Synthesis and pharmacological evaluation of N-(6-chlorobenzo[d]thiazol-2- yl)hydrazine carboxamide derivatives of benzothiazole

Article abstract of DOI:10.1007/s00044-012-0329-y

A series of 6-substituted-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole (6a-g) and 1,3,4-oxadiazole (7a-g, 8) derivatives of benzothiazole were synthesized in satisfactory yield and pharmacologically evaluated for their anti-inflammatory, analgesic, ulcerogenic, and lipid peroxidation activities by known experimental models. All the synthesized compounds were in good agreement with elemental and spectral data. Some of the synthesized compounds have significant anti-inflammatory and analgesic activities. Ulcerogenic and irritative action on the gastrointestinal mucosa, in comparison with standard are low.

Full text of DOI:10.1007/s00044-012-0329-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:3316–3328
DOI 10.1007/s00044-012-0329-y
ORIGINAL RESEARCH
Synthesis and pharmacological evaluation
of N-(6-chlorobenzo[d]thiazol-2-yl)hydrazine carboxamide
derivatives of benzothiazole
•
Sadaf J. Gilani Suroor A. Khan
Received: 21 June 2012 / Accepted: 6 November 2012 / Published online: 21 November 2012
Ó Springer Science+Business Media New York 2012
Abstract A series of 6-substituted-1,2,4-triazolo-[3,4-b]-
1,3,4-thiadiazole (6a–g) and 1,3,4-oxadiazole (7a–g, 8)
derivatives of benzothiazole were synthesized in satisfactory
yield and pharmacologically evaluated for their anti-
inflammatory, analgesic, ulcerogenic, and lipid peroxidation
activities by known experimental models. All the synthe-
sized compounds were in good agreement with elemental
and spectral data. Some of the synthesized compounds have
significant anti-inflammatory and analgesic activities.
Ulcerogenic and irritative action on the gastrointestinal
mucosa, in comparison with standard are low.
prostaglandins. Prostaglandins are derived from arachi-
donic acid, which originates from cell membrane phos-
pholipids through the action of phospholipase A2. The
metabolism of arachidonic acid to prostaglandins and leu-
kotrienes is catalyzed by cyclo-oxygenase and 5-lipoxy-
genase pathways, respectively (Tripathi, 2003; Brunton
et al., 2006).
Two related, but unique isoforms of cyclo-oxygenase,
designated COX-1 and COX-2 have been demonstrated in
mammalian cells (Gilani et al., 2011a; Kumar et al., 2008).
COX-1 is constitutively expressed and generates PGs
believed to be involved in GI mucosal protection (Habeeb
et al., 2001), whereas at the sites of inflammation throughout
the body, COX-2 is induced to generate PGs, believed to
mediate inflammation and pain. The anti-inflammatory
effects of non selective NSAIDs therefore seen to be med-
iated via inhibition of COX-2 (Almansa et al., 2003),
whereas deleterious effects in GI tract such as gastroduo-
denal ulceration, occurs as a result of inhibition of COX-1.
Traditional NSAIDs such as aspirin, diclofenac, flurbi-
profen, and ibuprofen are non selective; however, they
show greater selectivity for COX-1 than COX-2 (Jackson
and Hawkey, 1999; DeWitt, 1999; Mitchell et al., 1993).
Therefore chronic use of NSAIDs may elicit appreciable
GI irritation, bleeding, and ulceration. The discovery of
COX-2 provided the rationale for the development of drugs
devoid of GI disorders while retaining clinical efficacy as
anti-inflammatory agents. But the recent reports showed
that selective COX-2 inhibitors (coxibs) could lead to
adverse cardiovascular effects (Dogne et al., 2005).
Therefore, development of novel compounds having anti-
inflammatory and analgesic activity with improved safety
profile is still a necessity.
Keywords Triazolo-thiadiazole ꢀ 1,3,4-Oxadiazole ꢀ
Anti-inflammatory ꢀ Analgesic ꢀ Ulcerogenic ꢀ
Lipid peroxidation
Introduction
Non steroidal anti-inflammatory drugs (NSAIDs) are the
drugs with analgesic, anti-pyretic, and anti-inflammatory
effects. The anti-inflammatory activity of the NSAIDs is
mediated chiefly through inhibition of biosynthesis of
S. J. Gilani (&)
Department of Pharmaceutical Chemistry, KIET School
of Pharmacy, Ghaziabad 201206, Uttar Pradesh, India
e-mail: gilanisadaf@gmail.com
S. J. Gilani ꢀ S. A. Khan
Despite numerous attempts to develop new structural
prototype in the search for more effective NSAIDs,
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Jamia Hamdard (Hamdard University), New Delhi 110062, India
123

Med Chem Res (2013) 22:3316–3328
3317
benzothiazole still remain as one of the most versatile
class of compounds against inflammation and therefore,
are useful substructures for further molecular exploration.
Benzothiazole’s literature is enriched with progressive
findings of the moiety in respect of anti-inflammatory
(Paramashivappa et al., 2003) and analgesic (Rana et al.,
2007) activities. In addition, triazolo-thiadiazoles nucleus
constitutes the active part of several biologically active
compounds, including anti-bacterial (Gilani et al., 2011b),
anti-fungal (Gilani et al., 2011c), anti-tumor (Ibrahim,
2009), anti-inflammatory (Mathew et al., 2006), analgesic
(Amir et al., 2007) and so on. Moreover, 1,3,4-oxadiazole
also reported significant anti-microbial (Padmavathi et al.,
2009), anti-inflammatory (Amir and Shikha, 2004), and
analgesic activities (Gilani et al., 2010). Apart from above
important biological 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., thiazolo-triazoles, triazolo-
thiadiazoles, triazolo-thiazines, triazolo-thiazepines, and
triazolo-thiadiazines (Husain and Naseer, 2011). A triaz-
olo-thiadiazole system may be viewed as a cyclic analog
of two very important components-thiosemicarbazide
(Khalil, 2007) and biguanide (Mathew et al., 2007), which
often display diverse biological activities. Inspired by
these observations a composite system was investigated,
which combine these two biolabile components in a ring
together to give a compact and planar structure, and
screened for their biological activities. The prime objective
for the current study is to develop novel derivatives of
benzothiazole and finally screen them against anti-
inflammatory analgesic, ulcerogenic, and lipid peroxida-
tion activities.
Materials and methods
Chemistry
All the chemicals were purchased from Merck Chemical
Company, S.D. Fine (India) and Qualigens (India). Melting
points were determined in open capillary tubes in a Hicon
melting point apparatus and are uncorrected. IR (KBr)
spectra were recorded on a Nicolet, 5PC FTIR spectrometer
1
(mmax in cm-1), and H NMR spectra were recorded in
DMSO-d6 on a Bruker DRX-300 (300 MHz FT NMR)
spectrometer using tetramethylsilane (TMS) as internal
standard. Chemical shifts (d) are expressed in parts per
million (ppm); coupling constants (J) are reported in hertz
and refer to apparent peak multiplicities, which may not
necessarily be true coupling constants. Mass spectra were
recorded using Jeol SR-102 (FAB) mass spectrometer. The
homogeneity of the compounds was checked by thin layer
chromatography (TLC) on silica gel G (Merck) coated
plates using toluene:ethylacetate:formic acid (5:4:1) as
solvent system. Iodine chamber and UV lamp were used for
the visualization of TLC spots. Spectral data (1H NMR, IR
and mass) of the synthesized compounds were in full
agreement with the proposed structures. Elemental data
were performed on Perkin Elmer models 240 CHN analyzer
and found to be within 0.4 % of the theoretical values.
Synthesis of 6-chloro-1,3-benzothiazole-2-amines (1)
A mixture of aniline (0.01 mol) and potassium thiocyanate
(0.01 mol) in glacial acetic acid (g.a.a., 10 %) was cooled
and stirred. To this solution bromine (0.01 mol) was added
dropwise at such a rate as to keep the temperature below
10 °C throughout the addition. Stirring was continued for
an additional 3 h and the separated hydrochloride salt was
filtered, washed with acetic acid, and dried. It was dis-
solved in hot water and neutralized with aqueous ammonia
solution (25 %), filtered, washed with water and dried,
recrystallized with benzene.
Therefore, in continuation of our interest in the synthesis
of heterocycles containing benzothiazole moiety it was
planned to synthesize hybrid compounds incorporating
benzothiazole moiety with triazolo-thiadiazole and oxadi-
azole ring systems through different linkages. This combi-
nation was suggested in an attempt to investigate the
influence of such hybridization and structure variation on
the anticipated biological activities, hoping to add some
synergistic biological significance to the target molecules.
The substitution pattern of triazolo-thiadiazole and oxadi-
azole rings was carefully selected so as to confer different
electronic environment to the molecules. Hence, to discover
new and useful agents for treatment of aforementioned
diseases, we have replaced the hydrazide group of N-(6-
chlorobenzo[d]thiazol-2-yl)hydrazine carboxamide with
additional heterocycles, which have been found to possess
an interesting profile of anti-inflammatory and analgesic
activity with significant reduction in their ulcerogenic risks
in the stomach.
Yield 81 %; m.p. 208–210 °C; Rf-value 0.70; IR (KBr)
m cm-1: 817 (C–Cl), 1570 (C=N), 3480 (NH); H NMR
1
(DMSO-d6) d (ppm): 6.12 (s, 2H, NH2), 6.61–6.64 (m,
J = 9 Hz, 3H, Ar–H).
Synthesis of 1-(6-chloro-1,3-benzothiazol-2-yl) urea (2)
To the solution of sodium cyanate in minimum quantity of
water, glacial acetic acid (5 mL) was added. This solu-
tion was heated with 2-amino-6-chloro-benzothiazole (1,
0.01 mol) in alcohol till the contents of mixture become
turbid and volume remained half of the original volume.
The contents were added to ice cool water. The solid
obtained was filtered off and dried.
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Med Chem Res (2013) 22:3316–3328
Synthesis of 6-chloro-N-(6-substituted[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazol-3-yl) benzo[d]thiazol-2-
amine (6a–g) general procedure
Yield 76 %; m.p. 230–232 °C; Rf-value 0.76; IR (KBr)
m cm-1: 830 (C–Cl), 1560 (C=N), 1628 (C=O), 3310 (NH);
1H NMR (DMSO-d6) d (ppm): 6.34 (s, 2H, NH2),
6.68–6.70 (m, J = 6 Hz, 3H, Ar–H), 8.10 (s, 1H, NHC=O).
An
equimolar
mixture
of
4-amino-5-(6-chloro-
benzo[d]thiazol-2-ylamino)-4H-1,2,4-triazole-3-thiol (5,
0.10 mol) and aromatic acids in phosphorus oxychloride
(10 mL) was refluxed for 5 h. The reaction mixture was
cooled to room temperature and then gradually poured on
to crushed ice with stirring. The mixture was allowed to
stand overnight and the solid separated out was filtered,
treated with dilute sodium hydroxide solution and washed
thoroughly with cold water. The compound so obtained
was dried and recrystallized from ethanol.
Synthesis of N-(6-chlorobenzo[d]thiazol-2-yl)hydrazine
carboxamide (3)
To the warm hydrazine hydrate solution of compound 2 in
alcohol, conc. NaOH was added and refluxed for 6 h.
Reaction mixture was poured into crushed ice and solid
obtained was filtered off and dried. The solid collected out
was recrystallized from suitable solvent.
Yield 82 %; m.p. 241–243 °C; Rf-value 0.71; IR (KBr)
m cm-1: 657 (C–S–C), 817 (C–Cl), 1588 (C=N), 1660
(C=O), 3300 (NH); 1H NMR (DMSO-d6) d (ppm): 7.28 (s,
1H, NHNH2), 7.70–7.74 (m, J = 12 Hz, 3H, Ar–H), 9.12
(s, 1H, NHC=O).
6-Chloro-N-(6-phenyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-
3-yl)benzo[d]thiazol-2-amine (6a): Yield 74 %; m.p.
238–240 °C; Rf-value 0.70; IR (KBr) m cm-1 610 (C–S–C
benzothiazole), 674 (C–S–C triazolo-thiadiazole), 837
(C–Cl), 1269 (N–N=C triazolo-thiadiazole), 1416 (C–N
benzothiazole), 1518 (C=C aromatic), 3084 (C–H aro-
matic), 3314 (N–H); 1H NMR (DMSO-d6) d (ppm):
7.41–7.44 (m, J = 9 Hz, 8H, Ar–H), 8.06 (s,1H, NH); 13C
NMR (DMSO-d6) d (ppm): 118.3 (C-4, benzothiazole),
121.2 (C-7, benzothiazole), 125.8 (C-5, benzothiazole),
128.7 (C-40), 129.2 (C-30 & C-50), 129.8 (C-6, benzothia-
zole), 130.9 (C-20 & C-60), 132.3 (C-1a, benzothiazole),
133.5 (C-10), 143.3 (C-6, triazolo-thiadiazole), 151.3 (C-4a,
benzothiazole), 157.2 (C-4, triazolo-thiadiazole), 167.6 (C-3,
triazole), 174.5 (C-2, benzothiazole); MS: m/z 384 (M?),
385 (M??1), 386 (M??2); Anal. Calc. for C16H9ClN6S2
(384.87): C 49.93, H 2.36, N 21.84, S 16.66, Cl 9.21.
Found: C 49.96, H 2.40, N 21.88, S 16.69, Cl 19.24.
Synthesis of potassium dithiocarbazinate (4)
Potassium hydroxide (0.03 mol) was dissolved in absolute
ethanol (50 mL). The solution was cooled in ice bath and
acid hydrazide (3; 0.02 mol) was added with stirring. To
this carbon disulfide (0.025 mol) was added in small por-
tions with constant stirring. The reaction mixture was
agitated continuously for 12 h at room temperature. The
precipitated potassium dithiocarbazinate was collected by
filtration, washed with anhydrous ether (100 mL) and dried
in vacuum. The potassium salt thus obtained was in
quantitative yield and was used in the next step without
further purification.
Synthesis of 4-amino-5-(6-chlorobenzo[d]thiazol-2-
ylamino)4H-1,2,4-triazole-3-thiol (5)
6-Chloro-N-(6-(2-chlorophenyl)-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazol-3-yl)benzo[d]thiazol-2-amine (6b): Yield
71 %; m.p.254–256 °C; Rf-value 0.60; IR (KBr) m cm-1 616
(C–S–C benzothiazole), 688 (C–S–C triazolo-thiadiazole),
821 (C–Cl), 1274 (N–N=C triazolo-thiadiazole), 1422 (C–N
benzothiazole), 1524 (C=C aromatic), 3114 (C–H aromatic),
3318 (N–H); 1H NMR (DMSO-d6) d (ppm): 7.36–7.40 (m,
J = 12 Hz, 7H, Ar–H), 8.01 (s, 1H, NH); 13C NMR (DMSO-
d6) d (ppm): 118.3 (C-4, benzothiazole), 121.2 (C-7, ben-
zothiazole), 125.8 (C-5, benzothiazole), 127.3 (C-50), 128.9
(C-60), 129.3 (C-30), 129.8 (C-6, benzothiazole), 130.1
(C-40), 132.2 (C-20), 132.3 (C-1a, benzothiazole), 136.9
(C-10), 151.3 (C-4a, benzothiazole), 157.2 (C-4, triazolo-
thiadiazole), 167.6 (C-3, triazole), 174.1 (C-6, triazolo-
thiadiazole), 174.5 (C-2, benzothiazole); MS: m/z 419 (M?),
417 (M?-2); Anal. Calc. for C16H8Cl2N6S2 (419.31): C
45.83, H 1.92, N 20.04, S 15.29, Cl 16.91. Found: C 45.86, H
1.94, N 20.07, S 15.31, Cl 16.94.
A suspension of potassium dithiocarbazinate (4; 0.02 mol)
in water (50 mL) and hydrazine hydrate (99 %, 0.04 mol)
was refluxed for 18–20 h with occasional shaking. The
color of the reaction mixture changed to green, with evo-
lution of hydrogen sulfide gas. A homogenous reaction
mixture was obtained during the reaction process. The
reaction mixture was cooled to room temperature and
diluted with water (20 mL). On acidification with acetic
acid, the required triazole was precipitated out. It was
filtered, washed thoroughly with cold water, dried, and
recrystallized from ethanol.
Yield 74 %; m.p. 198–200 °C; Rf-value 0.68; IR (KBr)
m cm-1: 635 (C–S–C), 817 (C–Cl), 1528 (C=N), 2518
1
(SH), 3338 (NH2); H NMR (DMSO-d6) d (ppm): 5.15 (s,
2H, NH2), 7.28–7.32 (m, J = 12 Hz, 3H, Ar–H), 9.29 (s,
1H, NH), 13.18 (s,1H, SH).
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3319
167.6 (C-3, triazole), 169.0 (C-200), 174.1 (C-6, triazolo-thia-
diazole), 174.5 (C-2, benzothiazole); MS: m/z 442 (M?), 444
(M??2); Anal. Calc. for C18H11ClN6O2S2 (442.90): C 48.81,
H 2.50, N 18.97, S 14.48, Cl 8.00. Found: C 48.84, H 2.52, N
18.99, S 14.51, Cl 8.04.
6-Chloro-N-(6-(2,4-dichlorophenyl)-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazol-3-yl)benzo[d]thiazol-2-amine (6c) Yield
68 %; m.p. 248–250 °C; Rf-value 0.90; IR (KBr) m cm-1
:
614 (C–S–C benzothiazole), 686 (C–S–C triazolo-thiadia-
zole), 822 (C–Cl), 1275 (N–N=C triazolo-thiadiazole),
1424 (C–N benzothiazole), 1526 (C=C aromatic), 3112
(C–H aromatic), 3317 (N–H); 1H NMR (DMSO-d6) d
(ppm): 7.40–7.44 (m, J = 12 Hz, 6H, Ar–H), 8.03 (s, 1H,
NH); 13C NMR (DMSO-d6) d (ppm): 118.3(C-4, benzothi-
azole), 121.2 (C-7, benzothiazole), 125.8 (C-5, benzothia-
zole), 128.8 (C-60), 129.8 (C-6, benzothiazole), 130.2 (C-40),
130.3 (C-20), 130.7 (C-30), 132.9 (C-50), 132.3 (C-1a, ben-
zothiazole), 138.3 (C-10), 151.3 (C-4a, benzothiazole), 157.2
(C-4, triazolo-thiadiazole), 167.6 (C-3, triazole), 174.1 (C-6,
triazolo-thiadiazole), 174.5 (C-2, benzothiazole); MS: m/z 453
(M?), 451(M?-2), 455 (M??2); Anal. Calc. for C16H17
Cl3N6S2 (453.76): C 42.35, H 1.55, N 18.52, S 14.13, Cl
23.44. Found: C 42.37, H 1.58, N 18.56, S 14.15, Cl 23.47.
6-Chloro-N-(6-pheoxy-[1,2,4]triazolo[3,4-b][1,3,4]thia-
diazol-3-yl)benzo[d]thiazol-2-amine (6f) Yield 76 %;
m.p. 218–220 °C; Rf-value 0.60; IR (KBr) m cm-1: 626
(C–S–C benzothiazole), 692 (C–S–C triazolo-thiadiazole),
836 (C–Cl), 1274 (N–N=C triazolo-thiadiazole), 1434
(C–N benzothiazole), 1532 (C=C aromatic), 3123 (C–H
aromatic), 3318 (N–H); 1H NMR (DMSO-d6) d (ppm):
7.29–7.31 (m, J = 6 Hz, 8H, Ar–H), 8.10 (s, 1H, NH); 13
C
NMR (DMSO-d6) d (ppm): 118.3 (C-4, benzothiazole),
121.2 (C-7, benzothiazole), 122.7 (C-60), 124.6 (C-40),
125.8 (C-5, benzothiazole), 129.7(C-30 & C-50), 129.8
(C-6, benzothiazole), 132.3 (C-1a, benzothiazole), 151.3
(C-4a, benzothiazole), 155.2 (C-10), 157.2 (C-4, triazolo-
thiadiazole), 167.6 (C-3, triazole), 174.1 (C-6, triazolo-thia-
diazole), 174.5 (C-2, benzothiazole); MS: m/z 400 (M?), 401
(M??1), 403 (M??3); Anal. Calc. for C16H9ClN6OS2
(400.87): C 47.94, H 2.26, N 20.96, S 16.00, Cl 8.84. Found:
C 47.97, H 2.30, N 20.98, S 16.04, Cl 8.86.
6-Chloro-N-(6-o-tolyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadia-
zol-3-yl)benzo[d]thiazol-2-amine (6d) Yield 71 %; m.p.
282–284 °C; Rf-value 0.80; IR (KBr) m cm-1: 618 (C–S–C
benzothiazole), 691 (C–S–C triazolo-thiadiazole), 829
(C–Cl), 1281 (N–N=C triazolo-thiadiazole), 1427 (C–N
benzothiazole), 1531 (C=C aromatic), 3119 (C–H aro-
matic), 3319 (N–H); 1H NMR (DMSO-d6) d (ppm): 2.34 (s,
3H, CH3), 7.35–7.37 (m, J = 6 Hz, 7H, Ar–H), 8.07 (s, 1H,
6-Chloro-N-(6-(4-nitrophenyl)-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazol-3-yl)benzo[d]thiazol-2-amine (6g) Yield
13
NH); C NMR (DMSO-d6) d (ppm): 18.7 (C-100), 118.3
63 %; m.p. 116–118 °C; Rf-value 0.70; IR (KBr) m cm-1
:
(C-4, benzothiazole), 121.2 (C-7, benzothiazole), 125.8 (C-5,
benzothiazole), 126.2 (C-50), 127.4 (C-60), 128.6 (C-40),
129.5 (C-30), 129.8 (C-6, benzothiazole), 132.3 (C-1a,
benzothiazole), 136.9 (C-20), 137.2 (C-10), 151.3 (C-4a,
benzothiazole), 157.2 (C-4, triazolo-thiadiazole), 167.6
(C-3, triazole), 174.1 (C-6, triazolo-thiadiazole), 174.5 (C-2,
benzothiazole); MS: m/z 398 (M?), 400 (M??2); Anal.
Calc. for C16H17Cl3N6S2 (398.89): C 51.19, H 2.78, N
21.07, S 16.08, Cl 8.89. Found: C 51.21, H 2.82, N 21.10, S
16.12, Cl 8.92.
621 (C–S–C benzothiazole), 689 (C–S–C triazolo-thiadia-
zole), 830 (C–Cl), 1280 (N–N=C triazolo-thiadiazole),
1369 (NO2), 1429 (C–N benzothiazole), 1527 (C=C aro-
matic), 3118 (C–H aromatic), 3320 (N–H); 1H NMR
(DMSO-d6) d (ppm): 7.39–7.41 (m, J = 6 Hz, 7H, Ar–H),
8.12 (s, 1H, NH); 13C NMR (DMSO-d6) d (ppm): 118.3
(C-4, benzothiazole), 121.2 (C-7, benzothiazole), 124.4
(C-50), 125.8 (C-5, benzothiazole), 128.4 (C-60), 129.8 (C-6,
benzothiazole), 132.3(C-1a, benzothiazole), 139.6 (C-10),
143.3 (C-6, triazolo-thiadiazole) 147.9 (C-40), 151.3 (C-4a,
benzothiazole), 157.2 (C-4, triazolo-thiadiazole), 167.6
(C-3, triazole), 174.5 (C-2, benzothiazole); MS: m/z 429
(M?), 428 (M?-1), 430 (M??1); Anal. Calc. for C16H17
Cl3N6S2 (429.86): C 48.80, H 2.49, N 18.97, S 14.48, Cl
8.25. Found: C 48.84, H 2.52, N 18.99, S 14.51, Cl 8.27.
2-(3-(6-Chlorobenzo[d]thiazol-2-ylamino)-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazol-6-yl)phenyl acetate (6e) Yield
66 %; m.p. 286–288 °C; Rf-value 0.70; IR (KBr) m cm-1
:
626 (C–S–C benzothiazole), 684 (C–S–C triazolo thiadia-
zole), 817 (C–Cl), 1267 (N–N=C triazolo-thiadiazole),
1436 (C–N benzothiazole), 1527 (C=C aromatic), 3106 (C–H
aromatic), 3324 (N–H); 1H NMR (DMSO-d6) d (ppm):
2.51 (s, 3H, OCOCH3), 7.24–7.28 (m, J = 12 Hz, 7H,
Ar–H), 8.10 (s, 1H, NH); 13C NMR (DMSO-d6) d (ppm):
20.3 (C-100), 118.3 (C-4, benzothiazole), 121.2 (C-7, benzo-
thiazole), 123.2 (C-30), 125.8 (C-5, benzothiazole), 126.0
(C-50), 127.9 (C-60), 129.1 (C-40), 129.4 (C-10), 129.8 (C-6,
benzothiazole), 132.3 (C-1a, benzothiazole), 151.1 (C-20),
151.3 (C-4a, benzothiazole), 157.2 (C-4, triazolo-thiadiazole),
Synthesis of N-(6-chlorobenzo[d]thiazol-2-yl)-5-
substituted phenyl-1,3,4-oxadiazol-2-amine (7a–g)
general procedure:
Compound 3 (0.001 mol) and appropriate aromatic acid
(0.001 mol) was dissolved in phosphorus oxychloride and
refluxed for 20 h. The reaction mixture was slowly poured
over crushed ice and kept overnight. The solid thus
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Med Chem Res (2013) 22:3316–3328
separated out was filtered, washed with water, dried, and
recrystallized from ethanol.
N 14.09, S 8.06, Cl 26.75. Found: C 45.32, H 1.79, N
14.11, S 8.08, Cl 26.77.
N-(6-Chlorobenzo[d]thiazol-2-yl)-5-phenyl-1,3,4-oxadia-
zol-2-amine (7a) Yield 67 %; m.p. 262–264 °C; Rf-value
0.8; IR (KBr) m cm-1: 622 (C–S–C benzothiazole), 1454
(C–N benzothiazole), 1488 (C–O–C oxadiazole), 1518
(C=C aromatic), 3318 (N–H); 1H NMR (DMSO-d6) d
(ppm): 7.24–7.28 (m, J = 12 Hz, 8H, Ar–H), 8.10 (s,1H,
N-(6-Chlorobenzo[d]thiazol-2-yl)-5-o-tolyl-1,3,4-oxadia-
zol-2-amine (7d) Yield 65 %; m.p. 204–206 °C; Rf-value
0.80; IR (KBr) m cm-1: 632 (C–S–C benzothiazole), 812
(C–Cl), 1467 (C–N benzothiazole), 1496 (C–O–C oxadi-
azole), 1514 (C=C aromatic), 3331 (N–H); 1H NMR
(DMSO-d6) d (ppm): 2.21(s, 3H, CH3), 7.64–7.68 (m,
J = 12 Hz, 7H, Ar–H), 8.12 (s, 1H, NH); 13C NMR
(DMSO-d6) d (ppm): 18.7 (C-200), 118.3 (C-4, benzothia-
zole), 121.2 (C-7, benzothiazole), 125.8 (C-5, benzothia-
zole), 126.2(C-50), 127.4 (C-60), 128.6 (C-40), 129.5 (C-30),
129.8 (C-6, benzothiazole), 132.3 (C-1a, benzothiazole),
136.9 (C-20), 137.2 (10), 151.3 (C-4a, benzothiazole), 164.5
(C-5, oxadiazole), 169.3 (C-2, oxadiazole), 174.5 (C-2,
benzothiazole); MS: m/z 342 (M?), 344 (M??2); Anal.
Calc. for C16H11 ClN4OS (342.80): C 56.04, H 3.21, N
16.32, S 9.33, Cl 10.34. Found: C 56.06, H 3.23, N 16.34, S
9.35, Cl 10.37.
13
NH); C NMR (DMSO-d6) d (ppm): 126.1 (C-10), 118.3
(C-4, benzothiazole), 121.2 (C-7, benzothiazole), 125.8
(C-5, benzothiazole), 127.5 (C-20-& 60), 129.2 (C-30& 50),
128.7 (C-40), 129.8 (C-6, benzothiazole), 132.3 (C-1a,
benzothiazole), 151.3 (C-4a, benzothiazole), 160.3 (C-5,
oxadiazole), 169.3 (C-2, oxadiazole), 174.5 (C-2,
benzothiazole);
MS: m/z 328 (M?), 330 (M??2); Anal. Calc. for C15H9
ClN4OS (328.78): C 54.80, H 2.76, N 17.04, S 9.75, Cl
10.78. Found: C 54.82, H 2.78, N 17.08, S 9.77, Cl 10.81.
N-(6-Chlorobenzo[d]thiazol-2-yl)-5-(2-chlorophenyl)-1,3,4-
oxadiazol-2-amine (7b) Yield 64 %; m.p. 234–236 °C;
Rf-value 0.60; IR (KBr) m cm-1: 624 (C–S–C benzothia-
zole), 821 (C–Cl), 1456 (C–N benzothiazole), 1491 (C–O–C
2-(5-(6-Chlorobenzo[d]thiazol-2-ylamino)-1,3,4-oxadia-
zol-2-yl)phenyl acetate (7e) Yield 64 %; m.p. 244–
246 °C; Rf-value 0.70; IR (KBr) m cm-1: 638 (C–S–C
benzothiazole), 824 (C–Cl), 1468 (C–N benzothiazole),
1489 (C–O–C oxadiazole), 1517 (C=C aromatic), 3336
(N–H); 1H NMR (DMSO-d6) d (ppm): 2.59 (s, 3H, OCOCH3),
1
oxadiazole), 1512 (C=C aromatic), 3317 (N–H); H NMR
(DMSO-d6) d (ppm): 7.22–7.26 (m, J = 12 Hz, 7H,
Ar–H), 8.14 (s,1H, NH); 13C NMR (DMSO-d6) d (ppm):
118.3 (C-4, benzothiazole), 121.2 (C-7, benzothiazole),
125.8 (C-5, benzothiazole), 127.3 (C-50), 128.9 (C-60),
129.3 (C-30), 129.8 (C-6, benzothiazole), 130.1 (C-40),
132.2 (C-20),132.3 (C-1a, benzothiazole), 136.9 (C-10),
151.3 (C-4a, benzothiazole), 164.5 (C-5, oxadiazole),
169.3 (C-2, oxadiazole), 174.5 (C-2, benzothiazole);MS:
m/z 363 (M?), 361 (M?-2); Anal. Calc. for C15H8 Cl2N4OS
(363.22): C 49.60, H 2.22, N 15.42, S 8.83, Cl 19.52.
Found: C 49.64, H 2.24, N 15.45, S 8.85, Cl 19.54.
7.24–7.28 (m, J = 12 Hz, 7H, Ar–H),8.16 (s, 1H, NH); 13
C
NMR (DMSO-d6) d (ppm): 20.3 (C-100), 117.8 (C-10),
118.3 (C-4, benzothiazole), 121.2 (C-7, benzothiazole),
123.2 (C-30), 125.8 (C-5, benzothiazole), 126.0 (C-50),
129.1 (C-40), 129.8 (C-6, benzothiazole), 132.3 (C-1a,
benzothiazole), 137.1 (60), 151.1 (C-20), 151.3 (C-4a,
benzothiazole), 164.5 (C-5, oxadiazole), 169.0 (C-200),
169.3 (C-2, oxadiazole), 174.5 (C-2, benzothiazole); MS:
m/z 386 (M?), 388 (M??2); Anal. Calc. for C17H11
ClN4O3S (386.81): C 52.81, H 2.84, N 14.46, S 8.27, Cl
9.17. Found: C 52.79, H 2.87, N 14.48, S 8.29, Cl 19.20.
N-(6-Chlorobenzo[d]thiazol-2-yl)-5-(2,4-dichlorophenyl)-
1,3,4-oxadiazol-2-amine (7c) Yield 66 %; m.p. 257–
259 °C; Rf-value 0.70; IR (KBr) m cm-1: 628 (C–S–C
benzothiazole), 826 (C–Cl), 1461 (C–N benzothiazole),
1497 (C–O-C oxadiazole), 1516 (C=C aromatic), 3321
(N–H). 1H NMR (DMSO-d6) d (ppm): 7.26–7.30 (m,
J = 12 Hz, 6H, Ar–H), 8.20 (s, 1H, NH); 13C NMR
(DMSO-d6) d (ppm):118.3 (C-4, benzothiazole), 121.2
(C-7, benzothiazole), 125.8 (C-5, benzothiazole), 127.4 (C-50),
129.8 (C-6, benzothiazole), 130.3 (C-60), 130.9(C-30),
130.7, 132.3 (C-1a, benzothiazole), 133.6 (C-20), 135.0
(C-10), 135.7 (C-40), 151.3 (C-4a, benzothiazole), 164.5 (C-5,
oxadiazole), 169.3 (C-2, oxadiazole), 174.5 (C-2, benzo-
thiazole); MS: m/z 397 (M?), 395 (M?-2), 399 (M??2);
Anal. Calc. for C15H7 Cl3N4OS (397.67): C 45.30, H 1.77,
N-(6-Chlorobenzo[d]thiazol-2-yl)-5-phenoxy-1,3,4-oxadia-
zol-2-amine (7f) Yield 73 %; m.p. 264–266 °C; Rf-value
0.80; IR (KBr) m cm-1: 634 (C–S–C benzothiazole), 824
(C–Cl), 1464 (C–N benzothiazole), 1492 (C–O–C oxadi-
azole), 1518 (C=C aromatic), 3326 (N–H); 1H NMR
(DMSO-d6) d (ppm): 7.25–7.28 (m, J = 9 Hz, 8H, Ar–H),
8.14 (s, 1H, NH); 13C NMR (DMSO-d6) d (ppm): 118.3
(C-4, benzothiazole), 121.2 (C-7, benzothiazole), 122.7
(C-20& 60), 124.6 (C-40), 125.8 (C-5, benzothiazole), 129.7
(C-30& 50), 129.8 (C-6, benzothiazole), 132.3 (C-1a, ben-
zothiazole), 151.3 (C-4a, benzothiazole), 155.2 (C-10),
158.7 (C-5, oxadiazole), 169.3 (C-2, oxadiazole), 174.5
(C-2, benzothiazole); MS: m/z 344 (M?), 345 (M??1), 346
(M??2); Anal. Calc. for C15H9ClN4O2S (344.78): C 52.22,
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Med Chem Res (2013) 22:3316–3328
3321
H 2.61, N 16.23, S 9.27, Cl 10.28. Found: C 52.25, H 2.63,
N 16.25, S 9.30, Cl 10.30.
The animals were randomly allocated into groups at the
beginning of all the experiments. The experimental proto-
col was approved by the animal ethics committee of
Hamdard University. All the test compounds and the ref-
erence drug were administered orally suspended in 0.5 %
carboxymethyl cellulose (CMC) solution.
N-(6-Chlorobenzo[d]thiazol-2-yl)-5-(4-nitrophenyl)-1,3,4-
oxadiazol-2-amine (7g) Yield 67 %; m.p. 223–225 °C;
Rf-value 0.60; IR (KBr) m cm-1: 637 (C–S–C benzothia-
zole), 828 (C–Cl), 1378 (NO2), 1468 (C–N benzothiazole),
1496 (C–O–C oxadiazole), 1521 (C=C aromatic), 3336
Anti-inflammatory activity
1
(N–H); H NMR (DMSO-d6) d (ppm): 7.21–7.24 (m, J =
9 Hz, 7H, Ar–H), 8.16 (s, 1H, NH); 13C NMR (DMSO-d6)
d (ppm): 118.3 (C-4, benzothiazole), 121.2 (C-7, benzo-
thiazole), 128.8 (30& 50), 125.8 (C-5, benzothiazole), 129.8
(C-6, benzothiazole), 130.9 (C-20& 60), 132.2 (C-10),147.9
(40), 151.3 (C-4a, benzothiazole), 164.5 (C-5, oxadiazole),
169.3 (C-2, oxadiazole), 174.5 (C-2, benzothiazole); MS:
m/z 373 (M?), 374 (M??1), 375 (M??2); Anal. Calc.
for C15H8ClN5O3S (373.77): C 48.23, H 2.18, N 18.76, S
8.60, Cl 9.49. Found: C 48.20, H 2.16, N 18.74, S 8.58, Cl
9.51.
The synthesized compounds were evaluated for their anti-
inflammatory activity using the carrageenan induced hind
paw edema method (Winter et al., 1962). The animals
were randomly allocated into groups of six animals each
and were fasted for 24 h before the experiment with free
access to water. Control group received only 0.5 % car-
boxymethyl cellulose solution. Standard drug ibuprofen
was administered orally at a dose of 70 mg/kg. The test
compounds were administered orally at an equimolar oral
dose relative to 70 mg/kg ibuprofen. Into the sub plantar
region of the right hind paw of each rat, 0.1 mL of 1 %
carrageenan solution in saline was injected subcutane-
ously, 1 h after the administration of the test compounds
and standard drug. The right hind paw volume was
measured before and after 3 and 4 h of carrageenan
treatment by means of a plethysmometer. The percent
edema inhibition was calculated from the mean effect in
the control and treated animals according to the following
equation:
Synthesis of 5-(6-chlorobenzo[d]thiazol-2-ylamino)-
1,3,4-oxadiazole-2-thiol (8)
A mixture of 3 (0.005 mol), KOH (0.005 mol) and carbon
disulfide (5 mL) in ethanol (50 mL) was refluxed on a
steam bath for 12 h. The solution was then concentrated,
cooled, and acidified with dilute HCl. The solid mass that
separated out was filtered, washed with ethanol, dried, and
recrystallized from ethanol.
Percent edema inhibition ¼ ðVc ꢁ Vt= VcÞ ꢂ 100
where, Vt represents the mean increase in paw volume in
rats treated with test compounds and Vc represents the
mean increase in paw volume in control group of rats.
Yield 74 %; m.p. 192–194 °C; Rf-value 0.80; IR (KBr)
m cm-1: 614 (C–S–C benzothiazole), 826 (C–Cl), 1421 (C–N),
1505 (C–O–C oxadiazole), 1514 (C=C), 1614 (C=O), 2516
(SH), 3316 (N–H); 1H NMR (DMSO-d6) d (ppm):
7.17–7.19 (m, J = 6 Hz, 3H, Ar–H), 8.05 (s, 1H, NH),
13.11 (s, 1H, SH); 13C NMR (DMSO-d6) d (ppm): 118.3
(C-4, benzothiazole), 121.2 (C-7, benzothiazole), 125.8
(C-5, benzothiazole), 129.8 (C-6, benzothiazole), 132.3 (C-1a,
benzothiazole), 151.3 (C-4a, benzothiazole), 169.3 (C-5,
oxadiazole), 171.6 (C-2, oxadiazole), 174.5 (C-2, benzo-
thiazole); MS: m/z 284 (M?), 283 (M?-1), 285 (M??1).
Anal. Calc. for C9H5ClN4OS2 (284.75): C 37.96, H 1.77,
N 19.68, S 22.52, Cl 12.45. Found: C 37.98, H 1.79, N
19.71, S 22.56, Cl 12.47.
Analgesic activity
Analgesic activity was evaluated by tail immersion method
(Adeyemi et al., 2004). Swiss albino mice allocated into
different groups consisting of six animals in each, of either
sex, weighing 25–30 g were used for the experiment.
Analgesic activity was evaluated after oral administration
of the test compounds at an equimolar dose relative to
70 mg/kg ibuprofen. Test compounds and standard drugs
were administered orally as suspension in carboxymethyl
cellulose solution in water (0.5 % w/v). The analgesic
activity was assessed before and after 4 h interval of the
administration of test compounds and standard drugs. The
lower 5 cm portion of the tail was gently immersed into
thermostatically controlled water at 55 0.5 °C. The time
in seconds for tail withdrawal from the water was taken as
the reaction time with a cut-off time of immersion, set at
10 s for both control as well as treated groups of animals.
Pharmacological evaluation
Adult Wistar strain rats of either sex, weighing 150–200 g
were used for anti-inflammatory, ulcerogenic, and lipid
peroxidation activities, whereas swiss albino mice weigh-
ing 25–30 g were used for analgesic activity. The animals
were allowed food and water ad libitum except during the
experiments. They were housed in a room at 25 2 °C,
and 50 5 % relative humidity with 12 h light/dark cycle.
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Med Chem Res (2013) 22:3316–3328
Acute ulcerogenicity
allowed to clot for 45 min at room temperature and serum
was separated by centrifugation at 2,500 rpm for 15 min
and analyzed for various biochemical parameters.
Acute ulcerogenicity was determined according to the
standard method (Cioli et al., 1979). The animals were
allocated into different groups consisting of six animals in
each group. Ulcerogenic activity was evaluated after oral
administration of the test compounds at an equimolar dose
relative to 210 mg/kg ibuprofen. Control group received
only 0.5 % carboxymethyl cellulose solution. 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 sacrificed. 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.
Assessment of liver function
Liver functions such as serum glutamate oxaloacetate
transaminase (SGOT) and serum glutamate pyruvate
transaminase (SGPT) were assessed by a reported method
(Reitman and Frankel, 1957). The alkaline phosphatase,
total protein, and total albumin were measured according to
the reported procedures (King and Armstrong, 1934). All
data are recorded in Table 1.
Histopathological studies of liver
The histopathological studies were carried out by a repor-
ted method (Luna, 1968). The rats were killed under light
ether anesthesia after 24 h of the last dosage, the livers
were removed and washed with normal saline and stored in
formalin solution. Sections of 5–6 mm in thickness were
cut, stained with hematoxylin and eosin and then studied
under a light microscope (Figs. 1, 2, 3, 4).
Lipid peroxidation
Lipid peroxidation in the gastric mucosa was determined
according to the method given in literature (Ohkawa et al.,
1979). After screening for ulcerogenic activity, the gastric
mucosa was scraped with two glass slides, weighed
(100 mg) and homogenized in 1.8 mL of 1.15 % ice cold
KCl solution. The homogenate was supplemented with
0.2 mL of 8.1 % sodium dodecyl sulfate (SDS), 1.5 mL of
acetate buffer (pH 3.5) and 1.5 mL of 0.8 % thiobarbituric
acid (TBA). The mixture was heated at 95 °C for 60 min.
After cooling the reactants were supplemented with 5 mL
of the mixture of n-butanol and pyridine (15:1 v/v), shaken
vigorously for 1 min and centrifuged for 10 min at
4,000 rpm. The supernatant organic layer was taken out and
absorbance was measured at 532 nm on UV spectropho-
tometer. The results were expressed as nmol MDA/100 mg
Statistical analysis of data
All the statistical analyses were carried out using the soft-
ware SigmaStat 4.0 using ANOVA followed by dunnett’s
multiple comparison tests and the results are expressed in
mean SEM.
Results and discussion
Synthesis
N-(6-Chlorobenzo[d]thiazol-2-yl) hydrazine carboxamide
(3) have been synthesized through a multi step reaction as
depicted in Scheme 1. The 6-chloro-1,3-benzothiazole-2-
amine (1) was synthesized by reacting 4-chloroaniline with
potassium thiocyanate in a satisfactory yield. In FT-IR
spectrum bands at 3,480 and 1,570 cm-1 confirms the
presence of NH2 and C=N stretching vibrations, respec-
tively. The 1H NMR spectrum showed a singlet at d
6.12 ppm due to NH2 protons (D2O exchangeable).
tissue, using extinction coefficient 1.56 9 105 cm-1 M-1
.
Hepatotoxic studies
The study was carried out on Wistar albino rats of either
sex weighing 150–200 g. Animals were divided into three
groups, six rats in each. Group 1 was kept as control and
received only vehicle (0.5 % w/v solution of carboxy-
methyl cellulose in water), rest of the groups received test
compounds, at an equimolar oral dose relative to 210 mg/kg
ibuprofen in 0.5 % w/v solution of carboxymethyl cellu-
lose in water once in a day for 15 days. After the treatment
(15 days) blood was obtained from all groups of rats by
puncturing the retro-orbital plexus. Blood samples were
The benzothiazole-2-yl urea (2) were prepared by treating
6-chloro-1,3-benzothiazole-2-amine (1) with sodium cya-
nate in the presence of glacial acetic acid. Thebenzothiazole-
2-yl-urea (2) was then refluxed with hydrazine hydrate to
yield the product N-(6-chlorobenzo[d]thiazol-2-yl)hydra-
zine carboxamide (3) as outlined in Scheme 1. The infrared
spectra showed characteristic absorption bands at
3,300 cm-1 due to NH2 function beside the C=O at
123

Med Chem Res (2013) 22:3316–3328
3323
Table 1 Effect of selected compounds on serum enzymes, total proteins, and total albumin
Compounds
SGOT (units/mL)a
SGPT (units/mL)a
Alkaline phosphatasea
Total protein (g/dL)a
Total albumin (g/dL)a
Control
143.71 1.10
140.14 1.12
131.18 2.12**
138.42 2.11**
32.46 0.76
34.26 0.72
28.33 0.60**
30.74 0.54
12.17 0.16
11.67 0.18
11.09 0.11
12.11 0.14
1.72 0.02
1.74 0.06
1.79 0.17
1.75 0.10
1.63 0.02
1.71 0.01
1.74 0.22
1.70 0.11
Ibuprofen
6f
7c
a
Relative to control and data were analyzed by ANOVA followed by Dunnett’s multiple comparison test, for n = 6
** P \ 0.01
Fig. 1 Control; section of liver showing portal triad structures and
normal hepatic parenchyma. PV portal vein, Hep hepatocyte (9100)
Fig. 3 Compound 6f; section of liver showing hepatocytes with
clearing of cytoplasm and compression of sinusoidal spaces. BD bile
duct, Hep hepatocyte (9100)
Fig. 2 Ibuprofen; section of liver showing hepatocytes with clearing
of cytoplasm and compression of sinusoidal spaces. Hep hepatocyte
(9100)
Fig. 4 Compound 7c; section of liver showing hepatocytes with
clearing of cytoplasm and compression of sinusoidal spaces. PT portal
triad, Hep hepatocyte (9100)
1,660 cm-1, respectively. Similarly, the 1H NMR spectra of
the synthesized compounds showed singlet characteristic
peaks at d 9.12 cm-1 and the other at d 7.28 cm-1 due to
NHC=O (D2O exchangeable) and NHNH2 (D2O exchange-
able) proton, respectively.
to afford the corresponding intermediate potassium
dithiocarbazinate (4). This salt underwent ring closure
with an excess of 99 % hydrazine hydrate to give 4-amino-
5-(6-chlorobenzo[d]thiazol-2-ylamino)-4H-1,2,4-triazole-3-
thiol (5) in good yield. The resultant triazole (5) was
The acid hydrazide (3) was allowed to react with carbon
disulfide in the presence of potassium hydroxide in ethanol
123

3324
Med Chem Res (2013) 22:3316–3328
N
S
g.a.a.
Br2
NH2
(1)
Cl
NH2
KSCN
Cl
g.a.a
NaOCN
N
O
O
N
S
NH2NH2.H2O
NaOH/EtOH
NH
NH2
NH
NH2
(2)
NH
Cl
S
(3)
Cl
KOH/CS2/C2H5OH Stirring;Room temp.
N
NH
O
SH
S
O
S
(8)
Cl
N
N
S
N
S-K+
NH
NH
NH
(4)
Cl
N
NH2NH2.H2O Reflux
NH
O
R
S
Cl
NH2
N
N
N
SH
N
(7a-g)
N
N
N
H
S
Cl
(5)
RCOOH/POCl3
Reflux
NH
R
N
N
S
N
S
N
N
Cl
(6a-g)
6,7 R : [a = C6H5-, b = 2-ClC6H4-, c = 2,4-ClC6H3-, d = 2-CH3C6H4-, e = 2-OCOCH3C6H4-, f = OC6H5-, g = 4-
NO2C6H4-]
Scheme 1 Synthetic route for the title compounds
further converted to 6-chloro-N-(6-substituted-[1,2,4]triaz-
olo[3,4-b][1,3,4]thiadiazol-3-yl)benzo[d]thiazol-2-amine
(6a–g) through one-pot reaction by condensation with
appropriate aromatic acids in the presence of phosphorus
oxychloride, as outlined in Scheme 1. Phosphorus oxy-
chloride was necessary for this condensation, which acti-
vate the carbonyl group of aromatic acids and increases its
electrophilicity to enhance the addition of triazole to it.
The structure of 4-amino-5-(6-chlorobenzo[d]thiazol-2-
ylamino)-4H-1,2,4-triazole-3-thiol (5) was confirmed by
FT-IR,1H NMR spectral data and microanalysis. The
infrared spectra showed two characteristic absorption
bands, one of which appearing at 2,518 cm-1, was attrib-
uted to SH and the other at 3,338 cm-1, was assigned to
NH2, which were disappeared by the formation of the
triazolo-thiadiazoles. The 1H NMR spectra showed a
downfield D2O exchangeable singlet at d 13.18, attributed
to the SH group, while the NH2 group appeared as a singlet
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Med Chem Res (2013) 22:3316–3328
3325
at d 5.15. The absence of signals due to NH2 and SH
protons confirmed that the triazole was converted into
triazolo-thiadiazoles (6a–g) by reacting with group of
aromatic acids.
possess highest activity (89.60 1.90, 88.81 2.34 and
89.67 2.58 %, respectively) comparable to that of ibu-
profen (82.69 1.65 %). Furthermore, it was observed
that the presence of 2-chlorophenyl moiety (6b) at C-6
showed decrease of activity (50.78 4.70 %) and
replacement of this group by acetyl phenyl group (6e)
resulted in sharp decrease of anti-inflammatory activity
(20.62 2.93 %). Compounds 6a and 6g showed moder-
ate activity (57.93 4.15 and 66.66 2.74 %).
5-(6-Chlorobenzo[d]thiazol-2-ylamino)-1,3,4-oxadiazole-
2-thiol (8) was prepared by reacting hydrazide (3) with carbon
disulfide in alkaline medium and N-(6-chlorobenzo[d]thiazol-
2-yl)-5-substituted phenyl-1,3,4-oxadiazol-2-amine (7a–g)
were prepared by treatment of hydrazide (3) with appropriate
aromatic acids in the presence of phosphorus oxychloride
(Scheme 1). IR spectra of the compound 8 showed band at
1,505 cm-1 due to C–O–C stretching vibration of oxadiazole
and at 2,516 cm-1 corresponding to SH group, whereas
compounds 7a–g also showed bands at 1,488 cm-1 due to
C–O–C stretching vibration of oxadiazole but lacks SH
stretchingvibration. The structure was further supported byits
1H NMR spectrum which showed a singlet of SH proton at d
13.11 for compound 8.
The anti-inflammatory activity of 1,3,4-oxadiazole deriv-
atives were found between 32.53 3.56 and 88.81
1.73 %. The highest activity (88.81 1.73 %) was found in
the oxadiazole derivative 7f having phenoxy group at second
position. When this group was replaced by methyl and 4-nitro
phenyl group (7d and 7g) the activity was found to be
decreased (37.69 5.23 and 32.53 3.56 %). It was
observed that compounds having phenyl (7a) and 2,4-
dichlorophenyl group (7c) at second position showed equiv-
alent or better activity (88.27 1.73 and 87.14 1.09 %)
than the standard drug. When the 2-substituted phenyl group
was replaced by 2-mercapto group (8) the activity was found
to be good (69.76 4.55 %). Rest of the oxadiazole deriva-
tives showed moderate activity.
The elemental analysis and molecular ion peaks of
compounds 6a–g, 7a–g & 8 were consistent with the
assigned structure. The numbering pattern on the basic
nucleus is assigned in Scheme 2.
Pharmacological evaluation
Analgesic activity
Anti-inflammatory activity
The compounds 6c, 6d, 6f, 7a, 7c, 7f, and 8 which showed
comparable or equal anti-inflammatory activity to that of
standard reference drug, were further tested for their anal-
gesic activity at an equimolar oral dose relative to 70 mg/kg
ibuprofen (Table 3). The compounds showed analgesic
activity ranging from 52.00 2.21 to 79.03 1.62 %,
whereas the standard drug ibuprofen 73.50 1.10 %
inhibition. The highest analgesic activity (79.03 1.62 %)
was shown by oxadiazole derivative 7c, which also has
significant anti-inflammatory activity (87.14 1.09 %). It
was noted that compound 6f, a triazolo-thiadiazole deriva-
tive showing the highest anti-inflammatory activity, also
exhibited significant analgesic activity (75.00 1.42 %).
The remaining compounds showed reduced analgesic
activity. These compounds were further screened for their
acute ulcerogenic activity. The compounds were tested at an
equimolar oral dose relative to 210 mg/kg ibuprofen.
The anti-inflammatory activity of the synthesized com-
pounds 6a–g, 7a–g & 8 were evaluated by the carrageenan
induced paw edema method (Winter et al., 1962). The
compounds were tested at an equimolar oral dose relative
to 70 mg/kg ibuprofen. The percentage inhibition was
calculated both after 3 and 4 h, and since it was found to be
more after 4 h, this was made the basis of discussion. The
tested compounds showed anti-inflammatory activity
ranging from 20.62 2.93 to 89.67 2.58 %, whereas
the standard drug ibuprofen showed 82.69 1.65 %
inhibition, after 4 h (Table 2).
The anti-inflammatory activity of triazolo-thiadiazole
derivatives were in the range of 20.62 2.93 to
89.67 2.58 %. It was observed that the triazolo-thiadia-
zole derivatives having 2,4-dichlorophenyl (6c), methyl
phenyl (6d), and phenoxy group (6f) at sixth position
R
6
3
3
N
5
N
N
3
N
1
O
1
O
N
S
1
b
NH
d
NH
NH
4
d
SH
R
d
S
1
S
Cl
6
Cl
6
5
2
N
N
2
1
S
1
5
Cl
6
N
4
N N
N
3
3
4
3
(8)
(6a-g)
(7a-g)
Scheme 2 Numbering pattern on basic nucleus
123

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Med Chem Res (2013) 22:3316–3328
Table 2 Anti-inflammatory activity of the synthesized compounds
Compounds
Paw volume
0 h (Basal)
% Inhibition SEMa
Potency
After 3 h
After 4 h
After 3 h
After 4 h
6a
0.34 0.017
0.36 0.011
0.40 0.012
0.41 0.010
0.28 0.014
0.43 0.015
0.38 0.009
0.44 0.013
0.35 0.011
0.38 0.015
0.33 0.021
0.44 0.012
0.44 0.016
0.36 0.014
0.44 0.010
0.36 0.016
0.35 0.012
0.56 0.017
0.61 0.018
0.48 0.029
0.50 0.019
0.76 0.015
0.50 0.011
0.57 0.019
0.53 0.022
0.56 0.013
0.46 0.024
0.65 0.019
0.71 0.016
0.55 0.024
0.68 0.019
0.59 0.015
0.45 0.012
0.75 0.014
0.51 0.018
0.57 0.014
0.44 0.016
0.45 0.029
0.61 0.017
0.47 0.024
0.45 0.022
0.49 0.015
0.53 0.021
0.43 0.016
0.60 0.014
0.67 0.017
0.50 0.019
0.64 0.017
0.56 0.015
0.29 0.018
0.77 0.013
48.33 5.42*
41.66 3.57*
80.00 1.82
77.5 1.70*
10.00 2.23
78.33 3.07
53.33 3.07*
78.83 3.06
53.00 5.59
80.00 1.82
23.91 8.18*
31.00 4.96*
74.16 3.00**
19.16 3.74*
61.66 4.01*
78.16 1.08
–
57.93 4.15**
50.78 4.70*
89.60 1.90
88.81 2.34
20.62 2.93*
89.67 2.58
66.66 2.74*
88.27 1.73
53.17 2.584*
87.14 1.09
37.69 5.23*
50.78 7.44*
88.81 2.34
32.53 3.56*
69.76 4.55*
82.69 1.65
–
0.70
0.61
1.08
1.07
0.24
1.08
0.80
1.06
0.64
1.05
0.45
0.61
1.07
0.39
0.84
1.00
–
6b
6c
6d
6e
6f
6g
7a
7b
7c
7d
7e
7f
7g
8
Ibuprofen
Control
a
Relative to their respective standard and data were analyzed by ANOVA followed by dunnett’s multiple comparison test for n = 6;
dose = 70 mg/kg
* P \ 0.01
** P \ 0.05
Acute ulcerogenicity
nanomoles of malondialdehyde (MDA)/100 mg of gastric
mucosa tissue. Ibuprofen exhibited maximum tissue lipid
peroxidation 7.51 0.16, whereas control group showed
3.26 0.01. It was found that all the cyclized derivatives
showing less ulcerogenic activity also showed reduction in
lipid peroxidation (Table 3). Thus, these studies showed
that the synthesized compounds have inhibited the induc-
tion of gastric mucosal lesions and the results further
suggested that their protective effect might be related to the
inhibition of lipid peroxidation in the gastric mucosa.
The tested compounds showed significant reduction in
ulcerogenic activity ranging from 0.666 0.166 to
2.166 0.357, whereas standard drug ibuprofen showed
high severity index of 2.315 0.18. The maximum
reduction in ulcerogenic activity (0.666 0.166 and
0.917 0.22) was found in compounds 6f and 7c,
respectively. The rest of the compounds also showed better
GI safety profile as compared to ibuprofen, as illustrated in
Table 3. Thus, the results showed that substitution of the
hydrazide group by the bioisosteric groups triazolo-thia-
diazole and 1,3,4-oxadiazole, has resulted in significant
anti-inflammatory and analgesic activities along with
reduced ulcerogenic potential.
Hepatotoxic and histopathological studies of liver
The compounds 6f and 7c, derivatives of triazolo-thiadia-
zole and 1,3,4-oxadiazole, respectively, showing potent
anti-inflammatory and analgesic activities with reduced
ulcerogenicity and lipid peroxidation, were further studied
for their hepatotoxic effect in comparison with standard
ibuprofen drugs. Both compounds were studied for their
effect on biochemical parameters (Reitman and Frankel,
1957; King and Armstrong, 1934) (serum enzymes, total
proteins and total albumin), and liver histopathological
(Luna, 1968) testing were also carried out. As shown in
Table 1 activities of liver enzymes serum glutamate oxa-
loacetate transaminase (SGOT), serum glutamate pyruvate
transaminase (SGPT), alkaline phosphatase, total protein,
Lipid peroxidation
It has been reported that compounds showing less ulcero-
genic activity also showed reduced malondialdehyde
(MDA) content, a byproduct of lipid peroxidation (Pohle
et al., 2001). Therefore, an attempt was made to correlate
the decrease in ulcerogenic activity of the compounds with
that of lipid peroxidation. All the compounds screened for
ulcerogenic activity were also analyzed for their effect on
lipid peroxidation. Lipid peroxidation is measured as
123

Med Chem Res (2013) 22:3316–3328
3327
Table 3 Analgesic, ulcerogenic and lipid peroxidation activities of selected compounds
Compounds Analgesic activitya
Ulcerogenic activity
(severity index SEM)b
Nanomoles of MDA
content SEM/100 mg tissueb
Pre-treatment/normal Post-treatment/ % Inhibition
0 h (s) (normal)
Potency
after 4 h (s)
6c
1.26 0.047
1.60 0.033
1.24 0.046
0.90 0.036
0.24 0.055
1.25 0.062
1.30 0.031
1.43 0.009
–
2.21 0.054
2.83 0.035
2.17 0.045
1.45 0.032
2.22 0.048
1.90 0.032
2.24 0.047
2.60 0.01
–
75.39 1.24
76.87 2.01
75.00 1.42
0.92
0.93
0.91
0.925 0.325*
1.583 0.436*
0.666 0.166**
1.667 0.13*
0.917 0.22*
1.083 0.327*
2.166 0.357*
2.315 0.18
5.74 0.14*
7.44 0.12*
3.04 0.31**
4.47 0.19*
3.29 0.15**
9.10 0.14*
9.04 0.24*
7.51 0.16
6d
6f
7a
61.11 2.73* 0.74
79.03 1.62 0.96
7c
7f
52.00 2.21* 0.63
72.30 2.10** 0.88
8
Ibuprofen
Control
a
73.50 1.10
–
1.00
–
0.000 0.00
3.26 0.01
Relative to normal and data was analyzed by paired student’s t test for n = 6; dose = 70 mg/kg; ** P \ 0.0001, * P \ 0.005
b
Relative to their respective standard and data were analyzed by ANOVA followed by dunnett’s multiple comparision test for n = 6;
dose = 210 mg/kg; ** P \ 0.05, * P \ 0.01
Jamia Hamdard, for providing Wistar rats and Dr. A. Mukherjee,
M.D., Department of Pathology, All India Institute of Medical Sci-
ences (AIIMS), New Delhi, for carrying out histopathological studies.
and total albumin were almost identical with control val-
ues, whereas ibuprofen showed slight difference with
control values. The studies of the liver samples do not show
any significant pathological changes in comparison to
control group (Figs. 1, 2, 3, 4). No hepatocyte necrosis or
degeneration was seen in any of the samples. The hepa-
tocytes are swollen and compression of sinusoidal spaces is
seen. Ibuprofen also showed hepatocytes with clearing of
cytoplasm and compression of sinusoidal spaces.
Conflict of interest None.
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