Synthesis, characterization and anti-inflammatory activity of 2-(4-chlorobenzyl)-5-(di(5- substituted-1h-indol-3-yl)methyl)-6-(4-substituted phenyl)imidazo[2,1-b][1,3,4]thiadiazoles

Article abstract of DOI:10.14233/ajchem.2020.22830

In present study, the anti-inflammatory potential of a series of 2-(4-chlorobenzyl)-5-(di(5-substituted-1H-indol-3-yl)methyl)-6-(4-substituted phenyl)imidazo[2,1-b][1,3,4]thiadiazoles (5a-e) were evuluated after characterizing through 1H NMR, 13C NMR spec

Full text of DOI:10.14233/ajchem.2020.22830

Asian Journal of Chemistry; Vol. 32, No. 10 (2020), 2577-2582
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2020.22830
Synthesis, Characterization and Anti-inflammatory Activity of 2-(4-chlorobenzyl)-5-(di(5-
substituted-1H-indol-3-yl)methyl)-6-(4-substituted phenyl)imidazo[2,1-b][1,3,4]thiadiazoles
1
2
3
AYESHA SIDDIQ , SADASHIVAMURTHY SHAMANTH , MOHAMMED SALMAN ,
3
2
4,*
K.K. DHARMAPPA , MANTELINGU KEMPEGOWDA andSHANKAR JAYARAMA
1Post-Graduate Department of Biotechnology, Teresian College, Siddhartha Nagar, Mysuru-570011, India
2Department of Studies in Chemistry, Manasagangotri, University of Mysore, Mysuru-570006, India
3North Medical LLC, (CP) Qiagen, Al Reem Tower, Dafna, Doha, Qatar
4Department of Studies and Research in Food Technology, Davangere University, Davangere-577007, India
*Corresponding author: E-mail: shankarbio@gmail.com
Received: 18 May 2020;
Accepted: 6 July 2020;
Published online: 25 September 2020;
AJC-20072
In present study, the anti-inflammatory potential of a series of 2-(4-chlorobenzyl)-5-(di(5-substituted-1H-indol-3-yl)methyl)-6-(4-substituted
phenyl)imidazo[2,1-b][1,3,4]thiadiazoles (5a-e) were evuluated after characterizing through 1H NMR, 13C NMR spectral studies. Initial
toxicity and anti-inflammatory efficacy of synthesized compounds is evaluated against RAW264.7 macrophages cell line to derive at their
IC50 values. Compounds 5a-e are further tested for their anti-inflammatory activity in Swiss albino rats using the 1% carrageenan-induced
paw edema model. Compound 5a is taken as the lead compound and is further considered for behavioural evaluations such as stair
climbing and motility exercises. Paw edema is analyzed at time intervals of 0, 1, 2, 3, 4 and 24 h and stair climbing and motility tests are
surveyed after 24 h of exposure to the test compound. The results of present study show that the compound 5a significantly reduces the
paw thickness by 68% at p < 0.001 and increased the stair climbing (1.5 folds) and motility (1.6 folds) when compared to the 1%
carrageenan rat group. Conclusively the results of present study show that compound 5a exhibits strong anti-inflammatory potential that
can be used as a preclinical template for further investigations as an alternative therapy to the current NSAIDs.
Keywords: Imidazo[2,l-b][1,3,4]thiadiazole derivative, Anti-inflammatory activity, Paw edema, Stair climbing.
anti-inflammatory properties [9-11]. The imidazo[2,1-
b][1,3,4]thiadiazole construct are widely explored as a reliable
source for discovering novel biologically active molecules
since the 1,3,4-thiadiazole moiety is pharmaceutically suitable
as a “hydrogen binding domain” while the imidazole nucleus
is responsible for the biological activities of compounds con-
taining it [12-15].
Thus taking leads from the above broad-spectrum benefits
of imidazo[2,1-b][1,3,4]thiadiazole scaffold, we synthesized
imidazo[2,l-b][1,3,4]thiadiazole derivatives (5a-e) for their
proposed anti-inflammatory potential after having characteri-
zing them through LCMS and NMR. Among the synthesized
compounds, 5a displayed a good correlation in the analytical
and spectral data to the proposed structures. Compounds of
the 5a-e series were further studied for their cytotoxic potential
in mouse macrophage cell line RAW 264. Compound 5a was
INTRODUCTION
Inflammation is a complex defensive mechanism of the
body tissue that cover features of redness, pain and swelling
[1].Although non-steroidal anti-inflammatory drugs (NSAIDs)
are the most favoured line of treatment they can lead to gastro-
intestinal complications during long term use leading to
suppression of physiological prostaglandin production in these
tissues [2,3]. Since NSAIDs cause immune suppression after
long use research in this area, should address alternatives that
are safer and come with minimal side effects [4-7].
Imidazo[2,1-b][1,3,4]thiadiazole derivatives exhibit wide
range of biological activities [8]. The imidazo[2,1-b][1,3,4]-
thiadiazole scaffold makes up the core structure of pharma-
cologically active molecules possessing antimicrobial, anti-
convulsant, diuretic, anticancer, antitubercular, analgesic and
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2578 Siddiq et al.
Asian J. Chem.
selected as the lead compound for anti-inflammatory investi-
gations based upon its IC50 value 35 µM. 5a was orally gavaged
in Swiss albino rats using 1% carrageenan-induced paw edema.
The edema was compared to standard anti-inflammatory drug
indomethacin. Paw thickness was assessed at various time
points as 1, 2, 3, 4 and 24 h. Further stair climbing and motility
tests were performed after 24 h of exposure to 1% carrageenan.
The results of current study show that compound 5a signifi-
cantly reduces the paw thickness and increases the climbing
and motility in the animal after injection of compound 5a.
Conclusively the results of present study show that compound
5a exhibits strong anti-inflammatory potential that can be used
as a preclinical template for further investigation as an alter-
native line of treatment to NSAID therapy.
absorbance was read at a wavelength 560 nm. The results are
expressed as % inhibition vs. concentration
Animals and care: Male Swiss albino female rats weighing
around 120-150 g were used. They received standard mainte-
nance conditions with (12:12 h light/dark cycles, 50% 5%
humidity and temperature of 25 2 °C) and were housed in
polypropylene cages. Pellet diet was ad libitum with free access
to water. All the animal investigations were permitted by the
InstitutionalAnimal Ethics Committee (IAEC), (Approval No:
BCP/IAEC/EXTP/04/2018) Bharathi College of Pharmacy,
Bharathi Nagara, Mandya District, India. The experiments
were carried in agreement with the Committee for the purpose
of control and supervision of experiments on animals (CPCSEA)
guidelines for laboratory animal facility.
Synthesis of 2-(4-chlorobenzyl)-5-(di(5-substituted-1H-
indol-3-yl)methyl)-6-(4-substituted phenyl)imidazo[2,1-b]-
[1,3,4]thiadiazole (5a-e): 5-(4-Chlorobenzyl)-1,3,4-thiadiazol-
2-amine (1) was prepared from 4-chlorophenyl-acetic acid and
thiosemicarbazide using H2SO4 as solvent at 75 °C for 6 h. 2-
(4-Chlorobenzyl)-6-(4-substituted phenyl)imidazo[2,1-b]-
[1,3,4]thiadiazole derivatives (3a-d) were synthesized by reflu-
xing 5-(4-chlorobenzyl)-1,3,4-thiadiazol-2-amine (1) with the
appropriate phenacyl bromide (2) in ethanol for 8 h and quenching
with aqueous sodium carbonate. The various phenacyl bromides
(2a-d) were purchased from Sigma-Aldrich. 2-(4-Chlorobenzyl)-
6-(4-substituted phenyl)imidazo-[2,1-b][1,3,4]thiadiazole-5-
carbaldehydes (4a-d) were synthesized by using Vilsmeier-
Haack condition (POCl3 and DMF) on the corresponding 2-
(4-chlorobenzyl)-6-(4-substituted phenyl)imidazo[2,1-b]-
[1,3,4]thiadiazole. 2-(4-Chlorobenzyl)-5-(di(5-substituted-1H-
indol-3-yl)methyl)-6-(4-substituted phenyl)imidazo[2,1-b]-
[1,3,4]thiadiazoles (5a-e) were obtained by treating correspon-
ding aldehydes (4a-d) and two equivalence of 5-substituted
indoles in presence of iodine in acetonitrile at 60 ºC for 6 h
[16,17] (Scheme-I).
EXPERIMENTAL
Chlorophenyl acetic acid, thiosemicarbazide, phenacyl
bromide, ethanol, aqueous sodium carbonate and all the other
chemicals involved in the chemical synthesis were procured
from (Sigma-Aldrich, USA).Carrageenan was acquired from
(Hi-Media, India). Standard anti-inflammatory drug indomethacin
was acquired from (Recon, Bangalore, India). RAW 264.7 mouse
macrophage cell line was obtained fromATCC (American type
culture collection); Manassas, USA and cultured with Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10%
heat inactivated fetal bovine serum (FBS), 100 µg/mL strepto-
mycin and 100 µg/mL penicillin (Gibco BRL), maintained at
37 ºC in a 5% CO2 incubator.
Cell cytotoxicity:All the compounds 5a-e were screened
for in vitro cytotoxic potential using RAW 264.7 cell line. Cells
were seeded at a density of 1 × 106 cells/well in a 96-well plate
and grown at standard conditions (37 °C with 5% CO2) for 24 h
and further exposed to increasing concentrations of synthesized
compounds 5a-e from a concentration range of 6.25 µM to
200 µM. Compounds that were synthesized were dissolved in
DMSO as treatment group, while vehicle group were given
DMSO alone. After 24 h of exposure, cells were treated with
MTT (5 mg/mL) for 4 h. Insoluble formazan crystals are formed
in the process that was treated with 50 µL of DMSO and the
Spectral data
5-(4-Chlorobenzyl)-1,3,4-thadiazol-2-amine (1):Yield
65%; m.p.: 181-183 °C; brown solid; 1H NMR (400 Hz, DMSO-
O
N
H
N
O
N
N
(a)
(b)
N
N
+
Br
S
S
S
N
Cl
N
R1
Cl
Cl
NH2
1
R1
3
2
R1
4
(c)
R1
= 4-CH3-Ph
5b = 4-CH3-Ph
5c = 4-Cl-Ph
R2
R2
R2
Reagents and conditions:
(a)
5a
Br
H
H
H
H
Ethyl alcohol, sodium carbonate, reflux
(b) DMF, POCl3, 80-90 °C
(c)
HN
N
Iodine, acetonitrile, room temp.
5d
= 4-Br-Ph
5e = Ph
N
NH
S
N
Cl
R1
5(a-e)
Scheme-I: Synthesis of imidazo[2,1-b][1,3,4]thiadiazole derivative 5(a-e)

Vol. 32, No. 10 (2020)
Synthesis, Characterization and Anti-inflammatory Activity of Substituted Thiadiazoles 2579
d6): δ ppm: 4.123 (s, 2H, -CH2-), 7.022 (s, 2H, -NH2), 7.260-
7.80 (d, 2H, ar, J = 8 Hz), 7.348-7.367 (d, 2H, ar, J = 7.6 Hz);
13C NMR (400 Hz, DMSO-d6) δ ppm: 35.123, 129.002,
130.949, 131.998, 137.420, 157.520, 169.382; HRMS-(ESI):
m/z calculated for [C9H8ClN3S+H+] = 227.0127: found =
227.0129.
6-(4-Bromophenyl)-2-(4-chlorobenzyl)imidazo[2,1-b]-
[1,3,4]thiadiazole (3a): Yield 58%; m.p.: 165-167 °C; pale
yellow solid; 1H NMR (400 Hz, DMSO-d6): δ ppm: 4.430 (s,
2H, -CH2-), 7.412 (s, 4H, arom.), 7.547-7.569 (d, 2H, arom.,
J = 8.8 Hz), 7.756-7.777 (d, 2H, arom., J = 8.4 Hz), 8.672 (s,
1H, -CH=); 13C NMR (400 Hz, DMSO-d6) δ ppm: 36.533,
111.205, 120.558, 127.037, 129.257, 131.495, 132.017, 132.652,
133.601, 135.413, 144.197, 145.544, 164.715; HRMS-(ESI):
m/z calculated for [C17H11BrClN3S + H+] = 405.7113: found =
405.7115.
δ ppm: 4.523 (s,2H, -CH2-), 7.392-7.437 (t, 4H, arom., J =
8.8 Hz), 7.506-7.528 (d, 2H, arom., J = 8.8 Hz), 7.926-7.947
(d, 2H, arom., J = 8.4 Hz), 9.935 (s, 1H, -CHO); 13C NMR (400
Hz, DMSO-d6): δ ppm: 36.411, 123.865, 129.168, 129.304,
130.860, 131.473, 132.787, 134.810, 135.307, 150.990, 152.527,
167.393, 177.560; HRMS-(ESI): m/z calculated for [C18H11Cl2-
N3OS+ H+] = 389.2704: found = 389.2701.
2-(4-Chlorobenzyl)-6-phenylimidazo[2,1-b][1,3,4]thia-
diazole-5-carbaldehyde (4c): Yield 60%; m.p.: 148-150 °C;
pale yellow solid; 1H NMR (400 Hz, DMSO-d6): δ ppm: 4.532
(s, 2H, -CH2-), 7.425-7.484 (t, 7H, arom., J = 6.4 Hz), 7.878-
7.898 (t, 2H, arom., J = 6.0 Hz), 9.926 (s, 1H,-CHO); 13C NMR
(400 Hz, DMSO-d6): δ ppm: 36.411, 123.856, 129.177,
129.323, 130.014, 131.493, 132.631, 132.758, 135.394, 151.204,
154.541, 167.150, 177.587; HRMS-(ESI): m/z calculated for
[C18H12ClN3OS + H+] = 354.8254: found = 354.8259.
2-(4-Chlorobenyl)-6-(4-chlorobenzyl)imidazo[2,1-b]-
[1,3,4]thiadiazole (3b): Yield 63%; m.p.: 167-170 °C; pale
yellow solid; H NMR (400MHz-DMSO-d6): δ ppm : 4.428
2-(4-Chlorobenzyl)-6-(p-tolyl)imidazo[2,1-b][1,3,4]-
thiadiazole-5-carbaldehyde (4d):Yield 66%; m.p.: 158-160
ºC; pale yellow solid; 1H NMR (400 Hz, DMSO-d6): δ ppm:
2.317 (s, 3H, -CH3), 4.503 (s, 2H, -CH2-), 7.246-7.265 (d, 2H,
arom., J = 7.6 Hz), 7.403 (s, 4H, arom.), 7.754-7.774 (d, 2H,
1
(s, 2H, -CH2-), 7.409-7.434 (d, 6H, ar, J = 6 Hz), 7.814-7.835
(d, 2H, ar, J = 8.4 Hz), 8.662 (s, 1H, -CH=); 13C NMR (400 Hz,
DMSO-d6): δ ppm: 36.537, 111.149, 126.716, 129.090, 129.236,
131.473, 132.038, 132.660, 133.234, 135.384, 144.180, 145.522,
164.640; HRMS-(ESI): m/z calculated for [C17H11Cl2N3S+ H+]
= 361.2603: found = 361.2605.
2-(4-Chlorobenyl)-6-phenylimidazo[2,1-b][1,3,4]thia-
diazole (3c): Yield 55%; m.p.: 155-157 °C; pale yellow solid;
1H NMR (400 Hz, DMSO-d6): δ ppm : 4.421 (s, 2H, -CH2-),
7.210-7.247 (t, 1H, ar, J = 7.6 Hz), 7.339-7.377 (t, 2H, ar, J =
8 Hz), 7.406 (s, 4H, ar), 7.803-7.822 (d, 2H, ar, J = 7.6 Hz),
8.612 (s, 1H, -CH=); 13C NMR (400 Hz, DMSO-d6): δ ppm:
36.508, 110.741, 125.042, 127.689, 129.080, 129.245, 131.493,
132.631, 134.305, 135.462, 145.337, 164.406; HRMS-(ESI):
m/z calculated for [C17H12ClN3S + H+] = 326.8153: found =
326.8150.
2-(4-Chlorobenyl)-6-(p-tolyl)imidazo[2,1-b][1,3,4]thia-
diazole (3d): Yield 68%; m.p.: 165-167 °C; pale yellow solid;
1H NMR (400 Hz, DMSO-d6): δ ppm : 2.273 (s, 3H, -CH3),
4.416 (s, 2H, -CH2-), 7.156-7.176 (d, 2H, arom., J = 8 Hz), 7.405
(s, 4H, arom.), 7.689-7.709 (d, 2H, arom., J = 8 Hz), 8.537 (s, 1H,
-CH=); 13C NMR (400 Hz, DMSO-d6): δ ppm: 21.232, 36.523,
110.218, 125.026, 129.238, 129.627, 131.467, 131.590, 132.634,
135.479, 136.912, 145.525, 164.117; HRMS-(ESI): m/z calcu-
lated for [C18H14ClN3S+ H+] = 340.8419: found = 340.8422.
6-(4-Bromophenyl)-2-(4-chlorobenzyl)imidazo[2,1-b]-
[1,3,4]thiadiazole-5-carbaldehyde (4a): Yield 62%; m.p.:
138-140 °C; pale brown solid; 1H NMR (400 Hz, DMSO-d6):
δ ppm: 4.532 (s, 2H, -CH2-), 7.424 (s, 4H, arom.), 7.657-7.679
(d, 2H, arom., J = 8.8 Hz), 7.866-7.887 (d, 2H, arom., J = 8.4
Hz), 9.946 (s, 1H, CHO); 13C NMR (400 Hz, DMSO-d6): δ ppm:
36.447, 123.594, 123.897, 129.313, 131.116, 131.486, 131.865,
132.102, 132.804, 135.309, 151.017, 152.601, 167.390, 177.577;
HRMS-(ESI): m/z calculated for[C18H11BrClN3OS + H+] =
433.7214: found = 433.7215.
13
arom., J = 8 Hz), 9.897 (s, 1H, -CHO); C NMR (400 Hz,
DMSO-d6): δ ppm: 21.336, 123.660, 129.190, 129.285, 129.702,
129.835, 131.438, 132.785, 135.327, 139.719, 151.121, 154.698,
166.858, 177.407; HRMS-(ESI): m/z calculated for [C19H14Cl-
N3OS + H+] = 368.8520: found = 368.8524.
5-(Bis(5-bromo-1H-indol-3-yl)methyl)-2-(4-chloro-
benzyl)-6-(p-tolyl)imidazo[2,1-b][1,3,4]thiadiazole (5a):
Yield 55%; m.p.: 228-230 ºC; white solid; 1H NMR (400 Hz,
DMSO-d6): δ ppm: 2.313 (s, 3H, -CH3-), 4.263 (s, 2H, -CH2-),
6.251 (s, 1H, -CH-), 7.095-7.141 (t, 8H, arom., J = 13.6), 7.233-
7.252 (d, 2H, arom., J = 7.6 Hz), 7.302-7.348 (t, 4H, arom., J
= 18.4 Hz), 7.460-7.480 (d, 2H, arom., J = 8 Hz), 11.177 (s, 2H,
-NH-); 13C NMR (400 Hz, DMSO-d6): δ ppm: 26.031, 35.466,
41.024, 116.317, 118.798, 118.891, 125.710, 128.653, 129.730,
131.084, 132.755, 133.196, 133.781, 134.437, 135.729, 136.898,
137.134, 140.221, 141.872, 146.251, 148.876, 167.202; HRMS-
(ESI): m/z calculated for [C35H24Br2ClN5S + H+] = 742.9246:
found = 742.9248.
2-(4-Chlorobenzyl)-5-(di(1H-indol-3-yl)methyl)-6-(p-
tolyl)imidazo[2,1-b][1,3,4]Thiadiazole (5b): Yield 62%;
m.p.: 202-204 °C; white solid; 1H NMR (400 Hz, DMSO-d6):
δ ppm: 2.306 (s, 3H, -CH3-), 4.279 (s, 2H, -CH2-), 6.350 (s, 1H,
-CH-), 6.786-6.824 (t, 2H, arom., J = 15.2 Hz), 6.985-7.045
(m, 6H, arom.), 7.108-7.129 (d, 2H, arom., J = 7.6 Hz), 7.208-
7.227 (d, 2H, arom., J = 7.6 Hz), 7.311-7.365 (m, 4H, arom.),
7.498-7.518 (d, 2H, arom., J = 8 Hz), 10.915 (s, 2H, -NH-);
13C NMR (400 Hz, DMSO-d6): δ ppm: 21.243, 30.770, 36.338,
112.050, 114.747, 118.378, 118.942, 121.362, 124.644, 125.823,
126.736, 127.915, 129.033, 129.586, 131.032, 132.365, 135.493,
136.714, 136.888, 141.164, 143.790, 162.166; HRMS-(ESI):
m/z calculated for [C35H26ClN5S + H+] = 585.1324: found =
585.1328.
2-(4-Chlorobenzyl)-6-(4-chlorophenyl)-5-(di(1H-indol-
3-yl)methyl)imidazo[2,1-b][1,3,4]Thiadiazole (5c): Yield
54%; m.p.: 182-184 °C; white solid; 1H NMR (400 Hz, DMSO-
d6): δ ppm: 4.293 (s, 2H, -CH2-), 6.371 (s, 1H, -CH-), 6.794-
2-(4-Chlorobenzyl)-6-(4-chlorophenyl)imidazo[2,1-b]-
[1,3,4]thiadiazole-5-carbaldehyde (4b): Yield 55%; m.p.:
157-160 °C; pale yellow solid; 1H NMR (400 Hz, DMSO-d6):

2580 Siddiq et al.
Asian J. Chem.
6.831 (t, 2H, arom., J = 14.8 Hz), 6.988-7.037 (m, 6H, arom.),
7.118-7.139 (d, 2H, arom., J = 8.4 Hz), 7.315-7.367 (m, 4H,
arom.), 7.437-7.458 (d, 2H, arom., 8.4 Hz), 7.607-7.628 (d,
2H, arom., J = 8.4 Hz), 10.938 (s, 2H, -NH-); 13C NMR (400
Hz, DMSO-d6): δ ppm: 30.944, 36.338, 112.071, 114.307,
118.439. 119.003, 121.413, 124.756, 126.387, 126.695,
128.992, 129.053, 129.535, 131.063, 132.222, 132.407, 133.965,
135.421, 136.755, 139.913, 144.148, 162.802; HRMS-(ESI):
m/z calculated for [C34H23Cl2N5S + H+] = 605.5509: found =
605.5516.
6-(4-Bromophenyl)-2-(4-chlorobenzyl)-5-(di(1H-indol-
3-yl)methyl)imidazo[2,1-b][1,3,4]Thiadiazole (5d): Yield
57%; m.p.: 178-180 °C; white solid; 1H NMR (400 Hz, DMSO-
d6): δ ppm: 4.291 (s, 2H, -CH2-), 6.373 (s, 1H, -CH-), 6.794-
6.832 (t, 2H, arom., J = 15.2 Hz), 6.990-7.038 (m, 6H, arom.),
7.116-7.138 (d, 2H, arom., J = 8.8 Hz), 7.315-7.367 (m, 4H,
arom.), 7.544-7.596 (m, 4H, arom.), 10.938 (s, 1H, -NH-), 10.942
(s, 1H, -NH-); 13C NMR (400 Hz, DMSO-d6): δ ppm: 30.953,
36.333, 112.083, 114.292, 118.446, 119.001, 120.820, 121.414,
124.760, 126.414, 126.696, 129.061, 129.849, 131.065, 131.901,
132.407, 134.324, 135.423, 136.756, 139.938, 144.180, 162.820;
HRMS-(ESI): m/z calculated for [C34H23BrClN5S + H+] =
650.0019: found = 650.0023.
2-(4-Chlorobenzyl)-5-(di(1H-indol-3-yl)methyl)-6-
phenylimidazo[2,1-b][1,3,4]thiadiazole (5e): Yield 63%;
m.p.: 169-171 °C; white solid; 1H NMR (400 Hz, DMSO-d6): δ
ppm: 4.283 (s, 2H, -CH2-), 6.370 (s, 1H, -CH-), 6.782-6.818
(t, 2H, arom., J = 14.4 Hz), 6.972-7.033 (m, 6H, arom.), 7.108-
7.128 (d, 2H, arom., J = 8 Hz), 7.312-7.428 (m, 7H, arom.),
7.608-7.626 (d, 2H, arom., J = 7.2 Hz), 10.929 (s, 2H, -NH-);
13C NMR (400 Hz, DMSO-d6): δ ppm: 30.788, 36.304, 112.064,
114.603, 118.349, 118.952, 121.365, 124.673, 126.103, 126.696,
127.669, 127.971, 129.002, 129.031, 131.045, 132.349, 135.141,
135.491, 136.688, 141.066, 143.975, 162.412; HRMS-(ESI):
m/z calculated for [C34H24ClN5S + H+] = 571.1059: found =
571.1063.
Stair climbing activity test: Stair climbing activity test
was done following a previous protocol [18]. Briefly, rats were
previously acclimatized to climb a staircase that had steps at a
distance of 5, 10 and 15 cm. The climbing ability of rats was
evaluated based on a rewards placed on the steps at different
distances with step 2 at 10 cm having water and step 3 at 15 cm
having food. A score of 0 was awarded to the rat if it did not
climb; 1, for climbing onto step 1; 2, for climbing onto step 2
and 3, if the rat could climb all the three steps.
Motility test:A motility test was done following a previous
protocol [19,20]. Briefly, rats were observed for 5 min and
scored according to the following scorecard:
0- If the rat walked with difficulty and avoided touching
the toes of the inflamed paw to the floor.
1- If the rat walked with little difficulty, but with toe
touching the floor.
2- If the rat walked easily.
3- If the rat could climb all three steps.
Statistical analysis: Data were reported as Mean SEM
and analyzed statistically with one and two wayANOVA follo-
wed by Turkeys multiple-comparison test with significance at
p < 0.001.
RESULTS AND DISCUSSION
Design and chemistry: All compounds that were synthe-
sized displayed a good correlation in the spectral and analytical
data to the proposed structures.
Cell cytotoxicity: Compounds of series 5(a-e) were
screened in vitro for their anti-inflammatory properties against
RAW 264.7 cell line. IC50 was prepared by nonlinear regression
analysis and graph plotted by percentage inhibition against
concentration of series from 6-200 µM. The results of our study
showed that among all the compounds tested 5a induced the
highest anti-inflammatory potential with an IC50 value of 35
µM. Molecule 5b, 5c, 5d and 5e displayed IC50 values of 43,
68, 147 and 135 µM. Therefore, 5a was taken as lead compound
(Fig. 1).
Experimental layout: 20 male Swiss albino female rats
were distributed into 4 groups with 5 animals in each (n = 5):
Group A– Control (oral gavaging with saline)
Group B –1% carrageenan (100 µL)
Group C –Animals were orally gavaged with indomethacin
(10 mg/kg b.wt)
100
5a
5b
80
5c
Group D – Animals were orally gavaged with compound
5a (150 mg/kgb.wt).
60
40
20
0
5d
5e
Induction of acute inflammation: This was conducted
following protocols from the reported literature [16,17]. In short,
anti-inflammatory activity was determined by the carrageenan-
induced rat paw edema test. Acute inflammation (edema) was
induced 1 h after the oral administration of 5a, by intraplantar
injection of 100 µL of 1% carrageenan (Hi-Media, India) saline
solution into the left hind paw of the rats. The paw volume
(mL) was measured using plethysmometre (UgoBasile 7140,
Varese, Italy) at 1, 2, 3, 4 and 24 h after carrageenan injection.
Edema volume:
0.0
0.5
1.0
1.5
2.0
2.5
Conc. (µM) (log X)
5a
1.549
35.39
5b
5c
5d
5e
log IC50
IC50
1.637
43.37
1.832
68.00
2.167
146.9
2.129
134.6
Edema volume (mL) = Vt – Vo
where Vo is the mean paw volume before carrageenan intra-
plantar injection, Vt is the mean paw volume at t hours.
Fig. 1. Effect of (5a-e) on the toxicity of RAW 264.7 cell line. IC50 was
prepared by nonlinear regression analysis and graph plotted by percen-
tage inhibition against concentration of series from 6-200 µM

Vol. 32, No. 10 (2020)
Synthesis, Characterization and Anti-inflammatory Activity of Substituted Thiadiazoles 2581
4
Carrageenan induced paw edema: In this study, 100 µL
of 1% carrageenan intraplantar injections were given to the
rear paw of rats to induce inflammatory responses (Group B).
Carrageenan was further superimposed with either indome-
thacin that served as a positive control (Group C) or with the
test compound 5a (Group D), 5b (Group E), 5c (Group F), 5d
(Group G), 5e (Group H) to derive at their anti-inflammatory
efficacy [21]. The results of our study show that edema was
highest at 4 h with various groups showing the following mean
edema scores
3
2
1
0
**
***
***
Group A: 0.44 0.012;
Group C: 0.544 0.007;
Group E: 1.32 0.093;
Group G: 1.304 0.114;
Group B: 1.37 0.053
Group D: 0.614 0.035
Group F: 1.26 0.0149
Group H: 1.320 0.035
A
B
D
C
Groups
However, after 24 h the edema scores decreased drastically
with (Group C) showing 71% and (Group D) exhibiting a 68%
decrease in inflammation rates as compared to 1% carrageenan
group (Group B) that served as a positive control. Responses
of (Group E, F, G and H) were comparable to 1% carrageenan
group (Group B) and displayed poor anti-inflammatory res-
ponses as compared to (Group D).Group A showed increased
edema with a mean value of 0.44 0.009 at p < 0.001 (Fig. 2).
Fig. 3. Drugs were administered half-hour orally before injecting inflamma-
tion. Stair climbing activity was learnt at the time of peak inflamma-
tion (4 h for carrageenan). Group A: normal; Group B: carrageenan
control; Group C: indomethacin; Group D: treated. Data are presented
as mean SEM (**p < 0.01; *** p < 0.001)
decreased by 51% (Group B), 15% (Group C) and 20% (Group
D) as compared to the control group. As compared to (Group
B) indomethacin (Group C) showed a recovery by 1.7 folds
and compound 5a (Group D) showed a recovery of 1.6 folds.
This is interesting as 5a shows comparable results to (Group
C). Compound 5a representing (Group D) was able to revert
the motility of the animal by 31% when compared to (Group
B). Compound 5a qualifies as an anti-inflammatory chemical
as it showed comparable responses to indomethacin, the standard
anti-inflammatory drug (Group C) (Fig. 4).
2.0
C
G
D
H
A
E
B
F
1.5
1.0
0.5
0.0
4
3
2
1
0
0
1
2
3
4
24
Time (h)
Fig. 2. Change in paw thickness at t = 0, 1, 2, 3, 4 and 24 h; n = 5 (signicant
at p < 0.001). Edema was induced by injecting 0.1 mL of 1% solution
of carrageenan into the subplantar surface of the left hind paw. Group
A: normal; Group B: carrageenan control; Group C: indomethacin;
Group D: 5a; Group E: 5b; Group F: 5c; Group G: 5d; Group H: 5e
Stair climbing activity: In this study, we analyzed the
anti-inflammatory potential of compound 5a through the stair
climbing capacity exercise. Rats were inflicted with 100 µL
of 1% carrageenan intraplantar injections given in the rear
paw of rats to induce inflammatory responses (Group B). The
results of present study showed that when compared to (Group
B) positive control, (Group C) exhibited 1.7 fold increase and
(Group D) 1.5 fold increase in the recovery for stair climbing
capacity of the rats. Therefore, compound 5a shows comparable
responses to indomethacin and also qualifies as an anti-inflam-
matory agent (Fig. 3).
A
B
C
D
Groups
Fig. 4. Motility score was learnt at the time of peak inflammation (4 h for
carrageenan); Group A: normal; Group B: carrageenan control;
Group C: indomethacin; Group D: treated. Data are presented as
Mean SEM (**p < 0.01; ***p < 0.001)
Motility: The aim of this experiment was to study the
motility responses of the animal after inflicting them with 1%
carrageenan (Group B) along with Group C. and Group D. The
results of present study show that motility of the animal was
Conclusion
The present study reveals that the synthesized compound
imidazo[2,1-b][1,3,4]thiadiazole derivatives (5a-e) show the

2582 Siddiq et al.
Asian J. Chem.
7. H.P. Huemer, Mediat. Inflammation, 2015, 349176 (2015);
strong anti-inflammatory potential. It successfully reduced the
edema scores in tested groups C and D when compared to group
B (1% carrageenan) that served as a positive control. These
results are further substantiated from the data received from
the stair climbing and motility exercise in the rats that comple-
ment its anti-inflammatory nature. Conclusively, compound
5a successfully qualifies as an anti-inflammatory chemical and
this study is valuable as 5a exhibits a promising approach for
the treatment of inflammatory diseases and its anti-inflam-
matory potential can be exploited for designing a novel class
of NSAID drugs shortly.
https://doi.org/10.1155/2015/349176
8. B.A. Bhongade, S. Talath, R.A. Gadad andA.K. Gadad, J. Saudi Chem.
Soc., 20, S463 (2016);
https://doi.org/10.1016/j.jscs.2013.01.010
9. H. Patel, A. Shirkhedkar, S. Bari, K. Patil, A. Arambhi, C. Pardeshi, A.
Kulkarni and S. Surana, Bull. Fac. Pharm. Cairo Univ., 56, 83 (2018);
https://doi.org/10.1016/j.bfopcu.2018.03.001
10. J. Ramprasad, N. Nayak, U. Dalimba, P.Yogeeswari, D. Sriram, R.Achur,
S.K. Peethambar, and H.S.S. Kumar, Eur. J. Med. Chem., 95, 49 (2015);
https://doi.org/10.1016/j.ejmech.2015.03.024
11. G. Tu, S. Li, H. Huang, G. Li, F. Xiong, X. Mai, H. Zhu, B. Kuang and
W.F. Xu, Bioorg. Med. Chem., 16, 6663 (2008);
https://doi.org/10.1016/j.bmc.2008.05.081
12. S.S. Karki, K. Panjamurthya, S. Kumar, M. Nambiar, S.A. Ramareddy,
K.K. Chiruvella and S.C. Raghavan, Eur. J. Med. Chem., 46, 2109 (2011);
https://doi.org/10.1016/j.ejmech.2011.02.064
13. J.E. Appleton, K.N. Dack, A.D. Green and J. Steele, Tetrahedron Lett.,
34, 1529 (1993);
https://doi.org/10.1016/S0040-4039(00)60337-4
14. B.P. Bandgar and K.A. Shaikh, Tetrahedron Lett., 44, 1959 (2003);
https://doi.org/10.1016/S0040-4039(03)00032-7
15. C.G. Wermuth, MedChemComm, 2, 935 (2011);
https://doi.org/10.1039/C1MD00074H
16. D. Iyer, S.V. Vartak,A. Mishra, G. Goldsmith, S. Kumar, M. Srivastava,
M. Hegde, V. Gopalakrishnan, M. Glenn, M. Velusamy, B. Choudhary,
N. Kalakonda, S.S. Karki, A. Surolia and S.C. Raghavan, FEBS J.,
283, 3408 (2016);
ACKNOWLEDGEMENTS
One of the authors, AS thank Directorate of Minorities
fellowship, Government of Karnataka, India for financial
support (DOM/Fellowship CR-62/2017-18).
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests
regarding the publication of this article.
REFERENCES
https://doi.org/10.1111/febs.13815
1. D. Mehta and D. Bhargava, Apollo Med., 7, 251 (2010);
https://doi.org/10.1016/S0976-0016(12)60018-0
2. M.P. Seed, Expert Opin. Investig. Drugs, 5, 1617 (1996);
https://doi.org/10.1517/13543784.5.12.1617
3. P. Rao and E.E. Knaus, J. Pharm. Pharm. Sci., 11, 81 (2008);
https://doi.org/10.18433/J3T886
17. A. Cristina, D. Leonte, L. Vlase, L. Bencze, S. Imre, G. Marc, B. Apan,
C. Mogosan and V. Zaharia, Molecules, 23, 2425 (2018);
https://doi.org/10.3390/molecules23102425
18. B. Antonsson, F. Conti, A. Ciavatta, S. Montessuit, S. Lewis, I. Martinou,
L. Bernasconi, A. Bernard, J.J. Mermod, G. Mazzei and K. Maundrell,
Science, 277, 370 (1997);
https://doi.org/10.1126/science.277.5324.370
19. O.O.Adeyemi, S.O. Okpo and O.O. Ogunti, Fitoterapia, 73, 375 (2002);
https://doi.org/10.1016/S0367-326X(02)00118-1
20. M. De Castro Costa, P. De Sutter, J. Gybels and J. Van Hees, Pain, 10,
173 (1981);
https://doi.org/10.1016/0304-3959(81)90193-7
21. Y. Wang, C. Huang, Y. Cao and J.S. Han, Life Sci., 67, 261 (2000);
https://doi.org/10.1016/S0024-3205(00)00625-1
4. S. Amdekar, P. Roy, V. Singh, A. Kumar, R. Singh and P. Sharma, Int.
J. Inflamm., 2012, 752015 (2015);
https://doi.org/10.1155/2012/752015
5. A.W. Schüttelkopf, L. Gros, D.E. Blair, J.A. Frearson, D.M.F. vanAalten
and I.H. Gilbert, Bioorg. Med. Chem., 18, 8334 (2010);
https://doi.org/10.1016/j.bmc.2010.09.062
6. A.L. Wills, P. Montagazza and E.W. Horton, Academic Press: London,
UK, pp 31-48 (1969).