3,5,2′,4′-Tetrahydroxychalcone, a new non-purine xanthine oxidase inhibitor

Article abstract of DOI:10.1016/j.cbi.2010.12.004

Xanthine oxidase is a key enzyme that catalyses hypoxanthine and xanthine to uric acid and the overproduction of uric acid will lead to hyperuricemia which is an important cause of gout. In the present study, three chalcone derivatives were synthesized and evaluated for inhibitory activity against xanthine oxidase in vitro. Of the compounds, only Compound 1, 3,5,2′,4′-tetrahydroxychalcone, exhibited a significant inhibitory activity on xanthine oxidase with an IC₅₀ value of 22.5 μM. Lineweaver-Burk transformation of the inhibition kinetics data demonstrated that it was a competitive inhibitor of xanthine oxidase and Ki value was 17.4 μM. In vivo, intragastric administration of Compound 1 was able to significantly reduce serum uric acid levels and inhibited hepatic xanthine oxidase activities of hyperuricemic mice in a dose-dependent manner. Acute toxicity study in mice showed that Compound 1 was very safe at a dose of up to 5 g/kg. These results suggest that Compound 1 is a novel competitive xanthine oxidase inhibitor and is worthy of further development.

Full text of DOI:10.1016/j.cbi.2010.12.004

Chemico-Biological Interactions 189 (2011) 161–166
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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
3,5,2ꢀ,4ꢀ-Tetrahydroxychalcone, a new non-purine xanthine oxidase inhibitor
Yanfen Niua, Huajie Zhub, Jia Liua, Huafang Fanb,c, Ling Suna, Wei Lua, Xu Liua, Ling Lia,∗
a Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University, Ren Min West Road 191#, Kunming 650031, PR China
b Organic Synthesis and Natural Product Laboratory, The State Key Laboratory of Phytochemistry and Plant Resources in West China,
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, PR China
c College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 August 2010
Received in revised form 2 December 2010
Accepted 7 December 2010
Available online 15 December 2010
Xanthine oxidase is a key enzyme that catalyses hypoxanthine and xanthine to uric acid and the overpro-
duction of uric acid will lead to hyperuricemia which is an important cause of gout. In the present study,
three chalcone derivatives were synthesized and evaluated for inhibitory activity against xanthine oxi-
dase in vitro. Of the compounds, only Compound 1, 3,5,2ꢀ,4ꢀ-tetrahydroxychalcone, exhibited a significant
inhibitory activity on xanthine oxidase with an IC50 value of 22.5 ␮M. Lineweaver–Burk transformation
of the inhibition kinetics data demonstrated that it was a competitive inhibitor of xanthine oxidase and
Ki value was 17.4 ␮M. In vivo, intragastric administration of Compound 1 was able to significantly reduce
serum uric acid levels and inhibited hepatic xanthine oxidase activities of hyperuricemic mice in a dose-
dependent manner. Acute toxicity study in mice showed that Compound 1 was very safe at a dose of up
to 5 g/kg. These results suggest that Compound 1 is a novel competitive xanthine oxidase inhibitor and
is worthy of further development.
Keywords:
3,5,2ꢀ,4ꢀ-Tetrahydroxychalcone
Xanthine oxidase
Hyperuricemia
Uric acid
© 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
oxidase inhibitors, especially inhibitors of novel chemical structure
type.
Xanthine oxidase is a key enzyme in purine metabolic pathway,
catalyzing the oxidation of hypoxanthine to xanthine and finally to
uric acid [1]. The overproduction or underexcretion of uric acid will
lead to hyperuricemia which is a key cause of gout [2]. In addition,
hyperuricemia has become an independent risk of chronic kidney
disease and cardiovascular disease [3,4]. Obviously, maintaining
uric acid in normal levels is the important therapy for prevention
of gout and other disorders. For most patients with primary gout,
overproduction is one of the major factors of hyperuricemia. Xan-
thine oxidase has been recognized as one of the promising targets
for treatment of hyperuricemia. Allopurinol, a potent xanthine oxi-
dase inhibitor with a purine backbone, has been used clinically
for more than 40 years [5]. Unfortunately, this drug has infre-
quent but severe side effects such as hypersensitivity syndrome
[6], Stevens–Johnson syndrome [7] and renal toxicity [8]. More
recently, febuxostat, a novel non-purine xanthine oxidase inhibitor,
has received approval for the treatment of hyperuricemia in gout
patients in USA and Europe [9]. Febuxostat is able to selectively
inhibit xanthine oxidase with a mode of mixed-type inhibition [10].
However, drugs available for treatment of gout patients with hype-
ruricemia are still limited. There is a need to develop other xanthine
Chalcones, present widely in medicinal plants such as Glycyrhiza
uralensis and Flos Carthami., have been reported to possess versatile
bioactivities including antitumor [11], free radical scavenging [12],
antibiosis [13], antivirus [14], antiulcer [15], spasmolysis [16], and
so on. However, there are few reports on the effects of chalcones on
xanthine oxidase activity. Recently, in our course of screening xan-
thine oxidase inhibitory agents from natural products, we found
that chalcones had potential inhibitory effect on xanthine oxidase
in vitro. Chalcones, as a kind of flavonoe compounds, have many
derivatives with different substituting groups at two benzene rings,
however the previous study indicate that two hydroxyl groups at
C-5 and C-7 in flavaonones is valid in inhibiting xanthine oxidase
activity with the presence of double bond of C C at C2 and C3
position [17,18]. Based on the findings, we have synthesized three
chalcone derivatives and evaluated their inhibitory effects of (Fig. 1)
on xanthine oxidase along with the precursor.
2. Materials and methods
2.1. Materials
Xanthine oxidase (EC 1.1.3.22) from bovine milk (0.4 units/mg
protein), whereas xanthine, sodium pyrophosphate, potassium
oxonate and allopurinol were purchased from Sigma and Aldrich
Chemical Co. (St. Louis, MO, USA). The biochemical kits used in the
∗
Corresponding author. Tel.: +86 0871 5922782; fax: +86 0871 5922780.
E-mail address: kmli62@yahoo.com.cn (L. Li).
0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2010.12.004

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Y. Niu et al. / Chemico-Biological Interactions 189 (2011) 161–166
2.3. Synthesis of chalcone derivatives
OMe
OH
Chalcone derivatives 1, 2, 3 were prepared through a facile
synthetic process (Scheme 1), by the Claisen–Schmidt condensa-
tion using the corresponding acetophenones and benzaldehydes
as described previously [19].
HO
OH
O
HO
OH
O
OMe
OH
2.4. Inhibitory activity of the chalcone derivatives on xanthine
oxidase
2
1
OMe
The inhibitory activity of xanthine oxidase in vitro was eval-
uated by the formation of uric acid levels which were measured
spectrophotometrically by following the increase in absorbance
at 295 nm [20]. The reaction mixture contained 0.3% sodium
pyrophosphate buffer (pH 8.3) and 8.35 U/L xanthine oxidase, with
or without the chalcone derivatives. Allopurinol was used as a pos-
itive control. After preincubation at 25 ◦C for 15 min, the formation
of uric acid in the reaction mixture was initiated by addition of
400 ␮M xanthine. In the enzyme kinetics tests, the concentrations
of xanthine used were 20, 30, 50, 70, 90, 100, 200, 400 and 600 ␮M,
respectively. The related inhibition type of the chalcone derivatives
on xanthine oxidase was identified from Lineweaver–Burk plots,
and Ki value was analyzed by the software GraphPad Prism 4.0.
HO
OH
O
MeO
OH
OMe
O
4
3
Fig. 1. Chemical structures of compounds.
experiments were products of Nanjing Jiancheng Bioengineering
Institute. All other reagents were commercially available and of
analytical grade.
2.5. Hypouricemic effects of 3,5,2ꢀ,4ꢀ-tetrahydroxychalcone in
hyperuricemic mice
2.2. Animals
The hyperuricemic mice were induced by an intraperitoneal
injection of uricase inhibitor potassium oxonate [21,22]. Male mice
were divided into several groups. The normal control mice and
hyperuricemic control mice were treated with 1.0% polyethylene
glycol 400 (PEG400), whereas other hyperuricemic mice were
administrated with 3,5,2ꢀ,4ꢀ-tetrahydroxychalcone (Compound 1)
and allopurinol dissolved in 1.0% PEG400, respectively. All drugs
were given intragastrically twice daily for five doses or once daily
for 1, 3, and 7 days. The last dosage was given at 1 h after injection
intraperitoneally with potassium oxonate (500 mg/kg) to increase
Adult Kunming mice weighing 18–22 g were obtained from the
Experimental Animal Center, Kunming Medical University (Certifi-
cate No. SCXK 2005-0008). The animals were housed on a constant
12-h light/dark cycle in a temperature- and humidity-controlled
room, and allowed free access to solid food and tap water for 1
week prior to the experiments. All procedures were carried out in
accordance with the Institute Ethical Committee for Experimental
Use of Animals.
HO
OH
HO
OH
O
R1O
OH
i
ii
OR2
O
resorcinol
R1O
OH
O
4
OR2
a R1 = Me
b R1 = MOM
5
v
3 R1 = Me, R2 = Me
8 R1 = MOM, R2 = MOM
9
R1 = MOM, R2 = Me
R2O
OR2
R2O
OR2
HO
OH
iv
iii
CHO
COOMe
a R1 = Me
b R1 = MOM
COOH
3,5-dihydroxybenzoic acid
OR3
vi
a R1 = Me
b R1 = MOM
6
7
HO
OH
OR3
O
1 R3 = H
2
R3 = Me
Scheme 1. Reagents and conditions: (i) 98% CH3COOH, ZnCl2 anhydrous, reflux; (ii) (CH3O)2SO2 (2a) or CH3OCH2Cl (2b), K2CO3 anhydrous, acetone, reflux; (iii) (3a)
(CH3O)2SO2, 30% NaOH aqueous, pH = 9, reflux; (3b) (1) CH3OH, p-toluene sulfonic acid, hexane, reflux; (2) CH3OCH2Cl, K2CO3 anhydrous, acetone, reflux; (iv) (1) 50%
NH2NH2 aqueous, reflux. (2) 27% K3[Fe(CN)6] aqueous, TBBA, toluene, 10–15 ◦C; (v) 14% KOH aqueous, EtOH, N2 protection r.t.; (vi) 3 M HCl aqueous, MeOH, reflux.

Y. Niu et al. / Chemico-Biological Interactions 189 (2011) 161–166
163
Table 1
Inhibition of chalcone derivatives 1, 2, 3 and intermediate 4 on xanthine oxidase.
100
A
Compound
Xanthine oxidase inhibition (%)
75
50
25
0
7.4
74
740 (␮M)
1
2
3
4
10.09 5.12
2.18 1.21
3.45 2.43
0.65 0.13
78.16 6.21
64.97 6.11
5.07 1.34
6.89 2.32
3.67 1.32
95.60 3.94
88.89 6.20
4.12 1.52
5.76 1.23
4.35 1.23
–
Allopurinol
Data are expressed as means S.E. for four independent experiments.
the serum uric acid level. Blood samples were collected from the
mice via orbit vein bleeding after exposure to ethylic ether. Serum
uric acid levels were determined by using the phosphotungstic acid
method [23]. Mouse liver was excised, frozen immediately and
stored at −20 ◦C for measurement of the xanthine oxidase activ-
ity using commercially available kits. Food was withdrawn from
the animals 6 h prior to collection of blood sample.
-5.5
-5.0
-4.5
-4.0
-3.5
-3.0
log10(3,5,2',4'-tetrahydroxychalcone) M
100
75
50
25
0
B
2.6. Acute toxicity experiment
Healthy Kunming mice of both sexes, weighing 18–22 g, were
divided into control and treated groups of 20 animals matched for
weight and size. The treated group received 5 g/kg Compound 1
intragastrically and the control group received 1.0% PEG400. Ani-
mals were observed carefully at 30, 60, 120, 240, and 360 min after
treatment. Mortality, body weights, and behavior of the mice were
observed and recorded daily for 14 days.
-7
-6
-5
-4
-3
log10(allopurinol) M
2.7. Statistical analysis
Fig. 2. Concentration-dependent inhibition of xanthine oxidase (XO) activity by
3,5,2ꢀ,4ꢀ-tetrahydroxychalcone (Compound 1) and allopurinol. The activity of xan-
thine oxidase was measured spectrophotometrically by following the increase of
absorbance at 295 nm. In this assay, medium contained 400 ␮M xanthine, 8.35 U/L
xanthine oxidase, and different concentrations of Compound 1 or allopurinol. Data
are expressed as mean S.E. from four independent experiments.
All data were expressed as mean S.E. IC50 values were deter-
mined from the concentration dependence curves. Data from the
in vivo experiments were subjected to one-way analysis of variance
followed by Dunnett’s multiple comparison tests using GraphPad
Prism version 4.0 software. Statistical significance was accepted at
the level of P < 0.05.
of 2.0 mg/kg twice daily for five times significantly attenuated
the serum uric acid levels and hepatic xanthine oxidase activities
in hyperuricemic mice, when compared to the untreated hyper-
uricemic mice (P < 0.05). The hypouricemic action of Compound
1 was as potent as that of allopurinol at the same molar dose
(P < 0.05).
Table 2 illustrates that the serum uric acid levels were time-
dependently decreased after the mice were administered with
Compound 1. A single dose of 2.0 mg/kg was able to reduce serum
uric acid to the normal, although it was not as effective as allopuri-
nol at the same molar dose (P < 0.05).
3. Results
3.1. Effects of the chalcone derivatives on xanthine oxidase
activity
The inhibitory activities of the chalcone derivatives (1–3) and
the precursor (4) on xanthine oxidase are shown in Table 1 and
Fig. 2, from which it can be seen that Compounds 2, 3 and 4 did
not show any activity even at a concentration of 740 ␮M. However,
Compound 1 significantly inhibited the activity of xanthine oxi-
dase in a concentration-dependent manner, similar to allopurinol.
The IC50 value was calculated to be 22.5 ␮M for Compound 1 and
3.57 ␮M for allopurinol.
To further identify the binding type of Compound 1 on xanthine
oxidase, Lineweaver–Burk double reciprocal plots were drawn. As
shown in Fig. 3, Compound 1 was a competitive inhibitor of xan-
thine oxidase (Fig. 3A) and the inhibition constant Ki value was
determined to be 17.4 ␮M (Fig. 3B).
3.3. Acute toxicity studies
An acute toxicity study showed that animals treated intra-
gastically with Compound 1 at a dose of 5.0 g/kg did not show
any significantly abnormal signs, behavioral changes, body weight
changes, or macroscopic findings at any time of observation. There
was no mortality observed during the 14 days of the experiments.
4. Discussion
3.2. Effects of 3,5,2ꢀ,4ꢀ-tetrahydroxychalcone on serum uric acid
level and hepatic xanthine oxidase activity in potassium
oxonate-induced hyperuricemic mice
Xanthine oxidase is a highly versatile flavoprotein enzyme,
ubiquitous among species (from bacteria to human) and within
the various tissues of mammals. The enzyme catalyses the oxida-
tive hydroxylation of purine substrates at the molybdenum center
to form uric acid. The hydroxylation mechanism catalyzed by the
molybdenum center of xanthine oxidase is depicted in Fig. 5, being
initiated by abstraction of a proton from the Mo–OH group by an
An intraperitoneal injection of potassium oxonate caused a
marked increase in serum uric acid level, and this increase could
be maintained for 5 h after the injection (data not shown). As
reported in Fig. 4, Compound 1 given intragastrically at a dose

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Y. Niu et al. / Chemico-Biological Interactions 189 (2011) 161–166
900
750
600
450
300
150
A
-0.01
0.00
0.01
0.02
1/[s](µM)-1
0.03
0.04
0.05
17500
B
12500
7500
2500
-2500
Fig. 4. Hypouricemic effects of intragastric 3,5,2ꢀ,4ꢀ-tetrahydroxychalcone (Com-
pound 1) on serum uric acid levels (A) and hepatic xanthine oxidase (XO) activities
(B) in mice pretreated with uricase inhibitor potassium oxonate. CON – normal con-
trol, M – hypouricemic control. Data are expressed as means S.E. for 10 mice in
each group. aP < 0.05 or less vs. control group; bP < 0.05 or less vs. hyperuricemic
group; P > 0.05 vs. allopurinol group (Dunnett’s test).
-20
-Ki
-10
10
20
30
40
3,5,2',4'-tetrahydroxychalcone (µM)
Fig. 3. Inhibition kinetics of xanthine oxidase by 3,5,2ꢀ,4ꢀ-tetrahydroxychalcone
(Compound 1). A, Lineweaver–Burk plots in the absence of Compound 1 (ꢀ) or at
the different concentrations of Compound 1: 3.7 (ꢁ), 18.5 (ꢂ), and 24.0 (ꢃ) ␮M.
Secondary plots to calculate the inhibition constant is shown in B (Ki). The assay
medium contained 8.35 U/L xanthine oxidase, different concentrations of xanthine,
and the indicated concentrations of Compound 1. Each point is the average value
from four independent experiments.
activity on xanthine oxidase in a concentration-dependent man-
ner, with an IC50 value of 22.5 ␮M. The potency of inhibition of the
compound was moderate when compared with allopurinol (IC50
3.57 ␮M), approximately 6-fold lower than that of allopurinol. The
inhibition kinetics of Compound 1 on xanthine oxidase showed
its competitiveness, which was the same as allopurinol. This indi-
cated that Compound 1 could compete with xanthine for the active
site of xanthine oxidase to form xanthine oxidase-inhibitor com-
plex, leading to the suppression of uric acid production. In order to
investigate structure–activity relationships, two analogs of Com-
pound 1 (2 and 3) and an intermediate (4) were designed and
synthesized. But they did not suppressed the activity of xanthine
oxidase, implying that the inhibitory activity might be related to
the hydroxyl groups present both on aromatic rings A and B and
phenyl ring B. The suggestion is also supported by the Kong et al.
[20] who discovered that isoliquiritigenin markedly inhibited the
activity of xanthine oxidase. As shown in Fig. 6, isoliquiritigenin
has hydroxyl groups both on aromatic rings A and B. However,
active site glutamate residue that is universally conserved in this
family of molybdenum-containing enzymes. Subsequent to depro-
tonation, nucleophilic attack on substrate and concomitant hydride
transfer to the metal center leads to an intermediate having prod-
uct coordinated to the reduced Mo center via the newly introduced
hydroxyl group. From the molybdenum center, electrons are passed
sequentially via two [2Fe–2S] clusters to an FAD site, where they
are finally passed to NAD+ or O2 [24].
Allopurinol, a competitive xanthine oxidase inhibitor, is hydrox-
ylated by the enzyme to alloxanthine (oxypurinol), which, once
formed, coordinates tightly to the reduced form of the molybdenum
center specifically, replacing the Mo–OH group of native enzyme. In
the current study, only Compound 1 showed a significant inhibitory
Table 2
Hypouricemic effects of intragastric Compound 1 on serum uric acid levels in the hyperuricemic mice induced by uricase inhibitor potassium oxonate: a time-course study.
Group
Dose
Serum uric acid (␮mol/L)
(mg/kg)
(␮mol/kg)
1-Day
3-Day
7-Day
Control
Hyperuricemia
Hyperuricemia + 1
Hyperuricemia + allopurinol
1.0% PEG400
1.0% PEG400
88.8 19.1
88.8 19.1
88.8 19.1
143.5 37.6a
109.2 22.7b
86.0 34.0b
143.5 37.6a
94.2 30.0b,c
62.6 10.2b
143.5 37.6a
103.6 19.6b,c
61.5 11.8b
2.0
1.0
7.4
7.4
Data are expressed as means S.E. for 10 mice in each group.
a
P < 0.05 or less vs. control group.
P < 0.05 or less vs. hyperuricemic group.
P < 0.05 or less vs. allopurinol group (Dunnett’s test).
b
c

Y. Niu et al. / Chemico-Biological Interactions 189 (2011) 161–166
165
Fig. 5. Catalytic mechanism of xanthine oxidase conversion of xanthine to uric acid at the molybdenum-containing active site.
Conflict of interest statement
OH
3'
4' OH
5'
2'
The authors declare there are no conflicts of interest.
HO
OH
O
HO
OH
O
OH
1'
Acknowledgments
The work is supported by the funds from NSFC (30960453
and 30873141), Yunnan Provincial Foundation (20080C021,
2009M018) and 973 Program (2009CB522300).
Isoliquiritigenin
1
Fig. 6. Chemical structures of Compound 1 and isoliquiritigenin.
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