Design, characterization, computational studies, and pharmacological evaluation of substituted-N′-[(1E) substituted-phenylmethylidene] benzohydrazide analogs

Article abstract of DOI:10.1007/s00044-012-0270-0

A series of substituted-N′-[(1E)-substituted-phenylmethylidene] benzohydrazide analogs were synthesized and authenticated by TLC, UV-Visible, FTIR, and NMR spectroscopic techniques. The physicochemical similarity of the new analogs with standard drugs was assessed by calculating from a set of ten physicochemical properties using software programs. The information so obtained can be related to prediction of biological activity for important targets. All the target compounds 4a-n were evaluated for their antioxidant, anti-inflammatory, and antimicrobial activity using different in vitro models. The test compounds demonstrated good similarity values with respect to the standard drugs. The compounds 4c, 4d, and 4e have emerged as important lead compounds showing potential anti-inflammatory; and 4b, 4f, and 4c having antioxidant profile. While studying MIC against bacterial strains 4c, 4f, 4i, 4k, and 4m were most active among all the target compounds. All compounds were found to have very good antifungal activity. The compounds having nitro substitution at the arylidene moiety i.e., 4c and 4f showed the most potent antifungal as well as antibacterial activities. While studying total antioxidant activity, all target compounds were found to have good antioxidant activity.

Full text of DOI:10.1007/s00044-012-0270-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2755–2767
DOI 10.1007/s00044-012-0270-0
ORIGINAL RESEARCH
Design, characterization, computational studies,
and pharmacological evaluation of substituted-N0-[(1E)
substituted-phenylmethylidene]benzohydrazide analogs
•
Suman Bala Goldie Uppal Sunil Kamboj
•
•
•
Vipin Saini D. N. Prasad
Received: 11 April 2012 / Accepted: 29 September 2012 / Published online: 19 October 2012
Ó Springer Science+Business Media New York 2012
Abstract A series of substituted-N0-[(1E)-substituted-
phenylmethylidene]benzohydrazide analogs were synthe-
sized and authenticated by TLC, UV–Visible, FTIR, and
NMR spectroscopic techniques. The physicochemical
similarity of the new analogs with standard drugs was
assessed by calculating from a set of ten physicochemical
properties using software programs. The information so
obtained can be related to prediction of biological activity
for important targets. All the target compounds 4a–n were
evaluated for their antioxidant, anti-inflammatory, and
antimicrobial activity using different in vitro models. The
test compounds demonstrated good similarity values with
respect to the standard drugs. The compounds 4c, 4d, and 4e
have emerged as important lead compounds showing
potential anti-inflammatory; and 4b, 4f, and 4c having
antioxidant profile. While studying MIC against bacterial
strains 4c, 4f, 4i, 4k, and 4m were most active among all the
target compounds. All compounds were found to have very
good antifungal activity. The compounds having nitro
substitution at the arylidene moiety i.e., 4c and 4f showed
the most potent antifungal as well as antibacterial activities.
While studying total antioxidant activity, all target com-
pounds were found to have good antioxidant activity.
Keywords 4-Chlorobenzoic acid ꢀ
o-Benzoylbenzoic acid ꢀ Hydrazone ꢀ Antioxidant ꢀ
Anti-inflammatory activity ꢀ Antimicrobial activity
Introduction
Hydrazone is a versatile moiety that exhibits a wide variety
of biological activities. A hydrazone is a class of organic
compounds with the structure R1R2C=NNH2. They are
related to ketones and aldehydes by the replacement of the
oxygen with the –NNH2 functional group. Hydrazones are
formed usually by the action of hydrazine on ketones or
aldehydes. A hydrazone is an intermediate in the Wolff–
Kishner reduction (Corey, 1976; Corey and Enders, 1976).
Hydrazone compounds are not only intermediates but they
are also very effective organic compounds. There are many
clinically effective hydrazide–hydrazones such as nifuroxazide
(intestinal antiseptic), iproniazide, and isocarboxazide (anti-
tubercular) (Rollas and Kucukguzel, 2007). The most signifi-
cant reactivity of the hydrazones is their nucleophilicity. Thus,
reactions like Mannich reaction, coupling reaction, and halo-
genations have taken place readily at such carbon. It was
reported that hard nucleophiles attack preferentially nitrogen
atom, while soft ones attack at carbon atom (Atlan et al., 2000).
Non-steroidal anti-inflammatory drugs (NSAIDs) exerts
their effects by inhibition of prostaglandin production. The
pharmacological target of NSAIDs is cyclooxygenase (COX),
which catalyzes the first committed step in arachidonic acid
metabolism. It was reported that crystal structures of COX
enzymes with carboxylic acid containing NSAIDs shows that
the inhibitors are positioned in a similar fashion with their
S. Bala ꢀ G. Uppal ꢀ S. Kamboj ꢀ V. Saini (&)
M. M. College of Pharmacy, Maharishi Markandeshwar
University, Mullana, Ambala, Haryana 133207, India
e-mail: vipinsaini31@rediffmail.com
S. Bala
e-mail: sumankmj7@gmail.com
G. Uppal
e-mail: uppalgoldie@gmail.com
S. Kamboj
e-mail: sunilmmcp@gmail.com
D. N. Prasad
Shivalik College of Pharmacy, Nangal, Punjab, India
123

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Med Chem Res (2013) 22:2755–2767
carboxylates co-ordinates to Arg-120 and their aromatic func-
tionality projecting into the cyclooxygenase active site toward
Tyr-385. These studies suggest that the presence of hydrazone
moiety in some compounds have such a pharmacophoric effect
which leads to inhibition of COX (Eissa et al., 2009;Singhet al.,
1992; Elassar et al., 2007; Kurumbail et al., 1996). Mizushima
and Kobayashi, 1968 have reported protein denaturation as
in vitro screening model for determining anti-inflammatory
activity of compounds (Mizushima and Kobayashi, 1968).
Hydrazones are also found to have considerable anti-
oxidant activity and can be expressed as total antioxidant
activity and hydrogen peroxide scavenging activity. Oxi-
dative stress is a well-known mechanism that is responsible
for the development of vascular damage. Reactive oxygen
species (ROS) are normally produced throughout oxygen
metabolism and play a major role in physiological and
pathological cell redox signaling. Oxidative stress results
from an imbalance due to overproduction of ROS by the
different cellular sources. Antioxidants inhibit the genera-
tion of ROS and the subsequent formation of lipid perox-
idation products, thereby preventing both oxidative and
carbonyl stress. Most antioxidants prevent LDL oxidation
in cell-dependent and cell-free systems (Picot et al., 1994;
Sreenivasa et al., 2009; Melnyk et al., 2006; Belkheiri
et al., 2010; Sies, 1991; Stocker, 1999; Rao et al., 2005;
Lingnert et al., 1979; Chanda and Dave, 2009).
emergence of multi-drug resistant strains of Gram-positive
bacterial pathogens such as methicillin-resistant Staphylo-
coccus aureus and Staphylococcus epidermis and vanco-
mycin-resistant Enterococcus is a problem of ever-
increasing significance (Sriram et al., 2010; Mohamed
et al., 2007). There are two ways to counterbalance this
challenge: one is the controlled use of the currently mar-
keted antibiotics and the other is the development of novel
antimicrobial agents.
Material and method
Experimental
Melting points of newly synthesized compounds 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
compounds were confirmed by spectral data. The kmax was
calculated using double beam UV–Visible 1800 Shimadzu
spectrophotometer. The IR spectra were recorded on FTIR-
Shimadzu spectrometer using Nujol method. 1H NMR
spectra were recorded on BRUKER AVANCE II 400 NMR
spectrometer using DMSO as solvent and TMS as internal
standard, values were expressed in d ppm. Structural sim-
ilarity studies were performed using Chem 3D (2012)
(Nikolova and Jaworska 2003).
The dramatically rising prevalence of multi-drug resis-
tant microbial infections in the past few decades has
become a serious health care problem. In particular, the
Table 1 Physical data of synthesized compounds
Compounds
Molecular formula
% Age yield
Molecular weight
Solubility
Melting point (°C)
kmax
Rf value
2
C9H9ClO2
99.70
93.20
99.90
90.00
99.80
99.80
99.80
99.80
98.7
184.623
170.599
274.708
304.734
303.706
272.735
324.768
303.706
226.233
254.287
240.263
328.372
362.817
373.37
EtOH, MeOH
40.00
213.00
240.00
241.00
235.00
233.00
270.00
220.00
128.00
48.00
239
237
243
248
255
240
276
244
240
246
242
248
249
257
246
247
250
252
250
0.65
0.37
0.54
0.42
0.47
0.68
0.40
0.35
0.55
0.67
0.76
0.56
0.45
0.43
0.38
0.53
0.42
0.31
0.53
3
C7H7ClN2O
EtOH, MeOH
4a
4b
4c
4d
4e
4f
6
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
14H11ClN2O2
15H13ClN2O3
14H10ClN3O3
15H13ClN2O
18H13ClN2O2
14H10ClN3O3
14H10O3
DMSO, MeOH, CHCl3
DMSO, MeOH, CHCl3
DMSO, MeOH, CHCl3
DMSO, MeOH, CHCl3
DMSO, MeOH, CHCl3
DMSO, MeOH, CHCl3
EtOH
7
16H14O3
80.00
95.60
90.40
94.90
84.80
98.90
99.00
99.00
80.00
71.33
EtOH, MeOH
8
14H12N2O2
21H16N2O2
21H15ClN2O2
21H15N3O4
22H18N2O2
22H18N2O4
21H16N2O3
21H15N3O4
21H16N2O3
MeOH
299.00
256.00
278.00
259.00
266.00
270.00
265.00
270.00
273.00
4g
4h
4i
4j
4k
4l
4m
4n
DMSO, CHCl3
DMSO, CHCl3
DMSO, CHCl3
DMSO, CHCl3
DMSO, CHCl3
DMSO, CHCl3
DMSO, CHCl3
DMSO, CHCl3
342.399
374.398
344.372
373.37
344.372
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2757
EtO2C
CO2H
Reflux
EtOH
Conc. H2SO4
Cl
2
Cl
1
Reflux
NH2NH2 . H2O
H
N
CONHNH2
O
C
N
Ar
Reflux
Ar-CHO
Cl
3
Cl
4a-f
Scheme 1 Synthesis of 4-chloro-N’-[(1E)-substituted-phenylmethylidene]benzohydrazide analogs (4a–f)
General methods
in a round bottom flask. Contents were refluxed for 40 min,
cooled, and added chilled water. Excess of acid was neu-
tralized by addition of saturated sodium bicarbonate solu-
tion. The mixture was cooled and ester was filtered out in
good yield (Lingnert et al., 1979).
A series of 4-chlorobenzoic acid and o-benzoylbenzoic acid
hydrazone analogs were synthesized as per the Schemes 1
and 2. Fischer esterification of substituted benzoic acid with
methanol in the presence of concentrated sulfuric acid yiel-
ded the corresponding ester which on further reaction with
hydrazine hydrate in ethanol afforded the corresponding
4-chlorobenzoic acid hydrazide. The hydrazide so formed
was further reacted with aromatic aldehydes in the presence
of few drops of glacial acetic acid to yield the 4-chloro-N0-
[(1E)-substituted-phenylmethylidene]benzohydrazide (4a–f),
o-benzoyl-N0-[(1E)-substituted-phenylmethylidene]ben-
zohydrazide(4g–n). The synthesized substituted-N0-[(1E)-
substituted phenyl methylidene]benzohydrazide were
characterized onthe basis ofthe spectraland analyticalstudies
(Rajput and Rajput, 2009; Sunil et al., 2010).
General procedure for the synthesis of
4-chlorobenzohydrazide, (3)
Hydrazide was prepared by refluxing hydrazine hydrate
(0.05 mol) with ethyl-4-chlorobenzoate (0.05 mol) in etha-
nol (5.0 ml) at 80 °C for 4 h. Crystals were separated out by
filtration on cooling and can be recrystallized from ethanol.
General procedure for the synthesis of
4-chloro-N’-[(1E)-substituted-phenylmethyl-
idene]benzohydrazides, (4a–f)
General procedure for synthesis of ethyl-4-chlorobenzoate,
(2)
4-Chloro benzohydrazide 3 (0.0025 mol) was dissolved in
methanol, added (0.0025 mol) substituted aromatic alde-
hyde and three drops of glacial acetic acid, mixture was
refluxed for 30 min. The crude product was recrystallized
by a mixture of cold water and alcohol.
4-Chlorobenzoic acid (0.05 mol) was dissolved in ethanol
(10 ml) along with 4–5 drops of concentrated sulfuric acid
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O
O
CO2H
AlCl3
+
7
O
5
6
EtOH
Conc. H2SO4
O
O
CO2Et
CONHNH2
NH2NH2 . H2O
8
9
ArCHO
Reflux
O
C
O
N
Ar
N
H
4g-n
Scheme 2 Synthesis of o-benzoyl-N’-[(1E)-substituted-phenylmethylidene]benzohydrazide analogs (4g–n)
General procedure for the synthesis of o-benzoylbenzoic
acid, (6)
General procedure for synthesis of ethyl-
o-benzoylbenzoate, (8)
Anhydrous aluminum chloride (0.15 M) was suspended in
benzene 5 (0.51 M) in round bottom flask with a provision
for reflux condenser. To the mixture added phthalic anhy-
dride 6 (0.07 M) in small proportions with constant shaking.
The reaction mixture was slightly warmed after addition of
first installment of phthalic anhydride and cooled on ice
bath when became vigorous. After the addition of phthalic
anhydride the reaction mixture was refluxed for 3 h under
anhydrous conditions (CaCl2 tube was attached to the end of
the condenser) until the evolution of hydrochloride gas
ceased. Contents of flask were cooled and to this ice cold
2.5 % hydrochloric acid was added. Reaction mixture was
subjected to steam distillation to remove unreacted ben-
zene. Reaction mixture was filtered out while hot, cooled,
and crude o-benzoylbenzoic acid was separated out. Crude
product was dissolved in anhydrous sodium carbonate
solution, filtered and acidified with hydrochloric acid to
separate out the o-benzoylbenzoic acid in pure form.
4-Benzoylbenzoic acid (0.05 mol) was dissolved in ethanol
(10 ml) along with 4–5 drops of concentrated sulfuric acid
in a round bottom flask. Contents were refluxed for 40 min.
Cooled and chilled water was added. Excess of acid was
neutralized by addition of saturated sodium bicarbonate
solution. The mixture was cooled, and ester was filtered out
in good yield.
General procedure for the synthesis of
o-benzoylbenzohydrazide, (9)
Hydrazide was prepared by refluxing hydrazine hydrate
(0.05 M) with ethyl-o-benzoylbenzoate 7 (0.05 M) in
ethanol (5.0 ml) at 80 °C for 4 h. The reaction was mon-
itored by TLC. The white colored crystals were separated
out by filtration on cooling and can be recrystallized from
ethanol.
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2759
General procedures for the synthesis of o-benzoyl-
N0-[(1E)-substituted-phenylmethylidene]benzohydrazides,
(4g–n)
4-Chloro-N0-[(1E)-2-hydroxy-naphthalen-1-ylmethylene)-
hydrazide, (4e) IR: 3290.00, 3200.00, 2978.09, 1606.70,
1650.00, 1554.44, 937.40, 611.00 cm-1; 1H NMR (DMSO):
d 8.0 (s, NH), 7.89 (d, 2H of Ph), 7.86 (d, 2H of Ph), 7.63 (m,
o-Benzoylbenzohydrazide 8 (0.0025 M) was dissolved in 1Hof Ph), 7.45 (d, 2H of Ph), 7.30 (m, 1H of Ph), 7.21 (m, 1H
methanol. In this substituted aromatic aldehyde (0.0025 M) of Ph), 6.8 (s, CH), 5.0 (s, OH); MS: m/z 324.05, 325.05
with three drops of glacial acetic acid was added. Mixture (M ? 1), 326.05 (M ? 2).
was refluxed for 4–5 h. The reaction was monitored by
TLC. The white-colored crude product was filtered out and 4-Chloro-N0-[(1E)-3-nitro-benzylidene)-hydrazide, (4f) IR:
recrystallized by a mixture of cold water and ethanol.
3201.63, 2953.00, 1651.00, 1665.00, 1529.55, 1558.00,
921.97, 663.00 cm-1;1H NMR (DMSO): d 8.4 (d, 2H of
Ph), 8.2 (d, 2H of Ph), 8.0 (s, of NH), 7.89 (d, 2H of Ph),
7.60 (m, 1H of Ph), 7.45 (d, 2H of Ph), 7.0 (s, CH); MS: m/
z 303.04, 304.04 (M ? 1), 305.04 (M ? 2).
Spectral data
Ethyl-4-chlorobenzoate IR: 3034.03, 1726.29, 1421.5,
1
837.11, 756.10 cm-1; H NMR (DMSO): 7.91 (d, 2H of
o-Benzoylbenzoic acid, (7) IR: 3360.00, 2930.00,
1
1680.00, 1530.00, 930.00; H NMR (DMSO): d 11 (s, 1H,
Ph), 7.38 (d, 2H of Ph), 4.29 (q, 2H of CH2), 1.30 (t, 3H of
CH3).
OH), 8.43 (d, 1H, Ar–H), 8.18 (d, 1H, Ar–H), 7.95 (d, 1H,
Ar–H), 7.80 (t, 1H, Ar–H), 7.63 (d, 2H, Ar–H), 7.61 (t, 1H,
Ar–H), 7.51 (t, 2H, Ar–H).
4-Chlorobenzohydrazide IR: 3277.06, 3159.40, 1456.21,
866.04, 738.74 cm-1; 1H NMR (DMSO): 9.9 (s, NH), 7.8-
7.9 (d, 2H of Ph), 7.4-7.6 (d, 2H of Ph), 4.6 (s, of NH2).
Ethyl-o-benzoylbenzoate, (8) IR: 3035.96, 1735.93,
1
1570.00, 1271.00, 947.05; H NMR (DMSO): d 8.33 (d,
1H, Ar–H), 8.02 (d, 1H, Ar–H), 7.82 (t, 1H, Ar–H), 7.70 (t,
1H, Ar–H), 7.64 (d, 2H, Ar–H), 7.62 (t, 1H, Ar–H), 7.52 (t,
2H, Ar–H), 4.31 (q, 2H, CH2), 1.30 (t, 3H, CH3).
4-Chloro-N0-[(1E)-2-hydroxyphenylmethylidene]benzohyd-
razides, (4a) IR: 3398.57, 3232.70, 3072.60, 1680.70,
1638.00, 1600.92, 860.25, 636.51 cm-1
;
1H NMR
(DMSO): d 7.9 (s, of NH), 7.8 (d, 2H of Ph), 7.7 (d, 2H of
Ph), 7.5 (d, 2H of Ph), 7.37 (d, 2H of Ph), 7.3 (s, CH), MS:
m/z 274.05, 275.05 (M ? 1), 276.05 (M ? 2).
o-Benzoylbenzohydrazide, (9) IR: 3487.00, 3053.32,
1541.00, 1843.00, 790.81; 1H NMR (DMSO): d 8.40 (d, 1H,
Ar–H), 7.86 (t, 1H, Ar–H), 7.79 (t, 1H, Ar–H), 7.77 (d, 1H, Ar–
H), 7.65 (d, 2H, Ar–H), 7.64 (t, 1H, Ar–H), 7.53 (t, 2H, Ar–H),
6.85 (s, 1H, of NH), 3.34 (s, 1H, of NH2), 1.78 (s, 1H, of NH2).
4-Chloro-N0-[(1E)-4-hydroxy-3-methoxyphenylmethylidene]
benzohydrazides, (4b) IR: 3387.80, 3250.05, 3014.74,
2912.00, 1409.00, 1620.21, 1670.00, 1577.77, 1276.00,
854.57, 611.00 cm-1; 1H NMR (DMSO): d 8.9 (s, of CH),
7.9 (s, of NH), 7.5 (d, 2H of Ph), 7.4 (s, OH), 7.3 (d, 2H of
Ph), 7.0 (d, 2H of Ph), 3.42 (s, of OCH3); MS: m/z 274.05,
275.05 (M ? 1), 276.05 (M ? 2).
o-Benzoyl-N’-[(1E)-phenylmethylidene]benzohydrazide,
(4g) IR: 3331.00, 3072.00, 1635.64, 1630.00, 1581.63,
817.82;1H NMR (DMSO): d 12.50 (s, 1H, –CONH), 8.40
(d, 1H, Ar–H), 7.93 (d, 2H, Ar–H), 7.86 (t, 1H, Ar–H), 7.79
(m, 1H, Ar–H), 7.78 (m, 1H, Ar–H), 7.63 (d, 2H, Ar–H),
7.61 (t, 1H, Ar–H), 7.58 (m, 3H, Ar–H), 7.52 (m, 2H, Ar–
H), 7.37 (s, 1H, CH), MS: m/z 328.12, 329.12 (M ? 1),
330.13 (M ? 2).
4-Chloro-N0-[(1E)-4-nitrophenylmethylidene]benzohydrazides,
(4c) IR: 3381.21, 3059.10, 1649.14, 1620.00, 1548.84,
1578.00, 821.68, 707.88 cm-1; 1H NMR (DMSO): d 8.1 (s,
of NH), 7.98 (d, 2H of Ph), 7.96 (d, 2H of Ph), 7.94 (d, 2H
of Ph), 7.7 (d, 2H of Ph), 7.1 (s, CH); MS: m/z 303.04,
304.04 (M ? 1), 305.04 (M ? 2).
o-Benzoyl-N’-[(1E)-4-chlorophenylmethylidene]benzohyd-
razide, (4h) IR: 3282.84, 3035.96, 1664.57, 1644.00,
1521.84, 885.33, 671.23; 1H NMR (DMSO): d 11.5 (s, 1H,
–CONH), 8.40 (d, 1H, Ar–H), 7.86 (t, 1H, Ar–H), 7.82 (d, 2H,
Ar–H), 7.80 (t, 1H, Ar–H), 7.64 (m, 2H, Ar–H), 7.61 (m, 1H,
Ar–H), 7.53 (t, 2H, Ar–H), 7.47 (d, 2H, Ar–H), 7.37(d, 1H,
Ar–H); MS: m/z 362.08, 363.09 (M ? 1), 364.08 (M ? 2).
4-Chloro-N0-[(1E)-4-methylphenylmethylidene]benzohyd-
razides, (4d) IR: 3402.43, 3107.00, 2974.00, 1629.85,
1627.00, 1563.00, 1344.00, 821.68, 726.00 cm-1; 1H NMR
(DMSO): d 7.8 (s, of NH), 7.7 (d, 2H of Ph), 7.6 (d, 2H of
Ph), 7.45 (d, 2H of Ph), 7.4 (d, 2H of Ph), 7.3 (s, CH), 2.4
(s, of CH3); MS: m/z 272.07, 273.07 (M ? 1), 274.07
(M ? 2).
o-Benzoyl-N’-[(1E)-4-nitrophenylmethylidene]benzohydrazide,
(4i) IR: 3429.00, 3072.00, 1655.00, 1633.71, 1556.50,
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1
1519.00, 1049.00; H NMR (DMSO): d 11.5 (s, 1H, –
CONH), 8.48 (d, 1H, Ar–H), 8.27 (d, 2H, Ar–H), 7.97 (d, 2H,
Ar–H), 7.86 (t, 1H, Ar–H), 7.79 (t, 1H, Ar–H), 7.76 (d, 1H,
Ar–H), 7.65 (m, 2H, Ar–H), 7.63 (m, 1H, Ar–H), 7.51 (t, 2H,
Ar–H), 7.30 (s, 1H, CH); MS: m/z 373.11, 374.11 (M ? 1),
375.11 (M ? 2).
(t, 1H, Ar–H), 6.93 (m, 1H, Ar–H), 6.91 (m, 1H, Ar–H), 5.18 (s,
1H, OH); MS: m/z 344.12, 345.12 (M ? 1), 346.12 (M ? 2).
Similarity studies
Assessments of structural similarity of test compounds
4a–n were compared to that of standard compounds.
Assessment of structural similarity studies was performed
by means of physico-chemical and steric similarity
between the standard drugs and new analogs designed.
These studies helped us to determine good spatial com-
patibility and effective binding to enzymatic site as lock
and key model. The information was used for prediction of
biological activity of important target compounds. There-
fore, we calculated a number of parameters for the test
compounds using Chem 3D and compared them to the
values obtained for standard compounds.
o-Benzoyl-N’-[(1E)-4-methylphenylmethylidene]benzohyd-
razide, (4j) IR: 3311.00, 3018.00, 2792.93, 1676.00,
1
1668.00, 1560.00, 898.93; H NMR (DMSO): d 11.80 (s,
1H, –CONH), 8.38 (d, 1H, Ar–H), 7.91 (d, 2H, Ar–H), 7.86
(t, 1H, Ar–H), 7.79 (m, 1H, Ar–H), 7.77 (m, 1H, Ar–H),
7.63 (m, 2H, Ar–H), 7.61 (m, 1H, Ar–H), 7.51 (t, 2H, Ar–
H), 7.40 (d, 2H, Ar–H), 7.37 (s, 1H, CH), 2.41 (s, 3H,
CH3); MS: m/z 342.14, 343.14 (M ? 1), 344.14 (M ? 2).
o-Benzoyl-N’-[(1E)-4-hydroxy-3-methoxyphenylmethylide
-ne]benzohydrazide, (4k) IR: 3427.00, 3327.00, 3070.00,
1
2912.00, 1637.00, 1558.48, 1535.00, 1296.00, 916.00; H
The standard drug used for assessment of similarity with
target compounds were taken from the literature as:
nifuroxazide (intestinal antiseptic), tosufloxacin tosylate,
pyridoxal salicyloyl hydrazone, and cefixime (anti-micro-
bial agent). Various set of parameters were used for cal-
culations as given in Table 2. The distance di of a
particular target compound i can be presented as
NMR (DMSO): d 11.60 (s, 1H, –CONH), 8.37 (d, 1H, Ar–
H), 7.93 (s, 1H, CH), 7.84 (m, 1H, Ar–H), 7.81 (m, 1H, Ar–
H), 7.76 (d, 1H, Ar–H), 7.64 (m, 2H, Ar–H), 7.62 (m, 1H,
Ar–H), 7.53 (t, 2H, Ar–H), 7.37 (m, 1H, Ar–H), 7.34 (m,
1H, Ar–H), 6.88 (d, 1H, Ar–H), 5.79 (s, 1H, OH), 3.83 (s,
3H, CH3); MS: m/z 344.13, 345.13 (M ? 1), 346.13
(M ? 2).
ꢀ
ꢁ
2
n
X
1 ꢁ Xi ; j=Xi;standard
di2 ¼
n
j¼1
o-Benzoyl-N’-[(1E)-4-hydroxyphenylmethylidene]benzohy
-drazide, (4l) IR: 3280.00, 3179.00, 3051.00, 1629.85,
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; n is the total number of the considered
molecular parameters.
1
1529.00, 1485.00, 810.00; H NMR (DMSO): d 12.2 (s,
1H, –CONH), 8.47 (d, 1H, Ar–H), 7.93 (s, 1H, CH), 7.86
(t, 1H, Ar–H), 7.78 (m, 1H, Ar–H), 7.75 (m, 1H, Ar–H),
7.67 (m, 2H, Ar–H), 7.65 (m, 2H, -CH), 7.62 (m, 1H, Ar–
H), 7.54 (t, 2H, Ar–H), 6.85 (d, 2H, Ar–H), 4.98 (s, 1H,
OH); MS: m/z 344.12, 345.12 (M ? 1), 346.12 (M ? 2).
The similarity of the compounds can be calculated as:
% Age similarity ¼ ð1 ꢁ RÞ ꢂ 100
where R is quadratic mean also known as the root mean
o-Benzoyl-N’-[(1E)-2-nitrophenylmethylidene]benzohyd-
razide, (4m) IR: 3361.00, 2984.00, 1660.70, 1654.00,
square and R can be calculated as:
p
R ¼ di2
1
1465.26, 1359.82, 910.40; H NMR (DMSO): d 12.4 (s,
1H, –CONH), 8.4 (d, 1H, Ar–H), 8.08 (d, 1H, Ar–H), 7.95
(d, 1H, Ar–H), 7.9 (s, 1H, CH), 7.86 (t, 1H, Ar–H), 7.79
(m, 1H, Ar–H), 7.77 (m, 1H, Ar–H), 7.69 (t, 1H, Ar–H),
7.64 (m, 2H, Ar–H), 7.62 (m, 1H, Ar–H), 7.59 (m, 1H, Ar–
H), 7.51 (t, 2H, Ar–H); MS: m/z 373.11, 374.11 (M ? 1),
375.11 (M ? 2).
Assessment of structural similarities of target compounds
with standard drugs showed that compound 4a, 4b, 4c, 4d,
and 4f has good % age similarity except 4e (Table 3).
Quantitative structure–activity relationship-QSAR
The term quantitative structure–activity relationship (QSAR)
implies the empirical relationships that use molecular param-
eters to quantify a pharmacological or chemical property for a
set of molecules.
o-Benzoyl-N’-[(1E)-2-hydroxyphenylmethylidene]benzohy
-drazide, (4n) IR: 3300.00, 3267.00, 3010.00, 1649.85,
1
1539.00, 1490.00, 860; H NMR (DMSO): d 10.7 (s, 1H,
–CONH), 8.42 (d, 1H, Ar–H), 8.34 (s, 1H, CH), 7.86 (t, 1H,
Ar–H), 7.79 (m, 1H, Ar–H), 7.75 (m, 1H, Ar–H), 7.68 (m,
2H, Ar–H), 7.65 (m, 1H, Ar–H), 7.53 (t, 3H, Ar–H), 7.32
The logarithm of the octanol/water partition coefficient
has been widely used as the sole physicochemical property
123

Med Chem Res (2013) 22:2755–2767
2761
Table 2 Calculation of various molecular properties of target compounds
3
SAS (A )
3
MS (A )
3
CSEV (A )
˚
˚
˚
Compounds
Ovality
MR (cm3/mol)
MTI
5,893
WI
826
BI
207,525
MW
Log P
4a
486.988
535.536
529.718
512.973
529.190
537.939
586.111
609.866
637.035
617.156
640.929
594.132
626.359
591.876
487.133
570.267
537.301
580.224
249.021
276.697
275.376
263.596
286.118
276.885
313.857
328.62
198.182
222.644
225.459
210.681
241.515
221.998
266.645
280.869
296.904
283.424
295.399
270.606
288.675
270.660
192.36
1.5148
1.5591
1.5372
1.5394
1.5255
1.5616
1.5666
1.5844
1.6019
1.5950
1.6151
1.5752
1.5995
1.5711
1.5242
1.5303
1.5699
1.46146
7.6716
8.2885
8.3074
7.9823
9.3596
8.3074
10.037
10.529
10.826
10.501
10.808
10.190
10.826
10.190
7.183
274.708
304.734
303.706
272.735
324.768
303.707
328.372
362.817
373.370
342.399
374.398
344.372
373.370
344.372
275.222
404.350
301.303
437.46
3.532
3.406
3.133
4.409
4.530
3.133
4.573
5.132
3.259
5.061
4.058
4.184
3.259
4.184
–
4b
7,684
8,014
1,104
1,142
850
337,123
348,452
213,402
377,501
337,752
552,411
674,005
980,727
674,005
957,097
674,005
934,720
660,855
272,224
748,946
399,912
1,046,550
4c
4d
6,277
4e
9,625
1,324
1,106
1,654
1,817
2,359
1,817
2,300
1,871
2,245
1,833
981
4f
7,783
4g
12,737
13,772
17,462
14,426
16,192
13,990
16,700
13,774
6,809
4h
4i
344.787
332.831
346.431
518.705
337.859
317.924
245.631
318.963
278.198
331.272
4j
4k
4l
4m
4n
Std. 1*
Std. 2*
Std. 3*
Std. 4*
282.947
221.39
9.792
13,519
8,433
2,039
1,195
2,353
2.419
1.801
–
8.2029
10.691
320.889
15,459
SAS Connolly solvent accessible surface area, MS Connolly molecular surface area, CSEV Connolly solvent excluded volume, MR molar
refractivity MTI molecular topological index, WI Wiener index, BI Balaben index, MW molecular weight
to define various types of biological activity, due to its
relationship with membrane permeability. In order to
identify substituent effect on antimicrobial activity, quan-
titative structure–activity relationship (QSAR) studies of
title compounds were performed. Biological activity data
i.e., minimum inhibitory concentration (MIC) was con-
verted to the logarithmic value (pMIC) in lg/ml as listed in
Tables 4 and 5. The compounds were analyzed by physi-
cochemical-based QSAR (Hansch) approach using differ-
ent physicochemical parameters as independent and pMIC
values as dependent variables. These QSAR descriptors of
substituted hydrazides were calculated using the Sigmastat
package 3.5. The 14 test compounds are listed in Tables 4
and 5 along with their pMIC log P and ovality values
considered for QSAR development.
obtained by relating pMICsa, pMICec, and pMICca to log P
values by polynomial regression analysis (Table 4). Log P is a
measure of hydrophobicity, which is important for the pene-
tration, distribution as well as for the interaction of the drug
with receptors. Therefore, it can be suggested that lipophilic
Table 3 % Age similarities of target compounds with standard drugs
Compounds Nifuroxazide Tosufloxacin Pyridoxal
(1 - R) 100 tosylate salicyloyl
Cefexime
(1 - R)
100
(1 - R) 100 hydrazone
(1 - R) 100
4a
4b
4c
4d
4e
4f
85.79
61.36
57.67
96.86
42.49
60.63
87.46
81.96
78.99
83.88
78.62
85.10
79.68
85.25
90.86
89.05
92.94
77.23
74.70
92.47
71.23
62.05
85.45
62.05
74.40
75.65
85.92
75.77
72.58
71.92
77.07
55.63
52.55
76.56
48.84
37.26
68.53
38.09
54.27
54.81
69.16
54.97
82.24
87.32
86.91
83.52
88.48
87.38
92.02
96.06
98.38
94.88
98.68
93.72
97.77
93.57
The basic structure of test compounds is:
R'
O
Ar
4g
4h
4i
HN
N
4j
A number of QSAR equations were developed by taking
4k
4l
into consideration of various parameters and relating them to
MICvaluesofthetestcompoundsagainstvariousbacterialand
fungal strains. Among Staphylococcus aureus, Escherichia
coli and Candida albicans, the best QSAR equations were
4m
4n
123

2762
Med Chem Res (2013) 22:2755–2767
Table 4 Comparison of logarithmic values (pMIC) of minimum inhibitory concentration (MIC) to log P
Compounds
R0
Ar
pMICsa
PMICec
pMICca
Log P
4a
4b
4c
4d
4e
4f
4-Cl
2-OHC6H4
1.255
1.255
1.301
1.477
1.477
1.301
1.477
1.531
1.531
1.531
1.531
1.531
1.255
1.301
1.301
1.531
1.531
2.264
1.924
1.301
1.924
1.924
1.397
2.225
1.698
1.903
1.301
1.531
1.740
1.924
1.301
1.301
1.531
1.531
2.000
2.301
1.924
1.698
1.301
2.000
1.397
2.000
3.532
3.406
3.133
4.409
4.53
4-Cl
3-OCH3, 4-OHC6H3
4-NO2ꢀC6H4
4-CH3ꢀC6H4
2-OHC10H6
3-NO2C6H4
–C6H5
4-Cl
4-Cl
4-Cl
4-Cl
3.133
4.573
5.132
3.259
5.061
4.058
4.184
3.259
4.184
4g
4h
4i
2-COC6H5
2-COC6H5
2-COC6H5
2-COC6H5
2-COC6H5
2-COC6H5
2-COC6H5
2-COC6H5
4-ClꢀC6H4
4-NO2ꢀC6H4
4-CH3ꢀC6H4
3-OCH3, 4-OHC6H3
4-OHC6H4
4j
4k
4l
4m
4n
2-NO2ꢀC6H4
2-OHC6H4
Table 5 Comparison of logarithmic values (pMIC) of minimum inhibitory concentration (MIC) to ovality (X)
Compounds
R0
Ar
pMICse
pMICpa
pMICan
Ovality (X)
4a
4b
4c
4d
4e
4f
4-Cl
2-OHC6H4
1.53
2.00
0.00
2.30
2.00
1.69
1.69
1.69
1.47
2.30
2.30
1.92
1.47
1.53
1.09
1.40
1.30
0.81
1.40
1.40
0.81
1.09
1.30
1.40
1.69
1.40
1.40
1.09
1.00
1.69
0.81
0.81
2.00
0.81
0.81
1.30
1.40
0.81
1.40
1.92
1.09
1.53
1.5148
1.5591
1.5372
1.5394
1.5255
1.5616
1.5666
1.5844
1.6019
1.5950
1.6151
1.5752
1.5995
1.5711
4-Cl
3-OCH3, 4-OHC6H3
4-NO2ꢀC6H4
4-CH3ꢀC6H4
2-OHC10H6
3-NO2C6H4
–C6H5
4-Cl
4-Cl
4-Cl
4-Cl
4g
4h
4i
2-COC6H5
2-COC6H5
2-COC6H5
2-COC6H5
2-COC6H5
2-COC6H5
2-COC6H5
2-COC6H5
4-ClꢀC6H4
4-NO2ꢀC6H4
4-CH3ꢀC6H4
3-OCH3, 4-OHC6H3
4-OHC6H4
4j
4k
4l
4m
4n
2-NO2ꢀC6H4
2-OHC6H4
2
properties have to be checked for designing of potent anti-
bacterial agents as these are vital factors for its activity.
QSAR model for antibacterial activity against Staphy-
lococcus aureus
pMICsa ¼ ꢁ6:030 ðLog PÞ þ 21:78 Log P ꢁ 14:58 ðiiÞ
n = 13, r = 0.826, q2 = 0.683, s = 0.120, s2 = 0.014,
F = 24.372, here and thereafter, q2 cross validated r2
obtained by leave one out (LOO) technique.
2
pMICsa ¼ ꢁ19:98 ðLog PÞ þ 59:53 Log P ꢁ 40:53
ðiÞ
This equation showed an improved relationship between
MIC values and log P e.g., value of r was 0.826 (vs 0.682
in Eq. i) and s value was 0.120 (vs 0.122 in Eq. i).
The negative coefficient of Log P in the Eq. (i) indicates
that there is a negative correlation between the antibacterial
activity of substituted hydrazides and Log P. This is evi-
denced by the antibacterial activity data of substituted
hydrazides and their Log P values.
n = 14, r = 0.682, r2 = 0.466, s = 0.122, s2 = 0.015,
F = 10.064
Here and thereafter, r = coefficient of correlation,
r2 = coefficient of determination, s2 = sum of squares of
deviations of sample values (variance), s = standard devia-
tion i.e., square root of variance, F = Fischer statistics,
n = number of test compounds
QSAR model for antibacterial activity against Esche-
richia coli
On exclusion of compound GS9, Eq. (ii) was obtained
with better statistical validation:
123

Med Chem Res (2013) 22:2755–2767
2763
2
2
pMICec ¼ ꢁ1:126 ðLog PÞ þ 5:730 Log P ꢁ 2:389 ðiiiÞ
pMICca ¼ 0:191ðXÞ ꢁ 0:550 ðXÞ þ 1:197
ðixÞ
n = 14, r = 0.865, r2 = 0.758, s = 0.121, s2 = 0.014,
F = 31.918
On exclusion of compound GS4, Eq. (iv) was obtained
n = 14, r = 0.634, r2 = 0.403, s = 0.262, s2 = 0.068,
F = 0.0208
On exclusion of compound GS10, Eq. (ix) was obtained
with better statistical validation:
with better statistical validation:
2
2
pMICec ¼ ꢁ0:149 ðLog PÞ þ 1:626 Log P ꢁ 0:846
ðivÞ
pMICca ¼ 0:210ðXÞ ꢁ 0:613 ðXÞ þ 1:146
ðxÞ
n = 13, r = 0.894, q2 = 0.801, s = 0.118, s2 = 0.013,
F = 44.228
n = 13, r = 0.575, q2 = 0.758, s = 0.259, s2 = 0.067,
F = 0.00617
This equation showed an improved relationship between
MIC values and log P e.g., value of r was 0.894 (vs 0.865
in Eq. iii) and s value was 0.118 (vs 0.121 in Eq. iii).
QSAR model for antibacterial activity against Candida
albicans
This equation showed an improved relationship between
MIC values and log P e.g., value of r was 0.758 (vs 0.634
in Eq. ix), and s value was 0.259 (vs 0.262 in Eq. ix).
All the considered regression models must pass a test
based on the value of Fisher statistics of all their regression
coefficients. Good values of the F-test indicate that the
model is statistically significant, but in Eq. (x) as the value
of F-ratio is low indicates this model is less significant.
QSAR model for antibacterial activity against Asper-
gillus niger
2
pMICca ¼ ꢁ2:161 ðLog PÞ þ 6:673 Log P ꢁ 8:871
ðvÞ
n = 14, r = 0.462, r2 = 0.214, s = 0.323, s2 = 0.104,
F = 0.902
On exclusion of compound GS10, Eq. (vi) was obtained
with better statistical validation:
2
pMICca ¼ 0:111ðXÞ ꢁ 0:216 ðXÞ þ 1:657
ðxiÞ
2
pMICca ¼ ꢁ6:030 ðLog PÞ þ 21:78 Log P ꢁ 14:58
n = 14, r = 0.489, r2 = 0.240, s = 0.254, s2 = 0.064,
F = 2.565
ðviÞ
n = 13, r = 0.637, q2 = 0.407, s = 0.303, s2 = 0.091,
F = 0.483
On exclusion of compound GS5, Eq. (xi) was obtained
with better statistical validation:
This equation showed an improved relationship between
MIC values and log P e.g., value of r was 0.637 (vs 0.462
in Eq. v) and s value was 0.303 (vs 0.323 in Eq. v).
Similarly, anumberofQSARequationsweredeveloped by
taking into consideration of various parameters and relating
them to MIC values of the test compounds against Staphylo-
ccocus epidermidis, Pseudomonas aeruginosa, and Asper-
gillus niger. Among these, the best QSAR equations were
obtained by relating pMICse, pMICpa and pMICan to ovality
(X) values by polynomial regression analysis (Table 5).
QSAR model for antibacterial activity against Staphy-
lococcus epidermidis
2
pMICca ¼ 0:093ðXÞ ꢁ 0:168 ðXÞ ꢁ 1:620
ðxiiÞ
n = 13, r = 0.586, q2 = 0.344, s = 0.247, s2 = 0.061,
F = 2.520
This equation showed an improved relationship between
MIC values and log P e.g., value of r was 0.637 (vs 0.462
in Eq. xi), and s value was 0.303 (vs 0.323 in Eq. xi).
The statistical data so obtained demonstrates the role of the
parameter ovality (X) in antimicrobial activity. The parameter
ovality is a physicochemical parameter and it involves a
combined effect of size and surface area of the substituents. It
characterizes deformation of molecular electrons distribution.
The equations also showed the direct relationship between
ovality and pMIC i.e., an increase in ovality (X) enhances the
antimicrobial activity of compounds.
2
pMICse ¼ 0:179 ðXÞ ꢁ 0:678ðXÞ þ 2:193
ðviiÞ
n = 14, r = 0.602, r2 = 0.363, s = 0.210, s2 = 0.044,
F = 5.792
On exclusion of compound GS4, Eq. (viii) was obtained
with better statistical validation:
Pharmacological evaluation
2
pMICec ¼ 0:247ðXÞ ꢁ 0:915ðXÞ þ 2:395
ðviiiÞ
In vitro anti-inflammatory activity of synthesized
compounds (4a–n)
n = 13, r = 0.685, q2 = 0.456, s = 0.193, s2 = 0.037,
F = 1.294
This equation showed an improved relationship between
MIC values and log P e.g., value of r was 0.685 (vs 0.602
in Eq. vii), and s value was 0.193 (vs 0.210 in Eq. vii).
QSAR model for antibacterial activity against Pseudo-
monas aeruginosa
The synthesized compounds were screened for anti-
inflammatory activity using inhibition of albumin denatur-
ation technique which was studied according to Muzushima
and Kabayashi with slight modification (Panneerselvam et al.,
2010; Rao et al., 2005; Chanda and Dave 2009). The standard
123

2764
Med Chem Res (2013) 22:2755–2767
drug and test compounds were dissolved in minimum amount
of dimethyl formamide (DMF) and diluted with phosphate
buffer (0.2 M, pH 7.4). Final concentration of DMF in all
solutions was less than 2.5 %. Test solution (1 ml) containing
different concentrations of drug was mixed with 1 ml of
1 mM albumin solution in phosphate buffer and incubated at
27 1 °C in BOD incubator for 15 min.
Denaturation was induced by keeping the reaction
mixture at 60 10 °C in water bath for 10 min. After
cooling, the turbidity was measured at 660 nm by UV–
Visible spectrophotometer.
ability of synthesized compounds to scavenge hydrogen
peroxide was determined according to the method of Ruch
et al., (1989) (Panneerselvam et al., 2010). A solution of
hydrogen peroxide (40 mM) was prepared in phosphate
buffer (50 mM pH 7.4). The concentration of hydrogen
peroxide was determined by absorption at 230 nm using
UV–Visible spectrophotometer.
The sample solution (20–60 lg/ml) in distilled water was
added to hydrogen peroxide and absorbance at 230 nm was
determined after 10 min against a blank solution that contained
drug solution in phosphate buffer without hydrogen peroxide.
The IC50 values of the synthesized compounds 4a–n were
calculated as given in Table 6. These experiments suggest
that the nitro, alkyl, and hydroxyl substituent were one of the
key groups to enhance the antioxidant activity mainly due to
its redox property through one electron transfer mechanism.
Percentage of inhibition of denaturation was calculated
from control (without drug) using the following formula:
% age inhibition ¼ ðA0 ꢁ A1= A0Þ ꢂ 100
where, A0 is the absorbance of control and A1 is the
absorbance of test. Diclofenac was used as standard.
Each experiment was done in triplicate and average was
taken. Also it was found that 4a, 4c, 4d, and 4e compounds
lead to considerable inhibition of denaturation. Out of these,
4e was most active. The IC50 values of the synthesized
compounds 4a–n were calculated as given in Table 6. The
activity of all the compounds was compared with diclofenac
(standard). The compounds containing nitro, alkyl, and
b-naphthyl substituent showed maximum activity.
Total antioxidant activity The antioxidant activity of the
compound 4a–n was evaluated by the phosphomolybdenum
method according to the procedure of Prieto et al., (1999).The
assaywasbasedonthereductionofMo(VI)–Mo(V)bythetest
solution and subsequent formation of a green phosphate/Mo
(V) complex at acidic pH. Compounds were dissolved in
CHCl3:ethanol (2:1) and different concentrations of com-
pounds (250–1,000 lg/ml) were combined with 3 ml of
reagent solution (0.6 M sulfuric acid, 28 mM sodium phos-
phate, and 4 mM ammonium molybdate). The tubes containing
the reaction solution were incubated at 95 °C for 60 min.
Then the absorbance of the solution was measured at
695 nm using double beam UV–Visible spectrophotometer
against blank (with 3 ml of reagent solution without using test
In vitro antioxidant activity of synthesized compounds
(4a–n)
Hydrogen peroxide radical scavenging (H2O2) assay In
˙
the body, H2O2 led to production of hydroxyl radicals (OH)
Table 7 IC50 (lg/ml) values of antioxidant activity of the different
synthesized hydrazones
which cause lipid peroxidation and DNA damage. The
Table 6 IC50 (lg/ml) values of anti-inflammatory activity the dif-
ferent synthesized hydrazones (4a–n)
Compounds Total antioxidant activity H2O2 scavenging activity
IC50 (lg/ml)
IC50 (lg/ml)
S. no.
Compounds
IC50 (lg/ml)
4a
4b
4c
4d
4e
4f
50.00
32.00
45.00
42.00
120.00
30.00
49.00
20.00
18.00
5.00
34.00
17.00
01.80
04.30
69.00
22.00
152.00
109.00
125.00
84.00
79.00
91.00
101.00
132.00
46.00
00.50
1
4a
95.00
160.00
85.00
86.00
80.00
124.00
105.00
54.00
17.00
12.00
10.00
20.00
75.00
35
2
4b
3
4c
4
4d
5
4e
6
4f
4g
4h
4i
7
4g
8
4h
19
10
11
12
13
14
15
4i
4j
4j
4k
4l
70.00
100.00
42.00
15.00
55.00
41.00
4k
4l
4m
4n
4a
4b
4m
4n
Diclofenac (standard)
71.00
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Med Chem Res (2013) 22:2755–2767
2765
antimicrobial activity against the tested bacteria in com-
parison to cefixime as positive control.
Table 8 Minimum inhibitory concentration (MIC) in lg/ml of syn-
thesized compounds
Compounds Minimum inhibitory concentration (MIC) in lg/ml
EC
PA
SE
SA
CA
AN
Result and discussion
4a
20
34
34
184
84
20
84
84
25
168
50
80
20
34
–
12.5
25
34
18
18
20
30
30
20
30
34
34
34
34
34
18
20
–
55
84
20
20
34
34
100
200
84
50
20
100
25
100
–
10
50
10
10
100
10
10
20
25
10
25
84
10
34
–
Chemistry
4b
4c
100
100
200
100
50
20
The target compounds (4a–n) were prepared as outlined in
Schemes 1 and 2. The key intermediate was obtained by
Fischer esterification of 4-chlorobenzoic acid with the absolute
ethanol in the presence of concentrated sulfuric acid, and
hydrazide was obtained by refluxing ester with hydrazine
hydrate. Further hydrazone was obtained by refluxing hydra-
zide with substituted aromatic aldehyde in the presence of gla-
cial acetic acid. Finally, the new target compounds 4a–n were
obtained, in good yields (71–99 %), by condensing hydrazides
with the corresponding appropriate aldehydes in absolute eth-
anol. The purity and authenticity of compounds were estab-
lished by chromatographic and spectroscopic means. The
structures assigned to 4a–n were supported by IR spectra
showing absorption bands at 3,427–3,280 cm-1 and
3,402–3,200 cm-1 due to O–H and N–H stretching, respec-
tively. Band at 1,676–1,620 cm-1 indicated C=O stretch.
Stretching vibrations due to C=N and C=C (aromatic) were
4d
4e
6.5
25
4f
25
4g
6.5
12.5
20
50
4h
4i
50
30
4j
25
200
200
84
4k
4l
50
25
4m
4n
Control
25
30
12.5
–
34
–
Amoxycillin 12.5
200
–
100
–
12.5
–
–
–
Fluconazole
Cefixime
–
12.5
–
400
–
50
400
400
50
EC Escherichia coli; PA Pseudomonas aeruginosa; SE Staphylo-
coccus epidermidis; SA Staphylococcus aureus; CA Candida albi-
cans; AN Aspergillus niger
observed at 1,680–1,409 cm-1 and 1,620–1,500 cm-1
,
respectively. Band at 1,529.55–1,456 cm-1 was appeared due
to asymmetric N=O stretch. Bands at 1,296–1,276 cm-1 indi-
cated C–O–C stretch. Bands at 854–785 cm-1 indicated out of
plane aromatic stretch. A characteristic strong band at
726–611 cm-1 was due to the aliphatic C–Cl stretching
vibrations. The proton NMR of these compounds revealed the
presence of singlet 12.50–10.70 ppm for –NH–N=. The
–N=CH– proton was observed as broad singlet 8.56–6.8 ppm.
A singlet due to proton of –OH appeared at d 5.80. A 3 proton
singlet at d 3.83 appeared corresponding to methoxy protons. A
3 proton singlet at d 2.40 appeared corresponded to the protons
of methyl group. All the other aliphatic and aromatic protons
were observed within the expected regions.
solution) after cooling to room temperature. The antioxidant
activity was compared with tannic acid used as standard.
The IC50 values of the synthesized compounds 4a–n were
calculated and given in Table 7. These experiments sug-
gested that the alkyl, hydroxyl, and 3-nitro substituent were
one of the key groups to enhance greatly the antioxidant
activity of substituted hydrazones mainly due to its redox
property through the one electron transfer mechanism that
may attribute the antioxidant properties. Compound 4b and
4f showed maximum antioxidant activity.
Antimicrobial activity
Biological activity
The antibacterial activities of the compounds (4a–n) were
tested against Gram-positive bacteria Staphylococcus aur-
eus, Staphylococcus epidermidis and Gram-negative bac-
teria Escherchia coli, Pseudomonas aeruginosa. The
antifungal activities of the compounds (4a–n) were tested
against Candida albicans and Aspergillus niger. The
results of in vitro antimicrobial activities of the hydrazones
are listed in Table 8. Minimum inhibitory concentration
(MIC) of target compounds was measured against amox-
ycillin, fluconazole, and cefixime (reference drugs) as
described in Table 8. All the compounds showed good
Antimicrobial activity of synthesized hydrazone analogs
was carried out by agar diffusion method and the average
radius of zone of inhibition (in mm) and MIC (in lg/ml)
was recorded. All the synthesized derivatives were
screened for antimicrobial activity and compared with the
standard drugs amoxycillin, cefixime (for antibacterial
activity), and fluconazole (for antifungal activity).
Antifungal activity of all the compounds was good but
nothing was found to be more active compared to fluco-
nazole (reference drug). While studying MIC against bac-
terial strains 4c, 4f, 4i, 4k, and 4n were most active among
123

2766
Med Chem Res (2013) 22:2755–2767
all the target compounds. The compound 4d was found to
be more active against Staphylococcus aureus than
Staphylococcus epidermidis. All target compounds were
found to be active against Staphylococcus aureus and
Pseudomonas aeruginosa where as some of them were
found to be less active against Escherichia coli and
Staphylococcus epidermidis. The compound 4m was found
to be least active in Escherichia coli, Staphylococcus
aureus, and Candida albicans, but exhibited good activity
against the remaining bacterial and fungal strains.
percentage similarity except 4e. All the synthesized com-
pounds showed good structural similarity with cefixime
and tosufloxacin tosylate as compared to other standard
drugs. It was found that synthesized compounds have least
similarity to pyridoxal salicyloyl hydrazone. Least struc-
tural similarity of target compounds to pyridoxal salicyloyl
hydrazone was due to the absence of pyridine moiety in
target compounds. Further compounds 4g and 4n have very
low percentage similarity due to the presence of o-benzoyl
on aromatic ring.
Whilestudyingtheantifungal activity4c, 4d, 4e, 4f, and 4k
were found to be moderately active. The 4i was found to be
highly active against all the target compounds. Among all the
targetcompounds4e, 4g, and 4i were least active. Itwasfound
that target compounds were more active against Aspergillus
niger than Candida albicans. Activity against Aspergillus
niger was even more than standard drug. It was found that
hydrazone derivatives with 2-nitro and 4-nitrophenyl substi-
tution showed good antibacterial property. The presence of
methyl and methoxy group on phenyl ring was found to have
no impact in improving the antibacterial activity.
Quantitative structure activity relationship
From the analysis of structures and the activity, it was dis-
played that some structure–activity relationships can be
extracted in target compounds. The structural requirements
for antibacterial and antifungal activity are different for
substituted hydrazides. This was evidenced by the fact that
the some active antibacterial compounds 4g, 4i, and 4n
compounds showed least antifungal activity and compounds
4d and 4j being the most active antifungal compounds have
shown least antibacterial activity. The presence of electron-
withdrawing groups (–NO2, –Cl, –Br, –OH) on aromatic ring
improved the antimicrobial activity of compounds.
All the target compounds were also studied for their
antioxidant activity using two in vitro antioxidant activity
models; total antioxidant activity and hydrogen peroxide
scavenging activity.
The analysis of structures of most active antibacterial
compounds may be concluded that among the electron-
withdrawing groups (–NO2, –Cl, –Br), the halo groups on
the aromatic ring are necessary for the antimicrobial
activity. Further, it may be possible that the introduction of
electron-withdrawing halo group on the second aromatic
ring may further improve the antimicrobial activity of these
compounds. This indicated that the introduction of –Cl and
–NO2 group may improve antibacterial and antifungal
activity respectively.
While studying total antioxidant activity, all target
compounds were found to have good antioxidant activity.
Among all the target compounds 4a, 4b, 4c, 4d, 4f, 4g,
4h, 4i, 4j, 4k, 4i, 4m, and 4n were found to be active than
ascorbic acid (standard). Out of all the active compounds
4b, 4f, 4g, 4h, 4i, 4j, and 4n were even active than second
reference drug tannic acid. While studying the hydrogen
peroxide scavenging activity, we found that 4a, 4b, 4c, 4d,
and 4f were more active than ascorbic acid (standard).
None of them was active than second standard tannic acid.
These studies suggested that the nitro, alkyl, and hydroxyl
substituent were one of the key groups to enhance the
antioxidant activity mainly due to its redox property
through the one electron transfer mechanism.
Conclusion
Assessment of structural similarities of target compounds
with standard drugs showed that all compounds have good %
age similarity except 4e. All the synthesized compounds
showed good structural similarity with cefixime and tosu-
floxacin tosylate as compared to other standard drugs. It was
found that synthesized compounds have least similarity to
pyridoxal salicyloyl hydrazone. Least structural similarity of
target compounds to pyridoxal salicyloyl hydrazone was due
to the absence of pyridine moiety in target compounds.
Further, compounds 4g and 4n have very low percentage
similarity due to the presence of o-benzoyl on aromatic ring.
From the analysis of structures and activity, it was dis-
played that some structure–activity relationships can be
extracted in target compounds. The structural requirements
for antibacterial and antifungal activity are different for
The compounds were screened for their in vitro anti-
inflammatory activity, and this was done by means of
studying albumin denaturation studies. It was found that 4a,
4c, 4d, and 4e compounds lead to considerable inhibition of
denaturation. IC50 of the compounds were also calculated as
showed in Table 4. Compounds 4c, 4d, and 4e demonstrated
better activity which was very close to that of diclofenac
(standard). Out of these, 4e showed maximum activity.
Structure similarity studies
Assessment of structural similarities of target compounds
with standard drugs showed that all compounds have good
123

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2767
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substituted hydrazides. This was evidenced by the fact that
some active antibacterial compounds 4g, 4k, and 4n
showed least antifungal activity and compounds 4d and 4j
being the most active antifungal compounds have shown
least antibacterial activity. The presence of electron-
withdrawing groups (–NO2, –Cl, –Br, –OH) on aromatic
ring improved the antimicrobial activity of compounds.
The results provided basic information to establish that
the nature of the substituent on aromatic ring was very
essential for various types of activities for example in
present study the presence of 4-methyl (4d), 3-nitro (4f),
and 4-nitro (4c) leads to good antioxidant activity whereas
the presence of 4-hydroxy (4a) do not contribute much for
antioxidant activity. These studies suggested that the nitro,
alkyl, and hydroxyl substituent were one of the key groups
to enhance the antioxidant activity mainly due to its redox
property through the one electron transfer mechanism.
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Acknowledgments Authors are thankful to M. M. College of
Pharmacy, M. M. University, Mullana, Ambala for providing support
and facility to carry out research.
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