Synthesis, xanthine oxidase inhibition, and antioxidant screening of benzophenone tagged thiazolidinone analogs

Article abstract of DOI:10.1002/ardp.201400058

A series of novel 2-(diaryl methanone)-N-(4-oxo-2-phenyl-thiazolidin-3-yl)- acetamides were synthesized by various Schiff bases of (4-benzoyl-phenoxy)-aceto hydrazide with thioglycolic acid. The structures of the newly synthesized compounds were confirmed

Full text of DOI:10.1002/ardp.201400058

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
1
Full Paper
Synthesis, Xanthine Oxidase Inhibition, and Antioxidant
Screening of Benzophenone Tagged Thiazolidinone Analogs
1
1
1
2
V. Lakshmi Ranganatha , A. Bushra Begum , P. Naveen , Farhan Zameer ,
2
1
Raghavendra Hegdekatte , and Shaukath Ara Khanum
1
Department of Chemistry, Yuvaraja’s College (Autonomous), University of Mysore, Mysore, Karnataka, India
Mahajana Life Science Research Laboratory, Department of Biotechnology, Microbiology and Biochemistry,
Mahajana Research Foundation, Pooja Bhagavat Memorial Mahajana Post Graduate Centre, Affiliated to
University of Mysore, Metagalli, Mysore, Karnataka, India
2
A series of novel 2-(diaryl methanone)-N-(4-oxo-2-phenyl-thiazolidin-3-yl)-acetamides were synthesized
by various Schiff bases of (4-benzoyl-phenoxy)-aceto hydrazide with thioglycolic acid. The structures of
1
the newly synthesized compounds were confirmed by IR, H NMR, mass spectra, and C, H, N analysis.
Further, all the synthesized compounds 9a–n were evaluated for xanthine oxidase (XO) inhibition and
antioxidant properties. Among all the tested compounds, 9f, 9m, and 9n demonstrated potent XO
inhibition of 52, 76, and 26%, respectively, compared to the standard drug allopurinol, which is evident
from in vitro and in silico analysis. On the other hand, compounds 9c, 9d, and 9k exhibit potent
antioxidant properties.
Keywords: Antioxidants / Schiff base / Synthesis / Thiazolidinone / Xanthine oxidase
Received: February 16, 2014; Revised: March 23, 2014; Accepted: April 2, 2014
DOI 10.1002/ardp.201400058
Introduction
sitivity syndrome [8], and adverse drug reactions such as toxic
epidermal necrolysis syndrome [9]. Recently, a number of
studies have reported the important role of active oxygen
species in the evolution of more than 50 human diseases [10].
Active oxygen species are formed continuously in the human
body. They are removed by enzymatic and non-enzymatic
antioxidative defense systems under normal conditions [11].
One of the most important sources of active oxygen species is
xanthine oxidase. Reactive oxygen species (ROS), including
free radicals such as superoxide anion radicals, hydroxyl
radicals, and singlet oxygen are various forms of activated
oxygen and often generated by oxidation products of
biological reactions [12]. The ROS have stimulated significant
interest among scientists in the past decade due to their broad
range of effects in biological and medicinal systems. The ROS
can cause lipid peroxidation (LPO), which leads to deteriora-
tion of the food [13]. These ROS contribute to the oxidative
stress on the organisms and they are involved in many
pathological processes such as inflammation, atherosclerosis,
cancer, and aging [14].
XO is responsible for catalyzing the oxidation of hypoxan-
thine to xanthine and then to uric acid [1]. Elevated
concentrations of uric acid in the blood stream of human
body leads to formation of gout [2]. The treatment of gout
entails the use of therapeutic agents, which are mainly
xanthine oxidase inhibitors (XOI) [3]. The XOI interaction is by
blocking the biosynthesis of uric acid from purine in the body
and it is believed that either by increasing the excretion of
uric acid or reducing the uric acid production it helps to
reduce the risk of gout [4]. Allopurinol is one of the known
synthetic XOI, which is widely used in the therapeutic and
clinical management of gout [5]. However, some people
develop rashes as they are allergic to allopurinol due to the
generation of superoxides [6, 7]. Severe reactions also occur,
including liver function abnormalities, allopurinol hypersen-
Correspondence: Dr. Shaukath Ara Khanum, Department of Chemistry,
Yuvaraj’s College, University of Mysore, Mysore 570 005, Karnataka,
India.
E-mail: shaukathara@yahoo.co.in
Fax: þ91-821 2419239
Thiazolidinone is considered as a biologically important
active scaffold that possesses the majority of biological
activities. In the current scenario, thiazolidinone moiety is
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2
V. Lakshmi Ranganatha et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
used in ralitoline (anticonvulsant), etozoline (diuretic), and
pioglitazone (antidiabetic), which are standard drugs [15].
They demonstrate xanthine oxidase inhibition [16], antitu-
bercular [17], antiinflammatory [18], and as antivirals [19]. It is
well documented that benzophenone nucleus is associated
with a variety of pharmacological actions [20]. This diversity
efforts to discover potentially active new XO inhibitory
agents [23] and novel antioxidants [24], we have synthesized
2-(diaryl methanone)-N-(4-oxo-2-phenyl-thiazolidin-3-yl)-acet-
amide derivatives (9a–n) and evaluated their XO inhibitory
and antioxidant properties.
in the biological response profile has attracted the attention Results and discussion
of many researchers to explore benzophenones integrated
thiazolidinone skeleton towards multiple potential biological
activities [21]. Recent studies have revealed that the burden of
gout is increasing worldwide [22] and more research is
focused on synthesis of heterocyclic analogs as XOI as well as
antioxidants. In view of above mentioned facts and our initial
Chemistry
The reaction sequence for different title compounds 9a–n is
outlined in the Scheme 1. The starting materials substituted
phenyl benzoates (3a and 3b) were synthesized according to a
reported procedure through the reaction of o-cresol (1) with
HO
OH
COCl
CH3
R
CH3
+
R
1
0 % NaOH
AlCl3
150−170°C
O
O
0
°C, Stirring, 3 h
R
CH3
1
2a,b
O
R3
3a,b
4a,b
R4
R2
R1
K2CO3 Dry acetone
ClCH2COOC2H5 Reflux
−10 h
8
H
N
H
N
O
O
O
O
O
N
H
NH2
O
O
H
O
CH3
CH3
R
CH3
R
R1
NH2-NH2.H2O
Ethanol/Stirring
at rt, 2−3 h
Ethanol
Reflux, 8−10 h
AcOH
+
R
R4
R
2
O
R3
O
O
8
a
−
n
7a
−
g
6a,b
5a,b
1
,4-Dioxane
O
Reflux, 8−10 h
HS
Anhyd-ZnCl2
OH
a: R=R =R =R =H, R =Cl
2
3
4
1
b: R=R =R =H, R =R =OCH
1
4
2
3
3
c: R=R =R =R =H, R =OH
1
2
4
3
CH3
d: R=R =R =R =H, R =CH
1
3
4
2
3
O
O
O
R
O
e: R=R =R =H, R =NO , R =Cl
2 3 1 2 4
f: R=R =R =R =H, R =OCH
3
2
3
4
1
S
N
g: R=R1=R3 =R4=H, R2=Br
N
=R =R =H, R =Cl
h: R=Cl, R
2
3
4
1
H
R1
i: R=Cl, R =R =H, R =R =OCH
1
4
2
3
3
j: R=Cl, R =R =R =H, R =OH
1
2
4
3
k: R=Cl, R =R =R =H, R =CH
1
3
4
2
3
R2
l: R=Cl, R =R =H, R =NO , R =Cl
2
3
1
2
4
R4
m: R=Cl, R =R =R =H, R =OCH
R3
2
3
4
1
3
9a
−
n
n: R=Cl, R =R =R =H, R =Br
1 3 4 2
Scheme 1. Synthesis of 2-(diaryl methanone)-N-(4-oxo-2-phenyl-thiazolidin-3-yl)-acetamide analogs.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
Synthesis and Biological Screening of Benzophenone Analogs
3
Table 1. Comparative enzyme inhibitory activities of compounds
site and thiazolidinone ring may get bound to the peripheral
site of the enzyme and transfers electrons to molybdenum
center. Further, compounds 9a–n were tested for their ability
to block the XO activity for the substrate xanthine. The order
of in vitro inhibitory activity of the title compounds against
the rat liver XO is found to be 9m > 9f > 9n > 9l > 9h > 9e
9
a–n against rat liver xanthine oxidase in percentage inhibition.
Compounds
Rat liver (%)
9
9
9
9
9
9
9
9
9
9
9
9
9
9
a
b
c
d
e
f
g
h
i
j
k
l
m
n
22
09
11
ND
15
52
ND
15
19
14
20
18
76
(Table 1). However, other compounds in the series were
shown poor XO inhibition. The present study helps to
understand the structure–activity relationship, mode of
interaction and extent of inhibition of the compounds 9a–
n against rat liver (in vitro) and human milk (in silico) (Fig. 1).
Further the mode of interaction was analyzed by in silico
models using human milk XO (PDB Code – 2CKJ) on selected
active sites of amino acids of chain C and molybdenum.
Among the title compounds the best three entities 9m, 9f, and
9n were subjected to structure–activity relationship and their
structures permitted various modes of interaction with the
active sites of amino acids. The potentiality of the compounds
26
Allopurinol
100
ND, not detected.
Values in bold indicate potent inhibitors.
9m, 9f, and 9n is mainly due to the presence of the chloro
group in the benzoyl ring of the benzophenone and the
methoxy group in ortho position of phenyl ring attached to
thiazolidinone; the presence of the methoxy group at ortho
position of phenyl ring attached to thiazolidinone nucleus
and chloro group in ortho position of benzoyl ring of the
benzophenone and presence of the bromo group at meta
position of phenyl ring attached to thiazolidinone nucleus,
respectively. These in vitro results strongly correlate with
in silico models and it has shown relatively better activity
against human milk XO (76% inhibition, Fig. 1 and Table 2).
In order to evaluate the free radical-scavenging ability of the
compounds 9a–n, they were tested for in vitro antioxidant
property by DPPH radical scavenging assay, LPO assay,
reducing power assay, hydroxyl radical scavenging, and
metal chelating assays. Compounds 9c, 9d, and 9k as shown
moderately potent DPPH radical scavenger that is known to
abstract the labile hydrogen atom. In all the above five
different acid chlorides (2a and 2b) in the presence of 10%
sodium hydroxide. Compounds 3a and 3b on subjection to
Fries rearrangement afforded substituted diaryl methanones
commonly known as hydroxy benzophenones (4a and 4b).
Compounds 4a and 4b on reaction with ethyl chloroacetate
offered ethyl (2-aroyl-4-methylphenoxy) acetates (5a and 5b) in
excellent yield. Further, compounds 5a,b on being treated
with 99% hydrazine hydrate in the presence of ethyl alcohol
gave 2-(diaryl methanone)-aceto hydrazides (6a and 6b). Again,
compounds 6a,b were treated with different substituted
benzaldehydes (7a–g) in the presence of ethanol as a solvent to
deliver compounds (8a–n) in a pure state. Finally, compounds
8
a–n were treated with thioglycolic acid in the presence of 1,4-
dioxane as a solvent and anhydrous zinc chloride as a catalyst
furnished 2-(diaryl methanone)-N-[2–phenyl-4-oxo-thiazolidin-
3-yl]-acetamide derivatives (9a–n) in excellent yield.
3 3
methods due to the presence of –OH, –CH , and Cl with –CH
Pharmacology
groups, respectively, attached to the benzene ring when
compared with the standard synthetic antioxidant BHA
whereas the other compounds displayed mild activity. The
IC50 value of the standard BHA for DPPH method was found to
be 13.81 mg/mL whereas the IC50 values of the compounds 9c,
9d, and 9k found to be 17.88, 26.15, and 20.12 mg/mL,
respectively (Fig. 2). Radical scavenging action is an important
feature of antioxidants; however, hydrogen donating ability
of the antioxidant molecule contributes to its free radical
scavenging nature. In LPO, the initiation of the peroxidation
sequence in a membrane is due to the abstraction of a
hydrogen atom from a double bond in the fatty acid. The LPO
activities of compounds 9c, 9d, 9k, and BHA with the IC50
values of 20.12, 28.93, 24.36, and 13.72 mg/mL, respectively
(Fig. 3).
Novel 2-(diaryl methanone)-N-(4-oxo-2-phenyl-thiazolidin-3-yl)-
acetamide analogs were screened for the XOI and antioxidant
activity. The rate of formation of uric acid from the oxidation
of xanthine in the presence of compounds 9a–n inhibitors
against the rat liver XO was studied. The inhibitory activity of
the compounds 9a–n against XO was compared with standard
drug allopurinol (Table 1). Further on to understand the
binding mode of the newly synthesized compounds 9a–n with
XO, molecular docking studies of the most potent compounds
9
m, 9f, and 9n were carried out. The structure of XO (PDB
entry code 2CKJ) was obtained from protein data bank and the
protein was prepared by removing the ligands and the
substrate. However, active sites of amino acids were retained.
It is possible that benzophenone moiety binds to the active
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4
V. Lakshmi Ranganatha et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
Figure 1. Molecular docking of the three best title compounds 9f, 9m, and 9n against human milk xanthine oxidase (PDB: 2CKJ).
It is believed that antioxidant activity and reducing power
are interrelated. Reductones inhibit the LPO by donating a
hydrogen atom and thereby terminating the free radical
chain reaction [25]. Compounds 9a–n have revealed the
concentration-dependent activity (Fig. 4). In addition, com-
pounds 9c, 9d, 9k, and BHA displayed hydroxyl radical
DNA strand breakage, which contributes to carcinogenesis,
mutagenesis, and cytotoxicity. Further, metal chelating
ability was also accessed for the compounds 9c, 9d, 9k, and
ethylenediamine tetraacetic acid (EDTA) with IC50 values of
20.93, 25.4, 23.54, and 13.97 mg/mL, respectively (Fig. 6).
scavenging activity with IC50 values of 18.22, 24.22, 20.75, and Conclusion
4.59 mg/mL, respectively (Fig. 5). The hydroxyl radical is an
1
extremely reactive free radical formed in biological systems
and it has been implicated as a highly damaging species in
free radical pathology and capable of damaging biomolecules
in the living cells. Hydroxyl radical has the capacity to cause
In conclusion, the identification of the 2-(diaryl methanone)-N-
(4-oxo-2-phenyl-thiazolidin-3-yl)-acetamide analogs as scaffold
for a new class of XOI and antioxidants. This potentiality of
the compounds has encouraged us to synthesize a rationally
Table 2. Hydrogen bonding between the docked top ranked pose of potent compounds and standard against human milk XO.
Description of
the compounds
No. of H bonding and
their distance (Å)
Atomic contact
energy values
Sl. no
Residues in H bonding
His 213
Gly 329, Phe 515, Lys 518, Phe 550
Phe 421, Lys 526, Leu 548
Val 290, Asn 288
1
2
3
4
9m
9f
9n
2 (3.52, 5.72)
ꢀ252.45
ꢀ217.97
ꢀ190.59
ꢀ280.71
5 (2.6, 3.2, 2.64, 2.56, 3.34)
4 (3.48, 2.9, 2.3, 3.4)
3 (3.53, 2.92, 3.27)
Std (allopurinol)
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
Synthesis and Biological Screening of Benzophenone Analogs
5
Figure 5. In vitro % inhibition of hydroxyl radical scavenging assay
of compounds 9c, 9d, and 9k.
Figure 2. In vitro free radical scavenging assay of compounds 9c,
9
d, and 9k against DPPH.
Figure 6. In vitro % inhibition of metal ion chelation assay of
compounds 9c, 9d, and 9k.
Figure 3. In vitro % inhibition of lipid peroxidation by compounds 9c,
d, and 9k.
9
designed series of biologically active molecules. From the
results of the present study, compounds 9f, 9m, and 9n
demonstrated potent inhibitory activities against XO. Further,
the compounds 9c, 9d, and 9k showed admirable antioxidant
properties. Additional research on in vivo mice models is in
progress in our laboratory.
Experimental
Chemistry
All solvents and reagents were purchased from Sigma–Aldrich
Chemicals Pvt Ltd. Melting points were determined on an
electrically heated VMP-III melting point apparatus. The FT-IR
spectra were recorded using KBr discs and Nujol on FT-IR Jasco
1
4
100 infrared spectrophotometer. H NMR spectra were recorded
using Bruker DRX 400 spectrometer at 400 MHz with TMS as an
internal standard. Mass spectra were recorded on LC–MS (API-
4000) mass spectrometer. Further elemental analysis of the
Figure 4. In vitro reducing power assay of compounds 9c, 9d,
and 9k.
compounds was performed on a Perkin Elmer 2400 elemental
analyzer.
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V. Lakshmi Ranganatha et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
General procedure for the preparation of phenyl benzoates
a and 3b
Anal. calcd. for C18
H, 6.02%.
Ethyl [4(2-chlorobenzoyl)-2-methyl phenoxy]acetate 5b. Yield 86%, m.p.
18 4
H O (298): C, 72.48; H, 6.04. Found: C, 72.46;
3
Substituted benzoates (3a and 3b) were synthesized by benzoy-
lation of o-cresol (1, 0.001 mol) with corresponding benzoyl
chlorides (2a and 2b, 0.001 mol) using 10% sodium hydroxide
solution. The reaction mixture was stirred for 2–3 h at 0°C, and
monitored by TLC using 4:1 n-hexane/ethyl acetate solvent
mixture. After completion of the reaction the oily product was
extracted with ether (3 ꢁ 20 mL). Ether layer was washed with 10%
sodium hydroxide solution (3 ꢁ 50 mL) followed by water
ꢀ
1
1
5
2–55°C. IR (Nujol): 1675 (C–O), 1755 cm (ester, C–O). H NMR
DMSO): d 1.22 (t, 3H, CH of ester), 2.4 (s, 3H, CH ), 4.15 (q, 2H, CH
of ester), 4.6 (s, 2H, OCH ), 6.9–7.8 (m, 7H, Ar–H). MS: m/z 334
Mþ1). Anal. calcd. for C18 17ClO (333): C, 64.97; H, 5.15. Found:
C, 64.99; H, 5.19%.
(
3
3
2
2
(
H
4
General procedure for the preparation of 2-(diaryl
methanone)-acethydrazides 6a and 6b
To compounds 5a and 5b (0.01 mol) in ethanol (10 mL) 99%
hydrazine hydrate (0.01 mol) was added dropwise and continu-
ously stirred for 2 h at room temperature to achieve compounds
(
3 ꢁ 30 mL) and then dried over anhydrous sodium sulfate and
evaporated the solvent to afford compounds 3a and 3b [26].
-Methyl phenyl benzoate 3a. Yield 90%, pale yellow liquid. IR
2
ꢀ
1
1
(
7
(
Nujol): 1715 cm (C–O). H NMR (DMSO): d 2.45 (s, 3H, Ar–CH
.0–7.8 (m, 9H, Ar–H). MS: m/z 213 (Mþ1). Anal. calcd. for C14
212): C, 79.22; H, 5.70. Found: C, 79.18; H, 5.76%.
-Methyl phenyl 2-chlorobenzoate 3b. Yield 84%, color less liquid. IR
3
),
H O
12 2
6
a and 6b as white solid. The solid was crystallized with methanol
to get pure product (6a and 6b) [29].
2
4
-Benzoyl-2-methylphenoxy acethydrazide 6a. Yield 80%, m.p. 125–
ꢀ
1
1
(
7
C
Nujol): 1710 cm (C–O). H NMR (DMSO): d 2.33 (s, 3H, Ar–CH
.1–8.0 (m, 8H, Ar–H). MS: m/z 248 (Mþ1). Anal. calcd. for
11ClO (247): C, 68.16; H, 4.49. Found: C, 68.19; H, 4.53%.
3
),
ꢀ1
1
(
28°C. IR (Nujol): 1630 (C–O), 1670 (amide,C–O), 3120–3220 cm
1
NH–NH
2
). H NMR (DMSO): d 2.2 (s, 3H, Ar–CH
), 4.6 (s, 2H, CH ), 7.2–7.8 (m, 8H, Ar–H), 9.35 (bs, 1H, CONH).
(285): C, 67.59;
H, 5.67; N, 9.85. Found: C, 67.65; H, 5.74; N, 9.91%.
-(2-Chlorobenzoyl)-2-methylphenoxy acethydrazide 6b. Yield 85%, m.p.
40–142°C. IR (Nujol): 1636 (C –– O), 1676 (amide, C –– O), 3115–
3
), 3.55 (bs, 2H,
14
H
2
NH
2
2
MS: m/z 286 (Mþ1). Anal. calcd. for C16
16 2 3
H N O
General procedure for the preparation of substituted
-hydroxy benzophenones 4a and 4b
4
4
1
3
(
Substituted 4-hydroxy benzophenones (4a and 4b) were synthe-
sized by Fries rearrangement. Compounds 3a and 3b (0.001 mol)
were treated with anhydrous aluminum chloride (0.002 mol) as a
catalyst and heated at 150–170°C temperature under neat
condition for about 2–3 h. Then the reaction mixture was cooled
to room temperature quenched with 6 N HCl in the presence of
ice water, and stirred for about 2–3 h. The solid was filtered
and recrystallized with methanol to obtain compounds 4a
and 4b [27].
ꢀ1
1
210 cm (NH–NH
), 4.62 (s, 2H, CH
CONH). MS: m/z 319 (Mþ1). Anal. calcd. for C16
0.29; H, 4.74; N, 8.79. Found: C, 60.32; H, 4.77; N, 8.83%.
2
). H NMR (DMSO): d 2.12 (s, 3H, Ar–CH
), 7.2–7.7 (m, 7H, Ar–H), 9.45 (bs, H,
15ClN (318): C,
3
), 3.45
1
bs, 2H, NH
2
2
H
2 3
O
6
General procedure for the preparation of 2-(diaryl
methanone)-aceto benzylidene-hydrazides 8a–n
To a solution of compounds 6a and 6b (0.01 mol) in absolute
ethanol (50 mL), a catalytic amount of acetic acid and equimo-
lecular amount of corresponding aldehydes (7a–g) was added. The
reaction mixture was refluxed for 8–10 h and after the
completion of the reaction, the reaction mixture was cooled to
room temperature, poured into crushed ice, filtered, washed,
dried, and crystallized from acetonitrile to yield compounds 8a–n
in a good yield. Compounds 8a and 8h were taken as
representative examples to explain characterization data.
(4-Benzoyl-2-methyl-phenoxy)-acetic acid(2-chloro-benzylidene)-hydrazide
8a. Yield 80%, m.p. 170–172°C. IR (Nujol): 1630 (C–O), 1670
(
4-Hydroxy-3-methyl-phenyl)phenyl methanone 4a. Yield 72%, m.p.
ꢀ
1
1
1
(
10–112°C. IR (Nujol): 1640 (C–O), 3510–3600 cm (OH). H NMR
DMSO): d 2.35 (s, 3H, CH 6.71–7.70 (m, 8H, Ar–H), 12.0 (bs, 1H,
OH). MS: m/z 213 (Mþ1). Anal. calcd. for C14 (212.08): C,
9.22; H, 5.70. Found: C, 72.23; H, 5.69%.
3 ,
)
–
12 2
H O
7
(
4-Hydroxy-3-methyl-phenyl)-2-chlorophenyl methanone 4b. Yield
ꢀ
1
7
(
8%, m.p. 120–122°C. IR (Nujol): 1645 (C–O), 3520–3650 cm
1
OH). H NMR (DMSO): d 2.33 (s, 3H, CH
3
)
,
6.73–7.71 (m, 7H, Ar–H),
1
2.10 (bs, 1H, –OH). MS: m/z 248 (Mþ1). Anal. calcd. for C14
H11ClO
2
(247): C, 68.16; H, 4.49. Found: C, 68.19; H, 4.53%.
amide, C–O), 3130–3210 (NH–N), 1630 cm (N–CH). 1H NMR
DMSO): d 2.12 (s, 3H, Ar–CH ), 4.6 (s, 2H, CH ), 7.2–7.8 (m, 12H,
3 2
ꢀ
1
(
(
General procedure for the preparation of ethyl-2-(diaryl
methanone)-aceto esters 5a and 5b
Compounds 5a and 5b were obtained by refluxing a mixture of
compounds 4a and 4b (0.013 mol) and ethyl chloroacetate
Ar–H), 8.45 (s, 1H, N–CH), 9.55 (bs, 1H, CONH). MS: m/z 408 (Mþ1).
Anal. calcd. for C23 19ClN (407): C, 67.90; H, 4.71; N, 6.89.
H
2 3
O
Found: C, 67.94; H, 4.74; N, 6.92%.
[4-(2-Chloro-benzoyl)-2-methyl-phenoxy]-acetic acid(2-chloro-benzylidene)-
hydrazide 8h. Yield 85%, m.p. 160–163°C. IR (Nujol): 1630 (C–O),
(0.026mol) in dry acetone (50 mL) and anhydrous potassium
–
ꢀ
1
carbonate (0.019 mol) for 8–9 h. The reaction mixture was cooled
and solvent was removed by distillation. The residual mass was
triturated with cold water to remove potassium carbonate, and
extracted with ether (3 ꢁ 50 mL). The ether layer was washed with
–
–
1
670 (amide, C–O), 3130–3210 (NH–N), 1645 cm
(N–CH).
1
H NMR (DMSO): d 2.15 (s, 3H, Ar–CH ), 4.5 (s, 2H, CH ), 7.1–7.9
–
m, 11H, Ar–H), 8.65 (s, 1H, N–CH), 9.35 (bs, 1H, CONH). MS: m/z
3
2
(
443 (Mþ1). Anal. calcd. for C23H18Cl N O (442): C, 62.60; H, 4.11;
2
2 3
1
0% sodium hydroxide solution (3 ꢁ 50 mL) followed by water
N, 6.35. Found: C, 62.63; H, 4.14; N, 6.37%.
(
3 ꢁ 30 mL) and then dried over anhydrous sodium sulfate and
evaporated to dryness to obtain crude solid, which on recrystalli-
zation with ethanol afforded compounds 5a and 5b [28].
Ethyl (4-benzoyl-2-methyl phenoxy)acetate 5a. Yield 90%, m.p. 49–
General procedure for the preparation of 2-(diaryl
methanone)-N-(4-oxo 2-phenyl-thiazolidin-3-yl)-
acetamides 9a–n
Compounds 8a–n (0.01 mol) were dissolved in 1,4-dioxane
ꢀ
1
1
5
(
2°C. IR (Nujol): 1664 (C–O), 1760 cm (ester, C–O). H NMR
DMSO): d 1.2 (t, 3H, CH of ester), 2.3 (s, 3H, CH ), 4.1 (q, 2H, CH of
), 7.1–7.7 (m, 8H, Ar–H). MS: m/z 299 (Mþ1).
3
3
2
ester), 4.5 (s, 2H, OCH
2
2
(40 mL) with a catalytic amount of anhydrous ZnCl . Further,
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
Synthesis and Biological Screening of Benzophenone Analogs
7
C–O), 1655 (HN–N), 3140 cm 1 (amide, N–H). H NMR (DMSO):
d 2.15 (s, 3H, Ar–CH ), 4.20 (s, 2H, S–CH ), 4.3 (s, 2H, O–CH ), 6.15
(s, 1H, N–CH), 7.2–7.9 (m, 12H, Ar–H), 8.55 (bs, 1H, CO–NH). MS:
21BrN S (526): C, 57.15;
ꢀ
1
thioglycolic acid (0.015 mol) was added with constant stirring and
the reaction mixture was refluxed for about 8–10 h. After the
completion of the reaction, which was monitored by TLC, the
reaction mixture was cooled to room temperature, the excess
solvent was removed by distillation and the reaction mixture
poured into sodium bicarbonate solution. The solid product was
filtered, washed with cold water, and the resulting solid was
crystallized in ethanol to afford compounds 9a–n in a pure state.
3
2
2
m/z 527 (Mþ1). Anal. calcd. for C25
H
2 4
O
H, 4.03; N, 5.33; S, 6.10. Found: C, 57.18; H, 4.06; N, 5.36; S, 6.14%.
2-[4-(2-Chloro-benzoyl)-2-methyl-phenoxy]-N-[2-(2-chloro-phenyl)-4-oxo-
thiazolidin-3-yl]-acetamide 9h. Yield 80%, m.p. 179–180°C. IR (Nujol):
1630 (benzophenone, C–O), 1670 (amide, C–O), 1710 (thiazoli-
ꢀ
1
1
2
-(4-Benzoyl-2-methyl-phenoxy)-N-[2-(2-chloro-phenyl)-4-oxo-thiazolidin-3-
yl]-acetamide 9a. Yield 82%, m.p. 185–187°C. IR (Nujol): 1630
benzophenone, C–O), 1670 (amide, C–O), 1710 (thiazolidinone,
dinone, C–O), 1630 (HN–N), 3160 cm (amide, N–H). H NMR
(DMSO): d 2.4 (s, 3H, Ar–CH ), 4.4 (s, 2H, S–CH ), 4.6 (s, 2H, O–CH ),
6.10 (s, 1H, N–CH), 7.1–7.9 (m, 11H, Ar–H), 8.5 (bs, 1H, CO–NH).
S (516): C, 58.26;
3
2
2
(
ꢀ
1
1
C–O), 1630 (HN–N), 3160 cm (amide, N–H). H NMR (DMSO):
d 2.4 (s, 3H, Ar–CH ), 4.4 (s, 2H, S–CH ), 4.6 (s, 2H, O–CH ), 6.10
s, 1H, N–CH), 7.1–7.9 (m, 12H, Ar–H), 8.5 (bs, 1H, CO–NH). MS: m/z
MS: m/z 517 (Mþ1). Anal. calcd. for C25
2 2 4
H20Cl N O
3
2
2
H, 3.91; N, 5.44; S, 6.22. Found: C, 58.28; H, 3.95; N, 5.46; S, 6.24%.
2-[4-(2-Chloro-benzoyl)-2-methyl-phenoxy]-N-[2-(4-methoxy-phenyl)-4-oxo-
thiazolidin-3-yl]-acetamide 9i. Yield 76%, m.p. 150–153°C. IR (Nujol):
1635 (benzophenone, C–O), 1670 (amide, C–O), 1710 (thiazoli-
(
4
82 (Mþ1). Anal. calcd. for C25
2 4
H21ClN O S (481): C, 62.43; H, 4.40;
N, 5.82; S, 6.67. Found: C, 62.46; H, 4.43; N, 5.85; S, 6.69%.
-(4-Benzoyl-2-methyl-phenoxy)-N-[2-(4-methoxy-phenyl)-4-oxo-thiazolidin-
-yl]-acetamide 9b. Yield 70%, m.p. 160–162°C. IR (Nujol): 1635
benzophenone, C–O), 1670 (amide, C–O), 1710 (thiazolidinone,
ꢀ
1
1
2
dinone, C–O), 1640 (HN–N), 3155 cm (amide, N–H). H NMR
(DMSO): d 2.3 (s, 3H, Ar–CH ), 3.5 (s, 6H, O–CH ), 4.3 (s, 2H, S–CH ),
4.5 (s, 2H, O–CH ), 6.15 (s, 1H, N–CH), 7.3–8.0 (m, 10H, Ar–H),
8.6 (bs, 1H, CO–NH). MS: m/z 543 (Mþ1). Anal. calcd. for
25ClN S (542): C, 59.94; H, 4.66; N, 5.18; S, 5.93. Found:
3
3
3
2
(
2
ꢀ
1
1
C–O), 1640 (HN–N), 3155 cm (amide, N–H). H NMR (DMSO):
d 2.3 (s, 3H, Ar–CH ), 3.5 (s, 6H, O–CH ), 4.3 (s, 2H, S–CH ), 4.5
), 6.15 (s, 1H, N–CH), 7.3–8.0 (m, 11H, Ar–H), 8.6
bs, 1H, CO–NH). MS: m/z 508 (Mþ1). Anal. calcd. for C27
507): C, 64.02; H, 5.17; N, 5.53; S, 6.33. Found: C, 64.05; H, 5.19;
3
3
2
C
27
H
2 6
O
(
(
(
s, 2H, O–CH
2
C, 59.96; H, 4.67; N, 5.19; S, 5.95%.
H
26
N
2
O
6
S
2-[4-(2-Chloro-benzoyl)-2-methyl-phenoxy]-N-[2-(4-hydroxy-phenyl)-4-oxo-
thiazolidin-3-yl]-acetamide 9j. Yield 70%, m.p. 200–202°C. IR (Nujol):
1645 (benzophenone, C–O), 1675 (amide, C–O), 1715 (thiazoli-
N, 5.57; S, 6.34%.
-(4-Benzoyl-2-methyl-phenoxy)-N-[2-(4-hydroxy-phenyl)-4-oxo-thiazolidin-
-yl]-acetamide 9c. Yield 78%, m.p. 205–207°C. IR (Nujol): 1645
benzophenone, C–O), 1675 (amide, C–O), 1715 (thiazolidinone,
ꢀ
1
1
2
dinone, C–O), 1635 (HN–N), 3150 cm (amide, N–H). H NMR
(DMSO): d 2.15 (s, 3H, Ar–CH ), 4.3 (s, 2H, S–CH ), 4.5 (s, 2H,
O–CH ), 6.15 (s, 1H, N–CH), 7.2–7.8 (m, 10H, Ar–H), 8.1 (bs, 1H,
O–H), 8.6 (bs, 1H, CO–NH). MS: m/z 498 (Mþ1). Anal. calcd. for
21ClN S (497): C, 60.42; H, 4.26; N, 5.64; S, 6.45. Found: C,
3
3
2
(
2
ꢀ
1
1
C–O), 1635 (HN–N), 3150 cm (amide, N–H). H NMR (DMSO):
d 2.15 (s, 3H, Ar–CH ), 4.3 (s, 2H, S–CH ), 4.5 (s, 2H, O–CH ), 6.15
s, 1H, N–CH), 7.2–7.8 (m, 11H, Ar–H), 8.1 (bs, 1H, O–H), 8.6 (bs, 1H,
S (463):
3
2
2
C
25
H
2 5
O
(
60.44; H, 4.28; N, 5.67; S, 6.48%.
CO–NH). MS: m/z 464 (Mþ1). Anal. calcd. for C25
H
22
N
2
O
5
2-[4-(2-Chloro-benzoyl)-2-methyl-phenoxy]-N-[2-(3-methyl-phenyl)-4-oxo-
thiazolidin-3-yl]-acetamide 9k. Yield 75%, m.p. 220–222°C. IR (Nujol):
1640 (benzophenone, C–O), 1665 (amide, C–O), 1725 (thiazoli-
C, 64.92; H, 4.79; N, 6.06; S, 6.93. Found: C, 64.94; H, 4.80; N, 6.08;
S, 6.98%.
ꢀ
1
1
2
-(4-Benzoyl-2-methyl-phenoxy)-N-[2-(3-methyl-phenyl)-4-oxo-thiazolidin-3-
yl]-acetamide 9d. Yield 72%, m.p. 215–218°C. IR (Nujol): 1640
benzophenone, C–O), 1665 (amide, C–O), 1725 (thiazolidinone,
dinone, C–O), 1645 (HN–N), 3155 cm (amide, N–H). H NMR
(DMSO): d 2.22 (s, 6H, Ar–CH ), 4.15 (s, 2H, S–CH ), 4.3 (s, 2H,
O–CH ), 6.20 (s, 1H, N–CH), 7.15–7.85 (m, 11H, Ar–H), 8.6 (bs, 1H,
23ClN S (495):
3
2
(
2
ꢀ
1
1
C–O), 1645 (HN–N), 3155 cm (amide, N–H). H NMR (DMSO):
d 2.22 (s, 6H, Ar–CH ), 4.15 (s, 2H, S–CH ), 4.3 (s, 2H, O–CH ), 6.20
s, 1H, N–CH), 7.15–7.85 (m, 12H, Ar–H), 8.6 (bs, 1H, CO–NH). MS:
m/z 462 (Mþ1). Anal. calcd. for C26 S (461): C, 67.81; H,
.25; N, 6.08; S, 6.96. Found: C, 67.81; H, 5.25; N, 6.08; S, 6.96%.
-(4-Benzoyl-2-methyl-phenoxy)-N-[2-(2-nitro-5-chloro-phenyl)-4-oxo-thiazo-
lidin-3-yl] acetamide 9e. Yield 80%, m.p. 233–235°C. IR (Nujol): 1630
benzophenone, C–O), 1655 (amide, C–O), 1735 (thiazolidinone,
CO–NH). MS: m/z 496 (Mþ1). Anal. calcd. for C26
H
2 4
O
3
2
2
C, 63.09; H, 4.68; N, 5.66; S, 6.48. Found: C, 63.12; H, 4.69; N, 5.68;
S, 6.50%.
(
H
24
N
2
O
4
2-[4-(2-Chloro-benzoyl)-2-methyl-phenoxy]-N-[2-(2-nitro-5-chloro-phenyl)-4-
oxo-thiazolidin-3-yl]-acetamide 9l. Yield 82%, m.p. 230–231°C. IR
(Nujol): 1630 (benzophenone, C–O), 1655 (amide, C–O), 1735
5
2
ꢀ
1
(thiazolidinone, C–O), 1625 (HN–N), 3140 cm (amide, N–H).
1
(
H NMR (DMSO): d 2.25 (s, 3H, Ar–CH
(s, 2H, O–CH ), 6.20 (s, 1H, N–CH), 7.25–7.90 (m, 10H, Ar–H),
8.55 (bs, 1H, CO–NH). MS: m/z 461 (Mþ1). Anal. calcd. for
25 2 3 6
C H19Cl N O S (461): C, 53.58; H, 3.42; N, 7.50; S, 5.72. Found: C,
3 2
), 4.15 (s, 2H, S–CH ), 4.2
ꢀ
1
1
C–O), 1625 (HN–N), 3140 cm (amide, N–H). H NMR (DMSO):
d 2.25 (s, 3H, Ar–CH ), 4.15 (s, 2H, S–CH ), 4.2 (s, 2H, O–CH ), 6.20
s, 1H, N–CH), 7.25–7.90 (m, 11H, Ar–H), 8.55 (bs, 1H, CO–NH). MS:
m/z 427 (Mþ1). Anal. calcd. for C25 20ClN S (426): C, 57.09; H,
.83; N, 7.99; S, 6.10. Found: C, 57.10; H, 3.85; N, 7.99; S, 6.14%.
-(4-Benzoyl-2-methyl-phenoxy)-N-[2-(2-methoxy-phenyl)-4-oxo-thiazolidin-
-yl]acetamide 9f. Yield 85%, m.p. 150–153°C. IR (Nujol): 1635
benzophenone, C–O), 1645 (amide, C–O), 1730 (thiazolidinone,
2
3
2
2
(
H
3
O
6
53.59; H, 3.45; N, 7.55; S, 5.74%.
3
2-[4-(2-Chloro-benzoyl)-2-methyl-phenoxy]-N-[2-(2-methoxy-phenyl)-4-oxo-
thiazolidin-3-yl]-acetamide 9m. Yield 74%, m.p. 155–156°C. IR (Nujol):
1635 (benzophenone, C–O), 1645 (amide, C–O), 1730 (thiazoli-
2
3
ꢀ
1
1
(
dinone, C–O), 1620 (HN–N), 3145 cm (amide, N–H). H NMR
(DMSO): d 2.22 (s, 3H, Ar–CH ), 3.2 (s, 3H, O–CH ), 4.25 (s, 2H,
S–CH ), 4.4 (s, 2H, O–CH ), 6.22 (s, 1H, N–CH), 7.3–7.8 (m, 11H,
Ar–H), 8.55 (bs, 1H, CO–NH). MS: m/z 512 (Mþ1). Anal. calcd. for
23ClN S (511): C, 61.11; H, 4.54; N, 5.48; S, 6.28. Found: C,
ꢀ
1
1
C–O), 1620 (HN–N), 3145 cm (amide, N–H). H NMR (DMSO):
d 2.22 (s, 3H, Ar–CH ), 3.2 (s, 3H, O–CH ), 4.25 (s, 2H, S–CH ), 4.4
), 6.22 (s, 1H, N–CH), 7.3–7.8 (m, 12H, Ar–H), 8.55
bs, 1H, CO–NH). MS: m/z 478 (Mþ1). Anal. calcd. for C26
477): C, 65.53; H, 5.08; N, 5.88; S, 6.73. Found: C, 65.55; H, 5.09;
3
3
3
3
2
2
2
(
(
(
s, 2H, O–CH
2
H
24
N
2
O
5
S
C
26
H
2 5
O
61.14; H, 4.56; N, 5.49; S, 6.30%.
N, 5.90; S, 6.76%.
-(4-Benzoyl-2-methyl-phenoxy)-N-[2-(3-bromo-phenyl)-4-oxo-thiazolidin-3-
yl]-acetamide 9g. Yield 75%, m.p. 220–223°C. IR (Nujol): 1630
benzophenone, C–O), 1640 (amide, C–O), 1735 (thiazolidinone,
2-[4-(2-Chloro-benzoyl)-2-methyl-phenoxy]-N-[2-(3-bromo-phenyl)-4-oxo-
thiazolidin-3-yl]-acetamide 9n. Yield 84%, m.p. 208–210°C. IR (Nujol):
1630 (benzophenone, C–O), 1640 (amide, C–O), 1735 (thiazoli-
2
ꢀ
1
1
(
dinone, C–O), 1655 (HN–N), 3140 cm (amide, N–H). H NMR
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
V. Lakshmi Ranganatha et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
(
DMSO): d 2.15 (s, 3H, Ar–CH
3
), 4.20 (s, 2H, S–CH
), 6.15 (s, 1H, N–CH), 7.2–7.9 (m, 11H, Ar–H), 8.55 (bs, 1H,
20BrClN
560): C, 53.63; H, 3.60; N, 5.00; S, 5.73 Found: C, 53.66; H, 3.64;
2
), 4.3 (s, 2H,
Lipid peroxidation assay
O–CH
2
LPO inhibitory activity of compounds 9a–n was measured
according to Kulkarni et al. [33]. Egg lecithin (3 mg/mL in
phosphate buffer, pH 7.4) was sonicated (Hielscher GmbH UP 50H
ultra-challprocessor sonicator) for 30 min to obtain small
membrane liposome vesicles. Different concentrations of the
compounds were added to 0.5 mL of liposome mixture. LPO was
CO–NH). MS: m/z 561 (Mþ1). Anal. calcd. for C25
H
2 4
O S
(
N, 5.04; S, 5.75%.
Pharmacological screening
Xanthine oxidase enzyme inhibition assay
The XO inhibitory activity was monitored spectrophotometrically
3
induced by adding 10 mL of 400 mM FeCl and 10 mL of 200 mM L-
ascorbic acid. After 60 min of reaction at 37°C, the reaction was
stopped by the addition of 1 mL of 0.25 N HCl containing 15%
thiazolidine-4-carboxylic acid (TCA) and 0.375% 2,4,6-tribromo-
anisole (TBA) and incubation in a boiling water bath for 15 min.
After centrifugation at 10,000 rpm, absorbance of the superna-
tant was measured at 532 nm. The scavenging effect was
calculated using the equation as described for DPPH.
(UV-1800, Japan) by following the absorbance of uric acid at
2
92 nm under aerobic condition [30]. Briefly, rat liver was
homogenized in 0.01 M Tris–HCl (pH 8.0) containing 1 mM EDTA.
The homogenate was centrifuged and the supernatant was used
as a source of enzyme. It was stored at ꢀ80°C until use and the
protein content was determined by the Lowry’s method [31],
using bovine serum albumin (BSA) as standard.
Reducing power assay
The enzyme assay mixture consisted of 20 mM potassium
phosphate buffer (pH 7.4) containing 0.3 mM EDTA and the
enzyme source in a total volume of 2 mL. In dose-dependent
inhibition studies, the reaction was initiated by the addition
of xanthine (50 mM) as the substrate to the above assay
mixture and the test compounds. The absorption rate at a
wavelength of 292 nm indicates the formation of uric acid
at 10 min intervals at ambient temperature. Duplicate assays
were repeated thrice. Allopurinol was used as positive control
and dimethyl sulfoxide (DMSO) was used as blank. The
inhibitory activity of each test compound against XO was
indicated by their percentage inhibition values. The percentage
inhibition of XO activity was calculated using the following
formula.
The reducing power was measured according to Yen and
Chen [34]. In this method, incubating the reaction mixture
(1 mL) containing the samples in the phosphate buffer (0.2 M, pH
6.6) with potassium ferricyanide (1 g/100 mL water) at 50°C for
20 min. The reaction was terminated by adding TCA (10 g/100 mL
water), the mixture was centrifuged at 3000 rpm for 10 min and
the supernatant was mixed with ferric chloride (0.1 g/100 mL of
water); the absorbance was measured at 700 nm. Increased
absorbance of the reaction mixture indicated increased reducing
power.
Hydroxyl radical-scavenging activity
The determination of the scavenging effect of compounds 9a–n
on hydroxyl radicals was carried out as described by Halliwell
et al. [35]. The reaction mixture, containing compounds
Xanthine oxidase inhibitionð%Þ
abs control ꢀ abs sample
(
at different concentration) was incubated with deoxyribose
¼
ꢁ 100
(15 mM), H O2 (10 mM), FeCl3 (500 mM), EDTA (1 mM), and
2
abs control
ascorbic acid (1 mM) in potassium phosphate buffer
(
100 mM) pH 7.4 for 60 min at 37°C. The reaction was terminated
where abs control is the absorbance of the control reaction
containing all reagents except the test compound) and abs
sample is the absorbance of the test compound.
by adding 1 mL TBA (1% w/v) and then heating tubes in a boiling
water bath for 15 min. The contents were cooled and the
absorbance of the mixture was measured at 535 nm against the
reagent blank.
(
Antioxidant screening
Compounds 9a–n were tested for antioxidant property by 1-
diphenyl-2-picryl-hydrazyl (DPPH), LPO, reducing power, hydroxyl
radical, and metal ion chelating methods.
Metal ion chelating assay
2ꢀ
The Fe -chelating ability of the compounds 9a–n was measured
by the ferrous iron–ferrozine complex at 562 nm described by
Decker and Welch [36]. The reaction mixture containing FeCl
2 mM/L) and ferrozine (5 mM/L) along with extracts was adjusted
2
DPPH radical scavenging assay
(
The DPPH radical scavenging assay was done according to Yen
and Duh [32]: Briefly, 1 mL of DPPH solution [0.1mM, in 95%
ethanol (v/v)] was incubated with different concentrations of the
compounds. The reaction mixture was shaken and incubated for
to a total volume of 0.8 mL with methanol, mixed and incubated
for 10 min at room temperature. The absorbance of the mixture
was read at 562 nm against a blank. EDTA was used as positive
control. The ability of the extract to chelate ferrous ion was
calculated using the equation described for DPPH.
2
5
0 min at room temperature and the absorbance was read at
17nm against a blank. The antioxidant BHA was used as a
positive control in all the assays. The radical scavenging activity
was measured as a decrease in the absorbance of DPPH and
calculated using the following equation:
Statistical analysis
All experiments were carried out in triplicates and repeated in
three independent sets of experiments. Data are shown as
means ꢂ standard deviation (SD). The SPSS10.0.5 version for
Windows (SPSS software, Inc., USA) computer program was
used for statistical analysis. The significance of the study was
assessed by one-way ANOVA, followed by hoc comparison test.
Correlations between quantitative properties were evaluated by
ꢀ
ꢁ
Acontrol ꢀ Asample
Scavenging effectð%Þ ¼
ꢁ 100
Acontrol
where A is the absorbance.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
Synthesis and Biological Screening of Benzophenone Analogs
9
calculating the Duncan and Dunnett’s co-efficient. The statisti-
cal significance value was set at p < 0.05.
[4] M. Umamaheswari, K. Asok Kumar, A. Somasundaram,
T. Sivashanmugam, V. Subhadradevi, T. K. Ravi, J. Ethno-
pharmacol. 2007, 109, 547–551.
Molecular modeling and docking studies
[5] M. Fields, C. G. Lewis, M. D. Lure, Free Radic. Biol. Med. 1996, 20,
595–600.
The human milk XO (EC: 1.17.3.2) is mainly associated with gout
disorder and subsequent other diseases. The crystal structure of
human milk xanthine oxidase was studied with PDB: 2CKJ was
built using CPH Models server 3.0. Energy computations were
performed on the molecule using GROMOS 96 implementation of
the Swiss-PDB viewer. Electrostatic point charges on the
molecules were calculated. The structures of XO inhibitors of
the current study were constructed using the public domain web
server Dundee PRODRG Server [37], which optimizes the
conformation of the side chains and minimizes the energy.
The minimum energy conformers of ligands were interactively
docked into close proximity with the enzyme active site pocket.
The possibility of binding, precise location of binding sites and
the mode of ligand binding was carried out using an automated
docking software, Molegro Virtual Docker 2008, version 3.2.1
[6] C. E. Berry, J. M. Hare, J. Physiol. 2004, 555, 589–606.
[
[
7] L. D. Kong, Y. Zhang, X. Pan, R. X. Tan, C. H. K. Cheng, Cell. Mol.
Life Sci. 2000, 57, 500–505.
8] A. Umpierrez, J. Cuesta-Herranz, M. De Las Heras, M. Lluch-
Bernal, E. Figueredo, J. Sastre, J. Allergy Clin. Immunol. 1998,
1
01, 286–287.
9] P. Pacher, A. Nivorozhkin, C. Szabo, Pharmacol. Rev. 2006, 58,
7–114.
[
8
[10] B. Halliwell, Drugs 1991, 42, 569–605.
[11] B. P. Yu, Biol. Rev. 1994, 74, 139–162.
[
[
[
12] M. Oktay, I. Gulcin, O. Kufrevioglu, LWT Food Sci. Technol. 2003,
6, 263–271.
13] N. Miller, A. Diplock, C. Rice-Evans, J. Agric. Food Chem. 1995,
3, 1794–1801.
14] B. Halliwell, J. M. C. Gutteridge, C. E. Cross, J. Lab. Clin. Med.
992, 119, 598–620.
3
(Molegro ApS, Aarhus, Denmark, http://molegro.com), that is
4
based on guided differential evolution and a force field-based
screening function [38]. Possible binding conformation and
orientations were analyzed by clustering methods, embedded in
Molegro Molecular Viewer 2008, version 1.2.0. Docking studies
were carried out using the human milk XO model. The enzyme
was visualized using sequence option, A and B chain, salicylic
acid, water, co-factors except molybdenum were deleted. The C
chain as well as a molybdenum co-factor was retained and
subjected to docking studies. The binding site was computed
within spacing such that the binding site was well sampled with a
grid resolution of 0.3 Å. The ligand was docked into this grid using
the MolDock Optimizer algorithm and its interactions monitored
using detailed energy estimates. A maximum population of 100
and maximum interactions of 10,000 were used for each run and
the fve best poses were retained. The software Molegro Molecular
Viewer 2008, version 1.2.0 (http://molegro.com) was utilized to
identify hydrogen bonds and hydrophobic interactions between
residues at the active site and the ligand.
1
[15] K. J. Abhishek, V. Ankur, R. Veerasamy, K. Sushil, R. K.
Kashaw, R. K. Agrawal, Bioorg. Med. Chem. 2012, 20, 3378–
3395.
[
[
[
16] C. Buvana, A. Mahalaksmi, M. Bijo Mathew, M. Sukumar,
Int. J. ChemTech Res. 2011, 3, 671–675.
17] H. H. Parekh, K. A. Parikh, A. R. Parikh, J. Sci. 2004, 15, 143–
148.
18] A. Kumar, C. S. Rajput, Eur. J. Med. Chem. 2009, 44, 83–90.
[19] R. K. Rawal, R. Tripathi, P. Christophe, S. B. Katti, E. De
Clercq, Bioorg. Med. Chem. 2007, 15, 1725–1730.
[20] S. A. Khanum, B. A. Bushra, V. Girish, F. K. Noor, Int. J. Biomed.
Sci. 2010, 6, 60–65.
[
21] W. Cunico, C. R. B. Gomes, W. T. Vellasco, J. Mini Rev. Org.
Chem. 2008, 5, 336–344.
[
22] H. M. Kramer, G. Curhan, Am. J. Kidney Dis. 2002, 40, 37–42.
V. Lakshmi Ranganatha gratefully acknowledges the financial support
provided by the Department of Science and Technology, New Delhi,
under INSPIRE-Fellowship scheme [IF110555]. And all authors are
thankful to the principal, Yuvaraja’s College, University of Mysore, and
the Director of Mahajana Research Foundation, Mahajana Education
Society and Head of the Department of Biotechnology, for their support
and encouragement throughout the execution of this work. Finally,
we extend our sincere thanks to Prof. Aravind Balaji for language
corrections.
[
23] K. R. Sathisha, S. A. Khanum, J. N. Narendra Sharath
Chandra, F. Ayisha, S. Balaji, K. Gopal Marathe, G. Shubha,
K. S. Rangappa, Bioorg. Med. Chem. 2011, 19, 211–220.
[24] S. M. Sadiq, S. A. Khanum, J. Rajesha, Free Radic. Antioxid. 2011,
1, 31–38.
[25] S. Gupta, L. M. Rodrigues, A. P. Esteves, A. M. F. Oliveira-
Campos, N. Bras, Eur. J. Med. Chem. 2008, 43, 771–780.
[26] S. A. Khanum, B. A. Begum, V. Girish, F. K. Noor, Int. J. Biomed.
Sci. 2010, 6, 60–65.
[27] S. A. Khanum, S. Sheena, A. V. Deepak, Bioorg. Chem. 2004, 32,
11–222.
2
The authors have declared no conflict of interest.
[
28] V. Lakshmi Ranganatha, N. Vinutha, S. A. Khanum, S. Anthal,
D. Revannasiddaiah, Rajnikant, G. K. Vivek, X-Ray Struct. Anal.
Online 2012, 28, 27–28.
References
[
29] S. A. Khanum, S. Shashikanth, S. Umesha, R. Kavitha, Eur. J.
Med. Chem. 2005, 40, 1156–1162.
[
1] P. L. Owen, T. Johns, J. Ethnopharmacol. 1999, 64, 149–160.
[30] R. S. Kadam, K. R. Iyer, Indian J. Pharm. Sci. 2007, 69, 41–45.
[
2] A. K. Tausche, K. Richter, A. Grässler, S. Hansel, B. Roch,
H. E. Schroder, Ann. Rheum. Dis. 2004, 63, 1351–1352.
[31] O. H. Lowry, N. J. Rosebrough, A. L. Farr, R. J. Randall, J. Biol.
Chem. 1951, 193, 265–275.
[
3] L. D. Kong, Z. Abliz, C. X. Zhou, L. J. Li, C. H. K. Cheng,
R. X. Tan, Phytochemistry 2001, 58, 645–651.
[32] G. C. Yen, P. D. Duh, J. Agric. Food Chem. 1994, 42, 629–632.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

1
0
V. Lakshmi Ranganatha et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
[
33] A. P. Kulkarni, S. M. Aradhya, S. Divakar, Food Chem. 2004, 87,
51–557.
[36] E. A. Decker, B. Welch, J. Agric. Food Chem. 1990, 38, 674–677.
5
[37] A. W. Schüttelkopf, D. M. F. V. Aalten, Acta Crystallogr. 2004,
[
34] G. C. Yen, H. Y. Chen, J. Agric. Food Chem. 1995, 43, 27–32.
35] B. Halliwell, J. M. C. Gutteridge, O. I. Aruoma, Anal. Biochem.
68, 1355–1363.
[
[38] R. Thomsen, M. H. Christensen, M. H. MolDock, J. Med. Chem.
2006, 49, 3315–3321.
1987, 165, 215–219.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com