Synthesis and biological evaluation of 2,5-disubstituted 1,3,4-oxadiazole derivatives with both COX and LOX inhibitory activity

Article abstract of DOI:10.3109/14756366.2010.550890

Dual cyclooxygenase/lipoxygenase (COX/LOX) inhibitors constitute a valuable alternative to classical nonsteroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors for the treatment of inflammatory diseases. A series of 3-(5-phenyl/phenylami

Full text of DOI:10.3109/14756366.2010.550890

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(6): 767–776
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.550890
RESEARCH ARTICLE
Synthesis and biological evaluation of 2,5-disubstituted
1,3,4-oxadiazole derivatives with both COX and LOX
inhibitory activity
Mymoona Akhter1, Nayeema Akhter2, M. M. Alam1, M. S. Zaman1, Rikta Saha1, and A. Kumar3
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard University, New Delhi, India,
2Chemistry Division, Indian Institute of Integrated Medicine, CSIR, Srinagar, Kashmir, and 3Department of
Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center,
Kingsville, TX, USA
Abstract
Dual cyclooxygenase/lipoxygenase (COX/LOX) inhibitors constitute a valuable alternative to classical nonsteroidal
anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors for the treatment of inflammatory diseases. A
series of 3-(5-phenyl/phenylamino-[1,3,4]oxadiazol-2-yl)-chromen-2-one and N-[5-(2-oxo-2H-chromen-3-yl)-[1,3,4]
oxadiazol-2-yl]-benzamide derivatives were synthesized and screened for anti-inflammatory, analgesic activity. All
the derivatives prepared are active in inhibiting oedema induced by carrageenan. Compound 4e was found more
potent with 89% of inhibition followed by compound 4b (86%). Compounds with >70% of anti-inflammatory activity
were tested for analgesic, ulcerogenic, and lipid peroxidation profile. Selected compounds were also evaluated for
inhibition of COXs (COX-1 and COX-2) and LOXs (LOX-5, LOX-12, and LOX-15). Compound 4e was comparatively
selective for COX-2, LOX-5, and LOX-15. Study revealed that these derivatives were more effective than ibuprofen
with reduced side effects. It can be suggested that these derivatives could be used to develop more potent and safer
NSAIDs.
Keywords: Cyclooxygenase, lipoxygenase, oxadiazole, anti-inflammatory agents
Introduction
carboxylate function that like one of the AA forms an ion
Inflammation is a normal reaction to injury or infec-
tion. It is caused by the body release of hormone-like
substances called prostaglandins (PGs) and leukot-
rienes (LTs). ese inflammatory-inducing agents are
synthesized from arachidonic acid (AA) by the enzymes
cyclooxygenase (COX) and lipoxygenase (LOX). COX
enzymes catalyse the first committed step in the bio-
synthesis of PGs and thromboxanes, and are the phar-
macological targets of nonsteroidal anti-inflammatory
drugs (NSAIDs)1,2. NSAIDs have been subdivided into
two classes: (1) classical NSAIDs and (2) selective
COX-2 inhibitors. Ibuprofen, indomethacin, diclofenac,
naproxen (Figure 1) comes under first category. Despite
an extensive chemical diversity, they all possess a
pair with Arg-120 at the bottom of the COX active site3.
ey therefore share common side effects like gastroin-
testinal (GI) lesions and renal toxicity, leading at high
doses to erosions, ulcerations, bleedings, and even to
death4. is is because of their nonspecific inhibition of
both COX isoforms. Classical NSAIDs reduce the produc-
tion of proinflammatory PGs at sites of injury and also the
formation of physiological PGs in the stomach and the
kidney. ese observations provided a rationale for the
development of COX-2 selective inhibitors like celecoxib,
rofecoxib that should retain the potent anti-inflamma-
tory and analgesic effects of classical NSAIDs with less
GI adverse effects5. ey were shown to preferentially
inhibit the inducible isoform, that is, COX-2. Currently,
Address for Correspondence: Mymoona Akhter, Drug Design and Medicinal Chemistry Lab, Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Jamia Hamdard University, New Delhi–110062, India. Tel: + 91 11 26059688, extn 5645. Fax: +91 11 26059663. E-mail:
mymoonaakhter@gmail.com
(Received 13 October 2010; revised 18 December 2010; accepted 22 December 2010)
767

768 M. Akhter et al.
Abbreviations
HETE
AA
BDH
bs
CDCl3
COX
d
Hydroxy eicosatetraenoic acid
KCl
potassium chloride
low-density lipoprotein
lipoxygenase
leukotriene B4
multiplet
arachidonic acid
British Drug House
broad singlet
deuterated chloroform
cyclooxygenase
doublet
LDL
LOX
LTB4
m
MDA
NMR
NSAIDs
PBML
PGs
ppm
s
malondialdehyde
nuclear magnetic resonance
nonsteroidal anti-inflammatory drugs
peripheral blood mononuclear cell leukocyte
prostagladins
parts per million
singlet
DMSO
dimethyl sulphoxide
DMSO-d6 deuterated dimethyl sulphoxide
EDTA
EIA
GI
ethylenediaminetetraacetic acid
enzyme immunoassay
gastro intestinal
HCl
Hz
hydrochloric acid
hertz
SDS
SS1
sodium dodecyl sulphate
solvent system 1
IC50
dose that causes half-maximal inhibition (median
inhibition concentration)
coupling constants
SS2
TBA
TLC
solvent system 2
thiobarbituric acid
thin-layer chromatography
J
>500 COX-2-specific inhibitors have been designed. e
main structural features of these compounds are the
absence of the carboxylate group, characteristic of clas-
sical NSAIDs, and generally, the presence of a sulphone
(–SO2–) or sulphonamide (–SO2NH2) moiety (Figure 1),
which can interact with Arg-513 in the hydrophilic side
pocket of the COX-2 active site6. But the development
of selective COX-2 inhibitors did not solve the purpose
completely due to certain associated reasons, for exam-
ple, COX-2 is constitutively expressed in the kidney and
the reproductive tract, also cyclic hormonal induction of
COX-2 plays an important role in ovulation6. Second, the
adverse cardiovascular effects7 (by decreasing vasodila-
tory and antiaggregatory PGI2 production) have resulted
in the voluntary withdrawal of Vioxx® (rofecoxib) world-
wide. In these cases, the anti-inflammatory efficacy of
selective COX-2 inhibitors was only observed at doses
that inhibited COX-18. In conclusion, it appears that
selective COX-2 inhibitors do not fully satisfy the search
for new safer anti-inflammatory agents. LOX is a family of
non-haeme iron-containing dioxygenases, and exists in
three isoforms: LOX-5, LOX-12, and LOX-15. e LOX-5
pathway, which is the second major metabolic pathway
of AA, plays an important role in the pathophysiology of
several inflammatory9,10 and allergic diseases and gener-
ates products particularly important in inflammation
(LTs), and is up-regulated during COX blockade. is
up-regulation of LOX has been associated with various
adverse effects, such as asthma. LOX-5 has been shown
to be involved in the production of LTs, which are known
to contribute to the progression of osteoarthritis, asthma,
and inflammation11–14. LOX-15 has been implicated in
COOH
COOH
Cl
H
N
HO
COOH
N
O
O
H3CO
H3CO
Cl
Cl
Indomethacine
Naproxen
Diclofenac
Ibuprofen
H2NO2S
H3CO2S
O
N
O
CF3
N
N
H
O
H2NO2S
Celecoxib
Figure 1. Classical and nonclassical nonsteroidal anti-inflammatory drugs (NSAIDs).
Journal of Enzyme Inhibition and Medicinal Chemistry
Rofecoxib
Valdecoxib

Synthesis and biological evaluation of 2,5-disubstituted 1,3,4-oxadiazole derivatives 769
the oxidation of low-density lipoprotein (LDL), which
were used without further purification. Celecoxib and
ibuprofen were obtained as gift sample from Cadila
Pharma, India and Zydus Cadila, India, respectively.
Proton nuclear magnetic resonance (1H-NMR) spectra
were determined in deuterated chloroform (CDCl3), or
deuterated dimethyl sulphoxide (DMSO-d6) solution
on a Bruker Avance 300 spectrometer. Proton chemical
shifts (δ) are expressed in parts per million (ppm) rela-
tive to tetramethylsilane as an internal standard. Spin
multiplicities are given as s (singlet), d (doublet), bs
(broad singlet), and m (multiplet). Coupling constants
(J) are given in hertz (Hz). Liquid chromatography was
performed with a forced flow (flash chromatography)
with Merck Grade Silica gel (70–230 mesh). e solvents
used for elution varied depending on the compound
and included either one or a combination of the fol-
lowing: toluene:ethylacetate:formic acid (SS1) and
chloroform:methanol (SS2). Analytical thin-layer chro-
matography (TLC) was performed with Sigma-Aldrich
0.25-mm silica gel plates (60 Å), visualized under
254 nm ultraviolet light or iodine spray. e compounds
were purified by Combiflash Retrieve, Septech. All
yields were of purified product and were not optimized.
Melting points were determined in an open capillary
tube using a Decibel digital melting point apparatus
and are uncorrected.
ultimately causes atherosclerosis15,16. Most recently, it
has also been demonstrated that increased expression
of LOX-12/LOX-15 causes heart failure, in transgenic
mice17. erefore, agents that inhibit both the enzymes
viz. COX and LOX may arrive as winners in this race as
exemplified by Licofelone (ML-3000), a dual COX/LOX-5
inhibitor and a potent anti-inflammatory agent without
GI side effects18. erefore, dual inhibition of COX and
LOX is an interesting alternative to provide safer NSAIDs1.
Compounds with dual COX/LOX inhibitory activity have
recently emerged as potential anticancer agents19,20
.
Five-membered heterocyclic compounds like oxazole,
pyrazole, indole, triazole, oxadiazole have been widely
explored for their biological activities especially for anti-
inflammatory activity, for example, celecoxib bears pyra-
zole nucleus, phenyl butazones have pyrazole- 2,5-dione,
rofecoxib has furanone, and valdecoxib has isoxazole
nucleus. 1,3,4-Oxadiazoles forms an important class of
heterocyclic compounds with broad spectrum of biologi-
cal activities. Substituted 1,3,4-oxadiazoles have revealed
antibacterial21, antimycobacterial22, antifungal23, anti-in-
flammatory and analgesic24–26, anticonvulsant27, antihy-
perglycemic28, anticancer29, anti-HIV-130, and tyrosinase
inhibitory activity31.
1,2,4-Oxadiazole derivatives have been reported as
potent anti-inflammatory agents with selectivity for
COX-232–34
.
3-Phenyl-1,2,4-oxadiazole derivative has
Chemistry
been reported to exhibit analgesic activity far superior
than aspirin35. Compounds containing coumarin moi-
ety have also been reported to possess wide spectrum
of biological activity like anti-inflammatory, antican-
cer, antirheumatic, and so on. It has been reported that
styryl carbonyl derivatives possess appreciable anti-
inflammatory activity36,37. erefore, coumarin nucleus
that incorporates the styryl carbonyl moiety into a rigid
framework was selected to be a part of newly synthesized
compounds. Furthermore, coumarin/chromone and
related derivatives are recognized as inhibitors of both
the mediators of inflammation, that is, LOX and COX
Carbethoxy coumarin (1) and aryl isothiocyanates
were prepared by reported methods42,43, the 2-oxo-2H-
chromene-3-carboxylic acid hydrazide (2), substituted
2-oxo-2H-chromene-3-carboxylic acid benzylidene-
hydrazide (3a–e), and 3-(5-amino-[1,3,4]oxadiazol-2-
yl)-chromen-2-one (7) were synthesized by previously
reported procedures41. Compounds (5a–f) were prepared
by condensing compound 2 with aryl isothiocyanates
following reported procedures44.
General procedure for synthesis of oxadiazole (4a–e)
To a solution of compound 3 (0.01 mol) in acetic acid
(8 mL) was added ferric chloride (0.1 g) and water (4 mL)
and stirred for 1 h at room temperature followed by dilu-
tion with water to 100 mL. e reaction mixture was left
overnight at room temperature when a solid mass sepa-
rated was collected by filtration on suction pump and
washed with water thoroughly. e solid so obtained
was dried and purified to get the desired compound.
3-(5-Phenyl-[1,3,4]oxadiazol-2-yl)-chromen-2-one
(4a): Yield: 48%; m.p.: 151–153°C; Rf (SS1): 0.67; IR
(cm−1): 1060 (C-O-C), 1450, 1504, 1602, 1645 (ring stretch-
ing of oxadiazole ring), 1705 (C=O); 1H-NMR (ppm):
6.89–6.92 (m, 2H, H6,8), 7.48–7.54 (m, 2H, H5,7), 7.64–7.71
(m, 5H, ArH), 8.06 (s, 1H, H4); Anal. Calcd. for C17H10N2O3:
C, 70.34; H, 3.47; N, 9.65. Found: C, 70.29; H, 3.49; N,
9.63.
pathways of AA metabolism33,38
.
Considering the proinflammatory properties of LTs
and prostanoids, agents that are able to block equally the
synthesis of both eicosanoids (dual inhibitors) should
not only present a superior anti-inflammatory profile but
also fewer side effects than NSAIDs and selective COX-2
inhibitors39. In continuation to our work to develop
agents to treat inflammatory conditions40,41, we described
in this article the synthesis and biological evaluation of
a class of 3-(5-phenyl/phenylamino-[1,3,4]oxadiazol-2
-yl)-chromen-2-one and N-[5-(2-oxo-2H-chromen-3-
yl)-[1,3,4]oxadiazol-2-yl]-benzamide derivatives as dual
inhibitors of COXs and LOXs.
Materials and methods
3-[5-(4-Chloro-phenyl)-[1,3,4]oxadiazol-2-yl]-
chromen-2-one (4b): Yield: 52%; m.p.: 200–202°C; Rf
(SS2): 0.61; IR (cm−1): 1710 (C=O), 1650, 1615, 1480, 1380,
Reagents and solvents were purchased from local
suppliers of Sigma, British Drug House (BDH) and
© 2011 Informa UK, Ltd.

770 M. Akhter et al.
1
1070 (C-O-C), 760 (C-Cl); H-NMR (ppm): 7.2–7.24 (m,
2H, H6,8), 7.5 (d, 2H, J= 8.1 Hz, ArH), 7.6–7.65 (m, 2H, H5,7),
8.05 (s, 1H, H4), 8.18 (d, 2H, J= 8 Hz, ArH); Anal. Calcd. for
C17H9ClN2O3: C, 62.88; H, 2.79; N, 8.63. Found: C, 62.79;
H, 2.58; N, 8.60.
(cm−1): 1705 (CO), 1615 (C=N), 1075 (C-O-C), 3318 (NH),
1579, 1506, 1455; H-NMR (ppm): 2.51 (s, 3H, CH3), 7.72
(d, 2H, J= 8.2 Hz, ArH), 6.92–6.95 (m, 2H, H6,8) 8.26 (d, 2H,
J= 8 Hz, ArH) 7.61–7.68 (m, 2H, H5,7) 8.97 (br, s, 1H, NH),
8.40 (s, 1H, H4); Anal. Calcd. for C18H13N3O3: C, 67.71; H,
4.10; N, 13.16. Found: C, 67.57; H, 4.07; N, 13.04.
1
3-(5-p-Tolyl-[1,3,4]oxadiazol-2-yl)-chromen-2-one
(4c): Yield: 51%; m.p.: 160–162°C; Rf (SS1): 0.68; IR
(cm−1): 1070 (C-O-C), 1454, 1500, 1517, 1633 (C=N), 1660
(CO), 1712 (C=O); 1H-NMR (ppm): 2.16 (s, 3H, CH3),
7.04–7.10 (m, 2H, H6,8), 7.59–7.63 (m, 2H, H5,7), 7.43 (d,
2H, J= 8 Hz, ArH), 7.89 (d, 2H, J= 8.2 Hz, ArH), 8.12 (s, 1H,
H4); Anal. Calcd. for C18H12N2O3: C, 71.05; H, 3.97; N, 9.21.
Found: C, 71.13; H, 3.89; N, 9.12.
3-[5-(4-Methoxyphenyl)-[1,3,4]oxadiazol-2-yl]-
chromen-2-one (4d): Yield: 49%; m.p.: 168–171°C; Rf
(SS1): 0.71; IR (cm−1): 1066 (C-O-C), 1150 (OCH3), 1455,
1506, 1577, 1613 (C=N), 1696 (C=O); 1H-NMR (ppm): 3.47
(s, 3H, OCH3), 7.27–7.3 (m, 2H, H6,8), 7.53 (d, 2H, J= 8 Hz,
ArH), 7.6–7.64 (m, 2H, H5,7), 7.71 (d, J= 7.8 Hz, 2H, ArH),
8.05 (s, 1H, H4); Anal. Calcd. for C18H12N2O4: C, 67.50; H,
3.78; N, 8.75. Found: C, 67.49; H, 3.85; N, 8.68.
3-[5-(2,4-Dichloro-phenyl)-[1,3,4]oxadiazol-2-yl]-
chromen-2-one (4e): Yield: 58%; m.p.: 194–196°C; Rf
(SS2): 0.65; IR (cm−1): 771 (C-Cl), 1065 (C-O-C), 1455,
1506, 1577, 1610 (C=N), 1705 (C=O); 1H-NMR (ppm):
7.27–7.31 (m, 2H, H6,8), 7.42–7.49 (m, 2H, ArH) 7.56–7.59
(m, 2H, H5,7), 7.65 (d, J= 2.2, 1H, ArH), 8.13 (s, 1H, H4);
Anal. Calcd. for C17H8Cl2N2O3: C, 56.85; H, 2.25; N, 7.80.
Found: C, 56.94; H, 2.20; N, 7.67.
3-[5-(4-Methoxy-phenylamino)-[1,3,4]oxadiazol-2-
yl]-chromen-2-one (6d): Yield: 53%; m.p.: 168–170°C;
Rf (SS1): 0.69; IR (cm−1): 1713 (CO), 1597 (C=N), 1115
1
(C-O-C), 3317 (NH), 1577, 1500, 1454; H-NMR (ppm):
3.65 (s, 3H, OCH3), 6.83 (d, 2H, J= 8.0 Hz, ArH), 6.97–7.06
(m, 2H, H6,8), 7.46 (d, 2H, J= 8.1 Hz, ArH), 7.61–7.67 (m,
2H, H5,7), 10.24 (br, s, 1H, NH), 8.21 (s, 1H, H4); Anal.
Calcd. for C18H13N3O4: C, 64.47; H, 3.91; N, 12.53. Found:
C, 64.35; H, 3.86; N, 12.36.
3-(5-o-Tolylamino-[1,3,4]oxadiazol-2-yl)-chromen-2-
one (6e): Yield: 55%; m.p.: 161–163°C; Rf (SS1): 0.58; IR
(cm−1): 1065 (C-O-C), 1450 (oxa), 1504, 1589, 1613 (C=N),
1708 (CO), 3320 (NH); 1H-NMR (ppm): 2.38 (s, 3H, CH3),
7.20–7.22 (m, 2H, ArH), 6.96–7.08 (m, 2H, H6,8),7.36–7.41
(m, 2H, H5,7), 7.71–7.75 (m, 2H, Ar), 8.43 (s, 1H, H4), 10.3
(br, s, 1H, NH); Anal. Calcd. for C18H13N3O3: C, 67.71; H,
4.10; N, 13.16. Found: C, 67.83; H, 4.14; N, 13.10.
3-[5-(3-Chloro-phenylamino)-[1,3,4]oxadiazol-2-
yl]-chromen-2-one (6f): Yield: 38%; m.p.: 148–150°C;
Rf (SS2): 0.72; IR (cm−1) 1710 (CO), 1096 (C-O-C), 3409
1
(NH), 1625, 1579, 1501, 1451; H-NMR (ppm): 6.95–7.03
(m, 4H, ArH), 7.18–7.21 (m, 2H, H6,8), 7.57–7.61 (m, 2H,
H5,7), 10.02 (br, s, 1H, NH), 8.39 (s, 1H, H4); Anal. Calcd.
for C18H13N3O3: C, 67.71; H, 4.10; N, 13.16. Found: C, 67.59;
H, 4.21; N, 13.09.
General procedure for synthesis of oxadiazole (6a–f)
Compound (5) (0.01 mol) dissolved in ethanol (20 mL)
was added 15 mL of 6 N NaOH and 10% iodine solution
(in potassium iodide) drop wise until the colour of iodine
persisted. e reaction mixture was refluxed for 5–7 h. On
completion of reaction, the contents were cooled to room
temperature. A solid mass separated was collected and
thoroughly washed with water and purified to obtain the
product.
General procedure for the synthesis of N-[5-(2-oxo-2H-
chromen-3-yl)-[1,3,4]oxadiazol-2-yl]-benzamide (8a–d)
To
a solution of 3-(5-amino-[1,3,4]oxadiazol-2-yl)-
chromen-2-one (0.01 mol) in absolute ethanol was
added substituted aromatic acid chloride and heated
gently under reflux for 6–8 h. After complexion of reac-
tion, the solvent was removed till small volume was left.
is was poured onto crushed ice; after cooling to room
temperature, solid separated was filtered, washed with
water, dried, and purified to get the title product.
N-[5-(2-Oxo-2H-chromen-3-yl)-[1,3,4]oxadiazol-2-yl]-
benzamide (8a): Yield: 44%; m.p.: 174–176°C; Rf (SS1):
0.66; IR (cm−1): 1159 (C-O-C), 1633 (C=N), 1730 (CO),
3284 (NH); 1H-NMR (ppm): 7.20–7.23 (M, 1H, ArH),
7.28–7.32 (m, 2H, H6,8), 7.43–7.47 (m, 2H, H5,7), 7.56–7.6
(m, 3H, ArH), 8.17 (s, 1H, H4), 9.92 (br, s, 1H, NH); Anal.
Calcd. for C18H11N3O4: C, 64.86; H, 3.33; N, 12.61. Found:
C, 64.74; H, 3.41; N, 12.53.
3-(5-Phenylamino-[1,3,4]oxadiazol-2-yl)-chromen-2-
one (6a): Yield: 55%; m.p.: 128–130°C; Rf (SS1): 0.65; IR
(cm−1): 1125 (C-O-C), 1451, 1505, 1588, 1615, 1720 (C=O),
1
3318 (NH); H-NMR (ppm): 6.8–6.85 (m, 2H, H6,8), 7.18–
7.22 (m, 3H, ArH), 7.41–7.46 (m, 2H, H5,7), 7.53 (d, J= 7.4
Hz, 2H, ArH), 8.2 (s, 1H, H4) 9.37 (br, s, 1H, NH); Anal.
Calcd. for C17H11N3O3: C, 66.88; H, 3.63; N, 13.76. Found:
C, 66.72; H, 3.67; N, 13.54.
3-[5-(4-Chloro-phenylamino)-[1,3,4]oxadiazol-2-yl]-
chromen-2-one (6b): Yield: 48%; m.p.: 175–177°C; Rf
(SS2): 0.67; IR (cm−1): 778 (C-Cl), 1071 (C-O-C), 1454,
1
1505, 1577, 1630 (C=N), 1728 (CO), 3317 (NH); H-NMR
4-Chloro-N-[5-(2-oxo-2H-chromen-3-yl)-[1,3,4]oxadi-
azol-2-yl]-benzamide (8b): Yield: 52%; m.p.: 203–204°C;
Rf (SS2): 0.65; IR (cm−1): 772 (C-Cl), 1140 (C-O-C), 1725
(ppm): 6.91–6.96 (m, 2H, H6,8) 7.22 (d, 2H, J= 8.0 Hz,
ArH), 7.32 (d, 2H, J= 8.2 Hz, ArH), 7.48–7.51 (m, 2H,
H5,7), 9.92 (br, s, 1H, NH), 8.46 (s, 1H, H4); Anal. Calcd. for
C17H10ClN3O3: C, 60.10; H, 2.97; N, 12.37. Found: C, 60.18;
H, 2.92; N, 12.45.
3-(5-p-Tolylamino-[1,3,4]oxadiazol-2-yl)-chromen-2-
one (6c): Yield: 51%; m.p.: 181–183°C; Rf (SS1): 0.71; IR
1
(CO), 3281 (NH); H-NMR (ppm): 6.98 (d, 2H, J= 8.0 Hz,
ArH), 7.12 (br, s, 1H, NH), 7.18–7.23 (m, 2H, H6,8), 7.56–
7.61 (m, 2H, H5,7), 7.45 (d, 2H, J= 8.2 Hz, ArH), 8.21 (s, 1H,
H4); Anal. Calcd. for C18H10ClN3O4: C, 58.79; H, 2.74; N,
11.43. Found: C, 58.70; H, 2.68; N, 11.35.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and biological evaluation of 2,5-disubstituted 1,3,4-oxadiazole derivatives 771
4-Methyl-N-[5-(2-oxo-2H-chromen-3-yl)-[1,3,4]oxadi-
solution. e homogenate was supplemented with
0.2 mL of 8.1% sodium dodecyl sulphate (SDS), 1.5 mL of
acetate buffer (pH 3.5), and 1.5 mL of 0.8% thiobarbituric
acid (TBA). e mixture was heated at 95°C for 60 min.
e cooled reactants were shaken vigorously for 1 min
and centrifuged for 10 min at 4000 rpm after supplement-
ing with 5 mL of a mixture of n-butanol and pyridine
(15:1 v/v). e supernatant organic layer was collected
and absorbance was measured at 532 nm on UV spec-
trophotometer. e results are expressed as nmoles of
malondialdehyde (MDA)/100 mg tissue, using extinction
coefficient 1.56 × 105 per cm/M.
azol-2-yl]-benzamide (8c): Yield: 58%; m.p.: 180–188°C;
Rf (SS1): 0.69; IR (cm−1): 1162 (C-O-C), 1614 (C=N), 1721
1
(CO), 3210 (NH); H-NMR (ppm): 2.3 (s, 3H, CH3), 7.38
(d, J= 8.1 Hz, 2H, ArH), 7.28–7.35 (m, 2H, H6,8), 7.45–7.49
(m, 2H, H5,7), 7.54 (d, J= 8.0 Hz, 2H, ArH), 8.05 (s, 1H, H4),
8.55 (br, s, 1H, NH); Anal. Calcd. for C19H13N3O4: C, 65.70;
H, 3.77; N, 12.10. Found: C, 65.79; H, 3.65; N, 12.16.
4-Methoxy-N-[5-(2-oxo-2H-chromen-3-yl)-[1,3,4]
oxadiazol-2-yl]-benzamide (8d): Yield: 54%; m.p.:
168–170°C; Rf (SS1): 0.66; IR (cm−1): 1162 (C-O-C), 1618
1
(C=N), 1738 (CO), 3212 (NH); H-NMR (ppm): 3.71 (s,
3H, OCH3), 6.94 (d, J= 7.8 Hz, 2H, ArH), 7.16–7.20 (m, 2H,
H6,8), 7.54 (d, J= 8 Hz, 2H, ArH), 7.64–7.7 (m, 2H, H5,7),
8.12 (s, 1H, H4), 10.3 (br, s, 1H, NH); Anal. Calcd. for
C19H13N3O5: C, 62.81; H, 3.61; N, 11.57. Found: C, 62.76;
H, 3.54; N, 11.45.
COX inhibition studies
Selected 1,3,4-oxadiazole derivatives (4a, 4b, 4c, 4e,
6b, 6e, 8b) were tested for their ability to inhibit COX-1
and COX-2 using a COX-(ovine) inhibitor screening kit
(Catalogue No. 560101; Cayman Chemical, Ann Arbor,
MI). Stock solutions of test compounds were dissolved in
a minimum volume of dimethyl sulphoxide (DMSO) to
obtain 0.001, 0.01, 0.1, 1, 10, 100, and 500 μM in a final
volume of 1 mL. In brief, haematin reconstituted purified
COX-1 and COX-2 enzymes in a reaction buffer contain-
ing Tris–hydrochloric acid (HCl) (0.1 M, pH 8.0), 5 mM
ethylenediaminetetraacetic acid (EDTA), and 2 mM phe-
nol were pre-incubated at room temperature for 1 h with
test compounds followed by the addition of AA (100 μM)
for 2 min at 37°C. Reactions were terminated by adding
50 μL of 1 M HCl followed by the addition of 100 μL of
stannous chloride. e final product PGF2α formed was
measured by enzyme immunoassay (EIA) and the dose
that causes half-maximal inhibition (IC50) values were
determined following the instructions given in the kit
manual. Percent inhibition was calculated by compari-
son of compound treated to various control incubations.
e concentration of the test compound causing 50%
inhibition (IC50, μM) was calculated from the concentra-
tion–inhibition response curve.
Anti-inflammatory assay
e test compounds (4a–e, 6a–f, and 8a–d) were evalu-
ated using the in vivo rat carrageenan-induced foot
paw oedema at 20 mg/kg body weight as reported in
literature45.
Analgesic assay
Compounds (4a, 4b, 4c, 4e, 6b, 6e, 8b) that showed
70% and above inhibition in carrageenan-induced
oedema were screened for analgesic activity at 20 mg/
kg body weight using the 4% sodium chloride-induced
writhings (abdominal constriction) assay as reported in
literature46.
Acute ulcerogenesis assay
Compounds (4a, 4b, 4c, 4e, 6b, 6e, 8b) were tested for
acute ulcerogenic studies by reported method of Cioli
et al47. e studies were carried out on healthy Wistar rats
(150–200 g) at a dose three times the anti-inflammatory
dose viz. 60 mg/kg. e animals were divided into dif-
ferent groups of six each, group I served as control and
received vehicle only and group II received pure ibu-
profen 60 mg/kg. Other groups were administered test
compounds in dose molecularly equivalent to 60 mg/
kg of ibuprofen. e animals were fasted 8 h prior to a
single dose of each of the vehicle, standard and test
compounds, respectively, and sacrificed 17 h later dur-
ing which period food and water were available. e
animals were sacrificed and gastric mucosa of the rats
was examined for lesions and ulcers by means of a 4×
binocular magnifier. e scoring was done according to
the reported procedure47.
LOX inhibition assay
LOX-549, LOX-1250, and LOX-1551 activities were carried
out on 4a, 4c, 4e following the procedures as described
in literature. e enzyme sources for LOX-5, LOX-12,
and LOX-15 are human peripheral blood mononuclear
cell leukocyte (PBML) cells, human platelets, and rab-
bit reticulocytes, respectively. e substrates for LOX-5,
LOX-12, and LOX-15 used in the assays were endogenous
AA, 30 mM AA, and 256 mM linoleic acid, respectively.
e enzyme inhibition was quantitated by measuring
LTB4, 12-hydroxy eicosatetraenoic acid (12-HETE), and
15-HETE by EIA and spectrophotometrically.
Lipid peroxidation assay
Lipid peroxidation studies were carried out according to
the method of Ohkawa et al48. After scoring the gastric
mucosa of animals for ulcerogenic effect of synthesized
drugs, the gastric mucosa of animals was scraped with
two glass slides, weighed (100 mg), and homogenized
in 1.8 mL of 1.15% ice-cold potassium chloride (KCl)
Results and discussion
Chemistry
e synthetic route used to synthesize the compounds is
outlined in Scheme 1. 2,5 Disubstituted 1,3,4-oxadiazole
(4a–e) derivatives were obtained from Schiff bases
© 2011 Informa UK, Ltd.

772 M. Akhter et al.
O
O
O
O
O
O
H
N
O
NH2
(1)
(2)
CHO
R
NCS
CNBr
O
R
O
O
O
O
R
O
O
H
N
S
O
H
N
R
N
NH2
NH
N
H
N
R
(7)
N
O
(3a-e)
(5a-f)
FeCl3
O
COCl
KI/I
EtOH KOH
O
O
O
N
R
O
N
O
O
R
O
NH
O
N
O
N
NH
R
N
N
(4a-e)
a, R=H
a, R=H
b, R=4-Cl
c, R=4-CH3
d, R=4-OCH3
e, R=2,4-diCl
b, R=4-Cl
c, R=4-CH3
d, R=4-OCH3
e, R=2-CH3
f, R=3-Cl
a, R=H
(8a-d)
(6a-f)
b, R=4-Cl
c, R=4-CH3
d, R=4-OCH3
Scheme 1. Synthetic route for the title compounds.
(3a–e) of compound (2) by cyclization using aqueous
ferric chloride solution. Aryl semicarbazides (5a–f) were
obtained by treating compound (2) with different aryl
isothiocyanates in alcohol followed by cyclization to
corresponding 1,3,4-oxadiazole (6a–f) derivatives using
alkaline iodine solution. 2-(Coumarin-3yl)-5-amino-1-
,3,4-oxadiazole (7) was condensed with various acid
chlorides in pyridine to yield N-substituted 1,3,4-oxadi-
azoles (8a–d). e structures of synthesized compounds
were confirmed on the basis of different spectral studies.
e physical data, FTIR and 1H-NMR spectral data for all
the synthesized compounds are reported in experimen-
tal protocols.
the compounds (3a–e), the singlet signals belonging to
benzylidene (=CH-Ar) group were observed at aromatic
region, and disappearance of the signals belonging to
–NHNH2 indicated functionalization of hydrazide (2) to
hydrazone (3a–e). Disappearance of resonance due to
benzylidene group supported the formation of oxadiaz-
ole nucleus (4a–e), –NH– proton was observed as broad
singlet between 8 and 11 ppm (probably due to their
ability to get exchanged with D2O) each signal showing
integration for one proton. H4 of coumarin was located as
singlet between 8 and 8.5 ppm.
Biological activity
e FTIR spectra of the compounds synthesized
exhibited very similar features and showed the expected
bands for the characteristic groups that are present in the
compounds such as C=O and the C=N stretching vibra-
tions. In case of 1,3,4-oxadiazole derivatives, the presence
of C=N stretching band at 1645–1597 cm−1 and (C-O-C)
stretching band at 1170–1065 cm−1 is an evidence of ring
closure. e N-H stretching bands of the compounds
were observed between 3410 and 3210 cm−1. In the pro-
ton NMR spectral data, all protons were seen according
to the expected chemical shift and integral values. e
aromatic protons appeared in the range 6.6–8.5ppm. For
We have designed some new 1,3,4-oxadiazole derivatives
containing coumarin moiety and evaluated for their anti-
inflammatory activity. Anti-inflammatory activity was
checked by their ability to inhibit carrageenan-induced
inflammation in vivo. Compounds that showed 70% and
above inhibition in oedema were further screened for
analgesic activity, acute ulcerogenicity, as well as lipid
peroxide profile. Selected compounds were screened for
their ability to inhibit COX-1, COX-2, LOX-5, LOX-12, and
LOX-15 enzymes in vitro.
e anti-inflammatory activity was carried out by
Winter et al45. method, analgesic activity by Fukawa et al46.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and biological evaluation of 2,5-disubstituted 1,3,4-oxadiazole derivatives 773
method, acuteulcerogenicactivitybyCiolietal47. method,
4e towards COX-2 (COX-1 IC50 = 41.6 μM; COX-2 IC50 = 0.4
μM, SI for COX-2 = 104) (Table 2). Compound 4e was also
good in inhibiting LOX enzyme in in vitro analysis com-
pared with standard.
and lipid peroxidation studies by Ohkawa et al48. method.
e data are presented in Table 1. IC50 values for inhibi-
tion of COX-1 and COX-2 enzymes by these compounds
were determined by an EIA. Inhibition of human LOX-5
from human PBML cells, LOX-12 from human platelets,
and LOX-15 from rabbit reticulocytes were determined
by EIA and spectrophotometric quantitation.
Structure–activity relationship
A comparison of the structure–activity relationship (SAR)
data for the 2,5 disubstituted 1,3,4-oxadiazole derivatives
showed that the presence of a substitution with positive
hydrophobicity and electronic effect are good for anti-in-
flammatory activity like chloro substitution at p-position
of phenyl ring at fifth position of oxadiazole (4b). It was
observed that changing hydrophobicity (σ) or electronic
(π) parameter adversely affected the activity, decrease
in activity was observed by replacing chloro group with
methyl (4c) (+π and −σ) or methoxy group (4d) (– π and
–σ). Accordingly the activity should then increase with
increase in the πand σvalues. erefore, effect of addition
of another chlorine group in the molecule was observed.
e results showed that the activity indeed increased by
adding another chloro group into the nucleus (4e) con-
firming the above observation.
e anti-inflammatory activity of all the oxadiazole
derivatives (4a–e, 6a–f, and 8a–d) synthesized is pre-
sented in Table 1. e percent oedema inhibition relative
to control was measured after 2 and 3h of the treatment
and the inhibition of swelling in carrageenan-induced
oedema in rat paw brought about by oral administration
of the drugs is reported as paw volume SEM and per-
centage inhibition in oedema (Table 1). e percentages
of inhibition in swelling by the compounds were calcu-
lated using Equation (1).
Inhibition (%)






[(Vt −Vo ) control −(Vt −Vo ) treated]
(Vt −Vo )control
=
×100
(1)
Compounds (6a–f and 8a–d) exhibited less activity
comparatively, which may be due to increase the dis-
tance between the oxadiazole nucleus and phenyl ring
due to presence of –NH–, and –NHCO– group between
the two rings. e presence of single bond also results
in formation of large number of conformers, which
may be another reason for less activity of these deriva-
tives. Compounds (6b, 6e, and 8b) showed inhibition
of oedema by 74%, 72%, and 77%, respectively, but they
were nonselective in nature in inhibiting COX-1 and COX
-2 (COX-1 IC50 >100, >100, >100 μM; COX-2 IC50 > 100,
36.6, >100 μM, respectively) in in vitro analysis (Table 2).
Vt and Vo relates to the average volume in the hind paw
of the rats (n= 6) before any treatment and after anti-
inflammatory agent treatment, respectively.
e inhibition of oedema observed was in the range
of 35% to 89%. Compound 4e was the most active com-
pound (89% inhibition of oedema) and was found potent
than ibuprofen and equivalent to celecoxib in inhibit-
ing the oedema at 3 h. Compounds (4a, 4b, 4c, 4e, 6b,
6e, 8b) with percentage inhibition >70% were tested for
their ability to inhibit COX and LOX enzymes in vitro.
e in vitro COX inhibition assay showed selectivity of
Table 1. Biological evaluation of synthesized 2,5-disubstituted 1,3,4-oxadiazole derivatives.
Inhibition in oedema
(%)
Analgesic activity
(% Protection)
Ulcerogenic activity
[severity index
(SI) SEM]
Paw volume
nmol MDA content
SEM/100mg tissue
Entry
4a
4b
4c
4d
4e
6a
6b
6c
6d
6e
6f
8a
8b
8c
8d
Cel.
Ibu.
C
2 h
3 h
2 h
59
63
57
39
63
25
49
39
15
37
32
29
52
40
48
67
64
—
3 h
80
86
75
63
89
61
74
69
53
72
57
35
77
67
65.4
90
86
—
3 h
68.9
70
0.31 0.055
0.28 0.037
0.32 0.051
0.46 0.035
0.28 0.027
0.56 0.080
0.38 0.049
0.46 0.065
0.64 0.049
0.47 0.045
0.51 0.043
0.53 0.036
0.36 0.034
0.45 0.038
0.39 0.059
0.25 0.041
0.27 0.063
0.75 0.043
0.16 0.012
0.11 0.050
0.20 0.026
0.30 0.025
0.09 0.040
0.32 0.045
0.21 0.031
0.25 0.037
0.38 0.033
0.23 0.080
0.35 0.044
0.53 0.109
0.19 0.073
0.27 0.068
0.28 0.127
0.08 0.076
0.11 0.078
0.81 0.070
0.42 0.08
3.04 0.53
4.12 0.29
4.62 0.36
—
0.66 0.10
58.5
—
0.71 0.19
—
73.5
—
0.33 0.15
2.82 0.25
—
—
62.7
—
0.83 0.24
4.82 0.5
—
—
—
—
—
51.4
—
0.66 0.16
4.42 0.29
—
—
—
—
—
60.4
—
0.75 0.25
3.78 0.9
—
—
—
—
—
—
—
71.9
—
2.0 0.13
00
6.8 0.58
1.96 0.31
SI, the mean score of each treated group minus the mean score of the control group was considered as the “severity index” of gastric.
Cel., Celecoxib; Ibu., ibuprofen, C, control.
© 2011 Informa UK, Ltd.

774 M. Akhter et al.
LOX inhibition studies of 4a, 4c, and 4e showed that
these molecules have a moderate to good activity towards
LOX-5 and LOX-15 and very low activity towards LOX-12.
Compound 4e was the most active against LOX-5 and
LOX-15 with 48% and 19% inhibition at 10 μM, respec-
tively, and was found also more effective than the stan-
dard in inhibiting LOX-5 and LOX-15 (Table 3).
Compounds 4a, 4b, 4c, 4e, 6b, 6e, 8b were screened
for analgesic activity, acute ulcerogenicity, as well as lipid
peroxide profile. e analgesic activity of the compounds
was done at the same dose as used for anti-inflammatory
activity. e percent protection in mice brought about
by administration of the drugs is shown in Table 1. e
compounds tested showed analgesic activity in the range
of 51–74%. e percent protection was calculated using
Equation (2).
4c, 4e, 6b, 6e, 8b) was appreciably less than ibuprofen.
Less number of ulcers was seen in animals treated with
test compounds compared with the animals treated
with ibuprofen. e tested compounds showed severity
index ranging from 0.33 to 0.83, whereas the standard
drug ibuprofen showed severity index of 2.0 (Table 1).
Compounds 4a and 4e showed severity index of 0.42 and
0.33, respectively, which is less than one-fourth of the
value of ibuprofen. ese findings support the statement
that these compounds are relatively selective for COX-2.
Compounds that are less irritant to gastric mucosa are
also reported to show reduced MDA content, a by-prod-
uct of lipid peroxidation48. erefore, by determining the
MDA levels it can be ascertained that the compounds are
actually less irritant to gastric mucosa. To correlate the
ulcerogenic profile of compounds, the lipid peroxidation
values were also determined. e lipid peroxidation was
measured as nmoles of MDA/100 mg of tissue. Animals
treated with ibuprofen exhibited 6.8, whereas control
group showed 1.96 and the groups treated with synthe-
sized compounds showed lipid peroxidation in the range
of 2.5–5 (Table 1), suggesting that these derivatives are
less irritant to gastric mucosa.
Protection (%)


number of writhings in test
= 100 −
(2)




number of writhing in control ×100
Compound 4e showed 73.5% of protection against
sodium chloride-induced writhings compared with
71.9% protection with ibuprofen. Similar SAR pattern
in analgesic effect of compounds was observed as was
seen in anti-inflammatory activity. Compound bearing
electronegative group on phenyl ring directly attached
to the oxadiazole nucleus exhibited good analgesic effect
compared with other derivatives.
Conclusion
e studies showed that the dual inhibition of COX and
LOX as promising strategy for treating inflammatory con-
ditions due to their less associated side effects. Fifteen
new 2,5-disubstituted 1,3,4-oxadiazoles were success-
fully synthesized and characterized. All the compounds
showed anti-inflammatory profile with fewer side effects
and also exhibited protection against sodium chloride-
induced writhings. Compounds 4a, 4b, and 4e were most
active compounds. Compound 4a was also found selec-
tive for COX and LOX both.
Oxadiazole derivatives therefore present an opportu-
nity to develop new NSAIDs with reduced side effects.
e capacity to block both COX and LOX appears advan-
tageous as this will help in developing agents with less
unwanted effects because of increased expression of LOX
otherwise. ese observations suggest that these deriva-
tives could be used to develop leads for more potent dual
inhibitors of COX and LOX as nonsteroidal anti-inflam-
matory agents.
e acute ulcerogenic effect of synthesized com-
pounds was studied at 60 mg/kg in rats. It was observed
that the ulcerogenic effect of test compounds (4a, 4b,
Table 2. In vitro COX inhibition data for 1,3,4-oxadiazole
derivatives (4a, 4b, 4c, 4e, 6b, 6e, 8b).
IC50 (µM)
COX-2 selectivity
Entry
COX-1a
>100
35
COX-2a
indexb
>12
29
4a
8.7
4b
1.2
4c
>100
41.6
1.18
0.4
>100
36.6
>100
0.09
>85
104
0
4e
6b
>100
>100
>100
30.5
6e
3
8b
0
Celecoxib
339
aValues are means of two determinations acquired using an
ovine COX-1/COX-2 assay kit (Catalogue No. 560101; Cayman
Chemicals Inc., Ann Arbor, MI) and the deviation from the mean
is <10% of the mean value.
Declaration of interest
bIn vitro COX-2 selectivity index (COX-1/COX-2 IC50).
e authors are thankful to UGC for financial support.
e authors are also grateful to Janab Abdul Mueed Sb.
for providing infrastructure and facility to carry out this
work.
Table 3. In vitro LOX inhibition data at 10 µM for 1,3,4-
oxadiazole derivatives (4a, 4b, 4c).
Entry
LOX-5
86
LOX-12
LOX-15
37
4a
9
4
1
7
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