Design, synthesis and biological evaluation of 1,3,4-oxadiazoles as promising anti-inflammatory agents

Article abstract of DOI:10.1007/s00044-016-1740-6

A series of 1,3,4-oxadiazole derivatives were designed, synthesized and evaluated for radical scavenging and anti-inflammatory properties. Molecular docking simulation studies onto the proteins cyclooxygenase-1 (PDB: 1CQE) and cyclooxygenase-2 (PDB: 3LN1) to visualize the probable binding affinity towards anti-inflammatory importance and in silico studies, towards their appreciable ADME & probable toxicity property were screened. The best-ranked molecules; N-((5-substituted-1,3, 4-oxadiazol-2-yl)methyl) benzo[d]thiazol-2-amine (5a–5j) were synthesized from 2-(benzo[d]thiazol-2-ylamino)acetohydrazide (4) on reaction with aryl/heteroaryl/aliphatic carboxylic acid derivatives via acid catalyzed dehydrative cyclization. N-((5-mercapto-1,3,4-oxadiazol-2-yl)methyl) benzo[d]thiazol-2-amine (5k) was synthesized by base catalyzed condensation of hydrazide derivative 4 and with carbon disulfide. The newly synthesized compounds were characterized and established on the basis of elemental analysis, IR, ¹H NMR, ¹³C NMR and mass studies. The 1,3,4-oxadiazoles were evaluated for in vitro antioxidant property by 2,2′-diphenyl-1-picryl hydrazyl radical scavenging assay method and in vivo anti-inflammatory activity by carrageenan induced paw edema method. The radical scavenging activity indicated that the 1,3,4-oxadiazoles at 25 μM test concentration exhibited significant radical scavenging property ranging from 32.0 to 87.3 % in comparison to 76.0 % radical scavenging activity obtained for the reference drug, ascorbic acid. The results of the in vivo anti-inflammatory activity highlighted that the 1,3,4-oxadiazoles at 25 mg Kg^?1 test dose exhibited significant edema inhibition with a mean value ranging from 23.6 to 82.3 % in comparison to 48.3 % edema inhibition obtained for the reference drug, indomethacin. The compound 5h with mean edema inhibition value of 82.3 % and potent among the series was further evaluated for in vitro COX inhibition and was found to more selective towards COX-2. The acute ulcerogenic evaluation of compound 5h indicated it to be safe at the dose of 50 mg Kg^?1.

Full text of DOI:10.1007/s00044-016-1740-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1740-6
ORIGINAL RESEARCH
Design, synthesis and biological evaluation of 1,3,4-oxadiazoles as
promising anti-inflammatory agents
Vishwanathan Balasubramanya Iyer1 Bannimath Gurupadayya1
●
●
Venkata Sairam Koganti1 Bharthkumar Inturi1 Ravandur Shivanna Chandan1
●
●
Received: 24 February 2016 / Accepted: 31 October 2016
© Springer Science+Business Media New York 2016
Abstract A series of 1,3,4-oxadiazole derivatives were
designed, synthesized and evaluated for radical scavenging
and anti-inflammatory properties. Molecular docking
simulation studies onto the proteins cyclooxygenase-1
(PDB: 1CQE) and cyclooxygenase-2 (PDB: 3LN1) to
visualize the probable binding affinity towards anti-
inflammatory importance and in silico studies, towards
their appreciable ADME & probable toxicity property were
screened. The best-ranked molecules; N-((5-substituted-1,3,
4-oxadiazol-2-yl)methyl) benzo[d]thiazol-2-amine (5a–5j)
were synthesized from 2-(benzo[d]thiazol-2-ylamino)acet-
ohydrazide (4) on reaction with aryl/heteroaryl/aliphatic
carboxylic acid derivatives via acid catalyzed dehydrative
cyclization. N-((5-mercapto-1,3,4-oxadiazol-2-yl)methyl)
benzo[d]thiazol-2-amine (5k) was synthesized by base
catalyzed condensation of hydrazide derivative 4 and with
carbon disulfide. The newly synthesized compounds were
characterized and established on the basis of elemental
25 µM test concentration exhibited significant radical
scavenging property ranging from 32.0 to 87.3 % in com-
parison to 76.0 % radical scavenging activity obtained for
the reference drug, ascorbic acid. The results of the in vivo
anti-inflammatory activity highlighted that the 1,3,4-oxa-
diazoles at 25 mg Kg−1 test dose exhibited significant
edema inhibition with a mean value ranging from 23.6 to
82.3 % in comparison to 48.3 % edema inhibition obtained
for the reference drug, indomethacin. The compound 5h
with mean edema inhibition value of 82.3 % and potent
among the series was further evaluated for in vitro COX
inhibition and was found to more selective towards COX-2.
The acute ulcerogenic evaluation of compound 5h indicated
it to be safe at the dose of 50 mg Kg−1.
●
●
Keywords 1,3,4-Oxadiazole Anti-inflammatory
●
●
Benzothiazole Radical scavenging Ulcerogenic
evaluation
1
analysis, IR, H NMR, 13C NMR and mass studies. The
1,3,4-oxadiazoles were evaluated for in vitro antioxidant
property by 2,2′-diphenyl-1-picryl hydrazyl radical scaven-
ging assay method and in vivo anti-inflammatory activity
by carrageenan induced paw edema method. The radical
scavenging activity indicated that the 1,3,4-oxadiazoles at
Introduction
Prostaglandins (PGs), thromboxanes and leukotrienes are
classified under derivatives of eicosanoid, which are bio-
synthesized by several enzymatic steps in the arachidonate
cascade from arachidonic acid and essential fatty acid. PGs
have been involved in multiple biological functions,
responsible of numerous physiological processes (Nathan
2002). The prostaglandin H synthase (PGHS) enzyme is an
integral membrane protein and is the dedicated step in the
conversion of arachidonic acid to PGG2. PGG2 is further
reduced to PGH2 catalyzed by peroxidase activity of PGHS.
PGHS is sequentially the limiting step in the synthesis of
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1740-6) contains supplementary material,
which is available to authorized users.
* Vishwanathan Balasubramanya Iyer
bvishwanathan@jssuni.edu.in
1
Department of Pharmaceutical Chemistry, JSS College of
Pharmacy, JSS University, Sri Shivarathreeshwara Nagar,
Mysuru, Karnataka 570 015, India

Med Chem Res
other PGs, as PGH2 is consequently converted to prosta-
noids, thromboxane A2 by thromboxane synthases and
other PGs including PGE2 by prostaglandin synthases
(Williams et al. 2002). PGHS is primarily located in the
endoplasmic reticulum, PGHS is bi functional in nature, the
cyclooxygenase (COX) activity which converts the arachi-
donic acid to PGG2 and the peroxidase activity responsible
for the conviction of PGG2 to PGH2. The PGs have sig-
nificant role in tissue injury to exude inflammatory signals.
Inflammationis a protective and defense mechanism to an
injury caused by factors like physical trauma, microbial
infections, injurious stimuli or even a chemical burn.
Equally, the PGs have considerable role in the physiology
involved in release of renin and aggregation of platelet, and
have been implicated in cardiovascular disorders (Gund and
Shen 1977).
The COX activity of the PGHS is targeted by the non-
steroidal anti-inflammatory drugs (NSAIDs). NSAIDs are
first line agents for the management of osteoarthritis,
arthritis and other clinical condition associated with pain
and inflammation (Talley et al. 2000). NSAIDs are widely
prescribed, easily accessible as over the counter drugs and
are also commonly used as self-medication for the man-
agement of any pain and inflammation related clinical dis-
orders. NSAIDs act by inhibiting the enzyme prostaglandin
endoperoxidase; COX, thus suppressing prostaglandin bio-
synthesis.(Warner et al. 1999). Two isoforms of PGHS have
been identified and characterized, PGHS-1 (COX-1), and
PGHS-2 (COX-2).The physiological importance of both the
PGHS isoenzyme are extensively investigated, both PGHS-
1 and PGHS-2 differ for regulation, expression and dis-
tribution (Dannhardt and Kiefer 2001). PGHS-1 isoenzyme
is involved in cell–cell signaling, regulation of the mucus
formation and thromboxane production, maintaining tissue
homeostasis and provides cytoprotection in the gastro-
intestinal (GI) tract. The PGHS-2 isoenzyme expression
occurs in a limited number of cell types, mediates inflam-
matory signals and is regulated by specific stimulatory
signals indicative that PGHS-2 positive role in prostanoid
biosynthesis involved in mitogenesis and inflammation.
Prostaglandin biosynthesis has be eventually targeted in the
management of inflammatory conditions (Habeeb et al.
2001). Most of the clinical available NSAIDs are non-
selective COX inhibitor or exhibit greater selectivity for
COX-1 than COX-2 (Kujubu et al. 1991; Smith et al. 1998).
The long term and chronic usage of these non-selective
NSAIDs has led to the precipitation of secondary clinical
condition like dyspepsia, gastroduodenal ulcers, gastritis
bleeding and nephrotoxicity (Sondhi et al. 2007).
production of the ROS produces a state of oxidative stress
and this oxidative stress is also an equally important com-
ponent for GI ulceration. The ROS have substantial role in
the biosynthesis of PGs and in the COX and lipoxygenase
mediated conversion of arachidonic acid into proin-
flammatory intermediates. Signifying that, a molecule
which has free radical scavenging potential is more likely to
possess anti-inflammatory activity too. The management of
ROS with antioxidant abolishes the production of series of
proinflammatory cytokinins (El-Gazzar et al. 2009). Thus
highlighting that, the management of inflammatory condi-
tion could be benefited from the use of drugs that combine
antioxidant and anti-inflammatory activities. Tolfenamic
acid is an evident example under commercially available
NSAIDs with dual antioxidant and anti-inflammatory
activities. In view of the above facts, anti-inflammatory
investigations are always associated with preliminary anti-
oxidant evaluation for the tested compounds (Sakat et al.
2010).
Literature studies highlight that 1,3,4-oxadiazole have
been widely explored for its anti-inflammatory property
(Kucukguzel et al. 2007; Singh et al. 2013; Ravindra et al.
2006; Amir et al. 2007; Burbuliene et al. 2004; Kadi et al.
2007). 1,3,4-oxadiazole moiety provides a scaffold on
which pharmacophores can be arranged to afford potent and
selective drugs. 1,3,4-oxadiazole being a toxophoric moiety
(N-C-O), highlighting its importance as a potential biody-
namic molecule (Somani and Bhanushali 2011), has been
widely explored and reported for its biological activities like
anticonvulsant (Almasirad et al. 2004), antidepressant
(Zarghi et al. 2005), anticancer (Jin et al. 2006; Padmavathi
et al. 2009), analgesic (Kumar et al. 2008), anti-HIV (El-
Emam et al. 2004) antibacterial (Naveena et al. 2010; Sahin
et al. 2002), antifungal (Liu et al. 2008), antimycobacterial
(Ali and Shaharyar 2007; Kumar et al. 2010), anthelmintic
(Bharathi et al. 2010), hypoglycemic (Shingalapur et al.
2010) and antiangiogenic (Kumar et al. 2009) properties.
The pursuit for better therapeutic agents with clinical
advantage has always been the core of pharmaceutical
research. In view of the above facts and in continuation of
our study on 1,3,4-oxadiazoles (Vishwanathan et al. 2016),
we report here the synthesis of a small library of 1,3,4-
oxadiazoles and their anti-inflammatory efficacy.
Results and discussion
Chemistry
At the same time, clinical studies have indicated sig-
nificant correlation between reactive oxygen species (ROS)
and generation of free radicals during the expression of
inflammatory responses (Galanakis et al. 2004). Increased
Synthesis of the intermediates and the target compounds
5a–5k were accomplished according to the steps depicted in
Scheme 1. The starting material benzo[d]thiazol-2-amine
(2) was synthesized as per the reported method (Giorgioni

Med Chem Res
N
S
iii / iv
NH
CH2CONHNH2
S
N
S
CH2COOC2H5
NH
N
S
ii
i
NH
N
O
NH2
N
N
R
5a - 5k
4
3
2
R =C6H5(15a), 2-NO2C6H4(15b), 3,5-(NO2)2C6H3(15c), 2-NH2C6H4(15d), 4-NH2C6H4(15e)
Reagents and reaction condition:
i) ClCH2COOC2H5, K2CO3, CH3COCH3, reflux 9 h,
ii) NH2NH2.H2O, C2H5OH, reflux 16 h,
2-OHC6H4(15f), 4-OHC6H4(15g), 3-C5H4N(15h), H(15i), CH3(15j), SH(15k)
iii) POCl3, RCOOH (15a - 15j), C2H5OH, reflux 14 h,
iv) CS2, KOH (15k), reflux 7h
Scheme 1 Schematic representation for the synthesis of oxadiazole derivatives
et al. 2005) by condensation of aniline with potassium
thiocyanate to obtain 1-phenyl thiourea and cyclized in the
presence of bromine. The 2-amino benzothiazole derivative
2 was condensed with ethyl 2-chloroacetate in the presence
of anhydrous potassium carbonate to yield ethyl-2-(benzo
[d]thiazol-2-yl amino) acetate (3).The nucleophilic addition
of the ester derivative 3 with hydrazine monohydrate in
ethanol gave 2-(benzo[d]thiazol-2-ylamino)acetohydrazide
(4). The hydrazide derivative 4 on condensation with
appropriate heteroaryl/aryl/aliphatic carboxylic acids in the
presence of phosphorous oxychloride to undergo dehy-
drative cyclization and to yield N-((5-substituted-1,3,4-
oxadiazol-2-yl)methyl) benzo[d]thiazol-2-amine (5a–5j).
The acyl hydrazide derivative 4 on condensation with
carbon disulphide and potassium hydroxide yielded N-
((5-mercapto-1,3,4-oxadiazol-2-yl)methyl) benzo[d]thiazol-
2-amine (5k) (Bhandari et al. 2008).
the C2 position of the benzothiazole moiety and the
methylene bridge, respectively. The mass spectrum char-
acterization of the compound 5a exhibits an ion peak at 309
m/z which can be designated as the M+1 ion peak. The ion
peak at 159 and 149 m/z can be attributed to the 2-phenyl-5-
methyl-1,3,4-oxadiazole and 2-aminomethyl benzothiazole
fragments respectively.
In the proton NMR spectrum of N-[((5-(2-amino)phenyl)
1,3,4-oxadiazol-2-yl) methyl] benzo[d]thiazol-2-amine
(5d), the signal at δ 4.34 ppm was assigned to the amino
proton of the 2-aminophenyl moiety substitution at position
C5 of the oxadiazole nucleus. The eight aromatic protons of
benzothiazole and 2-aminophenyl moiety have resonated at
δ 7.23–8.02 ppm as multiplet. The signals at δ 3.29 and
5.81 ppm can be assigned to methylene protons and amino
proton respectively. The mass spectrum characterization of
the compound 5d exhibits an ion peak at 324 m/z which can
be designated as the M+1 ion peak. The ion peak at 174 m/z
can be attributed to the 2-(2-aminophenyl)-5-methyl-1,3,4-
oxadiazole fragment.
In the proton NMR spectrum of N-[((5-(2-hydroxy)phe-
nyl) 1,3,4-oxadiazol-2-yl) methyl] benzo[d]thiazol-2-amine
(5f), the singlet at δ 10.29 ppm was assigned to the hydroxy
group proton of the 2-hydroxyphenyl moiety at position C5
of the oxadiazole. The eight aromatic protons of ben-
zothiazole and 2-hydroxyphenyl moiety have resonated at δ
7.12–7.71 ppm as multiplet. The signals at δ 3.34 and 5.69
ppm can be assigned to methylene protons and amino
proton respectively. The ion peak at 324 m/z can be desig-
nated as the M+ molecular ion peak and a major ion peaks at
175 and 149 m/z, corresponding to the 2-phenol-1,3,4-oxa-
diazole and 2-aminobenzothiazole fragments respectively.
The IR spectrum characterization of compound 5-
[(benzo[d]thiazol-2-ylamino)methyl]-1,3,4-oxadiazole-2-
thiol (5k) indicated by a shift of absorption band from 1721
to 1230 cm−1, corresponding to the carbonyl group
stretching. This can be correlated to the ring stretching of
oxadiazole moiety. In the proton NMR spectrum of com-
pound 5k, the mercapto proton has resonated as a singlet at
δ 12.83 ppm and the multiplet at δ 7.54–7.38 ppm value can
be attributed to the aromatic protons of benzothiazole
1
In the H NMR spectrum of the acyl hydrazide deriva-
tive, 2-(benzo[d]thiazol-2-yl amino) acetohydrazide (4) two
isolated singlets at δ 11.73 and 4.08 ppm can be attributed
to the NH and NH2 protons of the hydrazide function
respectively and the multiplet between δ 7.03 to 7.64 ppm
was assigned to the four protons of the benzothiazole
moiety. The singlet at δ 5.58 ppm was assigned to the amino
proton and the methylene protons between the amino group
and the carbohydrazide function resonated at δ 3.25 ppm as
a singlet.
In the proton NMR spectrum of N-((5-phenyl-1,3,4-
oxadiazol-2-yl)methyl)benzo[d]thiazol-2-amine (5a), the
signals at δ 3.33 and 5.78 ppm values can be assigned to
methylene protons and amino proton at position C2 of the
benzothiazole moiety respectively. The multiplet at δ
7.02–7.59 ppm in the proton NMR spectrum of compound
5a accounts for nine aromatic protons of benzothiazole and
phenyl moiety out of the twelve protons. The absence of
hydrazide protons at δ 11.73 and 4.08 ppm in the proton
NMR spectrum confirms for the oxadiazole ring formation.
In the 13C NMR spectrum of compound 5a, the chemical
signal δ 174.7 and 167.5 ppm were assigned to the C5 and
C2 positions of 1,3,4-oxadiazole moiety respectively. The
chemical signals at δ 154.4 and 38.4 ppm were attributed to

Med Chem Res
Table 1 The in silico molecular properties of the designed compounds
Comp % ABSa TPSAb n-ROTBb n-HBAb n-HBDb mi logPb Molecular weight n violationsb Log Sc Drug likenessc Drug scorec
5a
5b
5c
5d
5e
5f
5g
5h
5i
87
71
55
78
78
80
80
83
87
87
87
63.84
109.67
155.49
89.87
89.87
84.07
84.07
76.73
63.84
63.84
63.84
4
5
6
4
4
4
4
4
3
3
3
5
8
1
1
1
3
3
2
2
1
1
1
1
3.17
3.08
3.02
2.61
2.25
2.91
2.69
2.1
308.37
353.36
398.36
323.38
323.38
324.37
324.37
309.35
232.27
246.29
264.33
0
0
1
0
0
0
0
0
0
0
0
−4.98
−5.44 −4.66
−5.9
2.98
0.65
0.32
0.28
0.53
0.61
0.68
0.64
0.77
0.88
0.89
0.66
11
6
−7.86
0.23
1.44
2.5
−5.06
−5.06
−4.68
−4.68
−4.18
−2.83
−2.46
−3.71
6
6
6
1.49
3.19
2.45
2.42
0.17
6
5
1.25
0.79
1.77
5j
5k
a
5
5
Data calculated as per the equation 1
b
c
Molecular property as obtained from Molinspiration online property calculation toolkit
Molecular property as obtained from OSIRIS property explorer software
moiety. The methylene protons and amino proton have
resonated at δ 3.36 and 5.85 ppm respectively. All the other
newly synthesized compounds were also characterized
similarly and were found in agreement with the proposed
structures.
Molecular docking results
The molecular modelling technique was used to explore,
predict and understand the protein/enzyme interactions with
our designed 1,3,4-oxadiazole library and also to visualize
the probable binding. The docking study was performed
using the SYBYL-X 2.1. Wherein, the designed 2,5-dis-
ubstituted-1,3,4-oxadiazoles were docked into the active
site of the selected enzymes. The crystal structure of the
enzyme along with its co-crystallized ligand was down-
loaded from Brookhaven Protein Data Bank (www.rcsb.
org) and was used for docking studies. The docking results
were validated by aligning the designed ligands with the co-
crystallized reference standards for the respective enzymes.
The reference standards employed were drawn and sub-
jected for the docking protocol along with the designed
series of oxadiazoles onto to the binding site to visualize the
binding affinity and the strength of binding to ascertain the
docking process and validate the same.
In silico ADME results
All the compounds had appropriate values towards Lipinski
parameters for biological efficacy with zero to one Lipinski
violation. The in silico results indicated that the compounds
agree to the Lipinski rules of the five and theoretically, the
designed compounds were presenting a positive oral bioa-
vailability. The compounds with a percentage of absorption
(% ABS) values ranging between 55 and 87 % highlighting
a moderate to high predicted oral availability. The mole-
cular weight of the designed compounds was within the
range of 232–398 D. The lipophilicity score of the designed
compounds were encouraging as the lipophilicity data
suggested that the compounds were optimally lipophilic in
nature ranging from 0.79 to 3.17.The topological polar
surface area (TPSA) values of the designed compounds
were less than 140 Å (except compound 5c), a TPSA value
less than 140 Å indicates a good permeability of the
molecule in the plasma membrane. The designed molecules
exhibited a positive value in the drug score calculation and
the values ranged from 0.28 to 0.89. The positive drug
scores highlight that the designed compounds have potential
to be considered as a new drug candidate, and the drug
score highlights that the screened compounds have pre-
dominant pharmacophoric groups, which are often found in
the drug molecules. The ADMET results were encouraging,
and calculated % ABS, TPSA, and Lipinski parameters of
the designed compounds are presented in Table 1.
Molecular docking studies on COX-I enzyme (1CQE)
The docking study onto the active site of the enzyme COX-1
(PDB: 1CQE) of the designed 2,5-disubstituted 1,3,4-oxa-
diazoles was encouraging. The co-crystallized ligand, flur-
biprofen in the B chain of the protein showed a docking
score of 6.5498 Kcal M−1 highlighting the hydrophobic
interactions with the surrounding amino acid residues
Leu352, Trp387, Val349, Ser530 and Ala527, resulting in
its stabilization, and Vanderwall’s interaction with Tyr355,
Ser353, and Arg120 residues, and strongly contributing to
its inhibitory effect. The carboxylic acid group as carbox-
ylate ion exhibited hydrogen bond interaction with the
residue Ser353 at the distance of 2.392 Å. The docking

Med Chem Res
celecoxib exhibited a docking score of 9.6857 Kcal M−1,
exhibiting the hydrophobic interactions with the surround-
ing amino acid residues Ser339, Gly512, Leu338, and
Val509, strongly contributed to the stabilization. The
Tyr371, Trp373, and Val335 residues were involved in
Vanderwall’s interaction with the alkyl/halo alkyl chain
present in the celecoxib. The hydrogen bonding interaction
of the amino group protons of the celecoxib was with the
residues Leu338 and Gln178 at a distance of 2.109 and
2.689 Å, respectively. The sulfonyl oxygen of the ligand
celecoxib, showed hydrogen bonding interaction with
Arg499 and Phe504 residues at a distance of 2.246 and
2.495 Å, respectively.
Figure 1 Molecular docking interaction of compound 5j along with
the reference standard flurbiprofen interacting with 1CQE
The docking study was promising and the result indi-
cated that our designed 1,3,4-oxadiazole molecules exhib-
ited a considerable affinity towards 3LN1. The 1,3,4-
oxadiazoles 5a–5k exhibited docking score in the range of
4.99–7.79 Kcal M−1. Compound 5g (4-hydroxyphenyl)
exhibited a docking score of 7.79 Kcal M−1, highest among
the series and compound 5k (mercapto) exhibited the lowest
docking score (4.99 Kcal M−1). The hydrogen bond inter-
action between nitrogen at position C4 of the 1,3,4-oxa-
diazole and Ser516 residue was the major interaction
exhibited by designed1,3,4-oxadiazoles. Docking result
indicated that the compounds showed a lower crash score
for 3LN1 to that of 1CQE. Table 2 represents the molecular
docking results of designed compounds; 5a–5k along with
their crash score and polar score (Fig. 2).
The docking studies towards anti-inflammatory efficacy
highlighted that the compounds have a significant affinity to
inhibit COX-2 (3LN1) enzyme than the COX-1 enzyme,
with a higher docking score in comparison, the docking
results highlight that the compounds are exhibiting higher
selectivity towards COX-2 than COX-1. Moreover, most of
the designed oxadiazoles were exhibiting a higher crash
score for COX-1 than that of COX-2 indicating the diffi-
culties in penetration and inhibiting the COX-1 enzyme.
This higher selectivity towards COX-2 is advantageous in
preventing secondary clinical conditions like gastro-
duodenal ulcers, gastritis bleeding etc., associated with the
inhibition of COX-1.
study revealed that the designed 1,3,4-oxadiazole deriva-
tives were exhibiting a considerable affinity towards 1CQE,
as the compounds 5a–5k exhibited a promising docking
score, ranging from 2.54 to 6.61 Kcal M−1. Compound 5j
(methyl) showed a docking score of 6.6107 Kcal M−1,
highest among the series and compound 5c (3,5-dini-
trophenyl) with the lowest docking score (2.5377 Kcal
M−1). The compound 5j (methyl) exhibited a good docking
score of 6.6107 Kcal M−1, higher than that of the co-
crystallized ligand flurbiprofen indicating a better interac-
tion with the protein COX-1 and highlighting its ability to
inhibit the same and produce a significant anti-inflammatory
action (Fig. 1). The compound 5j was well aligned in the
hydrophobic core of the 1CQE. The nitrogen at position 3
of the 1,3,4-oxadiazole moiety of compound 5j showed
hydrogen bond interaction with Ser530 residue at a distance
of 2.182 Å. Compound 5j exhibited a crash score of
−0.7027 Kcal M−1, indicating the ease of penetration of the
designed ligand into the binding site of the target enzyme,
resulting in better interaction with the amino acid residues.
The hydrogen bond interaction between the nitrogen at
position 4 of the 1,3,4-oxadiazole with Ser530 residue was
the major interaction exhibited by the designed1,3,4-oxa-
diazoles. Among the series, some of the designed com-
pounds exhibited a higher crash score highlighting that the
ligand were not able to stabilize in the binding site of the
target enzyme; 1CQE, and indicating an inappropriate
penetration. Table 2 represents the molecular docking
results of designed compounds; 5a–5k along with their
crash score and polar score (Fig. 2).
Docking validation
The reference standards employed were drawn and sub-
jected to the docking protocol along with the designed
series of 1,3,4-oxadiazoles. In order to visualize the binding
affinity and strength to ascertain the docking process and
validate the same. The docking pose was compared with the
co-crystallized binding pose of flurbiprofen and celecoxib
for 1CQE and 3LN1 protein targets, respectively. The
validation of the docking process indicated by the root
mean square deviation (RMSD) value and poses for
Molecular docking studies on COX-2 enzyme (3LN1)
The docking results of our designed 1,3,4-oxadiazoles onto
the active site of the enzyme COX-2 (3LN1) was con-
siderable. The 3LN1 protein, representing a typical
cylindrical core of 23 Å between Ser516, Ser339, and
Asp510 accounting the hydrophobic interaction. The ligand,

Med Chem Res
Table 2 Molecular docking
results of the compounds 5a–5k
onto the target enzymes
Sl. No.
Docking score towards 1CQEa
Docking score towards 3LN1a
Compound
Total score Crash score Polar score Total score Crash score Polar score
1.
5a
5b
5c
5d
5e
5f
3.8538
3.9529
2.5377
6.2515
5.0101
4.2473
4.7278
5.0651
4.9779
6.6107
4.0659
−4.816
0.491
6.6834
6.7892
5.4541
7.1129
7.4284
7.2264
7.7898
7.5655
5.4017
5.9914
4.9993
−1.4289
−1.3142
−4.39
0.1711
0.8868
1.9631
1.0318
0.7364
1.2273
2.4121
0.8615
1.0396
1.0067
0.2921
2.
−4.9012
−7.3806
−3.9454
−5.7075
−5.2355
−4.7015
−3.2302
−0.8156
−0.7027
−1.7477
−0.5917
0.0444
1.1621
1.9183
0.0085
0.0046
1.0538
0.3336
1.0589
1.0664
0.8714
0.1284
3.
4.
−1.504
5.
−1.546
6.
−1.3352
−1.2447
−0.8683
−0.3331
−0.5833
−0.8089
7.
5g
5h
5i
8.
9.
10.
11.
12.
13.
a
5j
5k
Flurbiprofen 6.5498
Celecoxib
9.6857
−0.5373
2.1673
Data expressed in Kcal M−1as obtained from the docking utility of SYBYL 2.1 software
5e (4-aminophenyl), 5g (4-hydroxyphenyl), 5d (2-amino-
phenyl) and 5h (pyridin-3-yl) exhibited better radical
scavenging activity, higher than that of the reference stan-
dard ascorbic acid employed.
The structure–activity relationship that can be drawn
from radical scavenging results, highlight that compounds
with electron donating group substitution on the phenyl
moiety were exhibiting a higher radical scavenging property
than that of other tested 1,3,4-oxadiazoles. The better
radical scavenging efficacy of these derivatives; 5d, 5e, 5f,
and 5g may be attributed to the possible stable resonance
hybrid formation with loss of the proton and thus neu-
tralizing the supra-radical. The substantial antioxidant
activity of the compound 5h (pyridin-3-yl) with a radical
scavenging property of 81.0 % may be attributed to the
N-oxide formation. Whilst, 5b (2-nitrophenyl) and 5c
(3,5-dinitrophenyl) derivatives were exhibiting a radical
savaging efficacy 38.6 and 36.6 % respectively, the com-
paratively lower radical savaging property may be indicated
to the electron-withdrawing property of the nitro function.
The radical scavenging activity highlighted the importance
of electron donating group substitution at the ortho/para
position of the phenyl moiety at position C5 of the 1,3,4-
oxadizole molecule. The statistical analysis of the com-
pounds 5a–5k for its antioxidant property by repeated
measures Dunnett’s ANOVA method indicate that the tested
compounds exhibited significance value of P < 0.001
highlighting the confidence interval of 99.9 % with respect
to the reference standard ascorbic acid. The radical savaging
data of the test compounds 5a–5k at 25 µM concentrations
were statistically analyzed using the repeated measures
ANOVA with Dunnett’s test and the data are expressed as
the mean SEM in Table 3.
Figure 2 Molecular docking interaction of compound 5g along with
the reference standard celecoxib interacting with 3LN1
the co-crystalized and docked ligand were 0.95 and 0.59 Å
for flurbiprofen and celecoxib, respectively. The RSMD
values are affirmative that the overall docking study is
corroborated, as the tolerance limit for RMSD value is 1.5
Å. Hence, the docking results are acceptable and indicative
that the docking protocol is validated.
In vitro radical scavenging evaluation
The 1,3,4-oxadiazole derivatives, 5a–5k were evaluated for
their free radical scavenging activity at 25 µM strength by
DPPH radical assay method using ascorbic acid as standard.
The compounds 5a–5k exhibited significant scavenging
activity ranging from 32.0 to 87.3 % in comparison to
76.0 % antioxidant activity obtained for the reference drug,
ascorbic acid. The analysis of the compounds antioxidant
activity indicate that the compounds 5f (2-hydroxyphenyl),

Med Chem Res
In vivo anti-inflammatory activity
inflammatory screening at the end of four hours after the
administration of carrageenan were promising, the tested
compounds exhibited substantial anti-inflammatory activity
with an edema inhibition ranging from 23.55 to 82.27 % in
comparison to 48.26 % edema inhibition exhibited by the
reference standard indomethacin employed.
The newly synthesized 1,3,4-oxadiazole derivatives 5a–5k
were evaluated for in vivo anti-inflammatory activity at a
dose of 25 mg Kg−1 by carrageenan induced paw edema
method. Indomethacin at 25 mg Kg−1 was used as reference
standard and CMC as the control. The result of the anti-
A
correlation between the structure and anti-
inflammatory activity data indicates that the compounds
with aryl substitutions at position C2 of the 1,3,4-oxadiazole
moiety (5a–5h) were exhibiting higher edema inhibition
than that of 1,3,4-oxadiazole derivatives with aliphatic/thiol
substitution at C2 position (5i–5k). The compound 5h with
pyridin-3-yl substitution at position C2 of the 1,3,4-oxa-
diazole moiety was found to be the most potent, with edema
inhibition value of 82.27 % in comparison to the other
tested 1,3,4-oxadiazole derivatives with edema inhibition
value ranging from 23.55 to 47.67 % and higher than that of
the reference standard indomethacin employed (48.26 %).
While compound 5j (methyl) exhibited the least anti-
inflammatory efficacy with an edema inhibition value of
only 23.55 %. Interestingly compounds with nitro sub-
stitution, compound 5b (2-nitro phenyl) and 5c (3,5-dinitro
phenyl) were exhibiting substantial anti-inflammatory effi-
cacy with edema inhibition of 44.19 and 47.67 % respec-
tively, similar to that of compound 5a and 5d–5g (phenyl/
phenyl substituted derivatives), contradicting to their radical
scavenging efficacy. The percentage edema inhibition
values of tested compounds 5a–5k at 25 mg Kg−1 strength
at the end of four hours and the mean difference in paw
Table 3 The radical scavenging efficacy of the compounds 5a–5k
Sl No. Compounds
Percentage free radical scavenging activitya,b
Trial 1 Trial 2 Trial 3 Mean SEM
1.
5a
5b
5c
5d
5e
5f
55.19
36.68
38.71
83.63
86.00
87.36
85.78
81.04
35.33
32.05
43.79
54.47
35.67
37.73
83.37
85.78
87.16
85.55
80.73
34.29
30.96
42.89
75.69
55.74
37.46
39.46
83.84
86.18
87.51
85.95
81.27
36.12
32.89
44.48
76.37
55.13 0.37***
36.60 0.52***
38.63 0.50***
83.61 0.14***
85.99 0.12***
87.34 0.10***
85.76 0.12***
81.01 0.16***
35.25 0.53***
31.97 0.56***
43.72 0.46***
76.04 0.20
2.
3.
4.
5.
6.
7.
5g
5h
5i
8.
9.
10.
11.
12.
a
5j
5k
Ascorbic acid 76.07
Results are expressed as the mean values from three parallel
experiments SEM
b
Data was analyzed by Dunnet’s test compared with reference drug
ascorbic acid. n = 3; (***) equals P < 0.001
Table 4 The percentage edema
inhibition values of tested
compounds 5a–5k
Sl no. Compounds Mean difference in paw volume SEMa,b
Edema
inhibition
(%)
0.5 h
1.0 h
2.0 h
4.0 h
1.
Control
5a
0.35 0.01 0.59 0.02
0.66 0.02
0.57 0.03
2.
0.35 0.02 0.52 0.02*** 0.53 0.02***†† 0.35 0.02***
39.83
44.19
47.67
42.73
43.31
39.24
38.37
3.
5b
5c
0.33 0.01 0.48 0.01*** 0.48 0.01***
0.33 0.01 0.44 0.01*** 0.45 0.00***
0.35 0.00 0.48 0.00*** 0.49 0.01***
0.34 0.01 0.48 0.01*** 0.48 0.01***
0.32 0.02***
0.30 0.01***
0.33 0.01***
0.33 0.01***
4.
5.
5d
5e
6.
7.
5f
0.35 0.01 0.48 0.01*** 0.52 0.01***† 0.35 0.02***
0.35 0.01 0.48 0.01*** 0.50 0.02*** 0.35 0.01***
8.
5g
9.
5h
5i
0.31 0.02 0.43 0.01***† 0.40 0.01***†† 0.10 0.01***††† 82.27
0.34 0.01 0.52 0.01*** 0.51 0.01*** 0.42 0.01***††† 26.45
0.35 0.01 0.53 0.01**† 0.53 0.01***†† 0.44 0.01***††† 23.55
10.
11.
12.
13.
5j
5k
0.34 0.01 0.49 0.01*** 0.47 0.01***
0.35 0.01***
0.30 0.01***
38.37
48.26
Indomethacin 0.31 0.01 0.48 0.01*** 0.46 0.01***
a Data was analyzed by Dunnet’s test compared with the negative control. n = 3; (***) equals P < 0.001, (**)
equals P < 0.01, (*) equals P < 0.05
b
Data was analyzed by Dunnet’s test compared with reference drug indomethacin n = 3; (†††) equals P <
0.001, (††) equals P < 0.01, (†) equals P < 0.05

Med Chem Res
Table 5 The mean percentage values of compound 5h for in vitro COX-1 and COX-2 enzymes inhibition at the three different test strength along
with its IC50 values
Compound Mean percentage COX-1
inhibition SEMa
Mean IC50
value SEM
Mean percentage COX-2
inhibition SEMa
Mean IC50
value SEM
6.25 µM
12.50 µM
25.00 µM
6.25 µM
12.50 µM
25.00 µM
5h
10.19 0.41 42.32 0.34 64.50 0.44 16.73 0.11
45.09 0.48 68.77 0.51 86.64 0.38 7.19 0.10
a
Results are expressed as the mean values from three parallel experiments SEM
volume SEM at regular time intervals are presented in
Table 4.
In vitro COX inhibition assay
While, compound 5d (2-amino phenyl), 5e (4-amino
phenyl), 5f (2-hydroxy phenyl) and 5g (4-hydroxy phenyl)
substitution which were exhibiting significant radical
scavenging property exhibited moderate anti-inflammatory
activity with edema inhibition value ranging from 38.37 to
43.31 % similar to compound 5a (2-phenyl derivative) with
edema inhibition value of 39.83 %, indicating that the
amino and hydroxyl functional group substitution at the
ortho/para position is not considerably contributing toward
the anti-inflammatory efficacy.
The statistical analysis of the edema inhibition data at the
end of 30 min highlighted that the compounds 5a–5k did
not exhibit a significant reduction in mean edema volume
with respect to both control and reference drug indo-
methacin. At the end of 1 h, compound 5a–5i, 5k, and
indomethacin exhibited a significance value of P < 0.001
highlighting the confidence interval of 99.9 % and com-
pound 5j (methyl) exhibited a significance value of P < 0.01
indicating the confidence interval of only 90.0 % with
respect to control. Compound 5h (pyridin-3-yl) exhibited a
significance value of P < 0.05 highlighting the confidence
interval of only 95.0 % with respect to reference standard
indomethacin. All the 1,3,4-oxadiazoles 5a–5k and
indomethacin exhibited a significance value of P < 0.001
highlighting the confidence interval of 99.9 % with respect
to control at the end of the second and fourth hour,
respectively.
It is most obvious that carrageenan induced edema in
animal models involves expressing COX-2, wherein COX-2
mRNA and COX-2 protein are induced (Willoughby et al.
2001). Literature study on anti-inflammatory activity of
compound 7-hydroxy-3,4-dihydrocadalin (HDC) evaluated
for its in vitro COX-1, COX-2 inhibition assay and corre-
lation to its in vivo carrageenan-induced edema inhibition
screening highlights that the compound HDC inhibited
COX-1 to a higher degree than COX-2, and the carrageenan
induced edema in rat paw was impervious (Segura et al.
2000). In view of the same and affirmative about our
compounds to inhibit COX-2 enzyme, the compound 5h
potent among the series was also evaluated for in vitro COX
inhibition towards its selectivity and potential to inhibit the
target proteins COX-1 and COX-2.
The results of the in vitro COX enzymes inhibition were
encouraging and was determined using a COX inhibitor
screening assay kit. The test compound 5h exhibited a
substantial inhibition of COX-2 and a moderate inhibition
of COX-1. The result highlighted the ability of our test
compound 5h to inhibit the enzyme COX-2 from catalyzing
the conversion of arachidonic acid to PGH2. The enzyme
inhibition data indicated that compound 5h exhibited a
higher IC50 value for COX-1 than to that of COX-2. The
study also indicated that the compound 5h exhibited a
concentration dependent inhibition of the target enzyme.
Compound 5h at the lowest test concentration (6.25 µM)
exhibited a mean enzyme inhibition values of 10.2 and
45.1 % for COX-1 and COX-2 respectively. Signifying that
compound 5h has significant affinity to inhibit COX-2 than
that of COX-1. The in vitro COX inhibition activity of the
test compound 5h for both COX-1 and COX-2 at the three
different test concentrations by enzymes immunoassay
(EIA) are expressed as the mean
SEM, along with the
IC50 values obtained by regression analysis are presented in
Table 5.
In vivo acute ulcerogenic activity
Acute ulcerogenicity of the test compound 5h was eval-
uated after a single dose oral administration to 24 h fasting
rats. The results indicated that the compound 5h to be
moderately safe at the tested strength of 50 mg Kg−1 with a
score of 1+ indicting the compound to produce redness
(hyperemia) in 66.7 % of the tested animals (n = 6).
Conclusions
A new class of oxadiazole derivatives were designed, effi-
ciently synthesized, characterized and evaluated for radical
scavenging property and anti-inflammatory efficacy. The
result of the study highlights, that the designed compounds
have optimal physicochemical parameters to be considered
as the potential candidate for drug development with opti-
mal values towards the ADMET screening. The molecular
docking results were encouraging as the compounds

Med Chem Res
exhibited substantial score towards enzyme inhibition. The
compounds exhibited a higher affinity to inhibit COX-2
enzyme than COX-1, which is beneficial in minimizing
nephrotoxicity and gastrointestinal damages. The radical
scavenging evaluation was considerable, as the tested
compounds exhibited significant scavenging property, and
compounds 5d, 5e, 5f, 5g, and 5h showed >80 % anti-
oxidant activity with respect to the reference standard
ascorbic acid employed. All the tested oxadiazoles showed
promising anti-inflammatory activity. The mean edema
inhibition data indicated that the compounds with aryl
substitution are significantly active. Compound 5h has
exhibited a significantly higher edema inhibition in com-
parison to the reference standard indomethacin employed.
The in vitro COX enzyme inhibition assay of compound 5h
was considerable. Compound 5h exhibited a higher affinity
to inhibit COX-2 than that of COX-1. The acute ulcerogenic
study indicated that compound 5h to be moderately safe.
The newly synthesized compound 5h may be a possible hit
as therapeutic agents. It can be concluded that this class of
compounds certainly holds great promise towards good
active leads in medicinal chemistry.
3D using the ligand preparation utility of SYBYL-X 2.1
application and by applying molecular mechanics force
fields (MMFF94s), the ligands were energy minimized and
charged using Gasteiger-Marsili method. The docking
simulation highlights the basic receptor-ligand interactions
and the strength of the ligand binding onto to the core. The
docking of the optimized 3D-structures of the molecules
was performed within 10 Å radius to appreciate and to
optimize the most probable binding pose of each ligand. A
series of hierarchical searches is performed by the docking
algorithm to realize the possible ligand affinity within the
binding site of the enzyme core. The docked poses of
geometrically optimized ligands are ranked with total
scores, highlighting the affinity of the ligand towards the
enzyme core, and also visualizing the most optimal binding
pose of each ligand. The essential parameters in terms of
hydrogen bonds and hydrophobic interactions, which in-
turn governs binding disparities for the each ligand in the
series is understood inthe form of score functions, the score
of the docked molecules indicate the affinity of the ligands
towards the enzyme core.
In order to visualize the importance of possible anti-
inflammatory efficacy of the molecules, the compounds
were docked onto COX-1(PDB ID: 1CQE) and COX-2
(PDB ID: 3LN1). Crystallographic data of the 1CQE and
3LN1 were used for docking studies since the data on these
have been well established and reported stimulation studies
to understand the anti-inflammatory efficacy (Eleftheriou
et al. 2012). The protein structure of 1CQE and 3LN1 is
well recognized with both hydrophobic active site and the
proteins were determined at 3.1 and 2.4 Å resolution,
respectively. The bound conformations of co-crystallized
ligands, flurbiprofen and celecoxib were used as controls in
order to define the active site in COX-1 and COX-2,
respectively. The compounds were docked along with the
co-crystallized ligands, celecoxib for COX-2 enzyme and
flurbiprofen in the case of a COX-1 enzyme, respectively
and the interactions like hydrogen bonding were visualized.
Material and methods
Molecular docking studies
The SYBYL-X 2.1 docking program was employed in the
molecular docking studies. The docking algorithm was to
achieve imminent information regarding the binding mode
and affinity of the designed compounds and explicate the
impact of structural differences in the selected protein
towards their anti-inflammatory activity. Crystallographic
data of the selected enzyme with its co-crystallized ligand
were downloaded from Brookhaven Protein Data Bank
(www.rcsb.org) and employed in the docking studies. The
x-ray crystal structure of protein enzymes was defined and
established; the hydrophobic active site and hydrogen bond
interaction amino acid residuals were distinctly understood.
Using the protein preparation module in SYBYL-X 2.1
program, the target protein was prepared, and bond orders
were assigned, water and other residues were removed,
hydrogen atoms were added and the protein model was
charged with AMBER7 FF99. The designed oxadiazoles
and the reference standards; flurbiprofen (flurbiprofen
available in the market as a COX-1 inhibitor) and celecoxib
(celecoxib available in the market as a COX-2 inhibitor) as
ligands were drawn in ChemDraw and converted into *.sdf
format. The reference standards; flurbiprofen and celecoxib
were also docked along with oxadiazole molecules to
validate the docking protocol for the COX-1 and COX-2
enzymes, respectively. The 2D molecules were converted to
In silico ADME studies
Poor ADME properties are one of the primary reasons for
rejection of drugs candidates in clinical trials, highlighting
the importance of screening for drug likeliness and ADME
properties of a molecule towards drug development. Oral
bioavailability plays a considerable role in the development
of bioactive compounds as medicinal agents, which is
essential for rational drug design. According to Lipinski
rule of five, molecules that breach more than one of Lipinski
rules may have complications with bioavailability. The
Lipinski rule of five establishes some structural parameters
in relevance to the oral bioavailability profile of the mole-
cules and is extensively adapted in designing compounds

Med Chem Res
(Zhao et al. 2002). The in silico studies were performed for
Lipinski parameters, as well as for the TPSA, % ABS and
the drug-likeness and drug score were included for the
designed 1,3,4-oxadiazoles in order to verify that these
compounds exhibit an optimal theoretical oral bioavail-
ability. The drug-likeness and drug score were calculated
using the OSIRIS property explorer software and the lipo-
philicity and TPSA were calculated using Molinspiration
uncorrected. The IR spectra were recorded using Shimadzu
FTIR-8400 instrument by KBr disc pellet technique/liquid
sampling using NaCl cells and only noteworthy absorption
1
levels (cm−1) are listed. H NMR and 13C NMR spectra
were recorded using Bruker AC-400 MHz FT NMR spec-
1
trophotometer at 400 MHz H (100 MHz 13C) with deu-
teriated dimethyl sulfoxide (DMSO-d6) as solvent and tetra
methyl silane (TMS) as internal standard (chemical shifts in
δ, ppm). The splitting patterns were designated as follows:
s: singlet; d: doublet; q: quartet; m: multiplet. The LCMS
were recorded using Shimadzu LCMS-2010A instrument
by ESI. The elementary analysis was recorded using
Thermo Finnigan FLASH EA 1112 CHNS analyzer and all
the compounds gave satisfactory elemental analysis.
online
property
calculation
toolkit
(http://www.
molinspiration.com). TPSA was also calculated using
Molinspiration online property calculation toolkit according
to the fragment-based method reported. Polar surface area,
together with lipophilicity is an important property of a
molecule which indicates the ease of transport across bio-
logical membranes. A high TPSA value indicates poor
absorption of a drug and, in turn, highlights low bioavail-
ability. The % ABS was estimated according to the Eq. 1.
Synthesis of benzo[d]thiazol-2-amine (2)
% ABS ¼ 109 ꢀ ð0:345 ´ TPSAÞ
Eq. 1: Calculation of percentage of absorption
ð1Þ
The mixture of aniline (25 mL, 0.25 M) and potassium
thiocyanate (25 g, 0.25 M) in glacial acetic acid (50 mL)
was cooled to a temperature less than 10 °C. To this solu-
tion bromine (40 mL, 0.25 M) was added dropwise with
stirring while maintaining the temperature less than 10 °C
throughout the addition. The mixture was further stirred for
an additional 3 h, and the separated hydrochloride salt was
filtered, washed with acetic acid and dried. Later, the salt
was dissolved in hot water and neutralized with ammonia
solution to precipitate the free amine derivative 2. The
product was filtered, washed with water and dried, recrys-
tallized using rectified spirit to obtain solid product 2.Yield:
87 %; M.P: 125–127 °C; Rf: 0.54; IR (KBr): ṽ3467 (NH2),
3059 (C=C), 1599 (C=N), 736 (C-S-C) cm−1; 1HNMR
(400 MHz, DMSO-d6): δ 6. 9–7.5 (m, 4H, Ar–H), 5.5 (s,
2H; NH2) ppm.
Experimental section
Molecular docking simulations were performed using
SYBYL-X 2.1, chemical structures and synthetic scheme
was drawn using ChemDraw and statistical analyses were
performed using GraphPad Prism 5.0 on the windows-7
professional operating system. The synthetic routes for the
preparation of target molecules were planned through ret-
rosynthetic approach. Most facile synthetic routes were
selected based on the percentage yield of molecules of each
reaction step. Percentage yield of some reaction steps was
improved by careful selection of starting materials and
catalyst system. While designing the synthetic routes, at
most priority was given for high yielding reproducible
reaction steps rather than concentrating on novel synthetic
routes and complex chemistry. All the reaction steps even if
reported in published literature were selected based on our
in-house laboratory facilities. Handling and disposal of all
reagents and catalysts to be used in the reaction steps were
studied thoroughly from the corresponding Material Safety
Data Sheets (MSDS).
All the chemicals and solvents used in this study were
purchased from Sigma-Aldrich Chemical Co., Spectrochem
Ltd., and Sd fine chemicals of LR grade, the solvents were
purified by distillation prior to use. All commercially
available reagents procured were used without further pur-
ification. TLC was used to assess the progress/completion
of reactions and the purity of the synthesized compounds
using aluminum backed sheets of silica gel 60 GF254
(Merck), ethyl acetate and hexane (4:1) as a solvent system
and iodine vapors as the visualizing agent. Melting points
were determined in open glass capillaries and are
Synthesis of ethyl-2-(benzo[d]thiazol-2-yl amino)
acetate (3)
A mixture of amine derivative 2 (3 g, 0.02 M), ethyl 2-
chloroacetate (6 mL, 0.05 M) and activated anhydrous
potassium carbonate (4 g) in dry acetone (30 mL) were
heated to reflux for 9 h. The progress of the reaction was
monitored by TLC. On completion, the mixture was filtered
and the filtrate was concentrated and poured into the cru-
shed ice to obtain the solid ester derivative 3. The product
was recrystallized using rectified spirit as the solvent.Yield:
69 %; M.P: 136–138 °C; Rf: 0.62 (ethyl acetate and hexane,
4:1); IR (KBr): ṽ3454 (NH2), 3030 (C=C), 1737 (C=O),
1633 (C=N), 760 (C–S–C)cm−1; 1HNMR (400 MHz,
DMSO-d6): δ 7.2–7.6 (m, 4H, Ar–H), 5.6 (s, 1H, NH), 4.2
(q, 2H, COOCH2), 3.4 (d, 2H, CH2COO), 1.3 (t, 3H, CH3)
ppm.

Med Chem Res
1
Synthesis of 2-(benzo[d]thiazol-2-yl amino)
acetohydrazide (4)
(C=N), 1264 (C–O–C), 1526 (NO, NO2) cm−1; HNMR
(400 MHz, DMSO-d6, ppm): δ 7.2–6.8 (m, 8H, Ar–H), 5.8
(s, 1H,NH), 3.4 (s, 2H,CH2); 13CNMR (400 MHz, DMSO-
d6, ppm): δ 168.3 (1C, C5-oxadizole), 162.5 (1C, C2-oxa-
dizole), 151.3 (1C, C2-benzothiazole), 148.4 (1C, C2-
nitrophenyl), 146.4 (1C, C9-benzothiazole), 142.5 (1C,
C1-nitrophenyl), 139.1 (1C, C8-benzothiazole), 133.2
(1C, C4-nitrophenyl), 132.1 (1C, C5-benzothiazole), 129.3
(1C, C6-benzothiazole), 128.6 (1C, C7-benzothiazole),
127.1 (1C, C4-benzothiazole), 126.4 (1C, C6-phenyl),
124.2 (1C, C3-phenyl), 121.9 (1C, C5-phenyl), 38.3 (1C,
NHCH2); MS m/z (%): 354 [M+1] (80); anal. calcd. for
C16H11N5O3S: C, 54.38; H, 3.14; N, 19.82; S, 9.07. Found:
C, 54.39; H, 3.13; N, 19.81; S, 9.11.
The mixture of ester derivative 3 (5 g, 0.02 M) and hydra-
zine monohydrate (10 mL, 0.2 M) in ethanol (30 mL) were
heated to reflux for 16 h. The progress of the reaction was
monitored by TLC. On completion, the mixture was
allowed to cool in refrigerator overnight to precipitate the
acyl hydrazide derivative 3. The product was recrystallized
using rectified spirit as the solvent. Yield: 56 %; M.P:
189–191 °C; Rf: 0.74(ethyl acetate and hexane, 4:1); IR
(KBr): ṽ3398, 3450 (NH2, NH), 3045 (C=C), 1721 (C=O),
1627 (C=N), 752 (C–S–C) cm−1; 1HNMR (400 MHz,
DMSO-d6): δ 11.7 (s, 1H, CONH), 7.6–7.0 (m, 4H, Ar–H),
5.6 (s, 1H, NH), 4.1 (s, 2H, NH2), 3.3 (s, 2H, CH2) ppm.
N-[(5-(3,5-dinitro phenyl) 1,3,4-oxadiazol-2-yl) methyl]
benzo[d]thiazol-2-amine (5c)
General procedure for the synthesis of N-((5-substituted-
1,3,4-oxadiazol-2-yl)methyl) benzo[d]thiazol-2-amine
(5a–5j)
Yield: 81 %; M.P: 255–256 °C; Rf: 0.68 (ethyl acetate and
hexane; 4:1); IR (KBr): ṽ3253 (NH), 3019 (C=C), 1663
(C=N), 1256 (C–O–C), 1519, 1522 (NO, NO2) cm−1;
1HNMR (400 MHz, DMSO-d6, ppm): δ 7.6–6.4 (m, 7H,
Ar–H), 5.8 (s, 1H, NH), 3.4 (s, 2H, CH2); 13CNMR (400
MHz, DMSO-d6, ppm): δ 172.2 (1C, C5-oxadizole), 165.3
(1C, C2-oxadizole), 155.3 (1C, C2-benzothiazole),148.7
(2C, C3,5-dinitrophenyl), 146.3 (1C, C9-benzothiazole),
139.4 (1C, C1-dinitrophenyl), 131.3 (1C, C4-dini-
trophenyl), 128.9 (1C, C8-benzothiazole), 128.3 (1C, C5-
benzothiazole), 127.1 (1C, C6-benzothiazole), 124.7 (1C,
C7-benzothiazole), 123.2 (1C, C4-benzothiazole), 121.8
(2C, C2,6-dinitrophenyl), 39.1 (1C, NHCH2); MS m/z (%):
398 [M+] (84); anal. calcd. for C16H10N6O5S: C, 48.24; H,
2.49; N, 21.10; S, 8.05. Found: C, 48.18; H, 2.49; N, 21.11;
S, 8.11.
To the solution of acyl hydrazide 4(2.2 g, 0.01 M) and car-
boxylic acid (0.01 M) in ethanol (25 mL), phosphorous
oxychloride (0.01 M) was added slowly in an exhaustion
chamber and heated to reflux for 8–14 h. The progress of the
reaction was monitored by TLC. On completion, the reaction
mixture was cooled to room temperature and crushed ice was
added to obtain oxadiazole derivative (5a–5j). The product
was recrystallized using rectified spirit as the solvent.
N-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)benzo[d]thiazol-
2-amine (5a)
Yield: 68 %; M.P: 215–216 °C; Rf: 0.60 (ethyl acetate and
hexane, 4:1); IR (KBr): ṽ3242 (NH), 3026 (C=C), 1662
1
(C=N), 1271 (C–O–C) cm−1; HNMR (400 MHz, DMSO-
d6, ppm): δ7.6–7.0 (m, 9H, Ar–H), 5.8 (s, 1H, NH), 3.3 (s,
2H, CH2); 13CNMR (400 MHz, DMSO-d6, ppm): δ 174.7
(1C, C5-oxadizole), 167.5 (1C, C2-oxadizole), 154.4 (1C,
C2-benzothiazole), 133.8 (1C, C9-benzothiazole), 131.7
(1C, C1-phenyl), 130.2 (1C, C8-benzothiazole), 130.1 (1C,
C5-benzothiazole), 129.3 (1C, C6-benzothiazole), 128.6
(1C, C4-phenyl), 128.5 (1C, C7-benzothiazole), 127.1 (1C,
C4-benzothiazole), 122.5 (1C, C2-phenyl), 121.5 (1C, C6-
phenyl), 120.1 (1C, C5-phenyl), 119.6 (1C, C3-phenyl),
38.4 (1C, NHCH2); MS m/z (%): 309 [M+1] (85); anal.
calcd. for C16H12N4OS: C, 62.32; H, 3.92; N, 18.17; S,
10.40. Found: C, 62.24; H, 3.89; N, 18.19; S, 10.22.
N-[(5-(2-amino phenyl) 1,3,4-oxadiazol-2-yl) methyl] benzo
[d]thiazol-2-amine (5d)
Yield: 62 %; M.P: 187–189 °C; Rf: 0.85 (ethyl acetate and
hexane; 4:1); IR (KBr): ṽ3318, 3251 (NH, NH2), 3024
1
(C=C), 1659 (C=N), 1276 (C–O–C) cm−1; HNMR (400
MHz, DMSO-d6, ppm): δ 8.0–7.2 (m, 8H, Ar–H), 5.8 (s,
1H, NH), 4.3 (s, 2H, NH2), 3.3 (s,2H, CH2); 13CNMR (400
MHz, DMSO-d6, ppm): δ 171.3 (1C, C5-oxadizole), 164.3
(1C, C2-oxadizole), 154.1 (1C, C2-benzothiazole),147.6
(1C, C2-aminophenyl), 145.9 (1C, C9-benzothiazole),
144.5 (1C, C1-aminophenyl), 139.1 (1C, C4-aminophenyl),
137.8 (1C, C8-benzothiazole), 134.1 (1C, C5-benzothia-
zole), 131.8 (1C, C6-benzothiazole), 128.7 (1C, C7-
benzothiazole), 127.1 (1C, C4-benzothiazole), 124.4 (1C,
C6-aminophenyl), 123.2 (1C, C3-aminophenyl), 119.8
(1C, C5-aminophenyl), 38.7 (1C, NHCH2); MS m/z (%):
324 [M+1] (70); anal. calcd. for C16H13N5OS: C, 59.43; H,
N-[(5-(2-nitro phenyl) 1,3,4-oxadiazol-2-yl) methyl] benzo
[d]thiazol-2-amine (5b)
Yield: 74 %; M.P: 237–239 °C; Rf: 0.72 (ethyl acetate and
hexane, 4:1); IR (KBr): ṽ3263 (NH), 3016 (C=C), 1653

Med Chem Res
4.05; N, 21.66; S, 9.92. Found: C, 59.41; H, 4.01; N, 21.67;
S 9.97.
C5-oxadizole), 166.2 (1C, C2-oxadizole), 157.1 (1C,
C2-benzothiazole), 149.4 (1C, C4-phenol), 147.8 (1C, C9-
benzothiazole), 142.7 (1C, C1-phenol), 139.4 (1C,
C8-benzothiazole), 134.7 (1C, C5-benzothiazole), 133.1
(1C, C6-benzothiazole), 129.6 (1C, C7-benzothiazole),
127.9 (1C, C4-benzothiazole), 126.4 (1C, C2-phenol),
124.2 (1C, C6-phenol), 123.8 (1C, C3-phenol), 123.3 (1C,
C5-phenol), 37.4 (1C, NHCH2); MS m/z (%): 324 [M+]
(60); anal. calcd. for C16H12N4O2S: C,59.25; H, 3.73; N,
17.27; S, 9.89.Found:C, 59.31; H, 3.79; N, 17.32; S, 9.94.
N-[(5-(4-amino phenyl) 1,3,4-oxadiazol-2-yl) methyl] benzo
[d]thiazol-2-amine (5e)
Yield: 68 %; M.P: 178–179 °C; Rf: 0.78(ethyl acetate and
hexane; 4:1); IR(KBr): ṽ3324, 3249 (NH, NH2), 3021
1
(C=C), 1612 (C=N), 1273 (C–O–C) cm−1; HNMR (400
MHz, DMSO-d6, ppm): δ 8.9–7.8 (m, 8H, Ar-H), 5.7 (s,
1H, NH), 4.7 (s, 2H, NH2), 3.8 (s, 2H; CH2); 13CNMR
(400 MHz, DMSO-d6, ppm): δ 167.2 (1C, C5-oxadizole),
164.1 (1C, C2-oxadizole), 154.8 (1C, C2-benzothiazole),
149.1 (1C, C4-aminophenyl), 147.2 (1C, C9-benzothia-
zole), 139.6 (1C, C1-aminophenyl), 138.5 (1C, C8-ben-
zothiazole), 133.7 (1C, C5-benzothiazole), 131.2 (1C, C6-
benzothiazole), 128.4 (1C, C7-benzothiazole), 127.3 (1C,
C4-benzothiazole), 124.2 (1C, C2-aminophenyl), 121.8
(1C, C6-aminophenyl), 121.7 (1C, C3-aminophenyl), 119.3
(1C, C5-aminophenyl), 38.3 (1C, NHCH2); MS m/z (%):
323 [M+] (40); anal. calcd. for C16H13N5OS: C, 59.43; H,
4.05; N, 21.66; S, 9.92. Found: C, 59.46; H, 4.07; N, 21.71;
S 10.07.
N-((5-(pyridin-3-yl)-1,3,4-oxadiazol-2-yl)methyl)benzo[d]
thiazol-2-amine (5h)
Yield: 69 %; M.P: 246–247 °C; Rf: 0.72 (ethyl acetate and
hexane; 4:1); IR(KBr): ṽ3245, 3215 (NH), 3023 (C=C),
1663 (C=N), 1278 (C–O–C) cm−1; 1HNMR (400 MHz,
DMSO-d6, ppm): δ 7.8–7.4 (m, 8H, Ar–H), 5.7 (s, 1H, NH),
3.3 (s, 2H, CH2); 13CNMR (400 MHz, DMSO-d6, ppm): δ
169.1(1C, C5-oxadizole), 164.6 (1C, C2-oxadizole), 147.5
(1C, C2-benzothiazole), 147.3 (1C, C6-pyridin-3-yl), 146.9
(1C, C4-pyridin-3-yl), 143.6 (1C, C1-pyridin-3-yl), 141.4
(1C, C9-benzothiazole), 138.3 (1C, C8-benzothiazole),
133.5 (1C, C5-benzothiazole), 131.3 (1C, C6-benzothia-
zole), 124.1 (1C, C7-benzothiazole), 123.7 (1C, C2-pyridin-
3-yl), 123.4 (1C, C3-pyridin-3-yl), 121.1 (1C, C4-ben-
zothiazole), 40.4 (1C, NHCH2); MS m/z (%): 309 [M+]
(100); anal. calcd. for C15H11N5OS: C, 58.24; H, 3.58; N,
22.64; S, 10.37. Found:C, 58.32; H, 3.56; N, 22.61; S, 10.22.
N-[((5-(2-hydroxy)phenyl) 1,3,4-oxadiazol-2-yl) methyl]
benzo[d]thiazol-2-amine (5f)
Yield: 47 %; M.P: 213–214 °C; Rf: 0.65(ethyl acetate and
hexane; 4:1); IR (KBr): ṽ3321 (OH), 3245 (NH), 3023
(C=C), 1663 (C=N), 1334 (C–O) 1278 (C–O–C)cm−1;
1HNMR (400 MHz, DMSO-d6, ppm): δ 10.3 (s, 1H, OH),
7.7–7.1 (m, 8H, Ar-H), 5.7 (s, 1H, NH), 3.3 (s, 2H, CH2);
13CNMR (400 MHz, DMSO-d6, ppm): δ 169.1 (1C, C5-
oxadizole), 164.6 (1C, C2-oxadizole), 156.3 (1C, C2-ben-
zothiazole), 148.9 (1C, C2-phenol), 147.1 (1C, C9-ben-
zothiazole), 141.6 (1C, C1-phenol), 138.3(1C, C4-phenol),
134.1(1C, C8-benzothiazole), 131.6 (1C, C5-benzothia-
zole), 127.3 (1C, C6-benzothiazole), 126.8 (1C, C7-ben-
zothiazole), 124.9 (1C, C4-benzothiazole), 124.3 (1C, C6-
phenol), 123.8 (1C, C3-phenol), 119.5 (1C, C5-phenol),
38.2 (1C, NHCH2); MS m/z (%): 324 [M+] (100); anal.
calcd. for C16H12N4O2S: C, 59.25; H, 3.73; N, 17.27; S,
9.89. Found: C, 59.23; H, 3.71; N, 17.22; S, 9.76.
N-[(1,3,4-oxadiazol-2-yl) methyl] benzo[d]thiazol-2-amine
(5i)
Yield: 94 %; M.P: 204–206 °C; Rf: 0.65(ethyl acetate and
hexane; 4:1); IR(KBr): ṽ3248 (NH), 3028 (C=C), 1661
1
(C=N), 1274 (C–O–C) cm−1; HNMR (400 MHz, DMSO-
d6, ppm): δ 8.6 (s, 1H, C5-oxadiazole),7.9–7.4 (m, 4H,
Ar–H), 5.6 (s, 1H, NH), 3.2 (s, 2H, CH2); 13CNMR (400
MHz, DMSO-d6, ppm): δ 167.6(1C, C2-oxadizole), 163.6
(1C, C5-oxadizole), 148.7 (1C, C2-benzothiazole), 147.3
(1C, C9-benzothiazole), 146.9 (1C, C8-benzothiazole),
139.7 (1C, C5-benzothiazole), 133.4 (1C, C6-benzothia-
zole), 131.3 (1C, C7-benzothiazole), 124.1 (1C, C4-ben-
zothiazole), 39.3 (1C, NHCH2); MS m/z (%): 233 [M+1
]
N-[((5-(4-hydroxy)phenyl) 1,3,4-oxadiazol-2-yl) methyl]
benzo[d]thiazol-2-amine (5g)
(100); anal. calcd. for C10H8N4OS: C, 51.71; H, 3.47;
N, 24.12; S, 13.81. Found: C, 51.72; H, 3.49; N, 24.15;
S, 13.86.
Yield: 53 %; M.P: 192–193 °C; Rf: 0.75 (ethyl acetate and
hexane, 4:1); IR (KBr): ṽ3343 (OH), 3239 (NH), 3027
(C=C), 1661 (C=N), 1331 (C–O) 1272 (C–O–C) cm−1;
1HNMR (400 MHz, DMSO-d6, ppm): δ 10.6 (s, 1H, OH),
8.6–7.7 (m, 8H, Ar–H), 5.8 (s, 1H, NH), 3.3 (s, 2H, CH2);
13CNMR (400 MHz, DMSO-d6, ppm): δ 171.4 (1C,
N-[((5-methyl) 1,3,4-oxadiazol-2-yl) methyl] benzo[d]
thiazol-2-amine (5j)
Yield: 87 %; M.P: 213–214 °C; Rf: 0.76(ethyl acetate and
hexane; 4:1); IR(KBr): ṽ3245 (NH), 3023 (C=C), 1663

Med Chem Res
1
(C=N), 1278 (C–O–C)cm−1; HNMR (400 MHz, DMSO-
d6, ppm): δ 8.8–7.8 (m, 4H), 5.8 (s, 1H, NH), 3.3 (s, 2H,
CH2), 1.2 (s, 3H, CH3); 13CNMR (400 MHz, DMSO-d6,
ppm): δ 168.4 (1C, C2-oxadizole), 164.2 (1C, C5-oxadi-
zole), 147.5 (1C, C2-benzothiazole), 146.8 (1C, C9-ben-
zothiazole), 145.4 (1C, C8-benzothiazole), 139.1 (1C, C5-
benzothiazole), 134.2 (1C, C6-benzothiazole), 133.6 (1C,
C7-benzothiazole), 124.6 (1C, C4-benzothiazole), 40.1 (1C,
NHCH2), 12.6 (1C, CH3); MS m/z (%): 246 [M+] (100);
anal. calcd. for C11H10N4OS: C, 53.64; H, 4.09; N, 22.75;
S, 13.02. Found: C, 53.62; H, 4.06; N, 22.76; S, 12.87.
percent free radical scavenging activity.
% Radical Scavenging ¼
Control absorbance ꢀ Test absorbance
´ 100
ð2Þ
Eq. 2: Calculation of percentage radical scavenging
Control absorbance
activity
In vivo anti-inflammatory activity
All the animal experiments adapted in the study were
reviewed and approved by the Institutional Animal Ethics
Committee (Ref No. 059/2010). The experimental proce-
dures and protocols were in accordance with the guidelines
of the CPCSEA, Ministry of Forests and Environment,
Govt. of India. The animals were obtained from the JSS
Medical College, Mysuru, India, and were maintained in
colony cages at 25 2 °C, relative humidity of 45–55 %,
under a 12 h light and dark cycle; they were fed with
standard animal feed. All the animals were acclimatized for
a week before use. Animals were deprived of food 12 h
prior to the experiment and only water was allowed ad
libitum. Acute toxicity studies were performed to estimate
the main study dose value of the synthesized compounds
5a–5k as per the organization for economic co-operation
and development (OECD) guidelines (TG 420 and 425).
The in vivo anti-inflammatory activity of the newly
synthesized 1,3,4-oxadiazoles were evaluated by
carrageenan-induced paw edema test method (Kucukguzel
et al. 2007) using groups of Albino rats of Wistar strain of
either sex, weighing 100–120 g each and the animals were
randomly divided into groups of six. The first group
received only 0.5 % carboxymethyl cellulose (CMC) solu-
tion and served as the untreated control. The second group
received 25 mg Kg−1 indomethacin orally and served as the
positive control. The other groups received the test com-
pounds suspended in 0.5 % CMC and given to the rats
orally at a dose of 25 mg Kg−1. After 1 h of the adminis-
tration of the test compounds and the standard drug, the
animals were injected with 0.1 mL of carrageenan (1 %
solution in normal saline) in the subplantar region of the
right hind. The right hind paw volume (mL) was measured
before and after 0.5, 1, 2 and 4 h of carrageenan treatment
by using mercury displacement technique with the help
of a digital plethysmograph (7141 UGO Basile, Italy).
The reduction in edema volume of the carrageenan-treated
paw at regular time intervals of the test group were com-
pared with the negative control and the reference standard
indomethacin and the data were statistically analyzed using
the repeated measures ANOVA with Dunnett’s test. Equa-
tion 3 presents the formula for the calculation of the percent
Synthesis of N-((5-mercapto-1,3,4-oxadiazol-2-yl)methyl)
benzo[d]thiazol-2-amine (5k)
To a solution of the acyl hydrazide4 (2.22 g, 0.01 M), dis-
solved in ethanol (25 mL), equimolar quantities of carbon
disulphide (0.01 M) and potassium hydroxides (0.01 M)
were added to the solution. The contents were heated to
reflux for 7 h. The progress of the reaction was monitored
by TLC. On completion, the mixture was neutralized with
dilute HCl to obtain the solid product 5k. The product
separated out was filtered and recrystallized using rectified
spirit as the solvent.Yield: 72 %; M.P: 217–219 °C; Rf: 0.76
(ethyl acetate and hexane; 4:1); IR(KBr): ṽ3238 (NH), 3087
(C=C), 2587 (SH), 1620 (C=N), 1230 (C–O–C) cm−1;
1HNMR (400 MHz, DMSO-d6, ppm): δ 12.8 (s, 1H, SH),
7.5–7.4 (m, 4H, Ar–H), 5.9 (s, 1H, NH), 3.4 (s, 2H, CH2);
13CNMR (400 MHz, DMSO-d6, ppm): δ 179.7 (1C, C5-
oxadizole), 169.2 (1C, C5-oxadizole), 156.9 (1C, C2-ben-
zothiazole), 147.3 (1C, C9-benzothiazole), 143.8 (1C, C8-
benzothiazole), 126.7 (1C, C5-benzothiazole), 125.7 (1C,
C6-benzothiazole), 122.4 (1C, C7-benzothiazole), 121.4
(1C, C4-benzothiazole), 42.7 (1C, NHCH2); MS m/z (%):
264 [M+] (40); anal. calcd. for C10H8N4OS2: C, 45.44;
H,3.05; N, 21.20; S, 24.26. Found: C, 45.42; H, 3.03; N,
21.23; S, 24.18.
In vitro radical scavenging activity
The in vitro radical scavenging activity of the newly syn-
thesized 1,3,4-oxadiazoles were screened by DPPH radical
scavenging assay method (Ma et al. 2013).To the 2 mL
solutions of synthesized compounds (25 µM), 2 mL DPPH
solution (25 µM) was added into the test tube. The solution
was incubated at 37 °C for 30 min and the absorbance of
each solution was measured at 517 nm against the reagent
blank solution. The ascorbic acid (25 µM) was used as the
reference standard. The experimental values obtained for
DPPH radical scavenging assays were statistically analyzed.
Equation 2 presents the formula for the calculation of the

Med Chem Res
anti-inflammatory activity.
gently in normal saline. The mucosal damage was examined
by means of a magnifying glass. Mucosal damage was
assessed for each stomach according to the following
scoring system: 1+ redness (petechiae); 2+ hemorrhagic
streaks (hemorrhage with moderate erosion) and 3+ ulcers
(hemorrhage with extensive and severe lesions).
ꢀ
ꢁ
a ꢀ x
% Inhibition ¼
´ 100
ð3Þ
a
Eq. 3: Calculation of percentage inhibition of edema
Where ‘a’ is the mean increase in edema in the control
group of animals and ‘x’ is the mean increase in edema in
the test group at regular interval of time. The mean percent
inhibition of edema by the reference standard indomethacin
and the test compounds at 25 mg Kg−1 concentrations were
compared with the negative control using the repeated
measures ANOVA with Dunnet’s test.
Acknowledgment The authors are thankful to Principal, JSS Col-
lege of Pharmacy, Mysuru and JSS University for providing us all
necessary facilities. We sincerely extend our gratitude to chairman and
staff, NMR research center, Mysore University, Mysore for providing
NMR and mass spectra. Authors also beholden to DBT for the
financial assistance (Ref. No: BT/PR5594/MED/29/540/20l2).
In vitro COX inhibition assay
Compliance with ethical standards
The enzyme inhibitory activity of the oxadiazole derivative
5h on both COX-1 (ovine) and COX-2 (human recombi-
nant) was measured calorimetrically by enzymes immu-
noassay (EIA) kit using COX inhibitor screening assay
purchased from Cayman Chemicals (Cayman Chemical
Co., Ann Arbor, MI, USA). The test compound 5h was
screened at 6.25, 12.5 and 25.0 µM strength for the inhi-
bition studies and procedure following manufacturer’s pro-
tocol. The absorbance at 415 nm was read by using a
microtitre plate reader (Automated Microplate Reader BIO-
TEK; ELX800). In briefly, heme and COX (COX-1/2)
enzyme were added to tubes containing COX reaction
buffer. The mixture was vortex mixed and exposed to
DMSO (negative control) or the test compound 5h in
DMSO for 5 min at 37 °C. Later, arachidonic acid (20 µM)
was added and further incubated for 5 min. This was fol-
lowed by the addition of hydrochloric acid (1 M) to stop the
COX reaction and followed by chemical reduction with
stannous chloride. The COX activity is proportionate to the
amount of COX derived PGH2. The PGH2 is reduced by
stannous chloride in the final step to produce PGF2α in the
reaction tube. The so generated PGF2α is measured by the
EIA kit.
Conflict of interest The authors declare that they have no com-
peting interests.
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