Studies on fused heterocyclic 3,6-disubstituted-1,2, 4-triazolo-1,3,4- thiadiazoles: Synthesis and biological evaluation

Article abstract of DOI:10.1007/s00044-009-9281-x

In this study, a series of 3,6-disubstituted-1,2, 4-triazolo-[3,4-b]-1,3,4- thiadiazoles (5a-t) were synthesized by condensing 4-amino-3-mercapto-(4H)-1,2, 4-triazoles (4a-c) with different aromatic or aroyl acids through one-pot reaction. The compounds were evaluated for their anti-inflammatory, analgesic, ulcerogenic, and lipid peroxidation actions. Some of the newly synthesized compounds showed very good anti-inflammatory activity with low GI toxicity and reduced lipid peroxidation. These compounds also showed interesting profile of analgesic activity in acetic acid-induced writhing test. The findings of the study indicate that the synthesized compounds have superior GI safety profile along with reduction in lipid peroxidation as compared to that of the standard. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-009-9281-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:47–54
DOI 10.1007/s00044-009-9281-x
ORIGINAL RESEARCH
Studies on fused heterocyclic 3,6-disubstituted-1,2,
4-triazolo-1,3,4-thiadiazoles: synthesis and biological evaluation
•
Asif Husain Md. Arif Naseer
Received: 31 March 2009 / Accepted: 3 December 2009 / Published online: 24 December 2009
Ó Springer Science+Business Media, LLC 2010
Abstract In this study, a series of 3,6-disubstituted-1,2,
4-triazolo-[3,4-b]-1,3,4-thiadiazoles (5a–t) were synthe-
sized by condensing 4-amino-3-mercapto-(4H)-1,2,4-tria-
zoles (4a–c) with different aromatic or aroyl acids through
one-pot reaction. The compounds were evaluated for their
anti-inflammatory, analgesic, ulcerogenic, and lipid per-
oxidation actions. Some of the newly synthesized com-
pounds showed very good anti-inflammatory activity with
low GI toxicity and reduced lipid peroxidation. These
compounds also showed interesting profile of analgesic
activity in acetic acid-induced writhing test. The findings
of the study indicate that the synthesized compounds have
superior GI safety profile along with reduction in lipid
peroxidation as compared to that of the standard.
2007; Hafez et al., 2008; Sharma et al., 2008). There are
many marketed drugs having the 1,2,4-triazole group, e.g.,
Triazolam, Alprazolam, Etizolam, and Furacylin (Kare-
goudar et al., 2008). In addition to these important bio-
logical applications, mercapto-1,2,4-triazoles are also of
great utility in preparative/synthetic organic chemistry, for
example, in the presence of various reagents, undergo
different types of reactions to yield other heterocyclic
compounds, e.g., thiazolotriazoles, triazolothiadiazoles,
triazolothiazines, triazolothiazepines, and triazolothiadia-
zines. Derivatives of 1,2,4-triazole and 1,3,4-thiadiazole
condensed nucleus systems (triazolothiadiazoles) found to
have diverse pharmacological activities including unique
anti-inflammatory, anti-edema, and analgesic properties
(Mathew et al., 2006, 2009; Karthikeyan et al., 2007;
Karegoudar et al., 2008; Prasad et al., 2009). Several
triazolothiadiazole derivatives have been prepared from
different non-steroidal anti-inflammatory agents and found
to possess improved pharmacological profile (Udupi et al.,
2004; Metwally et al., 2007; Amir et al., 2007).
Keywords Fused heterocycles ꢀ Triazolothiadiazoles ꢀ
Anti-inflammatory ꢀ Ulcerogenic
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are the
most commonly prescribed medications in the world. They
are used for the treatment of pain, fever, and inflammation,
particularly arthritis (Moore et al., 2006). The most com-
mon side effects of the use of NSAIDs are the occurrence
of gastrointestinal damage with gastric upset and irritation
being the major problems (Cioli et al., 1979; Allison et al.,
1992). The search for safer NSAIDs continues with the
failure of anticipated ‘‘Ideal’’ anti-inflammatory agents, the
coxibs, on long-term usage (Verrico et al., 2003; Woll-
heim, 2003).
The chemistry of 1,2,4-triazoles and their fused heterocy-
clic derivatives has received considerable attention in
recent years owing to their synthetic and effective medic-
inal importance. Heterocyclic compounds bearing a sym-
metrical triazoles or 1,3,4-thiadiazole moiety have been
reported to have a broad spectrum of pharmacological
activities including potential anti-inflammatory and anal-
gesic activities (Silvia et al., 2006; Kucukguzel et al.,
A. Husain (&) ꢀ Md. A. Naseer
In the design of new drugs, the development of hybrid
molecules through the combination of different pharma-
cophores in a single molecule may lead to compounds with
improved pharmacological profile. In this study, prompted
Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Jamia Hamdard University,
Hamdard Nagar, New Delhi 110062, India
e-mail: drasifhusain@yahoo.com
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Synthesis of potassium dithiocarbazinate derivatives (3a–
c) (Mathew et al., 2006) General procedure: Potassium
hydroxide (0.03 mol) was dissolved in absolute ethanol
(50 ml). The solution was cooled in ice bath and acid
hydrazide (3a–c; 0.02 mol) was added with stirring. To this
mixture, carbon disulfide (0.025 mol) was added in small
portions with constant stirring. The reaction mixture was
stirred continuously for 12 h at room temperature. The
precipitated potassium dithiocarbazinate was collected by
filtration, washed with anhydrous ether, and dried in vac-
uum. The potassium salt thus obtained was used in the next
step without further purification.
by these observations, the synthesis and biological evalu-
ation of 3,6-disubstituted-1,2,4-triazolo-1,3,4-thiadiazoles
as hybrid molecules including different pharmacophores
are aimed at. These compounds were evaluated of their
anti-inflammatory, analgesic, ulcerogenic, and lipid per-
oxidation activities.
Materials and methods
Chemistry
All the solvents were of LR-grade and were obtained from
Merck (Mumbai, India), CDH (New Delhi, India), and
S.D.fine (Mumbai, India). Melting points were determined
in open glass capillary tubes and are uncorrected. Thin
layer chromatography (TLC) was performed on Silica gel
G (Merck) in the solvent system—Toluene: Ethyl acetate:
Formic acid (5:4:1, v/v/v); iodine chamber, and UV lamp
were used for visualization of TLC spots. The IR spectra
were recorded in KBr pellets on (BIO-RAD FTS) FT-IR
spectrophotometer. 1H NMR spectra were recorded on
Bruker DPX-300 NMR spectrometer or BRUKER 400
Ultra ShieldTM in DMSO-d6 using tetramethylsilane as an
internal standard. The chemical shifts are reported in d ppm
scale. The splitting pattern abbreviations are as follows: s,
singlet; br s, broad singlet; t, triplet; m, multiplet. The
synthetic pathway is given in Scheme 1. Elemental data for
C, H, and N were within 0.4% of the theoretical values.
Synthesis of 4-amino-5-substituted-4H-1,2,4-triazole-3-
thiol (4a–c) (Mathew et al., 2006) General procedure: A
suspension of potassium dithiocarbazinate derivative
(3a–c) (0.1 mol), hydrazine hydrate (0.3 mol), and water
(5 ml) was heated under reflux for 8 h, hydrogen sulfide
was evolved during the reaction and clear solution resulted.
Dilution of the reaction mixture with cold water (50 ml)
and subsequent acidification with dil. HCl gave a white
precipitate. It was filtered, washed with water, and crys-
tallized from aq. ethanol to get TLC pure compound 4a–c.
4-Amino-5-b-enzyl-4H-1,2,4-triazole-3-thiol
(4a):
Yield 76%; m.p. 170°C; FT-IR (KBr) cm-1: 3317 (NH2),
3068 (CH), 2596 (SH), 1590 (C=N); 1H NMR (DMSO-d6)
d ppm: 3.98 (s, 2H, CH2), 5.65 (s, 2H, NH2), 7.08–7.31 (m,
5H, phenyl), 13.82 (br s, SH); C9H10N4S; Calc. N 27.16;
Found N 27.20.
4-Amino-5-phenoxymethyl-4H-1,2,4-triazole-3-thiol
(4b): Yield 74%; m.p. 157°C; FT-IR (KBr) cm-1: 3293
(NH2), 3071 (CH), 2608 (SH), 1605 (C=N); 1H NMR
(DMSO-d6) d ppm: 5.11 (s, 2H, OCH2), 5.72 (s, 2H, NH2),
7.11–7.36 (m, 5H, phenyl), 13.91 (br s, SH); C9H10N4OS;
Calc. N 25.21; Found N 25.24.
Synthesis of the compounds
Synthesis of aryl esters (1a–c) and their hydrazides (2a–c):
The aryl esters (1a–c) and their hydrazides (2a–c) were
synthesized according to the literature method (Mathew
et al., 2006).
C2H5OH
Scheme 1 Protocol for
synthesis of the title compounds
(5a–t)
NH2NH2.H2O
R-COOH
R-COOC2H5
(1a-c)
R-CONHNH2
(2a-c)
S
N
N
NH2NH2.H2O
CS2/KOH
+
R-CONHNH-C- S K
R
SH
N
(3a-c)
NH2
(4a-c)
N
N
R1COOH
POCl3
R
N
S
N
(5a-t)
R1
R = C6H5CH2-, C6H5OCH2-, 2-OHC6H4-
R1= C6H5CONHCH2-, 2-BrC6H4-, 3-BrC6H4-, 4-BrC6H4-,
2-C6H5CO-C6H4-, C10H7CH2-, C8H6NCH2-, C6H5COCH2CH2-
123

Med Chem Res (2011) 20:47–54
49
4-Amino-5-(2-hydroxyphenyl-4H-1,2,4-triazole-3-thiol
(4c): Yield 81%; m.p. 205°C; FT-IR (KBr) cm-1: 3310
(NH2), 3085 (CH), 2603 (SH), 1598 (C=N); 1H NMR
(DMSO-d6) d ppm: 4.98 (s, 1H, OH), 5.58 (s, 2H, NH2),
7.03–7.37 (m, 5H, phenyl), 13.87 (br s, SH); C8H8N4OS;
Calc. N 26.91; Found N 26.75.
(DMSO-d6) d ppm: 4.41 & 4.50 (s, each, 29 CH2),
6.75–7.47 (complex m, 10H, Ar–H), 8.58 (s, 1H, NH);
C19H15N5S; Calc. N 20.47; Found N 20.22.
3-Benzyl-6-(1-naphthylmethyl)[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazole (5g): Yield 62%; m.p. 145–148°C; Rf-
value 0.64; FT-IR (KBr) cm-1: 3047 (CH), 1592 (C=N),
1
1461 (C–N), 718 (C–S–C); H NMR (DMSO-d6) d ppm:
3,6-Disubstituted-[1,2,4]-triazolo-[3,4-b][1,3,4]-thiadiazoles
(5a–t) General procedure: A mixture of 4-amino-5-ben-
zyl-4H-1,2,4-triazole-3-thiol (4a–c) (0.01 mol), aryl/aroyl
acid (0.01 mol), and POCl3 (10 ml) was refluxed for
8–12 h. After completion of reaction, the reaction mixture
was cooled to room temperature and then poured onto
crushed ice. The crude product so obtained was filtered and
neutralized with aq. ammonia. It was washed with water
and crystallized from DMF–ethanol mixture (1:1) to get
pure compound (5a–t).
4.44 & 4.98 (s, 29 CH2), 7.35–8.16 (complex m, 12H,
Ar–H); C21H16N4 S; Calc. N 15.72; Found N 16.10.
3-Phenoxymethyl-6-phenylcarboxamidomethyl[1,2,
4]-triazolo-[3,4-b][1,3,4]-thiadiazole (5h): Yield 54%;
m.p. 126–128°C; Rf-value 0.72; FT-IR (KBr) cm-1: 3309
(NH), 3033 (CH), 1697 (C=O), 1583 (C=N), 1474 (C–N),
1
723 (C–S–C); H NMR (DMSO-d6) d ppm: 4.31 (s, 2H,
CH2), 5.10 (s, 2H, OCH2), 6.85–7.43 (m, 10H, Ar H), 8.33 (s,
1H, CONH); C18H15N5O2S; Calc. N 19.40; Found N 19.67.
3-Phenoxymethyl-6-(2-bromophenyl)[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazole (5i): Yield 66%; m.p. 80–82°C;
Rf-value 0.81; FT-IR (KBr) cm-1: 2990 (CH), 1597
3-Benzyl-6-phenylcarboxamidomethyl-[1,2,4]-triazolo-
3,4-b][1,3,4]-thiadiazole (5a): Yield 67%; m.p.
106–108°C; Rf-value 0.75; FT-IR (KBr) cm-1: 3034 (CH),
1
(C=N), 1485 (C–N), 690 (C–S-C), 543 (C–Br); H NMR
1
1640 (C=O), 1603 (C=N), 1489 (C–N), 721 (C–S-C); H
(DMSO-d6) d ppm: 5.64 (s, 2H, OCH2), 7.04 (m, 1H, H-4,
o-disubstituted phenyl), 7.19 (m, 2H, H-5,6, o-disubstituted
phenyl), 7.39 (m, 2H, H-2,6, phenyl), 7.68 (m, 2H, H-4,
phenyl ? H-3, o-disubstituted phenyl), 7.98 (m, 2H, H-3,5,
phenyl); C16H11BrN4OS; Calc. N 14.47; Found N 14.61.
3-Phenoxymethyl-6-(3-bromophenyl)[1,2,4]triazolo
[3,4b][1,3,4] thiadiazole (5j): Yield 62%; m.p. 110–112°C;
Rf-value 0.34; FT-IR (KBr) cm-1: 3028 (CH), 1609 (C=N),
1479 (C–N), 709 (C–S–C), 550 (C–Br); 1H NMR (DMSO-
d6) d ppm: 5.12 (s, 2H, OCH2), 6.83–7.46 (m, 9H, Ar H);
C16H11BrN4OS; Calc. N 14.47; Found N 14.53.
NMR (DMSO-d6) d ppm: 3.92 & 4.33 (s, each, 29 CH2),
7.09–7.78 (m, 10H, Ar H), 8.31 (s, 1H, CONH);
C18H15N5OS; Calc. N 21.04; Found N 20.88.
3-Benzyl-6-(2-bromophenyl)[1,2,4]triazolo[3,4-b][1,3,
4]thiadiazole (5b): Yield 70%; m.p. 170–172°C;
Rf-value 0.66; FT-IR (KBr) cm-1: 3060 (CH), 1636
1
(C=N), 1489 (C–N), 721 (C–S–C), 545 (C–Br); H NMR
(DMSO-d6) d ppm: 4.31 (s, 2H, CH2), 7.13–7.73 (m, 9H,
Ar–H); C16H11BrN4S; Calc. N 15.09; Found N 15.25.
3-Benzyl-6-(3-bromophenyl)[1,2,4]triazolo[3,4-b][1,3,
4]thiadiazole (5c): Yield 61%; m.p. 152–155°C; Rf-value
0.58; FT-IR (KBr) cm-1: 3044 (CH), 1590 (C=N), 1489
3-Phenoxymethyl-6-(4-bromophenyl)[1,2,4]triazolo
[3,4-b][1,3,4] thiadiazole (5k): Yield 60%; m.p. 82–85°C;
Rf-value 0.46; FT-IR (KBr) cm-1: 3037 (CH), 1615
1
(C–N), 716 (C–S–C), 541 (C–Br); H NMR (DMSO-d6) d
ppm: 3.96 (s, 2H, CH2), 7.17–7.72 (m, 9H, Ar H);
1
(C=N), 1486 (C–N), 716 (C–S–C), 544 (C–Br); H NMR
C16H11BrN4S; Calc. N 15.09; Found N 15.17.
(DMSO-d6) d ppm: 5.10 (s, 2H, OCH2), 6.81–7.49 (m, 9H,
Ar H); C16H11BrN4OS; Calc. N 14.47; Found N 14.62.
3-Phenoxymethyl-6-(2-benzoylphenyl)[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazole (5l): Yield 65%; m.p. 101–
103°C; Rf-value 0.63; FT-IR (KBr) cm-1: 3080 (CH),
3-Benzyl-6-(4-bromophenyl)[1,2,4]triazolo[3,4-b][1,3,
4]thiadiazole (5d): Yield 68%; m.p. 172–174°C; Rf-value
0.56; FT-IR (KBr) cm-1: 3051 (CH), 1581 (C=N), 1476
1
(C–N), 721 (C–S–C), 568 (C–Br); H NMR (DMSO-d6) d
1
ppm: 4.49 (s, 2H, CH2), 7.20–7.44 (m, 5H, phenyl),
7.88–7.96 (m, 4H, p-disubstituted phenyl); C16H11BrN4S;
Calc. N 15.09; Found N 15.17.
1747 (C=O), 1655 (C=N), 1466 (C–N), 692 (C–S–C); H
NMR (DMSO-d6) d ppm: 5.65 (s, 2H, OCH2), 7.02–7.85
(complex m, 14H, Ar–H); C23H16N4O2S; Calc. N 13.58;
Found N 13.53.
3-Benzyl-6-(2-benzoylphenyl)[1,2,4]triazolo[3,4-b][1,3,
4]thiadiazole (5e): Yield 65%; m.p. 112–115°C; Rf-value
0.60; FT-IR (KBr) cm-1: 3034 (CH), 1747 (C=O), 1590
(C=N), 1469 (C–N), 716 (C–S–C); 1H NMR (DMSO-d6) d
ppm: 3.94 (s, 2H, CH2), 7.13–7.81 (m, 14H, Ar H);
C23H16N4OS; Calc. N 14.13; Found N 14.35.
3-Phenoxymethyl-6-(1H-2-Indolylmethyl)[1,2,4]triaz-
olo[3,4-b][1,3,4]thiadiazole (5m): Yield 70%; m.p.
126–128°C; Rf-value 0.66; FT-IR (KBr) cm-1: 3373 (NH,
indole), 3016 (CH), 1614 (C=N), 1456 (C–N), 706 (C–S–
1
C); H NMR (DMSO-d6) d ppm: 4.56 (s, 2H, CH2,), 5.29
3-Benzyl-6-(1H-2-indolylmethyl)[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazole (5f): Yield 65%; m.p. 156–158°C; Rf-
value 0.76; FT-IR (KBr) cm-1: 3380 (NH, indole), 3028
(s, 2H, OCH2), 6.97–7.53 (m, 10H, Ar–H), 8.63 (s, 1H,
NH); C19H15N5OS; Calc. N 19.65; Found N 19.38.
3-Phenoxymethyl-6-(1-naphthylmethyl)[1,2,4]triazol-
o[3,4-b][1,3,4]thiadiazole (5n): Yield 62%; m.p. 172–
1
(CH), 1608 (C=N), 1490 (C–N), 743 (C–S–C); H NMR
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Med Chem Res (2011) 20:47–54
175°C; Rf-value 0.73; FT-IR (KBr) cm-1: 3024 (CH), 1598
(C=N), 1450 (C–N), 698 (C–S–C); H NMR (DMSO-d6) d
access to food and water ad libitum. The studies were
undertaken with prior approval from the Institutional
Animal Ethics Committee (IAEC) and utmost care was
taken to insure that the animals were treated in the most
humane and acceptable manner. Animals were obtained
from Central Animal House Facility, Hamdard University,
New Delhi, India. Registration number and date of reg-
istration of Animal House Facility: 173/CPCSEA, 28,
January 2000.
1
ppm: 4.56 (s, 2H, CH2), 5.29 (s, 2H, OCH2), 7.06–7.56 (m,
12H, Ar–H); C21H16N4OS; Calc. N 15.04; Found N 15.37.
3-Phenoxymethyl)-6-(3-oxo-3-phenylpropyl)[1,2,4]-
triazolo-[3,4-b][1,3,4]-thiadiazole (5o): Yield 58%; m.p.
98–100°C; Rf-value 0.46; FT-IR (KBr) cm-1: 3052 (CH),
1
1753 (C=O), 1608 (C=N), 1467 (C–N), 701 (C–S–C); H
NMR (DMSO-d6) d ppm: 3.08 & 3.85 (s, each, 29 CH2),
5.14 (s, 2H, OCH2), 6.40–7.36 (m, 10H, Ar–H); C19H16N4
O2S; Calc. N 15.67; Found N 15.37.
Anti-inflammatory activity
3-(2-Hydroxyphenyl)-6-phenylcarboxamidomethyl-
[1,2,4]-triazolo-[3,4-b][1,3,4]-thiadiazole (5p): Yield
The synthesized compounds were evaluated for their anti-
inflammatory activity following carrageenan-induced rat
paw edema method (Winter et al., 1962). The rats were
randomly divided into groups of six. One group was kept as
control, and received only 0.5% carboxymethyl cellulose
(CMC) solution and the other groups were treated with test
compounds and standard drug (Indomethacin) at a dose
level of 20 mg/kg p.o. Carrageenan solution (0.1% in sterile
0.9% NaCl solution) in a volume of 0.1 ml was injected
subcutaneously into the sub-plantar region of the right hind
paw of each rat, 30 min after the administration of the test
compounds, and standard drugs. The paw volume was
measured by saline displacement shown on screen of digital
Plethysmometer (Ugo Basile, Italy) at 2 and 3 h after car-
rageenan injection. Thus, the edema volume in control
group (Vc) and that of in groups treated with test compounds
(Vt) was measured and the percentage inhibition of edema
was calculated using the formula: Anti-inflammatory
activity (% inhibition) = [(Vc - Vt)/Vc] 9 100.
60%; m.p. 136–138°C; Rf-value 0.67; FT-IR (KBr) cm-1
3652 (OH), 3054 (CH), 1606 (C=N), 1482 (C–N), 707
:
1
(C–S–C); H NMR (DMSO-d6) d ppm: 3.08 (s, 2H, CH2),
4.86 (s, 1H, OH), 6.92–8.13 (m, 9H, Ar H), 8.31 (s, 1H,
CONH); C17H13N5O2S; Calc. N 19.93; Found N 20.20.
3-(2-Hydroxyphenyl)-6-(2-bromophenyl)[1,2,4]-triaz-
olo-[3,4-b][1,3,4]-thiadiazole (5q): Yield 64%; m.p.
166–168°C; Rf-value 0.56; FT-IR (KBr) cm-1: 3695 (OH),
3029 (CH), 1598 (C=N), 1489 (C–N), 689 (C–S–C), 571
1
(C–Br); H NMR (DMSO-d6) d ppm: 4.95 (s, 1H, OH),
6.94–8.18 (m, 8H, Ar–H); C15H9BrN4OS; Calc. N 15.01;
Found N 15.26.
3-(2-Hydroxyphenyl)-6-(3-bromophenyl)[1,2,4]-triaz-
olo-[3,4-b][1,3,4]-thiadiazole (5r): Yield 58%; m.p.
92–95°C; Rf-value 0.78; FT-IR (KBr) cm-1: 3657 (OH),
3005 (CH), 1607 (C=N), 1473 (C–N), 693 (C–S–C), 589
1
(C–Br); H NMR (DMSO-d6) d ppm: 4.95 (s, 1H, OH),
6.86–7.95 (m, 8H, Ar–H); C15H9BrN4OS; Calc. N 15.01;
Found N 15.21.
3-(2-Hydroxyphenyl)-6-(4-bromophenyl)[1,2,4]-triaz-
olo-[3,4-b][1,3,4]-thiadiazole (5s): Yield 63%; m.p.
183–185°C; Rf-value 0.56; FT-IR (KBr) cm-1: 3690 (OH),
3023 (CH), 1585 (C=N), 1457 (C–N), 688(C–S–C), 572
Analgesic activity
Analgesic activity was carried out by acetic acid-induced
writhing method (Collier et al., 1968) in mice. The com-
pounds, which exhibited good anti-inflammatory activity
([50%), were screened for their analgesic activity. A 1%
aqueous acetic acid solution (i.p. injection; 0.1 ml) was
used as writhing-inducing agent. Mice were kept individ-
ually in the test cage, before acetic acid injection and
habituated for 30 min. Screening of analgesic activity was
performed after i.p. administration of test drugs and the
reference drug (aspirin) at the dose of 25 mg/kg. All the
compounds were injected as CMC suspension (1%). One
group was kept as control and received 1% CMC. After
20 min of drug administration, 0.10 ml of 1% acetic acid
solution was given to mice intraperitoneally. The severity
of writhing response (stretching movements consisting of
arching of the back, elongation of body, and extension of
hind limbs) was recorded for 20 min after administration
of acetic acid solution. The analgesic activity was pre-
sented as % protection.
1
(C–Br); H NMR (DMSO-d6) d ppm: 4.94 (s, 1H, OH),
6.83–7.86 (m, 8H, Ar–H); C15H9BrN4OS; Calc. N 15.01;
Found N 15.23.
3-(2-Hydroxyphenyl)-6-(1-naphthyl)[1,2,4]-triazolo-
[3,4-b][1,3,4]-thiadiazole (5t): Yield 65%; m.p. 202–
204°C; Rf-value 0.70; FT-IR (KBr) cm-1: 3660 (OH),
1
3011 (CH), 1589 (C=N), 1475 (C–N), 701 (C–S–C); H
NMR (DMSO-d6) d ppm: 4.03 (s, 2H, CH2), 4.91 (s, 1H,
OH), 7.11–8.14 (m, 11H, Ar H); C20H14N4OS; Calc. N
15.63; Found N 15.90.
Biological evaluation
Wistar rats and albino mice of either sex weighing
180–200 and 22–25 g, respectively, were used. The ani-
mals were housed in groups of six at room temperature of
25 2°C under 12 h light/12 h dark cycle with free
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Med Chem Res (2011) 20:47–54
51
% Analgesic activity = (n - n0/n) 9 100, where n and
n0 are the mean number of writhes of control and test
groups, respectively.
After screening for ulcerogenic activity, the gastric mucosa
was scrapped with two glass slides, weighed (100 mg), and
homogenized in 1.8 ml of 1.15% cold potassium chloride
solution. The homogenate was supplemented with 0.2 ml
of 8.1% sodium dodecyl sulfate, 1.5 ml of acetate buffer
(pH 3.5), and 1.5 ml of 0.8% thiobarbituric acid. The
mixture was heated at 95°C for 1 h. After cooling, the
reactants were supplemented with 5 ml of the mixture of
n-butanol:pyridine (15:1 v/v), shaken vigorously for 1 min,
and centrifuged for 10 min at 4000 rpm. The supernatant
organic layer was taken out and absorbance was measured
at 532 nm on UV spectrophotometer. The results were
expressed as nmol MDA/100 mg tissue, using extinction
Acute ulcerogenesis
Ulcerogenic activity (Cioli et al., 1979) evaluated after p.o.
administration of test compounds or indomethacin at the
dose of 60 mg/kg. The rats (150–200 g) were divided into
different groups consisting of six animals in each group.
Control rats received p.o. administration of vehicle (sus-
pension of 1% methyl cellulose). Food but not water was
removed 24 h before administration of the test compounds.
After the drug treatment, the rats were fed with normal diet
for 17 h and then killed. The stomach was removed and
opened along the greater curvature, washed with distilled
water, and cleaned gently by dipping in normal saline. The
mucosal damage was examined by means of a magnifying
glass. For each stomach, the mucosal damage was assessed
according to the following scoring system: 0.5, redness;
1.0, spot ulcers; 1.5, hemorrhagic streaks; 2.0, ulcers [ 3
but B5; 3.0, ulcers [ 5. The mean score of each treated
group minus the mean score of control group was regarded
as severity index of gastric mucosal damage.
coefficient 1.56 9 105cm-1 M-1
.
Results and discussion
Synthesis
The preparation of 3,6-disubstituted-[1,2,4]-triazolo-[3,
4-b][1,3,4]-thiadiazole derivatives (5a–t) (Table 2) is
depicted in Scheme 1. The structures of 4-amino-3-
substituted-5-mercapto-(4H)-1,2,4-triazoles (4a–c) were
1
confirmed by IR, H NMR spectral data, and microanaly-
Lipid peroxidation
sis. The 1H NMR spectra showed a downfield broad singlet
at around d 13.8 attributed to SH group, whereas the NH2
group appeared as a singlet at around d 5.6. The absence of
signals for NH2 and SH protons confirmed that the triazoles
Lipid peroxidation in the gastric mucosa was determined
according to the reported method (Okhawa et al., 1979).
Table 1 Biological evaluation data of the title compounds
Compound
Anti-inflammatory activitya
(% inhibition)
Analgesic activitya
(% protection)
Ulcerogenic activitya
(severity index)
Lipid
peroxidationa,b
After 2 h
After 3 h
5a
21.23 1.96**
40.97 2.22
36.73 2.76**
35.88 2.58**
54.78 2.56
32.06 2.5**
18.47 3.26**
56.47 1.48
24.42 1.7**
28.03 1.97**
–
28.03 2.52**
56.26 3.03*
40.98 1.63**
37.15 2.07**
63.29 2.50
50.53 2.58**
24.63 2.98**
70.91 2.16
32.27 2.19**
35.03 1.43**
–
5e
30.43 1.59**
0.91 0.35**
3.26 0.23**
5f
5h
5l
43.48 1.25**
40.22 3.11**
1.25. 0.21*
0.83 0.25**
4.42 0.52**
3.15 0.46**
5m
5n
5o
65.58 1.72
1.16 0.25**
3.95 0.85**
5p
5t
Control
–
0.00
2.59 0.20**
7.95 0.65
Indomethacin
50.74 3.86
66.87 1.83
2.25 0.21
Acetylsalicylic acid
61.96 1.66
a
Values are represented as mean SEM. Relative to respective standard and data were analyzed by ANOVA followed by Dunnett’s multiple
comparison test for n = 6
b
Lipid peroxidation values are presented as nmol MDA content/100 mg tissue
** P \ 0.01, * P \ 0.05
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52
Med Chem Res (2011) 20:47–54
Table 2 Structures of the
synthesized compounds (5a–t)
N
N
R
N
S
N
R1
(5a-t)
No.
5k
No.
5a
R
R1
R
R1
O
OCH2
Br
C
H2
CH2HNC
5b
5l
OCH2
C
H2
O
Br
H
Br
5c
5d
5m
5n
N
C
H2
OCH2
OCH2
C
H2
C H 2
Br
C
H2
O
O
5e
5o
OCH2
OH
C
CCH2CH2C
H2
O
H
O
5f
5g
5h
5i
5p
5q
5r
5s
5t
N
C
C
H2
CH2HNC
H2
C H 2
OH
OH
OH
OH
C
H2
Br
O
Br
OCH2
OCH2
OCH2
CH2HNC
Br
Br
C H 2
Br
5j
were converted into triazolo–thiadiazoles (5a–t) by react-
ing with the –COOH group of acids.
kg oral dose and were compared with the standard drug
indomethacin at the same oral dose. The tested compounds
showed anti-inflammatory activity ranging from 24.63 to
70.91% (Table 1). Compound 5o presented the highest anti-
inflammatory activity (70.91%) better than the standard
drug indomethacin (66.87%). Another compound, 5l,
showed better anti-inflammatory activity (63.29%) than that
of the standard drug. Two more compounds, 5e and 5m,
showed significant activity with 56.26 and 50.53% inhibi-
tion, respectively. It was observed that the triazolo–thiadi-
azole derivatives having 3-oxo-3-phenylpropyl group (5o)
Biological evaluation
Anti-inflammatory activity
The in vivo anti-inflammatory activity of ten of the syn-
thesized compounds, 5a, 5e, 5f, 5h, 5l–p, and 5t, was
evaluated by carrageenan-induced rat paw edema method
(Winter et al., 1962). The compounds were tested at 20 mg/
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Med Chem Res (2011) 20:47–54
53
and 2-benzoylphenyl group (5l) at C-6 position possess high
activity. Replacement of these groups by 1H-2-indolylm-
ethyl (5m) resulted a decrease in anti-inflammatory activity.
It was also observed that the presence of benzyl (5a, 5e, and
5f) or 2-hydroxyphenyl moiety (5p and 5t) at C-3 showed
moderate anti-inflammatory activity. From the view point
of structure–activity relationship, it is clear that the triaz-
olo–thiadiazole derivatives of phenoxy acetic acid were
found to be more active than phenyl acetic acid and salicylic
acid derivatives. Similar results have been reported by
Mathew et al. (2006); among the synthesized different
series, substituted phenoxy acetic acid series showed
highest degree of anti-inflammatory and analgesic activity.
Test compounds 5e, 5l, 5m, and 5o were exhibited good
anti-inflammatory activity ([50%), and were further eval-
uated for their analgesic, ulcerogenic, and lipid peroxida-
tion actions.
ulcerogenic activity also showcase the reduction of MDA
content, a byproduct of lipid peroxidation. Therefore, an
attempt was made to correlate the low ulcerogenesis of the
compounds with that of lipid peroxidation. The lipid per-
oxidation was measured as nmol of MDA/100 mg of tissue
(Okhawa et al., 1979). The compounds, 5e, 5l, 5m, and 5o,
which were screened for ulcerogenic activity, were further
tested for their lipid peroxidation action. Indomethacin
(standard drugs) produced high lipid peroxidation, 7.95,
whereas the control group showed 2.59 nmol/100 mg of
tissue (Table 1). It was found that all compounds showing
low ulcerogenic activity also reduced lipid peroxidation.
Therefore, the study indicated that these fused heterocyclic
compounds have inhibited the induction of gastric mucosal
lesions, and the results further suggested that their pro-
tective effect might be related to the inhibition of lipid
peroxidation in the gastric mucosa.
Analgesic activity
Conclusion
The compounds 5e, 5l, 5m, and 5o evaluated for their
analgesic effects using acetic acid-induced writhing
method (Collier et al., 1968). The results of analgesic
activity (Table 1) indicated that compounds 5o having
3-oxo-3-phenylpropyl group at C-3 of triazolo–thiadiazole
ring showed better activity (65.58%) than that of standard
drug acetylsalicylic acid (aspirin) (61.96%). Replacement
of this group by 2-benzoylphenyl (5l) and 1H-2-indo-
lylmethyl (5m) resulted a decrease in activity (43.48 and
40.22%, respectively). These findings present an important
advantage of compounds 5o as anti-inflammatory with high
analgesic activity better than that of respective standards.
Twenty fused heterocyclic analogues (Table 2) were suc-
cessfully synthesized. Among the newer derivatives, two
compounds, 3-phenoxymethyl-6-(2-benzoylphenyl)[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole 5l and 3-phenoxymethyl-6-
(3-oxo-3-phenylpropyl)[1,2,4]-triazolo-[3,4-b][1,3,4]-thia-
diazole 5o emerged as lead compounds. These compounds
showed superior GI safety profile along with reduction in
lipid peroxidation as compared to that of indomethacin.
Biological evaluation results showed that the compounds are
promising anti-inflammatory and analgesic agents and could
be further modified to develop potential and safer anti-
inflammatory and analgesic agents.
Acute ulcerogenesis
Acknowledgments The authors are thankful to IIT, Delhi, and CIF,
Faculty of Science, Jamia Hamdard, New Delhi, for providing 1H
NMR and IR spectra of the compounds, respectively. Thanks are also
due to Mrs. Shaukat Shah, In-charge, Animal House, Jamia Hamdard
for providing animals.
The compounds which were screened for analgesic activity
were further tested for their ulcerogenic activity. Com-
pounds 5e, 5l, 5m, and 5o were tested according to the
method reported by Cioli et al. (1979). The tested com-
pounds showed low ulcerogenic activity (severity index)
ranging from 0.83 to 1.25, whereas the standard drug
indomethacin showed high severity index of 2.25. The
tested compounds exhibited better GI safety profile than
that of standard drug indomethacin (Table 1).
References
Allison MC, Howatson AG, Torrance CJ, Lee FD, Russell RIG (1992)
Gastrointestinal damage associated with the use of Nonsteroidal
Antiinflammatory drugs. N Engl J Med 327:749–754
Amir M, Kumar H, Javed SA (2007) Synthesis and pharmacological
evaluation of condensed heterocyclic 6-substituted-1,2,4-triazol-
o[3,4-b]-1,3,4-thiadiazole derivatives of naproxen. Bioorg Med
Chem Lett 17:4504–4508
Cioli V, Putzolu S, Rossi V, Sorza Barcellona P, Corradino C (1979)
The role of direct tissue contact in the production of gastro-
intestinal ulcers by anti-inflammatory drugs in rats. Toxicol Appl
Pharrmacol 50:283–289
Lipid peroxidation
Lipid peroxidation refers to the oxidative degradation of
lipids. This process proceeds by free radical chain reaction
in which free radicals steal electrons from the lipids in cell
membrane and consequently damages the cell. It often
affects polyunsaturated fatty acids forming malondialde-
hyde (MDA). It is evident that compounds showing low
Collier HO, Dinneen LC, Johnson CA, Schneider C (1968) The
abdominal constriction response and its suppression by analgesic
drugs in the mouse. Br J Pharmacol 32:295–310
123

54
Med Chem Res (2011) 20:47–54
Hafez HN, Hegab MI, Ahmed-Farag IS, Gazzar AB (2008) A facile
regioselective synthesis of novel spiro-thioxanthene and spiro-
xanthene-90,2-[1,3,4]thiadiazole derivatives as potential analge-
sic and anti-inflammatory agents. Bioorg Med Chem Lett
18:4538–4543
Karegoudar P, Prasad DJ, Ashok M, Mahalinga M, Poojary B, Holla
BS (2008) Synthesis, antimicrobial and antiinflammatory activ-
ities of some 1,2,4-triazolo[3,4-b][1,3,4]thiadiazoles and 1,2,4-
triazolo[3,4-b][1,3,4]thiadiazines bearing trichlorophenyl moi-
ety. Eur J Med Chem 43:808–815
Moore RA, Derry S, Phillips CJ, McQuay HJ (2006) Nonsteroidal
anti-inflammatory drugs (NSAIDs), cyclooxygenase-2 selective
inhibitors (coxibs) and gastrointestinal harm: review of clinical
trials and clinical practice. BMC Musculoskelet Disord 7:79–91
Okhawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in
animal tissues by thiobarbituric acid reaction. Anal Biochem
95:351–358
Prasad DJ, Ashok M, Karegoudar P, Poojary B, Holla BS, Kumari NS
(2009) Synthesis and antimicrobial activities of some new
triazolothiadiazoles bearing 4-methylthiobenzyl moiety. Eur J
Med Chem 44:551–557
Karthikeyan MS, Holla BS, Kulkuraya B, Kumari NS (2007)
Biological studies of some 2,4-dichloro-5-fluorophenyl contain-
ing triazolothiadiazoles. Monatshette fur chemie 138:
1309–1316
Sharma R, Sainy J, Chaturvedi SC (2008) 2-Amino-5-sulfanyl-1,3,4-
thiadiazoles: a new series of cyclooxygenase-2 inhibitors. Acta
Pharm 58:317–326
Kucukguzel SG, Kucukguzel I, Tatar E, Rollas S, Sahin F, Gulluce M,
DeClercq E, Kabasakal L (2007) Synthesis of some novel
heterocyclic compounds derived from diflunisal hydrazide as
potential anti-infective and anti-inflammatory agents. Eur J Med
Chem 42:893–901
Mathew V, Keshavayya J, Vaidya VP (2006) Heterocyclic system
containing bridgehead nitrogen atom: synthesis and pharmaco-
logical activities of some substituted-1,2,4-triazolo[3,4-b]-1,3,4-
thiadiazoles. Eur J Med Chem 41:1048–1058
Mathew V, Giles D, Keshavayya J, Vaidya VP (2009) Studies on
synthesis and pharmacological activities of 1,2,4-triazolo[3,4-b]-
1,3,4-thiadiazoles and their dihydro analogues. Arch Pharm
342:210–222
Metwally KA, Yaseen SH, Lashine SM, El-Fayomi HM, El-Sadek
ME (2007) Non-carboxylic analogues of arylpropionic acids:
synthesis, antiinflammatory activity and ulcerogenic potential.
Eur J Med Chem 42:152–160
Silvia S, Chiara B, Olga B, Francesco B, Angelo R, Walter F, Barbara
R, Annalisa C, Giuseppe F (2006) New 1,3,4-thiadiazole
derivatives endowed with analgesic and anti-inflammatory
activities. Bioorg Med Chem 14:1698–1705
Udupi RH, Rajeeva B, Srinivasulu N, Pasha TY, Setty SR, Bhat AR
(2004) Synthesis and biological evaluation of some 4-amino-
3[40-isobutylphenyl]-ethyl-5-mercapto-1,2,4-triazoles. Indian J
Heterocycl Chem 13:233–236
Verrico MM, Weber RJ, McKaveney TP, Ansani NT, Towers AL
(2003) Adverse drug events involving COX-2 inhibitors. Ann
Pharmacother 37:1203–1213
Winter CA, Risley ET, Nuss GW (1962) Carrageenan-induced edema
in hind paw of the rat as an assay for antiinflammatory drugs.
Proc Soc Exp Biol Med 3:544–547
Wollheim FA (2003) New studies of COX-inhibitors, yet issues
remain. Lakartidningen 100:2927–2931
123