Design, synthesis, pharmacological evaluation and molecular docking studies of substituted oxadiazolyl-2-oxoindolinylidene propane hydrazide derivatives

Article abstract of DOI:10.5935/0103-5053.20160090

The manuscript describes design and synthesis of novel oxadiazolyl-2-oxoindolinylidene propane hydrazides as amide tethered hybrids of indole and oxadiazole and their evaluation for antiinflammatory and analgesic activity. The compounds were synthesized following five step reaction to yield fifteen derivatives as 3-(5-substituted-1,3,4-oxadiazol-2-yl)-N′-[2-oxo-1,2-dihydro-3Hindol-3-ylidene]propane hydrazides. The final derivatives 3-[5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-yl]-N′-[2-oxo-1,2-dihydro-3H-indol-3-ylidene]propane hydrazide and 3-[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl]-N′-[2-oxo-1,2-dihydro-3H-indol-3-ylidene]propane hydrazide were found to be highly promising molecules with severity index of 0.35 and 0.56, respectively, which is promising for an analgesic compound. The hydroxy and methyl substitution on phenyl ring system provided with active anti-inflammatory compounds having increase in reaction time of 84.11 and 83.17percent, respectively compared to standard drug at 85.84percent. Molecular docking studies exhibit comparable interaction with synthesized derivatives and standard drug having a dock score of -4.44 by the K-nearest neighbour genetic algorithm method.

Full text of DOI:10.5935/0103-5053.20160090

http://dx.doi.org/10.5935/0103-5053.20160090
J. Braz. Chem. Soc., Vol. 27, No. 11, 1998-2010, 2016.
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Article
Design, Synthesis, Pharmacological Evaluation and Molecular Docking Studies of
Substituted Oxadiazolyl-2-Oxoindolinylidene Propane Hydrazide Derivatives
Deweshri Kerzare,a,b Rupesh Chikhale,*,a,b,c Ratnadeep Bansode,d Nikhil Amnerkar,b
Nazira Karodia,e Anant Paradkard and Pramod Khedekara
aDepartment of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University,
Amravati Road, 440 033 Nagpur-MS, India
bKamala Nehru College of Pharmacy, Butibori, 441 108 Nagpur-MS, India
cAdvanced Centre for Treatment, Research and Education in Cancer (ACTREC),
Tata Memorial Centre, Kharghar, 410 210 Navi Mumbai-MS, India
dCentre for Pharmaceutical Engineering Science, Faculty of Life Sciences, University of Bradford,
BD7 1DP Bradford, United Kingdom
eSchool of Biology, Chemistry and Forensic Science, University of Wolverhampton,
WV1 1LY Wolverhampton, United Kingdom
The manuscript describes design and synthesis of novel oxadiazolyl-2-oxoindolinylidene
propane hydrazides as amide tethered hybrids of indole and oxadiazole and their evaluation for anti-
inflammatory and analgesic activity. The compounds were synthesized following five step reaction
to yield fifteen derivatives as 3-(5-substituted-1,3,4-oxadiazol-2-yl)-N’-[2-oxo-1,2-dihydro-3H-
indol-3-ylidene]propane hydrazides. The final derivatives 3-[5-(4-hydroxyphenyl)-1,3,4-oxadiazol-
2-yl]-N’-[2-oxo-1,2-dihydro-3H-indol-3-ylidene]propane hydrazide and 3-[5-(4-methylphenyl)-
1,3,4-oxadiazol-2-yl]-N’-[2-oxo-1,2-dihydro-3H-indol-3-ylidene]propane hydrazide were found
to be highly promising molecules with severity index of 0.35 and 0.56, respectively, which is
promising for an analgesic compound. The hydroxy and methyl substitution on phenyl ring system
provided with active anti-inflammatory compounds having increase in reaction time of 84.11 and
83.17%, respectively compared to standard drug at 85.84%. Molecular docking studies exhibit
comparable interaction with synthesized derivatives and standard drug having a dock score of
−4.44 by the K-nearest neighbour genetic algorithm method.
Keywords: hybrid approach, indole, anti-inflammatory, analgesic, molecular docking
Introduction
results in form of newer and safer molecules. One of such
approach is hybrid approach.2 It involves development
of better, synergistic molecules on hybridization of two
or more active biomolecules or ligands to develop newer
derivative that possess good pharmacological activity.
The success of this approach has been unequivocal with
reports of development of anticancer hybrid agents,
several nonsteroidal anti-inflammatory drugs (NSAIDs)
and antiviral agents. Many drugs that are developed on
basis of hybrid approach are used in therapy today and
many more are in pipelines and yet to come on market.
The success of hybrid approach is depicted by
several molecules such as cediranib, which is a hybrid
of quinazoline and indole; ateviridine, a hybrid of
pyridine and indole; indalpine, a hybrid of indole; and
Discovery of new molecules within a short possible time
has become a focal point in current medicine. Need for new
and better drugs with less toxicity and more selectivity are
major criteria for the designing of a molecule. Recently,
many new approaches are observed to be practiced for
development of newer biologically active molecules. Special
focus is given on structure based drug discovery, fragment
based drug discovery, protein-protein interaction inhibitor
study, proteomics and pharmacogenomics and these are
becoming more and more popular to medicinal chemists.1
Still the earlier methods are persistent and yielding good
*e-mail: rupeshchikhale7@gmail.com

Vol. 27, No. 11, 2016
Kerzare et al.
1999
Figure 1. Examples describing success story of hybrid approach in drug discovery.
piperazine and pravadoline, which is a hybrid of indole
and morpholine, respectively (Figure 1). The success of
this approach is well defined in discovery of NSAIDs such
as the pravadoline, which acts as cyclooxygenase (COX)
inhibitor but also exhibit a potent cannabinoid agonist
effect.3 The need for efficient and nontoxic NSAIDs
has made us to design and develop newer and better
molecules. The unwanted gastrointestinal (GI) ulceration,
bleeding, nephrotoxicity observed in conventional COX
inhibitors and the failure of selective COX inhibitors due
to adverse cardiac effects suggest for better drugs devoid
of these severe side effects.4 In view of this scenario, it was
decided to employ the hybrid approach for development of
novel molecules for their analgesic and anti-inflammatory
activity. To achieve this goal, we selected to design and
synthesize a hybrid molecule from indole and oxadiazole
nuclei. Indole derivatives have been found to exhibit
variety of pharmacological activities and have raised
considerable interest because of their potential beneficial
effects on human health.5,6 They have been reported
to possess antibacterial,7 anticonvulsant,8 antifungal,9
antiviral,10 anticancer11 and anti-inflammatory12 properties.

2000
Design, Synthesis, Pharmacological Evaluation and Molecular Docking Studies
J. Braz. Chem. Soc.
Indole derivative such as indomethacin is an established
NSAID but it cause severe gastrointestinal side effect.
The oxadiazole is found to poses versatile activity
like the antiviral, anticancer and anticystic activity;
compounds bearing 1,3,4-oxadiazole nucleus are also
known to exhibit unique antiedema and anti-inflammatory
potential.13 In the past, many indole and 1,3,4-oxadiazole
derivatives showed potential for anti-inflammatory and
analgesic activity in animal model of inflammation
and pain.14-18
Compound
R
49
50
p-OH p-CH3 m-NO2 o-OCH3, m’-OCH3 p-Cl p-NO2 p-COCH3 p-F o-Cl o-NO2 m-Cl o-F p-Br m-Br
51
52
53
54
55
56
57 58
59
60 61 62
63
H
Scheme 1. Synthesis of N’-(2-oxoindolin-3-ylidene)-3-(5-substituted phenyl-1,3,4-oxadiazol-2-yl)propane hydrazide derivatives 49-63.

Vol. 27, No. 11, 2016
Kerzare et al.
2001
The need for newer drug molecule and success of
hybrid approach compelled us for development of some
new NSAIDs. The target moieties selected for formation
of a hybrid drug was based on our previous literature
study, and it was found that indole and oxadiazole nuclei
possess analgesic and anti-inflammatory properties and
thus it was found worth to prepare a hybrid derivative from
these moieties. To achieve this goal, we synthesized fifteen
different hybrids of indole and oxadiazole as 5-substituted-
1,3,4-oxadiazole-2-yl-(2-oxo-1,2-dihydro-indol-3-ylidene)
propane hydrazide derivatives. Scheme 1 explains the
synthesis of these derivatives that involves two different
steps. In the first step, substituted aryl acid hydrazides
were obtained from various substituted benzoic acids with
formation of intermediate benzoates. In the second step, the
indole-2,3-dione was reacted with hydrazine hydrate to form
isatin-3-hydrazone, which in the subsequent reaction with
succinic anhydride produces indolyl hydrazinyl butonic acid.
This acid on reaction with substituted aryl acid hydrazides
obtained from earlier steps yields the final derivatives as
substituted oxadiazolyl indolyl propane hydrazides.
These newly synthesized compounds were tested
initially for their in vitro characteristic as active or inactive
molecules by the egg albumin denaturation test.19 Further,
these compounds were subjected for their analgesic and anti-
inflammatory activity by the carrageenan induced rat paw
edema model20 and Eddy’s hot plate method,21 respectively.
After evaluation for pharmacological studies, it becomes
necessary to determine the safety of synthesized compounds
with respect to ulcerogenesis study or the determination
of severity index with respect to reference drug such as
indomethacin. Further, to determine the possible interaction
of most potent compound and receptor in silico, studies need
to be carried out. This was achieved by molecular docking
studies of the ligands on the COX-II receptor.22,23
The series of final derivatives N’-(2-oxoindolin-3-
ylidene)-3-(5-substituted phenyl-1,3,4-oxadiazol-2-yl)
propane hydrazides 49-63 as described in this study
is outlined in Scheme 1. The products were obtained
following two different steps; in the first step various
substituted benzoic acids 1-15 were obtained commercially;
these substituents were reacted with ethanol in presence
of sulphuric acid to derive corresponding benzoates 16‑30
under conditions of nucleophilic substitution reaction.
These benzoates were further treated with hydrazine
hydrate to yield various substituted aryl acid hydrazides 31-
45, which were further employed in the reaction system. In
the second step to the reaction scheme, isatin 46 was treated
with hydrazine hydrate under ambient conditions to yield
the isatin-3-hydrazone 47. It was further substituted by
succinic anhydride to yield the oxoindolyidene hydrazinyl
butanoic acid 48. Up to this moiety, the step two provides
with reactant for the final reaction, in which the various
substituted aryl acid hydrazides 31-45 and oxoindolyidene
hydrazinyl butanoic acid 48 react to undergo the cyclization
reaction in presence of phosphorous oxychloride, yielding
various N’-(2-oxoindolin-3-ylidene)-3-(5-substituted
phenyl-1,3,4-oxadiazol-2-yl) propane hydrazides 49-63
in 40-80% yield.
Experimental
Materials and methods
Chemicals were obtained from Sigma-Aldrich
(St. Louis, MO, USA), S D Fine-Chem (Mumbai, MH,
India) and Merck (Darmstadt, Germany), unless specified.
Melting points (m.p.) were detected with open capillaries
using ThermoNik precision melting point cum boiling point
apparatus (model C-PMB-2, Mumbai, MH, India) and are
uncorrected. Infrared (IR) spectra (KBr) were recorded
on FTIR-8400s spectrophotometer (Shimadzu, Tokyo,
Japan) at the Department of Pharmaceutical Sciences,
Rashtrasant Tukadoii Maharai (RTM) Nagpur University.
Proton (1H) and carbon 13 (13C) nuclear magnetic resonance
(NMR) were obtained using a Bruker Avance II 400 MHz
spectrometer (Billerica, MA, USA), using tetramethylsilane
(TMS) as internal standard. All chemical shift values were
recorded as d (ppm), coupling constant value J is measured
in hertz, the peaks are presented as s (singlet), d (doublet),
t (triplet), brs (broad singlet), dd (double doublet), m
(multiplet). The purity of compounds was controlled by
thin layer chromatography (silica gel HF254e361, type
60, 0.25 mm; Merck, Darmstadt, Germany). Electrospray
ionization mass spectrometry (ESI-MS) was recorded at
Waters Q-TOF spectrometer (Waters, Milford, MA, USA)
at the Sophisticated Analytical Instrumentation Facility
(SAIF), Punjab University (Chandigarh, PB, India).
Synthesis
General methods for synthesis of substituted ethyl
benzoates (16-30)24
To a solution of substituted benzoic acid (1-15)
(0.246 mol) in dry ethanol (2.5 mol), concentrated sulphuric
acid (0.5 mL) was added. The reaction mixture was refluxed
for 8 h. Excess of ethanol was distilled off and the content
was allowed to cool. The residue was poured into separating
funnel containing 60 mL of water. Carbon-tetrachloride
(5-10 mL) was added to obtain sharp separation of aqueous
and ester layer. Ester layer was washed with sodium
hydrogen carbonate solution. The esters (16-30) were

2002
Design, Synthesis, Pharmacological Evaluation and Molecular Docking Studies
J. Braz. Chem. Soc.
collected and recrystallized from ethanol. Details of these
compounds are available in Supplementary Information.
phosphorous oxychloride (15-20 mL), compound (48)
(0.01 mol) was added. The reaction mixture was refluxed
for 5 h. The mixture was cooled to room temperature and
poured onto crushed ice and neutralized the contents with
sodium hydroxide solution (1 mol L−1). The product was
filtered, washed with water, dried and recrystallized using
methanol to get 49-63.
General methods for synthesis of substituted aryl acid
hydrazides (31-45)24
The substituted ethyl benzoates (16-30) (0.01 mol)
dissolved in dry ethanol (25 mL), hydrazine hydrate (99%,
0.01 mol) was added and the mixture was refluxed for 6 h.
The reaction mixture was cooled and the solid obtained was
filtered and recrystallized from dilute ethanol or from water.
Details of these compounds are available in Supplementary
Information.
3-(5-Phenyl-1,3,4-oxadiazol-2-yl)-N’-[2-oxo-1,2-dihydro-3H-
indol-3-ylidene]propane hydrazide (49)
Yield: 2.03 g (78%); m.p. 208-210 ºC; Rf 0.83
(chloroform); λmax 448 nm; IR (KBr) ν / cm−1 3254,
3059, 2916, 1655, 1618; 1H NMR (DMSO) d 8.91 (s, 1H,
Ar−H), 8.59 (s, 1H,Ar−H), 8.0 (s, 1H,Ar−H), 7.98 (d, 2H,
J 7.9 Hz,Ar−H), 7.76 (t, 2H, J 8.3 Hz,Ar−H), 7.45 (dd, 1H,
J 7.4 Hz,Ar−H), 7.32 (dd, 1H, J 7.6 Hz,Ar−H), 3.58 (s, 2H,
CH2), 2.38 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6)
d 160.53, 156.17, 148.33, 131.52, 126.20, 123.76, 121.78,
120.72, 33.98; EI-MS m/z [M + H]+ 362.58; anal. calcd.
for C19H15N5O3: C, 63.1; H, 4.15; N, 19.37; found: C, 63.3;
H, 4.17; N, 19.39.
Procedure for synthesis of 3-hydrazinylidene-1,3-dihydro-
2H-indol-2-one (47)25
A mixture of isatin (46) (1 mmol) and hydrazine hydrate
(99%, 0.055 g, 1.1 mmol) in absolute methanol (25 mL) was
refluxed for 1 h and then cooled to room temperature. The
precipitate of hydrazones was filtered and dried. The crude
productwasrecrystallizedfromethanoltogivehydrazones(47).
Yield: 1.0 g (70%); m.p. 248-250 ºC; Rf 0.39
(methanol:toluene, 1:4); IR (KBr) ν / cm−1 3411, 2916,
1655, 1618; 1H NMR (DMSO) d 7.63 (s, 1H, Ar−H), 7.45
(s, 1H, Ar−H), 7.31 (s, 1H, Ar−H), 7.20 (s, 1H, Ar−H),
7.0 (s, 1H, NH), 3.34 (s, 2H, NH); 13C NMR (100 MHz,
DMSO-d6) d 166.25, 152.64, 130.73, 125.24, 120.75,
120.61, 117.55, 78.81; EI-MS m/z [M + H]+ 162.24.
3-[5-(4-Hydroxyphenyl)-1,3,4-oxadiazol-2-yl]-N’-[2-oxo-1,2-
dihydro-3H-indol-3-ylidene]propane hydrazide (50)
Yield: 2.08 g (80%), m.p. 210-212 ºC; Rf 0.8
(chloroform); λmax 442 nm; IR (KBr) ν / cm−1 3585, 3211,
2849, 2978, 1711, 1605; 1H NMR (DMSO) d 9.49 (s, 1H,
OH), 7.72 (t, 2H, J 6.9 Hz, Ar−H), 7.40 (t, 2H, J 8.1 Hz,
Ar−H), 7.21 (t, 1H, J 6.9 Hz, Ar−H), 7.18 (d, 1H, J 7.4 Hz,
Ar−H), 6.65 (t, 1H, J 7.8 Hz, Ar−H), 6.36 (s, 1H, Ar−H),
2.31 (s, 2H, CH2), 1.23 (s, 2H, CH2); 13C NMR (100 MHz,
DMSO-d6) d 166.11, 162.44, 157.29, 153.76, 137.59,
132.14, 128.25, 126.72, 122.81, 115.18, 112.36, 102.89,
50.84, 23.17; EI-MS m/z [M + H]+ 378.11; anal. calcd. for
C19H15N5O4: C, 63.4; H, 4.13; N, 19.33; found: C, 63.6; H,
4.17; N, 19.35.
Procedure for synthesis of 4-oxo-4-[2-(2-oxo-1,2-dihydro-
3H-indol-3-ylidene)-hydrazinyl]butanoic acid (48)26
The mixture of compound (47) (0.01 mol), succinic
anhydride (0.01 mol) and trimethylamine (1 mmol) in
dichloromethane (DCM) was stirred at room temperature
for 3 h and diluted with DCM. The solution was washed
with sodium bicarbonate and brine twice. The product
thus obtained was dried over sodium sulphate and the
excess of solvent was distilled off. The resulting solid was
recrystallized from ethanol.
Yield: 0.08 g (80%); m.p. 238-240 ºC; Rf 0.58 (toluene);
IR (KBr) ν / cm−1 3394, 3059, 2916, 1655, 1618; 1H NMR
(DMSO) d 12.44 (s, 1H, OH), 7.99 (t, 1H, J 6.7 Hz, Ar−H),
7.97 (s, 1H, Ar−H), 7.44 (d, 1H, J 8.2 Hz, Ar−H), 7.31
(d, 1H, J 7.2 Hz,Ar−H), 6.30 (s, 1H, NH), 3.78 (s, 2H, CH2),
2.23 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d 166.22,
157.59, 148.41, 131.35, 126.12, 123.58, 121.71, 120.58,
118.78, 106.41, 51.01, 30.31; EI-MS m/z [M + H]+ 262.04.
3-[5-(4-Methylphenyl)-1,3,4-oxadiazol-2-yl]-N’-[2-oxo-1,2-
dihydro-3H-indol-3-ylidene]propane hydrazide (51)
Yield: 1.82 g (70%); m.p. 200-202 ºC; Rf 0.47
(chloroform); λmax 357 nm; IR (KBr) ν / cm−1 3249, 2849,
2918, 1614, 1598; 1H NMR (DMSO) d 7.75 (t, 1H, J 8.1 Hz,
Ar−H), 7.46 (t, 1H, J 7.6 Hz, Ar−H), 7.34 (t, 1H, J 5.9 Hz,
Ar−H), 7.19 (d, 1H, J 7.8 Hz, Ar−H), 7.02 (m, 2H, J 7.9 Hz,
Ar−H), 6.82 (t, 1H, J 8.4 Hz, Ar−H), 6.43 (s, 1H, Ar−H),
3.34 (s, 4H, CH2), 2.32 (s, 3H, CH3); 13C NMR (100 MHz,
DMSO-d6) d166.37, 166.05, 162.55, 158.97, 153.97, 152.79,
137.52, 133.74, 130.88, 128.22, 126.76, 122.81, 120.82,
117.68, 113,56, 112.36, 102.79, 55.90, 54.94, 23.20; EI-MS
m/z [M + H]+ 376.31; anal. calcd. for C20H17N5O3: C, 64.1;
H, 4.6; N, 18.7; found: C, 64.6; H, 4.8; N, 18.91.
General procedure for synthesis of 3-(5-substituted-1,3,4-
oxadiazol-2-yl)-N’-[2-oxo-1,2-dihydro-3H-indol-3-ylidene]
propane hydrazide derivatives (49-63)27
To a solution of compound (31-45) (0.01 mol) in

Vol. 27, No. 11, 2016
Kerzare et al.
2003
3-[5-(3-Nitrophenyl)-1,3,4-oxadiazol-2-yl]-N’-[2-oxo-1,2-
dihydro-3H-indol-3-ylidene]propane hydrazide (52)
(dd, 2H, J 8.4 Hz, Ar−H), 6.68 (s, 1H, Ar−H), 3.35 (s, 2H,
CH2), 2.51 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6)
d 165.71, 163.07, 155.16, 149.14, 147.20, 137.16, 128.28,
126.96, 123.96, 122.80, 112.24, 101.69, 55.87, 23.37; EI-MS
m/z [M + H]+ 407.16; anal. calcd. for C19H14N6O5: C, 56.3;
H, 3.48; N, 20.68; found: C, 56.6; H, 3.5; N, 20.69.
Yield: 1.87 g (72%); m.p. 248-250 ºC; Rf 0.72
(chloroform); λmax 447 nm; IR (KBr) ν / cm−1 3084, 2918,
1655, 1618, 1529; 1H NMR (DMSO) d 8.31 (s, 1H,Ar−H),
7.87 (d, 2H, J 7.3 Hz, Ar−H), 7.61 (t, 2H, J 8.4 Hz, Ar−H),
7.21 (t, 2H, J 7.9 Hz, Ar−H), 6.62 (s, 1H, Ar−H), 3.63
(s, 2H, CH2), 2.34 (s, 2H, CH2); 13C NMR (100 MHz,
DMSO-d6) d 165.75, 163.11, 155.20, 147.82, 143.43,
137.15, 133.34, 130.43, 127.01, 124.34, 123.33, 123.02,
122.80, 121.50, 112.37, 101.87, 51.03, 23.38; EI-MS m/z
[M + H]+ 407.72; anal. calcd. for C19H14N6O5: C, 56.3; H,
3.5; N, 20.77; found: C, 56.5; H, 3.8; N, 20.79.
3-[5-(4-Acetylphenyl)-1,3,4-oxadiazol-2-yl]-N’-[2-oxo-1,2-
dihydro-3H-indol-3-ylidene]propane hydrazide (56)
Yield: 2.08 g (80%); m.p. 166-170 ºC, Rf 0.65
(chloroform); λmax 525 nm; IR (KBr) ν / cm−1 3107,
2918, 1648, 1603, 1210, 2849; 1H NMR (DMSO): d 7.73
(t, 1H, J 7.4 Hz, Ar−H), 7.49 (d, 1H, J 7.9 Hz, Ar−H), 7.19
(t, 2H, J 8.5 Hz, Ar−H), 7.03 (d, 1H, J 8.3 Hz, Ar−H), 6.75
(d, 1H, J 7.4 Hz, Ar−H), 6.65 (s, 1H, Ar−H), 6.37 (s, 1H,
Ar−H), 3.71 (s, 2H, CH2), 3.35 (s, 2H, CH2), 2.50 (s, 3H,
CH3); 13C NMR (100 MHz, DMSO-d6) d 200.86, 166.12,
162.48, 153.70, 147.14, 146.49, 137.65, 132.70, 126.74,
123.93, 122.80, 119.31, 115.50, 112.50, 111.41, 102.84,
56.29, 55.58, 50.82, 23.13; EI-MS m/z [M + H]+ 404.09;
anal. calcd. for C21H17N5O4: C, 62.41; H, 4.23; N, 17.39;
found: C, 62.48; H, 4.28; N, 17.42.
3-[5-(2,3-Dimethoxyphenyl)-1,3,4-oxadiazol-2-yl]-N’-[2-
oxo-1,2-dihydro-3H-indol-3-ylidene]propane hydrazide (53)
Yield: 2.16 g (83%); m.p. 230-232 ºC; Rf 0.69
(chloroform); λmax 325 nm; IR (KBr) ν / cm−1 3293, 2918,
1655, 1618, 2835, 1425; 1H NMR (DMSO) d 7.73 (s, 1H,
Ar−H), 7.21 (d, 2H, J 8.4 Hz, Ar−H), 7.19 (s, 1H, Ar−H),
7.05 (s, 1H, Ar−H), 6.76 (t, 1H, J 7.9 Hz, Ar−H), 6.66
(s, 1H, Ar−H), 4.07 (t, 2H, J 7.9 Hz, CH2), 3.34 (t, 2H,
J 8.4 Hz, CH2), 2.50 (s, 3H, O−CH3), 1.22 (d, 3H, J 7.8 Hz,
O−CH3); 13C NMR (100 MHz, DMSO-d6) d 165.64,
162..36, 153.50, 147.05, 146.48, 137.67, 132.76, 126.71,
122.80, 119.49, 115.51, 112.52, 111.62, 102.97, 56.38,
55.57, 23.08, 14.13; EI-MS m/z [M + H]+ 422.14; anal.
calcd. for C21H19N5O5: C, 59.9; H, 4.52; N, 16.65; found:
C, 59.5; H, 4.8; N, 16.69.
3-[5-(4-Fluorophenyl)-1,3,4-oxadiazol-2-yl]-N’-[2-oxo-1,2-
dihydro-3H-indol-3-ylidene]propane hydrazide (57)
Yield: 2.14 g (82 %); m.p. 182-184 ºC, Rf 0.65
(chloroform), λmax 398 nm, IR (KBr) ν / cm−1 2992,
1
2916, 1655, 1605, 1097; H NMR (DMSO) d 8.10
(d, 2H, J 8.3 Hz, Ar−H), 7.68 (t, 2H, J 7.6 Hz, Ar−H), 7.26
(dd, 1H, J7.6 Hz,Ar−H), 7.24 (dd, 1H, J7.3 Hz,Ar−H), 6.81
(t, 2H, J 6.9 Hz, Ar−H), 3.73 (s, 2H, CH2), 2.50 (s, 2H,
CH2); 13C NMR (100 MHz, DMSO-d6) d 164.52, 163.31,
161.31, 160.36, 135.94, 129.96, 129.88, 127.96, 124.35,
124.17, 115.94, 115.72, 114.50, 103.80, 99.49, 57.01,
51.68; EI-MS m/z [M + H]+ 380.12; anal. calcd. for
C19H14FN5O3: C, 60.31; H, 3.71; N, 18.48; found: C, 60.38;
H, 3.78; N, 18.52.
3-[5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl]-N’-[2-oxo-1,2-
dihydro-3H-indol-3-ylidene]propane hydrazide (54)
Yield: 1.12 g (43%); m.p. 240-244 ºC; Rf 0.66
(chloroform); λmax 452 nm; IR (KBr) ν / cm−1 3239, 2843,
1713, 1547, 2902; 1H NMR (DMSO) d 8.98 (s, 1H,Ar−H),
8.59 (s, 1H, Ar−H), 8.0 (d, 2H, J 8.1 Hz, Ar−H), 7.75
(t, 2H, J 7.9 Hz,Ar−H), 7.45 (dd, 1H, J 7.4 Hz,Ar−H), 7.32
(dd, 1H, J 7.9 Hz, Ar−H), 3.50 (s, 2H, CH2), 2.31 (s, 2H,
CH2); 13C NMR (100 MHz, DMSO-d6) d 160.56, 156.15,
148.33, 131.50, 126.72, 126.21, 123.77, 121.79, 120.72,
115.25, 51.01, 33.92; EI-MS m/z [M + H]+ 397.32; anal.
calcd. for C19H14ClN5O3: C, 57.62; H, 3.58; N, 17.71; found:
C, 57.68; H, 3.62; N, 18.1.
3-[5-(2-Chlorophenyl)-1,3,4-oxadiazol-2-yl]-N’-[2-oxo-1,2-
dihydro-3H-indol-3-ylidene]propane hydrazide (58)
Yield: 1.15 g (43%); m.p. 244-246 ºC; Rf 0.61
(chloroform); λmax 455 nm; IR (KBr) ν / cm−1 3235, 2843,
1713, 1537, 2902, 725; 1H NMR (DMSO) d 8.76 (s, 1H,
Ar−H), 8.31 (s, 1H, Ar−H), 7.92 (d, 2H, J 8.4 Hz, Ar−H),
7.70 (t, 2H, J 7.6 Hz,Ar−H), 7.43 (dd, 1H, J 7.5 Hz,Ar−H),
7.30 (dd, 1H, J 7.8 Hz, Ar−H), 3.40 (s, 2H, CH2), 2.53
(s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d 164.16,
154.17, 149.38, 133.60, 125.12, 125.26, 123.77, 121.80,
121.76, 116.27, 55.10, 34.82; EI-MS m/z [M + H]+ 397.43;
anal. calcd. for C19H14ClN5O3: C, 57.62; H, 3.58; N, 17.71;
found: C, 57.68; H, 3.62; N, 18.1.
3-[5-(4-Nitrophenyl)-1,3,4-oxadiazol-2-yl]-N’-[2-oxo-1,2-
dihydro-3H-indol-3-ylidene]propane hydrazide (55)
Yield: 2.32 g (89%); m.p. 240-244 ºC; Rf 0.71
(chloroform); λmax 448 nm; IR (KBr) ν / cm−1 3121, 2916,
1
1655, 1618, 2849, 1522; H NMR (DMSO) d 8.15 (s,
1H, Ar−H), 7.74 (q, 1H, J 7.9 Hz, Ar−H), 7.46 (t, 1H,
J 7.5 Hz, Ar−H), 7.31 (dd, 2H, J 8.2 Hz, Ar−H), 7.21

2004
Design, Synthesis, Pharmacological Evaluation and Molecular Docking Studies
J. Braz. Chem. Soc.
3-[5-(2-Nitrophenyl)-1,3,4-oxadiazol-2-yl]-N’-[2-oxo-1,2-
dihydro-3H-indol-3-ylidene]propane hydrazide (59)
Yield: 2.22 g (89%); m.p. 242-244 ºC; Rf 0.71
(chloroform); λmax 438 nm; IR (KBr) ν / cm−1 3121, 2916,
1645, 1615, 2749, 1522; 1H NMR (DMSO) d 8.31 (s, 1H,
Ar−H), 7.87 (d, 1H, J 7.6 Hz,Ar−H), 7.78 (d, 1H, J 7.4 Hz,
Ar−H), 7.61 (dd, 2H, J 5.3 Hz,Ar−H), 7.21 (t, 2H, J 7.4 Hz,
Ar−H), 6.72 (t, 1H, J 7.1 Hz,Ar−H), 3.63 (s, 2H, CH2), 2.34
(s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d 165.75,
163.11, 155.20, 147.82, 143.43, 137.15, 133.34, 130.43,
127.01, 124.34, 123.33, 123.02, 122.80, 121.50, 112.37,
101.87, 51.11, 23.38; EI-MS m/z [M + H]+ 407.4; anal.
calcd. for C19H14N6O5: C, 56.3; H, 3.48; N, 20.68; found:
C, 56.6; H, 3.5; N, 20.69.
CH2); 13C NMR (100 MHz, DMSO-d6) d 160.53, 156.17,
148.33, 131.52, 126.20, 123.76, 121.78, 120.72, 33.98; EI-MS
m/z [M + H]+ 441.32; anal. calcd. for C19H14BrN5O3: C, 60.31;
H, 3.71; N, 18.48; found: C, 60.38; H, 3.78; N, 18.52.
3-[5-(3-Bromophenyl)-1,3,4-oxadiazol-2-yl]-N’-[2-oxo-1,2-
dihydro-3H-indol-3-ylidene]propane hydrazide (63)
Yield: 2.15 g (82%); m.p. 175-177 ºC, Rf 0.65
(chloroform), λmax 395 nm, IR (KBr) ν / cm−1 3392, 2816,
1635, 1605, 1097; 1H NMR (DMSO) d 8.71 (s, 1H,Ar−H),
8.61 (s, 1H, Ar−H), 8.21 (s, 1H, Ar−H), 7.88 (d, 1H,
J 7.9 Hz,Ar−H), 7.66 (t, 2H, J 8.3 Hz,Ar−H), 7.42 (dd, 1H,
J 7.4 Hz,Ar−H), 7.22 (dd, 1H, J 7.6 Hz,Ar−H), 3.20 (s, 2H,
CH2), 2.35 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6)
d 164.53, 155.17, 148.30, 130.52, 126.25, 125.78, 122.60,
118.74, 42.61, 33.98; EI-MS m/z [M + H]+ 441.09; anal.
calcd. for C19H14BrN5O3: C, 60.31; H, 3.71; N, 18.48; found:
C, 60.33; H, 3.75; N, 18.51.
3-[5-(3-Chlorophenyl)-1,3,4-oxadiazol-2-yl]-N’-[2-oxo-1,2-
dihydro-3H-indol-3-ylidene]propane hydrazide (60)
Yield: 1.1 g (43%); m.p. 235-237 ºC; Rf 0.66
(chloroform); λmax 432 nm; IR (KBr) ν / cm−1 3229, 2743,
1713, 1538, 2902, 725; 1H NMR (DMSO) d 8.56 (s, 1H,
Ar−H), 8.14 (s, 1H, Ar−H), 7.87 (d, 2H, J 8.4 Hz, Ar−H),
7.65 (t, 2H, J 7.6 Hz,Ar−H), 7.45 (dd, 1H, J 7.5 Hz,Ar−H),
7.25 (dd, 1H, J 7.8 Hz, Ar−H), 3.35 (s, 2H, CH2), 2.80
(s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d 172.16,
159.17, 144.48, 136.60, 125.16, 123.26, 123.17, 121.85,
121.70, 117.27, 56.10, 33.83; EI-MS m/z [M + H]+ 397.02;
anal. calcd. for C19H14ClN5O3: C, 57.62; H, 3.58; N, 17.71;
found: C, 57.68; H, 3.62; N, 18.1.
Pharmacology
In vitro evaluation for anti-inflammatory activity by protein
albumin denaturation method
All synthesized compounds were screened for
anti-inflammatory activity by using in vitro method
reported earlier by Mizushima and Kobayashi19 with
slight modification. Accordingly, inhibition of albumin
denaturation technique was studied, a 5.0 mL reaction
mixture was prepared consisting of 0.2 mL of egg albumin
(obtained from fresh hen’s egg), 2.8 mL phosphate buffered
saline (pH 6.4) and 2.0 mL of varying concentration of test
compounds, so that final concentrations become 25, 50, 100
and 200 μg mL−1. Similar volume of double distilled water
served as control. Then, the mixtures were incubated at
37 2 °C in an incubator for 15 min and then heated at 70 ºC
for 5 min. After cooling, their absorbance was measured at
660 nm by using vehicle as blank. Indomethacin with final
concentration of 50 and 100 μg mL−1 was used as reference
drug and treated similarly for determination of absorbance.
The reading for this activity was taken on the same day and
percent inhibition of protein denaturation was calculated
by equation 1, as follows:
3-[5-(2-Fluorophenyl)-1,3,4-oxadiazol-2-yl]-N’-[2-oxo-1,2-
dihydro-3H-indol-3-ylidene]propane hydrazide (61)
Yield: 2.11 g (82 %); m.p. 180-182 ºC, Rf 0.65
(chloroform), λmax 368 nm, IR (KBr) ν / cm−1 3297,
1
2916, 1655, 1605, 1097; H NMR (DMSO) d 8.10
(d, 2H, J 8.3 Hz, Ar−H), 7.68 (t, 2H, J 7.6 Hz, Ar−H), 7.26
(dd, 1H, J 7.6 Hz,Ar−H), 7.24 (dd, 1H, J 7.3 Hz,Ar−H), 6.81
(t, 2H, J 6.9 Hz,Ar−H), 3.73 (s, 2H, CH2), 2.50 (s, 2H, CH2);
13C NMR (100 MHz, DMSO-d6) d 164.52, 163.31, 161.31,
160.36, 135.94, 129.96, 129.88, 127.96, 124.35, 124.17,
115.94, 115.72, 114.50, 103.80, 99.49, 57.01, 51.68; EI-MS
m/z [M + H]+ 380.16; anal. calcd. for C19H14FN5O3: C, 60.31;
H, 3.71; N, 18.48; found: C, 60.38; H, 3.78; N, 18.52.
3-[5-(4-Bromophenyl)-1,3,4-oxadiazol-2-yl]-N’-[2-oxo-1,2-
dihydro-3H-indol-3-ylidene]propane hydrazide (62)
(1)
Yield: 2.64 g (82%); m.p. 174-176 ºC, Rf 0.65
(chloroform), λmax 398 nm, IR (KBr) ν / cm−1 2982, 2906,
1655, 1685; 1H NMR (DMSO) d 8.91 (s, 1H, Ar−H), 8.59
(s, 1H,Ar−H), 8.0 (s, 1H,Ar−H), 7.98 (d, 1H, J7.9 Hz,Ar−H),
7.76 (t, 2H, J 8.3 Hz, Ar−H), 7.45 (dd, 1H, J 7.4 Hz, Ar−H),
7.32 (dd, 1H, J 7.6 Hz,Ar−H), 3.58 (s, 2H, CH2), 2.38 (s, 2H,
In vivo evaluation
Anti-inflammatory activity
The anti-inflammatory activity was carried out
according to the method of Halen et al.20 and employed

Vol. 27, No. 11, 2016
Kerzare et al.
2005
with some modifications.18 Sprague Dawley rats were used
for this study; seventeen groups with six rats per group
were formed. All test samples were administered to
animals at 100 mg kg−1 dosage, as suspension in 0.5%
carboxymethyl cellulose in water. The samples were
administered orally; after 60 min of drug dose, injection
of 0.1 mL solution of carrageenan (0.5 mg 25 mL−1)
was injected into the sub-plantar tissue of the left hind
paw of each rat. Out of this, one group was treated with
standard drug indomethacin (100 mg kg−1). The initial
volume of paw was measured within 30 s after carrageenan
injection. Later on paw volume was measured after 1-5 h,
respectively. The relative increase in the paw volume
was calculated in the individual animal of the control,
test, and standard groups, respectively. The percentage
of inhibition of edema was calculated by the equation 2,
as follows:
all synthesized compounds were quantified in vivo by
Eddy’s hot plate method using analgesiometer. Albino
wistar mice were used for this study; seventeen groups with
six rats per group were formed. All the test compounds
were suspended in 0.5% of carboxymethylcellulose
sodium (CMC) and administered orally. The albino wistar
rats were treated with the newly synthesized derivatives
(100 mg kg−1, p.o.) and standard drug indomethacin
(100 mg kg−1, p.o.). The animals were individually placed
on the hot plate maintained at 55 °C, one hour after their
respective treatments. The response time was noted as the
time at which animals reacted to the pain stimulus either
by paw licking or jump response. The relative increase in
reaction time was measured at an interval of 0, 30, 60 and
90 min in the individual animal of the control, test and the
standard group (Table 1). The percent increase in reaction
time was calculated using equation 3 as follows,
(2)
(3)
where Dt means relative change in paw volume in test
group and Dc means relative change in paw volume in
control group.
where It is the reaction time at time t and Io is the reaction
time at time zero (0 min).
Ulcerogenic activity
Analgesic activity
Acute ulcerogenesis was done according to Cioli et al.28
and modifications standardized in our laboratory.18 Albino
rats (150-200 g) were divided into seven different groups
Analgesic activity test was performed following the
method of Eddy and Leimbach.21 Analgesic activities of
Table 1. Anti-inflammatory, analgesic, ulcerogenic index and molecular docking studies of synthesized compounds (49-63)
Anti-inflammatory activity
Analgesic activity
In vitro
In vivo
Inhibition of paw edemab / %
time / h
Ulcerogenic
index
Dock
score
Compound
Increase in reaction / %
time / min
Inhibition of
denaturation / %
Absorbancea
1
2
3
30
60
90
49
0.0401
0.0409
0.0407
0.0403
0.0402
0.0398
0.0403
0.0401
0.0402
0.0403
0.0397
0.0373
0.0403
0.0404
0.0402
0.0510
61.64
66.59
66.19
65.58
65.07
62.13
65.63
61.64
63.18
65.87
61.58
54.12
65.12
64.16
62.13
82.14
11.6
35.8
38.4
13.4
30.3
20.4
20.2
35.1
35.5
21.2
33.34
22.5
23.1
28.5
23.5
28.5
13.7
40.9
43.1
14.1
30.5
21.2
21.4
37.6
40.0
22.6
35.56
25.8
25.2
31.8
24.8
28.7
13.9
42.7
45.5
14.9
30.6
23.9
21.7
38.1
40.5
24.4
36.12
28.2
33.9
38.2
25.2
46.6
19.45
35.86
21.86
11.92
24.55
27.57
26.69
21.38
44.91
20.34
27.16
21.34
11.96
21.34
23.34
64.27
66.81
77.11
76.14
70.35
29.72
28.52
23.91
57.81
51.12
25.33
29.12
23.33
58.35
23.38
24.39
82.26
71.98
84.11
83.17
81.11
35.00
40.11
26.69
59.79
54.29
27.13
32.13
27.13
70.11
25.13
27.13
85.84
0.24
0.35
0.56
0.52
NT
−3.91
−4.44
−4.37
−3.45
−4.01
−3.33
−3.05
−3.27
−3.63
−1.85
−2.48
−0.16
−2.20
−4.04
−4.46
−4.47
50
51
52
53
54
NT
55
NT
56
NT
57
NT
58
NT
59
NT
60
NT
61
0.39
NT
62
63
NT
Standard drugc
1.25
aReadings at 660 nm; bincrease in paw volume measured after 3 h from administration of samples; cindomethacin. NT: Not treated.

2006
Design, Synthesis, Pharmacological Evaluation and Molecular Docking Studies
J. Braz. Chem. Soc.
consisting of six animals in each one. Ulcerogenic activity
was evaluated after p.o. administration of test compounds
or indomethacin at the dose of 50 mg kg−1. Control rats
received p.o. administration of vehicle (suspension of 1%
methyl cellulose in water). Food but not water was removed
24 h before administration of the test compounds. After
the drug treatment, the rats were fed normal diet for 17 h
and then sacrificed. The stomach was removed and opened
along the greater curvature, washed with distilled water
and cleaned gently by dipping in saline. Gastric mucosa of
the rats was examined by means of a four times binocular
magnifier and the lesions were counted according to earlier
reported method.18 Accordingly, the mean score of each
treated group minus the mean score of the control group
was considered to determine the severity index of gastric
damage for compounds under study.
two different steps were carried out to provide with
substituted aryl acid hydrazides and indolidene hydrazinyl
butanoic acids, which were further reacted to yield the final
derivatives. In the first step, various substituted benzoic
acids 1-15 esterifies in presence of ethanol and concentrated
sulphuric acid to yield corresponding ethyl benzoates 16-30.
These compounds were treated with hydrazine hydrate
to yield the substituted aryl acid hydrazide 31-45. These
compounds were not characterised on basis of NMR and
mass analysis because they are reported in literature; hence,
the physicochemical parameters were used and carry out the
confirmation of these compounds.All the compounds 16-45
were analysed on the basis of melting point and TLC and
found to be in understanding with the reported molecules.
In the second step, isatin (1H-indole-2,3-dione) 46 was
reacted with hydrazine hydrate in presence of methanol
under conditions of reflux to yield the 3-hydrazinylidene-
1,3-dihydro-2H-indol-2-one 47. NMR spectra of this
compound exhibited prominent signals at d 3.34 ppm
corresponding to the secondary amide proton, the aromatic
protons belonging to fused benzene ring was exhibited
around d 7.0 to 7.7 ppm presenting four protons. The
major spectral change was observed in the IR spectrum
which provides with an appearance of primary amine
functional group at 3411.12 cm−1. This structure was
further confirmed on basis of mass spectral study which
reflects the molecular weight of 162.24 on the ESI-MS in
positive mode. In the next step, 47 was reacted with succinic
anhydride under basic condition in an aprotic solvent
such as dichloromethane to obtain 4-oxo-4-[2-(2-oxo-1,2-
dihydro-3H-indol-3-ylidene)-hydrazinyl]butanoic acid 48.
This compound was confirmed on the basis of spectral
studies; 1H NMR exhibited singlets at d 3.78 and 2.23 ppm
for four protons belonging to two methylene carbons. The
proton of hydroxyl group was observed at d 12.44 ppm,
whereas the protons of secondary amides were observed
at d 6.30 ppm. The four aromatic protons were reflected
around d 7.30 to 8.0 ppm for the benzene ring system. This
was further confirmed from mass spectra in positive mode
with molecular weight 262.02.
Molecular docking study
For docking purpose, the three-dimensional structure
of COX-2 (protein data bank, PDB code 4Z0L) was
obtained from RCSB PDB.29 The receptor molecule was
refined and validated on the basis of Ramchandran plot
using Biopredicta© module on the Vlife MDS Molecular
Modeling software, version 4.3.1.22 The Vlife MDS suit
uses k-nearest neighbour genetic algorithm (KNN-GA)
method for molecular docking.22 The scoring function is
based on the basis of best suited moiety for the target with
respect to energy and inter molecular interactions. The
ligands that were already present within the receptor in
bound form were removed to allow for docking protocol.
All the ligands were prepared and docked for this study in
flexible docking mode and atoms located within a range
of 5.0 Å from the amino acid residues were selected in
the active site. Standard drug indomethacin was also
included into the series of compounds under study to
make comparison with respect to in silico analysis. The
docking calculations and energy minimization were set in
the Biopredicta© module, most of the parameters were set
default with 10000 cycles per molecule for the active site
cavity No. 1. Since this receptor is a homodimer nearly
all amino acid residues are common in it, the active site
cavity consisted of amino acid residues GLN374, LEU145,
TRP139, TYR373, PRO127, ASN375, PRO538, SER541,
GLY536, ASN537 and PHE142.
The final derivatives of this series 3-(5-substituted-
1,3,4-oxadiazol-2-yl)-N’-[2-oxo-1,2-dihydro-3H-indol-
3-ylidene]propane hydrazides 49-63 were synthesized
by carrying out cyclization of compounds 31-45 with 48
in presence of phosphorous oxychloride. These products
were obtained in satisfactory yield and purity as studied on
the thin layer chromatography and melting point studies.
The structural confirmation was carried out on the basis
of spectral studies, the IR spectra of these compounds
exhibited absorbance for important functional groups,
such as secondary amide at 3154 cm−1; the carbonyl group
Results and Discussion
The reported investigation deals with synthesis and
characterization of several hybrid derivatives from indole
and 1,3,4-oxadiazole nucleuses linked via hydrazide
chain to form final fifteen derivatives. To achieve these,

Vol. 27, No. 11, 2016
Kerzare et al.
2007
is indicated at 1655 cm−1 and the C=N bond is reflected
around 1618 cm−1. These groups are common to all the
molecules from final derivatives. The 1H NMR spectra of
these compounds exhibited several characteristic NMR
shifts. The ethylene protons were observed around d 2.00
to 3.85 ppm; the aromatic protons were observed within the
range of d 7.00 to 8.50 ppm, representing the phenyl ring
and the indole aromatic nucleus. Compound 50 presented
with a hydroxyl proton at d 9.49 ppm. These compounds
were also analysed for their mass spectral characteristic
and was found to be in good agreement with the results
obtained for compound 50, which exhibited molecular
mass of [M + H]+ 378.11 in the electron spray ionisation
with positive mode of mass spectrometry.
All the synthesized compounds of the final series 49-63
were screened for in vitro anti-inflammatory activity based
on assay developed by Mizushima and Kobayashi.19 This
assay is also known as the protein albumin denaturation test
as it involves the determination of percentage protection from
denaturation of protein and thus its stabilization ultimately
the inhibition of inflammation. The relativity of absorbance
between test samples (compounds 49-63) with respect to
control, designated the stabilization of protein and thus the
inhibition of heat-induced protein (albumin) denaturation or
the inhibition by derivatives and reference drug indomethacin.
The increase in absorbance in the test compounds indicated
better stabilization of proteins compared to standard drug
indomethacin and the blank. Compounds 50 and 51 exhibited
absorbance of 0.0409 and 0.0407 accounting for a 66.59 and
66.19% of denaturation, respectively. These are highest in
this series of compounds followed by 65.87% (0.0403) for
58, then 65.63% (0.0403) for 55, and 65.07% (0.0402) for 53
(Table. 1). Other derivatives have also shown better percent
of denaturation, which is in the range of 54.12 to 64.16%;
the standard drug indomethacin has exhibited percent of
denaturation of 82.14% (0.0510). These results indicate
that all the compounds have potential anti-inflammatory
activity since the least active compound is also exhibiting a
percent denaturation of 54.12%, with more than fifty percent,
whereas the most active compound presents inhibition of
66.59%. In vitro assay displayed some features of this series
with respect to substitutions; methyl, hydroxy, methoxy,
chloro and nitro substituted derivatives, such as 50, 51,
58 and 55, possess good anti-inflammatory activity. Since
these compounds were studied at various concentrations
(25, 50, 100 and 200 μg mL−1), it was decided to perform
in vivo activity on all the derivatives to determine their anti-
inflammatory activity.
by the Institutional Animal Ethics Committee (IAEC).
The compounds were tested at 100 mg kg−1 oral dose and
were compared with the standard drug indomethacin at
100 mg kg−1 oral dose. The tested compounds showed
anti-inflammatory activity ranging from 13.9 to 45.5% after
3 h (Table 1). The anti-inflammatory results revealed that
compounds 50, 51 and 58 exhibited good anti-inflammatory
activity whereas compounds 53 and 55 showed moderate
activity, and compounds 63, 62, 57, 54 and 49 showed
low activities when compared with standard drug
(indomethacin). These results illustrate that compounds
substituted with methyl 51, hydroxy 50, methoxy 53 and
nitro 55 at position 5 of the 1,3,4-oxadiazole ring system
showed good anti-inflammatory activity, having maximum
percentage of inhibition in edema. Surprisingly, the halogen
derivatives such as chloro 54, fluoro 57, bromo 63 and
acetyl 56 showed low activity. This indicates that the
compound having p-substituted electron withdrawing
groups may enhance anti-inflammatory activity and
electron releasing groups diminished the activity. The
activity declines with replacement of electronegative
group by electropositive group. It is noteworthy that NO2
substitution of 3-nitrophenyl in 52 and 4-nitrophenyl in 55
induce remarkable change in the activity, i.e., compound
substituted with 3-nitrophenyl 52 showed low activity while
compound substituted with 4-nitrophenyl 55 showed good
activity. So, p-substituted derivatives favour good activity
than m-substituted derivatives.
The analgesic activity of the synthesized compounds
was evaluated by hot plate test according to Eddy and
Leimbach.21 The compounds were tested at 100 mg kg−1
oral dose and were compared with the standard drug
indomethacin at 100 mg kg−1 oral dose. The tested
compounds showed analgesic activity ranging from
25.13 to 84.11% (Table 1) after 90 min time cycle for
the series of compounds under study. The analgesic
screening results revealed that compounds 50, 51 and
52 showed good analgesic activity whereas compounds
49 and 61 showed moderate activity and compounds
56 and 57 showed low activity when compared with
standard drug. The results illustrate that compounds
substituted with 3-nitrophenyl 52 at position 5 of the
1,3,4-oxadiazole ring system showed good analgesic
activity having elevated percentage increase in reaction
time following the compounds 50 and 51 with maximum
activity. In addition, compounds substituted with
phenyl 49 and 2-fluorophenyl 61 showed moderate
activities while compound substituted with 2,3-dimethoxy
53, 4-chlorophenyl 54 and 4-nitrophenyl 55 showed
low activity. This indicates that the compounds having
p-substituted and m-substituted electron withdrawing
Compounds were evaluated for their in vivo anti-
inflammatory activity by carrageenan induced paw edema
method. The protocol of animal experiments was approved

2008
Design, Synthesis, Pharmacological Evaluation and Molecular Docking Studies
J. Braz. Chem. Soc.
groups may enhance analgesic activity and electron
releasing groups diminish the activity. From the above
discussion, it is clear that compound substituted with
4-fluorophenyl 57 was found to be a fair anti-inflammatory
agent with poor analgesic activity. These results suggest
that unlike the substitution on indomethacin, the
1,3,4-oxadiazole ring system behaves differently and
suggests for substitution with electron donating group
(EDG) on the ring system.
The ulcerogenic activity was performed according to
Cioli et al.28 and reports published earlier from our lab.18
Those compounds, which showed good results in anti-
inflammatory and analgesic activity, were screened for
their ulcerogenic activity. The tested compounds exhibited
activity in the range of 0.14 to 0.56, whereas standard drug
indomethacin presented very high severity index of 1.25.
Results of this activity indicate that compound 50 and
51 have a severity index of 0.35 and 0.56, respectively.
Compounds 49 and 61 exhibit severity index of 0.24 and
0.39, respectively, which is the lowest compared to other
tested compounds; these compounds lack good anti-
inflammatory activity. This experiment clearly indicates
that 50 and 51 are better tolerated derivative of this series
and are far safer than the standard drug indomethacin with
respect to the ulcerogenic effect.
Indole is a very well-known nucleus and its derivative
indomethacin is known to possess anti-inflammatory
activity and used in therapy since last forty years.
Accordingly, we proposed and derived several hybrids
consisting of indole and oxadizole nuclei as inhibitor of
the COX receptor, which is the target receptor for nearly
all the NSAIDs. On the basis of in vitro and in vivo
studies, it was found that compounds 50 and 51 exhibit
good anti-inflammatory and analgesic activity. In order
to investigate the molecular interactions and binding
modes of some of the synthesized derivatives like 50,
51, 53, 55 and 62, we docked these derivatives with
COX enzyme (PDB code 4Z0L)29 using licensed version
of Vlife MDS 4.32 software tools.22 This COX-2 receptor
was used for our study because of two reasons: first, this
complex provides with a indole derivative, which can act
as a reference molecule for docking; and the second reason
was the similarity with our animal model. Prior to carrying
out docking the COX-2 receptor was prepared for docking
by removing the ligands in complex but the water molecules
were retained for this particular study. The receptor was
prepared by removing the already bound ligand and then
repaired with for missing residues by Biopredicta© tool.
COX-2 is a homodimer and contains identical residues,
active sites and channels; therefore, we employed a single
chain for our study. This model was validated on the basis of
Ramchandran Plot analysis and was found to be satisfactory
with the core and allowed count of about 98% including
glycine and proline residues. This model was saved and
further employed for carrying out docking studies.
The docking was carried out for all synthesized
compounds. Figure 2 exhibits the docking mode of
compound 50 and 51 with all three important interactions
like hydrogen bonding, hydrophobic interaction and the
Van derWaals forces interactions. Three compounds, 50, 51
and 53, have exhibited good activity with 50 and 51 being
most active followed by the other. This was also observed
with the results of molecular docking; the energies for 50
and 51 were found to be −4.44 and −4.37, respectively,
which is highest in the series of synthesized derivatives
and comparable to standard drug indomethacin with score
of −4.47 (Table 1). Compound 50 revealed that it binds to
the active site of COX-2 receptor by forming hydrogen
bond with GLY536 (bond length: 2.125 Å) and TYR373
(bond length: 2.325 Å; Figure 2a, 50). Hydrophobic
interactions were found to be mostly between GLN374
with bond length of 4.635, 4.538 and 4.045, ARG376
(Figure 2b, 50). It also formed multiple Van der Waal
interactions with the receptor amino acids having bond
length between 2 to 3.9 Å (Figure 2c, 50). Compound 51
prominently displayed that it binds to the active site of
COX-2 receptor by forming two hydrogen bonds with
ASN375 (bond length: 2.108 and 2.286 Å; Figure 2a, 51).
Hydrophobic interactions were found to be mostly among
GLN374A, GLN374B, ASN375B, TYR373B, PRO127A,
SER541B and ASN537B with bond length between 2.342
and 4.828 (Figure 2b, 51). It also formed multiple Van der
Waal interactions with the receptor amino acids having
bond length between 2.0 and 3.9 Å (Figure 2c, 51).
The Van der Waal interactions were observed between
the receptor and indole nucleus plus its substituted
oxadiazole moiety. This molecule seemed to lay on both
the chains as A and B of the homodimer, it is illustrated
by fact that interactions were observed with GLY536A,
PHE142B, TRP139B, PRO538A, ASN537A, GLN374A,
ASN375A, ARG376B, ASN373B, GLY536B, TYR373B,
PRO127A, PRO538B and ASN537B with bond length
from 2.1 to 3.9 Å. Compounds 53 and 55 were found to
show good dock score of −4.01 and −3.05, respectively;
these compounds also had interaction with TRP139B,
PRO538A,ASN537A, GLN374A,ASN375A,ARG376B,
ASN373B and ASN 537B, forming the hydrophobic and
Van der Waals interactions. In case of compounds 62 and
63, the dock score was found to be comparatively high
viz, −4.04 and −4.46, respectively, but they did not show a
good biological activity profile in wet lab studies. So, this
dock score might be due to the presence of halogens, such

Vol. 27, No. 11, 2016
Kerzare et al.
2009
Figure 2. Molecular docking results for compound 50 (above) and 51 (below): (a) hydrogen bond interaction; (b) hydrophobic interaction; (c) Van der
Waals interaction.
as the bromine. It is observed that presence of methyl or
hydroxyl groups causes rise in activity as well as it can be
seen that nitro substitution on the 1,3,4-oxadiazole causes
increase in the activity. The docking of newly designed
molecules has also presented their entry into deep sited
region of the receptor, probably on the confluence of both
the chains. Thus, it is considered that presence of indole
and oxadiazole ring in the single molecule is beneficial for
the activity, but the presence of halogen such as fluorine
or chlorine on the 1,3,4-oxadiazole reduces the activity.
Further, it is also noted that the position of substitution on
the phenyl ring has great contribution towards the activity
as an anti-inflammatory and analgesic compound.
In this preliminary communication, we have presented
synthesis and pharmacological evaluation of novel N’-
(2-oxoindolin-3-ylidene)-3-(5-substituted phenyl-1,3,4-
oxadiazol-2-yl)propane hydrazide derivatives 49-63,
obtained following a five step reactions. The reactions
were simple and follow-up procedure resulted in pure
compounds with satisfactory yield. In vitro and in vivo anti-
inflammatory activity revealed that compounds 50 and 51
exhibit comparable inhibition to standard drug indomethacin
with added advantage of very less possibility of ulceration.
These compounds were found to be good analgesic agents
also when compared with standard drug. These molecules
exhibited that methyl and hydroxy substituted molecules
are better tolerated as well as present better activity when
compared to their chloro and fluoro counterparts. The
molecular docking studies on these molecules also verified
the wet lab results, compounds 50 and 51 exhibited hydrogen
bonding, hydrophobic interactions as well as Van der Waals
interactions. The dock scores and binding energy were found
to be in good agreement with the pharmacological results.
This work need to be further elaborated with respect to
number of molecules, their versatility, animal models, and
structure activity relationship studies and most importantly
with respect to the pK/pD studies.
Conclusions
In conclusion, we have described the design and
synthesis of novel oxadiazolyl-2-oxoindolinylidene
propane hydrazides as amide tethered hybrids of indole
and oxadiazole and their evaluation for anti-inflammatory
and analgesic activity. The compounds were successfully
synthesised following five step reaction to yield fifteen

2010
Design, Synthesis, Pharmacological Evaluation and Molecular Docking Studies
J. Braz. Chem. Soc.
derivatives as 3-(5-substituted-1,3,4-oxadiazol-2-yl)-N’-[2-
oxo-1,2-dihydro-3H-indol-3-ylidene]propane hydrazides.
In vitro and in vivo studies have exhibited final derivatives
50 and 51 as highly promising molecules with severity
index of 0.35 and 0.56, respectively, encouraging for an
analgesic compound. The hydroxy and methyl substitutions
on phenyl ring system provided with active compounds
having percentage of inhibition of 84.11 and 83.17%,
respectively, compared to standard the drug at 85.84%.
Molecular docking studies are also in agreement with
the pharmacological evaluation with potent compounds
exhibiting dock score of −4.44. It can be stated that these
compounds can be further studied for their structure-
activity relationship (SAR) studies and developed into
potential lead molecules.
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Published online: March 22, 2016