Fenbufen based 3-[5-(substituted aryl)-1,3,4-oxadiazol-2-yl]-1-(biphenyl-4-yl)propan-1-ones as safer antiinflammatory and analgesic agents

Article abstract of DOI:10.1016/j.ejmech.2009.04.009

The synthesis of a series of 3-[5-(substituted aryl)-1,3,4-oxadiazol-2-yl]-1-(biphenyl-4-yl)propan-1-ones derived from 4-oxo-4-(biphenyl-4-yl)butanoic acid (fenbufen) is described. The structures of these compounds were supported by IR, ¹H NMR, mass spectrometric data and elemental analysis. These compounds were tested for their antiinflammatory, analgesic, ulcerogenic and lipid peroxidation actions. A few compounds were found to have very good antiinflammatory activity in carrageenan induced rat paw edema test, while a fair number of compounds showed significant analgesic activity in acetic acid induced writhing test. The newly synthesized compounds showed very low ulcerogenic action with reduced malondialdehyde (MDA) content, which is one of the byproducts of lipid peroxidation. In vitro COX-1 and COX-2 isozyme inhibition studies were also performed on some of the selected compounds. Compound 4i and 4h were found to be more selective towards COX-2 as indicated by COX-2 selectivity index of 36.06 and 29.05 (COX-2 IC₅₀ = 1.5 μM and 1.8 μM) respectively.

Full text of DOI:10.1016/j.ejmech.2009.04.009

European Journal of Medicinal Chemistry 44 (2009) 3798–3804
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Fenbufen based 3-[5-(substituted aryl)-1,3,4-oxadiazol-2-yl]-1-
(biphenyl-4-yl)propan-1-ones as safer antiinflammatory and analgesic agents
,
b
,
,
,
,4
Asif Husain a , Ausaf Ahmad 1, M.M. Alam a 2, Mohd. Ajmal a 3, Priyanka Ahuja a
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, New Delhi 110062, India
b Department of Biochemistry and Biophysics, Box 712, University of Rochester Medical Center, Rochester, NY 14642, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a series of 3-[5-(substituted aryl)-1,3,4-oxadiazol-2-yl]-1-(biphenyl-4-yl)propan-1-ones
derived from 4-oxo-4-(biphenyl-4-yl)butanoic acid (fenbufen) is described. The structures of these
compounds were supported by IR, 1H NMR, mass spectrometric data and elemental analysis. These
compounds were tested for their antiinflammatory, analgesic, ulcerogenic and lipid peroxidation actions.
A few compounds were found to have very good antiinflammatory activity in carrageenan induced rat
paw edema test, while a fair number of compounds showed significant analgesic activity in acetic acid
induced writhing test. The newly synthesized compounds showed very low ulcerogenic action with
reduced malondialdehyde (MDA) content, which is one of the byproducts of lipid peroxidation. In vitro
COX-1 and COX-2 isozyme inhibition studies were also performed on some of the selected compounds.
Compound 4i and 4h were found to be more selective towards COX-2 as indicated by COX-2 selectivity
Received 30 March 2008
Received in revised form
26 March 2009
Accepted 2 April 2009
Available online 14 April 2009
Keywords:
1,3,4-Oxadiazole
Fenbufen
Antiinflammatory
Analgesic
Ulcerogenic
index of 36.06 and 29.05 (COX-2 IC50 ¼ 1.5
m
M and 1.8
m
M) respectively.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Lipid peroxidation
1. Introduction
modification have been taken with the aim of improving NSAIDs’
safety profile [7–12]. Studies by us [7,8] and others [9–12] have
shown that derivatization of the carboxylate function of some
NSAIDs resulted in an increased antiinflammatory activity with
reduced ulcerogenic effect.
During recent years there has been a large investigation on
different classes of oxadiazole compounds many of which were
found to possess an extensive spectrum of pharmacological
activities. In particular, compounds bearing 1,3,4-oxadiazole
nucleus are known to exhibit unique antiedema and antiin-
flammatory activities [13–15]. Differently substituted oxadiazole
moiety has also been found to have other interesting activities
such as analgesic, antibacterial, antifungal, anticonvulsant, anti-
cancer, etc. [16–20].
In our attempt to discover safer agents for treatment of
inflammatory conditions, we have replaced the carboxylic acid
group of fenbufen with an additional heterocycle i.e. 1,3,4-oxadia-
zole. These new compounds have been found to possess potential
antiinflammatory and analgesic activities with significant reduc-
tion in their ulcerogenic effect.
Non-steroidal antiinflammatory drugs (NSAIDs) are the most
commonly prescribedmedications in theworld. Theyare used for the
treatment of pain, fever and inflammation, particularly arthritis [1,2].
The most prevalent side effects of the use of non-steroidal antiin-
flammatory drugs are the occurrence of gastrointestinal damage
with gastric upset and irritationbeing the major problems [3,23]. The
search for safer NSAIDs continues with the failure of anticipated
‘Ideal’ antiinflammatory agents, the coxibs, on long-term usage [4,5].
Fenbufen 3 is an example of a well-known aroylpropanoic acid
class of antiinflammatory drugs and available in the market under
the name of Cinopal [6]. Aroylpropanoic acids including fenbufen
are good antiinflammatory agents but produce gastrointestinal
side effects [6,7]. Synthetic approaches based upon chemical
* Corresponding author. Tel.: þ9111 26059681/26059688x5647, mobile: þ91 989
1116086; fax: þ91 11 16988874.
E-mail address: drasifhusain@yahoo.com (A. Husain).
1
Tel.: þ1 585 275 6642. ausaf_ahmad@urmc.rochester.edu.
2
Tel.: þ91 11 26059681/26059688x5645, mobile: þ91 986811258; fax: þ91 11
16988874. hamlucky@rediffmail.com.
2. Chemistry
3
Tel.: þ91 11 26059681/26059688x5614, mobile: þ91 9927550544; fax: þ91 11
16988874. ajmal562003@yahoo.co.in.
4
3-[5-(Substituted
aryl)-1,3,4-oxadiazol-2-yl]-1-(biphenyl-4-
Tel.: þ91 11 26059681/26059688x5614, mobile: þ91 9891443528; fax: þ91 11
16988874. priyanka_amiable2000@yahoo.co.in.
yl)propan-1-ones 4a–l described in this study are shown in Table 1,
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.04.009

A. Husain et al. / European Journal of Medicinal Chemistry 44 (2009) 3798–3804
3799
Table 1
Biological activities of the compounds 4a–l.
N
N
O
R
O
Compound
Antiinflammatory activity
(% inhibition)
Analgesic activity
(% protection)
Ulcerogenic activity
(severity index)
Lipid peroxidationa
No.
R
4a
43.7 ꢀ 1.6**
46.8 ꢀ 1.0**
53.1 ꢀ 1.3
51.3 ꢀ 0.4**
66.1 ꢀ 0.6**
61.9 ꢀ 0.2**
56.3 ꢀ 0.4**
0.4 ꢀ 0.1**
0.5 ꢀ 0.1**
0.6 ꢀ 0.1**
0.7 ꢀ 0.1**
4.3 ꢀ 0.2**
Cl
4b
4c
4d
5.7 ꢀ 0.1**
5.8 ꢀ 0.1**
5.8 ꢀ 0.1**
Cl
AcO
43.7 ꢀ 1.1**
NO2
F
4e
4f
56.2 ꢀ 1.1
53.1 ꢀ 1.3
50.0 ꢀ 1.1*
59.4 ꢀ 2.3
45.8 ꢀ 0.3**
72.5 ꢀ 0.2**
45.1 ꢀ 0.1**
51.3 ꢀ 0.0**
0.5 ꢀ 0.2**
0.6 ꢀ 0.2**
0.5 ꢀ 0.2**
0.5 ꢀ 0.1**
4.3 ꢀ 0.2**
4.6 ꢀ 0.1**
5.8 ꢀ 0.2**
6.1 ꢀ 0.1**
CH3
OCH3
OCH3
4g
4h
4i
62.5 ꢀ 1.1*
57.3 ꢀ 0.0**
0.7 ꢀ 0.1**
4.9 ꢀ 0.1**
5.8 ꢀ 0.1**
OCH3
CH2
4j
50.0 ꢀ 1.1*
53.1 ꢀ 1.2
54.1 ꢀ 0.6
0.5 ꢀ 0.2**
0.6 ꢀ 0.1**
4k
55.8 ꢀ 0.0**
5.8 ꢀ 0.1**
CH2O
(continued on next page)

3800
A. Husain et al. / European Journal of Medicinal Chemistry 44 (2009) 3798–3804
Table 1 (continued )
Compound
No.
Antiinflammatory activity
(% inhibition)
Analgesic activity
(% protection)
Ulcerogenic activity
(severity index)
Lipid peroxidationa
R
CH2O
4l
59.4 ꢀ 1.4
68.6 ꢀ 0.0**
0.6 ꢀ 0.2**
5.9 ꢀ 0.1**
Diclofenac
Fenbufen
Control
65.6 ꢀ 1.3
56.3 ꢀ 1.3
70.3 ꢀ 0.1
54.1 ꢀ 0.0
–
2.3 ꢀ 0.2
1.5 ꢀ 0.2
0.00
8.9 ꢀ 0.1
6.8 ꢀ 0.1
3.8 ꢀ 0.1
*P < 0.05 compared to the parent drug (fenbufen), and data were given in mean ꢀ SEM and analyzed by ANOVA.
**P < 0.01 compared to the parent drug (fenbufen), and data were given in mean ꢀ SEM and analyzed by ANOVA.
a
Expressed as nmol MDA content/100 mg tissue.
and the reaction sequence for the preparation is outlined in Scheme
1. The required 4-oxo-4-(biphenyl-4-yl)butanoic acid (fenbufen) 3
was prepared by condensing biphenyl with succinic anhydride in
the presence of anhydrous aluminium chloride following Friedel–
Craft’s acylation reaction conditions [7]. Reaction between fenbu-
fen and aryl acid hydrazides 2a–l in phosphorous oxychloride
(reaction time varies from 2 to 5 h) afforded title compounds 4a–l
in 56–71% yield. The purity of compounds was checked by TLC and
elemental analysis.
revealed bands at 2975–2945 (CH2), 1650–1665 (C]O) and 1440–
1420 (C–N).
3. Pharmacological results and discussion
3.1. Antiinflammatory activity
Compounds 4a–l were evaluated for their in-vivo antiin-
flammatory activity by carrageenan induced paw edema method
[21]. The protocol of animal experiments has been approved by the
Institutional Animal Ethics Committee (IAEC). The compounds
were tested at 10 mg/kg oral dose and were compared with the
standard drug diclofenac and the parent drug fenbufen at the same
oral dose. The tested compounds showed antiinflammatory activity
ranging from 43.75% to 62.50% (Table 1). Compounds 4c, 4e, 4f, 4h,
4k and 4l were equipotent to fenbufen while 4i proved to be more
potent than fenbufen, its antiinflammatory activity being compa-
rable with that of diclofenac. Data show that the presence of 2-
naphtyloxymethyl, 4-methoxyphenyl or 3,4-dimethoxy phenyl
substitution at the 5 position of the oxadiazole ring can either
maintain or improve the antiinflammatory activity in this series of
fenbufen derivatives.
Analytical and spectral data (1H NMR, IR, mass spectra) of the
synthesized compounds were in full agreement with the proposed
structures. In general, The 1H NMR spectral data of the title
compounds showed two triplets of two protons each approxi-
mately at around
d 2.5 and d 3.5 ppm, respectively which are
assigned to two methylene protons (–CH2–CH2–). Other signals
were observed at appropriate places. The mass spectra showed
acylium fragments containing biphenyl and aryl moieties as major
peaks followed by peaks with loss of CO besides the molecular ion
peaks in reasonable intensities supporting the structure. In the
case of aryl group having chlorine atom as a substituent, the
molecular ion or other related ions produced the appropriate
isotopic abundances. The infrared spectral data (cmꢁ1
; KBr)
H2SO4
NH2NH2.H2O
R-COOH + C H OH
R-COOC H
R-CONHNH2
2a-l
2
5
2
5
1a-l
Succinic anhydride
Anhyd. AlCl3
O
OH
O
3
R-CONHNH2 (2a-l)
N
N
POCl3
O
R
O
4a-l
R=C6H5-, 2-Cl-C6H4-, 4-Cl-C6H4-, 2-OAc-C6H4-, 4-NO2-C6H4-,4-F-C6H4-, 4-CH3-C6H4-
-C H -,3,4-(OCH ) -C H -, C H CH -, C H -OCH -, 2-C H -OCH -
4-OCH3
6
4
3 2
6
3
6
5
2
6
5
2
10
7
2
Scheme 1.

A. Husain et al. / European Journal of Medicinal Chemistry 44 (2009) 3798–3804
3801
3.2. Analgesic activity
more favorable for COX-2 blocking. The parent drug fenbufen
showed selectivity index of 0.48.
The analgesic activity of the synthesized compounds was eval-
uated by acetic acid induced writhing test [22]. The newly
synthesized compounds showed activity ranging from 45.12% to
72.52%. The activity showed that compound 4f exhibited maximum
analgesic activity (72.52%) and its activity was better than that of
the standard drug diclofenac (70.32%). Compound 4l showed good
activity (68.57%). Seven compounds (4b, 4c, 4d, 4f, 4i, 4k and 4l)
out of twelve new compounds have been found to possess better
analgesic activity (55.80–72.52%) than that of fenbufen (54.12%).
The results are presented in Table 1.
4. Experimental protocols
4.1. Chemistry
Melting points were determined in open capillary tubes and are
uncorrected. Microanalysis of the compounds was done on Perkin–
Elmer model 240 analyzerand the values were found within ꢀ0.4% of
the theoretical values. The IR spectra were measured as potassium
bromide pellets using a Perkin–Elmer 1725X spectrophotometer. 1H
NMR spectra were recorded on Bruker spectropsin DPX-300 MHz
with tetramethylsilane (TMS) as an internal standard. The splitting
pattern abbreviations are as follows: s, singlet; d, doublet; dd, double
doublet; t, triplet; q, quartet; m, multiplet. Mass spectra were recor-
ded on a Jeol JMS-D 300 instrument fitted with a JMS 2000 data
systemat70 eV. Spectraldataareconsistentwithassignedstructures.
The progress of each reaction was monitored on silica gel G plates
using iodine vapors as visualizing agent.
It is concluded that the presence of 4-fluorophenyl or 2-naph-
thyloxymethyl or chlorophenyl substitution at position 5 in the
oxadiazole ring causes significant increase in analgesic activity.
3.3. Acute ulcerogenesis
All compounds were screened for their ulcerogenic activity
according to Cioli et al. [23]. The tested compounds showed
significant reduction in ulcerogenic activity ranging from 0.417 to
0.666, whereas the standard drug diclofenac and the parent drug
fenbufen showed high severity index, 2.332 and 1.500 (Table 1).
The results indicate that compounds showed very low ulcerogenic
potential. Thus it is concluded that transformation of the carboxylic
group of fenbufen into oxadiazole nucleus resulted in marked
decrease in ulcerogenic activity while retaining their high antiin-
flammatory and analgesic properties.
4.1.1. Aryl acid ethyl esters (1a–l) and their hydrazides (2a–l)
These compounds were obtained by the method reported in
literature [27].
4.1.1.1. Benzoic acid hydrazide (2a). Yield: 68%, m.p. 94–96 ꢂC. 1H
NMR (CDCl3)
d 4.29 (s, 2H, NH2), 7.47 (m, 3H, H-3,4,5), 7.63 (m, 2H,
H-2,6), 8.10 (s, 1H, CONH). MS: m/z 136 (Mþ), 105, 78, 77.
3.4. Lipid peroxidation study
4.1.1.2. 2-Chlorobenzoic acid hydrazide (2b). Yield: 72%, m.p. 88 ꢂC.
1H NMR (CDCl3)
d 4.50 (s, 2H, NH2), 7.19–7.25 (m, 4H, phenyl), 8.21
(s, 1H, CONH). MS: m/z 170 (Mþ), 105, 77.
Lipid peroxidation refers to the oxidative degradation of lipids.
This process proceeds by free radical chain reaction in which free
radicals abstract electrons from the lipids in cell membrane and
consequently damages the cell. It most often affects poly-
unsaturated fatty acids forming malondialdehyde (MDA). It is
evident that compounds showing less ulcerogenic activity also
showed reduced malondialdehyde (MDA) content, a byproduct of
lipid peroxidation [24]. Therefore, an attempt was made to corre-
late the low ulcerogenesis of the compounds with that of lipid
peroxidation. The lipid peroxidation was measured as nmol of
MDA/100 mg of tissue [25]. Diclofenac and fenbufen (standard and
parent drugs) produced high lipid peroxidation, 8.911 and 6.842
respectively, whereas the control group showed 3.788 nmol/
100 mg of tissue (Table 1). It was found that all compounds showing
low ulcerogenic activity also reduced lipid peroxidation. Therefore,
the study indicated that these oxadiazole derivatives have inhibited
the induction of gastric mucosal lesions and the results further
suggested that their protective effect might be related to the inhi-
bition of lipid peroxidation in the gastric mucosa.
4.1.1.3. 4-Chlorobenzoic acid hydrazide (2c). Yield: 71%, m.p. 98–
100 ꢂC. 1H NMR (CDCl3)
d
4.57 (s, 2H, NH), 7.41 and 7.52 (d, each, 2ꢃ
A2B2, p-disubstituted phenyl), 8.27 (s,1H, CONH). MS: m/z 170 (Mþ),
139, 105, 77.
4.1.1.4. 2-Acetoxybenzoic acid hydrazide (2d). Yield: 85%, m.p.
82 ꢂC. 1H NMR (CDCl3)
d 4.03 (s, 3H, OCH3), 4.88 (s, 2H, NH), 7.17–
7.21 (m, 4H, phenyl), 8.42 (s, 1H, CONH). MS: m/z 194 (Mþ), 105, 77.
4.1.1.5. 4-Nitrobenzoic acid hydrazide (2e). Yield: 75%, m.p. 70 ꢂC.
1H NMR (CDCl3)
d
4.79 (s, 2H, NH), 7.73 and 7.91 (d, each, 2ꢃ A2B2,
p-disubstituted phenyl), 8.60 (s, 1H, CONH). MS: m/z 181 (Mþ), 135,
105, 77.
4.1.1.6. 4-Fluorobenzoic acid hydrazide (2f). Yield: 78%, m.p. 96–
98 ꢂC. 1H NMR (CDCl3)
d
4.60 (s, 2H, NH), 7.48 and 7.79 (d, each, 2ꢃ
A2B2, p-disubstituted phenyl), 9.01 (s, 1H, CONH). MS: m/z 154
(Mþ), 105, 77.
3.5. In vitro cyclooxygenase (COX) inhibition assays
4.1.1.7. 4-Methylbenzoic acid hydrazide (2g). Yield: 70%, m.p. 74 ꢂC.
The compounds (4e, 4h, 4i and 4l), which showed significant
antiinflammatory activity (>55% of inhibition in edema), were
further evaluated for their ability to inhibit cyclooxygenase (COX)
according to the method described by Jashim Uddin et al. [26]. In
vitro COX-1 and COX-2 enzyme inhibition data showed that the
compounds were more selective towards COX-2 than COX-1.
Compound 4i and 4h with 4-methoxy and 3,4-dimethoxy substi-
tution showed a selectivity index of 36.1 and 29 for COX-2 vs COX-1.
1H NMR (CDCl3)
d 1.77 (s, 3H, CH3), 4.08 (s, 2H, NH), 6.88 and 7.53
(d, each, 2ꢃ A2B2, p-disubstituted phenyl), 8.19 (s, 1H, CONH). MS:
m/z 150 (Mþ), 119, 91, 77.
4.1.1.8. 4-Methoxybenzoic acid hydrazide (2h). Yield: 68%, m.p.
78 ꢂC. 1H NMR (CDCl3)
d 3.87 (s, 3H, OCH3), 4.14 (s, 2H, NH), 6.96
and 7.60 (d, each, 2ꢃ A2B2, p-disubstituted phenyl), 8.21 (s, 1H,
CONH). MS: m/z 166 (Mþ), 135, 107, 77.
These compounds (4i and 4h) have IC50 value (COX-2) of 1.5
1.8 M and IC50 value (COX-1) of 54.1 M and 52.3 M, respectively,
suggesting that methoxy group helps the compound in orientation
mM and
m
m
m
4.1.1.9. 3,4-Dimethoxybenzoic acid hydrazide (2i). Yield: 74%, m.p.
71 ꢂC. 1H NMR (CDCl3)
d
3.96 (two closely spaced singlets, 2ꢃ

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A. Husain et al. / European Journal of Medicinal Chemistry 44 (2009) 3798–3804
OCH3), 4.53 (s, 2H, NH), 6.98 (d, 1H, H-5), 7.13 (d, 1H, H-2), 7.31 (dd,
4.1.3.5. 3-[5-(4-Nitrophenyl)-1,3,4-oxadiazol-2-yl]-1-(biphenyl-4-
1H, H-6), 8.19 (s, 1H, CONH). MS: m/z 196 (Mþ), 166.
yl)propan-1-one (4e). Yield: 56%, m.p. 122 ꢂC. IR (cmꢁ1, KBr): 2965,
1663, 1433. 1H NMR (CDCl3)
d
2.61 and 3.57 (t, each, 2ꢃ CH2), 7.44
4.1.1.10. Phenylacetic acid hydrazide (2j). Yield: 68%, m.p. 82 ꢂC.
(m, 3H, H-3,4,5, phenyl), 7.62 (m, 2H, H-2,6, phenyl), 7.71 and 7.85
(d, each, 2ꢃ A2B2, p-nitro phenyl), 7.74 and 7.98 (d, each, 2ꢃ A2B2, p-
disubstituted phenyl). MS: m/z 399 (Mþ), 181, 153, 77. Anal.
C23H17N3O4 (C, H, N).
1H NMR (CDCl3)
d 3.58 (s, 2H, CH2), 4.15 (s, 2H, NH), 6.98–7.18
(m, 5H, phenyl), 8.25 (s, 1H, CONH). MS: m/z 150 (Mþ), 119, 78,
77.
4.1.1.11. Phenoxyacetic acid hydrazide (2k). Yield: 72%, m.p. 78 ꢂC.
4.1.3.6. 3-[5-(4-Fluorophenyl)-1,3,4-oxadiazol-2-yl]-1-(biphenyl-4-
1H NMR (CDCl3)
d
4.56 (s, 2H, OCH2), 5.08 (s, 2H, NH), 7.21–
yl)propan-1-one (4f). Yield: 71%, m.p. 136–138 ꢂC. IR (cmꢁ1, KBr):
7.28 (m, 5H, phenyl), 8.63 (s, 1H, CONH). MS: m/z 166 (Mþ),
2970, 1653, 1420. 1H NMR (CDCl3)
d
2.55 and 3.52 (t, each, 2ꢃ CH2),
135, 77.
7.48 (m, 3H, H-3,4,5, phenyl), 7.66 (m, 2H, H-2,6, phenyl), 7.43 and
7.54 (d, each, 2ꢃ A2B2, p-fluoro phenyl), 7.05 and 7.84 (d, each, 2ꢃ
A2B2, p-disubstituted phenyl). MS: m/z 372 (Mþ), 181, 153, 77. Anal.
C23H17FN2O2 (C, H, N).
4.1.1.12. 2-Naphthoxy acetic acid hydrazide (2l). Yield: 76%, m.p.
105–107 ꢂC. 1H NMR (CDCl3)
d 4.78 (s, 2H, OCH2), 4.97 (s, 2H, NH),
7.19 (m, 2H, H-1,3), 7.94 (m, 5H, H-4,5,6,7,8), 8.56 (s, 1H, CONH). MS:
m/z 216 (Mþ), 128, 127.
4.1.3.7. 3-[5-(4-Methylphenyl)-1,3,4-oxadiazol-2-yl]-1-(biphenyl-4-
yl)propan-1-one (4g). Yield: 58%, m.p. 144 ꢂC. IR (cmꢁ1, KBr): 2975,
4.1.2. 4-Oxo-4-(biphenyl-4-yl)butanoic acid (fenbufen) (3)
1655, 1428. 1H NMR (CDCl3)
d 2.13 (s, 3H, CH3), 2.53 and 3.37 (t,
Fenbufen was prepared by the method reported in literature [7].
each, 2ꢃ CH2), 6.64 and 7.78 (d, each, 2ꢃ A2B2, p-disubstituted
phenyl), 7.42 (m, 5H, phenyl), 6.91 and 7.63 (d, each, 2ꢃ A2B2, p-
methyl phenyl). MS: m/z 368 (Mþ), 181, 153, 91, 77. Anal.
C24H20N2O2 (C, H, N).
Yield: 70%, m.p. 180 ꢂC, 1H NMR (CDCl3)
d
2.82 and 3.37 (t, each, 2ꢃ
CH), 7.45 (m, 3H, H-3,4,5, phenyl), 7.64 (m, 2H, H-2,6, phenyl), 7.70
and 8.07 (d, each, 2ꢃ A2B2, p-substituted phenyl). MS: m/z 254
(Mþ), 181, 153, 77.
4.1.3.8. 3-[5-(4-Methoxyophenyl)-1,3,4-oxadiazol-2-yl]-1-(biphenyl-
4.1.3. General procedure for the synthesis of 3-[5-(substituted aryl)-
1,3,4-oxadiazol-2-yl]-1-(biphenyl-4-yl)propan-1-ones (4a–l)
Compounds 2a–l (0.001 mol) were dissolved in phosphorous
oxychloride (5 mL) and to it was added 3 (equimolar, 0.001 mol).
The reaction mixture, after refluxing for 2–5 h, was cooled to room
temp. and poured onto crushed ice. On neutralization of the
contents with sodium bicarbonate solution (20% w/v) a solid mass
separated out which was filtered, washed with water, dried and
recrystallized from methanol to give 4a–l.
4-yl)propan-1-one (4h). Yield: 65%, m.p. 154–156 ꢂC. IR (cmꢁ1
,
KBr): 2971, 1650, 1433. 1H NMR (CDCl3)
d 3.87 (s, 3H, OCH3), 2.61
and 3.19 (t, each, 2ꢃ CH2), 6.76 and 7.87 (d, each, 2ꢃ A2B2, p-
disubstituted phenyl), 7.53 (m, 5H, phenyl), 7.13 and 7.71 (d, each,
2ꢃ A2B2, p-methoxy phenyl). MS: m/z 384 (Mþ), 153, 77. Anal.
C24H20N2O3 (C, H, N).
4.1.3.9. 3-[5-(3,4-Dimethoxy phenyl)-1,3,4-oxadiazol-2-yl]-1-(biphe-
nyl-4-yl)propan-1-one (4i). Yield: 62%, m.p. 150 ꢂC. IR (cmꢁ1, KBr):
2970, 1655, 1425. 1H NMR (CDCl3)
d 3.95 (two closely spaced
4.1.3.1. 3-(5-Phenyl-1,3,4-oxadiazol-2-yl)-1-(biphenyl-4-yl)propan-
singlets, 6H, 2ꢃ OCH3), 2.57 and 3.45 (t, each, 2ꢃ CH2), 6.96 (d, 1H,
H-5, dimethoxy phenyl), 7.13 (d, 1H, H-2, dimethoxy phenyl), 7.34
(dd, 1H, H-6, dimethoxy phenyl), 7.46 (m, 3H, H-3,4,5, phenyl), 7.66
(m, 2H, H-2,6, phenyl), 7.71 and 8.17 (d, each, 2ꢃ A2B2, p-disubsti-
tuted phenyl). MS: m/z 414 (Mþ), 181, 153, 77. Anal. C25H22N2O4
(C, H, N).
1-one (4a). Yield: 62%, m.p. 148 ꢂC. IR (cmꢁ1, KBr): 2975, 1650,
1420. 1H NMR (CDCl3)
d
2.51 and 3.47 (t, each, 2ꢃ CH2), 7.48 (m, 6H,
H-3,4,5, 2ꢃ phenyl), 7.62 (m, 4H, H-2,6, 2ꢃ phenyl), 7.65 and 8.14 (d,
each, 2ꢃ A2B2, p-disubstituted phenyl). MS: m/z 354 (Mþ), 181, 105,
78, 77. Anal. C23H18N2O2 (C, H, N).
4.1.3.2. 3-[5-(2-Chlorophenyl)-1,3,4-oxadiazol-2-yl]-1-(biphenyl-4-
4.1.3.10. 3-(5-Benzyl-1,3,4-oxadiazol-2-yl)-1-(biphenyl-4-yl)propan-
yl)propan-1-one (4b). Yield: 58%, m.p. 158–160 ꢂC. IR (cmꢁ1, KBr):
1-one (4j). Yield: 56%, m.p. 158 ꢂC. IR (cmꢁ1, KBr): 2965, 1651
2967, 1650, 1435. 1H NMR (CDCl3)
d
2.55 and 3.43 (t, each, 2ꢃ CH2),
1430. 1H NMR (CDCl3)
d
2.39 and 3.58 (t, each, 2ꢃ CH2), 4.11 (s,
7.29 (m, 4H, H-3,4,5,6, o-chloro phenyl), 7.57 (m, 5H, phenyl), 7.73
and 7.84 (d, each, 2ꢃ A2B2, p-disubstituted phenyl). MS: m/z 388
(Mþ), 181, 153, 78, 77. Anal. C23H17N2O2Cl (C, H, N).
3H, CH2), 7.39 (m, 6H, H-3,4,5, 2ꢃ phenyl), 7.60 (m, 4H, H-2,6,
2ꢃ phenyl), 7.65 and 7.81 (d, each, 2ꢃ A2B2, p-disubstituted
phenyl). MS: m/z 368 (Mþ), 181, 153, 91, 77. Anal. C24H20N2O2
(C, H, N).
4.1.3.3. 3-[5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl]-1-(biphenyl-4-
yl)propan-1-one (4c). Yield: 61%, m.p. 146 ꢂC. IR (cmꢁ1, KBr): 2970,
4.1.3.11. 3-(5-Phenoxymethyl-1,3,4-oxadiazol-2-yl)-1-(biphenyl-4-
1655, 1430. 1H NMR (CDCl3)
d
2.51 and 3.57 (t, each, 2ꢃ CH2), 7.32
yl)propan-1-one (4k). Yield: 68%, m.p. 164 ꢂC. IR (cmꢁ1, KBr):
(m, 3H, H-3,4,5, phenyl), 7.38 (m, 2H, H-2,6, phenyl), 7.45 and 7.64
(d, each, 2ꢃ A2B2, p-disubstituted phenyl; biphenyl), 7.11 and 7.83
(d, each, 2ꢃ A2B2, p-chloro phenyl). MS: m/z 388 (Mþ), 181, 153, 77.
Anal. C23H17N2O2Cl (C, H, N).
2970, 1653, 1422. 1H NMR (CDCl3)
d
2.51 and 3.47 (t, each, 2ꢃ
CH2), 4.55 (s, 3H, OCH2), 7.46 (m, 6H, H-3,4,5, 2ꢃ phenyl), 7.66
(m, 4H, H-2,6, 2ꢃ phenyl), 7.71 and 7.88 (d, each, 2ꢃ A2B2, p-
disubstituted phenyl). MS: m/z 384 (Mþ), 181, 135, 77. Anal.
C24H20N2O3 (C, H, N).
4.1.3.4. 3-[5-(2-Acetoxyphenyl)-1,3,4-oxadiazol-2-yl]-1-(biphenyl-4-
yl)propan-1-one (4d). Yield: 66%, m.p. 138–140 ꢂC. IR (cmꢁ1
,
4.1.3.12. 3-[(5-(2-Naphthoxymethyl)-1,3,4-oxadiazol-2-yl)-1-(biphe-
KBr): 2955, 1660, 1425. 1H NMR (CDCl3)
d
2.39 (s, 3H, OCOCH3),
nyl-4-yl)propan-1-one (4l). Yield: 61%, m.p. 166 ꢂC. IR (cmꢁ1, KBr):
2.61 and 3.52 (t, each, 2ꢃ CH2), 7.48 (m, 3H, H-3,4,5, phenyl),
7.65 (m, 2H, H-2,6, phenyl), 7.25 (m, 4H, H-3,4,5,6, o-disubsti-
tuted phenyl), 7.77 and 7.84 (d, each, 2ꢃ A2B2, p-disubstituted
phenyl). MS: m/z 412 (Mþ), 181, 153, 93, 92, 78, 77. Anal.
C25H20N2O4 (C, H, N).
2965, 1655, 1440. 1H NMR (CDCl3)
d
2.51 and 3.47 (t, each, 2ꢃ CH2),
4.89 (s, 3H, OCH2), 7.19 (2H, H-1,3, naphthoxy), 7.48 (m, 3H, H-3,4,5,
phenyl), 7.67 (m, 2H, H-2,6, phenyl), 7.75 and 8.11 (d, each, 2ꢃ A2B2,
p-disubstituted phenyl), 7.94 (m, 5H, H-4,5,6,7,8, naphthoxy). MS:
m/z 434 (Mþ), 181, 153, 128, 77. Anal. C23H18N2O2 (C, H, N).

A. Husain et al. / European Journal of Medicinal Chemistry 44 (2009) 3798–3804
3803
4.2. Pharmacology
evaluating ulcerogenic activity, the gastric mucosa was scrapped
with two glass slides, weighed (100 mg) and homogenized in
1.8 mL of 1.15% cold potassium chloride solution. The homogenate
was supplemented with 0.2 mL of 8.1% sodium dodecyl sulfate,
1.5 mL of acetate buffer (pH 3.5) and 1.5 mL of 0.8% thiobarbituric
acid. The mixture was heated at 95 ꢂC for 1 h. After cooling, the
reactants were supplemented with 5 mL of the mixture of n-
butanol:pyridine (15:1 v/v), shaken vigorously for 1 min and
centrifuged for 10 min at 4000 rpm. The supernatant organic layer
was taken out and absorbance was measured at 532 nm on UV
spectrophotometer. The results were expressed as nmol MDA/
100 mg tissue, using extinction coefficient 1.56 ꢃ 105/cm/M.
4.2.1. Antiinflammatory activity
Antiinflammatory activity test was performed following the
method of Winter et al. [21]. Freshly prepared suspension of
carrageenan (0.05 mL, 1% w/v solution in 0.9% saline) was injected
under the plantar aponeurosis of the right hind paw of each rat.
Animals were divided in groups of six in each group. One group was
kept as control and the animals of other groups were pre-treated
with the test drugs suspended in 1% carboxymethylcellulose (CMC)
given orally 30 min before carrageenan injection. The paw volume
was measured before and after 4 h of carrageenan injection by
plethysmograph. The percentage inhibition of inflammation was
calculated by applying the following formula:
4.2.5. In vitro cyclooxygenase (COX) inhibition assays
The ability of the test compounds listed in Table 2 to inhibit
ovine COX-1 and COX-2 (IC50 value, mM) was determined using an
Antiinflammatoryactivity ð% inhibitionÞ ¼ ðVc ꢁ VtÞ=Vc ꢃ 100
enzyme immunoassay (EIA) kit (catalog no. 560101, Cayman
Chemical, Ann Arbor, MI, USA) according to the methodology
described by Jashim Uddin et al. [26]. Cyclooxygenase catalyzes the
where Vc ¼ edema volume in control group, Vt ¼ edema volume in
groups treated with test compounds.
first step in the biosynthesis of arachidonic acid (AA) to PGH2. PGF2
a
4.2.2. Analgesic activity
produced from PGH2 by reduction with stannous chloride is
measured by enzyme immunoassay (ACEÔ competitive EIA). Stock
solution of test compounds was dissolved in a minimum volume of
DMSO. Briefly, to a series of supplied reaction buffer solutions
Analgesic activity was carried out by acetic acid induced
writhing method [22] using albino mice (25–30 g) of either sex on
groups of six animals each. A 1% aqueous acetic acid solution (i.p.
injection; 0.1 mL) was used as writhing inducing agent. Mice were
kept individually in the test cage, before acetic acid injection and
habituated for 30 min. Screening of analgesic activity was per-
formed after p.o. administration of test drugs at the dose of 10 mg/
kg. All compounds were dissolved in 1% carboxymethylcellulose
(CMC) solution. One group was kept for the control experiment and
received p.o. administration of 1% CMC. Diclofenac and fenbufen
were used as reference drugs. After 1 h of drug administration
0.10 mL of 1% acetic acid solution was given to mice intraperito-
neally. Stretching movements consisting of arching of the back,
elongation of body and extension of hind limbs were counted for 5–
15 min of acetic acid injection. The analgesic activity was expressed
in terms of % inhibition. % Analgesic activity ¼ {(n–n0)/n} ꢃ 100
where n ¼ mean number of writhes of control group and n0 ¼ mean
number of writhes of test group. The percent protection in mice
brought about by administration of the drugs is shown in Table 1.
(960
phenol) with either COX-1 or COX-2 (10
of heme (10 L) were added 10 L of various concentrations of test
drug solutions (0.01, 0.1, 1, 10, 50 and 100 L in a final volume of
1 mL). These solutions were incubated for a period of 5 min at 37 ꢂC
after which 10 L of AA (100 M) solution were added and the COX
reaction was stopped by the addition of 50 L of 1 M HCl after
mL, 0.1 M Tris–HCl pH 8.0 containing 5 mM EDTA and 2 mM
mL) enzyme in the presence
m
m
m
m
m
m
2 min. PGF2 produced from PGH2 by reduction with stannous
a
chloride was measured by enzyme immunoassay. This assay is
based on the competition between PGs and PG–acetylcholine
esterase conjugate (PG tracer) for a limited amount of PG anti-
serum. The amount of PG tracer that is able to bind to the PG
antiserum is inversely proportional to the concentration of PGs in
the wells since the concentration of PG tracer is held constant while
the concentration of PGs varies. This antibody–PG complex binds to
a mouse anti-rabbit monoclonal antibody that had been previously
attached to the well. The plate is washed to remove any unbound
reagents and then Ellman’s reagent, which contains the substrate to
acetylcholine esterase, is added to the well. The product of this
enzymatic reaction produces a distinct yellow color that absorbs at
405 nm. The intensity of this color, determined spectrophotomet-
rically, is proportional to the amount of PG tracer bound to the well,
which is inversely proportional to the amount of PGs present in the
4.2.3. Acute ulcerogenesis
Acute ulcerogenesis test was done according to Cioli et al. [23].
Albino rats (150–200 g) were divided into different groups consist-
ing of six animals in each group. Ulcerogenic activity was evaluated
after p.o. administration of test compounds or diclofenac or fenbufen
at the dose of 30 mg/kg. Control rats received p.o. administration of
vehicle (suspension of 1% methylcellulose). Food but not water was
removed 24 h before administration of the test compounds. After the
drug treatment, the rats were fed normal diet for 17 h and then
sacrificed. The stomach was removed and opened along the greater
curvature, washed with distilled water and cleaned gently by
dipping in saline. The gastric mucosa of the rats was examined by
means of a magnifying glass. For each stomach, the severity of
mucosal damage was assessed by measuring severity index i.e.
severity of drug to cause mucosal damage. It is measured according
to the following scoring system: 0.5 – redness; 1.0 – spot ulcers; 1.5 –
hemorrhagic streaks; 2.0 – ulcers >3, but ꢄ5; 3.0 – ulcers > 5.
The mean score of each treated group minus the mean score of
the control group was considered as severity index of gastric
mucosal damage.
well during the incubation: Absorbance
a [Bound PG tracer] a1/
PGs. Percent inhibition was calculated by the comparison of
compound treated to various control incubations. The concentra-
tion of test compound causing 50% inhibition (IC50
,
mM) was
Table 2
In vitro COX-inhibition data for 4e, 4h, 4i and 4l.
Compound
COX-1a (IC50
,
m
M)
COX-2a (IC50
,
m
M)
COX-2 SIb
4e
4h
4i
4l
Celecoxib
Fenbufen
42.6
52.3
54.1
48.4
33.1
3.9
8.2
1.8
1.5
2.5
0.07
8.1
5.19
29.05
36.06
19.36
472
0.48
a
Values are means of two determinations acquired using an ovine COX-1/COX-2
assay kit (catalog no. 560101, Cayman Chemicals, MI, USA) and the deviation from
the mean is <10% of the mean value.
4.2.4. Lipid peroxidation study
Lipid peroxidation of the synthesized compounds was deter-
mined according to the method of Ohkawa et al. [25]. After
b
In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).

3804
A. Husain et al. / European Journal of Medicinal Chemistry 44 (2009) 3798–3804
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