Synthesis of pyrimidones and evaluation of their xanthine oxidase inhibitory and antioxidant activities

Article abstract of DOI:10.1002/ardp.201300240

The increasing prevalence of gout has been accompanied by a growing number of patients intolerant to or with disease refractory to the available urate-lowering therapies. This metabolic disease is a common disease with a higher prevalence in men older than 30 years and in women older than 50 years. These findings highlight the need for emerging treatments to effectively lower urate levels. In this view, we describe the xanthine oxidase (XO) inhibitory activities of the synthesized compounds 5a-j and also their antioxidant activities. Compounds 5c, 5d, 5f, 5h, and 5j exhibited good inhibitory activities against XO. On the other hand, compounds 5g and 5j exhibited moderate antioxidant activity. In order to find new urate-lowering compounds, the xanthine oxidase (XO) inhibitory and antioxidant activities of the newly synthesized pyrimidones 5a-j were evaluated. Compounds 5c, 5d, 5f, 5h, and 5j exhibited good inhibitory activities against XO. In addition, compounds 5g and 5j showed moderate antioxidant activity.

Full text of DOI:10.1002/ardp.201300240

Arch. Pharm. Chem. Life Sci. 2013, 346, 805–811
805
Full Paper
Synthesis of Pyrimidones and Evaluation of Their Xanthine
Oxidase Inhibitory and Antioxidant Activities
Handuvinahalli Devanna Gurupadaswamy1, Virupaksha Girish1, Farhan Zameer2,
Raghavendra Hegdekatte2, Jyoti Bala Chauhan2, and Shaukath Ara Khanum1
1
Department of Chemistry, Yuvaraja’s College Mysore, University of Mysore, Mysore, Karnataka, India
Mahajana Life Science Research Laboratory, Department of Biotechnology, Microbiology and Biochemistry,
2
Mahajana Research Foundation, Pooja Bhagavat Memorial Mahajana Post Graduate Centre, Affiliated to
University of Mysore, Metagalli, Mysore, Karnataka, India
The increasing prevalence of gout has been accompanied by a growing number of patients intolerant to
or with disease refractory to the available urate-lowering therapies. This metabolic disease is a common
disease with a higher prevalence in men older than 30 years and in women older than 50 years. These
findings highlight the need for emerging treatments to effectively lower urate levels. In this view, we
describe the xanthine oxidase (XO) inhibitory activities of the synthesized compounds 5a–j and also
their antioxidant activities. Compounds 5c, 5d, 5f, 5h, and 5j exhibited good inhibitory activities
against XO. On the other hand, compounds 5g and 5j exhibited moderate antioxidant activity.
Keywords: Antioxidant activity / Pyrimidones / Xanthine oxidase inhibitory activity
Received: June 24, 2013; Revised: August 13, 2013; Accepted: August 15, 2013
DOI 10.1002/ardp.201300240
Introduction
joints leading to severe and episodic painful inflammation [7].
Recent epidemiological studies revealed that the overall
disease burden of gout worldwide is increasing [8]. XOR is a
complex molybdoflavor enzyme present in two different
functional forms: dehydrogenase and XO [9]. XO is a critical
source of reactive oxygen species (ROS) that contribute to
vascular inflammation [10]. Under normal physiological
conditions, it is mainly found in the dehydrogenase form,
while in inflammatory situations, post-translational modifi-
cation converts the dehydrogenase form into XO. These
inflammatory conditions lead to an increase in XO levels and
thus an increased ROS generation by the enzymatic process,
finally resulting in alterations in vascular function [9]. It has
also been shown that XO secondarily leads to peroxy nitrite
formation. Peroxy nitrite is one of the most powerful ROS that
is produced by the reaction of nitric oxide and superoxide
radicals, and is considered to be a marker for reactive nitrogen
species, accompanied by oxidative stress [11, 12]. Both XO and
dehydrogenase form catalyze the removal of hydrogen from a
substrate using oxygen as hydrogen acceptor, and it gets
reduced. During reoxygenation (i.e., reperfusion phase) it
reacts with molecular oxygen, thereby releasing superoxide
anion radicals. The XO pathway has been implicated as an
important route in the oxidative injury to tissue, especially
after ischemia–reperfusion [13].
Enzyme inhibitors are molecules that bind to enzymes and
decrease their activity [1]. Since blocking an enzyme’s activity
can kill a pathogen or correct a metabolic imbalance, many
drugs are enzyme inhibitors [2]. The design of the inhibitors,
based on the structure of an enzyme active site, is helpful to
determine the 3D structure of the enzyme and of the enzyme
in complex with an inhibitor at high resolution. Precisely all
the molecule interactions that are necessary for a drug to bind
to its target site based on the architecture of enzymes active
site and the interactions that stabilize the inhibitor with
greater binding affinity can be designed with a shape that fits
better into the active site and with charge distributed suitable
for increased interaction energies. Also, it may involve
topographic studies [3, 4]. The enzyme xanthine oxidase
(XO), also known as xanthine oxidoreductase (XOR), helps in
the breakdown of purine to uric acid [5] and overproduction
of uric acid leads to hyperuricemia, which is linked to gout [6].
Gout is characterized by the deposition of uric acid in the
Correspondence: Dr. Shaukath Ara Khanum, Department of Chemistry,
Yuvaraja’s College, University of Mysore, Mysore 570 006, India.
E-mail: shaukathara@yahoo.co.in
Fax: þ91 821 2419239
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

806
H. D. Gurupadaswamy et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 805–811
Nitrogen heterocycles in general and pyrimidine and its
analogs in particular are the most explored fields in
heterocyclic chemistry due to their diverse biological
activities [14–16]. Pyrimidine, being an integral part of DNA
and RNA, plays a vital role in several biological processes, and
diverse biological properties have been associated with
numerous fused pyrimidines including XO inhibition [17],
anti-inflammatory [18], anti-allergic [19], hypnotic [20], anti-
viral [21], anti-microbial [22], and antioxidant activity [23]. The
above-mentioned biological importance of pyrimidones led us
to synthesize novel pyrimidine analogs comprising hydrazide
linkage. In continuation of our studies in the area of XO
inhibitors [24] and with a view to the synthesis of novel
pyrimidones, we herein report design, synthesis, and XO
inhibition and antioxidant activity of pyrimidones.
amides 5a–j 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 rat liver XO
is found to be 5c > 5d > 5f > 5h > 5j; however other com-
pounds in the series were very poor XO inhibitors and the
activity was not in a detectable range. The present study helps
to understand structure-activity relationship mode of inter-
action and extent of inhibition of compounds 5a–j against rat
liver (in vitro) and human milk (in silico) of XO.
A series of assays were then performed in order to evaluate
the antioxidant properties of the synthesized compounds 5a–
j. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging
assay is widely used to investigate the radical scavenging
activities of several natural as well as synthetic
compounds [28]. The scavenging activity has been studied
in the process of hydrogen atom transfer to the stable free
radical DPPH to compare the activity of compounds under
investigation with that of widely known antioxidant
parameter. Free radical scavenging activity of DPPH is a
stable free radical at room temperature and accepts an
electron or hydrogen radical to become a stable diamagnetic
molecule. The reduction capability of DPPH radicals was
determined by decreased absorbance at 517 nm, which is
induced by antioxidants. The recent study was carried out to
determine the hydrogen-donating capabilities of the com-
pounds 5a–j. The results are summarized in Fig. 1. Compound
5g with trifluoro methyl group and compound 5j with N,N-
dimethyl amino group showed moderate activity compared
with the standard synthetic antioxidant butylated hydroxyl
anisole (BHA) whereas the other compounds displayed mild
activity. The IC50 value of the standard BHA in DPPH method
was found to be 13.81 mg/mL, whereas the IC50 values of the
compounds 5g and 5j were found to be 35.81 and 17.45 mg/mL,
respectively (Fig. 1).
Results and discussion
The inhibition activity of synthesized compounds 5a–j was
tested against XO [25, 26]. The rate of formation of uric acid
from oxidation of xanthine in the presence of 5a–j inhibitors
against rat liver XO was studied. The inhibitory activity of the
compounds 5a–j against XO was compared with standard
drug allopurinol (Table 1). The known X-ray structure of
xanthine oxidoreductase bound molecule shows highly
specific binding pocket presenting a long narrow cavity
leading toward the Mo(IV) complex. Molybdenum protein
sites of both XO and xanthine dehydrogenase are structurally
equivalent. Further, to understand the binding mode of newly
synthesized compounds 5a–j with XO, molecular docking
studies of the most potent compounds (5c, 5d, and 5f) and the
structure of XO (PDB entry code: 2CKJ) was obtained and
analyzed [27]. It is possible that phenyl moiety binds to active
site and heterocyclic ring may bind to the peripheral site of
enzyme and transfer electrons to molybdenum center (a2)
electron acceptor from no site of XO. Different heterocyclic
Lipid peroxidation (LPO) refers to the oxidative degradation
of lipids. It is the process in which free radicals steal electrons
from the lipids in cell membranes, resulting in cell damage.
Table 1. Comparative inhibitory activities of compounds 5a–j
(15 mM) against rat liver xanthine oxidase in percentage inhibition.
BHA 5g 5j
Compounds
Rat liver
100
80
60
40
20
0
5a
5b
5c
5d
5e
5f
5g
5h
ND
ND
55%
45%
ND
40%
ND
32%
ND
5i
5j
15%
100%
5
10
15
20
25
Allopurinol
Concentration in μg/mL
ND, not detected.
Figure 1. DPPH scavenging assay of potent compounds 5g and 5j.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 805–811
XO Inhibitory and Antioxidant Activities of Pyrimidone Analogs
807
The LPO activities of compounds 5g, 5j, and BHA had the IC50
values of 33.69, 18.13, and 13.72 mg/mL, respectively (Fig. 2).
To substantiate the result of DPPH radical scavenging
activity the reducing power of the compounds 5a–j was also
evaluated. Reducing power assay was determined based on
the principle that substances that have reduction potential
react with potassium ferricyanide to form potassium
ferocyanide, which then reacts with ferric chloride to form
ferric–ferrous complex that has maximum absorption at
700 nm. At the concentration of 25 mg/mL compound 5g
showed threefold and compound 5j showed twofold lesser
reducing power ability than the standard (Fig. 3).
with IC50 values of 39.88, 23.85, and 14.49 mg/mL, respectively
(Fig. 4).
Chelation of transition metals prevents catalysis of
hydrogen peroxide decomposition and Fenton type reaction.
In presence of chelating agents, the complex formation is
disrupted with the result that the red color of the complex is
decreased. The transition metal ion Fe2þ possesses the ability
to move single electrons by virtue of which it can allow the
formation and propagation of many radical reactions. Metal
chelating ability was also accessed for the compounds 5g, 5j,
and EDTA with IC50 values of 31.25, 17.56, and 13.97 mg/mL,
respectively (Fig. 5).
The damaging action of hydroxyl radicals is the strongest
among free radicals. In biochemical systems, superoxide
radical is converted by superoxide dismutase to hydrogen
peroxide, which can subsequently generate extremely reac-
tive hydroxyl radicals in the presence of certain transition
metal ions. Further, hydroxyl radical has the capacity to cause
DNA strand breakage and leads to mutations. Compounds 5g,
5j, and BHA displayed hydroxyl radical scavenging activity
The mode of interaction was analyzed by docking using
human milk XO (PDB entry code: 2CKJ) on selected active site
of amino acids of chain C and molybdenum. Among the title
compounds the best three entities, namely, 5c, 5d, and 5f,
were subjected for structure–activity relationship and their
structure permitted various modes of interaction with active
site of amino acid. Compound 5c produced relatively better
activity against human milk XO with atomic contact energy of
ꢀ156.41 followed by 5d (ꢀ131.51), 5f (ꢀ127.55) than compared
to standard drug allopurinol (ꢀ280.71), respectively.
BHA 5g 5j
100
80
60
40
20
0
BHA 5g 5j
100
80
60
40
20
0
5
10
15
20
25
Concentration in μg/mL
5
10
15
20
25
Figure 2. Inhibition of lipid peroxidation by potent compounds 5g
and 5j.
Concentration in μg/mL
Figure 4. Hydroxyl radical scavenging assay of potent compounds
5g and 5j.
BHA
5g
5j
EDTA 5g 5j
1.2
1
100
80
60
40
20
0
0.8
0.6
0.4
0.2
0
0
5
10
15
20
25
30
5
10
15
20
25
Concentration in μg/mL
Concentration in μg/mL
Figure 3. Reducing power assay of potent compounds 5g and 5j.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 5. Metal ion chelation assay of potent compounds 5g an 5j.
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808
H. D. Gurupadaswamy et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 805–811
–
Conclusion
3b: Yield: 81%; mp: 189.8–190.5°C; IR (KBr): 1630 (amide, C O);
–
3720–3515 cmꢀ1 (NH–NH); 1H NMR (DMSO-d6) d: 4.20 (s, 2H, CH2),
7.71–7.74 (d, 2H, Ar-H); 7.80–7.82 (d, 2H, Ar-H); 10.40 (bs, 1H, NH);
10.63 (bs, 1H, NH); MS (EI): m/z (75%) Mþ 291; Anal. calcd. for
C9H8BrClN2O2 (291): C, 37.08; H, 2.77; Br, 27.41; Cl, 12.16; N, 9.61.
Found: C, 37.15; H, 2.82; Cl, 12.14; N, 9.57%.
From the results of the present study, it is concluded that a
series of novel pyrimidones were synthesized and their XO
inhibitory activities were evaluated. Compounds 5c, 5d,
5f, 5h, and 5j exhibited good inhibitory activities against XO.
On the other hand, compounds 5g and 5j exhibited good
antioxidant activity.
–
3c: Yield: 81%; mp: 221–222°C; IR (KBr): 1650 (amide, C O);
–
3750–3555 cmꢀ1 (NH–NH); 1H NMR (DMSO-d6) d: 4.20 (s, 2H, CH2),
7.64–7.66 (d, 2H, Ar-H); 7.88–7.91 (d, 2H, Ar-H); 10.1 (bs, 2H, NH);
10.55 (bs, 2H, NH); MS (EI): m/z (74%) Mþ 338; Anal. calcd. for
C9H8ClIN2O2 (338): C, 31.93; H, 2.38; Cl, 10.47; I, 37.49; N, 8.28; O,
9.45. Found: C, 31.85; H, 2.29; Cl, 10.58; N, 8.36%.
–
Experimental
3d: Yield: 80%; mp: 178–180°C; IR (KBr): 1640 (amide, C O);
–
3690–3535 cmꢀ1 (NH–NH); 1H NMR (DMSO-d6) d: 2.36 (s, 3H, CH3),
d: 4.19 (s, 2H, CH2), 7.29–7.31 (d, 2H, Ar-H); 7.76–7.79 (d, 2H, Ar-H);
10.2 (bs, 1H, NH); 10.43 (bs, 1H, NH); MS (EI): m/z (72%) Mþ 226;
Anal. calcd. for C10H11ClN2O2 (226): C, 52.99; H, 4.89; Cl, 15.64; N,
12.36. Found: C, 52.81; H, 4.75; Cl, 15.80; N, 12.25%.
–
Chemistry
Materials and methods
Chemicals were purchased from Sigma–Aldrich Chemical Co. TLC
was performed on aluminum-baked silica plates and visualized
by UV light. Melting points were determined on a Thomas Hoover
capillary melting point apparatus with a digital thermometer. IR
spectra were recorded on a FT-IR Shimadzu 8300 spectrophoto-
meter, 1H NMR spectra were recorded on a Bruker 400 MHz NMR
spectrophotometer in DMSO-d6, and chemical shift were recorded
in parts per million downfield from tetramethyl silane. Mass
spectra were obtained with a VG70-70H spectrophotometer, and
important fragments are given with the relative intensities in
brackets. Elemental analysis results are within 0.4% of the
calculated value.
3e: Yield: 73%; mp: 114–118°C; IR (KBr): 1615 (amide, C O);
–
3720–3515 cmꢀ1 (NH–NH); 1H NMR (DMSO-d6) d: 1.31 (s, 9H,
(CH3)3), 4.27 (s, 2H, CH2), 7.80–7.82 (d, 2H, Ar-H); 7.92–7.94 (d, 2H,
Ar-H); 10.3 (bs, 1H, NH); 10.62 (bs, 1H, NH); MS (EI): m/z (74%) Mþ
268; Anal. calcd. for C13H17ClN2O2 (268): C, 58.10; H, 6.38; Cl,
13.19; N, 10.42. Found: C, 58.22; H, 6.45; Cl, 13.30; N, 10.49%.
–
3f: Yield: 77%; mp: 198–199°C; IR (KBr): 1635 (amide, C O);
–
3685–3545 cmꢀ1 (NH–NH); 1H NMR (DMSO-d6) d: 3.83 (s, 3H, CH3),
d: 4.20 (s, 2H, CH2), 7.03–7.05 (d, 2H, Ar-H); 7.85–7.88 (d, 2H, Ar-H);
10.31 (bs, 1H, NH); 10.38 (bs, 1H, NH); MS (EI): m/z (74%) Mþ 242;
Anal. calcd. for C10H11ClN2O3 (242): C, 49.50; H, 4.57; Cl, 14.61; N,
11.54. Found: C, 49.55; H, 4.62; Cl, 14.75; N, 11.65%.
Synthesis and characterization of compounds 3a–3j
Synthesis of 4-chlorobenzoic acid-N-(2-chloro acetyl) hydrazide
(3a) (Scheme 1): Chloroacetic acid (2, 1.46 g, 12.9 mmol) was
dissolved in dichloromethane (20 mL), 2,6-lutidine (4.14 mL,
38.7 mmol), TBTU (4.24 g, 12.9 mmol) was added, and the
reaction mixture was stirred for 10 min at 25°C. To the reaction
mixture 4-chlorobenzoic acid hydrazide (1a, 2.0 g, 11.7 mmol)
was added dropwise and it was stirred at ambient temperature
for 3 h. After completion of the reaction, monitored by TLC, the
reaction mass was quenched with 10% sodium bicarbonate
solution (20 mL). The solid precipitated was filtered, washed
with water (3 ꢁ 20 mL), and dried to obtain compound 3a as white
solid.
–
3g: Yield: 74%; mp: 205–208°C; IR (KBr): 1630 (amide, C O);
–
3740–3515 cmꢀ1 (NH–NH); 1H NMR (DMSO-d6) d: 4.22 (s, 2H, CH2),
7.89–7.91 (d, 2H, Ar-H); 8.05–8.07 (d, 2H, Ar-H); 10.48 (bs, 1H, NH);
10.79 (bs, 1H, NH); MS (EI): m/z (74%) Mþ 280; Anal. calcd. for
C10H8ClF3N2O2 (280) C, 42.80; H, 2.87; Cl, 12.63; F, 20.31; N, 9.98.
Found: C, 42.80; H, 2.92; Cl, 12.68; F, 20.38; N, 10.05%.
–
3h: Yield: 81%; mp: 189–192°C; IR (KBr): 1640 (amide, C O);
–
3720–3545 cmꢀ1 (NH–NH); 1H NMR (DMSO-d6) d: 4.19 (s, 2H, CH2),
7.48–7.51 (d, 2H, Ar-H); 7.97–8.00 (d, 2H, Ar-H); 10.5 (bs, 2H, NH);
10.79 (bs, 1H, NH); MS (EI): m/z (75%) Mþ 296; Anal. calcd. for
C10H8ClF3N2O3 (296): C, 40.49; H, 2.72; Cl, 11.95; F, 19.21; N, 9.44.
Found: C, 40.58; H, 2.81; Cl, 12.05; F, 19.25; N, 9.55%.
–
–
–
3i: Yield: 82%; mp: 226–228°C; IR (KBr): 1650 (amide, C O);
Yield: 88%; mp: 188–190°C; IR (KBr): 1645 (amide, C O);
–
3670–3555 cmꢀ1 (NH–NH); 1H NMR (DMSO-d6) d: 4.23 (s, 2H, CH2),
8.09–8.12 (d, 2H, Ar-H); 8.35–8.37 (d, 2H, Ar-H); 10.45 (bs, 1H, NH);
10.85 (bs, 1H, NH); MS (EI): m/z (74%) Mþ 257; Anal. calcd. for
C9H8ClN3O4 (257): C, 41.96; H, 3.13; Cl, 13.76; N, 16.31. Found: C,
42.06; H, 3.24; Cl, 13.85; N, 16.42%.
3760–3525 cmꢀ1 (NH–NH); 1H NMR (DMSO-d6) d: 4.51 (s, 2H, CH2),
7.50–7.54 (d, 2H, Ar-H); 7.81–7.84 (d, 2H, Ar-H); 9.85 (bs, 1H, NH);
10.05 (bs, 1H, NH); MS (EI): m/z (75%) Mþ 247; Anal. calcd. for
C9H8Cl2N2O2 (247): C, 43.75; H, 3.26; Cl, 28.70; N, 11.34. Found: C,
43.88; H, 3.19; Cl, 28.64; N, 11.33%.
O
O
O
H
N
O
NH2
2,6-Lutidine/ TBTU
DCM
N
H
N
H
Cl HN
Cl
HO
+
O
+
R
R
N
4
_
3a j
_
2
1a j
O
O
N
f: R=OMe
g: R=CF 3
h: R=OCF 3
i: R=NO 2
j: R=NMe 2
a: R= Cl
b: R=Br
c: R=I
d: R=Me
e: R= tBu
H
N
N
H
N
K2CO3
O
Acetone
R
_
Scheme 1. Synthesis of pyrimidone derivatives.
5a j
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Arch. Pharm. Chem. Life Sci. 2013, 346, 805–811
XO Inhibitory and Antioxidant Activities of Pyrimidone Analogs
809
–
–
3j: Yield: 80%; mp: 188–191°C; IR (KBr): 1655 (amide, C O);
5f: Yield: 61%; mp: 253–253°C; IR (KBr): 1610 (C O); 1625
–
–
3750–3535 cmꢀ1 (NH–NH); 1H NMR (DMSO-d6) d: 2.97 (s, 6H,
2CH3), 4.18 (s, 2H, CH2), 6.69–6.72 (d, 2H, Ar-H); 7.72–7.75 (d,
2H, Ar-H); 10.14 (bs, 2H, NH); 10.5 (bs, 2H, NH); MS (EI): m/z
(74%) Mþ 255; Anal. calcd. for C11H14ClN3O2 (255): C, 51.67;
H, 5.52; Cl, 13.87; N, 16.43. Found: C, 51.80; H, 5.60; Cl, 13.95; N,
16.51%.
(amide, C O); 3720–3510 cm (NH–NH); 1H NMR (DMSO-d6) d:
ꢀ1
–
–
3.82 (s, 3H, OCH3); 4.73 (s, 2H, CH2); 6.1–6.3 (d, 1H, pyrimidone-H
at sixth position); 6.5–6.6 (d, 1H, pyrimidone-H at fifth position);
7.23–7.4 (d, 2H, Ar-H); 7.5–7.6 (s, 1H, pyrimidone-H at second
position); 7.72–7.83 (d, 2H, Ar-H); 10.7 (bs, 2H, NH); MS (EI): m/z
(72%) Mþ 303; Anal. calcd. for C14H14N4O4 (302): C, 55.63; H, 4.67;
N, 18.53. Found: C, 55.72; H, 4.78; N, 18.45%.
–
5g: Yield: 65%; mp: 265–267°C; IR (KBr): 1610 (C O); 1665
–
Synthesis and characterization of compounds 5a–5j
(amide, C O); 3695–3515 cm (NH–NH); 1H NMR (DMSO-d6) d:
ꢀ1
–
–
Synthesis of 4-chloro benzoic acid-N-[2-(6-oxo-6H-pyrimidin-1-yl)-
acetyl]-hydrazide (5a) (Scheme 1): To a solution of compound (3a,
1.0 g, 4.0 mmol) and 3H-pyrimidin-4-one (4, 0.39 g, 4.0 mmol) in
acetone (20 mL), potassium carbonate (1.68 g, 12.1 mmol) was
added and the reaction mixture was heated to 55°C for 12 h
under inert atmosphere. After the completion of reaction
monitored by TLC, the reaction mixture was cooled to 25°C,
filtered to remove potassium carbonate, and washed with acetone
(3 ꢁ 20 mL), and the mother liquor was concentrated. The crude
product was purified by column chromatography using silica gel
as stationary phase and hexane/ethyl acetate as mobile phase to
achieve compound 5a as white solid.
4.75 (s, 2H, CH2); 6.2–6.32 (d, 1H, pyrimidone-H at sixth position);
6.54–6.65 (d, 1H, pyrimidone-H at fifth position); 7.2–7.4 (d, 2H,
Ar-H); 7.55–7.63 (s, 1H, pyrimidone-H at second position); 7.75–
7.85 (d, 2H, Ar-H); 10.75 (bs, 2H, NH); MS (EI): m/z (74%) Mþ 340;
Anal. calcd. for C14H11F3N4O3 (340): C, 49.42; H, 3.26; F, 16.75; N,
16.47. Found C, 49.55; H, 3.33; F, 16.64; N, 16.38%.
–
5h: Yield: 63%; mp: 233–235°C; IR (KBr): 1620 (C O); 1675
–
ꢀ1
(amide, C O); 3720–3510 cm (NH–NH); 1H NMR (DMSO-d6) d:
–
–
4.73 (s, 2H, CH2); 6.41–6.43 (d, 1H, pyrimidone-H at sixth position);
6.6–6.69 (d, 1H, pyrimidone-H at fifth position); 7.25–7.41 (d, 2H,
Ar-H); 7.43–7.59 (s, 1H, pyrimidone-H at second position); 7.71–
7.82 (d, 2H, Ar-H); 10.6 (bs, 2H, NH); MS (EI): m/z (75%) Mþ 356;
Anal. calcd. for C14H11F3N4O4 (356): C, 47.20; H, 3.11; F, 16.00; N,
15.73. Found: C, 47.30; H, 3.25; F, 16.10; N, 15.62%.
–
–
Yield: 47%; mp: 245–247°C; IR (KBr): 1610 (C O); 1655 (amide,
–
C O); 3710–3505 cmꢀ1 (NH–NH); 1H NMR (DMSO-d6) d: 4.74 (s, 2H,
–
–
CH2); 6.1–6.24 (d, 1H, pyrimidone-H at sixth position); 6.57–6.64
(d, 1H, pyrimidone-H at fifth position); 7.35–7.42 (d, 2H, Ar-H);
7.5–7.55 (s, 1H, pyrimidone-H at second position); 7.8–7.85 (d, 2H,
Ar-H); 10.71 (bs, 2H, NH); MS (EI): m/z (75%) Mþ 306; Anal. calcd. for
C13H11ClN4O3 (306): C, 50.91; H, 3.61; Cl, 11.56; N, 18.27. Found: C,
51.02; H, 3.58; Cl, 11.48; N, 18.36%.
5i: Yield: 51%; mp: 265–267°C; IR (KBr): 1605 (C O); 1625
–
ꢀ1
(amide, C O); 3690–3505 cm (NH–NH); 1H NMR (DMSO-d6) d:
–
–
4.75 (s, 2H, CH2); 6.42–6.44 (d, 1H, pyrimidone-H at sixth position);
6.71–6.79 (d, 1H, pyrimidone-H at fifth position); 7.3–7.45 (d, 2H,
Ar-H); 7.51–7.6 (s, 1H, pyrimidone-H at second position); 7.8–7.89
(d, 2H, Ar-H); 10.8 (bs, 2H, NH); MS (EI): m/z (75%) Mþ 317; Anal.
calcd. for C13H11N5O5 (317): C, 49.22; H, 3.49; N, 22.07. Found: C,
49.35; H, 3.62; N, 22.17%.
–
5b: Yield: 41%; mp: 270–272°C; IR (KBr): 1615 (C O); 1615
–
(amide, C O); 3690–3525 cmꢀ1 (NH–NH); 1H NMR (DMSO-d6) d: 4.7
–
–
(s, 2H, CH2); 6.2–6.25 (d, 1H, pyrimidone-H at sixth position); 6.56–
6.66 (d, 1H, pyrimidone-H at fifth position); 7.33–7.45 (d, 2H,
Ar-H); 7.5–7.6 (s, 1H, pyrimidone-H at second position); 7.79–7.83
(d, 2H, Ar-H); 10.75 (bs, 2H, NH); MS (EI): m/z (74%) Mþ 351; Anal.
calcd. for C13H11BrN4O3 (351): C, 44.46; H, 3.16; Br, 22.75; N, 15.95.
Found: C, 44.59; H, 3.28; Br, 22.64; N, 15.85%.
–
–
5j: Yield: 48%; mp: 216.8–218.5°C; IR (KBr): 1615 (C O);
–
1665 cmꢀ1 (amide, C O); 3715–3500 cm
(NH–NH); 1H NMR
ꢀ1
–
–
(DMSO-d6) d: 2.9 (s, 6H, 2CH3); 4.73 (s, 2H, CH2); 6.39–6.48 (d, 1H,
pyrimidone-H at sixth position); 6.7–6.77 (d, 1H, pyrimidone-H at
fifth position); 7.28–7.4 (d, 2H, Ar-H); 7.45–7.55 (s, 1H, pyrimidone-
H at second position); 7.82–7.89 (d, 2H, Ar-H); 11.55 (bs, 2H, NH);
MS (EI): m/z (72%) Mþ 315; Anal. calcd. for C15H17N5O3 (315): C,
57.13; H, 5.43; N, 22.21. Found: C, 57.03; H, 5.55; N, 22.12%.
5c: Yield: 48%; mp: 282–284°C; IR (KBr): 1620 (C O); 1625
–
ꢀ1
(amide, C O); 3705–3535 cm (NH–NH);1H NMR (DMSO-d6) d:
–
–
4.65 (s, 2H, CH2); 6.2–6.25 (d, 1H, pyrimidone-H at sixth position);
6.55–6.7 (d, 1H, pyrimidone-H at fifth position); 7.3–7.45 (d, 2H,
Ar-H); 7.55–7.6 (s, 1H, pyrimidone-H at second position); 7.78–7.82
(d, 2H, Ar-H); 10.65 (bs, 2H, NH); MS (EI): m/z (74%) Mþ 398; Anal.
calcd. for C13H11IN4O3 (398): C, 39.22; H, 2.78; I, 31.87; N, 14.07.
Found: C, 39.29; H, 2.91; I, 31.97; N, 14.15%.
–
Biology
Xanthine oxidase inhibition studies
The XO inhibitory activity was monitored spectrophotometrically
following the absorbance of uric acid at 292 nm under aerobic
conditions [25, 26]. 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 Lowry’s method [29], using bovine serum albumin
(BSA) as standard.
5d: Yield: 55%; mp: 239–241°C; IR (KBr): 1610 (C O); 1635
–
ꢀ1
(amide, C O); 3725–3540 cm (NH–NH); 1H NMR (DMSO-d6) d:
–
–
2.38 (s, 3H, CH3); 4.65 (s, 2H, CH2); 6.2–6.25 (d, 1H, pyrimidone-H at
sixth position); 6.55–6.64 (d, 1H, pyrimidone-H at fifth position);
7.35–7.4 (d, 2H, Ar-H); 7.5–7.6 (s, 1H, pyrimidone-H at second
position); 7.75–7.82 (d, 2H, Ar-H); 10.74 (bs, 2H, NH); MS (EI): m/z
(73%) Mþ 286; Anal. calcd. for C14H14N4O3 (286): C, 58.73; H, 4.93;
N, 19.57. Found: C, 58.65; H, 5.05; N, 19.68%.
–
XO activity was recorded using a Shimadzu spectrophotometer
(UV-1800, Japan). 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
5e: Yield: 46%; mp: 214–216°C; IR (KBr): 1615 (C O); 1645
–
ꢀ1
(amide, C O); 3700–3505 cm (NH–NH); 1H NMR (DMSO-d6) d:
–
–
1.29 (s, 9H, tBu); 4.6 (s, 2H, CH2); 6.2–6.26 (d, 1H, pyrimidone-H at
sixth position); 6.52–6.6 (d, 1H, pyrimidone-H at fifth position);
7.3–7.45 (d, 2H, Ar-H); 7.54–7.6 (s, 1H, pyrimidone-H at second
position); 7.75–7.82 (d, 2H, Ar-H); 10.65 (bs, 2H, NH); MS (EI): m/z
(75%) Mþ 328; Anal. calcd. for C17H20N4O3 (328): C, 62.18; H, 6.14;
N, 17.06. Found: C, 62.06; H, 6.31; N, 17.18%.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

810
H. D. Gurupadaswamy et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 805–811
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:
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 [33].
Metal ion chelating assay
Xanthine oxidase Inhibition ð%Þ
Abs control ꢀ Abs sample
The Fe2þ-chelating ability of the extract was measured by the
ferrous iron–ferrozine complex at 562 nm [34]. The reaction
mixture containing FeCl2 (2 mM/L) and ferrozine (5 mM/L) along
with extracts was adjusted 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.
¼
ꢁ 100
Abs control
Abs control, absorbance of the control reaction (containing all
reagents except the test compound); Abs sample, absorbance of
the test compound.
Antioxidant activity
DPPH radical scavenging assay
DPPH radical scavenging assay was done according to Yen and
Duh [30]. Briefly, 1 mL of DPPH solution (0.1 mM, in 95% ethanol
v/v was incubated with different concentrations of the com-
pounds. The reaction mixture was shaken and incubated for
20 min at room temperature, and the absorbance was read at
517 nm against a blank. The antioxidant BHA was used as a
positive control in all the assays. The radical scavenging activity
was measured as decreased 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 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 post-hoc comparison test. Correlations between
quantitative properties were evaluated by calculating the Duncan
and Dennett’s coefficient. Statistical significance value was set at
p < 0.05.
ꢀ
ꢁ
Acontrol ꢀ Asample
Scavenging effect ð%Þ ¼
ꢁ 100
Acontrol
Molecular docking
The human milk XO (EC: 1.17.3.2) with PDB entry code: 2CKJ was
built using CPH Models server 3.0. The ligands were docked into
the active site using the molecular docking software Molegro
Molecular Viewer 2008, version 1.2.0 (http://molegro.com) [35]
with default parameters for calculating the docking modes of
small molecules into protein-binding sites based on their shape
complementarity. In this, we have used ChemScore, a scoring
function that is derived from regression against receptor–ligand
binding free energies [36].
Lipid peroxidation assay
LPO inhibitory activity was measured according to Kulkarni et al.
[31]. Egg lecithin (3 mg/mL in phosphate buffer, pH 7.4) was
sonicated (Hielscher GmbH UP 50H ultra-chill processor soni-
cator) 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 induced by adding 10 mL of 400 mM
FeCl3 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% TCA and 0.375% TBA and incubation
in a boiling water bath for 15 min. After centrifugation at
10,000 rpm, absorbance of the supernatant was measured at
532 nm. The scavenging effect was calculated using the equation
as described for DPPH.
One of the authors (S.A.K.) gratefully acknowledges the financial
support provided by Vision Group on Science and Technology,
Department of Information Technology, Biotechnology and Science &
Technology, Government of Karnataka, Bangalore, under the scheme
CISEE-programme.
Reducing power assay
The reducing power was measured by incubating the reaction
mixture (1 mL) containing the samples in the phosphate buffer
(0.2 M, pH 6.6) with potassium ferricynaide (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 [32]. Increased absorbance of the reaction mixture
indicated increased reducing power.
The authors have declared no conflict of interest.
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