Synthesis and analgesic activity of new 1,3,4-oxadiazoles and 1,2,4-triazoles

Article abstract of DOI:10.1007/s00044-010-9335-0

A series of new 1,3,4-oxadiazoles and 1,2,4- triazoles were synthesized in order to obtain new compounds with potential analgesic activity. Compounds were evaluated for their analgesic activities by formalin-induced nociception test. Mefenamic acid (as the reference drug) did not show any activity in the early phase of the formalin test, while compounds 7b, 7c, 8c, and 9a significantly reduced the nociception in this phase. However in the late phase of formalin test all of the target compounds and mefenamic acid showed analgesic activity in comparison to control.

Full text of DOI:10.1007/s00044-010-9335-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:435–442
DOI 10.1007/s00044-010-9335-0
ORIGINAL RESEARCH
Synthesis and analgesic activity of new 1,3,4-oxadiazoles
and 1,2,4-triazoles
•
Ali Almasirad Abbas Shafiee Mohammad Abdollahi
•
•
•
Amir Noeparast Nasir Shahrokhinejad Nasim Vousooghi
•
•
•
Sayyed Abbas Tabatabai Reza Khorasani
Received: 31 October 2009 / Accepted: 1 March 2010 / Published online: 18 March 2010
Ó Springer Science+Business Media, LLC 2011
Abstract A series of new 1,3,4-oxadiazoles and 1,2,4-
triazoles were synthesized in order to obtain new com-
pounds with potential analgesic activity. Compounds were
evaluated for their analgesic activities by formalin-induced
nociception test. Mefenamic acid (as the reference drug)
did not show any activity in the early phase of the formalin
test, while compounds 7b, 7c, 8c, and 9a significantly
reduced the nociception in this phase. However in the late
phase of formalin test all of the target compounds and
mefenamic acid showed analgesic activity in comparison to
control.
Introduction
Arachidonic acid (AA) contained in cellular membranes is
released by phospholipase A2 (PLA2) and further metabo-
lized by two major enzymatic pathways: 5-lipoxygenase
(5-LO) and cyclooxygenase (COX), leading to pro-
inflammatory leukotriens (LTs) and prostanoids, respec-
tively. LTB4 synthesized by 5-LO potently activates cell
migration and chemotaxis, as well as superoxide produc-
tion and lysosomal enzyme secretion in neutrophils.
Increased production of this eicosanoid has been involved
in hyperalgesic response and related with phathological
conditions such as rheumatoid arthritis and inflammatory
bowel disease (Nikfar et al., 1997; Hosseini-Tabatabaei
and Abdollahi 2008). It is interesting to note that COX
inhibition by conventional non-steroidal anti-inflammatory
drugs (NSAIDs) or by new COX-2 inhibitors leads to an
up-regulation of the 5-LO pathway, yielding various
adverse effects such as increase of gastrointestinal damage
and asthma (Celotti and Laufer 2001; Rainsford 1999;
Fosslien 1998). As a result, a new strategy is to minimize
gastric toxicity of NSAIDs considering the dual inhibition
of 5-LO and COX enzymes. In this scenario, various
structural families of dual inhibitors have been designed
and several compounds are currently undergoing preclini-
cal or clinical development (Charlier and Michaux 2003;
Moreau et al., 2006; Rao et al., 2005). Among these
compounds, some potent NSAIDs, fenamates, resulting
from the bioisosteric replacement of the carboxylic acid
moiety by a tetrazole, were first reported to inhibit COX
and to some extent, 5-LO. Then, several fenamates, among
which flufenamic acid 1, were converted to potent dual
COX/5-LOX inhibitors, after substitution of their carbox-
ylate moiety with other acidic heterocycles, namely 1,3,4-
oxadizole, 1,3,4-thiadiazole and 1,2,4-triazole. Compound
Keywords 1,3,4-Oxadiazole Á 1,2,4-Triazole Á
Analgesic activity Á Formalin test
A. Almasirad (&)
Department of Medicinal Chemistry, Pharmaceutical Sciences
Branch, Islamic Azad University, 1941933111 Tehran, Iran
e-mail: almasial@sina.tums.ac.ir
A. Shafiee
Department of Medicinal Chemistry, Faculty of Pharmacy and
Pharmaceutical Sciences Research Center, Tehran University of
Medical Sciences, 14174 Tehran, Iran
M. Abdollahi Á A. Noeparast Á N. Shahrokhinejad Á
R. Khorasani
Department of Pharmacology and Toxicology, Faculty of
Pharmacy, and Pharmaceutical Sciences Research Center,
Tehran University of Medical Sciences, 14174 Tehran, Iran
N. Vousooghi
Department of Pharmacology, Faculty of Medicine, Shahid
Beheshti University of Medical Sciences, Tehran, Iran
S. A. Tabatabai
Department of Medicinal Chemistry, School of Pharmacy,
Shahid Beheshti University of Medical Sciences, Tehran, Iran
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Med Chem Res (2011) 20:435–442
NH
activity of new analogues of compound 2 by bioisosteric
replacement of NH with S and oxidation of S to SO and
SO2 in order to make this moiety as a real hydrogen bond
acceptor similar to NH in compound 2 and in the hope of
obtaining additional inhibitors of AA metabolism (Alm-
asirad et al., 2005; Almasirad et al., 2006; Rineh et al.,
2007; Shafiee et al., 2009).
N
COOH
NH
Y
X
NH
CF3
CF3
1
2
Results and discussion
X=O, N, S
Y=S, O
Chemistry
Fig. 1 Dual COX/5-LO inhibitor derived from flufenamic acid
The designed compounds were synthesized according to
Schemes 1 and 2. Compounds 4 and 5 were prepared by
oxidation of ester 3a with hydrogen peroxide (Shafiee
et al., 1998; Brannigan et al., 1976). The key intermediate
hydrazides 6a–6d were prepared from the reaction of
hydrazine hydrate with compounds 3a, 3b, 4, 5,
2 (X=O, Y=S) was the most potent synthesized analogue in
these series (Fig. 1). (Boschelli et al., 1992; Boschelli
et al., 1993).
As a part of our ongoing research program to find novel
anti-inflammatory, analgesic, and anticonvulsant com-
pounds, herein, we describe the synthesis and analgesic
Scheme 1 a CH3COOH, H2O2,
rt; b CH3COOH, H2O2, reflux,
and c NH2–NH2.H2O, EtOH, rt
O
O
C
OCH3
C
OCH3
R
a
O
S
S
R
3a, R=H
3b, R=Cl
4, R=H
b
O
C
OCH3
O
S
O
R
5, R=H
O
C
NH NH2
X
6a, X=S, R=H
6b, X=SO, R=H
6c, X=SO2, R=H
6d, X=S, R=Cl
C
R
3a, 3b, 4, 5
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437
Scheme 2 a CS2, KOH, reflux;
b BrCN, Dioxane, rt; and c (1)
KSCN, HCl, H2O (2) NaOH
4%, reflux
O
C
N
NH
N
X
N
X
NH NH2
NH2
S
O
O
a
b
X
R
R
R
6
8
7
8a, X=S, R=H
8b, X=SO, R=H
8c, X=SO2, R=H
8d, X=S, R=Cl
7a, X=S, R=H
7b, X=SO, R=H
7c, X=SO2, R=H
7d, X=S, R=Cl
c
NH
N
X
S
N
H
9a, X=S, R=H
9b, X=SO, R=H
9c, X=SO2, R=H
9d, X=S, R=Cl
R
9
respectively. 5-Aryl-1,3,4-oxadiazole-2(3H)-thiones 7a–7d
were prepared by reaction of hydrazides 6a–6d with KOH
and CS2. Reaction of hydrazides 6a–6d with cyanogen
bromide gave 5-aryl-2-amino-1,3,4-oxadiazoles 8a–8d.
5-aryl-1,2,4-triazole-3-thiones 9a–9d were synthesized via
reaction of 6a–6d with potassium thiocyanate and hydro-
chloric acid followed by cyclization of thiosemicarbazide
intermediate with aqueous sodium hydroxide (Almasirad
et al., 2007).
Table 1 Analgesic activity of compounds (7a–9c) against the early
phase (0–10 min) in formalin test
Compounda
Nociceptionb (Mean SEM)
Inhibitionc (%)
Control
73.1 6.3
55.5 5.9
63.5 4.7
43.2 3.4
42.5 2.9
60.8 4.4
61 5.9
–
Mefenamic acid
24.08
13.13
40.9*
41.86*
16.83
16.55
29.41
40.49*
26.67
49.38*
31.66
31.19
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
Pharmacology
51.6 3.6
43.5 4.7
53.6 7.2
37 7.6
The analgesic effect of target compounds 7a–9c is shown
in Tables 1 and 2. As seen in Table 1, compounds 7b, 7c,
8c, and 9a showed significant reduction in the early phase
of formalin-induced nociception. Mefenamic acid was
ineffective on this phase (neurogenic pain). As can be
observed from Table 1, oxidation of sulfur in compound 7a
to sulfoxide 7b or sulfone 7c increased the activity. How-
ever, in triazole series 9, oxidation of sulfur decreased the
activities (compounds 9b and 9c). In addition bioisosteric
replacement of oxygen atom in compound 7a with NH
increased the activity. In fact, compound 9a was the most
active compound. The effect of synthesized compounds on
inflammatory pain is summarized in Table 2. All of
the target compounds 7a–9c as well as mefenamic acid
showed significant analgesic activity in the late phase of
formalin test whereas compound 7b was more active than
mefenamic acid. The synthesized compounds generally
50 5.9
50.3 4.9
* Differed from control group at P \ 0.01
a
All the compounds were tested at the dose of 30 lmol/kg
There were 6 animals in each group
b
c
Inhibition percentage was calculated by potential of nociception
inhibition of test compounds in comparison to that of control
exhibited more activity during the inflammatory phase
(57.95–86.12, inhibition range) in comparison to neuro-
genic phase (13.13–49.38, inhibition range).
The pharmacological evaluation of this study showed a
good analgesic profile in comparison to control and me-
fenamic acid. Our results suggest that compounds 7b, 7c,
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Med Chem Res (2011) 20:435–442
Relative activityd
Table 2 Analgesic activity of compounds (7a–9c) against the late phase (10–30 min) in the formalin test
Compounda
Nociceptionb (mean SEM)
Inhibitionc (%)
Control
193.8 11.4
48.6 4.8
55.56 5.1
26.9 6.8
81.5 3.7
37.8 5.2
70.3 4.5
39 3.9
–
–
1**
Mefenamic acid
74.92
71.33
86.12
57.95
80.49
63.73
79.88
68.27
67.07
79.62
63.31
64.91
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
0.95**
1.15**
0.77**
1.06**
0.85**
1.07**
0.91*
0.89*
1.06*
0.85**
0.87**
61.5 5.5
63.8 6.7
39.5 3.8
71.1 7.3
68 3.6
*, ** Represent difference from control group at P \ 0.05 and P \ 0.01, respectively
a
All the compounds were tested at the dose of 30 lmol/kg
b
There were six animals in each group
c
Inhibition percentage was calculated by potential of nociception inhibition of test compounds in comparison to that of control
Relative activity was calculated by potential of nociception inhibition of test compounds in comparison to that of mefenamic acid
d
8c and 9a, unlike mefenamic acid as the reference drug and
pain generally responds poorly to conventional analgesics
and its treatment can be difficult (Davies et al., 1994; Niv
and Devor 2006). Therefore, discovery of new drugs for
treatment of this kind of pain is clinically relevant.
most of other NSAIDs have an additional and interesting
analgesic action, in that these compounds were effective on
neurogenic pain (Vaz et al., 1996; Correa and Calixto
1993; Malmberg and Yaksh 1992). The analgesic effect of
these compounds during the neurogenic phase of the for-
malin test suggests that they have a different or additional
mechanism of action than classical NSAIDs. However,
further studies are needed to determine that mecha-
nism(s) responsible for the analgesic action of com-
pounds 7b, 7c, 8c and 9a. These four compounds are
among the 1,3,4-oxadiazole-2(3H)-thiones, 2-amino-1,3,4-
oxadiazoles and 1,3,4-triazole-2-thiones respectively, so all
of these scaffolds can be active in the early phase but
according to the results, sulfoxide and sulfone moieties are
the common functional groups between three of these four
compounds. In contrast to the early phase, all of the target
compounds and mefenamic acid were active in the late
phase that can be explained by inhibition of the arachidonic
acid metabolic pathway (Hunskaar et al., 1985).The most
active compound 7b in the late phase, similar to the
compound 2 (X=O, Y=S) as a dual COX/5-LO inhibitor,
has the 1,3,4-oxadiazole-2-(3H)-thione heterocyclic ring so
the dual inhibition of COX/5-LO by these compounds is
likely to happen (Boschelli et al., 1992).
Experimental
Chemicals were purchased from Merck chemical company
(Tehran, Iran), Compounds 3a, 3b, 6a, 7a, 8a, 9a were
prepared according to the previously described methods
(Almasirad et al., 2007; Bentrude and Martin 1962; Dub-
uisson and Dennis 1977).
Melting points were taken on a Kofler hot stage appa-
ratus (Reichert, Vienna, Austria) and are uncorrected.
1H-NMR spectra were obtained using a Brucker FT-80
spectrometer (Brucker, Rheinstetten, Germany). Tetra-
methylsilane was used as an internal standard. Mass spectra
were obtained using a Finnigan Mat TSQ-70 spectrometer
at 70 eV (Finnigan Mat, Bremen, Germany). The IR spectra
were obtained using a Nicolet FT-IR Magna 550 spectro-
graphs (KBr disks) (Nicolet, Madision, WI, USA). The
purity of compounds was confirmed by TLC using different
mobile phases. The results of the elemental analyses (C, H,
N) were within 0.4% of theoretical values for C, H, and N.
Various oxadiazole and triazole derivatives were pre-
pared with the objective of developing new analgesic
agents. In summary, synthesized compounds showed anal-
gesic activity in both phases of formalin test indicating
involvement of different mechanisms of action. Neurogenic
Methyl 2-(phenylsulfinyl)benzoate (4, R=H)
To a stirring solution of compound 3a (7 g, 28.7 mmol) in
acetic acid (4 ml) was added 30% hydrogen peroxide
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Med Chem Res (2011) 20:435–442
439
(3 ml) at room temperature. Four additional portions of
30% hydrogen peroxide (3 ml) were added after 2, 4, 6,
and 8 h. The stirring was continued for 1 h, and then the
mixture was diluted with water and neutralized with 10%
aqueous solution of sodium hydroxide. The organic layer
was dried (sodium sulfate) and concentrated in vacuo to
give 6.39 g (85%) of 4 (R=H), mp 76–77°C; IR (KBr)
(DMSO-d6): 9.81 (bs, 1H, NH), 8.20 (dd, 1H, J = 8.1,
1.8 Hz, aromatic), 7.92–7.25 (m, 8H, aromatic), 4.65 (bs,
2H, NH2). MS: m/z (%) 276 (M?, 18), 245 (99), 181 (10),
152 (100), 77 (41). Anal. Calcd. for C13H12N2O3S: C, 56.51;
H, 4.38; N, 10.14. Found: C, 56.73; H, 4.52; N, 10.36.
2-(2-Chlorophenylthio)benzoic acid hydrazide (6d)
1
m cm-1 1710 (C=O), 1031 (S=O); H-NMR(CDCl3): 8.78
(dd, 1H, J = 7.8, 1.3 Hz, aromatic), 8.09–7.26 (m, 8H,
aromatic), 3.87 (s, 3H, CH3). MS: m/z (%) 260 (M?, 15),
211(16), 180 (10), 166 (100), 151 (32), 77 (12). Anal.
Calcd. for C14H12O3S: C, 64.60; H, 4.65. Found: C, 64.26;
H, 4.87.
Yield 91%; mp 85–86 (ether). IR (KBr) m cm-1 3303, 3127
(NH2, NH), 1665 (C=O). H-NMR (CDCl3): 8.78 (bs, 1H,
1
NH), 7.67–7.17 (m, 8H, aromatic), 4.05 (bs, 2H, NH2).
MS: m/z (%) 280 (M??2, 5) 278 (M?, 15), 247 (100), 212
(27), 184 (48), 142 (24). Anal. Calcd. for C13H11ClN2OS:
C, 56.01; H, 3.98; N, 10.05. Found: C, 56.19; H,3.17; N,
10.26.
Methyl 2-(phenylsulfonyl)benzoate (5, R=H)
Compound 3a (4.52 g, 18.5 mmol) was dissolved in 65 ml
of acetic acid, and H2O2 (12 ml of 30% solution) was
added. The solution was heated under reflux for 20 h. The
mixture was cooled and diluted with water and worked up
similar to compound 4 to give compound (5, R=H). The
yield was 4.5 g (88%), mp 57–59°C; IR (KBr) m cm-1
1738 (C=O), 1311, 1158 (SO2); 1H-NMR (CDCl3):
8.19–7.92 (m, 3H, aromatic), 7.71–7.26 (m, 6H, aromatic),
3.92 (s, 3H, CH3). MS: m/z (%) 276 (M?, 10), 244 (93),
212 (100), 182 (95), 152 (85), 104 (30), 77 (34). Anal.
Calcd. for C14H12O4S: C, 60.86; H, 4.38. Found: C, 60.98;
H, 4.17.
General procedure for the preparation of 5-aryl-1,3,4-
oxadiazole-2(3H)-thiones (7b–7d)
A mixtue of hyrazide (6b–6d) (12.2 mmol), potassium
hydroxide (12.2, mmol), carbon disulfide (2.5 ml) and
ethanol (14 ml) was heated under reflux for 7 h. The sol-
vent was removed in vacuo and the residue was dissolved
in water and acidified with dilute hydrochloric acid. The
resulting precipitate was removed by filtration and purified
by crystallization or TLC.
5-[2-(Phenylsulfinyl)phenyl]-1,3,4-oxadiazole-2-(3H)-
thione (7b)
General procedure for the preparation of hyrazides
(6b–6d)
Yield 57%; mp 247–249°C (ethanol). IR (KBr) m cm-1
3121 (NH), 2530 (weak, SH), 1342 (C=S). 1H-NMR
(DMSO-d6): 14.22 (bs, 1H, NH), 8.22-7.52 (m, 9H, aro-
matic). MS: m/z (%) 302 (M?, 11), 213 (100), 184 (31),
152 (10). Anal. Calcd. for C14H10N2O2S2: C, 55.61; H,
3.33; N, 9.26. Found: C, 55.42; H, 3.14; N, 9.47.
To a stirring solution of compounds 3b, 4, or 5 (14 mmol)
in 7 ml ethanol at room temperature hydrazine hydrate
(14 ml, 280 mmol) was added. After 8 h, the content were
poured into water (15 ml), filtered and crystallized from
suitable solvents.
5-[2-(Phenylsulfonyl)phenyl]-1,3,4-oxadiazole-2(3H)-
2-(Phenylsulfinyl)benzoic acid hydrazide (6b)
thione (7c)
Yield 92%; mp 145–146°C (ethanol). IR (KBr) m cm-1
3482, 3380 (NH2), 3288 (NH), 1623 (C=O), 1024 (S=O).
1H-NMR (DMSO-d6): 9.95 (bs, 1H, NH), 8.12 (dd, 1H,
J = 7.9, 1.4 Hz, aromatic), 7.81–7.38 (m, 8H, aromatic),
4.55 (bs, 2H, NH2). MS: m/z (%) 260 (M?, 5), 229 (100),
184 (36), 152 (6), 77 (4). Anal. Calcd. for C13H12N2O2S:
C, 59.98; H, 4.65; N, 10.76. Found: C, 60.12; H, 4.52; N,
10.92.
Yield 86%; mp 140–142°C (ethanol). IR (KBr) m cm-1
3272 (NH), 1306 (C=S). H-NMR (DMSO-d6): 14.30 (bs,
1
1H, NH), 8.34 (d, 1H, J = 7.1 Hz, aromatic), 7.90–7.60
(m, 8H, aromatic). MS: m/z (%) 318 (M?, 93), 252 (61),
228 (75), 182 (100), 151 (38), 77 (33). Anal. Calcd. for
C14H10N2O3S2: C, 52.82; H, 3.17; N, 8.80. Found: C,
52.96; H, 3.07; N, 8.65.
5-[2-(2-Chlorophenylthio)phenyl]-1,3,4-oxadiazole-2-
2-(Phenylsulfonyl)benzoic acid hydrazide (6c)
(3H)-thione (7d)
Yield 70%; mp 165–167°C (ether). IR (KBr) m cm-1 3365,
Compound 7d was purified by TLC eluting with petroleum
ether-ethyl acetate (1:1) and then crystallized from ethanol,
3170 (NH2, NH), 1631 (C=O), 1324, 1160 (SO2). 1H-NMR
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Med Chem Res (2011) 20:435–442
yield 30%; mp 206–208°C. IR (KBr) m cm-1 3277 (NH),
1332 (C=S). H-NMR (DMSO-d6): 14.49 (bs, 1H, NH),
General procedure for the preparation of 5-aryl-1,2,4-
1
triazole-3-thiones (9b–9c)
7.88–7.37 (m, 7H, aromatic), 6.97–6.85 (m, 1H, aromatic);
MS: m/z (%) 322 (M??2, 40), 320 (M?, 100), 285 (16),
246 (18), 224 (32), 182 (23). Anal. Calcd. for
C14H9ClN2OS2: C, 52.41; H, 2.83; N, 8.73. Found: C,
52.60; H, 2.71; N, 8.64.
A suspension of hydrazide (6b–6d) (8.2 mmol), potassium
thiocyanate (41 mmol), hydrochloric acid (8 ml) and water
100 ml was refluxed for 3 h. After cooling the resulting
thiosemicarbazide was removed by filtration and washed
with water. It was dissolved in 4% sodium hydroxide
solution (5 ml) and refluxed for 4 h. The mixture after
charcoal treatment and filtration was acidified with
hydrochloric acid to pH 5–6 and the resulting precipitate
was filtered and purified by crystallization or TLC.
General procedure for the preparation of 2-amino-5-
aryl-1,3,4-oxadiazoles (8b–8d)
To a stirring solution of hydrazide (6b–6d) (3.71 mmol) in
dioxane (12 ml), sodium bicarbonate (3.71 mmol) in water
(4 ml) was added at room temperature. The mixture was
stirred at room temperature for 5 min and cyanogen bro-
mide (3.85 mmol) was added. After 4 h, water (30 ml) was
added to the mixture and the precipitate was removed by
filtration and purified by crystallization or TLC.
5-[2-(Phenylsulfinyl)phenyl]-2,4-dihydro-3H-1,2,4-
triazole-3-thione (9b)
Yield 47%; mp 247–249°C (ethanol). IR (KBr) m cm-1
3128 (NH), 2539 (weak, SH), 1320 (C=S). 1H-NMR
(DMSO-d6): 13.72 (bs, 1H, NH), 7.76 (dd, 1H, J = 7.3,
2.9 Hz, aromatic), 7.51-7.33 (m, 7H, aromatic), 7.05 (dd,
1H, J = 7.1, 2.4 Hz, aromatic). MS: m/z (%) 301 (M?,
12), 285 (16), 212 (100), 184 (43), 108 (23). Anal. Calcd.
for C14H11N3OS2: C, 55.79; H, 3.68; N, 13.94. Found: C,
55.93; H, 3.79; N, 13.78.
2-Amino-5-[2-(phenylsulfinyl)phenyl]-1,3,4-oxadiazole
(8b)
Yield 45%; mp 228-231°C (ethanol). IR (KBr) m cm-1
3320, 3275 (NH2), 1670 (NH2). 1H-NMR (DMSO-d6): 8.22
(dd, 1H, J = 7.4, 1.7 Hz, aromatic) 7.88–7.40 (m, 8H,
aromatic). MS: m/z (%) 285 (M?, 10), 213 (100), 183 (57),
151 (24). Anal. Calcd. for C14H11N3O2S : C, 58.93; H,
3.89; N, 14.73. Found: C, 59.12; H, 3.95; N, 14.55.
5-[2-(Phenylsulfonyl)phenyl]-2,4-dihydro-3H-1,2,4-
triazole-3-thione (9c)
Compound 9c was purified by TLC eluting with chloroform-
ethanol (5:1) and then crystallized from ethanol, yield 30%;
mp 133–135°C. IR (KBr) m cm-1 3132 (NH), 1306 (C=S).
1H-NMR (DMSO-d6): 13.4 (bs, 1H, NH), 8.26–7.53 (m, 9H,
aromatic). MS: m/z (%) 317 (M?, 45), 252 (100), 220 (20),
165 (17), 77 (10). Anal. Calcd. for C14H11N3O2S2: C, 52.98;
H, 3.49; N, 13.24. Found: C, 52.76; H, 3.65; N, 13.46.
2-Amino-5-[2-(Phenylsulfonyl)phenyl]-1,3,4-
oxadiazole (8c)
Yield 50%; mp 166–168°C (ethanol). IR (KBr) m cm-1
3441, 3344 (NH2), 1644 (NH2). 1H-NMR (DMSO-d6):
8.34–8.23 (m, 1H, aromatic), 7.98–7.52 (m, 8H, aromatic),
7.10 (bs, 2H, NH2). MS: m/z (%) 301 (M?, 10), 235 (100),
182 (95), 152 (60), 77 (32). Anal. Calcd. for C14H11N3O3S:
C, 55.80; H, 3.68; N, 13.95. Found: C, 55.96; H, 3.54; N,
13.76.
5-[2-(2-Chlorophenylthio)phenyl]-2,4-dihydro-3H-
1,2,4-triazole-3-thione (9d)
Compound 9d was purified by TLC eluting with chloro-
form-ethanol (12:1) and then crystallized from ethanol,
yield 45%; mp 233–235°C. IR (KBr) m cm-1 3201, 3172
2-Amino-5-[2-(2-Chlorophenylthio)phenyl]-1,3,4-
1
(NH), 2543 (weak, SH), 1303 (C=S). H-NMR (DMSO-
oxadiazole (8d)
d6): 13.15 (bs, 1H, NH), 7.75–7.21 (m, 8H, aromatic). MS:
m/z (%) 321 (M??2, 35), 319 (M?, 100) 284 (90), 225
(16), 209 (10). Anal. Calcd. for C14H9ClN2S2: C, 55.16; H,
2.98; N, 9.19. Found: C, 55.37; H, 3.14; N, 9.37.
Compound 8d was purified by TLC eluting with petroleum
ether-ethyl acetate (1:5) and then crystallized from ethanol,
yield 76%; mp 158–159°C; IR(KBr) m cm-1 3344, 3252
1
(NH2), 1640 (NH2). H-NMR (CDCl3): 7.89–7.78 (m, 1H,
aromatic), 7.51–6.92 (m, 7H, aromatic), 5.81 (bs, 2H,
NH2). MS: m/z (%) 305 (M??2, 5), 303 (M?, 12), 268
(100), 355 (28), 198 (64), 184 (40), 152 (31). Anal. Calcd.
for C14H10ClN3OS: C, 55.35; H, 3.32; N, 13.83. Found: C,
55.54; H, 3.26; N, 13.65.
Pharmacology
Male NMRI mice weighing 20–25 g (from the animal
house of the Faculty of Pharmacy, TUMS) were used. The
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Med Chem Res (2011) 20:435–442
441
activity of N-arylhydrazone derivatives of mefenamic acid.
J Pharm Pharm Sci 8(3):419–425
animals were housed in colony cages, condition of constant
temperature (22 2°C), a 12 h light/dark schedule, and
allowed free access to standard diet and tap water except
during the experiment. The animals were allowed to
habituate to the laboratory environment for 2 h before the
experiments were initiated. All ethical manners for use of
laboratory animals were considered carefully and the pro-
tocol of study was approved by TUMS ethical committee.
The compounds were administered intra-peritoneally (ip),
30 lmol/kg; 0.2 ml/20 g as a suspension in saline and
tween 80 (4% w/v) 30 min before formalin injection.
Mefenamic acid (Hakim Pharmaceutical Co., Tehran, Iran)
(30 lmol/kg, ip) was used as standard drug under the same
conditions. The control group received vehicle (0.2 ml/
20 g, ip) alone (Rineh et al., 2007).
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A (2007) Synthesis, anticonvulsant and muscle relaxant activites
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Formalin-induced test
Final compounds (7a–9c) were tested in the formalin-
induced pain test. Twenty-five microliters of formalin
(0.5%) was injected subcutaneously into the dorsal surface
of the right hind paw of the mouse using a micro syringe
with a 26-gauge needle. Immediately after the formalin
injection, animals were placed individually in glass cylin-
der (20 cm wide, 25 cm long) and a glass door and a mirror
were arranged at a 45° angle under the cylinder to allow
clear observation of the paws of the animals. The total time
in seconds that the animal spent licking or biting the
injected paw during the period of 0–10 min was considered
as indicator of neurogenic pain (early phase) and during the
10–30 min (late phase) represented the inflammatory pain
(Rineh et al., 2007; Dubuisson and Dennis 1977; Monsef
et al., 2004).
Dubuisson D, Dennis SG (1977) The formalin test: a quantitative
study of the analgesic effects of morphine, meperidine and brain
stem stimulation in rats and cats. Pain 4:161–174
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Statistics
The results were expressed as the mean SEM of 6 animals
per group. The data were statistically analyzed by one-way
analysis of variance (ANOVA) followed by Tukey multi-
comparison test. Differences with P \ 0.05 between exper-
imental groups were considered statistically significant.
Malmberg AB, Yaksh TL (1992) Antinociceptive actions of spinal
nonsteroidal anti-inflammatory agents on the formalin test in the
rat. J Pharmacol Exp Ther 263:136–146
Acknowledgements This work was supported by grants from
Tehran University of Medical Sciences Research Council and INSF
(Iran National Science Foundation).
Monsef HR, Ghobadi A, Iranshahi M, Abdollahi M (2004) Antino-
ciceptive effects of Peganum harmala L. alkaloid extract on
mouse formalin test. J Pharm Pharm Sci 65:65–69
Moreau A, Chen QH, Roa PNP, Knaus EE (2006) Design, synthesis
and biological evaluation of (E)-3-(4-methanesulfonylphenyl)-2-
(aryl)acrylic acids as dual inhibitors of cyclooxygenases and
lipoxygenase. Bioorg Med Chem 14:7716–7727
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