH3), 4.43 (2H, s, CH2),
and 100 m
g of streptomycin/ml (SigmaeAldrich, USA) at 37 ꢁC in
7.18 (2H, d, J ¼ 8), 7.28e7.33 (4H, m, ar), 7.94e7.98 (2H, m, ar). þESI:
a humidified atmosphere containing 5% CO2.
403.2 (402.28).
6.3.2. Trypan blue dye exclusion assay
6.2.8. Synthesis of the 2-aralkyl-5-formyl-6-(40-fluorophenyl)-
imidazo[2,1-b][1,3,4]thiadiazole derivatives (SCR3)
The effect of 1ae1e, 2ae2d, 3a and 3b and 4ae4c on cell viability
of T-cell leukemic cells was determined by Trypan blue dye exclusion
assay [4,21]. CEM cells were seeded at a density of 1 ꢂ 105 cells/ml,
cultured for 24 h and compounds were added at a concentration of
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 2-
aralkyl-6-(40-fluorophenyl)-imidazo[2,1-b][1,3,4]thiadiazole SCR1
(4 mmol) was added slowly to the Vilsmeier reagent while main-
taining 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 that separated was filtered, washed with
water, dried and recrystallized from EtOHeDMF with a yield
55e60%.
10, 50, 100 and 250 mM. DMSO (SigmaeAldrich, USA) treated cells
were used as vehicle control. 5-fluorouracil treated cells were used
as positive control. Cells were harvested at intervals of 24 h and
resuspended in 0.4% Trypan blue (SigmaeAldrich, USA) and the
viable cells were counted using hemocytometer. IC50 values (50%
inhibition of cell growth) were estimated after 48 and 72 h of
treatment. Experiment was repeated atleast 3 times and the values
obtained were plotted against respective time points.
6.3.3. MTT assay
6.2.8.1. 2-Benzyl-5-formyl-6-(40-fluorophenyl)-imidazo[2,1-b][1,3,4]
The effect of 1ae1e, 2ae2d, 3a and 3b and 4ae4c on cell
proliferation of CEM cells was determined by MTT assay as
described previously as in References [15,22]. In brief, cells were
treated with increasing concentrations of compounds (10, 50, 100
thiadiazole (3a). Yield 55%. FTIR (
l
cmꢀ1): 3081, 3028, 2951, 2864,
2777, 1676, 1595, 1522, 1482, 1315. 1H NMR
d
: 4.56 (2H, s, CH2),
7.32e7.45 (7H, m, ar), 8.01e8.05 (2H, m, ar), 9.98 (1H, s, CHO). þESI:
338 (337.37).
and 250
mM) and incubated for 24, 48 and 72 h. MTT reagent
(5 mg/ml, SigmaeAldrich, USA) was added and the color change
due to proliferating cells was estimated. 5-fluorouracil treated
cells were used as positive control while DMSO treated cells were
used as vehicle control. Each experiment was repeated
a minimum of 3 independent times and the values obtained were
plotted.
6.2.8.2. 2-(4-Chlorobenzyl)-5-formyl-6-(40-fluorophenyl)-imidazo
[2,1-b][1,3,4]thiadiazole (3b). Yield 60%. FTIR (
l
cmꢀ1): 3053, 2980,
2916, 2840, 1653, 1604, 1492, 1388, 1230, 1090. 1H NMR
d: 4.56 (2H,
s, CH2), 7.32e7.36 (2H, m, ar), 7.45 (4H, s, ar), 7.99e8.03 (2H, m, ar),
9.96 (1H, s, CHO). -ESI: 370 (371.82).

2116
S.S. Karki et al. / European Journal of Medicinal Chemistry 46 (2011) 2109e2116
6.3.4. Tritiated thymidine incorporation assay
start up grant for SCR. KPM is supported by IISc postdoctoral
fellowship programme, India.
DNA synthesis was monitored by labeling cells using [3H]
thymidine as described [21]. The 4a was added to CEM cells at
a concentration of 10 m mCi) was
M after 24 h. The [3H] thymidine (1
Appendix. Supplementary material
added after 8 h of treatment and was harvested after 12, 24 and
48 h of incubation, processed and used for scintillation counting.
The radioactivity count was expressed as disintegrations/min.
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.02.064.
6.3.5. Cell cycle analysis
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6.3.6. Measurement of mitochondrial membrane potential
Changes of mitochondrial transmembrane potential (djm) were
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tetrachloro-1, 1, 3, 30- tetraethylbenzimidazolcarbocyanamide
iodided; Calbiochem, USA) [15,23]. Cells were cultured, treated
with 4a (1, 5, 10 and 20
pended in PBS and incubated with JC-1 (0.5
m
M) for 48 h, harvested, washed, resus-
m
M) at 37 ꢁC for 15 min.
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CellQuest Pro software an excitation at 488 nm laser and emission
at 530/30, 580/610 nm. JC-1 monomers emit at 530 nm (FL-1) and J-
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treatment with 4a (1, 5, 10 and 20
genomic DNA was resolved on a 1% agarose gel [17] and visualized
by ethidium bromide staining and analyzed.
mM) for 48 h. The isolated
6.3.8. Statistical analysis
Values were expressed as mean ꢃ SEM for control and experi-
mental samples and statistical analysis was performed by Student’s
t-test. The values were considered statistically significant, if the p-
value was less than 0.05.
Acknowledgements
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659e670.
We thank Dr. Bibha Choudhary, Ms. Nishana M. and members of
the SCR laboratory for discussions and help. This work was sup-
ported by grant from Leukemia Research Foundation, USA, and IISc