Synthesis, characterization, andcomputational studies on phthalic anhydride-based benzylidene-hydrazide derivatives as novel,potential anti-inflammatory agents

Article abstract of DOI:10.1007/s00044-013-0848-1

A series of phthalic anhydride-based substituted benzylidene-hydrazide derivatives (3a-i) was synthesized. The synthesized derivatives were authenticated by TLC, UV-visible, FTIR, NMR, and mass spectroscopic techniques and further screened for in vivo anti-inflammatory and analgesic activities by carrageenan-induced rat paw oedema and tail immersion methods, respectively, using diclofenac sodium as standard drug. The derivatives 3d, 3e, and 3h were found to be most active anti-inflammatory and analgesic agents among all the synthesized derivatives. The physico-chemical similarity of the derivatives with standard drugs was assessed by calculating various physicochemical properties using software programs. The percent similarity of synthesized derivatives was found to be good except 3i. The derivatives were subjected to QSAR by multilinear regression using Analyze it version 3.0 software and two statistically sound models were developed with R² (0.933-0.960), R_adj² (0.595-0.762) and Q² (0.999) with good F (2.76-4.84) values. Molecular docking studies were performed by MVD software (version 2012.5.0.0). The derivative 3h has emerged out as most potent anti-inflammatory agent with highest dock score, i.e., -93.64. Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0848-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:2676–2689
DOI 10.1007/s00044-013-0848-1
ORIGINAL RESEARCH
Synthesis, characterization, and computational studies on phthalic
anhydride-based benzylidene-hydrazide derivatives as novel,
potential anti-inflammatory agents
•
•
•
Anu Kajal Suman Bala Sunil Kamboj
Vipin Saini
Received: 19 July 2013 / Accepted: 14 October 2013 / Published online: 31 October 2013
ꢀ Springer Science+Business Media New York 2013
Abstract A series of phthalic anhydride-based substi-
tuted benzylidene-hydrazide derivatives (3a–i) was syn-
thesized. The synthesized derivatives were authenticated
by TLC, UV–visible, FTIR, NMR, and mass spectroscopic
techniques and further screened for in vivo anti-inflam-
matory and analgesic activities by carrageenan-induced rat
paw oedema and tail immersion methods, respectively,
using diclofenac sodium as standard drug. The derivatives
3d, 3e, and 3h were found to be most active anti-inflam-
matory and analgesic agents among all the synthesized
derivatives. The physico-chemical similarity of the deriv-
atives with standard drugs was assessed by calculating
various physicochemical properties using software pro-
grams. The percent similarity of synthesized derivatives
was found to be good except 3i. The derivatives were
subjected to QSAR by multilinear regression using Ana-
lyze it version 3.0 software and two statistically sound
models were developed with R2 (0.933–0.960), Ra2dj
(0.595–0.762) and Q2 (0.999) with good F (2.76–4.84)
values. Molecular docking studies were performed by
MVD software (version 2012.5.0.0). The derivative 3h has
emerged out as most potent anti-inflammatory agent with
highest dock score, i.e., -93.64.
Introduction
Cyclooxygenase (COX) is the key enzyme in the synthesis
of main mediators (prostaglandins) of inflammation and
algesia (Botting, 2006). Inflammation is considered as a
protective response of our body to prevent infections and
by combating the foreign substances by releasing media-
tors (Borne, 2002). COX enzyme has two isoforms: COX-1
and COX-2. COX-2 is an inducible enzyme and most of the
classical NSAIDs actually inhibit COX-1 instead of COX-2
which results in gastrointestinal injury by gastric ulcera-
tion, suppression of TXA2 formation and platelet aggre-
gation (Husain et al., 2005). The gastric ulceration is due to
free carboxylic group in parent drugs (Akhter et al., 2010).
Studies suggest that the derivatization of carboxylic func-
tionality with hydrazone moiety in compounds have such a
pharmacophoric effect which leads to inhibition of COX
with minimal side effects like ulcerogenicity (Hunashal,
2011; Mehtap et al., 2009).
Within the scope of rational drug design, computational
methods have gain more and more importance to design
work flows that are faster, more efficient, and cheaper.
Computational studies have been developed as important
contributors in drug designing. The term quantitative
structure–activity relationship (QSAR) implies the empir-
ical relationships that use molecular parameters to quantify
a pharmacological or chemical property for a set of mol-
ecules. These studies permit complex biological systems to
be modeled successfully using simple structural parameters
and to predict substituent effects for a series of biologically
active compounds (Wang et al., 2006).
Keywords Phthalic anhydride ꢀ Benzylidene-hydrazide ꢀ
Analgesic activity ꢀ Anti-inflammatory activity ꢀ
QSAR ꢀ Molecular docking
Docking is defined as a method which predicts the
preferred orientation of one molecule to a second when
bound to each other to form a stable complex (Kitchen
et al., 2004). A main principle in drug discovery and
A. Kajal ꢀ S. Bala (&) ꢀ S. Kamboj ꢀ V. Saini
M. M. College of Pharmacy, Maharishi Markandeshwar
University, Mullana, Ambala, Haryana 133207, India
e-mail: sumankmj7@gmail.com
123

Med Chem Res (2014) 23:2676–2689
2677
development is the interaction between receptors and
enzymes with their ligands (Nunez et al., 2012). Docking
of a ligand is typically achieved by generating a number of
orientations (or poses) of a ligand within the active site and
scoring of poses, to identify one or more that closely
approximate the bioactive conformation determined by
X-ray crystallography (Regina et al., 2008). This is a
computational method to determine possible binding
modes of a ligand to the active site of a receptor. It makes
an image of the active site with interaction points known as
grid. Then it fits the ligand in the binding site either by grid
search or energy search.
obtained with Vg-11-250 J70S spectrophotometer at 70 eV
using electron ionization (EI source). For mass spectra,
solutions were made in HPLC grade methanol. Structural
similarity studies were performed by using Chem 3D Ultra
(version 10) (Nikolova and Jaworska, 2003). QSAR studies
were performed by multilinear regression using Analyze it
version 3.0 software. Molecular docking studies were
performed by Molegro Virtual Docker software (version
2012.5.0.0).
General methods
The present work describes the synthesis of benzyli-
dene-hydrazide derivatives and focused on the investiga-
tion of anti-inflammatory and analgesic activities of
synthesized derivatives. Additional efforts have been made
for computational studies which include QSAR studies and
in silico docking studies to the target COX-2 enzyme.
A series of phthalic anhydride-based substituted benzyli-
dene-hydrazide derivatives were synthesized as outlined in
Fig. 1. Phthalic anhydride and glycine were used as start-
ing materials. First of all, acid was synthesized by fusion of
both the starting materials. The acid was then subjected to
chlorination by using thionyl chloride. After chlorination,
ester (1) was synthesized by reacting with ethanol. The
ester so formed was reacted with hydrazine hydrate in
presence of ethanol to get corresponding hydrazide (2). The
hydrazide derivative was then reacted with different
substituted aromatic aldehydes in presence of methanol and
glacial acetic acid to yield the 2-(1,3-dioxo-1,3-dihydro-
2H-isoindol-2-yl)-N’-(substituted benzylidene)acetohyd-
razide derivatives (3a–i). The synthesized benzylidene-
hydrazide derivatives were characterized on the basis of the
spectral and analytical studies (Salimon et al., 2010;
Mehtap et al., 2009; Bhandari et al., 2008; Ahluwalia and
Aggarwal, 2000).
Materials and methods
Experimental
Melting points of newly synthesized benzylidene-hydra-
zide derivatives were determined on digital melting point
apparatus (Flora; Perfit India) and were found uncorrected.
Silica gel G plates of 3 9 15 cm were used for TLC and
spots were located by iodine chamber (Table 1). The
structures of the synthesized derivatives were confirmed by
spectral data. The kmax was calculated by using double
beam UV–visible 1800 Shimadzu spectrophotometer. The
IR spectra were recorded on FTIR-Shimadzu spectrometer
(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)acetic acid
1
using Nujol method. H NMR and 13C NMR spectra were
A mixture of phthalic anhydride (0.06 M) and glycine
(0.06 M) was placed in a 100-ml round bottom flask along
with calcium chloride drying tube. The reaction mixture
was fused for 20–30 min at 160–190 ꢁC. The product
recorded on BRUKER AVANCE II 400 NMR spectrom-
eter using DMSO as solvent and TMS as internal standard,
values were expressed in d ppm. Mass spectra were
Table 1 Physical data of synthesized compounds
Compounds
Molecular formula
%Age yield
Molecular weight
Solubility
kmax
Rf value
1
C12H11NO4
91.8
65.7
90.2
88.4
80.3
90.0
89.9
78.8
75.9
83.6
79.4
233.22
219.2
DMSO, EtOH, CHCl3
DMSO, EtOH, CHCl3
DMSO, EtOH, CHCl3
DMSO, EtOH, CHCl3
DMSO, EtOH, CHCl3
DMSO, EtOH, CHCl3
DMSO, EtOH, CHCl3
DMSO, EtOH, CHCl3
DMSO, EtOH, CHCl3
DMSO, EtOH, CHCl3
DMSO, EtOH, CHCl3
299
261
232
271
260
259
254
328
261
248
292
0.78
0.31
0.41
0.39
0.40
0.38
0.39
0.50
0.41
0.40
0.36
2
C
C
C
C
C
C
C
C
C
C
10H9N3O3
3a
3b
3c
3d
3e
3f
3g
3h
3i
17H13N3O3
17H12N4O5
17H12N4O5
17H12ClN3O3
18H15N3O4
17H13N3O4
18H15N3O3
18H15N3O5
20H19N3O6
307.31
352.308
352.308
341.755
337.337
323.31
321.337
353.336
397.389
123

2678
Med Chem Res (2014) 23:2676–2689
Fig. 1 Synthesis of (1,3-dioxo-
1,3-dihydro-isoindol-2-yl)-
acetic acid (substituted
benzylidene)-hydrazide
derivatives (3a–i)
O
O
Δ
NCH2COOH
NH2CH2COOH
glycine
+
O
160-190 oC
O
O
Phthalic anhydride
Phthaloylglycine
Reflux
SOCl2
O
O
ethanol
NCH2COOC2H5
NCH2COCl
O
O
1
Phthaloylglycine chloride
Reflux
NH2NH2.H2O
O
O
methanol
glacial acetic acid
NCH2CONHN
NCH2CONHNH2
RCHO
Reflux
CHR
O
2
O
3(a-i)
obtained was cooled and crystallized from water. 1H NMR
(DMSO-d6 400 MHz): 11.0 (s, 1H, OH), 7.99 (m, 2H, Ar–
H), 7.55 (m, 2H, Ar–H), 4.52 (s, 2H, –CH2); 13C NMR
(DMSO-d6); 167.0 (C-2, 5), 131.9 (C-3,4), 127.8 (C-6,9),
132.0 (C-7,8), 47.2 (C-10), 176.0 (C-11).
Ethyl(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)acetate (1)
Phthaloylglycine chloride obtained was dissolved in etha-
nol. The mixture was slightly warmed and filtered imme-
diately. Filterate was kept in undisturbed condition for the
formation of (1). Mp 78–80 ꢁC. IR (Nujol): 3011.93,
2832.59, 1737.94, 1480.43, 1405.20, 1329.20, 1216.17,
(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)acetyl chloride
1
705.01 cm-1; H NMR (DMSO-d6 400 MHz): 7.91 (m,
2H, Ar–H), 7.38 (m, 2H, Ar–H), 4.48 (s, 2H, –CH2), 4.12
(s, 2H, –CH2), 1.30 (t, 3H, –CH3); 13C NMR (DMSO-d6);
166.9 (C-2, 5), 132.3 (C-3,4), 127.4 (C-6,9), 132.9 (C-7,8),
44.9 (C-10), 171.0 (C-11), 59.2 (C-12), 13.6 (C-13).
(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)acetic
acid
(0.039 M) was placed in 250-ml round bottom flask
then thionyl chloride (16 ml) was added carefully in the
flask and reflux gently for 30–40 min having a calcium
chloride drying tube on the top. Contents of the flask
were shaken from time-to-time to ensure thorough
mixing. Excess thionyl chloride was removed by dis-
tillation under reduced pressure. The residual phtha-
loylglycine chloride was crystallized from petroleum
ether. 1H NMR (DMSO-d6 400 MHz): 7.92 (m, 2H,
Ar–H), 7.45 (m, 2H, Ar–H), 5.18 (s, 2H, –CH2); 13C
NMR (DMSO-d6 100 MHz); 166.7 (C-2, 5), 131.3 (C-
3,4), 127.0 (C-6,9), 132.1 (C-7,8), 58.4 (C-10), 172.5
(C-11).
2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)acetohydrazide
(2)
This compound was prepared by refluxing hydrazine
hydrate (0.05 M) with (1) (0.05 M) in ethanol (15 ml) at
75–80 ꢁC for 2–3 h. The reaction was monitored by TLC.
The white colored crystals were separated out by filtration
on cooling and recrystallized from ethanol. Mp
240–245 ꢁC. IR (Nujol): 3252.12, 3052.48, 2871.12,
123

Med Chem Res (2014) 23:2676–2689
2679
1727.33, 1540.23, 1500.68, 1305.86, 854.50 cm-1
;
1H
The orange-colored crude product was filtered out and re-
crystallized by ethanol. Mp 246–248 ꢁC. IR(Nujol):
3270.21, 3055.38, 2872.61, 1776.08, 1642.46, 1638.09,
NMR (DMSO-d6 400 MHz): 8.21 (br, s, 1H, –NH), 7.82
(m, 2H, Ar–H), 7.76 (m, 2H, Ar–H), 4.45 (s, 2H, –CH2),
3.8 (br, s, 2H, –NH2); 13C NMR (DMSO-d6); 166.4 (C-2,
5), 132.0 (C-3,4), 127.1 (C-6,9), 132.0 (C-7,8), 47.3 (C-
10), 170.3 (C-11).
1537.33,
1530.58,
1503.54,
1305.86,
1303.94,
859.32 cm-1; H NMR: 9.59 (s, 1H, –NH–N=), 8.2 (m,
1H, Ar–H), 8.01 (s, 1H, –N=CH–), 7.89 (m, 2H, Ar–H),
7.78 (m, 2H, Ar–H), 7.75 (m, 1H, Ar–H), 7.64 (m, 1H, Ar–
H), 7.53 (m, 1H, Ar–H), 4.68 (s, 2H,-CH2); 13C NMR
(DMSO-d6); 166.2 (C-2, 5), 132.5 (C-3,4), 127.1 (C-6,9),
132.9 (C-7,8), 47.4 (C-10), 173.1 (C-11), 154.7 (C-17),
126.3 (C-18), 129.9 (C-19), 134.7 (C-20), 131.7 (C-21),
123.7 (C-22), 148.9 (C-23); MS: m/z 352.08, 353.08
(M?1), 354.09 (M?2).
1
N’-Benzylidene-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-
yl)acetohydrazide (3a)
Hydrazide (2) (0.002 M) was dissolved in methanol
(15 ml). To this benzaldehyde (0.002 M) and a few drops
of glacial acetic acid were added and mixture was refluxed
for 2 h. The reaction was monitored by TLC. The white-
colored crude product was filtered out and recrystallized by
ethanol. Mp 243–246 ꢁC. IR(Nujol): 3256.63, 3056.34,
2838.12, 1758.19, 1658.54, 1544.08, 1502.61, 1023.28,
860.29 cm-1; 1H NMR (DMSO-d6 400 MHz): 9.62 (s, 1H,
–NH–N=), 8.21 (s, 1H, –N=CH-), 7.84 (m, 2H, Ar–H),
7.78 (m, 2H, Ar–H), 7.59 (m, 2H, Ar–H), 7.46 (m, 3H, Ar–
H), 4.56 (s, 2H, –CH2); 13C NMR (DMSO-d6); 167.9 (C-2,
5), 132.3 (C-3,4), 127.4 (C-6,9), 132.9 (C-7,8), 47.4 (C-
10), 173.0 (C-11), 154.7 (C-17), 131.2 (C-18), 129.0 (C-
19,23), 128.6 (C-20,22), 130.8 (C-23); MS: m/z 307.10,
308.10 (M?1), 309.10 (M?2).
2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-N’-(4-
chlorobenzylidene)acetohydrazide (3d)
Hydrazide (2) (0.002 M) was dissolved in methanol (15 ml).
To this 4-chlorobenzaldehyde (0.002 M) and a few drops of
glacial acetic acid was added and mixture was refluxed for
3–4 h. The reaction was monitored by TLC. The white-
colored crude product was filtered out and recrystallized by
ethanol. Mp 237–239 ꢁC. IR(Nujol); 3320.61, 3053.45,
2871.15, 1776.52, 1647.58, 1630.61, 1538.30, 1530.58,
1306.83, 862.22, 667.44 cm-1; 1H NMR: 9.72 (s, 1H, –NH–
N=), 7.98 (s, 1H, –N=CH–), 7.92 (m, 2H, Ar–H), 7.79 (m,
2H, Ar–H), 7.5 (d, 2H, Ar–H, J = 8.2 Hz), 7.41 (d, 2H, Ar–
H, J = 8.2 Hz), 4.48 (s, 2H, –CH2); 13C NMR (DMSO-d6);
166.2 (C-2, 5), 132.5 (C-3,4), 127.1 (C-6,9), 132.9 (C-7,8),
47.4 (C-10), 173.1 (C-11), 154.7 (C-17), 129.3 (C-18), 130.4
(C-19,23), 129.0 (C-20,22), 136.1 (C-21); MS: m/z 341.06,
343.05 (M?1), 342.06 (M?2).
2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-N’-(4-
nitrobenzylidene)acetohydrazide (3b)
Hydrazide (2) (0.002 M) was dissolved in methanol
(15 ml). To this 4-nitrobenzaldehyde (0.002 M) and a few
drops of glacial acetic acid was added and mixture was
refluxed for 3 h. The reaction was monitored by TLC. The
pale yellow-colored crude product was filtered out and
recrystallized by ethanol. Mp 245–247 ꢁC. IR(Nujol):
3302.06, 3056.34, 2830.16, 1755.30, 1662.30, 1656.92,
2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-N’-(3-
methoxybenzylidene)acetohydrazide (3e)
1585.55, 1544.08, 1322.26, 1302.01, 822.68 cm-1
;
1H
Hydrazide (2) (0.002 M) was dissolved in methanol
(15 ml). To this 3-methoxybenzaldehyde (0.002 M) and a
few drops of glacial acetic acid was added and mixture was
refluxed for 4 h. The reaction was monitored by TLC. The
white-colored crude product was filtered out and recrys-
tallized by ethanol. Mp 240–242 ꢁC. IR(Nujol); 3310.16,
3054.41, 2854.69, 1763.61, 1662.52, 1636.52, 1541.19,
NMR(DMSO-d6 400 MHz): 9.68 (s, 1H, –NH–N=), 8.39
(d, 2H, Ar–H, J = 8.3 Hz), 7.98 (s, 1H, –N=CH–), 7.9 (m,
2H, Ar–H), 7.7 (m, 2H, Ar–H), 7.32 (d, 2H, Ar–H,
J = 8.3 Hz), 4.75 (s, 2H, –CH2); 13C NMR (DMSO-d6);
166.4 (C-2,5), 132.5 (C-3,4), 127.4 (C-6,9), 132.0 (C-7,8),
47.6 (C-10), 173.0 (C-11), 154.3 (C-17), 137.3 (C-18),
129.9 (C-19,23), 123.7 (C-20,22), 150.7 (C-21); MS: m/
z 352.08, 353.08 (M?1), 354.09 (M?2).
1
1235.76, 1045.19, 863.18 cm-1; H NMR: 9.76 (s, 1H, –
NH–N=), 8.23 (s, 1H, –N=CH–), 7.9 (m, 2H, Ar–H), 7.74
(m, 2H, Ar–H), 7.21 (m, 1H, Ar–H), 7.11 (m, 1H, Ar–H),
6.99 (m, 1H, Ar–H), 6.79 (m, 1H, Ar–H), 4.56 (s, 2H, –
CH2), 3.82 (s, 3H, –OCH3); 13C NMR (DMSO-d6); 166.2
(C-2, 5), 132.5 (C-3,4), 127.1 (C-6,9), 132.9 (C-7,8), 47.4
(C-10), 173.1 (C-11), 154.7 (C-17), 132.2 (C-18), 114.6
(C-19), 162.1 (C-20), 116.4 (C-21), 129.6 (C-22), 121.3
(C-23), 56.0 (C-24); MS: m/z 337.11 338.11 (M?1),
339.11 (M?2).
2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-N’-(2-
nitrobenzylidene)acetohydrazide (3c)
Hydrazide (2) (0.002 M) was dissolved in methanol
(15 ml). To this 2-nitrobenzaldehyde (0.002 M) and a few
drops of glacial acetic acid was added and mixture was
refluxed for 1.5 h. The reaction was monitored by TLC.
123

2680
Med Chem Res (2014) 23:2676–2689
2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-N’-(4-
7.79 (m, 2H, Ar–H), 7.10 (m, 1H, Ar–H), 6.98 (m, 1H, Ar–H),
6.72 (m, 1H, Ar–H), 5.3 (s, 1H, –OH), 4.62 (s, 2H, –CH2),
3.81 (s, 3H, –OCH3); 13C NMR (DMSO-d6); 165.2 (C-2,5),
132.8 (C-3,4), 129.1 (C-6,9), 132.5 (C-7,8), 45.1 (C-10), 175
(C-11), 154.9 (C-17), 124.8 (C-18), 116.0 (C-19), 149.3
(C-20), 145.2 (C-21), 116.8 (C-22), 112.7 (C-23), 56.3
(C-26); MS: m/z 353.10, 354.10 (M?1), 355.11 (M?2).
hydroxybenzylidene)acetohydrazide (3f)
Hydrazide (2) (0.002 M) was dissolved in methanol
(15 ml). To this 4-hydroxybenzaldehyde (0.002 M) and a
few drops of glacial acetic acid was added and mixture was
refluxed for 4.5 h. The reaction was monitored by TLC.
The off white-colored crude product was filtered out and
recrystallized by ethanol. Mp 241–244 ꢁC. IR(Nujol):
3627.29, 3261.77, 3131.57, 2844.49, 1770.64, 1645.35,
2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-N’-(3,4,5-
trimethoxybenzylidene)acetohydrazide (3i)
1
1630.61, 1533.63, 1350.27, 860.29 cm-1; H NMR: 9.71
(s, 1H, –NH–N=), 8.25 (s, 1H, –N=CH–), 7.98 (m, 2H, Ar–
H), 7.81 (m, 2H, Ar–H), 7.31 (d, 2H, Ar–H, J = 8.1 Hz),
6.64 (d, 2H, Ar–H, J = 8.1 Hz), 5.2 (s, 1H, –OH), 4.52 (s,
2H, –CH2); 13C NMR (DMSO-d6); 166.2 (C-2, 5), 132.5
(C-3, 4), 127.1 (C-6, 9), 132.9 (C-7,8), 47.4 (C-10), 173.1
(C-11), 154.7 (C-17), 123.8 (C-18), 130.4 (C-19,23), 115.8
(C-20,22), 159.6 (C-21); MS: m/z 323.09, 324.09 (M?1),
325.10 (M?2).
Hydrazide (2) (0.002 M) was dissolved in methanol
(15 ml). To this 3,4,5-trimethoxybenzaldehyde (0.002 M)
and a few drops of glacial acetic acid was added and
mixture was refluxed for 2 h. The reaction was monitored
by TLC. The white-colored crude product was filtered out
and recrystallized by ethanol. Mp 246–247 ꢁC. IR(Nujol):
3330.12, 3053.45, 2843.63, 1764.22, 1647.28, 1640.81,
1538.30, 1528.65, 1248.11, 1040.32, 822.68 cm-1 1H
;
NMR: 9.68 (s, 1H, –NH–N=), 8.14 (s, 1H, –N=CH–), 7.8
(m, 2H, Ar–H), 7.73 (m, 2H, Ar–H), 6.62 (m, 2H, Ar–H),
4.43 (s, 2H, –CH2), 3.8 (s, 9H, –OCH3); 13C NMR
(DMSO-d6); 166.2 (C-2,5), 132.3 (C-3,4), 127.4 (C-6,9),
132.0 (C-7,8), 47.6 (C-10), 173 (C-11), 154.7 (C-17), 125.7
(C-18), 107.9 (C-19,23), 148.7 (C-20,22), 135.5 (C-21),
56.3 (C-26,28), 56.6 (C-29); MS: m/z 397.13, 398.13
(M?1), 399.13 (M?2).
2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-N’-(4-
methylbenzylidene)acetohydrazide (3g)
Hydrazide (2) (0.002 M) was dissolved in methanol
(15 ml). To this 4-methylbenzaldehyde (0.002 M) and a
few drops of glacial acetic acid was added and mixture was
refluxed for 3 h. The reaction was monitored by TLC. The
pale yellow-colored crude product was filtered out and
recrystallized by ethanol. Mp 242–243 ꢁC. IR(Nujol):
3255.02, 3056.34, 2853.64, 1783.27, 1646.32, 1632.35,
Pharmacological evaluation
1544.08, 1462.15, 1322.26, 1302.33, 862.68 cm-1 1H
;
Anti-inflammatory activity
NMR: 9.62 (s, 1H, –NH–N=), 8.14 (s, 1H, –N=CH–), 7.72
(m, 2H, Ar–H), 7.64 (m, 2H, Ar–H), 7.5 (d, 2H, Ar–H,
J = 7.8 Hz), 7.24 (d, 2H, Ar–H, J = 7.8 Hz), 5.2 (s, 2H, –
CH2), 2.7(s, 3H, -CH3); 13C NMR (DMSO-d6); 166.2 (C-2,
5), 132.5 (C-3, 4), 127.1 (C-6, 9), 132.9 (C-7,8), 47.4 (C-
10), 173.1 (C-11), 154.7 (C-17), 128.2 (C-18), 128.9 (C-
19,23), 129.3 (C-20,22), 140 (C-21), 20.9 (C-24); MS: m/
z 321.11, 322.11 (M?1), 323.12 (M?2).
The synthesized benzylidene-hydrazide derivatives (3a–i)
were screened for anti-inflammatory activity by carra-
geenan-induced rat paw oedema method. In carrageenan
model, rat paw edema was induced by injection of 0.1 ml
of freshly prepared carrageenan (1 % w/v) in subplantar
region of the left hind paw of rats and paw was marked at
the level of lateral malleolus (Winter et al., 1962). The
different groups of rats were pre-treated with their
respective doses. After 1 h, the rats were subjected to
injection of carrageenan and paw volume was measured by
plethysmometer (model pth-7070, sr.no.pt 070509, Medi-
cad system) after 1, 2, and 3 h. Mean SEM for treated
and control animal groups was calculated and compared for
each time interval and statistically analyzed. Percent inhi-
bition of inflammation after test/standard was calculated
using the formula,
2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-N’-(4-
hydroxy-3-methoxybenzylidene)acetohydrazide (3h)
Hydrazide (2) (0.002 M) was dissolved in methanol
(15 ml). To this 4-hydroxy-3-methoxy benzaldehyde
(0.002 M) and a few drops of glacial acetic acid was added
and mixture was refluxed for 6 h. The reaction was mon-
itored by TLC. The white-colored crude product was fil-
tered out and recrystallized by ethanol. Mp 248–249 ꢁC.
IR(Nujol): 3726.63, 3282.36, 3055.38, 2849.62, 1748.12,
1629.44, 1644.39, 1542.15, 1502.61,1321.30, 1238.21,
% inhibition ¼ Vc ꢁ Vt=Vc ꢂ 100;
ð1Þ
1218.21, 1042.51, 860.29 cm-1 1H NMR: 9.74 (s, 1H,
;
where Vt is the of paw oedema volume (ml) in test/standard
compound at corresponding time, and Vc is paw oedema
–NH–N=), 8.19 (s, 1H, –N=CH–), 7.96 (m, 2H, Ar–H),
123

Med Chem Res (2014) 23:2676–2689
2681
Table 2 Anti-inflammatory activity of synthesized benzylidene-hydrazide derivatives
Groups
Change in paw oedema volume (ml)
1 h
Percent
inhibition
2 h
Percent
inhibition
3 h
Percent
inhibition
Control
0.56 0.03
0.34 0.01
–
0.69 0.04
0.27 0.02
–
0.75 0.04
0.24 0.01
–
Diclofenac sodium
(10 mg/kg)
39.8
60.8
68.0
3a
3b
3c
3d
3e
3f
0.48 0.01
0.45 0.02
0.39 0.01*
0.41 0.01
0.40 0.02
0.43 0.01
0.46 0.02
0.38 0.02*
0.41 0.01
14.2
19.6
30.3
26.7
28.5
23.2
17.8
32.1
26.7
0.58 0.05
0.39 0.03*
0.35 0.01*
0.36 0.01*
0.35 0.01*
0.39 0.02*
0.52 0.03
0.32 0.01*
0.41 0.02*
15.9
43.4
49.2
47.8
49.2
43.4
24.6
53.6
40.5
0.62 0.05
0.34 0.02*
0.32 0.01*
0.31 0.01*
0.29 0.02*
0.37 0.02*
0.56 0.04
0.27 0.02*
0.38 0.01*
17.3
54.6
57.3
58.6
61.4
50.6
25.3
64.0
49.4
3g
3h
3i
All values represent mean SEM (n = 6)
* P \ 0.05 when experimental groups were compared with control
volume (ml) in control. The paw volume and percentage
inhibition data for synthesized benzylidene-hydrazide deriv-
atives at 1, 2, and 3 h are summarized in Table 2 and Fig. 2.
and Supervision of Experiments on Animals (CPCSEA),
Govt. of India (Protocol no. MMCP/IAEC/12/09).
Similarity studies
Analgesic activity
Assessments of structural similarity of benzylidene-hydra-
zide derivatives 3a–i was compared to that of standard
compounds. Assessment of structural similarity studies was
performed by means of physicochemical similarity between
the standard drugs and new analogs designed. The informa-
tion was used for prediction of biological activity of impor-
tant target compounds for novel drug discovery. Therefore,
we calculated a number of parameters for the synthesized
derivatives using Chem 3D Ultra (version 10) and compared
them to the values obtained for standard compounds.
The standard drugs used for assessment of similarity
with synthesized derivatives were taken from literature as:
sulindac, indomethacin, and aceclofenac. Various set of
parameters were used for calculations as given in Table 4.
The distance di of a particular target compound i can
presented as:
Analgesic activity of synthesized benzylidene-hydrazide
derivatives (3a–i) was carried out by using tail immersion
method. Swiss albino mice were divided into 11 groups
each containing six animals. The lower 5-cm portion of the
tail was marked. This part of the tail was immersed in a cup
of freshly filled water of exactly 55 ꢁC. Within a few
seconds, the mice reacted by withdrawing the tail. Tail
withdrawal from hot water was taken as end point. The
dependent variable in this test is the time taken by animal
to flick its tail. Normally, a mice withdraws its tail within 3
to 5 s and withdrawal time more than 5 s is considered as
positive response. A cut of period of 10–12 s was observed
to prevent damage to the tail. Then the animals were
treated with different doses. The animals were submitted to
the same procedure and reaction time was noted before and
after 30, 60, and 90 min. The average values of reaction
time after each time interval was calculated and compared
statistically between treated groups and control group
(Vogel, 2002). The relative latency of tail flick response for
synthesized benzylidene-hydrazide derivatives at 30, 60,
and 90 min are summarized in Table 3 and Fig. 3.
ꢀ
ꢁ
2
n
X
1 ꢁ Xi; j=Xi;standard
di2 ¼
;
ð2Þ
n
j¼1
where Xi.j is value of molecular parameters i for compound
j, Xi,standard is the value of the same molecular parameter
i for standard drug, and n is the total number of the con-
sidered molecular parameters.
The experimental protocol was approved by Institutional
AnimalsEthicalCommittee(IAEC)andanimalcarewasdone
as per the guidelines of Committee for the Purpose of Control
The similarity of the compounds can be calculated as:
123

2682
Med Chem Res (2014) 23:2676–2689
O2N
NO2
Cl
3a
3c
3b
3f
3d
OCH3
OCH3
OH
CH3
OH
3g
3e
3h
OCH3
OCH3
OCH3
3i
3a
3b
3c
3d
3e
3f
3g
3h
3i
Diclofenac
Fig. 2 Graphical representation of anti-inflammatory activity of synthesized benzylidene-hydrazide derivatives
Quantitative structure–activity relationship (QSAR)
%Age similarity ¼ ð1 ꢁ RÞ ꢂ 100;
ð3Þ
where R is quadratic mean also known as the root mean
square (RMS), and R can be calculated as:
The QSAR analysis was carried out on a series of nine
substituted benzylidene-hydrazide derivatives using multi-
linear regression. For this purpose, various physicochemi-
cal parameters were calculated and correlated with
biological activities, i.e., anti-inflammatory (Bhatia et al.,
2010) and analgesic activities to obtain QSAR models. The
physicochemical parameters were computed using Chem
3D Ultra after energy minimization to minimum RMS
p
R ¼ di2:
ð4Þ
Assessment of structural similarities of synthesized
derivatives with standard drugs showed that all the
derivatives have good % age similarity except 3i and 3d
was found to have excellent percentage of similarity
([90 %) with all the standard drugs (Table 5).
˚
gradient of 0.100 kcal/mole A by MOPAC software
123

Med Chem Res (2014) 23:2676–2689
2683
validates R2) and F (Fischer statistics), considering all the
parameters in the model significant only at 95 % confi-
dence level (p \ 0.05).
Table 3 Analgesic activity of synthesized benzylidene-hydrazide
derivatives
Groups
Reaction time (s)
Basic structure of synthesized substituted benzylidene-
(After
30 min)
(After
60 min)
(After 90 min)
3.87 0.10
hydrazide derivative
O
Control
2.13 0.03 3.01 0.04
C
HN
Diclofenac sodium
(10 mg/kg)
3.06 0.05 5.06 0.08 10.93 0.01
O
N
N
3a
3b
3c
3d
3e
3f
2.83 0.02 3.22 0.04 3.89 0.02
R
5.39 0.03 6.45 0.05* 6.83 0.01*
5.35 0.04 6.29 0.10* 6.88 0.06*
5.61 0.04 7.13 0.19* 8.91 0.21*
O
3.21 0.06 3.94 0.04
3.42 0.08 3.83 0.03
3.32 0.07 3.86 0.02
4.92 0.03
4.12 0.03
4.50 0.18
QSAR models for anti-inflammatory activity
3g
3h
3i
LogðP=100 ꢁ PÞ ¼ ꢁ0:04069ðlog PÞ þ 0:02529ðMWÞ
ꢁ 0:03073ðSASÞ þ 0:002801ðMRÞ
4.53 0.10 6.22 0.07* 9.09 0.03*
3.83 0.07 4.04 0.05 4.21 0.05
All values represent mean SEM (n = 6)
þ 23:04ðOvalityÞ ꢁ 27:41
N ¼ 9; R2 ¼ 0:642; Ra2dj ¼ 0:046; Press ¼ 0:002;
* P \ 0.05 when experimental groups were compared with control
Q2 ¼ 0:996; F ¼ 1:08; S ¼ 0:311
ð5Þ
package (Han et al., 2012). Out of all the physicochemical
parameters (Table 4), following five parameters were
selected for QSAR studies (Log P, connolly solvent
accessible surface area (SAS), molar refractivity (MR),
ovality and molecular weight (MW) which have highly
significant effect on biological activity.
Here and thereafter,
R2: coefficient of correlation, Ra2dj: coefficient of deter-
mination, F: Fischer statistics, N: number of test com-
pounds, Press: predictive error sum of squares, Q2: cross
validated R2, BA: Biological activity, S: Standard error of
estimation.
Biological activity data was converted to the logarithmic
values. For anti-inflammatory activity, percent inhibition
(P) was converted to log(P/100 - P) (Bhatia et al., 2010)
and for analgesic activity relative latency (%) for 90 min
was expressed as [(value of the drug - value of the con-
trol)/value of the control] 9 100 was taken as biological
activity for QSAR model development.
On exclusion of 3b and 3c, Eq. (4) was obtained with
better statistical validation:
LogðP=100 ꢁ PÞ ¼ 0:6998ðlog PÞ þ 0:06842ðMWÞ
ꢁ 0:02632ðSASÞ ꢁ 0:7417ðMRÞ
þ 133ðOvalityÞ ꢁ 153:6
N ¼ 7; R2 ¼ 0:960; Ra2dj ¼ 0:762; Press ¼ 0:0002;
Statistical analysis
Q2 ¼ 0:999; F ¼ 4:844; S ¼ 0:173
ð6Þ
The statistical significance of the models was assessed on
the basis of various parameters such as R2 (coefficient of
correlation), R2adj (coefficient of determination), Q2 (cross
The observed and predicted anti-inflammatory activity
of synthesized benzylidene-hydrazide derivatives is
summarized in Table 6, and plot of calculated and
Fig. 3 Graphical representation
of analgesic activity of
After 90 minutes
12
10
8
synthesized benzylidene-
hydrazide derivatives.
* p \ 0.05 when experimental
groups were compared with
control
*
*
*
*
6
4
2
0
Target compounds
123

2684
Med Chem Res (2014) 23:2676–2689
Table 4 Calculation of various physicochemical properties of synthesized benzylidene-hydrazide derivatives
a
2
SAS (A )
b
2
MSA (A )
c
3
SEV (A )
MRd
MTIe
WIf
BIg
MWh
Log P
˚
˚
˚
Compounds
Ovality
3a
548.748
595.471
584.965
572.5
285.398
315.85
308.19
300.2
232.695
264.148
254.099
247.109
257.202
236.958
250.143
261.027
308.473
282.584
284.198
264.743
1.5599
1.5865
1.5885
1.5764
1.5958
1.5685
1.5851
1.6015
1.6535
1.52626
1.52364
1.45068
84.22
10,029
14,118
13,458
10,910
12,526
11,104
11,492
13,637
18,064
10,917
10,017
8,482
1,351
1,980
1,878
1,544
1,727
1,544
1,544
1,927
2,565
1,517
1,424
1,278
384,784
307.31
2.0271
1.165
3b
–
714,244
678,468
477,303
577,890
477,303
477,303
695,793
1,144,506
506,021
474,843
465,387
352.308
352.308
341.755
337.337
323.31
3c
–
1.165
3d
89.03
90.69
85.92
89.27
92.38
103.61
99.250
96.211
86.31
2.585
3e
593.296
556.94
312.125
290.474
304.328
316.339
365.062
317.84
318.50
289.25
1.9007
1.6376
2.5142
1.5112
1.6479
2.2525
3.2216
3.6459
3f
3g
579.038
599.231
677.692
580.517
582.618
522.949
321.337
353.336
397.389
356.420
356.807
354.191
3h
3i
Std.1
Std.2
Std.3
a
Connolly solvent accessible surface area
Connolly molecular surface area
Connolly solvent excluded volume
Molar refractivity
b
c
d
e
f
Molecular topological index
Wiener index
g
h
Balaben index
Molecular weight, Std.1—sulindac, Std.2—indomethacin, Std.3—aceclofenac
Table 5 Assessment of structural similarity of synthesized benzyli-
dene-hydrazide derivatives with standard drugs
Table 6 Observed and predicted anti-inflammatory activity of syn-
thesized benzylidene-hydrazide derivatives
Compounds
Sulindac
(1 - R) * 100
Indomethacin
(1 - R) * 100
Aceclofenac
(1 - R) * 100
Compounds Anti-inflammatory activity
Activity % Observed
inhibition
(P)
Calculated Residuals
activity log(P/ activity
100 - P)
3a
3b
3c
3d
3e
3f
64.4
85
45.7
78.8
88.8
90.3
96.6
75.8
90.8
81.3
39.9
82.6
54.07
64.7
91.7
72.5
98.1
86.9
55
92.3
94.3
96.4
75.3
93.9
84.6
8.02
3a
3b
3c
3d
3e
3f
17.4
54.6
57.3
58.6
61.4
50.6
25.3
64.0
49.4
-0.67643
–
-0.59761 -0.07882
–
–
–
–
–
0.150897
0.201581
0.010424
-0.4702
0.249877
-0.01042
0.151309 -0.00041
3g
3h
3i
0.171784
-0.10816
-0.48822
0.029797
0.118586
0.018024
31.9
3g
3h
3i
0.342281 -0.0924
-0.02633 0.015911
observed anti-inflammatory activity is given in Fig. 4 for
Eq. (6).
Compounds are not included in QSAR model development
QSAR models for analgesic activity
Log BA ¼ 2:215ðlog PÞ þ 0:05131ðMWÞ ꢁ 0:2212ðSASÞ
ꢁ 0:1456ðMRÞ þ 304:6ðOvalityÞ ꢁ 361:8
Log BA ¼ 1:905ðlog PÞ þ 0:04127ðMWÞ ꢁ 0:0564ðSASÞ
ꢁ 0:3592ðMRÞ þ 127:8ððOvalityÞ ꢁ 154:2
N ¼ 7; R2 ¼ 0:933; Ra2dj ¼ 0:595; Press ¼ 0:002;
N ¼ 9; R2 ¼ 0:702; Ra2dj ¼ 0:205; Press ¼ 0:0026;
Q2 ¼ 0:999; F ¼ 2:76; S ¼ 0:209
ð8Þ
Q2 ¼ 0:999; F ¼ 1:41; S ¼ 0:679
ð7Þ
On exclusion of 3b and 3c, Eq. (7) was obtained with better
statistical validation:
The observed and predicted analgesic activity of synthesized
benzylidene-hydrazide derivatives is summarized in Table 7
and plot of calculated and observed biological activity is given
in Fig. 4 for Eq. (8).
123

Med Chem Res (2014) 23:2676–2689
2685
Fig. 4 Plot between the
calculated and observed
activities: a anti-inflammatory
activity and b analgesic activity
of synthesized benzylidene-
hydrazide derivatives
(b)
(a)
0.4
0.2
0
2.6
2.1
1.6
1.1
0.6
0.1
-0.4
-0.2
-0.4
-0.6
-0.8
y = 0.9612x - 0.0045
R² = 0.9603
y = 0.9327x + 0.086
R² = 0.9325
-0.8
-0.3
0.2
-0.4
0.1
0.6
1.1
1.6
2.1
Obs. activity
Obs. activity
The binding modes of active derivatives and standard
drugs with the active sites of enzyme are shown in Figs. 5
and 6, respectively.
Table 7 Observed and predicted analgesic activity of synthesized
benzylidene-hydrazide derivatives
Compounds
Analgesic activity
Observed
Calculated
Residual
Result and discussion
3a
3b
3c
3d
3e
3f
-0.28668
–
-0.04185
–
-0.24483
–
Chemistry
–
–
–
2.01867
1.433478
0.810229
1.21163
2.037601
0.943768
2.032896
1.357953
0.47634
1.493453
1.997297
0.905142
-0.01423
0.075526
0.333889
-0.28182
0.040305
0.038626
The structures of synthesized derivatives were supported
by means of chromatographic and spectroscopic methods.
1
Both analytical and spectral data (IR and H NMR) of all
3g
3h
3i
the synthesized derivatives were in full agreement with
the proposed structures. The structures of the synthesized
derivatives were proven by the spectroscopic method. The
structures assigned to (3a–i) were supported by IR spectra
showing absorption bands at 3,330–3,252 cm-1 due to
N–H stretching. Stretching vibrations due to phthalyl
C=O were observed at 1,783–1,748 cm-1. Carbonyl
stretch was observed at 1,658–1,629 cm-1. Stretching
vibrations due to C=N and C=C (aromatic) were observed
at 1,668–1,642 and 1,544–1,502 cm-1, respectively. Band
at 1,503–1,502 cm-1 was appeared due to asymmetric
N=O stretch. Bands at 1,238–1,235 cm-1 indicated C–O–
C stretch. Bands at 863–822 cm-1 indicated out of plane
aromatic stretch. The proton NMR of these derivatives
revealed the presence of singlet 9.59–9.76 ppm for –NH–
N=. The –N=CH– proton was observed as broad singlet
7.98–8.25 ppm. A 2 protons singlet at 4.42–4.68 ppm
appeared to methylene group. All the other aliphatic and
aromatic protons were observed within the expected
regions. This part concluded the synthesis of benzylidene-
hydrazide derivatives.
Compounds are not included in QSAR model development
Molecular docking studies
Docking studies of synthesized benzylidene-hydrazide
derivatives with (COX-2) enzyme
The synthesized benzylidene-hydrazide derivatives (3a–
i) were computationally designed and optimized for their
interaction with enzyme COX-2 (Pdb-1cx2). The result of
their interaction were compared with binding energies of
standard drugs (diclofenac sodium, sulindac and SC-S58,
where SC-S58 is a selective COX-2 inhibitor, co-crystallized
ligand belonging to the vicinal diaryl heterocyclic class and is
also used as reference ligand) and summarized in terms of
dock score in Table 8 by using following methodology:
•
•
Ligand preparation
Anti-inflammatory activity
Protein preparation and detecting cavities of protein
molecules
The main side effect associated with commonly available
NSAIDs is gastric ulceration, which is due to free car-
boxylic group in the parent drug. In the present study,
•
•
Executing a docking set up through docking wizard
panel
Poses of protein–ligand complex
123

2686
Med Chem Res (2014) 23:2676–2689
Table 8 Ligand–receptor
interaction of synthesized
benzylidene-hydrazide
derivatives compounds to target
COX-2 enzyme
Ligand
Docking score
(binding energy)
Distance
˚
(A)
Amino
acid
Group involved in
interaction with receptor
3d
-82.49
2.69
Lys 485
Carbonyl oxygen of five
membered ring of phthalic
anhydride
2.90
3.11
Glu 479
Lys 485
Nitrogen of –CONH group
3e
-91.87
Carbonyl oxygen of five
membered ring of phthalic
anhydride
3.34
2.80
3.07
Ser 477
Thr 476
Thr 476
Nitrogen of five membered
ring of phthalic anhydride
Carbonyl oxygen of –CONH
group
Carbonyl oxygen of five
membered ring of phthalic
anhydride
3.53
2.77
Ile 498
Oxygen of methoxy group
3h
-93.64
Lys 485
Carbonyl oxygen of five
membered ring of phthalic
anhydride
2.84
Thr 476
Carbonyl oxygen of five
membered ring of phthalic
anhydride
3.27
3.15
Thr 476
Ser 477
Carbonyl oxygen of –CONH
group
Nitrogen of five membered
ring of phthalic anhydride
3.28
2.84
3.06
2.57
3.15
2.69
Glu 479
Ser 496
Lys 492
Ser 477
Thr 476
Lys 492
Nitrogen of –CONH group
Oxygen of hydroxyl group
Oxygen of hydroxyl group
Oxygen of –SOCH3 group
Oxygen of –SOCH3 group
Sulindac
-77.424
Diclofenac
SC-S58
-58.259
-91.779
Carbonyl oxygen of
-COOH group
3.02
2.71
3.28
Ile 112
Arg 120
Ser 119
Nitrogen of –SO2NH2
Nitrogen of diazole ring
Nitrogen of diazole ring
derivatization of the carboxylate functionality by hydra-
zone moiety was carried out to get better anti-inflammatory
agents. The anti-inflammatory activity data in terms of
percent inhibition of benzylidene-hydrazide derivatives
(3a-i) is given in Table 2. The results obtained (Fig. 2)
indicated the effect of various substituents on the anti-
inflammatory activity of synthesized derivatives in given
order as 3h (p-hydroxy-m-methoxy phenyl) [ 3e (m-
methoxy phenyl) [ 3d (p-chloro phenyl) [ 3c (o-nitro
phenyl) [ 3b (p-nitro phenyl) [ 3f (p-hydroxy phe-
nyl) [ 3i (3,4,5-trimethoxy phenyl) [ 3g (tolyl) [ 3a
(phenyl).
3i possess good anti-inflammatory activity, while 3a and 3g
have not shown significant anti-inflammatory activity.
The detailed concern of activity and structure of the
synthesized derivatives resolved that:
•
Bulkier groups in test derivatives have directed to
increased lipophilicity which in turn enhanced the
permeability across the biological membrane to pro-
duce the desired action.
•
•
The electron withdrawing groups in test derivatives
have improved the biological activity.
The hydroxyl (–OH) and methoxy (–OCH3) groups
were found to be involved in terms of virtuous
hydrogen bond interaction with receptor.
The results suggested that the derivatives 3d, 3e, and 3h
have shown highest activity. The derivatives 3b, 3c, 3f, and
123

Med Chem Res (2014) 23:2676–2689
2687
Fig. 5 Binding modes of 3d,
3e, and 3h (3d, 3e, and 3h as
docking view and 3d0, 3e0, and
3h0 as interaction view) with
COX-2, where blue and red
lines represent hydrogen
bonding and favorable steric
interaction, respectively
Analgesic activity
Similarity studies
The analgesic activity data in terms of relative latency of
benzylidene-hydrazide derivatives (3a-i) is given in
Table 3. The reaction time (in s) for the derivatives 3d
(substituted with p-chloro phenyl) and 3h (substituted with
p-hydroxyl along with m-methoxy group) was found to be
8.91 – 0.21 and 9.09 – 0.03, respectively, after 90 min
which is comparable with reaction time of diclofenac
sodium (10.93 – 0.01) after 90 min. It indicated that these
derivatives possess potent analgesic activity. The deriva-
tives 3a, 3e, 3f, 3g, 3i were not found to be significantly
active.
Assessment of structural similarities of synthesized benzyl-
idene-hydrazide derivatives with anti-inflammatory and
analgesic drugs (sulindac, indomethacin, and aceclofenac)
indicated that all the derivatives have shown good percent-
age of similarity ranging from 54 to 98.1 % except 3i,
which has shown less percentage of similarity with all three
standard drugs ranging from (8.02 to 39.9 %) in Table 5.
The derivative 3d was found to have excellent percentage
of similarity (>90 %) with all standard drugs. The
structural similarity of all the derivatives with standard
drugs varies from one to another may be due to the more
123

2688
Med Chem Res (2014) 23:2676–2689
Fig. 6 Binding modes of
sulindac, diclofenac, and SC-
S58 (sulindac, diclofenac, and
SC-S58 as docking view and
sulindac0, diclofenac0, and SC-
S580 as interaction view) with
COX-2, where blue and red
lines represent hydrogen
bonding and favorable steric
interaction, respectively
difference between the values of physicochemical param-
eters calculated for the synthesized derivatives as well as
standard drugs. However, it is not always necessary that
good structural similarity of a compound lead to (Kubinyi,
1998) good therapeutic activity as derivative 3f has shown
an excellent percentage of similarity (75–98 %) but it
was found to be less active as analgesic agent.
structural features to the benzylidene-hydrazide derivatives
used in the study, various physicochemical properties were
calculated with the help of various chemoinformatic soft-
wares as listed in the experimental section. Log P is a
measure of lipophilicity, which is important for penetra-
tion, distribution as well as the interaction of drug with
receptor. Therefore, it is suggested that lipophillic property
has to be investigated for designing of potent anti-inflam-
matory and analgesic agents. Ovality is another physico-
chemical parameter which involves a combined effect of
size as well as surface area of the substituents present in
structure. It characterizes deformation of molecular elec-
tron distribution. Connolly solvent accessible surface area
(SAS) is indicative of surface properties, molar refractivity
(MR) is a measure of the volume occupied by an atom or
Quantitative structure activity relationship (QSAR)
analysis
The results suggested that percent inhibition (anti-inflam-
matory activity) and relative latency (analgesic activity)
were highly dependent on Log P, SAS, MR, ovality, and
MW. In order to quantify the contribution of these
123

Med Chem Res (2014) 23:2676–2689
2689
References
group and molecular weight (MW) parameter is an indic-
ative of the size of the molecule. All the developed models
have good coefficient of correlation (0.933–0.960), coeffi-
cient of determination (0.595–0.762) and cross validated R2
(0.999) with good Fischer statistics (2.76–4.84).
Ahluwalia VK, Aggarwal R (2000) Comprehensive practical organic
chemistry; preparation and qualitative analysis. Universities
Press (India), Hydrebad, pp 229–230
Akhter M, Husain A, Azad B, Ajmal M (2010) Aroylpropionic acid
based 2,5-disubstituted-1,3,4-oxadiazoles: synthesis and their
anti-inflammatory and analgesic activities. Eur J Med Chem
44:2372–2378
Molecular docking studies
Bhandari SV, Bothara KG, Raut MK, Patil AJ, Sarkate AP, Mokale VJ
(2008) Design, synthesis and evaluation of anti-inflammatory, anal-
gesic and ulcerogenicity studies of novel s-substituted phenacyl-1,3,4-
oxadiazole-2-thiol and schiff bases of diclofenac acid as nonulcero-
genic derivatives. Bioorg Med Chem 16:1822–1831
Bhatia NM, Mahadik KR, Bhatia MS (2010) Exploring the influence of
steric, electronic and lipophillic descriptors of 1,3-diarylpropenones on
their anti-inflammatory activity. DARU 18:230–236
Borne RF (2002) Non-steroidal anti-inflammatory drugs. In: Williams
DA, Lemke TL (eds) Foye’s principles of medicinal chemistry,
5th edn. Lippincott Williams & Wilkins, New York, pp 751–793
Botting RM (2006) Cyclooxygenase: past, present and future.
J Therm Biol 31:208–219
Han WS, Lee JK, Lee JS, Hahn HG, Yoon CN (2012) Study of
thiazoline derivatives for the design of optimal fungicidal
compounds using multiple linear regressions (MLR). Bull
Korean Chem Soc 33:1703–1706
In the present study, molecular docking studies were per-
formed using MVD software (Version 2012.5.0.0). The syn-
thesized derivatives were subjected to molecular docking
studies and possible interactions of target derivative with
cyclooxygenase-2 (COX-2) were studied. The derivative 3h
was found to be most potent anti-inflammatory compound
having high docking score (binding energy), i.e., -93.64 (high
as compared to all standard drugs) with hydrogen bond dis-
˚
tance 2.77–3.28 A (Table 8). The results revealed that
hydrogen bond distance is important in docking studies. The
˚
distance more than 3.2 A indicates weak hydrogen bonding
˚
between ligand and receptor, 2.6–3.2 A represent good
˚
hydrogen bonding and less than 2.5 A indicates strong
Hunashal RD (2011) Synthesis, anti-inflammatory and analgesic
activity of 2-[4-(substituted benzylideneamino)-5-(substituted
phenoxymethyl)-4H-1,2,4-triazol-3-yl thio] acetic acid deriva-
tives. Arab J Chem. doi:10.1016/j.arabjc.2011.01.003
Husain MSYK, Hasan SM, Alam MM (2005) 2-Arylidene-4-(4-
phenoxy-phenyl)but-3-en-4-olides: synthesis, reactions and bio-
logical activity. Eur J Med Chem 40:1394–1404
hydrogen bonding (Jeffrey 1997). Almost all the active
derivativeshaveshowngoodhydrogenbondingwithenzymes.
The hydroxyl and methoxy group in 3h act as donors played a
very important role for the formation of six hydrogen bonds
with COX-2 enzyme and emerged out as potential COX-2
inhibitor. These results could be used for the development of
novel, potent, and effective COX-2 inhibitors.
Jeffrey GA (1997) An introduction to hydrogen bonding. Oxford
University Press, New York
Kitchen DB, Decornez H, Furr JR, Bajorath J (2004) Docking and
scoring in virtual screening for drug discovery: methods and
applications. Nat Rev Drug Discov 3(11):935–949
Kubinyi H (1998) Similarity and dissimilarity: a medicinal chemist’s
view, in 3D QSAR in drug design. Kluwer, Dordrecht
Mehtap G, Semra U, Esra K (2009) Synthesis and analgesic and anti-
Conclusion
From the results of anti-inflammatory activity, it was con-
cluded that the derivatives 3d, 3e, and 3h have potent anti-
inflammatory activity. In analgesic activity, the derivatives
3d and 3h were found to be potent analgesic agents. The
derivatives 3b and 3c also have shown good analgesic
activity. The derivative 3d was found to have excellent per-
centage of similarity ([90 %) with all standard drugs. The
results of QSAR studies suggested that anti-inflammatory,
analgesic activities are highly dependent on physicochemical
parameters such as Log P, connolly solvent accessible sur-
face area (SAS), molar refractivity (MR), ovality, and
molecular weight (MW). The five parameters correlation
equations for anti-inflammatory and analgesic activities could
be used for the prediction of biological activities of unknown
and unavailable compounds of this class. The derived model
could be used in future for designing of more potent and
specific COX-2 inhibitors against inflammation and pain.
inflammatory
activities-6-substituted-3(2H)-pyridazinone-2-
acetyl-2-(p-substituted/nonsubstituted benzal) hydrazone deriva-
tives. Eur J Med Chem 44:3760–3764
Nikolova N, Jaworska J (2003) Approaches to measure chemical
similarity-a review. QSAR Comb Sci 22:1006–1026
Nunez S, Venhorst J, Kruse CG (2012) Target–drug interactions: first
principles and their application to drug discovery. Drug Discov
Today 17:10–22
Regina GL, Romano S, Valerio G, Antonio L, Ettore N, Olivia B,
Paola T, Enzo A (2008) Synthesis, structure–activity relationship
and molecular modeling studies of new indole inhibitors of
monoamine oxidase A and B. Bioorg Med Chem 22:9729–9740
Salimon J, Salih N, Yousif E, Hameed A, Kreem A (2010) Synthesis
and pharmacological evaluation of 9(10H)-acridone bearing
1,3,4-oxadiazole derivatives as antimicrobial agents. Arab J
Chem 3:205–210
Vogel HG (2002) Drug discovery and evaluation: pharmacological
assays. Springer, New York
Wang X, Tang Y, Xie Q, Qiu Z (2006) QSAR study of 4-phenylpi-
peridine derivatives as mu opioid agonists by neural network
method. Eur J Med Chem 41:226–232
Acknowledgments Authors are thankful to M. M. College of
Pharmacy, M. M. University, Mullana, Ambala for providing support
and facility to carry out research.
Winter A, Risley EA, Nuss GW (1962) Carrageenan-induced oedema
in hind paws of the rat as an assay for anti-inflammatory drugs.
Proc Soc Exp Biol 111:544–547
123