Novel arylpyridine-based 1,3,4-oxadiazoles: Synthesis, antibacterial, and anti-inflammatory evaluation

Article abstract of DOI:10.1002/jccs.201800248

In view of developing novel bioactive compounds, a series of 2-(5-[2-methyl-6-arylpyridin-3-yl]-1,3,4-oxadiazol-2-ylthio)-1-arylethanones (6a–n) were designed and synthesized in good yield. Novel compounds were evaluated for their antibacterial and anti-i

Full text of DOI:10.1002/jccs.201800248

Received: 28 June 2018
Revised: 22 November 2018
Accepted: 25 November 2018
DOI: 10.1002/jccs.201800248
A R T I C L E
Novel arylpyridine-based 1,3,4-oxadiazoles: Synthesis,
antibacterial, and anti-inflammatory evaluation
1
1
2
Sowmya Padejjar Vasantha | Boja Poojary | Revanasiddappa Bistuvalli Chandrashekarappa
1
Department of Chemistry, Mangalore University,
In view of developing novel bioactive compounds, a series of 2-(5-[2-methyl-
6-arylpyridin-3-yl]-1,3,4-oxadiazol-2-ylthio)-1-arylethanones (6a–n) were
Mangalagangothri, Karnataka, India
2
Department of Pharmaceutical Chemistry, NGSM
Institute of Pharmaceutical Sciences, Mangalore,
Karnataka, India
designed and synthesized in good yield. Novel compounds were evaluated for their
antibacterial and anti-inflammatory activities. All synthesized compounds were
screened for their antibacterial activity against Staphylococcus aureus, Bascillus
subtilis, Eschericia coli, and Pseudomonas aeruginosa strains. Compounds 6a, 6b,
6c, 6h, and 6i displayed the highest antibacterial activity with minimal inhibitory
concentration (MIC) values ranging from 6.25–12.5 μg/mL in comparison with the
standard Ciprofloxacin. The results of anti-inflammatory activity of carrageenan-
induced footpad edema assay indicated that tested compounds exhibited remark-
able anti-inflammatory activity with percentage of inhibition of 63.9–70.1%
Correspondence
Boja Poojary, Department of Chemistry,
Mangalore University, Mangalagangothri 574199,
Karnataka, India.
Email: bojapoojary@gmail.com
(potency 96.8–106.20% of indomethacin activity) after 3 hr. Particularly, 6c–e and
6j–l were found to be excellent inhibitors of inflammation, with potential higher
than that of the standard, Indomethacin.
KEYWORDS
bioorganic chemistry, medicinal chemistry, organic chemistry
[
2]
1
| INTRODUCTION
infection, and cancer through interacting and inhibiting the
[
3,4]
enzymatic activity.
For long-term use, NSAIDs are fre-
Antimicrobial agents have gained a lot of importance
because of life-threatening infectious diseases like diphthe-
quently associated with serious side effects like upper gas-
trointestinal (GI) irritation, bleeding, dyspepsia, ulceration,
and in some cases death. These side effects are due to non-
[1]
[5]
ria, typhoid, tuberculosis, cholera, HIV, plague etc caused
by microbes increased to an alarming level around the world.
This is mainly due to rapid development of multidrug-
resistant microbial pathogens. Hence, minimizing multidrug-
resistant pathogens may be an important approach to the
primary prevention or treatment of these diseases. These
clinical limitations such as drug resistance, toxicity, and side
effects had attracted a great deal of research interest in thera-
peutic antimicrobial-based drug formulations.
selective inhibition of COXs, thereby design and develop-
ment of NSAIDs with enhanced safety profile is still a
necessity and challenge for the pharmaceutical industry.
In this context, considerable interest has been focused on
1,3,4-oxadiazole derivatives that has occupied a unique
place in the field of medicinal chemistry. They are well
[
6]
[7]
[8]
known as antifungal,
anticancer,
fungicidal,
[
9]
[10]
[11]
antidepressant, hypoglycemic,
and anti-HIV
agents.
Inflammation is a normal reaction to injury or infection.
Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of
the most frequently used medications for the treatment of
pain and inflammation caused by chronic illnesses, including
diabetes, cardiovascular disease, arthritis, psoriasis,
Additionally, 1,3,4-oxadiazoles are very good bioisosteres of
esters and amides, which help substantially to enhance the
pharmacological activity by participating in hydrogen bond-
ing interactions with the receptors. Hence, they have moti-
vated countless researchers to study their synthesis and
©
2019 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
J Chin Chem Soc. 2019;1–13.
http://www.jccs.wiley-vch.de
1

2
SOWMYA ET AL.
pharmacological properties. Moreover, the presence of toxo-
phoric –N=C–O– linkage in the oxadiazole core contributes
to significant pharmacokinetic property like lipophilicity,
which in turn increases the ability of drug to reach the target
linker moieties are present and tried to develop potential bioac-
tive molecules. Herein, a variety of arylpyridine derivatives
bearing 1,3,4-oxadiazoles, containing a diverse set of aromatic
substitutions through S-alkyl linkage, were designed and
synthesized by placing the aryl pyridine moiety at the fifth
position and S-alkyl at the second position of the
[12]
by transmembrane diffusion.
Besides, the literature sur-
vey reveals that the presence of alkyl or acyl, carboxamide
linkage on the oxadiazole core plays an important role in
biological activity by increasing the ability of penetration
1,3,4-oxadiazole ring and screening the results of antibacterial
and anti-inflammatory activities.
[
13]
into the active site.
On the other hand, different substituted pyridines are
well documented in the literature as they have been associ-
ated with an extensive range of pharmaceutical and pesticide
2
2
| RESULTS AND DISCUSSION
.1 | Chemistry
activities
analgesic,
antidepressant,
inhibition,
including
anticancer,
anti-inflammatory,
[
14]
[15]
[16]
antimicrobial,
anticonvulsant,
anti-TB,
In the present work, the desired arylpyridine-based
1,3,4-oxodiazoles were geared up by the series of steps and
are depicted in Scheme 1. The enaminones 2a–b were
obtained by refluxing the 4-chloroacetophenone 1a and
4-bromoacetophenone 1b with dimethyl formamide dimethyl
acetal. In a one-pot, three-component heterocylocondensa-
tion process of 2a–b, diketoester and ammonium acetate in
glacial acetic acid media furnished corresponding
ethyl (6-aryl-2-methylpyridine)-3-carboxylates 3a–b. The
intermediates, 3a–b, were then subjected to hydrazinolysis
by hydrated hydrazine into corresponding arylpyridine
carbohydrazides 4a–b. 5-Arylpyridine-1,3,4-oxadiazol-
2-thiones 5a–b were synthesized by intramolecular cycliza-
tion of different arylpyridine carbohydrazides 4a–b with
carbon disulfide in the presence of sodium hydroxide in
absolute ethanol, as a solvent, and finally, the preparation of
target compounds was achieved via the nucleophilic substitu-
tion reaction of 5-arylpyridine-1,3,4-oxadiazol-2-thiones 5a–
b with substituted phenacyl bromides/chlorides in acetone
and triethylamine. The structures of 2-(5-[2-methyl-
[
17]
[18]
phosphodiestrase-3 inhibitor,
HIV
[
19]
[20]
antihypertensive agents,
selective inhibitors
[21]
[22]
of hepatitis C virus (HCV) , and insecticidal activities.
Additionally, Numerous studies pointed out the antimicro-
bial and anti-inflammatory properties of 1,3,4-oxadiazole
and pyridine derivatives as promising antimicrobial and anti-
[23–28]
inflammatory agents (Figure 1).
Additionally, there are
reports that incorporation of chlorine/bromine atoms into a
molecule provides compounds that exhibit superior anti-
[29–32]
inflammatory and antimicrobial activities.
Heterocyclic
compounds with a chloro-substituted/bromo-substituted phe-
nyl ring are reported as potent analgesic used in inflamma-
[33]
[34]
tory pain, for example, SC-558,
zomepirac
and
[35]
DuP-697. Furthermore, antimicrobial and anti-inflammatory
activities of 1,3,4-oxadiazoles clubbed with chloro-substituted/
bromo-substituted arylpyridine moieties are not even reported
in the literature. In the light of above findings, we considered it
of interest to synthesize a new class of heterocyclic molecules
in which all of these 1,3,4-oxadiazole, pyridine, and S-alkyl
O
O
O
O
(i)
CH3
(ii)
N
N
CH3
CH3
CH3
R
R
R
2
a,b
3a,b
1
a,b
(iii)
O
NH2
N
H
N
NH
(
iv)
N
CH3
O
S
R
N
R
CH3
4a,b
5
a,b
(
v)
N
N
R1
O
S
R
N
O
CH3
6a-n
SCHEME 1 Outline for the synthesis. Reagents and conditions: (i) DMF–
ꢀ
FIGURE 1 Literature reports for active 1,3,4-oxadiazole, pyridine and
S-alkyl linker-based anti-inflammatory and antimicrobial derivatives and
strategy employed for designing arylpyridine-based 1,3,4-oxadiazoles
DMA, 90 C, 5–6 hr. (ii) ethyl acetoacetate, NH OAc, AcOH, reflux, 3 hr.
4
(iii) N
2
H
4
, reflux, 5–6 hr. (iv) CS
2
, NaOH, EtOH, reflux 4 hr. (v) Phenacyl
bromide/chloride, Et
3
N, acetone, reflux, 5 hr

SOWMYA ET AL.
3
6
(
-arylpyridin-3-yl]-1,3,4-oxadiazol-2-ylthio)-1-arylethanones
6a–n) (Table 1) were ascertained by their spectral and
analytical data.
The formation of intermediates 5a–b from corresponding
the four protons of the 4-bromophenyl moiety, which further
13
confirmed the structure. Its C NMR further confirmed the
S-alkylated product. The spectrum of 6l displayed a highly
deshielded signal at 193.4 ppm due to the presence of car-
bonyl carbon, which was absent in previous
1,3,4-oxadiazole-2-thiones. Moreover, a signal correspond-
ing to C=S carbon of intermediate 5b was found to be miss-
ing and it showed additional signal at 42.2 ppm
carbohydrazides was confirmed by the presence of character-
istic stretching vibrations for NH, C=N, and C=S groups in
FT-IR. When compound 4b was converted to the corre-
sponding oxadiazole thione 5b, the absorption bands due to
amide carbonyl and NH/NH groups disappeared, instead
corresponding to the S–CH
carbon, which clearly indicates
2
2
new absorption bands for NH, C=N, and C=S groups were
the formation of target 6l. The shifting of stretching vibra-
−
1
−1
observed at 3,062, 1,581, 1,332 cm , respectively. In the
tion to a lower region (C–S) 1,191 cm
1,332 cm and the absence of vibration at 3,062 cm cor-
from (C=S)
1
−1
−1
H NMR spectrum of compound 5b, the presence of the pyr-
−
1
responding to the NH; while a new vibration at 1,681 cm
idine ring was affirmed by two doublets at 8.03 ppm
corresponding to C=O in its IR spectrum provide additional
support for the formation of target 6l from 1,3,4-oxadiazole-
(
J = 8.4 Hz) for C –H and 8.22 ppm (J = 8.4 Hz) for C -H
4
5
and a sharp singlet at 2.80 ppm attributable for the methyl
group on the pyridine unit. The signals resonating at 7.71
and 8.11 ppm as two doublets due to four protons supported
the presence of aryl nucleus in the molecule. Furthermore,
2
-thione derivative 5b. The mass spectrum provided support
to the structure of the compound 6l as it displayed (M − 1),
(M − 1) + 2], and [(M − 1) + 4] ion peaks at m/z 532.80,
34.10, 536.80, respectively, which is in agreement with its
molecular formula C H N BrCl O S, evidencing the for-
[
5
13
the C NMR spectrum of 5b confirmed the formation of the
1
1
1
,3,4-oxodiazole ring by displaying signals at 177.1 and
59.4 ppm due to C-2 and C-5 carbon atoms of the
,3,4-oxadiazole ring. The structure of new intermediate 5b
22 14
3
2 2
mation of compound.
was further confirmed by its mass spectrum that displayed
M + 1), (M + 2) ion peaks at m/z 347.25, 349.10, respec-
tively, which is in agreement with its molecular formula.
The formation of new arylpyridine-based
,3,4-oxodiazole derivatives (6a–n) was confirmed by their
2.2 | In vitro antibacterial activity
(
Antibacterial potency of newly synthesized compounds was
checked by performing their in vitro antibacterial activity
using agar disc-diffusion method against Eschericia coli and
Pseudomonas aeruginosa representative of Gram-positive
and Staphylococcus aureus, Bascillus subtilis representative
of Gram-negative bacterial strains. Ciprofloxacin was used
as the positive control. The results of the preliminary antimi-
crobial activity of compounds 6a–n are reported in Table 2.
The results revealed that they showed varying degrees of
inhibition against the tested microorganisms. In general,
compounds 6b, 6c, 6h, 6i, and 6n displayed the best antibac-
terial activity with zone of inhibition ranging between 13.6
and 16.5 mm toward Gram-positive bacteria B. subtilis and
were considered the most sensitive among the tested micro-
organisms, while the other compounds were weakly active
or completely inactive with the inhibition zone ≤12.5 mm.
In contrast, none of the tested compounds displayed an
observable inhibitory effect against S. aureus organism.
Compounds 6h and 6i produced good inhibiting potency
toward Gram-negative E. coli with the inhibition zone
1
spectral analysis (Supporting Information). In general, in the
FT-IR spectra (6a–n), the bands corresponding to NH and
C=S of the 1,3,4-oxadiazole-2-thione ring disappeared.
Also, new characteristic peaks corresponding to C=O and
C–S stretching vibrations were appeared, indicating the S-
1
alkylation. Additionally, in the H NMR spectra the presence
of a new signal between 5.03–5.20 ppm for the S–CH2
group clearly demonstrated the S-alkylation. Similarly, in
1
3
the C NMR spectra of (6a–n), the signal corresponding to
C=S of intermediates 5a–b was found to be missing, instead
the presence of a new signal corresponding to C=O is evi-
denced the formation of S-alkylated analogs. In particular,
1
for the prototype 6l, its H NMR showed additional doublets
at 7.80 and 7.95 ppm with coupling constants 8.4 and 2 Hz
corresponding to C –H and C –H of the 2,4-dichlorophenyl
3
6
ring, respectively. A doublet of doublet in the region
.63–7.65 ppm integrating for one proton with coupling
constants 8.4 and 2 Hz corresponding to C –H of the
7
>
13 mm, while the compound 6g was moderately active
5
with the inhibition zone 11.5 mm. Furthermore, compounds
6a, 6b, 6i, and 6n produced marked activity against Gram-
negative P. aeruginosa with the inhibition zone ranging
value between 12.5–14.5 mm, compared with other com-
pounds of the series. The compounds 6d, 6e, 6f, 6j, 6k, 6l,
and 6m were found to be active against all bacterial strains
but results were not appreciable. Based on the preliminary
screening, the most active compounds were further exam-
ined for their in vitro minimal inhibitory concentration
(MIC) against the same organisms using the microdilution
2,4-dichlorophenyl ring clearly confirmed the S-alkylated
derivative. In addition to this, the spectrum showed a singlet
at 5.03 ppm integrating two protons of the S–CH group,
2
which provides the additional support for the S-substitution.
The signal at 2.87 ppm attributed to the –CH3 group
attached to the pyridine moiety. Similarly, pyridine-C –H
4
and C –H appeared as doublets at 8.05 and 8.31 ppm with a
5
coupling constant of 8 Hz and doublet of doublets in the
region at 7.72–7.74 and 8.13–8.15 ppm were attributed to

4
SOWMYA ET AL.
TABLE 1 Physical data of the newly synthesized 2-(5-(2-methyl-6-arylpyridin-3-yl)-1,3,4-oxadiazol-2-ylthio)-1-arylethanones (6a–n) via Scheme 1
ꢀ
Compound
R
R
1
Mol. formula
Mol. weight
Yield (%)
Melting point ( C)
6
a
Cl
23
C H
18
N
3
ClO
2
S
435.08
90
180–181
6
b
Cl
Cl
C
22
H
15
N
3
BrO
2
S
498.98
451.08
91
98
212–213
198–199
Br
O
6
c
C
23
H
18
N
3
ClO
3
S
6
d
e
Cl
Cl
C
22
H
H
15
N
N
3
3
Cl
Cl
2
O
O
2
S
455.03
488.99
85
73
209–210
139–140
Cl
6
C
22
14
3
2
S
Cl
Cl
6
6
f
Cl
Cl
C
29
H
18
N
3
Cl
3
O
2
S
577.02
421.07
79
97
147–149
151–152
Cl
Cl
g
C
22
H
16
N
3
ClO
2
S
6
h
i
Br
Br
C
23
H
H
18
N
N
3
3
BrO
2
S
479.03
545.25
94
95
183–184
213–214
6
C
22
15
Br
2
O
2
S
Br
6
j
Br
23
C H
18
N
3
BrO
3
S
495.03
94
199–200
O
6
k
l
Br
Br
C
22
H
H
15
N
N
3
3
BrClO
2
S
498.98
532.94
98
83
217–218
144–146
Cl
6
C
22
14
2 2
BrCl O S
Cl
Cl

SOWMYA ET AL.
5
TABLE 1 (Continued)
ꢀ
Compound
R
R
1
Mol. formula
Mol. weight
Yield (%)
Melting point ( C)
6
m
Br
29
C H
18
N
3
BrCl
2
O
2
S
620.35
76
168–169
Cl
Cl
6
n
Br
22
C H
16
N
3
BrO
2
S
465.01
96
153–154
method. Results are summarized in Table 3. It was observed
that the results were in agreement with the preliminary
screening results. The tested compounds displayed moderate
to good inhibitory activity with the MIC values ranging from
P. aeruginosa and E. coli, respectively. Furthermore, among
the tested compounds, 6c (R = Cl and R = 4-methoxyphe-
1
nyl) exhibited highest inhibition against B. subtilis strain
with an MIC value of 6.25 μg/mL, which displayed reduc-
tion in inhibition activity toward P. aeruginosa and
S. aureus organisms with an MIC value 50 μg/mL. Com-
100 to 6.25 μg/mL. Among the tested compounds, 6i
(
R = Br and R = 4-bromophenyl) showed highest inhibition
1
with an MIC value of 6.25 μg/mL toward B. subtilis and
E. coli strain and good activity toward P. aeruginosa with
an MIC value of 12.5 μg/mL. Furthermore, tested 6b, 6h
and 6i exhibited good activity with an MIC value of
pound 6h (R = Br and R = 4-methylphenyl) displayed good
1
activity against B. subtilis and E. coli with an MIC value of
12.5 μg/mL and it showed moderate activity toward P. aeru-
ginosa. Replacement of bromine in 6h by chlorine, that is
12.5 μg/mL and compound 6g showed moderate activity
compound 6a (R = Cl and R = 4-methylphenyl) led to sig-
1
toward B. subtilis with an MIC value of 25 μg/mL. Replace-
ment of bromine by chlorine in 6i led to compound 6b
nificant loss of activity toward B. subtilis and E. coli. How-
ever, it showed increased activity against P. aeruginosa with
an MIC value of 12.5 μg/mL. Rest of the tested compounds
exhibited weak to moderate activity against the tested organ-
isms with MIC values ranging from 25–100 μg/mL. Thus, it
was clear that the introduction of chlorine did not improve
the potency. Additionally, activity results revealed that halo,
methoxy, or methylphenacyl substituents in the aryl part are
essential for the antibacterial activity.
(
R = Cl and R = 4- bromophenyl), whose activity was
1
reduced to MIC values of 12.5 and 100 μg/mL toward
TABLE 2 Antibacterial activity of synthesized compounds 6a–n using the
agar cup plate method
Antibacterial activity data of the target 6a–n in terms of the zone of
inhibition
Antibacterial activity diameter of growth inhibition
zone
2
.3 | In vivo anti-inflammatory activity
a
Gram positive
S. aureus
—
Gram negative
E. coli
Based on the antibacterial potency, compounds 6c–6e, 6g,
6j–6l, and 6n were evaluated for their anti-inflammatory
activity by employing the carrageenan-induced paw edema
bioassay in rats and acute toxicity. We used a well-known
anti-inflammatory agent, Indomethacin, as the standard.
Sample
Control
B. subtilis
—
P. aeruginosa
—
—
6
6
6
6
6
6
6
6
6
6
6
6
6
6
a
b
c
d
e
f
14 ꢁ 0
11.5 ꢁ 0.5
15.5 ꢁ 0.5
16.5 ꢁ 0.5
12.5 ꢁ 0.5
11.5 ꢁ 0.5
10.5 ꢁ 0.5
11 ꢁ 1
15.5 ꢁ 0.5
15.5 ꢁ 0.5
10.5 ꢁ 0.5
9.5 ꢁ 0.5
10.5 ꢁ 0.5
12 ꢁ 0
8 ꢁ 0
5 ꢁ 0
8 ꢁ 0
10 ꢁ 0
14.5 ꢁ 0
13.5 ꢁ 0.5
8.5 ꢁ 0.5
9.5 ꢁ 2.5
9 ꢁ 0
10.5 ꢁ 0.5
7.5 ꢁ 0.5
6.5 ꢁ 0.5
11.5 ꢁ 0.5
10.5 ꢁ 0.5
11 ꢁ 1
8.5 ꢁ 0.5
9.5 ꢁ 0.5
8.5 ꢁ 0.5
11.5 ꢁ 0.5
13 ꢁ 1
TABLE 3 Antibacterial activity of the selected target molecules in terms
of MIC
10 ꢁ 0
Antibacterial activity MIC in μg/mL
8 ꢁ 0
8 ꢁ 0
g
h
i
11.5 ꢁ 0.5
13 ꢁ 0
13.5 ꢁ 0.5
9.5 ꢁ 0.5
6.5 ꢁ 0.5
7 ꢁ 1
10.5 ꢁ 0.5
8.5 ꢁ 0.5
13.5 ꢁ 0.5
10.5 ꢁ 0.5
8.5 ꢁ 0.5
10.5 ꢁ 0.5
13 ꢁ 1
Sample
B. subtilis
—
E. coli
—
P. aeruginosa
Control
—
6a
50
100
100
50
12.5
12.5
50
j
6b
12.5
6.25
50
k
l
6c
6g
25
50
m
n
11 ꢁ 1
7 ꢁ 1
31 ꢁ 1
7 ꢁ 1
6h
12.5
12.5
25
12.5
6.25
100
2
50
13.5 ꢁ 0.5
17 ꢁ 1
7.5 ꢁ 0.5
12.5 ꢁ 0.5
6i
6.25
25
Ciprofloxacin
14.5 ꢁ 0.5
15.5 ꢁ 0.5
6n
Ciprofloxacin
2
4
a
Results are expressed as mean ꢁ SEM (n = 5).

6
SOWMYA ET AL.
TABLE 4 Anti-inflammatory effect of the selected compounds (50 mg/kg) and indomethacin (10 mg/kg) against carrageenan-induced paw edema in rats
Anti-inflammatory activity: Change in paw volume (mL) after drug treatment (ꢁSEM) (% inhibition)
a
Potency (%)
Sample
0 hr
1 hr
2 hr
3 hr
3 hr
Control
0.139 ꢁ 0.0077 (0)
ND
0.283 ꢁ 0.0161 (0)
ND
0.306 ꢁ 0.0186 (0)
ND
0.368 ꢁ 0.0136 (0)
ND
0
6
6
6
6
6
6
6
6
6
6
6
6
6
6
a
b
c
d
e
f
ND
ND
ND
ND
ND
ND
0.11 ꢁ 0.0037 (20.9)***
0.112 ꢁ 0.0031 (19.4)***
0.107 ꢁ 0.0033 (23.0)***
ND
0.135 ꢁ 0.0043 (52.3)***
0.135 ꢁ 0.0043 (52.3)***
0.158 ꢁ 0.007 (44.2)**
ND
0.11 ꢁ 0.0026 (64.1)***
0.12 ꢁ 0.0052 (60.8)***
0.117 ꢁ 0.0021 (61.8)***
ND
0.11 ꢁ 0.0045 (70.1)***
0.112 ꢁ 0.0031 (69.7)***
0.112 ꢁ 0.0017 (69.6)***
ND
106.2
105.6
105.5
ND
g
h
i
0.117 ꢁ 0.0021 (15.8)**
ND
0.161 ꢁ 0.0043 (42.4)
ND
0.158 ꢁ 0.0045 (48.3)*
ND
0.130 ꢁ 0.0021 (64.7)*
ND
98
ND
ND
ND
ND
ND
ND
j
0.112 ꢁ 0.004 (19.4)***
0.115 ꢁ 0.0043 (17.3)***
0.107 ꢁ 0.0033 (23.0)***
ND
0.143 ꢁ 0.0042 (49.5)***
0.132 ꢁ 0.0048 (53.4)***
0.13 ꢁ 0.0026 (54.1)***
ND
0.12 ꢁ 0.0026 (60.8)***
0.13 ꢁ 0.0068 (57.5)***
0.118 ꢁ 0.004 (61.4)***
ND
0.115 ꢁ 0.0022 (68.8)***
0.12 ꢁ 0.0026 (67.4)***
0.115 ꢁ 0.0043 (68.8)***
ND
104.2
102.1
104.2
ND
k
l
m
n
0.119 ꢁ 0.007(14.3)**
0.12 ꢁ 0.0059 (13.7)
0.164 ꢁ 0.0026 (42)
0.15 ꢁ 0.0072 (47)
0.162 ꢁ 0.0031(47)
0.15 ꢁ 0.0075 (51.0)
0.133 ꢁ 0.0043 (63.9)*
0.125 ꢁ 0.0067 (66.0)
96.8
100
Indomethacin
Differences between means at *p < 0.01; **p < 0.005; and ***p < 0.0001 were regarded significant. ND: not determined.
a
Potency is expressed as % of edema inhibition of the tested compounds relative to % of edema inhibition of indomethacin at 3 hr.
The results are summarized in Table 4, as for activity, the
tested compounds possess significant inhibition of inflam-
mation that is, paw edema, when compared to the normal
control group at a dose level of 50 mg/kg. The treatment
with doses of 50 mg/kg of 6c having the 4-chlorophenyl
moiety attached to the pyridine ring and the
more chloro substituent in 6d and 6k decreased their activ-
ity from 69.7 to 68.8%, i.e., compounds 6e and 6l dis-
played good anti-inflammatory activity, but lower than
that of the starting compounds 6d and 6k. Additionally,
among the tested compounds, animals treated with com-
pounds 6g and 6n with an unsubstituted phenyl ring exhib-
ited the lowest anti-inflammatory activity.
4
-methoxyphenyl ring attached to the other extreme end of
the 1,3,4-oxadiazole ring resulted a most active compound
by exhibiting significant inhibition of paw-edema 70.1%,
when compared to the normal control group and was more
effective than the standard-treated group 66% at a test dose
of 10 mg/kg, their potency being nearly equal to 106.2%
that of Indomethacin. Furthermore, replacing the
When we correlate the structures of tested compounds
and their activity, it is clear that anti-inflammatory activity
is sensitive to structural changes that is, the chlorophenyl
substituent on pyridine and electron donating
p-methoxyphenacyl spacer on other end of the oxadiazole
moiety imparted superior activity than the other phenacyl
spacer derivatives. Furthermore, derivatives with the chlor-
ophenyl substituent on the pyridine ring considerably
enhanced the anti-inflammatory activity when compared to
the oxadiazoles with bromophenyl on the pyridine ring.
While the addition of the dichlorophenacyl spacer
decreased the anti-inflammatory activity compared to the
monochlorophenacyl derivatives. Meanwhile, the unsub-
stituted phenacyl moiety did not affect the reduction in the
edema thickness.
4
-chlorophenyl group with 4-bromophenyl in 6c led to
compound 6j displaying a significant activity. It must be
noted that its inhibiting potency was decreased to 68.8%,
indicating that the 4-bromophenyl substituent was less
efficient when compared to the 4-chlorophenyl group.
Acute treatment with the compound 6d having the
4
-chlorophenyl substituent on both extreme ends emerged
as a good anti-inflammatory agent by exhibiting the per-
centage inhibition value of 69.7%, this effect was signifi-
cant (p < 0.001) when compared to the standard-treated
group. Meanwhile, replacing the 4-chlorophenyl substitu-
ent in the pyridine ring of 6d with the 4-bromophenyl sub-
stituent resulted in compound 6k, which emerged as a
potent compound with a percentage inhibition of 67.4%
notably, which promoted a decrease in the inhibition of
inflammation with their potency being nearly equal to
The acute toxicity study demonstrated that none of the
screened compounds induce any appreciable behavioral change
at the administered doses during the observation period.
3 | CONCLUSIONS
1
6
02.1% that of Indomethacin. However, when compared to
d, the activity was slightly less, which clearly indicates
In conclusion, a series of 2-(5-[2-methyl-6-arylpyridin-3-yl]-1,-
3,4-oxadiazol-2-ylthio)-1-arylethanones (6a–n) were prepared
in good yield and their anti-inflammatory and antimicrobial
activity were investigated. Evaluation of anti-inflammatory
the efficacy of the 4-chloro substituent on the anti-
inflammatory activity. Furthermore, introduction of one

SOWMYA ET AL.
7
activity revealed that most of the synthesized compounds
showed a significant decrease in inflammation induced by car-
rageenan in rat's paws. Compound 6c induced amazing anti-
inflammatory activity by exhibiting the highest percentage of
inhibition of 70.1% (with potency 106.2% to that of indometh-
acin), whereas the reference drug Indomethacin displayed per-
centage of inhibition of 66%. Furthermore, compounds 6d, 6e,
4.2 | Ethyl 6-(4-chlorophenyl)-2-methylpyridine-
3-carboxylate (3a)
ꢀ
Brown solid. Yield: 98%, mp. 42–43 C. FT IR (ATR,
−1
cm ): 1,723 (C=O), 1,641 (C=N), 1,579 (C=C), and
1
8
23 (C–Cl); H NMR (400 MHz, DMSO-d , δ, ppm): 1.34
6
(
t, 3H, J = 7.4 Hz, –CH ), 2.77 (s, 3H, –CH ), 4.29–4.34
3
3
1
(
q, 2H, J = 7.2 Hz, –CH ), 7.53–7.55 (dd, 2H, J = 8.8 Hz,
2
2
6k, and 6l exhibited significant anti-inflammatory activity
J = 2 Hz, C3 and C –H of 4-ClC H ), 7.95 (d, 1H,
5
6 4
compared to the standard drug. Moreover, the activity
results demonstrated that halogen atoms particularly
chlorophenyl on the pyridine ring and the methoxyphena-
cyl spacer have made good contribution to potency. In
addition to this, the in vitro antimicrobial profile evi-
denced that among the series, compounds 6c and 6i
emerged as more potent antibacterial agents. Furthermore,
compounds 6a, 6b, and 6h displayed good antibacterial
activity compared with the standard. The investigation
confirmed that the design is successful in generating
members of the antibacterial and anti-inflammatory agent
family.
J = 8.4 Hz, C –H of pyridine), 8.04–8.06 (dd, 2H,
4
1
2
J = 8.8 Hz, J = 2 Hz, C and C –H of 4-ClC H ), and
2
6
6 4
13
8
.24 (d, 1H, J = 8.4 Hz, C –H of pyridine); C NMR
4
(
100 MHz, DMSO-d , δ, ppm): 165.7, 158.6, 156.4, 139.2,
6
136.5, 135.7, 129.5, 128.1, 123.6, 117.4, 60.8, 24.6, and
14.1; MS: calculated for C H NClO is 275.07; found: (M
15
14
2
+) 275.75, (M + 2) 277.75 m/z; Anal. Calc. for
C H NClO : C, 65.34; H, 5.12; and N, 5.08%. Found: C,
15
14
2
65.49; H, 5.56; and N, 5.06%.
4
3
.3 | Ethyl 6-(4-bromophenyl)-2-methylpyridine-
-carboxylate (3b)
ꢀ
Brown solid. Yield: 98%, mp. 49–51 C. FT IR (ATR,
−
1
cm ): 1,724 (C=O), 1,641 (C=N), 1,581(C=C), and
1
7
(
75 (C–Br); H NMR (400 MHz, DMSO-d , δ, ppm): 1.34
4
| EXPERIMENTAL
6
t, 3H, J = 7.4 Hz, –CH ), 2.78 (s, 3H, –CH ), 4.30–4.35
(q, 2H, J = 7.2 Hz, –CH
J = 2 Hz, C
3
3
1
All the chemicals and reagents were purchased from Merck,
S.D. Fine, and Sigma-Aldrich Company and used as
received. The purity of compounds was checked by thin
layer chromatography (TLC) analyses performed on silica
gel-G-coated aluminum plates (Merck) and spots were visu-
alized by UV light. Melting points were determined in open
glass capillaries and are uncorrected. FT (ATR)-IR absorp-
tion spectra were acquired on apparatus Thermo Nicolet,
2
), 7.70–7.72 (dd, 2H, J = 8.8 Hz,
–H of 4-BrC ), 7.95 (d, 1H,
–H of pyridine), 8.08–8.10 (dd, 2H,
2
and C
H
6 4
3
5
J = 8.4 Hz, C
4
1
2
J = 8.8 Hz, J = 2 Hz, C
8.25 (d, 1H, J = 8.4 Hz, C
100 MHz, DMSO-d , δ, ppm): 165.8, 158.6, 156.4, 139.4,
and C
–H of 4-BrC1
H ), and
2
6
6 4
3
–H of pyridine); C NMR
5
(
1
(
3
6
36.5, 131.7, 128.9, 123.9, 123.6, 117.4, 60.9 (–CH ), 24.6
–CH ), and 14 (–CH ); MS: calculated for C H NBrO is
19.02; found: (M+) 319.20, (M + 2) 321.20 m/z; Anal.
2
3
3
15 14
2
−
1
1
13
Avatar 370 in the 4,000–400 cm range. The H and
NMR spectra were recorded for d -DMSO solution on Bru-
C
Calc. for C H NBrO : C, 56.27; H, 4.41; and N, 4.37%.
15 14
2
6
Found: C, 56.49; H, 4.56; and N, 4.20%.
ker Avance III, 400 and 100 MHz, respectively, with TMS
as the internal standard. Elemental analysis (C, H, and N) of
compounds was carried out using CHNS Elementar Vario
EL III. The mass spectra were recorded using a Water,
synapt G2 high detection mass spectrometer and are
uncorrected.
4.4 | Synthesis of 2-methyl-6-arylnicotinohydrazide
4
a–b
A mixture of hydrazine hydrate (99%, 6 mL) and ethyl
-methyl-6-arylnicotinate 3a–b (6 mmol) was heated under
2
reflux condition for 6 hr and allowed to cool to room tem-
perature. The resultant solid was filtered and washed with
water and dried, and recrystallized from dioxane.
4
.1 | Synthesis of ethyl 2-methyl-6-arylnicotinate
(3a–b)
A mixture of enaminones 2a–b (2 mmol), ethylacetoacetate
2.2 mmol), and ammonium acetate (40 mmol) was refluxed
4.5 | 6-(4-Chlorophenyl)-2-methylpyridine-
3-carbohydrazide (4a)
(
for 5 hr. in glacial acetic acid media. The progress of reac-
tion was monitored via TLC till single spot. After cooling,
poured into ice-cold water, residue formed was collected by
filtration and washed with petroleum ether and dried. The
residue was purified by recrystallization in ethanol to afford
compound 3a–b as brown solids.
ꢀ
White solid. Yield: 97%, mp. 179–180 C. FT IR (ATR,
−1
cm ): 3,325, 3,296 (NH ), 3,192 (NH), 1,640 (C=O), 1,585
C=N), 1,516 (C=C), and 824 (C–Cl); H NMR (400 MHz,
DMSO-d , δ, ppm): 2.60 (s, 3H, –CH ), 4.54 (s, 2H, –NH ),
.54 (d, 2H, J = 8.4 Hz, C and C –H of 4-ClC H ), 7.79
2
1
(
6
3
2
7
3
5
6 4
(
d, 1H, J = 8 Hz, C –H of pyridine), 7.87 (d, 1H,
4

8
SOWMYA ET AL.
J = 8.4 Hz, C –H of pyridine) 8.05 (d, 2H, J = 8.4 Hz, C
3.32; and N, 13.83%. Found: C, 55.49; H, 3.56;
and N, 13.06%.
5
2
1
3
and C –H of 4-ClC H ), and 9.64 (s, 1H, NH); C NMR
6
6 4
(
100 MHz, DMSO-d , δ, ppm): 167.1, 155.7, 154.3, 137.3,
6
1
36.5, 135.7, 130.7, 129.2, 128.2, 117.1, and 22.9 (–CH3);
4
1
.9 | 5-[6-(4-Bromophenyl)-2-methylpyridin-3-yl]-
,3,4-oxadiazole-2(3H)-thione (5b)
MS: calculated for C H N ClO is 261.07; found: (M+)
1
3 12 3
261.75,
(M + 2)
263.75 m/z;
Anal.
Calc.
for
ꢀ
Off White solid. Yield: 98%, mp. 2,111–212 C. FT IR
C H N ClO: C, 59.66; H, 4.62; and N, 16.06%. Found: C,
1
3 12 3
−
1
(ATR, cm ): 3,062 (NH), 1,581 (C=N), 1,508 (C=C),
59.49; H,4.56; and N, 16.06%.
1
1
,332 (C=S), 1,299 (C–O–C), and 761 (C–Br); H NMR
(
2
400 MHz, DMSO-d , δ, ppm): 2.80 (s, 3H, –CH ), 7.71 (d,
6
3
4
3
.6 | 6-(4-Bromoophenyl)-2-methylpyridine-
-carbohydrazide (4b)
H, J = 8.4 Hz, C and C –H of 4-BrC H ), 8.01(d, 1H,
3
5
6 4
J = 8.4 Hz, C –H of pyridine), 8.11 (d, 2H, J = 8.4 Hz, C2
and C –H of 4-BrC H ), and 8.22 (d, 1H, J = 8.4 Hz, C –H
of pyridine); C NMR (100 MHz, DMSO-d , δ, ppm):
1
1
for C H N BrOS is 346.97; found: (M+) 347.25, (M + 2)
3
2
4
ꢀ
White solid. Yield: 98%, mp. 182–183 C. FT IR (ATR,
cm ): 3,326, 3,294 (NH ), 3,195 (NH), 1,641 (C=O),
1
6
6
4
5
−
1
13
2
6
1
,583 (C=N), 1,506 (C=C), and 784 (C–Br); H NMR
77.1 (C=S), 159.4, 156.4, 155.7, 137.1, 136.3, 131.8,
(
400 MHz, DMSO-d , δ, ppm): 2.60 (s, 3H, –CH ), 4.54
6
3
28.8, 123.7, 117.6, 116.1, and 24.9 (–CH ); MS: calculated
3
(
s, 2H, NH ), 7.71 (d, 2H, J = 8.4 Hz, C and C –H of
2
3
5
14 10 3
4
-BrC H ), 7.79 (d, 1H, J = 8 Hz, C –H of pyridine), 7.87
6
4
4
49.10 m/z; Anal. Calc. for C H N BrOS: C, 48.29; H,
14 10 3
.89; and N, 12.07%. Found: C, 48.49; H, 2.56;
(
d, 1H, J = 8.4 Hz, C –H of pyridine) 8.07 (d, 2H,
5
J = 8.4 Hz, C and C –H of 4-BrC H ), and 9.63 (s, 1H,
NH); C NMR (100 MHz, DMSO-d , δ, ppm): 167.0,
2
6
6 4
and N, 12.06%.
1
3
6
1
1
55.7, 154.5, 137.1, 136.5, 131.7, 129.4, 128.6, 122.9,
4
2
2
.10 | General procedure for synthesis of
-(5-[2-methyl-6-arylpyridin-3-yl]-1,3,4-oxadiazol-
-ylthio)-1-arylethanone 6a–n
17.0, and 22.9 (–CH ); MS: calculated for C H N BrO
3
13 12 3
is 305.02; found: (M + 1) 306.02, [(M + 1) + 2]
3
3
08.02 m/z; Anal. Calc. for C H N BrO: C, 51.00; H,
13 12 3
.95; and N, 13.72%. Found: C, 50.49; H, 3.56;
Triethylamine (1 mmol) was added as a base to a suspension
of 1,3,4-oxadiazole-2-thione 5a–b (1 mmol) in acetone, stir-
red for 15 min. Then, appropriate phenacyl bromide/chloride
and N, 13.06%.
(1 mmol) was added and the mixture was heated under
4.7 | 5-[2-methyl-6-arylpyridin-3-yl]-1,3,4-oxadiazole-2
reflux for 3 hr. Completion of the reaction was monitored by
TLC. After completion, the reaction mixture was poured to
ice cold water, the resulting solid was collected and recrys-
tallized from ethanol.
(
3H)-thione 5a–b
2
-methyl-6-arylnicotinohydrazide 4a–b (1 mmol) was dis-
solved in absolute ethanol. Carbon disulfide (2 mmol) was
added, followed by the addition of sodium hydroxide
(
2 mmol). The mixture was heated until the evolution of
4
1
.11 | 2-({5-[6-(4-Chlorophenyl)-2-methylpyridin-3-yl]-
,3,4-oxadiazol-2-yl}thio)-1-(p-tolyl)Ethan-1-one (6a)
H S ceased (6–7 hr). After completion of the reaction, the
2
mixture was cooled, diluted with water, and was neutralized
by dilute hydrochloric acid. The precipitated product was fil-
tered off, washed with cold water and dried, and recrystal-
lized from ethanol-water.
ꢀ
Off White solid. Yield: 90%, mp. 180–181 C. FT IR (ATR,
cm ): 1,668 (C=O), 1,583 (C=N), 1,471 (C=C), 1,176 (C–
S–C), 1,091 (C–O), and 815 (C–Cl); H NMR (400 MHz,
−
1
1
DMSO-d , δ, ppm): 2.42 (s, 3H, –CH ), 2.86 (s, 3H, –CH ),
6
3
3
4
1
.8 | 5-[6-(4-Chlorophenyl)-2-methylpyridin-3-yl]-
,3,4-oxadiazole-2(3H)-thione (5a)
5.16 (s, 2H, –CH ), 7.42 (d, 2H, J = 8 Hz, C and C –H of
2
2
6
1
2
4-MeC H ), 7.61 (dd, 2H, J = 6.8 Hz, J = 2 Hz, C and
6
4
3
ꢀ
C –H of 4-ClC H ), 7.96 (d, 2H, J = 8 Hz, C and C –H of
5
6
4
3
5
Off White solid. Yield: 96%, mp. 218–220 C. FT IR (ATR,
−
1
4-MeC H ), 8.04 (d, 1H, J = 8.4 Hz, C –H of pyridine),
6
4
4
cm ): 3,092 (NH), 1,585 (C=N), 1,507 (C=C), 1,336
C=S), 1,287 (C–O–C), and 836 (C–Cl); H NMR
400 MHz, DMSO-d , δ, ppm): 2.81(s, 3H, –CH ), 7.59 (d,
1
2
1
8.22 (dd, 2H, J = 6.8 Hz, J = 2 Hz, C and C –H of
(
2
6
4
-ClC H ), and 8.32 (d, 1H, J = 8.4 Hz, C –H of pyridine);
(
6
4
5
6
3
1
3
C NMR (100 MHz, DMSO-d , δ, ppm): 193.4 (C=O),
2
H, J = 8.4 Hz, C and C –H of 4-ClC H ), 8.03 (d, 1H,
6
3
5
6 4
1
1
64.4, 163.2, 156.4, 155.3, 139.1, 137.8, 136.1, 134.8,
33.4, 131.6, 129.6, 129.2, 128.9, 128.4, 116.8, 42.3
J = 8.4 Hz, C –H of pyridine), 8.19 (d, 2H, J = 8.8 Hz, C2
and C –H of 4-ClC H ), and 8.24 (d, 1H, J = 8.4 Hz, C –H
of pyridine); C NMR (100 MHz, DMSO-d , δ, ppm):
77.6 (C=S), 159.9, 156.9,156.2, 137.6, 136.5, 135.3,
4
6
6
4
5
13
(–CH
), 25.9 (–CH
ClO S is 435.08; found: (M+) 435.55, (M + 2)
437.55 m/z; Anal. Calc. for C23 ClO S: C, 63.37; H,
), and 24.9 (–CH
); MS: calculated for
6
2
3
3
1
1
C
23
H
18
N
3
2
29.4, 129.1, 118.2, 116.9, and 25.4 (–CH ); MS: calculated
H
18
N
3
2
3
for C H N ClOS is 303.02; found: (M+) 303.77, (M + 2)
4.16; and N, 9.64%. Found: C, 63.49; H, 4.56;
and N, 9.06%.
14 10 3
3
05.77 m/z; Anal. Calc. for C H N ClOS: C, 55.35; H,
14 10 3

SOWMYA ET AL.
9
4
2
1
.12 | 1-(4-Bromophenyl)-2-({5-[6-(4-chlorophenyl)-
-methylpyridin-3-yl]-1,3,4-oxadiazol-2-yl} thio)Ethan-
-one (6b)
(d, 2H, J = 8 Hz, C and C –H of 4-ClC H ), 8.06 (d, 1H,
3
5
6 4
1
J = 8.4 Hz, C –H of pyridine), 8.12 (dd, 2H, J = 6.8 Hz,
4
2J = 2 Hz, C and C –H of 4-ClC H ), and 8.22 (d, 1H,
2
6
6 4
1
3
ꢀ
J = 8.4 Hz, C –H of pyridine); C NMR (100 MHz,
5
Off White solid. Yield: 91%, mp. 212–213 C. FT IR (ATR,
cm ): 1,660 (C=O), 1,581(C=N), 1,485 (C=C), 1,176 (C–
S–C), 1,091 (C–O), 830 (C–Cl) H NMR (400 MHz,
DMSO-d , δ, ppm): 2.86 (s, 3H, –CH ), 5.12 (s, 2H, –CH ),
−
1
DMSO-d , δ, ppm): 193.7 (C=O), 164.6, 163.2, 156.7,
6
1
155.6, 137.8, 136.4, 135.7, 134.8, 133.4, 131.6, 130.2,
1
29, 128.9, 128.4, 116.8, 42.2 (–CH ), and 24.9 (–CH );
2
3
6
3
2
1
2
MS: calculated for C H N Cl O S is 455.03; found:
22 15 3 2 2
7
4
4
4
8
4
.62 (dd, 2H, J = 6.8 Hz, J = 2 Hz, C and C –H of
-ClC H ), 7.78 (d, 2H, J = 8 Hz, C and C –H of
-BrC H ), 7.98 (d, 2H, J = 8 Hz, C and C –H of
-BrC H ), 8.06 (d, 1H, J = 8.4 Hz, C –H of pyridine),
3
5
(
M + 1) 455.97, (M + 2) 457.96, and (M + 4) 459.96 m/z;
6
4
3
5
Anal. Calc. for C H N Cl O S: C, 57.90; H, 3.31; and N,
2
2
15
3
2 2
6
4
2
6
9
.21%. Found: C, 56.98; H, 3.56; and N, 9.16%.
6
4
4
1
2
.22 (dd, 2H, J = 6.8 Hz, J = 2 Hz, C and C –H of
2
6
-ClC H ), and 8.34 (d, 1H, J = 8.4 Hz, C –H of pyridine);
4.15 | 2-({5-[6-(4-Chlorophenyl)-2-methylpyridin-3-yl]-
1,3,4-oxadiazol-2-yl}thio)-1-(2,4-dichloro phenyl)Ethan-
6
4
5
1
3
C NMR (100 MHz, DMSO-d , δ, ppm): 193.7 (C=O),
6
1
1
4
65.4, 163.7, 156.4, 155.7, 137.2, 136.7, 135.1, 133.5,
32.0, 131.7, 130.3, 128.7, 128.5, 124.0, 116.8,
2.3 (–CH ), and 24.9 (–CH ); MS: calculated for
1-one (6e)
Brown solid. Yield: 73%, mp. FT IR (ATR, cm−1):
2
3
ꢀ
1
1
39–140 C. 1,683 (C=O), 1,581 (C=N), 1,477 (C=C),
C H N BrO S is 498.98; found: (M+) 499.55, (M + 2)
1
2
2
15
3
2
,191 (C–S–C), 1,095 (C–O), and 833 (C–Cl); H NMR
500.55, and (M + 4) 502.55 m/z; Anal. Calc. for
(
(
400 MHz, DMSO-d , δ, ppm): 2.87 (s, 3H, –CH ), 5.03
s, 2H, –CH ), 7.60 (dd, 2H, J = 6.8 Hz, J = 2 Hz,
6
3
C H N BrO S: C, 52.76; H, 3.02; and N, 8.39%.
Found: C, 53.49; H, 3.06; and N, 8.26%.
1
2
2
2
15
3
2
2
C
and C –H of 4-ClC H ), 7.65 (dd, 1H,
3
5
2
6
4
1
J = 8.8 Hz, J = 2.4 Hz, C –H of 2,4-Cl C H ), 7.80
5
2
6
3
4
1
1
.13 | 2-({5-[6-(4-Chlorophenyl)-2-methylpyridin-3-yl]-
,3,4-oxadiazol-2-yl}thio)-1-(4-methoxy phenyl)Ethan-
-one (6c)
(
d, 1H, J = 2 Hz, C –H of 2,4-Cl C H ), 7.95 (d, 1H,
3
2
6
3
J = 8.4 Hz, C –H of 2,4-Cl C H ), 8.05 (d, 1H,
6
2
6
3
J = 8.4 Hz, C –H of pyridine), 8.22 (dd, 2H,
4
1
2
ꢀ
J = 6.8 Hz, J = 2 Hz, C and C –H of 4-ClC H ), and
Off White solid. Yield: 98%, mp. 198–199 C. FT IR (ATR,
cm ): 1,667 (C=O), 1,583 (C=N), 1,493 (C=C), 1,176 (C–
S–C), 1,091 (C–O), and 836 (C–Cl); H NMR (400 MHz,
DMSO-d , δ, ppm): 2.87 (s, 3H, –CH ), 3.79 (s, 3H, –
2
6
6
3
4
1
−
1
8
.31 (d, 1H, J = 8.4 Hz, C –H of pyridine); C NMR
5
1
(
100 MHz, DMSO-d , δ, ppm): 193.4 (C=O), 164.2,
6
1
1
4
63.1, 156.5, 155.7, 137.5, 137.1, 136.1, 134.8, 134.7,
31.8, 131.5,130.3, 128.9, 128.6, 127.6, 117.7, 116.9,
2.2 (–CH2), 24.9 (–CH ); MS: calculated for
6
3
OCH ), 5.14 (s, 2H, –CH ), 7.05 (d, 2H, J = 8 Hz, C and
3
2
3
1
C –H of 4-OMeC H ), 7.62 (dd, 2H, J = 6.8 Hz,
3
5
6 4
2
C H N Cl O S is 488.99; found: (M+) 489.30,
J = 2 Hz, C3 and C –H of 4-ClC H ), 7.98 (d, 2H,
22 14
3
3
2
5
6 4
(
M + 2) 491.35, and (M + 4) 492.80 m/z; Anal. Calc.
J = 8 Hz, C3 and C –H of 4-OMeC H ), 8.06 (d, 1H,
5
6 4
1
for C H N Cl O S: C, 53.84; H, 2.88; and N, 8.56%.
J = 8.4 Hz, C –H of pyridine), 8.21 (dd, 2H, J = 6.8 Hz,
22 14
3
3
2
4
2
J = 2 Hz, C and C –H of 4-ClC H ), and 8.30 (d, 1H,
Found: C, 53.49; H, 2.86; and N, 8.46%.
2
6
6 4
1
3
J = 8.4 Hz, C –H of pyridine); C NMR (100 MHz,
5
DMSO-d , δ, ppm): 194.4 (C=O), 163.4, 162.2, 160.4,
4.16 | 2-({5-[6-(4-Chlorophenyl)-2-methylpyridin-3-yl]-
1,3,4-oxadiazol-2-yl}thio)-1-(1,7-dichloro-9H-fluoren-
2-yl))Ethan-1-one (6f)
6
1
1
2
56.4, 152.3, 136.8, 136.1, 134.2, 131.6, 131.1, 128.8,
28.6, 128.4, 116.6, 114.1, 55.5(–OCH ), 42.5 (–CH ), and
3
2
5.9 (–CH ); MS: calculated for C H N ClO S is 451.08;
3
23 18
3
3
−1
Brown solid. Yield: 79.2%, mp. FT IR (ATR, cm ):
found: (M+) 451.93, (M + 2) 453.07 m/z; Anal. Calc. for
ꢀ
1
1
47–149 C. 1,668 (C=O), 1,583 (C=N), 1,482 (C=C),
C H N ClO S: C, 61.13; H, 4.01; and N, 9.30%.
2
3
18
3
3
1
,176 (C–S–C), 1,092 (C–O), and 836 (C–Cl); H NMR
Found: C, 61.49; H, 3.56; and N, 9.06%.
(
400 MHz, DMSO-d , δ, ppm): 2.86 (s, 3H, –CH ), 3.96 (s,
6
3
2
7
H, C –H of 2,7-dichloro-9H-fluorene), 5.16 (s, 2H, –CH ),
9
2
4
2
1
.14 | 1-(4-Chlorophenyl)-2-({5-[6-(4-chlorophenyl)-
-methylpyridin-3-yl]-1,3,4-oxadiazol-2-yl} thio)Ethan-
-one (6d)
.40–7.43 (m, 2H, C6 and C8-H of 2,7-dichloro-9H-fluor-
1
2
ene), 7.61 (dd, 2H, J = 6.8 Hz, J = 2 Hz, C and C –H of
3
5
4
-ClC H ), 7.71–7.75 (m, 2H, C , C –H of 2,7-dichloro-9H-
6
4
3
5
ꢀ
Off White solid. Yield: 85%, mp. 209–210 C. FT IR (ATR,
cm ): 1,666 (C=O), 1,585 (C=N), 1,483 (C=C), 1,176 (C–S–
C), 1,089 (C–O), and 821 (C–Cl); H NMR (400 MHz,
DMSO-d , δ, ppm): 2.87 (s, 3H, –CH ), 5.06 (s, 2H, –CH ),
fluorene), 8.04 (d, 1H, J = 8.4 Hz, C
4
–H of pyridine), 8.22
(dd, 2H, J = 6.8 Hz, J = 2 Hz, C and C –H of
4-ClC ), and 8.32 (d, 1H, J = 8.4 Hz, C –H of pyridine);
C NM R (100 MHz, DMSO-d , δ, ppm): 193.6 (C=O),
−1
1
2
2
6
1
H
6
4
5
13
6
3
2
6
7
.52 (d, 2H, J = 8 Hz, C and C –H of 4-ClC H ), 7.73 (dd,
165.6, 165.2, 156.5, 155.7, 143.6, 143.1, 143, 139.8, 137.5,
134.7, 134.5, 133.1, 132.9, 130.6, 128.9,128.7, 128.6,
3
5
6 4
1
2
2H, J = 6.8 Hz, J = 2 Hz, C and C –H of 4-ClC H ), 7.84
3
5
6 4

1
0
SOWMYA ET AL.
1
1
28.3, 127.8, 126.5, 116.9, 42.3 (–CH ), 36.7, 24.9 (–CH );
S–C), 1,071 (C–O), and 698 (C–Br); H NMR (400 MHz,
DMSO-d , δ, ppm): 2.86 (s, 3H, –CH ), 5.13 (s, 2H, –CH ),
2
3
MS: calculated for C H N Cl O S is 577.02; found:
2
9
18
3
3
2
6
3
2
1
2
(
(
M + 1) 578.09, (M + 2) 579.02, (M + 4) 581.02, and
M + 6) 583.03 m/z; Anal. Calc. for C H N Cl O S: C,
7.74 (dd, 2H, J = 6.8 Hz, J = 1.6 Hz, C and C –H of
4-BrC H ), 7.78 (d, 2H, J = 8 Hz, C3 and C –H
of 4-BrC H ), 7.98 (d, 2H, J = 8 Hz, C and C –H of
4-BrC H ) 8.03 (d, 1H, J = 8 Hz, C –H of pyridine), 8.16
3
5
2
9
18
3
3
2
6
4
5
60.17; H, 3.13; and N, 7.26%. Found: C, 60.19; H, 3.10;
6
4
2
6
and N, 7.16%.
6
4
4
1
2
(
dd, 2H, J = 6.8 Hz, J = 2 Hz, C2 and C –H of
6
4
-BrC H ), and 8.32 (d, 1H, J = 8 Hz, C –H of pyridine);
4
1
.17 | 2-({5-[6-(4-Chlorophenyl)-2-methylpyridin-3-yl]-
,3,4-oxadiazol-2-yl}thio)-1-phenylethan-1-one (6g)
6
4
5
1
3
C NMR (100 MHz, DMSO-d
, δ, ppm): 193.3 (C=O),
6
1
1
65.8, 165.5, 156.8, 155.5, 137.5, 134.7, 132.0,
ꢀ
Brown solid. Yield: 97%, mp. 151–152 C. FT IR (ATR,
31.8,130.3, 128.9, 124, 123.6, 116.9, 42.3 (–CH ), 24.9 (–
−
1
2
cm ): 1,670 (C=O), 1,583 (C=N), 1,452 (C=C), 1,180 (C–
S–C), 1,000 (C–O), and 819 (C–Cl); H NMR (400 MHz,
DMSO-d , δ, ppm): 2.85 (s, 3H, –CH ), 5.06 (s, 2H, –CH ),
.61 (dd, 2H, J = 6.8 Hz, J = 2 Hz, C and C –H of
1
CH
(
3
); MS: calculated for C22
M + 1) 546.35, (M + 2) 547.35, and (M + 4) 549.35 m/z;
Br S: C, 48.46; H, 2.77; and N,
H N Br O S is 545.25; found:
15 3 2 2
6
3
2
1
2
Anal. Calc. for C22
H
15
N
3
O
2 2
7
3
5
7
.71%. Found: C, 48.49; H, 2.86; and N, 7.66%.
4
-ClC H ), 7.71–7.75 (m, 3H, C , C and C –H of C H ),
6
4
3
4
5
6 5
8
.04 (d, 1H, J = 8.4 Hz, C –H of pyridine), 8.21 (dd, 2H,
4
1
2
4
1
1
.20 | 2-({5-[6-(4-Bromophenyl)-2-methylpyridin-3-yl]-
,3,4-oxadiazol-2-yl}thio)-1-(4-methoxy phenyl)Ethan-
-one (6j)
J = 6.8 Hz, J = 2 Hz, C2 and C –H of 4-ClC H ),
6
6 4
8.13–8.15 (m, 2H, C and C –H of C H ), and 8.32 (d, 1H,
2
6
6 5
3
1
J = 8.4 Hz, C –H of pyridine); C NMR (100 MHz,
DMSO-d , δ, ppm): 194.1 (C=O), 164.5, 163.3, 156.2,
1
1
calculated for C H N ClO S is 421.07; found: (M+)
4
C H N ClO S: C, 62.63; H, 3.82; and N, 9.96%.
Found: C, 62.49; H, 3.65; and N, 9.76%.
5
ꢀ
6
Off white solid. Yield: 94%, mp. 199–200 C. FT IR (ATR,
cm ): 1,668 (C=O), 1,583 (C=N), 1,471 (C=C), 1,176 (C–
S–C), 1,091 (C–O), and 712 (C–Br); H NMR (400 MHz,
DMSO-d , δ, ppm): 2.88 (s, 3H, –CH ), 3.79 (s, 3H, –
−
1
55.3, 137.8,136.6, 135.5, 134.8, 131.6, 128.9, 128.8,
28.6, 128.2, 127.9, 116.8, 42.3 (–CH ), 24.9 (–CH ); MS:
1
2
3
22
16
3
2
6
3
21.9,
(M + 2)
423.9 m/z;
Anal.
Calc.
for
OCH ), 5.13 (s, 2H, –CH ), 7.05 (d, 2H, J = 8 Hz, C and
3
2
3
1
2
2
16
3
2
C –H of 4-OMeC H ), 7.74 (dd, 2H, J = 6.8 Hz,
5
6 4
2
J = 1.6 Hz, C3 and C –H of 4-BrC H ), 7.98 (d, 2H,
5
6 4
J = 8 Hz, C3 and C –H of 4-OMeC H ), 8.05 (d, 1H,
5
6 4
1
4
1
.18 | 2-({5-[6-(4-Bromophenyl)-2-methylpyridin-3-yl]-
,3,4-oxadiazol-2-yl}thio)-1-(p-tolyl)Ethan-1-one (6h)
J = 8 Hz, C –H of pyridine), 8.15 (dd, 2H, J = 6.8 Hz,
4
2
J = 2 Hz, C and C –H of 4-BrC H ), and 8.31 (d, 1H,
2
6
6 4
13
ꢀ
J = 8 Hz, C –H of pyridine); C NMR (100 MHz, DMSO-
5
Cream solid. Yield: 94%, mp. 183–184 C. FT IR (ATR,
cm ): 1,664 (C=O), 1,583 (C=N), 1,481 (C=C), 1,176 (C–
S–C), 1,091 (C–O), and 715 (C–Br); H NMR (400 MHz,
DMSO-d , δ, ppm): 2.42 (s, 3H, –CH ), 2.87 (s, 3H, –CH ),
−
1
d6, δ, ppm): 193.4 (C=O), 165.8, 165.4, 160.4, 156.7, 155.8,
137.5, 134.7, 131.8, 131.1, 128.9, 128.7, 123.6, 116.9,
1
1
14, 55.5 (–OCH ), 42.2 (–CH ), and 24.9 (–CH ); MS: cal-
3
2
3
6
3
3
culated for C H N BrO S is 495.03; found: (M + 1)
5.05 (s, 2H, –CH ), 7.42 (d, 2H, J = 8 Hz, C and C –H of
23 18
3
3
2
2
6
1
2
4
96.35,
(M + 2)
497.35 m/z;
Anal.
Calc.
for
4-MeC H ), 7.74 (dd, 2H, J = 6.8 Hz, J = 1.6 Hz, C and
6
4
3
C H N BrO S: C, 55.65; H, 3.66; and N, 8.47%.
C –H of 4-BrC H ), 7.96 (d, 2H, J = 8 Hz, C and C –H of
4
23 18
3
3
5
6
4
3
5
Found: C, 55.49; H, 3.76; and N, 8.46%.
-MeC H ), 8.06 (d, 1H, J = 8 Hz, C –H of pyridine), 8.13
6
4
4
1 2
(dd, 2H, J = 6.8 Hz, J = 2 Hz, C2 and C –H of
6
4
-BrC H ), and 8.32 (d, 1H, J = 8 Hz, C –H of pyridine);
4.21 | 2-({5-[6-(4-Chlorophenyl)-2-methylpyridin-3-yl]-
1,3,4-oxadiazol-2-yl}thio)-1-(4-chlorophenyl)Ethan-
1-one (6k)
6
4
5
1
3
C NMR (100 MHz, DMSO-d , δ, ppm): 193.4 (C=O),
6
1
1
65.8, 165.4, 156.7, 155.8, 139.1,137.5, 134.7, 133.4,
31.8, 129.6, 129.2, 128.9, 123.6, 116.9, 42.2 (–CH ), 25.9
2
ꢀ
Off white solid. Yield: 98%, mp. 217–218 C. FT IR (ATR,
(
–CH3),
and 24.9 (–CH3); MS: calculated for
−1
cm ): 1,666 (C=O), 1,585 (C=N), 1,483 (C=C), 1,176 (C–
C H N BrO S is 479.03; found: (M + 1) 480.38, (M + 2)
4
3
2
3
18
3
2
1
S–C), and 1,089 (C–O), 712 (C–Br); H NMR (400 MHz,
81.38 m/z; Anal. Calc. for C H N BrO S: C, 57.51; H,
23 18 3 2
.78; and N, 8.75%. Found: C, 57.49; H, 3.86;
DMSO-d , δ, ppm): 2.83 (s, 3H, –CH ), 5.16 (s, 2H, –CH ),
6
3
2
7
2
7
.52 (d, 2H, J = 8 Hz, C and C –H of 4-ClC H ), 7.75 (dd,
3
5
6 4
and N, 8.76%.
1 2
H, J = 6.8 Hz, J = 1.6 Hz, C and C –H of 4-BrC H ),
3
5
6 4
.84 (d, 2H, J = 8 Hz, C and C –H of 4-ClC H ), 8.07 (d,
3
5
6 4
4
2
1
.19 | 1-(4-Bromophenyl)-2-({5-[6-(4-bromophenyl)-
-methylpyridin-3-yl]-1,3,4-oxadiazol-2-yl} thio)Ethan-
-one (6i)
1
H, J = 8 Hz, C –H of pyridine), 8.14 (dd, 2H,
4
1
2
J = 6.8 Hz, J = 2 Hz, C and C –H of 4-BrC H ), and
8
2
6
6 4
13
.32 (d, 1H, J = 8 Hz, C –H of pyridine); C NMR
5
ꢀ
Off white solid. Yield: 95%, mp. 213–214 C. FT IR (ATR,
cm ): 1,666 (C=O), 1,587 (C=N), 1,483 (C=C), 1,180 (C–
(100 MHz, DMSO-d
, δ, ppm): 193.4 (C=O), 165.9, 165.5,
6
−
1
156.6, 155.4, 137.5, 134.6, 135.7, 133.5, 131.5, 130.2,

SOWMYA ET AL.
11
1
29, 128.7, 123.6, 116.9, 42.2 (–CH ), 25.2 (–CH ); MS:
4.24 | 2-({5-[6-(4-Bromophenyl)-2-methylpyridin-3-yl]-
2
3
calculated for C H N BrClO S is 498.98; found: (M + 1)
1,3,4-oxadiazol-2-yl}thio)-1-phenylethan-1-one (6n)
22
15
3
2
4
99.95, (M + 2) 500.95, and (M + 4) 502.95 m/z; Anal.
ꢀ
Brown solid. Yield: 96%, mp. 153–154 C. FT IR (ATR,
Calc. for C H N BrClO S: C, 52.76; H, 3.02; and N,
8
−1
22
15
3
2
cm ): 1,681 (C=O), 1,581 (C=N), 1,475 (C=C), 1,176 (C–
S–C), 1,064 (C–O), and 719 (C–Br); H NMR (400 MHz,
.39%. Found: C, 52.74; H, 3.01; and N, 8.36%.
1
DMSO-d , δ, ppm): 2.85 (s, 3H, –CH ), 5.20 (s, 2H, –CH ),
6
3
2
7
.59–7.63 (m, 2H, C and C –H of C H ), 7.71–7.75 (m,
3
5
6 5
4
1
1
.22 | 2-({5-[6-(4-Bromophenyl)-2-methylpyridin-3-yl]-
,3,4-oxadiazol-2-yl}thio)-1-(2,4-dichloro phenyl)Ethan-
-one (6l)
3
H, J = 8 Hz, C and C –H of 4-BrC H and C –H of
3
5
6
4
4
C H ), 8.04 (d, 1H, J = 8 Hz, C –H of pyridine), 8.07–8.15
6
5
4
(m, 4H, C , C –H of 4-BrC H and C H ), and8.32 (d, 1H,
2
6
6
4
13
6 5
ꢀ
Brown solid. Yield: 83%, mp. 144–146 C. FT IR (ATR,
cm ): 1,681 (C=O), 1,581 (C=N), 1,475 (C=C), 1,191
J = 8 Hz, C –H of pyridine); C NMR (100 MHz, DMSO-
5
−
1
d6, δ, ppm): 193.4 (C=O), 165.8, 165.4, 156.7, 155.8, 137.5,
135.6, 134.7, 131.8, 130.1, 128.9, 128.7, 128.5, 123.6,
116.9, 42.2 (–CH ), 24.9 (–CH ); MS: calculated for
1
(
(
(
C–S–C), 1,064 (C–O), and 763 (C–Br); H NMR
400 MHz, DMSO-d , δ, ppm): 2.87 (s, 3H, –CH ), 5.03
s, 2H, –CH ), 7.63 (dd, 1H, J = 8.4 Hz, J = 2 Hz,
6
3
2
3
1
2
C
H
N
16
BrO
3
S is 465.01; found: (M+) 465.55, (M + 2)
2
2
22
1
C –H of 2,4-Cl C H ), 7.74 (dd, 2H, J = 6.8 Hz,
467.50 m/z; Anal. Calc. for C22
3.46; and N, 9.01%. Found: C, 56.49; H, 3.48;
and N, 9.02%.
H
16
N
3
BrO S: C, 56.66; H,
2
5
2
6
3
2
J = 1.6 Hz, C and C –H of 4-BrC H ), 7.80 (d, 1H,
3
5
6
4
J = 2 Hz, C –H of 2,4-Cl C H ), 7.95 (d, 1H, J = 8.4
3
2
6
3
Hz, C –H of 2,4-Cl C H ), 8.05 (d, 1H, J = 8 Hz, C –H
6
2
6
3
4
1
2
of pyridine), 8.15 (dd, 2H, J = 6.8 Hz, J = 2 Hz, C
4.25 | In vitro antimicrobial activity assay
2
and C –H of 4-BrC H ), and 8.31 (d, 1H, J = 8 Hz,
6
6
4
The newly synthesized compounds were screened for their
antibacterial activity against four bacterial strains namely
P. aeruginosa, E. coli representative of Gram Positive and
B. subtilis, S. aureus representative of Gram Negative bacte-
rial strains. Ciprofloxacin was used as positive control. The
primary screening was carried out using the Agar cup plate
1
3
C –H of pyridine); C NMR (100 MHz, DMSO-d , δ,
5
6
ppm): 193.4 (C=O), 165.8, 165.4, 156.7, 155.8, 137.5,
1
1
37.2, 134.7, 131.8, 131.5, 130.3, 128.9, 127.6, 125.1,
23.6, 117.7, 116.9, 42.2 (–CH ), 24.9 (–CH ); MS: calcu-
2
3
lated for C H N BrCl O S is 532.94; found: (M+) 490.35,
22
14
3
2 2
[
36]
(
M + 2) 491.35, and (M + 4) 493.35 m/z; Anal. Calc. for
method.
C H N BrCl O S: C, 49.37; H, 2.64; and N, 7.85%.
2
2
14
3
2
2
The sterilized nutrient agar medium was distributed
00 mL each in two 250 mL conical flasks and allowed to
Found: C, 49.49; H, 2.86; and N, 7.46%.
1
cool to room temperature. To these media, 18–24 hr grown
bacterial subcultures were added and shaken thoroughly to
ensure uniform distribution of organisms throughout the
medium. Then, this agar medium was distributed in equal
portions, in sterilized petri dishes, ensuring that each petri
dish contains about 45–50 mL of the medium. The medium
was then allowed for solidification. Then, cups were made
with the help of a sterile cork borer (6 mm diameter) punch-
ing into the set of agar media. The solutions of required con-
centrations (100 μg/mL) of test compounds were prepared
by dissolving the compounds in DMSO filled into the cups
with 1 mL of respective solution. Then, the petri dishes were
kept for incubation in an inverted position for 24–48 hr at
4
1
2
.23 | 2-({5-[6-(4-Bromophenyl)-2-methylpyridin-3-yl]-
,3,4-oxadiazol-2-yl}thio)-1-(1,7-dichloro-9H-fluoren-
-yl))Ethan-1-one (6m)
ꢀ
Pale yellow solid. Yield: 76%, mp. 168–169 C. FT IR
−
1
(ATR, cm ): 1,668 (C=O), 1,583 (C=N), 1,477 (C=C),
1
1,180 (C–S–C), 1,098 (C–O), and 736 (C–Br); H NMR
(400 MHz, DMSO-d , δ, ppm): 2.84 (s, 3H, –CH ), 3.96 (s,
6
3
2
7
H, C –H of 2,7-dichloro-9H-fluorene), 5.12 (s, 2H, –CH ),
.40–7.43 (m, 2H, C6 and C8–H of 2,7-dichloro-9H-fluor-
9
2
ene), 7.70–7.75 (m, 4H, C , C –H of 4-BrC H and
2
pyridine), 8.16 (dd, 2H, J = 6.8 Hz, J = 2 Hz, C and
3
5
6 4
,7-dichloro-9H-fluorene), 8.06 (d, 1H, J = 8 Hz, C –H of
4
ꢀ
1
2
37 C in an incubator. When growth inhibition zones were
2
developed surrounding each cup, their diameter in mm was
measured and compared with that of the standard drug. The
MIC for the potent compounds 6a, 6b, 6c, 6g, 6h, 6i, and 6n
against the same microorganisms used in the primary screen-
ing was used according to the reported Broth dilution
C –H of 4-BrC H ), and 8.32 (d, 1H, J = 8 Hz, C –H of
6
6
4
5
13
pyridine); C NMR (100 MHz, DMSO-d , δ, ppm): 193.6
6
(C=O), 165.6, 165.2, 156.5, 155.7, 143.6, 143.1,
1
1
2
6
6
43, 139.8, 137.5, 134.7, 133.1, 132.9, 131.8, 130.6, 128.9,
28.7, 128.3, 127.8, 126.5, 123.6, 116.9, 42.2 (–CH ), 36.7,
2
[37]
method.
4.9 (–CH ); MS: calculated for C H N BrCl O S is
3
29 18
3
2 2
20.35; found: (M+) 620.96, (M + 2) 622.90, (M + 4)
24.90, and (M + 6) 626.96 m/z; Anal. Calc. for
4
.26 | In vivo anti-inflammatory assay
C H N BrCl O S: C, 55.88; H, 2.91; and N, 6.74%.
Found: C, 55.79; H, 2.86; and N, 6.76%.
The rats were divided into ten groups of six animals each.
Group I (negative control) received 1 mL of normal saline,
2
9
18
3
2 2

1
2
SOWMYA ET AL.
Group II (Standard) received 10 mg/kg p.o. indomethacin,
and Group III-IX received synthetic compounds 50 mg/kg.
After 1 hr, the rats were challenged with subcutaneous injec-
tion of 0.1 mL of 1% w/v solution of carrageenan (Sigma
chemical co, St. Louis MO, USA) into the plantar side of the
left hind paw. The paw was marked in the cup of the appara-
tus with water. The digital plethysmograph apparatus was
used for the measurement of rat paw volume. The paw vol-
ume was measured every hour for 3 hr after injection of car-
rageenan to each group to determine the change in the paw
edema thickness. The difference between the initial and sub-
sequent reading gave the actual edema volume.
ACKNOWLEDGMENTS
The authors gratefully acknowledge DST-PURSE, Manga-
lore University, SAIF, Cochin, and MIT Manipal, for spec-
tral data. Authors thank the personnel of Department of
Microbiology, MM's NG. Halgekar Institute of Dental Sci-
ences and Research Centre, Belgaum for their valuable help
in antimicrobial studies. The authors also thank the Head,
Department of Pharmaceutical Chemistry, NGSM Institute
of Pharmaceutical Sciences, Paneer, Deralakatte for provid-
ing research facilities to carry out the anti-inflammatory
study.
CONFLICTS OF INTEREST
4.27 | Animals
The authors declare no potential conflict of interests.
Animal Wistar rats of either sex and of approximately the
same age, weighing about 125–150 g, were used for the
study and were housed in polypropylene cages and fed with
standard pellet diet and water ad libitum. The animals were
under alternate cycle of 12 hr of darkness and light each.
Before each test, the animals were fasted for at least 12 hr.
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