Microwave-mediated synthesis of triazolothiadiazoles as anti-inflammatory, analgesic, and anti-oxidant agents

Article abstract of DOI:10.1007/s00044-011-9569-5

Efficient approach to the synthesis of a series of triazolothiadiazole analogs of ibuprofen has been carried out using microwave energy. Thus a series of 1,2,4-triazolo[3,4-b]-thiadiazoles 5 were synthesized starting from 4-amino-3-[1-(4-isobutylphenyl)ethyl]-5-mercapto-1,2,4-triazole 3 and different aromatic acids using phosphorous oxychloride as cyclizing agent by microwave and conventional method. Microwave irradiation reduces both time and reaction endeavors along with reduced amount of phosphorous oxychloride. In addition, preliminary results of the biological evaluation of these compounds are also reported and they found to exhibit significant antioxidant, anti-inflammatory, and analgesic activities with reduced ulcerogenicity. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9569-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:543–551
DOI 10.1007/s00044-011-9569-5
ORIGINAL RESEARCH
Microwave-mediated synthesis of triazolothiadiazoles
as anti-inflammatory, analgesic, and anti-oxidant agents
•
K. V. Sujith Balakrishna Kalluraya
•
•
Adithya Adhikari K. Vijayanarayana
Received: 18 November 2010 / Accepted: 12 January 2011 / Published online: 26 January 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Efficient approach to the synthesis of a series of
triazolothiadiazole analogs of ibuprofen has been carried out
using microwave energy. Thus a series of 1,2,4-triazolo[3,4-
b]-thiadiazoles 5 were synthesized starting from 4-amino-3-
[1-(4-isobutylphenyl)ethyl]-5-mercapto-1,2,4-triazole 3 and
different aromatic acids using phosphorous oxychloride as
cyclizing agent by microwave and conventional method.
Microwave irradiation reduces both time and reaction
endeavors along with reduced amount of phosphorous
oxychloride. In addition, preliminary results of the biologi-
cal evaluation of these compounds are also reported and they
found to exhibit significant antioxidant, anti-inflammatory,
and analgesic activities with reduced ulcerogenicity.
our work on the synthesis of 1,2,4-triazoles derivatives of
ibuprofen (Sujith et al., 2009), a series of 1,2,4-triazolo
[3,4-b]-thiadiazoles were being synthesized having improved
anti-inflammatory and analgesic properties.
Ibuprofen is an important class of drug belongs to
NSAIDs with anti-inflammatory and analgesic activity
(Xiangguo et al., 2006). Long term use of NSAIDs like
ibuprofen has been associated with gastrointestinal com-
plications ranging from stomach irritation to life-threaten-
ing GI ulceration, bleeding, and nephrotoxicity (Allison
et al., 1992; Amir and Kumar, 2007). In this perspective
ibuprofen has been exploited, in fact this molecule has a
carboxylic group as functional group, common to most
NSAIDs which is responsible for its local irritation
(Hawkey et al., 2000). It has been reported that the
derivatization of the carboxyl function of representa-
tive NSAIDs, resulted in an increased anti-inflammatory
activity with reduced ulcerogenic effect (Kalgutkar et al.,
2000; Duflos et al., 2001; Metwally et al., 2007). This
paved the way for the development of variety of hetero-
cyclic compounds, such as triazole and thiadiazole. So we
decided to combine ibuprofen with 1,2,4-triazolo-[3,4-b]-
1,3,4-thiadiazole ring which has been formed as the result
of heterocyclization in order to obtain new derivative with
increased pharmacological activity. In the design of new
compounds, development of hybrid molecules through the
combination of different pharmacophores in one structure
may lead to compounds with increased biological activity.
These observations prompted us to synthesize new triaz-
olothiadiazole scaffold of ibuprofen. The synthesis of the
title compounds was carried out by both conventional
methods as well as by using microwave energy.
Keywords Microwave synthesis ꢀ Triazolothiadiazoles ꢀ
Anti-inflammatory ꢀ Analgesic ꢀ Antioxidant ꢀ
Ulcerogenic activities
Introduction
The quest for NSAIDs with improved efficacy or safety
profile has gained considerable attention to discover new
analogs. Prompted by the varied biological activities of 1,2,
4-triazoles and 1,3,4-thiadiazoles, and in continuation of
K. V. Sujith ꢀ B. Kalluraya (&) ꢀ A. Adhikari
Department of Studies in Chemistry,
Mangalore University, Mangalagangothri 574199,
Karnataka, India
e-mail: bkalluraya@gmail.com
K. Vijayanarayana
Department of Pharmacy,
Manipal College of Pharmaceutical Sciences,
Manipal 576104, Karnataka, India
Microwave irradiation dominates over usual conven-
tional methods due to its non-hazardous and eco-friendly
nature. The microwave induced organic reaction received
123

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Med Chem Res (2012) 21:543–551
considerable attention due to their simplicity and opera-
tional convenience. The use of microwave irradiation as a
source of heat in synthetic chemistry offers a promising
alternative (Jyothi et al., 2007; Kalluraya et al., 2008).
Microwave assisted heating under controlled conditions is
an invaluable technology because it often dramatically
reduces reactions times, with increased yield (Escalante
(BHT). The DPPH (1,1-diphenyl-2-picrylhydrazyl) assay is
used as a preliminary test to investigate the antioxidant
potential of the synthesized compounds. Compound show-
ing better DPPH free radical scavenging property was
investigated for further in vitro antioxidant studies like
hydroxyl radical scavenging, superoxide anion scavenging,
and nitric oxide radical scavenging activity. The DPPH free
radical scavenging activity of the compounds was deter-
mined by using the modified method (Brand-Williams et al.,
1995). The hydroxyl radical scavenging activity was deter-
mined according to the modified methods (Chung et al.,
1997; Nishikimi and Rao, 1972). The superoxide anion
scavenging activity was determined. Sodium nitroprusside
in aqueous solution at physiological pH spontaneously
produce nitric oxide, which reacts with oxygen to produce
nitrite ions, which can be determined by using the Griess
Illosvoy reaction (Garrat, 1964). Acute ulcerogenicity was
determined (Cioli et al., 1979) by evaluating after oral
administration of the test compounds (5c, 5d, and 5g) at an
equimolar dose relative to 200 mg/kg ibuprofen. All ulcer
[0.5 mm were counted and the overall total length was
designated as the ulcer index. Well diffusion method was
employed for the determination of antibacterial and anti-
fungal activity. Staphylococcus aureus (Gram ?ve) and
Eschericia coli (Gram -ve) are the bacteria used for the
antibacterial assay. Similarly Aspergillus was used for
the antifungal assay. But none of the compounds showed any
significant antibacterial or antifungal activity.
´
and Dıaz-Coutin˜o, 2009). Nowadays, microwave assisted
organic synthesis has become a new era in the field of
synthetic chemistry (Algul et al., 2009).
Results and discussion
Afteroptimizationofthe experimental conditions, 4-amino-3-
[1-(4-isobutylphenyl)ethyl]-5-mercapto-1,2,4-triazole 3 was
synthesized by following an easy one-pot protocol (Sche-
me 1). Similarly 6-substituted-3-[1-(4-isobutylphenyl)ethyl]-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles 5 were prepared by
the reaction of equimolar mixture of ibuprofen substituted
triazole 3 and aromatic acids 4 employing phosphorus
oxychloride as cyclizing agent under conventional heating
and microwave irradiation method. The main advantage of
microwave-mediated method in this regard is the minimum
use of the hazardous phosphorus oxychloride compared to
conventional method (Scheme 2).
Anti-inflammatory activity was determined by the car-
rageenan-induced paw edema method in Wistar albino rats
using plethysmography (Winter et al., 1962). Diclofenac at
an oral dose of 20 mg/kg served as the standard drug. For
further comparison ibuprofen was additionally used as a
second standard reference. Analgesic activities were eval-
uated on Swiss albino mice by tail-flick method (Di stasi
et al., 1988) in this method heat is used as a source of pain. A
dose level of 10 mg/kg pentazocine and ibuprofen served as
the standard drugs for comparison. The responses produced
in the animal were observed for 30 min intervals and
percentage in reaction time was calculated for analgesic
activity. The in vitro antioxidative potential for the synthe-
sized compounds was studied and the results were com-
pared with a standard antioxidant butylated hydroxytoluene
All the synthesized compounds 5 were tested for their
anti-inflammatory activity at an equimolar oral dose rela-
tive to 20 mg/kg diclofenac and ibuprofen. The compounds
showed anti-inflammatory activity ranging from 34 to 69%
(Table 1), whereas standard drugs diclofenac and ibupro-
fen showed 75 and 42% inhibition after 3 h, respectively.
Compounds 5a, 5d, 5f, and 5g displayed the highest anti-
inflammatory activity among the set of compounds tested
in this study. Test compounds that exhibited the most
potent anti-inflammatory activity were further evaluated
for their analgesic activity in mice. In general, the tested
compounds showed a better analgesic activity compared to
pentazocine as illustrated in Table 2.
Scheme 1 Reagents and
conditions: (a) Microwave, 1, 2,
irradiation at 160 W, 4–5 min;
(b) Fusion, 1, 2, reflux with
stirring 4-5 h, 160°C
CH3
H3C
COOH
a
CH3
CH3
N
N
H3C
(1)
+
SH
N
CH3
S
NH2
b
(3)
H2N NH
C
NH
NH2
(2)
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Med Chem Res (2012) 21:543–551
545
a
b
Scheme 2 Reagents and
conditions: (a) 3, 4, POCl3,
Microwave irradiation 160 W,
4–5 min; (b) 3, 4, POCl3, reflux
14–16 h
CH3
CH3
N
N
H3C
N
N
H3C
R
COOH
(4)
+
SH
N
N
S
R
CH3
CH3
NH2
N
(5)
(3)
R = CH3, C2H5, C3H7, 4-BrC6H5, 2-ClC6H4, 2-Cl-4-NO2C6H3,
O
Table 1 Anti-inflammatory activity of ibuprofen derivatives against carrageenan-induced rat paw edema model in rats
Compounds Change in paw volume in milliliter SEMa (% inhibition)
0.5 h
1 h
1.5 h
2 h
0.8 4.18 (76)
2.5 h
1
3 h
Diclofenac
0.2 2.38 (82)*
0.88 4.31 (20)
0.4 1.13 (64)
1.05 3.22 (5)
0.5 1.01 (65)*
0.6 2.56 (80)**
3.9 (73)*
1
0.89 (75)**
Ibuprofen
1.15 2.67 (18)** 2.17 2.69 (25)*
2.11 3.74 (36)*
1.4 2.08 (58)
1.8 4.41 (45)
2.4 1.15 (28)
1.2 0.97 (64)*
1.35 1.54 (59)**
1.1 2.64 (67)
1.35 0.85 (59)*
2.19 4.25 (39)
1.6 3.27 (56)
2.26 2.35 (42)*
1.5 4.08 (62)*
1.75 2.19 (55)*
2.6 4.19 (34)**
1.3 1.72 (67)*
1.6 3.13 (59)*
1.2 4.28 (69)
5a
5b
5c
5d
5e
5f
5g
a
0.8 3.02 (43)*
1.1 1.14 (22)
1.2 1.79 (15)*
0.6 1.64 (58)**
0.8 1.48 (43)*
1.35 1.71 (4)
1.1 1.67 (62)*
1.5 2.14 (49)**
2.2 1.74 (24)
0.9 3.37 (69)
1.25 3.05 (57)
1.2 2.84 (59)*
1.3 1.44 (55)
2
1.71 (44)
1
3.64 (10)
2.5 4.99 (31)*
1.15 2.31 (68)*
1.5 4.11 (58)**
1.2 5.14 (67)**
1.4 2.07 (61)
0.8 2.14 (28)
0.4 0.87 (64)
2.0 0.94 (–)
0.7 3.16 (36)* 1.15 3.26 (18)*
1.4 1.56 (64)**
Results are expressed as their mean values (n = 6)
* P \ 0.05, ** P \ 0.01; significant from the control
Table 2 Analgesic activity data of ibuprofen derivatives by tail-flick method
Compounds
Before treatment SEMa
After treatment SEMa (% increase in reaction time)
0.5 h
1.0 h
1.5 h
Pentazocine
1.81 0.21
2.13 0.26
1.50 0.21
1.53 0.17
1.62 0.25
1.56 0.15
1.73 0.23
1.41 0.27
2.18 0.14 (20.44)
2.38 0.29 (11.73)
1.90 0.42 (26.66)
1.64 0.11 (7.18)
1.98 0.19 (22.22)
1.86 0.38 (19.23)
2.03 0.37 (17.34)
1.54 0.12 (9.21)
2.54 0.28 (40.33)
2.61 0.38 (22.53)
1.98 0.14 (32.00)
1.78 0.45 (16.33)
2.39 0.23 (47.53)
2.18 0.48 (39.74)
2.28 0.33 (31.79)
1.98 0.21 (40.42)
3.21 0.16 (77.34)*
2.77 0.33 (30.04)*
2.14 0.19 (42.66)**
1.99 0.27 (30.06)*
2.65 0.18 (63.58)**
2.78 0.17 (78.20)*
2.45 0.24 (41.61)*
2.48 0.22 (75.88)*
Ibuprofen
5a
5b
5d
5e
5f
5g
a
Results are expressed as their mean values (n = 6)
* P \ 0.05, ** P \ 0.01; significant from the control
From the pharmacological activity results obtained, the
structure–activity relationship can be drawn for test com-
pounds 5a–g. The anti-inflammatory activity data showed
that the compound 5d having furyl group at position 6 of
triazolothiadiazole possesses highest overall activity.
Similarly tested compounds 5a, 5f, and 5g possessing
methyl, 4-bromophenyl, and 2-chlorophenyl, respectively,
showed excellent anti-inflammatory activity. Except com-
pound 5c all the tested compounds exhibited better anti-
inflammatory activity than the parent ibuprofen. The
compounds that showed better anti-inflammatory activity
were further tested for their analgesic activity at an equi-
molar oral dose relative to 10 mg/kg pentazocine. Com-
pounds showed analgesic activity ranging from 30.06 to
75.88%, whereas the standard drug pentazocine showed
77.34% and ibuprofen showed 30.04% inhibition after
1.5 h. It was noted that the compounds showing highest
anti-inflammatory activity also exhibited highest analgesic
activity. Among these compounds, 5e having 2-chloro-5-
nitrophenyl group at position 6 of triazolothiadiazole
showed activity (78.20%, P [ 0.05) more than that of
standard pentazocine, whereas compound 5g (75.88%,
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Med Chem Res (2012) 21:543–551
80
70
60
50
40
30
20
10
0
1 Hour
2 Hour
3 Hour
Diclo
Ibu
5a
5b
5c
5d
5e
5f
5g
Fig. 1 Anti-inflammatory activity data of compounds 5
90
0.5 Hour
80
1 Hour
70
Fig. 3 Comparison of antioxidant properties of 5c to BHT at 100 lg/ml
concentration
1.5 Hour
60
50
40
30
20
10
0
Where as other compounds failed to scavenge DPPH rad-
ical. The scavenging ability of 5c on DPPH is almost
similar to scavenging activity of standard antioxidant BHT.
So this compound is further tested for in vitro antioxidant
studies like hydroxyl radical scavenging, superoxide anion
scavenging, and nitric oxide radical scavenging activity.
The results of these studies are summarized in Table 4.
Compound 5c exhibited significant hydroxyl radical scav-
enging (62.35%), superoxide anion scavenging (58.57%),
and nitric oxide radical scavenging (52.57%) properties at
100 lg concentration. A bar diagram comparing hydroxyl
radical scavenging, superoxide anion scavenging, nitric
oxide radical, and DPPH radical scavenging activities at
100 lg concentration are shown in Fig. 3.
Pent
Ibu
5a
5b
5d
5e
5f
5g
Fig. 2 Analgesic activity data of compounds 5
P [ 0.05) and compound 5d (63.58%, P [ 0.01) showed
good analgesic activity. All the tested compounds exhibited
better analgesic activity than the starting compound ibu-
profen. Further comparison, is done by plotting bar dia-
gram for anti-inflammatory (Fig. 1) and analgesic activities
(Fig. 2).
Among the synthesized compounds used in the experi-
ment, compound 5c having propyl group at position 6
of triazolothiadiazole showed strong DPPH scavenging
activity compared to the standard (Table 3). At the con-
centration of 100 lg 5c and standard BHT exhibits 77.89
and 84.79% free radical scavenging activity, respectively.
Compounds which exhibited excellent antioxidant and
anti-inflammatory activities were further tested for their acute
ulcerogenic activity. The tested compounds showed low
ulcerogenic activity ranging from 8.54 0.28 to 14.12
1.90, compared to ibuprofen having high ulcer index
29.37 0.54. The maximum reduction in ulcerogenicity
Table 3 DPPH radical assay of triazolo[3,4-b]-1,3,4-thiadiazoles (5)
Compounds
5a
5b
5c
5d
5e
5f
5g
BHT
% Radical scavenged at 100 lg/ml
7.90
5.61
77.89
20.61
4.46
1.71
23.02
84.79
Table 4 Percentage inhibition
of hydroxyl, superoxide anion,
and nitric oxide radical
Concentration (lg/ml)
5c (% inhibition)
Hydroxyl radical
Superoxide anion
Nitric oxide radical
scavenging activity of 3-[1-(4-
isobutylphenyl)ethy]-6-propyl-
1,2,4-triazolo[3,4-b]-1,3,4-
thiadiazole (5c)
20
26.13 0.23
38.70 0.65
44.23 0.54
56.43 0.34
62.35 0.42
68.26 0.46
24.48 0.37
36.36 0.64
42.41 0.36
52.95 0.56
58.57 0.46
72.46 .48
14.27 0.45
26.67 0.54
38.27 0.68
46.69 0.44
52.57 0.86
62.24 .0.42
40
60
80
100
BHT (100 lg/ml)
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Med Chem Res (2012) 21:543–551
547
Table 5 Acute ulcerogenic activity of compounds 5c, 5f, and 5i
Compounds
5c
5d
5g
Ibuprofen
No. of Ulcers
Ulcer index
5.32 0.14**
8.54 0.28**
5.513 0.17**
12.57 0.47**
7.38 0.48**
14.12 1.90**
15.41 0.54
29.37 0.54**
The values are expressed in mean SEM and the P value ** P \ 0.01 is very significant
(8.54 0.28) was observed for compound 5c having pro-
pyl group attached to 6th position of triazolothiadiazole.
Other tested ibuprofen triazolothiadiazole derivatives 5d
(12.57 0.47) and 5g (14.12 1.90) also exhibited better
GI safety profile compared to the starting material ibuprofen
(Table 5).
method is depicted first followed by conventional yield in
the characterization data.
Conventional one-pot method and microwave method
(Scheme 1)
An equimolar mixture of ibuprofen (0.01 mol) and thio-
carbohydrazide (0.01 mol) taken in a 100 ml r.b. flask were
heated on an oil bath till the contents melted. The reaction
mixture was continuously stirred and maintained at a
temperature of 160°C for further half an hour. It was
allowed to cool and treated with dilute sodium bicarbonate
solution in order to remove any unreacted acid left. The
solid was filtered, washed with water, dried, and recrys-
tallized from ethanol to obtain the pure triazole. Yield
62%; m.p. 150–152°C.
An equimolar mixture of ibuprofen (0.01 mol) and
thiocarbohydrazide (0.01 mol) were ground together to get
a uniform mixture. This was zapped inside a 100 ml beaker
and subjected to microwave irradiation on a household
microwave oven operating at 160 W for about 5 min. It
was allowed to cool and treated with dilute sodium bicar-
bonate solution in order to remove any un-reacted acid
left. The solid was filtered, washed with water, dried, and
recrystallized from ethanol to obtain the pure triazole.
Yield 68%; m.p. 150–152°C.
Conclusion
A practical and high-yielding methodology to synthesize
ibuprofen triazole under microwave irradiation has been
developed for the first time. Herein we developed hybrid
molecules through the combination of different pharma-
cophores in one structure which lead to compounds with
increased pharmacological activity. We determined the
microwave-mediated conditions to obtain expected com-
pounds in good yields. Another advantage of microwave
method in the synthesis of triazolothiadiazole is the limited
use of hazardous phosphorous oxychloride compare to the
conventional method. The in-built heterocyclic ring sys-
tem of ibuprofen exhibited increased biological potency.
Compound 5c exhibited significant antioxidant properties.
It was interesting to note that compounds 5a, 5d, 5f, and 5g
were found to have significant anti-inflammatory properties
comparable with the standard drugs. Further these com-
pounds exhibited moderate to good analgesic activity. Most
of the derivatives exhibited better anti-inflammatory and
analgesic activities than the starting compound ibuprofen
with reduced ulcerogenic property.
Conventional method and microwave method
(Scheme 2)
A mixture of triazole (3) (0.01 mol), substituted acids (4)
(0.01 mol), and phosphorus oxychloride (20 ml) was
heated for reflux on an oil bath for 14 h. The resulting
reaction mass was poured into crushed ice with stirring.
The solid thus obtained was filtered, washed with dilute
sodium bicarbonate solution, followed by water, dried, and
recrystallized from ethanol.
Experimental
Melting points were determined in open capillary tubes and
are uncorrected. IR spectra were recorded by dispersing the
compounds in KBr pellets on a Shimadzu FT-IR 157
1
spectrophotometer. H NMR spectra were recorded on a
A mixture of triazole (3) (0.01 mol), substituted acids
(4) (0.01 mol), and phosphorus oxychloride (5 ml) taken in
a 100 ml r.b. flask was irradiated on a microwave oven
at 160 W for 3–5 min. The resulting reaction mass was
poured into crushed ice with stirring. The solid thus
obtained was filtered, washed with dilute sodium bicar-
bonate solution, followed by water, dried, and recrystal-
lized from ethanol.
400 MHz Bruker Avance II NMR spectrometer and all the
chemical shift values were reported as d (ppm). 13C NMR
spectra were recorded on a Bruker Avance II 400 NMR
spectrometer. Mass spectra were recorded on a JEOL
SX-102 (FAB) mass spectrometer. Both microwave and
conventional methods are employed for the synthesis of
compounds 3 and 5a–g. Yield obtained from microwave
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Med Chem Res (2012) 21:543–551
3-[1-(4-Isobutylphenyl)ethyl]-6-methyl-[1,2,4]-
triazolo[3,4-b]-[1,3,4]-thiadiazole (5a)
J = 8.08 Hz, ibu–Ar), 7.63 (d, 1H, Furyl); FABMS (m/z,
%): 354 (M? ? 2, 34.9), 353 (M? ? 1, 100), 352 (M?,
10.5), 161 (97.7). Anal. Calcd for C19H20N4SO: C, 64.74;
H, 5.71; N, 15.89; S, 9.09. Found: C, 64.68; H, 5.69; N,
15.92; S, 9.07.
M.P. 124–26°C, Yield 54 and 48%, IR (KBr) c/cm-1: 2949
1
(C–H), 1570 (C=N); H NMR (CDCl3) d: 0.87 (d, 6H,
J = 6.40 Hz, (CH3)2), 1.77–1.82 (m, 1H, CH), 1.84 (d, 3H,
J = 7.20 Hz, CHCH3), 2.43 (d, 2H, J = 7.20 Hz, CH2–
Ar), 2.63 (s, 3H, CH3), 4.59 (q, 1H, CHCH3), 7.09 (d, 2H,
J = 8.00 Hz, ibu–Ar), 7.30 (d, 2H, J = 8.00 Hz, ibu–Ar);
FABMS (m/z, %): 461 (89), 302 (M? ? 2, 16), 301
(M? ? 1, 100). Anal. Calcd for C16H20N4S: C, 63.96; H,
6.71; N, 18.64; S, 10.67. Found: C, 63.89; H, 6.77; N,
18.59; S, 10.63.
6-(2-Chloro-5-nitrophenyl)-3-[1-(4-isobutylphenyl)ethyl]-
[1,2,4]-triazolo[3,4-b]-[1,3,4]-thiadiazole (5e)
M.P. 110°C, Yield 80 and 71%, IR (KBr) c/cm-1: 2959
1
(C–H), 1570 (C=N); H NMR (CDCl3) d: 0.87 (d, 6H,
J = 6.60 Hz, (CH3)2), 1.79–1.86 (m, 1H, CH), 1.94 (d, 3H,
J = 7.28 Hz, CHCH3), 2.44 (d, 2H, J = 7.16 Hz, CH2–
Ar), 4.72 (q, 1H, CHCH3), 7.13 (d, 2H, J = 8.16 Hz, ibu–
Ar), 7.36 (d, 2H, J = 8.08 Hz, ibu–Ar), 7.76 (d, 1H,
J = 8.84 Hz, Ar), 8.35 (d, 1H, J = 8.88 Hz, Ar). 8.78 (s,
1H, Ar); FABMS (m/z, %): 443 (M? ? 2, 66.1), 442
(M? ? 1, 100), 441 (M?, 28.1), 406 (52.1), 161 (61.9).
Anal. Calcd for C21H20N5SO2Cl: C, 57.07; H, 4.56; N,
15.84; S, 7.25. Found: C, 56.98; H, 4.54; N, 15.80; S, 7.22.
6-Ethyl-3-[1-(4-isobutylphenyl)ethyl]-6-ethyl-[1,2,4]-
triazolo[3,4-b]-[1,3,4]-thiadiazole (5b)
M.P. 80°C, Yield 66 and 58%, IR (KBr) c/cm-1: 2956
1
(C–H), 1579 (C=N); H NMR (CDCl3) d: 0.88 (d, 6H,
J = 6.40 Hz, (CH3)2), 1.12–1.15 (t, 3H, CH3), 1.48–1.51
(q, 2H, CH2), 1.77 (d, 3H, J = 7.20 Hz, CHCH3),
1.77–1.86 (m, 1H, CH), 2.43 (d, 2H, J = 7.20 Hz, CH2–
Ar), 4.51 (q, 1H, CHCH3), 7.13 (d, 2H, J = 8.00 Hz, ibu–
Ar), 7.31 (d, 2H, J = 8.00 Hz, ibu–Ar); FABMS (m/z, %):
315 (M? ? 1, 100), 314 (M?, 57.1), 161 (14.6). Anal.
Calcd for C17H22N4S: C, 64.93; H, 7.05; N, 17.81; S,
10.19. Found: C, 64.87; H, 7.02; N, 17.78; S, 10.21.
6-(4-Bromophenyl)-3-[1-(4-isobutylphenyl)ethyl]-[1,2,4]-
triazolo[3,4-b]-[1,3,4]-thiadiazole (5f)
M.P. 156°C, Yield 91 and 93%, IR (KBr) c/cm-1: 2954
1
(C–H), 1586 (C=N); H NMR (CDCl3) d: 0.84 (d, 6H,
J = 6.64 Hz, (CH3)2), 1.76–1.80 (m, 1H, CH), 2.05 (d, 3H,
J = 7.24 Hz, CHCH3), 2.51 (d, 2H, J = 7.16 Hz, CH2–
Ar), 4.71 (q, 1H, CHCH3), 7.12 (d, 2H, J = 8.00 Hz, ibu–
Ar), 7.30 (d, 2H, J = 8.00 Hz, ibu–Ar), 7.83 (d, 1H,
J = 8.92 Hz, Ar), 7.86 (d, 1H, J = 8.88 Hz, Ar); 13C
NMR (CDCl3) d: 20.11, 22.61, 30.02, 36.29, 44.62, 127.52,
128.66, 129.35, 129.64, 133.13, 139.21, 140.29, 150.61,
153.27, 165.52; FABMS (m/z, %): 442 (M? ? 2, 82.9),
440 (M?, 100), 161 (37.3). Anal. Calcd for C21H21N4SBr:
C, 57.14; H, 4.79; N, 12.69; S, 7.26. Found: C, 57.08; H,
4.81; N, 12.65; S, 7.23.
3-[1-(4-Isobutylphenyl)ethyl]-6-propyl-[1,2,4]-
triazolo[3,4-b]-[1,3,4]-thiadiazole (5c)
M.P. 130°C, Yield 91 and 89%, IR (KBr) c/cm-1: 2925
1
(C–H), 1575 (C=N); H NMR (CDCl3) d: 0.86 (d, 6H,
J = 6.40 Hz, (CH3)2), 0.87–0.96 (m, 2H, CH2), 1.57–1.59
(t, 2H, CH2), 1.74 (d, 3H, J = 7.20 Hz, CHCH3),
1.76–1.81 (m, 1H, CH), 2.39 (d, 2H, J = 7.00 Hz, CH2–
Ar), 3.32 (s, 3H, CH3), 4.10 (q, 1H, CHCH3), 7.11 (d, 2H,
J = 8.08 Hz, ibu–Ar), 7.21 (s, 2H, J = 8.08 Hz, ibu–Ar);
FABMS (m/z, %): 329 (M? ? 1, 22.1), 328 (M?, 100), 272
(14.3), 161 (27.6). Anal. Calcd for C18H24N4S: C, 65.81;
H, 7.36; N, 17.05; S, 9.76. Found: C, 65.77; H, 7.33; N,
17.11; S, 9.72.
6-(2-Chlorophenyl)-3-[1-(4-isobutylphenyl)ethyl]-[1,2,4]-
triazolo[3,4-b]-[1,3,4]-thiadiazole (5g)
M.P. 129°C, Yield 72 and 64%, IR (KBr) c/cm-1: 2951
1
(C–H), 1591 (C=N); H NMR (CDCl3) d: 0.89 (d, 6H,
6-(2-Furyl)-3-[1-(4-isobutylphenyl)ethyl]-[1,2,4]-
triazolo[3,4-b]-[1,3,4]-thiadiazole (5d)
J = 6.60 Hz, (CH3)2), 1.79–1.86 (m, 1H, CH), 1.93 (d, 3H,
J = 7.28 Hz, CHCH3), 2.44 (d, 2H, J = 7.20 Hz, CH2–
Ar), 4.68 (q, 1H, CHCH3), 7.11 (d, 2H, J = 8.04 Hz, ibu–
Ar), 7.36 (d, 2H, J = 8.08 Hz, ibu–Ar), 7.40–7.44 (t, 1H,
Ar), 7.47–7.51 (t, 1H, Ar), 7.56 (d, 1H, J = 8.04 Hz, Ar),
7.88 (d, 1H, J = 7.82 Hz, Ar); FABMS (m/z, %): 398
(M? ? 2, 60), 397 (M? ? 1, 100), 161 (43). Anal. Calcd
for C21H21N4SCl: C, 63.54; H, 5.33; N, 14.11; S, 8.07.
Found: C, 63.48; H, 5.31; N, 14.08; S, 8.05.
M.P. 118–19°C, Yield 89 and 82%, IR (KBr) c/cm-1: 2968
1
(C–H), 1578 (C=N); H NMR (CDCl3) d: 0.88 (d, 6H,
J = 6.60 Hz, (CH3)2), 1.79–1.85 (m, 1H, CH), 1.89 (d, 3H,
J = 7.32 Hz, CHCH3), 2.43 (d, 2H, J = 7.16 Hz, CH2–
Ar), 4.66 (q, 1H, CHCH3), 6.62 (dd, 1H, Furyl), 7.10 (d,
2H, J = 8.12 Hz, ibu–Ar), 7.13 (d, 1H, Furyl), 7.34 (d, 2H,
123

Med Chem Res (2012) 21:543–551
Anti-inflammatory activity
549
activity was determined according to the modified method
of Chung et al. Using the method of Nishikimi et al. the
superoxide anion scavenging activity was determined.
Sodium nitroprusside in aqueous solution at physiological
pH spontaneously produce nitric oxide, which reacts with
oxygen to produce nitrite ions, which can be determined by
using the Griess Illosvoy reaction.
Anti-inflammatory activity was determined by the carra-
geenan-induced paw edema method in Wistar albino rats
by using plethysmography following the method of Winter
et al. Diclofenac at an oral dose of 20 mg/kg served as
the standard drug for comparison. The test compounds
(20 mg/kg) were administered 30 min prior to adminis-
tration of carrageenan (0.1 ml of 1% w/v) in the plantar
region of the paw. The paw volumes were measured at 1, 2,
and 3 h after carrageenan administration. The percentage
of inhibition was calculated by using the following equa-
tions and the results are presented in Table 1. All the
pharmacological data are expressed as mean SEM.
DPPH radical scavenging assay
The DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical
scavenging activity of all the triazolothiadiazoles (5a–g)
was determined. Freshly prepared 2 ml of 0.1 mM DPPH
solution in methanol was added to 1 ml of methanolic
solution of synthesized compounds (100 lg/ml), taken in
different test tubes and the volume was adjusted using
methanol. It was mixed and vortexed thoroughly and left in
dark for 30 min. The absorbance of stable DPPH was mea-
sured at 517 nm. The DPPH control was prepared using the
same procedure by replacing the sample by methanol. Rad-
ical scavenging activity was expressed as the inhibition
percentage and was calculated using the equation of DPPH
radical scavenging activity.
ꢀ
ꢁ
Vt
% Inhibition ¼ 1 ꢁ
ꢂ 100
Vc
where Vt average edema volume of test compounds; Vc
average edema volume of control.
Analgesic activity
Analgesic activities were evaluated on Swiss albino mice
by tail-flick method. In this method heat is used as a source
of pain. A dose level of 10 mg/kg pentazocine and ibu-
profen served as the standard drugs for comparison. The
responses produced in the animal were observed for 30 min
intervals and percentage in reaction time was calculated for
analgesic activity, from this percentage increase in reaction
time (as index of analgesia) at each time interval is cal-
culated using the following equation and the results are
presented in Table 2.
ðAc ꢁ AtÞ
% Radical scavenged ¼
ꢂ 100
Ac
where Ac absorbance of the control; At absorbance of the
test compounds.
Hydroxyl radical scavenging activity
The hydroxyl radical scavenging activity was determined
according to the modified method of Chung et al. The Fenton
reaction mixture containing 200 ll of 10 mM FeSO4ꢀ7H2O,
200 ll of 10 mM EDTA, and 200 ll of 10 mM 2-deoxyri-
bose was mixed with 1.2 ml of 0.1 M phosphate buffer (pH
7.4) containing 100 lg/ml concentration of 5c. Freshly
prepared 200 ll of 10 mM H2O2 was added to the mixture
and incubated for 4 h at 37°C. Later, 1 ml of 2.8% TCA and
1 ml of 1% TBA were added and placed in boiling water
bath for 10 min. The mixture was brought to room temper-
ature and centrifuged at 2000 rpm for 5 min and absorbance
was measured at 532 nm. The percentage of hydroxyl rad-
ical scavenging activity was calculated by employing the
following formula and compared with the standard BHT.
ꢂ
ꢃ
RTAT
RTBT
% Increase in reaction time ¼
ꢁ 1 ꢂ 100
where RTAT reaction time after treatment; RTBT reaction
time before treatment.
Antioxidant activity
The in vitro antioxidative potential for some of synthesized
compounds were studied and the results were compared
with a standard antioxidant butylated hydroxytoluene
(BHT). The DPPH (1,1-diphenyl-2-picrylhydrazyl) assay is
used as a preliminary test to investigate the antioxidant
potential of the synthesized compounds (Table 4). Com-
pound showing better DPPH free radical scavenging
property was investigated for further in vitro antioxidant
studies like hydroxyl radical scavenging, superoxide anion
scavenging activity and nitric oxide radical scavenging
activity. The DPPH free radical scavenging activity of the
compounds was determined by using the modified method
of Brand-Williams et al. The hydroxyl radical scavenging
% Hydroxyl radical scavenging
Absorbance of sample
¼
ꢂ 100
Absorbance of blank
Superoxide anion scavenging activity
Using the method of Nishikimi et al. the superoxide anion
scavenging activity was determined, wherein a mixture of
123

550
Med Chem Res (2012) 21:543–551
Acknowledgments The authors are thankful to UGC for the
financial assistance and to the Head, SAIF, CDRI, Lucknow for
1 ml of NBT (156 lM NBT in 100 mM phosphate buffer,
pH 7.4), 1 ml NADH (468 lM in 100 mM phosphate
buffer, pH 7.4), and 100 lg/ml concentration of 5c was
prepared. To this mixture 100 ll of PMS solution (60 lM
PMS in 100 mM phosphate buffer, pH 7.4) was added to
start the reaction. The reaction mixture was incubated at
25°C for 5 min, and the absorbance was measured at
560 nm against blank. Decreased absorbance of the reac-
tion mixture indicated increased superoxide anion scav-
enging activity. BHT was used as standard.
1
providing mass and H NMR spectral data.
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