Design, green synthesis, and anti-inflammatory activity of schiff base of 1,3,4-oxadiazole analogues

Article abstract of DOI:10.2174/15701808113109990041

Cyclooxygenase enzyme is a validated therapeutic target for designing drug molecules with anti-inflammatory activity. Herein, a series of various schiff base of 1,3,4-oxadiazole analogues were designed. Considering reasonable structural similarity of the target compounds with the commonly used anti-inflammatory drug indomethacin, it was decided to dock the target compounds into the active site of the molecular target of indomethacin. Prior to docking, the active sites of the proteins are identified. The docking study is performed using the UCSF DOCK 6.5 program. And also the utilization of principles involved in green chemistry is significantly reducing chemical waste and reaction times. To illustrate these advantages in the synthesis of bioactive oxadiazole derivatives, various environmentally benign protocols that involve greener alternatives were studied. The efficiency of microwave heating technology has resulted in remarkable reductions of reaction times (reduced from days and hours to minutes and seconds) with better product yield. The structures of newly synthesized compounds have been elucidated on the basis of IR, 1H NMR, 13C NMR, LC-MS and elemental analysis. An evaluation of the anti-inflammatory activity of the prepared compounds has indicated that some of them exhibited moderate to significant activity as compared to indomethacin.

Full text of DOI:10.2174/15701808113109990041

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82
Letters in Drug Design & Discovery, 2014, 11, 82-89
Design, Green Synthesis, and Anti-Inflammatory Activity of Schiff Base of
1,3,4-oxadiazole Analogues
Biswa Mohan Sahooa,*, Subas Chandra Dindaa, BVV Ravi Kumarb, Jnyanaranjan Pandab and Pathik
S Brahmkshatriyac
aSchool of Pharmaceutical Education & Research (SPER), Berhampur University, Odisha, India
bDepartment of Medicinal Chemistry, Roland Institute of Pharmaceutical Sciences, Odisha, India
cInstitute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic and Center for
Biomolecules and Complex Systems, Czech Republic
Abstract: Cyclooxygenase enzyme is a validated therapeutic target for designing drug molecules with anti-inflammatory
activity. Herein, a series of various schiff base of 1,3,4-oxadiazole analogues were designed. Considering reasonable
structural similarity of the target compounds with the commonly used anti-inflammatory drug indomethacin, it was de-
cided to dock the target compounds into the active site of the molecular target of indomethacin. Prior to docking, the ac-
tive sites of the proteins are identified. The docking study is performed using the UCSF DOCK 6.5 program. And also the
utilization of principles involved in green chemistry is significantly reducing chemical waste and reaction times. To illus-
trate these advantages in the synthesis of bioactive oxadiazole derivatives, various environmentally benign protocols that
involve greener alternatives were studied. The efficiency of microwave heating technology has resulted in remarkable re-
ductions of reaction times (reduced from days and hours to minutes and seconds) with better product yield. The structures
1
of newly synthesized compounds have been elucidated on the basis of IR, H NMR, 13C NMR, LC-MS and elemental
analysis. An evaluation of the anti-inflammatory activity of the prepared compounds has indicated that some of them ex-
hibited moderate to significant activity as compared to indomethacin.
Keywords: Anti-inflammatory activity, Design, Green synthesis, Oxadiazole, Schiff base, Spectroscopic Studies.
INTRODUCTION
which arachidonic acid is converted to prostaglandin H2 [7].
COX exists in two isomeric forms such as COX-1and COX-
2. Among the two isomeric forms, COX-2 mainly acts as
target of commonly used anti-inflammatory drugs [8,9].
Docking study was made by interacting oxadiazole deriva-
tives with the active site of target COX-2. In the present
study we have taken an attempt to synthesize some oxadia-
zole derivatives to develop safe and potent agent for the
treatment of inflammatory diseases. So, the various schiff
base of 1,3,4-oxadiazole analogues were prepared by micro-
wave heating as it offers advantages over conventional
methods in terms of reaction time, yield, purity, cost effec-
tive and environmental friendly. Thus the use of microwave
energy for the synthesis of oxadiazole follows the Green
chemistry approach [10-12]. The goal of green synthesis is to
incorporate a new approach to the synthesis, processing and
application of chemical substances in such a manner as to
reduce threats to human health and environment [13]. In the
current study, oxadiazole derivatives were prepared from
anthranillic acid on esterification with ethanol in the pres-
ence of conc. H2SO4 followed by treatment with hydrazine
hydrate, carbon disulphide in presence of anhydrous potas-
sium hydroxide. Finally the schiff base of oxadiazole was
synthesized by reacting with various substituted benzalde-
hydes under microwave irradiation heating method [14-16].
The newly synthesized compounds were characterized by
means of their IR, 1H NMR, LC-MASS and elemental analy-
sis. Finally the titled compounds were evaluated for anti-
Heterocycles containing oxadiazole ring system are
found to exhibit a wide spectrum of pharmacological activi-
ties such as analgesic, anti-inflammatory, antihyperlipi-
demic, antihypertensive, antihyperglycemic, anticonvulsant,
antiparkinsonian, anti-tubercular and anticancer [1-6]. In
modern drug discovery process, molecular docking is exten-
sively used for understanding the interaction between drug
and receptor. Docking refers to the optimal positioning of the
drug molecule with respect to the binding site of target pro-
tein. It is mainly used to predict the binding orientation of
drug molecule to their target protein to find out their affinity
and activity. When the docking program is used prior to ex-
perimental screening, it can be considered as powerful com-
putational technique to reduce time and cost required for the
effective drug development. Today docking study is helpful
in predicting potential inhibitors for the treatment of life
threatening diseases. So, molecular docking studies were
performed by using UCSF DOCK 6.5 program to determine
the interaction between oxadiazole analogues and active site
of cyclooxygenase (COX). Cyclooxygenases are the en-
dogenous enzyme system mainly responsible for cellular
prostaglandin biosynthesis and catalyze the reactions in
*Address correspondence to these authors at Biswa Mohan Sahoo, School
of Pharmaceutical Education & Research (SPER), Berhampur University,
Odisha-760 007, India; Tel: +91-9040442719, Fax: 0680-2243322;
E-mail: biswamohan81@gmail.com
1875-628X/14 $58.00+.00
©2014 Bentham Science Publishers

Synthesis, and Anti-Inflammatory Study of Oxadiazole Analogues
Letters in Drug Design & Discovery, 2014, Vol. 11, No. 1 83
Fig. (1). A close-up view of the docked ligand (compound 4i) in Cyclooxygenase-2 enzyme (PDB code 1CX2). a) Binding interactions of 4i
(in sticks) with the neighboring protein residues (in lines) b) The fit of the compounds 4i in the active site. Color coding: Ligand carbon in
green, protein carbon in cyan, nitrogen in blue, oxygen in red, sulfur in light brown, hydrogen in white.
inflammatory activity by carrageenan induced paw edema
method in Wistar rats.
affinity of some newly synthesized compounds with COX-2.
Indomethacin is a non-steroidal anti-inflammatory drug
which has analgesic, anti-inflammatory and antipyretic ac-
tivities. Its pharmacological action is thought to be mediated
through inhibition of the enzyme cyclooxygenase (COX), the
enzyme responsible for catalyzing the rate-limiting step in-
volved prostaglandin synthesis via arachidonic acid pathway
[17-19]. Due to structural similarity of the target compounds
with indomethacin, it was decided to perform docking of the
target compounds into the active site of the molecular target
of indomethacin. The crystallographic structure of COX-2
(PDB code 1CX2) was selected from Protein Data Bank
(PDB). The structures were prepared by the structure editing
tools DockPrep in UCSF Chimera (UCSF Chimera,Version
1, USA). UCSF DOCK 6.5 program was used for docking
study. Among all the tested compounds, compound 4i substi-
tuted with hydroxyl group showed good interaction with the
active site of Cyclooxygenase-2 enzyme (PDB code 1CX2)
and also with the neighboring protein residues. So, the order
of binding interaction of individual compounds is repre-
sented as 4i> 4h> 4b> 4e> 4a> 4d> 4j> 4f> 4c> 4g> 4k> 4l>
4m. Among the compounds with flouro and chloro substitu-
tion at para position of the nucleus, later derivative showed
higher affinity towards receptor. While considering chloro
and bromo derivatives, the chloro compounds provided sig-
nificant docking results. Moreover, the compounds with
electron withdrawing substituent exhibited good binding
interaction with target protein. From the docking study, it is
concluded that higher the score better the binding of drug
molecule with target protein (Fig. (1) and Table 1).
MATERIALS AND METHODS
All chemicals used in the synthetic experiment were sup-
plied by E. Merck and S.D. Fine Chemicals. Melting points
were determined in open capillary tube and are found uncor-
rected. The completion of organic reactions and purity of the
compounds were checked by TLC on pre-coated Silica gel-
aluminum plates using a mixture of chloroform and ethanol
(8:2, v/v) as an eluent. UV light or iodine vapor was used
for visualization. Infrared (IR) spectra were recorded (in KBr)
on a Fourier-transform IR, model IR Affinity-1 (SHIMA-
1
DZU), and the values are expressed in cm-1. The H NMR
and 13C NMR spectra were obtained on multinuclear FT
NMR Spectrometer, model Advance-II (Bruker), (400 MHz)
using CDCl3 as solvent, tetramethylsilane (TMS) as an inter-
nal standard. The chemical shift values are expressed as ppm
(parts per million) units, downfield from TMS. The Multi-
plicity of the NMR signals are designated as singlet (s),
doublet (d), triplet (t), quartet (q), and multiplet (m). The
LC-MS spectra were recorded on Waters Micromass Q-Tof
Micro (70 eV), a hybrid quadrupole time of flight mass spec-
trometer. The elemental analysis was performed on a Perkin
Elmer Eal 240 elemental analyser (Perkin Elmer, USA). The
microwave irradiated synthesis of compound 4a–j were car-
ried out in scientific microwave oven (Catalyst System, op-
erating between power level of 140-700W). For molecular
docking study, UCSF DOCK 6.5 program was used.
Molecular Docking Study
General Chemistry
Before carrying out the docking study, it is essential to
identify the binding site in the target protein. It was per-
formed to evaluate the biological activity of the titled com-
pounds. Prostaglandins are subgroup of the eicosanoid fam-
ily which acts as chemical messengers of inflammation and
pain. Among these prostaglandins, prostaglandin synthase-2
or Cyclooxygenase-2 formed via COX-2 pathways are
known to mediate inflammation. COX-2 enzyme is the ma-
jor molecular target of clinically used Nonsteroidal anti-
inflammatory drugs (NSAIDs). The present research work is
focused on the use of insilico methods to study the binding
Compounds containing an azomethine group (-N=CH-),
known as Schiff bases are formed by the condensation of a
primary amine with a carbonyl compound. Thus, structurally
Schiff base is a nitrogen analogue of an aldehyde or ketone
in which the carbonyl group (C=O) has been replaced by an
imine or azomethine group. The imine group present in such
compounds has been shown to be vital for their biological
activities. Schiff base reactions are useful in making carbon-
nitrogen bonds in case of new drug synthesis. The total num-
ber of hydrogen and carbons present in the above com-
1
pounds are conformed by H NMR and 13C NMR spectral

84 Letters in Drug Design & Discovery, 2014, Vol. 11, No. 1
Sahoo et al.
Table 1. Results of Molecular Docking by UCSF Dock 6.5.
hydes are relatively unstable and are readily polymerizable
while those of aromatic aldehydes, having an effective con-
jugation system, are more stable [20-24]. Initially anthranil-
lic acid 1 undergoes esterification with ethanol in presence
of conc. H2SO4 to produce ethyl-2-aminobenzoate 2. Com-
pound 2 undergoes reaction with hydrazine hydrate to pro-
duce 2-aminobenzohydrazide 3. 1,3,4-oxadiazole 4 is ob-
tained by the ring closure reaction of the 2-aminobenz-
ohydrazide with carbon disulphide in the presence of potas-
sium hydroxide. Finally the various schiff bases of 1,3,4-
oxadiazole 4a-j are synthesized by reacting with substituted
benzaldehydes as depicted in the scheme (1).
Comp. Code
DOCK_Score
4a
-21.00
-25.51
-18.74
-20.30
-22.17
-19.20
-15.89
-25.78
-33.55
-19.78
-14.83
-14.78
-8.39
4b
4c
4d
4e
4f
4g
Synthesis
4h
Various Schiff bases of 1,3,4-oxadiazole were synthe-
sized from an aromatic amine [5-(2-aminophenyl)-1,3,4-
oxadiazole-2(3H)-thione] and a carbonyl compound (substi-
tuted benzaldehyde) by nucleophilic addition reaction (Table
2 and 3).
4i
4j
4k
Step-I: General Synthesis of 5-(2-aminophenyl)-1,3,4-
oxadiazole-2-thione (4)
4l
4m
To the ethanolic solution of anthranillic acid (0.05mole,
6.85g), conc. sulphuric acid was added dropwise and shaked
for few min to get the white precipitate of ethyl-2-
aminobenzoate. To this, hydrazine hydrate (0.1mole, 4.85ml)
was added and the reaction mixture was refluxed for 6 h. To
the resulting mixture, potassium hydroxide (3g) and carbon
disulphide (10ml) were added and the reaction mixture was
refluxed until the evolution of H2S gas ceased. TLC was
monitored to check the completion of reaction. The resulting
mixture was cooled, poured into ice cold water and acidified
with dil. HCl (10%) adjusted to pH 5. The solid product was
filtered, washed with cold water, dried and recrystallized
from ethanol.
Indomethacin
-26.37
R
14
15
16
17
13
12
11 N CH
10
9
8
19
1
O
18
S
6
2
5
7
NH
3
N
4
Fig. (2). Numbering of schiff base of 1,3,4-oxadiazole derivatives
(4a-j).
data. The 13C NMR is generated based on the same funda-
mental as in case of proton NMR spectrum. Only 1.1 % of
naturally occurring carbon is 13C and actually an advantage
because of less coupling. The 13C NMR directly gives infor-
mation about the carbon skeleton not the proton attached to
the structure. The chemical shift tells about the hybridization
(sp3, sp2, sp) of each carbon. Schiff bases of aliphatic alde-
Step-II: Synthesis of schiff base of 1,3,4-oxadiazole de-
rivatives (4a-j)
Conventional Method
The solution of 5-(2-aminophenyl)-1,3,4-oxadiazole-2-
thione (0.01 mole, 1.93g) in ethanol (5ml) with substituted
benzaldehyde and few drops glacial acetic acid (GAA) were
NH2
NH2
NH2
NH2
C2H5OH
O
CS2/KOH
S
NH2.NH2.H2O
Conc.H2S
NH
CONHNH2
N
COOH O4
COOC2H5
4
3
1
2
R
Microwave
( 10-20 min, 210W)
or Conventional
( Reflux 5-6h)
OHC
R
N
CH
O
S
NH
N
(4a-j)
Scheme (1). Synthetic protocol used for the preparation of schiff base of 1,3,4-oxadiazole analogues. R: a = 2-Br b = 3-Br, c = 4-Br, d = 4-
OCH3, e = 4-F, f = 2-Cl, g = 3-Cl, h = 4-Cl, i = 4-OH, j = 2-NO2.

Synthesis, and Anti-Inflammatory Study of Oxadiazole Analogues
Letters in Drug Design & Discovery, 2014, Vol. 11, No. 1 85
Table 2. Comparison Between Conventional and Microwave-Assisted Method of Synthesis of schiff base of 1,3,4-oxadiazole Deriva-
tives in Terms of Yield and Reaction Time.
Conventional Synthesis
Microwave Synthesis
Time (min)
Compound
Time (h)
Yield (%)
Yield (%)
4a
4b
4c
4d
4e
4f
5
5
6
5
5
6
5
5
6
5
62
68
66
59
75
65
69
64
73
76
10
12
11
12
12
14
13
15
10
14
75
77
73
64
88
78
89
86
82
67
4g
4h
4i
4j
Table 3. Physical Data of Synthesized Compounds (4 a-j).
N
CH
O
R
S
NH
N
Comp. Code
R
MF
MW
mp (0C)
Rf
4a
4b
4c
4d
4e
4f
2-Br
3-Br
C15H10BrN3OS
C15H10BrN3OS
C15H10BrN3OS
C16H13N3O2S
C15H10FN3OS
C15H10ClN3OS
C15H10ClN3OS
C15H10ClN3OS
C15H11N3O2S
C15H10N4O3S
360.23
360.23
360.23
311.36
299.32
315.78
315.78
315.78
297.33
326.33
180-182
110-112
185-187
160-162
185-187
168-170
176-178
170-172
137-139
108-111
0.62
0.65
0.60
0.61
0.64
0.63
0.63
0.53
0.61
0.57
4-Br
4-OCH3
4-F
2-Cl
4g
4h
4i
3-Cl
4-Cl
4-OH
2-NO2
4j
refluxed for 5-6h. In between TLC was monitored for con-
firmation of completion of the reaction. The reaction mixture
was cooled and poured into ice cold water to get the solid
product. The solid mass was filtered, washed with cold wa-
ter, dried and recrystallized from ethanol.
to get the solid product. The product was filtered, washed
with cold water, dried and recrystallized from ethanol.
5-(2-(2-bromobenzylideneamino)phenyl)-1,3,4-oxadiazole-
2-thione (4a)
IR (KBr, cm-1): 3356 (-NH), 3056 (C-H, Ar), 2844 (-
N=CH-), 1624 (C=N), 1584 (C=C, Ar), 1502 (N=C), 1346
(C-N), 1057 (C=S), 756 (C-O-C), 506 (C-Br); H NMR
Microwave Irradiation Method
1
The solution of 5-(2-aminophenyl)-1,3,4-oxadiazole-2-
thione (0.01 mole, 1.93g) in ethanol (5ml) with substituted
benzaldehyde and few drops GAA were irradiated at power
level 210 W for 10-15 minutes. In between TLC was moni-
tored for confirmation of completion of the reaction. The
reaction mixture was cooled and poured into ice cold water
(CDCl3) ꢀ(ppm): 9.98 (s, 1H, NH), 8.50 (s, 1H, -N=CH-),
7.07-7.78 (m, 8H, Ar-H); 13C NMR: ꢀ 117.4 (C-6), 120.5 (C-
14), 122.2 (C-10), 126.4 (C-8), 127.8 (C-17), 130.3 (C-7),
131.3 (C-15), 131.1 (C-18), 131.5 (C-9), 133.4 (C-16), 135.2
(C-19), 153.2 (C-11), 154.4 (C-5), 156.3 (C-2), 160.4 (C-

86 Letters in Drug Design & Discovery, 2014, Vol. 11, No. 1
Sahoo et al.
13). Anal. Calc. for C15H10BrN3OS (%): C, 50.05; H, 2.80;
N, 11.68; found (%): C, 50.11; H, 2.82; N, 11.56.
(CDCl3) ꢀ(ppm): 9.84 (s, 1H, NH), 8.55 (s, 1H, -N=CH-),
7.44-7.87 (m, 8H, Ar-H); 13C NMR: ꢀ 118.2 (C-6), 122.4 (C-
10), 127.1 (C-17), 127.4 (C-8), 129.2 (C-15), 130.2 (C-7),
130.4 (C-18), 132.3 (C-9), 132.5 (C-16), 133.2 (C-19), 134.1
(C-14), 153.1 (C-11), 155.1 (C-5), 157.2 (C-2), 160.2 (C-
13). Mass (m/z): 316.8 [M+1]. Anal. Calc. for
C15H10ClN3OS (%): C, 57.05; H, 3.19; N, 13.31; found (%):
C, 57.02; H, 3.03; N, 13.23.
5-(2-(3-bromobenzylideneamino)phenyl)-1,3,4-oxadiazole-
2-thione (4b)
IR (KBr, cm-1): 3360 (-NH), 3043 (C-H, Ar), 2847 (-
N=CH-), 1628 (C=N), 1582 (C=C, Ar), 1507 (N=C), 1338
(C-N), 1064 (C=S), 768 (C-O-C), 508 (C-Br); 1H NMR
1
(CDCl3) ꢀ(ppm): H NMR: 9.94 (s, 1H, NH), 8.50 (s, 1H, -
5-(2-(3-chlorobenzylideneamino)phenyl)-1,3,4-oxadiazole-
2-thione (4g)
N=CH-), 7.03-7.72 (m, 8H, Ar-H); 13C NMR: ꢀ 118.4 (C-6),
122.5 (C-10), 123.4 (C-15), 127.6 (C-8), 128.4 (C-18), 130.4
(C-7), 131.2 (C-17), 132.1 (C-9), 132.5 (C-14), 134.2 (C-
16), 136.2 (C-19), 153.2 (C-11), 155.2 (C-5), 157.4(C-2),
160.3 (C-13). Mass (m/z): 360.8 [M+]. Anal. Calc. for
C15H10BrN3OS (%): C, 50.05; H, 2.80; N, 11.68; found (%):
C, 50.11; H, 2.82; N, 11.63.
IR (KBr, cm-1): 3367 (-NH), 3057 (C-H, Ar), 2858 (-
N=CH-), 1664 (C=N), 1600 (C=C, Ar), 1527 (N=C), 1325
(C-N), 1076 (C=S), 997 (C-Cl), 752 (C-O-C); 1H NMR
(CDCl3) ꢀ(ppm): 9.87 (s, 1H, NH), 8.56 (s, 1H, -N=CH-),
7.43-7.86 (m, 8H, Ar-H); 13C NMR: ꢀ 118.2 (C-6), 122.4 (C-
10), 127.2 (C-18), 127.4 (C-8), 129.2 (C-14), 130.2 (C-17),
130.4 (C-7), 131.3 (C-16), 132.5 (C-9), 134.3 (C-15), 135.1
(C-19), 153.1 (C-11), 155.2 (C-5), 157.1 (C-2), 160.2 (C-
13). Anal. Calc. for C15H10ClN3OS (%): C, 57.05; H, 3.19;
N, 13.21; found (%): C, 57.02; H, 3.09; N, 13.21.
5-(2-(4-bromobenzylideneamino)phenyl)-1,3,4-oxadiazole-
2-thione (4c)
IR (KBr, cm-1): 3365 (-NH), 3032 (C-H, Ar), 2841 (-
N=CH-), 1620 (C=N), 1587 (C=C, Ar), 1506 (N=C), 1336
(C-N), 1068 (C=S), 758 (C-O-C), 501 (C-Br); H NMR
1
5-(2-(4-chlorobenzylideneamino)phenyl)-1,3,4-oxadiazole-
2-thione (4h)
(CDCl3) ꢀ(ppm): 9.38 (s, 1H, NH), 8.59 (s, 1H, -N=CH-),
7.49-7.90 (m, 8H, Ar-H); 13C NMR: ꢀ 118.1 (C-6), 122.3 (C-
10), 125.2 (C-16), 127.6 (C-8), 130.4 (C-7), 131.4 (C-14),
131.4 (C-18), 131.6 (C-15), 131.6 (C-17), 132.5 (C-9), 132.8
(C-19), 153.2 (C-11), 155.2 (C-5), 157.1 (C-2), 160.2 (C-
13). Anal. Calc. for C15H10BrN3OS (%): C, 50.01; H, 2.80;
N, 11.66; found (%): C, 50.21; H, 2.82; N, 11.46.
IR (KBr, cm-1): 3360 (-NH), 3045 (C-H, Ar), 2856 (-
N=CH-), 1662 (C=N), 1604 (C=C, Ar), 1524 (N=C), 1322
(C-N), 1072 (C=S), 994 (C-Cl), 756 (C-O-C); 1H NMR
(CDCl3) ꢀ(ppm): 9.81 (s, 1H, NH), 8.58 (s, 1H, -N=CH-),
7.44-7.89 (m, 8H, Ar-H); 13C NMR: ꢀ 118.2 (C-6), 122.4 (C-
10), 127.4 (C-8), 129.2 (C-15), 129.2 (C-17), 130.2 (C-7),
130.4 (C-14), 131.3 (C-18), 131.5 (C-19), 132.3 (C-9), 136.5
(C-16), 153.1 (C-11), 155.2 (C-5), 157.1 (C-2), 160.2 (C-
13). Anal. Calc. for C15H10ClN3OS (%): C, 57.05; H, 3.19;
N, 13.21; found (%): C, 57.04; H, 3.29; N, 13.11.
5-(2-(4-methoxybenzylideneamino)phenyl)-1,3,4-oxadia-
zole-2-thione (4d)
IR (KBr, cm-1): 3348 (-NH), 3012 (C-H, Ar), 2837 (-
N=CH-), 1602 (C=N), 1529 (C=C, Ar), 1502 (N=C), 1355
(C-N), 1251 (C-OCH3), 1109 (C=S), 781 (C-O-C); 1H NMR
1
5-(2-(4-hydroxybenzylideneamino)phenyl)-1,3,4-oxadia-
zole-2-thione (4i)
(CDCl3) ꢀ(ppm): H NMR: 9.41 (s, 1H, NH), 8.58 (s, 1H, -
N=CH-), 7.50-7.91 (m, 8H, Ar-H), 2.43 (s, 3H, OCH3); 13C
NMR: ꢀ 55.7 (C-20), 114.2 (C-15), 114.2 (C-17), 118.2 (C-
6), 122.4 (C-10), 126.2 (C-19), 127.4 (C-8), 130.4 (C-14),
130.4 (C-18), 130.6 (C-7), 132.4 (C-9), 153.2 (C-11), 155.1
(C-5), 157.1 (C-2), 160.2 (C-13), 163.2 (C-16). Anal. Calc.
for C16H13N3O2S (%): C, 61.76; H, 4.24; N, 13.54; found
(%): C, 61.62; H, 4.11; N, 13.47.
IR (KBr, cm-1): 3325cm-1 (C-OH), 3240 (-NH), 3059 (C-
H, Ar), 2841 (-N=CH-), 1668 (C=N), 1575 (C=C, Ar), 1558
1
(N=C), 1371 (C-N), 1074 (C=S), 754 (C-O-C); H NMR
(CDCl3) ꢀ(ppm): 10.43 (s, 1H, OH), 9.53 (s, 1H, NH), 8.59
(s, 1H, -N=CH-), 7.50-7.91 (m, 8H, Ar-H); 13C NMR: ꢀ
116.2 (C-15), 116.2 (C-17), 118.2 (C-6), 122.4 (C-10), 126.5
(C-19), 127.4 (C-8), 130.2 (C-7), 130.4 (C-14), 130.4 (C-
18), 132.3 (C-9), 153.1 (C-11), 155.1 (C-2), 157.1 (C-2),
160.2 (C-13), 160.6 (C-16). Anal. Calc. for C15H11N3O2S
(%): C, 60.59; H, 3.73; N, 14.23; found (%): C, 60.49; H,
3.62; N, 14.14.
5-(2-(4-fluorobenzylideneamino)phenyl)-1,3,4-oxadiazole-
2-thione (4e)
IR (KBr, cm-1): 3178 (-NH), 3064 (C-H, Ar), 2953 (-
N=CH-), 1633 (C=N), 1604 (C=C, Ar), 1510 (N=C), 1321
(C-N), 1157 (C-F), 1097 (C=S), 796 (C-O-C); 1H NMR
(CDCl3) ꢀ(ppm): 9.78 (s, 1H, NH), 8.50 (s, 1H, -N=CH-),
7.03-7.74 (m, 8H, Ar-H); 13C NMR: ꢀ 115.4 (C-15), 115.4
(C-17), 118.2 (C-6), 122.4 (C-10), 127.4 (C-8), 129.2 (C-
19), 130.4 (C-7), 130.6 (C-14), 130.6 (C-18), 132.4 (C-9),
153.2 (C-11), 155.1 (C-5), 157.1 (C-2), 160.2 (C-13), 165.2
(C-16). Anal. Calc. for C15H10FN3OS (%): C, 60.16; H, 3.32;
N, 14.02; found (%): C, 60.04; H, 3.15; N, 14.14.
5-(2-(2-nitrobenzylideneamino)phenyl)-1,3,4-oxadiazole-2-
thione (4j)
IR (KBr, cm-1): 3292 (-NH), 3055 (C-H, Ar), 2866 (-
N=CH-), 1666 (C=N), 1647 (C=C, Ar), 1606 (N=C), 1523,
1
1357 (C-NO2), 1315 (C-N), 1047 (C=S), 742 (C-O-C); H
NMR (CDCl3) ꢀ(ppm): 9.44 (s, 1H, NH), 8.58 (s, 1H, -
N=CH-), 7.50-7.89 (m, 8H, Ar-H); 13C NMR: ꢀ 118.2 (C-6),
121.4 (C-15), 122.4 (C-10), 126.5 (C-19), 127.4 (C-8), 130.2
(C-18), 130.4 (C-7), 132.1 (C-16), 132.3 (C-9), 135.1 (C-
17), 148.7 (C-14), 153.1 (C-11), 155.1 (C-5), 157.2 (C-2),
160.2 (C-13). Anal. Calc. for C15H10N4O3S (%): C, 55.20; H,
3.07; N, 17.17; found (%): C, 55.12; H, 3.04; N, 17.12.
5-(2-(2-chlorobenzylideneamino)phenyl)-1,3,4-oxadiazole-
2-thione (4f)
IR (KBr, cm-1): 3362 (-NH), 3054 (C-H, Ar), 2853 (-
N=CH-), 1660 (C=N), 1607 (C=C, Ar), 1524(N=C), 1327
(C-N), 1078 (C=S), 992 (C-Cl), 758 (C-O-C); 1H NMR

Synthesis, and Anti-Inflammatory Study of Oxadiazole Analogues
Letters in Drug Design & Discovery, 2014, Vol. 11, No. 1 87
Table 4. Anti-Inflammatory Activity of the Synthesized Compounds (4a-j).
1st
h
2nd
h
3rd
h
4th
h
Mean
Edema
Volume
(mL)
Mean
Edema
Volume
(mL)
Mean
Edema
Volume
(mL)
Compounds
Mean Edema
Volume (mL)
% of Inhibition
% of Inhibition
% of Inhibition
% of Inhibition
Control
0.49
0.30
0
1.085
0.71
0.31
0
1.018
0.87
0.28
0
1.024
0.98
37.0
0
0.601
Indometha-
cin
38.78 0.645
56.34 0.706
67.82 0.484
76.53 0.456
4a
4b
4c
4d
4e
4f
0.45
0.39
0.37
0.41
0.40
0.43
0.42
0.41
0.35
0.44
08.16 0.849
20.41 1.085
24.49 1.050
16.33 1.010
18.37 0.965
12.24 1.118
14.19 0.768
16.33 1.148
28.57 0.913
10.2 1.270
0.52
0.44
0.41
0.48
0.46
0.5
26.76 0.872
38.03 1.018
42.25 0.976
32.29 1.119
35.21 0.929
29.58 1.134
30.99 0.953
33.8 1.089
46.48 0.802
28.17 1.072
0.57
0.46
0.42
0.52
0.49
0.53
0.51
0.50
0.40
0.54
34.48 0.637
47.13 0.902
51.72 0.758
40.23 0.788
43.68 0.724
39.08 0.953
41.38 0.685
42.53 0.920
54.02 0.637
37.93 0.839
0.59
0.47
0.43
0.54
0.51
0.55
0.52
0.51
0.41
0.98
39.80 0.645
52.04 0.601
56.12 0.543
44.90 0.742
47.96 0.601
43.88 0.812
46.94 0.645
47.96 0.682
57.65 0.574
41.88 0.812
4g
4h
4i
0.49
0.47
0.38
0.51
4j
expressed as mean SEM. Analyzing the anti-inflammatory
activity of synthesized derivatives 4a–j, the following struc-
ture activity relationship (SAR) was observed. It was found
that the compound 4i substituted with hydroxyl group at para
position of nucleus showed maximum anti-inflammatory
activity in comparison to F, Cl, Br, OCH3, NO2 group on the
whole level. The position of the substituents on the phenyl
ring greatly influenced the anti-inflammatory activity. In
case of bromo and chloro derivatives, the activity order is
para> meta> ortho. While considering compound substituted
with methoxy group at para position, moderate anti-
inflammatory activity was observed. Similarly compound
substituted with nitro group showed less anti-inflammatory
activity among tested compounds of the series. Whenever
the molecular docking was considered to correlate the anti-
inflammatory activity of synthesized compounds, it was ob-
served that most of the compounds with good binding inter-
action on active site of Cyclooxygenase-2 enzyme, showed
good anti-inflammatory activity.
PHARMACOLOGY
Based on the results obtained in molecular docking study,
it was decided to synthesize some selected oxadiazole ana-
logues which are showing good binding interaction with the
target protein. Then the newly synthesized compounds were
evaluated for their anti-inflammatory activity [25] by car-
rageenan induced paw edema method in Wistar rats. Wistar
rats of either sex (180-250 g) were randomly selected and
housed under standard laboratory conditions, given pellet
and tap water ad libitum and maintained under standard en-
vironmental conditions throughout the duration of experi-
mentation. The animals were divided into different groups of
six animals each (n=6) and fasted for 12h before the experi-
ment. Group I served as control and received vehicle, group
II served as standard and received Indomethacin (10 mg/kg,
p.o.) and other groups were served as test groups and re-
ceived the test compounds (200 mg/kg/p.o.) 1h prior to in-
jection of carrageenan. The vehicle, standard and test com-
pounds were administered in the suspension form using car-
boxy methyl cellulose to the respective groups. The initial
right hind paw volume of the rats were measured using a
digital plethysmometer and then 0.1 ml of 1% w/v car-
rageenan solution in normal saline was injected into the sub
planter region of the right hind paw. The volume of right
hind paw was measured at 1st, 2nd, 3rd, 4th h after carrageenan
injection by using digital plethysmometer. The data were
expressed as paw volume (mL) and were presented in Table
4 and graphically presented in Fig. (3). The experimental
protocols for the animal studies were approved by the insti-
tutional animal ethics committee prior to the conduct of the
animal experiments (Regd. No. 926/ab/06/CPCSEA). The
results of statistical analysis for animal experiments were
CONCLUSION
A series of schiff base containing 1,3,4-oxadiazole were
synthesized based on the results obtained from their molecu-
lar docking study. Both the conventional and microwave
assisted synthesis were carried out to compare their yield of
product and reaction time. With the help of microwave, the
yield of product was increased from 59 to 89% with lesser
reaction time as compared to conventional heating method.
The docking results indicated that most of the compounds
could have the potential to become a lead molecule and may
act as preferential inhibitors of COX-2 in comparison to the
standard drug. From the results of anti-inflammatory screen-

88 Letters in Drug Design & Discovery, 2014, Vol. 11, No. 1
Sahoo et al.
Fig. (3). Graphical representations of % of Inhibition (Mean SEM) of synthesized Compound (4a-j) and Indomethacin on the rat paw
edema induced by sub-plantar injection of carrageenan.
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CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
The authors are indebted to School of Pharmaceutical
Education & Research, Berhampur University and Roland
Institute of Pharmaceutical Sciences for providing necessary
facilities to carry out the research work.
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Received: May 24, 2013
Revised: June 27, 2013
Accepted: July 08, 2013