Synthesis and evaluation of anti-inflammatory and analgesic activity of 3-[(5-substituted-1,3,4-oxadiazol-2-yl-thio)acetyl]-2H-chromen-2-ones

Article abstract of DOI:10.1007/s00044-010-9494-z

A novel series of 3-[(5-substituted-1,3,4-oxadiazol- 2-yl-thio)acetyl]-2H- chromen-2-one (7a-i) were synthesized by the condensation between the appropriately substituted 5-substituted-1,3,4-oxadiazolyl-2-thione (4a-i) derived from various existing NSAIDs and 3-(2-bromoacetyl)- 2H-chromen-2-one (6) under reflux in the presence of sodium ethoxide. Structure of the synthesized compounds was established on the basis of physicochemical, elemental analysis, and spectral data. The title compounds were screened for in vivo acute anti-inflammatory and analgesic activities at a dose of 200 mg/kg bw. Among the series, four compounds 7c, 7e, 7f, and 7h were found to possess a significant anti-inflammatory and analgesic activity profile. In addition, these compounds were also found to possess a less degree of ulcerogenic potential as compared to standard NSAIDs. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9494-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:16–26
DOI 10.1007/s00044-010-9494-z
ORIGINAL RESEARCH
Synthesis and evaluation of anti-inflammatory and analgesic
activity of 3-[(5-substituted-1,3,4-oxadiazol-2-yl-thio)acetyl]-
2H-chromen-2-ones
•
Nista Ingale Veeresh Maddi Mahesh Palkar
•
•
•
Pradeepkumar Ronad Shivalingrao Mamledesai
•
•
A. H. M. Vishwanathswamy Darbhamulla Satyanarayana
Received: 4 August 2010 / Accepted: 29 October 2010 / Published online: 18 November 2010
Ó Springer Science+Business Media, LLC 2010
Abstract A novel series of 3-[(5-substituted-1,3,4-oxa-
diazol-2-yl-thio)acetyl]-2H-chromen-2-one (7a–i) were syn-
thesized by the condensation between the appropriately
substituted 5-substituted-1,3,4-oxadiazolyl-2-thione (4a–i)
derived from various existing NSAIDs and 3-(2-bromoacetyl)-
2H-chromen-2-one (6) under reflux in the presence of sodium
ethoxide. Structure of the synthesized compounds was estab-
lished on the basis of physicochemical, elemental analysis, and
spectral data. The title compounds were screened for in vivo
acute anti-inflammatory and analgesic activities at a dose of
200 mg/kg bw. Among the series, four compounds 7c, 7e, 7f,
and 7h were found to possess a significant anti-inflammatory
and analgesic activity profile. In addition, these compounds
were also found to possess a less degree of ulcerogenic
potential as compared to standard NSAIDs.
Introduction
Large numbers of non-steroidal anti-inflammatory drugs
(NSAIDs) are widely preferred class of drugs used in the
treatment of pain, fever, inflammatory diseases, and rheu-
matoid arthritis (Tally et al., 2000; Palomer et al., 2002).
The pharmacological activities of NSAIDs are related in
the suppression of prostaglandin biosynthesis from ara-
chidonic acid by inhibiting the enzyme prostaglandin
endoperoxidase, popularly known as cyclooxygenase and
5-lipoxygenase (Smith et al., 1998; Warner et al., 1996).
These enzymes catalyze the rate-limiting steps in the bio-
synthesis of prostaglandins and leukotrienes, respectively.
Cyclooxygenase exists in two isoforms, COX-1 and COX-
2, which are regulated and expressed differently. COX-1
provides cytoprotection in gastrointestinal tract, whereas
inducible COX-2 selectively mediates inflammatory sig-
nals (Amgad et al., 2001).
Keywords 3-Acetyl coumarin Á 1,3,4-Oxadiazole Á
NSAIDS Á Anti-inflammatory Á Analgesic
Long-term usages of NSAIDs have been associated with
dyspepsia, GI ulceration, and nephrotoxicity. The GI
damage from NSAIDs is generally attributed to two fac-
tors: local irritation by the carboxylic acid moiety common
to most NSAIDs (topical effect), and decreased tissue
prostaglandin production, which determines the physio-
logical role of cytoprotective prostaglandins in maintaining
the GI health and homeostasis (Amir and Shika, 2007). The
discovery of COX-2 isoforms has opened the possibility of
developing selective COX-2 inhibitors to act as an effec-
tive NSAID without the gastric side effects. Previously
marketed drugs like Celecoxib, Rofecoxib, and Etodolac,
etc., act by inhibiting COX-2 enzyme. However, these
COX-2 selective inhibitors have been reported to be
associated with cardiovascular side effects (Bombardier
et al., 2000). In order to overcome these side effects, most
studies are being focused on synthesis and identification of
N. Ingale Á V. Maddi (&) Á M. Palkar Á P. Ronad Á
S. Mamledesai
Department of Pharmaceutical Chemistry, KLES’ College
of Pharmacy, Vidyanagar, Hubli 580031, Karnataka, India
e-mail: veeresh_m@rediffmail.com
A. H. M. Vishwanathswamy
Department of Pharmacology, KLES’ College of Pharmacy,
Vidyanagar, Hubli 580031, Karnataka, India
D. Satyanarayana
Department of Pharmaceutical Chemistry, NGSM Institute
of Pharmaceutical Sciences, Paneer, Deralakatte, Mangalore,
Karnataka, India
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Med Chem Res (2012) 21:16–26
17
new chemical entities with minimal side effects and
excellent anti-inflammatory properties. Inflammation is
known not only as a symptom of great deal of common
diseases but also as an early phase of some serious diseases
such as cancer, heart vascular diseases, and Alzheimer’s
dementia. Thus, the discovery of novel anti-inflammatory
drugs has been attracting a lot of interests.
drops of conc. sulfuric acid to yield methyl esters of
NSAIDs (2a–i), which were further refluxed in absolute
ethanol for 15–20 h in the presence of hydrazine hydrate,
yielded the carbohydrazides of NSAIDs (3a–i). Further,
5-substituted-1,3,4-oxadiazolyl-2-thione (4a–i) were syn-
thesized by refluxing a mixture of carbohydrazides of
NSAIDs, in the presence of potassium hydroxide and car-
bon disulfide in methanol on a steam bath for 12 h.
Synthesis of 3-acetyl-2H-chromen-2-one (5) was carried
out by reacting salicylaldehyde with ethyl acetoacetate in
the presence of base piperidine as illustrated in Scheme 2.
The reaction is an example of the Knoevenagel reaction
(Knoevenagel, 1898), in which the active methylene
compounds reacts with 2-hydroxy benzaldehyde, has been
extensively used as the first step in the synthesis of
3-acetylcoumarin. Further, 3-acetyl-2H-chromen-2-one (5)
was brominated using bromine in presence of chloroform
to yield 3-(2-bromoacetyl)-2H-chromen-2-one (6).
The synthesis of coumarins and their derivatives has
attracted considerable attention from organic and medicinal
chemists for many years as a large number of natural and
synthetic products contain this heterocyclic nucleus. Sev-
eral coumarin derivatives have been reported to present
multiple biological activities (Egan et al., 1990) and
especially anti-inflammatory/antioxidant activities. Kon-
togiorgis and Hadjipavlou-Litina (2004) have reported the
synthesis and in vivo/in vitro anti-inflammatory/antioxidant
activities of several new coumarin derivatives with a
7-azomethine linkage.
The substituted 1,3,4-oxadiazoles are heterocyclic com-
pounds, which serve both as biomimetic as well as reactive
pharamacophores and are of considerable pharmaceutical
interest. 1,3,4-oxadiazole is an imperative scaffold since
several 1,3,4-oxadiazole derivatives are known to be asso-
ciated with multiple biological activities, such as pesticidal,
CNS stimulant, anti-inflammatory, hypotensive, bacterici-
dal, hypoglycemic, analgesic, anticonvulsive, antiemetic,
diuretic (Deshmukh et al., 1976). Recently, Bhandari et al.
(2008) have reported the synthesis and evaluation of anti-
inflammatory, analgesic, and ulcerogenicity studies of
novel S-substituted phenacyl-1,3,4-oxadiazol-2-thione and
schiff bases of diclofenac as non-ulcerogenic derivatives.
In view of the above facts and in continuation of our
research on novel anti-inflammatory agents (Bolakatti
et al., 2008; Khode et al., 2009; Ronad et al., 2008), we
report herein the synthesis of 3-[(5-substituted-1,3,4-oxa-
diazol-2-yl-thio)acetyl]-2H-chromen-2-ones (7a–i), which
have been found to possess an appealing profile of anti-
inflammatory and analgesic activities. In this study, we
have attempted to convert the carboxylic acid group of the
well-known NSAIDs into 1,3,4-oxadiazole that is con-
densed with coumarin nucleus, which possess an interest-
ing profile of anti-inflammatory activity along with
significantly lower ulcerogenic potential.
The synthesis of 3-[(5-substituted-1,3,4-oxadiazol-2-yl-
thio)acetyl]-2H-chromen-2-ones (7a–i) was carried out by
the condensation of 5-substituted-1,3,4-oxadiazolyl-2-thi-
one (4a–i) with 3-(2-bromoacetyl)-2H-chromen-2-one (6)
under reflux in ethanol in presence of sodium ethoxide (see
Scheme 3).
Pharmacological evaluation
The acute toxicity test was carried out according to Orga-
nization for Economic Co-operation and Development
(OECD) guidelines (OECD, Guidelines for Testing of
Chemicals, 2008) to determine toxicity of all the synthe-
sized compounds. The in vivo acute anti-inflammatory
activity for all the test compounds was evaluated on male
albino rats using carrageenan-induced rat paw edema
model by adopting the earlier reported method of Winter
et al. (1962). Further, analgesic activity and ulcerogenic
studies were carried out for most active compounds 7c, 7e,
7f, and 7h by the earlier reported method of Koster and
Anderson (1959) and Vogel (2002) (Drug Discovery and
Evaluation: Pharmacological Assays), respectively.
Results and discussion
Chemistry
Synthetic and spectral studies
The synthesis of 5-substituted-1,3,4-oxadiazolyl-2-thione
(4a–i) derivatives was achieved through convenient syn-
thetic route outlined in Scheme 1. The various well-known
NSAIDs, viz., Ibuprofen, Aceclofenac, Diclofenac, Keto-
profen, Indomethacin, Flurbiprofen, Ketorolac etc., were
refluxed for 8–10 h in presence of dry methanol and few
Structure of the title compounds (7a–i) was established on
the basis of physicochemical, elemental analysis, and
spectral data, which are summarized in experimental sec-
tion. All the newly synthesized compounds gave satisfac-
tory analysis for the proposed structures, which were
1
confirmed on the basis of their IR and H-NMR spectral
123

18
Med Chem Res (2012) 21:16–26
Scheme 1 Synthesis of
O
O
C
5-substituted-1,3,4-oxadiazol-
2-thione (4a–i). Reagents and
conditions: (A) Methanol, Conc.
H2SO4, reflux, 8–10 h; (B) ethyl
chloroacetate, dry acetone,
reflux, 6 h; (C) Hydrazine
hydrate, Absolute ethanol,
reflux, 15–20 h; (D) KOH,
Carbon disulfide in methanol,
reflux, steam bath, 12 h
(a)
H
(c)
R
COOH
N
R
C
NH2
R
O
CH3
Carbohydrazides of NSAIDs
Methyl esters of NSAIDs
Substituted NSAIDs
(3a-i)
(2a-h)
(1a-h)
(c)
(d)
H2
C
O
C
O
C
OC2H5
(b)
H
H
N
N
H3C
OH
H3C
O
C
N
N
O
2i
1i
R
SH
O
5-substituted-1,3,4-oxadiazolyl-2-thione
(4a-i)
Scheme 2 Synthesis of 3-(2-
bromoacetyl)-2H-chromen-2-
one (6). Reagents and
conditions: (A) Piperidine, stir,
rt, 20 min; (B) Br2, Chloroform,
warm, 15–20 min
O
O
C
O
O
C
H2
C
CH3
H
(a)
+
H3C
C
O
CH3
H2
O
O
OH
Ethyl acetoacetate
3-acetyl-2H-chromen-2-one
(5)
Salicylaldehyde
(b)
O
C
CH2Br
O
O
3-(2-bromoacetyl)-2H-chromen-2-one
(6)
data. The IR spectra of these compounds showed moder-
ately strong bands around 1711–1744 cm-1
1599–
restlessness. Therefore, 1/10th of the maximum tolerated
dose, i.e., 200 mg/kg bw. was chosen as therapeutic dose
for the various pharmacological evaluations.
,
1618 cm-1, and 2350–2367 cm-1, characteristic of the
C=O (lactone of coumarin), C=N and C–S groups,
1
respectively. In the H-NMR spectra, a characteristic sin-
In vivo acute anti-inflammatory activity
glet signal due to the fourth proton of coumarin ring
appeared at d 8.29–8.73. The most informative signal due
to the –S–CH2–C=O protons appeared as singlet at d
4.24–4.7. The signals due to the aromatic protons appeared
as multiplets at d 6.8–8.3.
Table 1 reveals the in vivo acute anti-inflammatory activity
of a novel series of 3-[(5-substituted-1,3,4-oxadiazol-2-yl-
thio)acetyl]-2H-chromen-2-one (7a–i) at a dose of 200 mg/
kg in carrageenan-induced paw edema method. As shown
in Table 1, the entire investigated compounds exhibited
moderate-to-good anti-inflammatory activity with the per-
centage inhibition of edema formation ranging from
37.5–81.3 and 26.3–71.3, while the reference drug dic-
lofenac (13.5 mg/kg) showed 79.5% and 75.0% inhibition
at third and fifth hour, respectively. Compounds 7e (74.9
and 61.6%), 7f (65.4 and 60.9%), and 7h (67.4 and 71.3%)
showed good inhibitory activity at 3rd and 5th hour,
respectively, while the most active compound 7c (81.3 and
64.5%) among the series, exhibited the excellent inhibitory
activity at 3rd and 5th hour, respectively. The anti-
Pharmacological screening
Acute toxicity study
From the preliminary toxicity studies, it was observed that,
all the test compounds have revealed good safety profile till
the uppermost dose (2,000 mg/kg). No mortality of animals
observed even after 24 h but there were few changes in the
behavioral response like alertness, touch response, and
123

Med Chem Res (2012) 21:16–26
19
Table 1 In vivo acute anti-inflammatory activity of a novel series of 3-[(5-substituted-1,3,4-oxadiazol-2-yl-thio)acetyl]-2H-chromen-2-one
(7a–i) derivatives by carrageenan-induced paw edema
O
C
N
N
R
S
CH2
O
O
O
(7a-i)
Compound
R
Anti-inflammatory activitya (%) Inhibition of edema ( SEM)
3rd h
5th h
Control
–
–
7a
45.0 ( 0.054)*
46.1 ( 0.028)*
CH3
CH
CH3
CH
CH2 CH3
7b
46.3 ( 0.039)*
48.7 ( 0.040)*
OH
Cl
7c
81.3 ( 0.010)**
64.5 ( 0.040)**
NH
Cl
Cl
7d
37.5 ( 0.045)*
26.3 ( 0.069)
NH
Cl
O
O
F
7e
74.9 ( 0.015)**
61.6 ( 0.040)**
H
H3C
123

20
Med Chem Res (2012) 21:16–26
Table 1 continued
Compound
R
Anti-inflammatory activitya (%) Inhibition of edema ( SEM)
3rd h
5th h
CH3
7f
65.4 ( 0.047)**
60.9 ( 0.010)**
O
O
7g
56.9 ( 0.005)*
51.3 ( 0.008)*
CH3
N
Cl
O
CH3
7h
67.4 ( 0.016)**
71.3 ( 0.018)**
N
O
7i
56.3 ( 0.035)*
79.5 ( 0.013)
43.4 ( 0.008)*
75.0 ( 0.017)
H2CO
NHCOCH3
Diclofenac
–
a
Inhibitory activity on carrageenan-induced rat paw edema. The results are expressed as mean SEM; Significance was calculated by using
one-way ANOVA with Dunnet t-test. The difference in results was considered significant when p \ 0.05. * p \ 0.05 vs. control at 200 mg/kg
bw.; ** p \ 0.01 vs. control at 200 mg/kg bw.; p \ 0.01 vs. control at 13.5 mg/kg bw
Ulcerogenic activity
inflammatory activity of those compounds was comparable
to that of the standard drug diclofenac sodium. Compounds
that showed significant anti-inflammatory activity profile
were further evaluated for analgesic activity.
The major drawback of NSAIDs is their gastric ulcer for-
mation due to gastric irritation. The extent of ulcerogenic
effect was evaluated for compounds 7c, 7e, 7f, and 7h in rat
stress model at the therapeutic dose (i.e., 200 mg/kg bw.).
The gastric ulcerogenic potential was evaluated by calcu-
lating the ulcer index in treated and control animals. Results
are given in Table 3 that indicates the ulcer index range of
compounds 7c (1.7–2.1), 7e (2.3–2.8), 7f (3.2–3.5), and 7h
(4.4–5.1) as compared to their standard reference drugs
Diclofenac (5.5–6.4), Flurbiprofen (5.3–6.0), Ketoprofen
(4.6–5.4), and Ketorolac (4.9–5.7), respectively. Therefore,
the test compounds cause less gastric ulceration and dis-
ruption of gastric epithelial cells at the above mentioned
oral dose. However, test compound 7h induces ulcer, which
is almost equipotent to its standard drug.
Analgesic activity
Abdominal construction response induced by acetic acid is a
sensitive procedure to establish of peripherally acting anal-
gesics. The compounds 7c, 7e, 7f, and 7h were tested for
analgesic activity at 200 mg/kg bw. in mice. The results of
analgesic activity indicated that all the tested compounds
exhibited good analgesic activity (Table 2). Compounds 7c
(68%), 7e (60%), 7f (73%), and 7h (54%) have shown almost
comparable activity to that of reference drug acetylsalicylic
acid (69%) in peripheral analgesic activity model.
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Med Chem Res (2012) 21:16–26
21
Table 2 Analgesic activity of compounds 7c, 7e, 7f, and 7h by acetic acid-induced writhing method
O
C
N
N
R
S
CH2
O
O
O
(7c, 7e, 7f and 7h)
Compound
Control
R
–
No. of wriths in 15 min (mean SEM)
44.9 0.21
% Protection
–
7c
7e
7f
14.2 0.59**
18.1 0.23*
12.3 0.35**
68.0
60.0
73.0
Cl
NH
Cl
F
H
H3C
CH3
O
7h
20.5 0.46*
54.0
N
O
Acetylsalicylic acid
–
13.8 0.57
69.0
The results are expressed as mean SEM (n = 6). Significance was calculated by using one-way ANOVA with Dunnet t-test. The difference in
results was considered significant when p \ 0.05. * p \ 0.05 vs. control at 200 mg/kg bw.; ** p \ 0.01 vs. control at 200 mg/kg bw.;
p \ 0.001 vs. control at 135 mg/kg bw
Conclusion
oxadiazol-2-yl-thio)acetyl]-2H-chromen-2-one (7a–i). These
novel compounds containing both 1,3,4-oxadiazole and
coumarin ring systems were prepared by the condensation
reaction between the appropriately 5-substituted-1,3,4-
In this paper, we report the synthesis and pharmacological
evaluation of a novel series of 3-[(5-substituted-1,3,4-
123

22
Med Chem Res (2012) 21:16–26
oxadiazolyl-2-thione (4a–i) derived from various NSAIDs
and 3-(2-bromoacetyl)-2H-chromen-2-one (6) as depicted
in Schemes 1–3.
Table 3 Ulcerogenic activity of compounds 7c, 7e, 7f and 7h in
comparison with their standard NSAIDs
Compound
Ulcer index range
The title compounds did not show toxic effects at doses
up to 2,000 mg/kg bw in acute toxicity experiments. The in
vivo acute anti-inflammatory effects of the test compounds
were assessed using the functional model of carrageenan-
induced rat paw edema. Four compounds 7c, 7e, 7f, and 7h
were found to possess a promising and significant anti-
inflammatory profile. These compounds were also found to
have significant analgesic activity in the acetic acid-
induced writhing model. In addition, the tested compounds
were also found to possess less degree of ulcerogenic
potential as compared to standard NSAIDs.
Control
7c
1.2–1.6
1.7–2.1
2.3–2.8
3.2–3.5
4.4–5.1
5.5–6.4
5.3–6.0
4.6–5.4
4.9–5.7
7e
7f
7h
Diclofenac
Flurbiprofen
Ketoprofen
Ketorolac
The results are expressed as ulcer index values from minimum to
maximum range
O
C
Experimental
All the research chemicals were purchased from Sigma–
Aldrich (St. Louis, MO, USA) or Lancaster Co. (Ward Hill,
MA, USA) and used as such for the reactions. Various
NSAIDs used in this study were received as a gift sample
from Fourrts (India) Lab. Pvt. Ltd. Chennai (Tamilnadu,
India). Solvents except laboratory reagent grade were dried
and purified, when necessary, according to the literature.
Reactions were monitored by thin-layer chromatography
(TLC) on pre-coated silica gel plates from Merck
(Darmstadt, Germany).
CH2Br
N
N
+
R
SH
O
O
O
5-substituted-1,3,4-oxadiazolyl- 3-(2-bromoacetyl)-2H-chromen-2-one
-2-thione (4a-i)
(6)
(a)
O
C
N
N
R
S
CH2
O
O
O
3-[(5-substituted-1,3,4-oxadiazol-2-yl-thio)acetyl]-2H-chromen-2-one
(7a-i)
Melting point of synthesized compounds was deter-
mined in Thermonik melting point apparatus (Thermonik,
Mumbai, India) and is uncorrected. IR spectrum was
recorded on Thermo Nicolet IR200 FT-IR Spectrometer
CH3
CH
7b: R =
OH
7a: R =
7c: R =
7e: R =
CH3
CH
CH2 CH3
Cl
1
(Madison, WI, USA) by using KBr pellets. The H-NMR
Cl
spectra were recorded on Bruker Avance II [400 MHz
(Bruker, Rheinstetten/Karlsruhe, Germany)] using CDCl3
and DMSO-d6 as solvent. Chemical shift are given in d
ppm units with respect to TMS. The purity of the com-
pounds was examined by TLC on silica gel G plate using
n-hexane and ethyl acetate (6:4) as mobile phase and iodine
vapors as visualizing agent. The compounds were purified
using flash chromatography SP-01 (Biotage, Sweden). The
anti-inflammatory activity was carried out using digital
plethysmometer (Ugo-Basile, Italy).
NH
NH
7d: R =
7f: R =
O
Cl
O
Cl
CH3
F
H
H3C
O
O
CH3
N
7g: R =
Cl
N
7h: R =
O
General procedure for the synthesis of methyl esters
of NSAIDs (2a–h)
O
CH3
O
To a mixture of 0.05 mol of NSAIDs (1a–h) and 20-ml
absolute methanol, few drops of Conc. sulfuric acid were
added. The reaction mixture was refluxed for 8–10 h (pro-
gress of reaction was constantly monitored by TLC). Then,
reaction mixture was cooled, and the solvent was distilled
7i: R =
O
C
CH3
C
NH
H2
Scheme 3 Synthetic route of 3-[(5-substituted-1,3,4-oxadiazol-2-yl-
thio)acetyl]-2H-chromen-2-one (7a–i). Reagents and conditions: (A)
absolute ethanol, sodium ethoxide, reflux, 25–30 h
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Med Chem Res (2012) 21:16–26
23
off on a rotary evaporator (Buchii). The residue was taken
into separating funnel containing 100 ml of distilled water
and was successively extracted with diethyl ether. The
methyl esters NSAIDs (2a–h) were separated rapidly at the
bottom of the separatory funnel. The lower layer of methyl
esters NSAIDs (2a–h) was carefully collected and was dried
over anhydrous sodium sulfate. Finally, the excess of die-
thyl ether was distilled off. The product was collected and
recrystallized from appropriated solvent.
(CDCl3, d, ppm): 8.51 (s, 1H, fourth proton of coumarin),
7.65–7.68 (d, 2H, Ar–H), 7.32–7.37 (d, 2H, Ar–H), 2.73 (s,
3H, –CH3).
General procedure for synthesis of 3-(2-bromoacetyl)-
2H-chromen-2-one (6)
To a solution of 3-acetyl-2H-chromen-2-one (5, 0.25 mol)
in 200 ml of ethanol-free chloroform was added bromine
(0.25 mol) in 25 ml chloroform with intermittent shaking
and warming. The mixture was heated for 15 min on water
bath to expel most of the hydrogen bromide, then cooled,
and filtered. The solid was washed with ether and recrys-
tallized from glacial acetic acid gave colorless needles
(Koelsch, 1950). Yield = 62%, mp 160–163°C. IR spectra
(KBr cm-1): 3060 (C–H str of Ar), 2895 (C–H str of CH2),
General procedure for the synthesis of 2i
A mixture of p-acetamido phenol (1i, 0.01 mol) and ethyl
chloroacetate (0.01 mol) was refluxed using dry acetone in
the presence of anhydrous potassium carbonate for 6 h.
The reaction mixture was cooled and poured into crushed
ice. The solid obtained was filtered, dried, and recrystal-
lized from glacial acetic acid (IIango et al., 2009).
1
1737 (C=O str of coumarin), 1685 (C=O str of acetyl). H
NMR (CDCl3, d ppm): 8.43 (s, 1H, fourth proton of cou-
marin), 7.54–7.6 (d, 2H, Ar–H), 7.41–7.45 (d, 2H, Ar–H),
4.73 (s, 2H, –CH2Br).
General procedure for synthesis of carbohydrazides
of NSAIDs (3a–i)
General procedure for synthesis of 3-[(5-substituted-
1,3,4-oxadiazol-2-yl-thio)acetyl]-2H-chromen-2-one
(7a–i)
Compound 2a–i (0.01 mol) and hydrazine hydrate (99%,
0.02 mol) were refluxed in absolute ethanol for 15–20 h
(monitored by TLC). The mixture was concentrated,
cooled, and poured the in ice cold water. The solid thus
separated was filtered, dried, and recrystallized from
appropriated solvent.
A mixture of 5-substituted-1,3,4-oxadiazol-2-thione (4a–i,
0.01 mol) was dissolved in ethanol (5 ml) and 1 ml of
sodium ethoxide (0.01 mol) and heated under reflux for
4 h. To the above reaction mixture, a solution of 3-(2-
bromoacetyl)-2H-chromen-2-one (6, 0.01 mol) in ethanol
was added and refluxed further for 25–30 h. (The reaction
was monitored by TLC). The reaction mixture was poured
into ice-cold water and the precipitate of 3-[(5-substituted-
1,3,4-oxadiazol-2-yl-thio)acetyl]-2H-chromen-2-one (7a–
i) was collected by filtration after washing with water and
dried. Further, the title compounds were purified by flash
chromatography. The physicochemical and spectral data of
each compound are given below.
General procedure for synthesis of 5-substituted-1,3,4-
oxadiazol-2-thione (4a–i)
A mixture of one of the carbohydrazides of NSAIDs (3a–
i) (0.05 mol), KOH (0.05 mol), and carbon disulfide (5 ml)
in methanol was refluxed on a steam bath for 12 h. The
solution was then concentrated, cooled, and acidified with
dil. HCl. The solid mass that separated out was filtered,
washed with ethanol, dried, and recrystallized from the
appropriated solvent (Amir and Shikha, 2004).
3-[2-{5-(1-(4-Isobutylphenyl)ethyl)-1,3,4-oxadiazol-2-
ylthio}acetyl]-2H-chromen-2-one (7a)
General procedure for synthesis of 3-acetyl-2H-
chromen-2-one (5)
Yield = 54%, mp 178–180°C. IR spectra (KBr, cm-1):
3033 (C–H str of Ar), 2947 (C–H str of CH3), 2822 (C–H
str of CH2), 2361 (C–S str), 1729 (C=O str of coumarin),
1688 (C=O str of S–CH2–C=O group), 1607 (C=N str),
To a cold mixture of salicylaldehyde (0.2 mol) and ethyl
acetoacetate (0.2 mol), 2 ml of piperidine was added by
rapid stirring. After 20 min, the yellowish solid separated
was filtered off subsequently washed with ethanol and was
recrystallized from water:ethanol (3:7) (Khode et al.,
2009). Yield = 79.3%, mp 120°C. IR spectra (KBr cm-1):
3075 (C–H str of Ar), 2929 (C–H str of CH3), 1740 (C=O
str of coumarin), 1695 (C=O str of acetyl). 1H NMR
1
1518 (C=C str). H NMR (CDCl3, d, ppm): 8.5 (s, 1H,
fourth proton of coumarin), 7.08–7.7 (m, 8H, Ar–H), 4.7 (s,
2H, S–CH2–C=O group), 4.2 (q, 1H, –CH of Ibuprofen),
2.72 (d, 2H, –CH2 of Ibuprofen), 1.93 (m, 1H, –CH, iso-
butyl group), 1.41 (d, 3H, –CH3 of Ibuprofen), 1.16 (dd,
123

24
Med Chem Res (2012) 21:16–26
6H, –CH3 of isobutyl group). Elemental (CHN) analysis
calculated for C25H24N2O4S: C, 66.86; H, 5.44; N, 6.21
(Found: C, 66.83; H, 5.50; N, 6.28).
str), 1729 (C=O str of coumarin), 1682 (C=O str of S–CH2–
1
C = O group), 1610 (C=N str), 1557 (C=C str). H NMR
(CDCl3, d, ppm): 8.6 (s, 1H, fourth proton of coumarin),
7.0–7.7 (m, 12H, Ar–H), 4.58 (q, 1H, –CH of flurbiprofen),
4.36 (s, 2H, S–CH2–C=O group), 1.25 (t, 3H, CH3 of
flurbiprofen). Elemental (CHN) analysis calculated for
C27H19FN2O4S: C, 66.65; H, 3.95; N, 5.75 (Found: C,
66.69; H, 3.94; N, 5.74).
3-[2-{5-(2-Hydroxyphenyl)-1,3,4-oxadiazol-2-yl-
thio}acetyl]-2H-chromen-2-one (7b)
Yield = 83%, mp 204–206°C. IR spectra (KBr cm-1):
3503 (br, O–H str), 3082 (C–H str of Ar), 2890 (C–H str of
CH2), 2361 (C–S str), 1711 (C=O str of coumarin), 1693
(C=O str of S–CH2–C=O group), 1599 (C=N str), 1552
3-[2-{5-(1-(3-Benzoylphenyl)ethyl)-1,3,4-oxadiazol-2-
ylthio}acetyl]-2H-chromen-2-one (7f)
1
(C=C str). H NMR (CDCl3, d, ppm): 8.36 (s, 1H, fourth
proton of coumarin), 6.75–7.5 (m, 8H, Ar–H), 5.29 (s, br,
1H, OH), 4.24 (s, 2H, S–CH2–C=O group). Elemental
(CHN) analysis calculated for C19H12N2O5S: C, 60.05; H,
3.15; N, 7.40 (Found: C, 59.97; H, 3.20; N, 7.44).
Yield = 70%, mp 170–172°C. IR spectra (KBr cm-1):
3024 (C–H str of Ar), 2952 (C–H str of CH3), 2902 (C–H
str of CH2), 2363 (C–S str), 1744 (C=O str of coumarin),
1710 (C=O str of ketoprofen), 1695 (C=O str of S–CH2–
1
C=O group), 1605 (C=N str), 1566 (C=C str). H NMR
3-[2-{5-(2-(2,6-Dichlorophenylamino)benzyl)-1,3,4-
oxadiazol-2-ylthio}acetyl]-2H-chromen-2-one (7c)
(DMSO, d, ppm): 8.47 (s, 1H, fourth proton of coumarin),
6.9–7.8 (m, 13H, Ar–H), 4.49 (s, 2H, S–CH2–C=O group),
1.33 (t, 3H, CH3 of ketoprofen). Elemental (CHN) analysis
calculated for C28H20N2O5S: C, 67.72; H, 4.05; N, 5.65
(Found: C, 67.79; H, 4.04; N, 5.67).
Yield = 70%, mp 138–140°C. IR spectra (KBr cm-1):
3294 (N–H str), 3073 (C–H str of Ar), 2915 (C–H str of
CH2), 2363 (C–S str), 1724 (C=O str of coumarin), 1682
(C=O str of S–CH2–C=O group), 1605 (C=N str), 1566
1
3-[2-{5-((1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl)methyl)-1,3,4-oxadiazol-2-yl-thio}acetyl]-2H-
chromen-2-one (7g)
(C=C str). H NMR (CDCl3, d, ppm): 8.88 (s, 1H, N–H of
diclofenac), 8.52 (s, 1H, fourth proton of coumarin),
6.93–7.85 (m, 11H, Ar–H), 4.37 (s, 2H, S–CH2–C=O
group), 2.24 (s, 2H, CH2 of diclofenac). Elemental (CHN)
analysis calculated for C26H17Cl2N3O4S: C, 58.04; H, 3.18;
N, 7.83 (Found: C, 58.09; H, 3.21; N, 7.84).
Yield = 64%, mp 118–120°C. IR spectra (KBr cm-1):
3055 (C–H str of Ar), 2970 (C–H str of CH3), 2892 (C–H
str of CH2), 2363 (C–S str), 1738 (=N–C=O str of indo-
methacin), 1717 (C=O str of coumarin), 1679 (C=O str of
[5-2-Oxo-2-(2-oxo-2H-chromen-3-yl)ethylthio)-1,3,4-
oxadiazol-2-yl)methyl 2-(2(2,6-
dichlorophenylamino}phenyl]acetate (7d)
1
S–CH2–C=O group), 1600 (C=N str), 1555 (C=C str). H
NMR (CDCl3, d, ppm): 8.43 (s, 1H, fourth proton of
coumarin), 6.8–8.3 (m, 11H, Ar–H), 4.81 (s, 2H, CH2 of
indomethacin), 4.64 (s, 2H, S–CH2–C=O group), 3.82 (s,
3H, OCH3 of Indomethacin), 1.9 (s, 3H, CH3 of indo-
methacin). Elemental (CHN) analysis calculated for
C31H22ClN3O6S: C, 62.07; H, 3.73; N, 6.98 (Found: C,
62.09; H, 3.78; N, 7.03).
Yield = 77%, mp 140–142°C. IR spectra (KBr cm-1):
3290 (N–H str), 3040 (C–H str of Ar), 2897 (C–H str of
CH2), 2350 (C–S str), 1730 (C=O str of coumarin), 1689
(C=O str of S-CH2-C=O group), 1607 (C=N str), 1565
(C=C str). 1H NMR (DMSO, d, ppm): 9.26 (s, 1H, N–H of
aceclofenac), 8.57 (s, 1H, fourth proton of coumarin),
6.84–7.77 (m, 11H, Ar–H), 4.44 (s, 2H, S–CH2–C=O
group), 3.61 (s, 2H, –OCH2–Het), 2.67 (s, 2H, –CH2–C=O
of aceclofenac). Elemental (CHN) analysis calculated for
C28H19Cl2N3O6S: C, 56.40; H, 3.22; N, 7.04 (Found: C,
56.39; H, 3.24; N, 7.04).
3-[2-{5-(5-Benzoyl-2,3-dihydro-1H-pyrrolizin-1-yl)-1,3,4-
oxadiazol-2-ylthio}acetyl]-2H-chromen-2-one (7h)
Yield = 61%, mp 184–186°C. IR spectra (KBr cm-1):
3080 (C–H str of Ar), 2878 (C–H str of CH2), 2360 (C–S
str), 1726 (C=O str of coumarin), 1705 (C=O str of ketor-
olac), 1688 (C=O str of S–CH2–C=O group), 1602 (C=N
str), 1568 (C=C str). 1H NMR (CDCl3, d, ppm): 8.29 (s, 1H,
fourth proton of coumarin), 7.4–8.1 (m, 9H, Ar–H), 7.1–7.3
(dd, 2H, pyrrzolo protons), 4.5 (s, 2H, S–CH2–C=O group),
3.52 (t, 1H, CH of ketorolac), 3.18–3.24 (dd, 2H, CH2 of
3-[2-{5-(1-(2-Fluorobiphenyl-4-yl)-1,3,4-oxadiazol-2-
ylthio}acetyl]2H-chromen-2-one (7e)
Yield = 64%, mp 176–178°C. IR spectra (KBr cm-1):
3070 (C–H str of Ar), 2910 (C–H str of CH2), 2359 (C–S
123

Med Chem Res (2012) 21:16–26
25
Acute anti-inflammatory activity
ketorolac), 2.83–2.9 (dd, 2H, CH2 of ketorolac). Elemental
(CHN) analysis calculated for C19H13N3O5S: C, 57.75; H,
3.30; N, 10.67 (Found: C, 57.79; H, 3.28; N, 10.73).
In vivo acute anti-inflammatory activity was evaluated
using carrageenan-induced rat paw edema assay model of
inflammation by adopting the method of Winter et al.
(1962) for the compounds listed in Table 1. Male albino
rats (170–220 g) were fasted with free access to water at
least 12 h prior to experiments and were divided randomly
into 11 groups of six each. Control group received 1 ml of
0.5% sodium carboxymethyl cellulose (sodium CMC),
standard group received 13.5 mg/kg of diclofenac and test
groups received 200 mg/kg of synthesized compounds
(7a–i). The rats were dosed orally, 1 h later; a subplantar
injection of 0.05 ml of 1% solution of carrageenan in
sterile distilled water was administered to the left hind
footpad of each animal. The paw edema volume was
measured with a digital plethysmometer at 0, 1st, 3rd, and
5th h after carrageenan injection. Paw edema volume was
compared with vehicle control group, and percent reduc-
tion was calculated as 1 - (edema volume in the drug-
treated group/edema volume in the control group) 9 100.
3-(2-{(5-(4-Hydroxyphenylamino)-1,3,4-oxadiazol-2-
yl}thio)acetyl)-2H-chromen-2-one (7i)
Yield = 72%, mp 224–226°C. IR spectra (KBr cm-1):
3433 (N–H str), 3014 (C–H str of Ar), 2862 (C–H str of
CH2), 2362 (C–S str), 1734 (C=O str of coumarin), 1689
(C=O str of S–CH2–C=O group), 1618 (C=N str), 1538
(C=C str). 1H NMR (DMSO, d, ppm): 9.60 (s, 1H, N–H of
paracetamol), 8.73 (s, 1H, fourth proton of coumarin),
6.87–7.85 (m, 8H, Ar–H), 5.22 (s, 2H, O–CH2 of para-
cetamol), 4.69 (s, 2H, S–CH2–C=O group), 1.96 (s, 2H,
O=C–CH3). Elemental (CHN) analysis calculated for
C27H19N3O5S: C, 65.16; H, 3.85; N, 8.45 (Found: C, 65.19;
H, 3.88; N, 8.47).
Pharmacology
Animals
Analgesic activity
Albino mice of either sex weighing 20–25 g were used for
acute toxicity studies and analgesic activity. Healthy male
albino adult rats weighing 170–220 g were used for various
pharmacological screening. Animals were procured from
Venkateshwara Enterprises, Bangalore, India (245/CPC-
SEA) and housed individually in polypropylene cages,
maintained under standard conditions of alternating 12-h
light/dark cycles at a constant temperature (25 2°C) and
35–60% relative humidity). The pharmacological evalua-
tions were conducted after obtaining ethical clearance from
Animal Ethics Committee of KLES College of pharmacy,
Hubli (India). Animals were fed with standard rat pellet
diet (Hindustan Lever Ltd, Mumbai, India) and water
ad libitum.
Twenty-four hours prior to actual testing a large number of
mice (20–25 g) received intraperitoneally 10 ml/kg 0.6%
glacial acetic acid. Animals were observed for writhing
movements. Only those showing one or other type of writhing
movements (positive responders) were chosen for the test on
the next day. On the test day, the responders received com-
pounds half an hour prior to glacial acetic acid challenge.
Compounds 7c, 7e, 7f, and 7h were orally administered at a
dose of 200 mg/kg as a suspension in 0.5% sodium CMC.
Standard drug used was acetylsalicylic acid at a dose of
30 mg/kg as a suspension in 0.5% sodium CMC. Each mouse
was then observed for the total number of stretching episodes
or writhing for 15 min following glacial acetic acid injection.
The mean value for each group was calculated.
Ulcerogenic activity
Acute toxicity
Albino rats of either sex were divided into control, stan-
dard, and different test groups of six animals each group
(170–220 g). They were starved for 48 h (water ad libitum)
before drug administration. Control group received only
0.5% sodium CMC solution, standard group was orally
administered with acetylsalicylic acid in sodium CMC
solution and test compounds 7c, 7e, 7f, and 7h were
administered orally at the dose of 200 mg/kg bw. All the
animals were sacrificed after 7 h of drug administration.
Stomach was removed and placed on saline-soaked filter
paper until inspection. A longitudinal incision along the
greater curvature was made with fine scissors. The stomach
The acute toxicity test was carried out according to the
OECD guidelines to establish the therapeutic dose of test
compounds. Albino mice of each six each weighing
between 20 and 25 g were grouped into nine groups of six
animals each, starved for 24 h with water and libitum prior
to test. On the day of experiment, animals were adminis-
tered with different compounds to different groups in an
increase dose of 10, 100, 200, 1000, and 2000 mg/kg body
weight orally. The animals were observed continuously for
3 h for general behavioral, neurological, and autonomic
profiles, and then every 30 min for next 3 h, and finally for
next 24 h or till death.
123

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Med Chem Res (2012) 21:16–26
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absence of gastric irritation was determined. The ulcer
index range for each group was determined according to a
previously reported method by counting the number of
lesions (x) in each of five size classes (y). The classes were
defined as y = 1 (pinpoint lesion), y = 2 (lesions \ 1 mm
diameter), y = 3 (lesions 1–2 mm diameter), y = 4
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Acknowledgments The authors are thankful to The Principal,
KLES College of Pharmacy, Hubli, for providing necessary facilities
to carry out this research study. We sincerely express our gratitude to
Dr. M. N. A. Rao, General Manager (R&D), Divi’s Laboratory,
Hyderabad, and Dr. Y. S. Agasimundin, Professor, K.L.E.S College
of Pharmacy Hubli, for their encouragement. We are grateful to The
Director, SAIF, Punjab University, for providing elemental and
spectral analysis. The authors sincerely thank the AICTE, New Delhi
(India), for their financial support under MODROBS Scheme (File
No. 8023/RID/BOR/MOD/201/7/8). The authors also express special
thanks to Fourrts (India) Lab. Pvt. Ltd., Chennai (Tamil Nadu, India),
and Elvina Pharmaceuticals Pvt. Ltd. Kotur, Dharwad, for providing
gift samples of NSAIDs.
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