Synthesis and evaluation of hydroxychalcones as multifunctional non-purine xanthine oxidase inhibitors for the treatment of hyperuricemia

Article abstract of DOI:10.1016/j.bmcl.2017.01.053

A series of hydroxychalcone derivatives have been designed, synthesized and evaluated for human xanthine oxidase (XO) inhibitory activity. Most of the tested compounds acted moderate XO inhibition with IC₅₀ values in the micromolar rang. Molecular docking and kinetic studies have been performed to explain the binding modes of XO with the selected compounds. In addition, in vitro antioxidant screening results indicated that some of the hydroxychalcones possessed good anti-free radical activities. Furthermore, the preferred compounds 16 and 18 were able to significantly inhibit hepatic xanthine oxidase activity and reduced serum uric acid level of hyperuricemic mice in vivo. In summary, compounds 16 and 18 with balanced activities of antioxidant, XO inhibition and serum uric acid reduction, which are promising candidates for the treatment of hyperuricemia and gout.

Full text of DOI:10.1016/j.bmcl.2017.01.053

Accepted Manuscript
Synthesis and evaluation of hydroxychalcones as multifunctional non-purine
xanthine oxidase inhibitors for the treatment of hyperuricemia
Zhaodi Xie, Xiaoting Luo, Zhuan Zou, Xiao Zhang, Feifei Huang, Ruishan Li,
Shijie Liao, Yun Liu
PII:
S0960-894X(17)30067-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.01.053
BMCL 24629
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 October 2016
15 January 2017
17 January 2017
Please cite this article as: Xie, Z., Luo, X., Zou, Z., Zhang, X., Huang, F., Li, R., Liao, S., Liu, Y., Synthesis and
evaluation of hydroxychalcones as multifunctional non-purine xanthine oxidase inhibitors for the treatment of
hyperuricemia, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.
2017.01.053
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Synthesis and evaluation of hydroxychalcones as
multifunctional non-purine xanthine oxidase inhibitors for
the treatment of hyperuricemia
Zhaodi Xie a, Xiaoting Luo a, Zhuan Zou a, Xiao Zhang a, Feifei Huang a, Ruishan Li a,
Shijie Liao *, b and Yun Liu *, b
a First Affiliated Hospital, Guangxi Medical University, Nanning, P. R. China
b
Department of Orthopedics, First Affiliated Hospital, Guangxi Medical University,
Nanning, P. R. China
*Corresponding Authors: E-mail: liuyun200450250@sina.com (Yun Liu);
274021690@qq.com (Shijie Liao).
Abstract
A series of hydroxychalcone derivatives have been designed, synthesized and
evaluated for human xanthine oxidase (XO) inhibitory activity. Most of the tested
compounds acted moderate XO inhibition with IC50 values in the micromolar rang.
Molecular docking and kinetic studies have been performed to explain the binding
modes of XO with the selected compounds. In addition, in vitro antioxidant screening
results indicated that some of the hydroxychalcones possessed good anti-free radical
activities. Furthermore, the preferred compounds 16 and 18 were able to significantly
inhibit hepatic xanthine oxidase activity and reduced serum uric acid level of
hyperuricemic mice in vivo. In summary, compounds 16 and 18 with balanced
activities of antioxidant, XO inhibition and serum uric acid reduction, which are
promising candidates for the treatment of hyperuricemia and gout.
Keywords: Xanthine oxidase, Hydroxychalcone derivatives, Antioxidant,
Hyperuricemia
1

Hyperuricemia is a common metabolic disease characterized by elevated serum uric
acid (UA) which is the final oxidation product of purine catabolism in human.1
Epidemiologic data indicate that the prevalence of hyperuricemia has been increasing
in recent decades, but its treatment is still limited.2, 3
In the process of the evolution, human and great apes lost the uricase enzyme
which can convert uric acid to 5-hydroxyisourate, and eventually to the more soluble
allantoin.4 As a consequence, serum uric acid level in human is higher and can be
altered more easily by various factors than that in other mammals.5 Hepatic
over-production or renal under-excretion of uric acid can both contribute to
hyperuricemia and further cardiovascular and metabolic disorders associated with
hyperuricemia.6, 7 Xanthine oxidase (XO) is a key enzyme that catalyzes the oxidation
of xanthine and hypoxanthine into uric acid. As overproduction of uric acid is the
primary cause of hyperuricemia, XO has always been considered as the most
promising target for treatment of this condition.8 Nowadays, allopurinol and
febuxostat are clinically available to reduce serum uric acid levels by inhibiting XO,
but more and more toxicity cases and side effects are reported during the use of them.9,
10
So, new XO inhibitors with more specific effects and fewer side effects than
allopurinol and febuxostat are needed for treating hyperuricemia and gout.
Hyperuricemia has been increasingly reported to be associated with the
pathogenesis of a wide range of chronic diseases such as hypertension, diabetes
mellitus, metabolic syndrome, and renal and cardiovascular disease.11, 12 Recent
research indicates that oxidative stress may play a major pathophysiological role in
2

the development of hyperuricemia involving diseases mentioned above, especially
renal and cardiovascular disease.13, 14 Uric acid, while being a potent antioxidant in
the extracellular environment, is a pro-oxidant inside the cell where it oxidizes lipids,
reduces nitric oxide availability in endothelial cells, and increases reactive oxygen
species (ROS).15 As a result, high levels of serum uric acid cause an increase of
cardiovascular and renal risk.16, 17 ROS overproduction may be also caused by the
enhancement of XO expression and activity. While hepatic XO catalyzes the
oxidation of hypoxanthine and xanthine to uric acid, superoxide ion and hydrogen
peroxide are also formed in this process.18 The production of radical species by XO
activity has been intensively studied in conjunction with kidney injuries and various
forms of ischemic and vascular injuries.19, 20 These mechanisms suggest that there is a
need for novel antioxidant approaches preventing ROS formation to attenuate renal
and cardiovascular disorders associated with hyperuricemia.
Chalcones, a group of naturally occurring compounds, have attracted increasingly
widespread attention in present-day society, since they possess a wide range of
pharmacological properties possess a variety of bioactivities, including antitumor, free
radical scavenging, antibiosis, antivirus, antiulcer and spasmolysis.21-25 In the past
years, some natural and synthetic hydroxychalcones have been reported as XO
inhibitors.26-30 Additionally, many flavone compounds such as rutin, quercetin and
mulberroside have been found with hypouricemic effects in experimental
hyperuricemia animals.31-35 Based on these findings, we deduce that the XO inhibition
and hypouricemic effect are impacted by the hydroxyl substitution in chalcone
3

skeleton. Accordingly, we have designed and synthesized a series of hydroxychalcone
derivatives and evaluated their XO inhibitory, antioxidant potency and the
structure-activity relationship (SAR) studies, as well as hypouricemic action in a
mouse model of hyperuricemia.
The hydroxychalcone derivatives (1-15) were synthesized along with the pathway
shown in Scheme 1 with two methods.36 The commercially available starting
materials hydroxyacetophenone reacted with the appropriate hydroxylbenzaldehydes
in the presence of 20% KOH (method A) or piperidine heated to 85 °C (method B) to
give the target compounds in moderate to good yields. Compounds 16-18 were
synthesized by a hydroxyl-protecting strategy in Scheme 2. Benzyl-protected
hydroxyacetophenone reacted with benzyl-protected hydroxylbenzaldehydes in the
presence of 20% KOH to afford the benzyloxychalcones, then benzyloxychalcones
were treated with BBr3 under ice-bath to give compounds 16-18. All compounds were
purified by column chromatography. The structures of the compounds were verified
by 1H NMR, 13C NMR and mass spectrometry as cited in the experimental section.
4

Scheme 1-2. Synthesis of the hydroxychalcone derivatives 1-18.
The XO inhibitory activities of compounds 1-18 were measured
spectrophotometrically under aerobic conditions using xanthine as the substrate by
following the increase in absorbance at 295 nm, with Allopurinol as a reference.28, 37
The corresponding IC50 values were demonstrated in Table 1. Based on the screening
data, it could be seen that most of the tested compounds were able to inhibit XO with
IC50 values in the micromolar range. Among the compounds, 2, 8, 9, 12, 15, 16 and
18 showed good inhibitory activities, indicating that hydroxyl-substitution at
2-position was in favor of XO inhibition. Compared with different
hydroxyl-substituted chalcones, the number of hydroxyl-substituted affected the
inhibitory activities toward XO, and trend of was tri-hydroxychalcones >
di-hydroxychalcones > mono-hydroxychalcones. It was worth noting that compounds
16 and 18 exhibited higher XO inhibition than the tri-hydroxychalcone 17.
5

Additionally, compounds 1, 4 and 14 were inactive at highest concentration tested
(1000 µM). It could be concluded that the position of hydroxyl-substitution impacted
XO inhibition. The results above also suggested that the hydroxychalcones could act
as XO inhibitors and be further investigated.
Table 1. In vitro XO inhibition and antioxidant activities of the synthesized
compounds
Compoun
XO
inhibition
ABTS assay
(trolox
DPPH assay
inhibitory
ds
IC50 (µM) equivalent) b)
rate c)
a)
1
2
3
4
5
6
7
N d)
71.3 8.6
165 10
N d)
145 6.6
228 11
159
N f)
18.4%
13.5%
11.7%
12.5%
10.8%
11.3%
25.7%
N f)
N f)
N f)
N f)
N f)
5
N f)
8
9
10
11
12
13
14
87.4 3.1
91.8 2.1
107 11
117 9
88.3 6.3
133 11
N d)
0.17
0.93
0.38
0.43
0.18
0.16
N f)
28%
108 7
197 16
35.9 %
24.3%
19.7%
10.8%
15
16
17
18
79.4 2.7
56.8 2.3
0.24
0.87
0.45
0.51
23.1%
128 10 µM
38.6%
108
8
47.3 4.1
287 17 µM
Allopuri
19.6 1.8
N f)
20.5%
nol
Resverat
rol
n.t. e)
0.65
95.1 5.3µM
a) IC50: 50% inhibitory concentration (means SEM of three experiments). b) Data are
c)
expressed as (mmol trolox)/(mmol tested compound).
Inhibitory rates of the
compounds at 2000 µM. d) Inactive at 1000 µM (highest concentration tested), at
higher concentrations the compounds precipitate. e) n.t.=not tested. f) trolox equivalent
lower than 0.1.
Compound 16 was used to further investigate the mode of XO inhibition. The type
of XO inhibition was determined by Michaelis-Menten kinetic experiments.28 The
6

catalytic rates were measured at five different allopurinol concentrations, and each
plot was constructed at four different concentrations of 16 (0, 25, 50 and 100 µM).
The overlaid reciprocal Lineweaver-Burk plots (Figure 2) showed that the plots for
different concentrations of 16 were linear and intersected at the second quadrant. This
pattern indicated that 16 was mixed type XO inhibitor, and this result further proved
that the hydroxychalcones were mixed type XO inhibitors.
Figure 2. Kinetic study on the mechanism of XO inhibition by 16. Overlaid
Lineweaver-Burk reciprocal plots of XO initial velocity at increasing xanthine
concentration in the absence of inhibitor and in the presence of 16 are shown. Enzyme
activity was expressed as change in absorbance at 295 nm per unit time. Lines were
derived from a weighted least-squares analysis of the data points.
In order to explain the binding modes of compounds 16 and 18, the molecular
docking study was performed using software package MOE. Since
molybdenum-pterion sites of both XO and bovine xanthine dehydrogenase (XDH) are
structurally equivalent, the X-ray crystal of the complex XDH/Febuxostat (PDB code
1N5X) used in the docking study was obtained from the RCSB Protein Data Bank
(PDB) database.37-39 The 3D and 2D binding pictures were illustrated in Figure 3. As
shown in Figure 3A-B, compound 16 located in the binding pocket of XO. There
were three hydrogen bonds formed between the hydroxyls of the ligand 16 and Tyr
7

1083, Ser 1082, Gln 1194. In addition, the similar interaction between 18 and XO was
shown in Figure 3C-D. Surprisingly, the hydroxyl groups of 18 exhibited extra
hydrogen bonds interaction with Arg 912, Ser 1080 and Gln 112, which might help 18
to anchor onto the binding site more tightly. These observations could provide a
rational explanation for the inhibitory potency between 16, 18 and XO, which also
indicated that introducing hydroxyl groups might be favorable for hydroxychalcone
analogues to display better XO inhibitory potency.
Figure 3. 3D and 2D docking models of compound 16 (A, B) and 18 (C, D) with XO
generated by MOE. Atom colors: yellow-carbon atoms of 16 and 18, gray-carbon
atoms of residues of XO, dark blue-nitrogen atoms, red-oxygen atoms. The dashed
lines represent the interactions between the protein and the ligand.
ROS overproduction has been associated with the enhancement of XO expression
8

and activity.15 Thus, the antioxidant may be an important therapeutic strategy for the
treatment of hyperuricemia and gout. DPPH radicals can be used in preliminary
screening of compounds capable of scavenging ROS, since these nitrogen radicals are
much more stable and easier to handle than oxygen free radicals.40 For comparison
purpose, resveratrol was used as the reference. The IC50 values of all
hydroxychalcones are summarized in Table 1. From the result, it could be seen that
some multi-hydroxychalcones (9, 10, 16 and 18) exhibited good scavenging activities
equivalent to resveratrol, while the mono-hydroxychalcones (2-7) and some
mono-hydroxychalcones (8, 11-15) showed lower radical scavenging activities. These
results indicated that the number and position of the OH groups were critical in
determination of scavenging activity. The free radical scavenging trend of the
hydroxychalcones was impacted by the number of OH groups (tri-hydroxychalcones >
di-hydroxychalcones > mono-hydroxychalcones).
Compounds 1-18 were also tested for their antioxidant activities by using the ABTS
method.41 Trolox, a water-soluble vitamin E analog, was used as a reference.
Resveratrol was also determined for comparison. The antioxidant activities were
provided as a trolox equivalent, with their relative potency compared with trolox. As
shown in Table 1, these compounds demonstrated moderate antioxidant activities.
Among them, compounds 9, 11 and 16-18 possessed the ability to scavenge the ABTS
radical with 0.93, 0.43, 0.87, 0.45, and 0.65 trolox equivalents respectively.
Interestingly, ortho- or para-position OH groups on the ring of benzaldehyde were
favorable to the ABTS radicals scavenging activity. The similar free radical
9

scavenging trend of the hydroxychalcones was observed between ABTS assay and
DPPH assay. According to that, the results indicated that polyphenol derivatives
possessed good antioxidant activities and can be considered as potential choices for
the development of anti-hyperuricemia drugs.
Therapeutic effects of compounds 16 and 18 on serum uric acid level and hepatic
xanthine oxidase activity were tested in potassium oxonate-induced hyperuricemic
37, 42, 43
mice
As shown in Table 2 and Figure 4A, intragastrical administration of
potassium oxonate significantly increased serum uric acid level (P < 0.001) in
hyperuricemia mice, compared with normal mice. After the treatment of
hydroxychalcone 16 at both low (10 mg/kg) and high (50 mg/kg) doses, serum uric
acid level of hyperuricemia mice can be significantly decreased at a dose-dependent
manner (P < 0.05, P < 0.01). The treatment of hydroxychalcone 18 has similar effect
on hyperuricemia mice and it remarkably decreased serum uric acid level at both 10
mg/kg and 50 mg/kg doses (P < 0.05, P < 0.01). Allopurinol at 10 mg/kg, as a positive
control, significantly reduced serum uric acid level of hyperuricemia mice (P < 0.001),
even lower than that of normal mice.
Table 2. Effects of compounds 16 and 18 on serum uric acid level and hepatic XO
activity in hyperuricemic mice.
Group
Dose
(mg/kg)
serum uric acid
(mg/dl)
XO activity
(U/g protein)
62.1 1.1
Control
Hyperuricemia
16
-
-
4.38 0.19
5.89 0.36###
5.21 0.28*
4.50 0.31**
5.14 0.25*
4.45 0.27**
4.14 0.18***
81.5 1.4###
73.2 2.2*
64.1 2.6**
70.5 2.3*
62.0 2.9**
50.9 0.9***
10
50
10
50
10
18
Allopurinol
###P<0.001, compared with normal group; P<0.05, P<0.01, ***P<0.001, compared
*
**
10

with hyperuricemia + vehicle group. Values were mean SEM (n = 8)
To evaluate whether hydroxychalcones 16 and 18 had effects on urate production in
mice, liver XO activity was determined (Table 2 and Figure 4B). As expected, the
hepatic XO activity of model hyperuricemia mice is much higher than that of normal
mice (P < 0.001). However, the hepatic XO activity of hyperuricemia mice treated
with hydroxychalcone 16 at 10 mg/kg and 50 mg/kg is significantly decreased (P <
0.05, P < 0.01), compared with model mice. Similarly, hydroxychalcone 18 at both
low（10 mg/kg） and high （50 mg/kg）doses notably inhibited liver XO activity of
hyperuricemia mice (P < 0.05, P < 0.01). Allopurinol at the dose of 10 mg/kg also
significantly suppressed hepatic XO activity of hyperuricemic mice (P < 0.001).
Figure 4. Hypouricemic effects of hydroxychalcones 16 and 18 on hepatic xanthine
oxidase (XO) activities (A) and serum uric acid level (B) in mice pretreated with
11

uricase inhibitor potassium oxonate. ###P<0.001, compared with normal group;
*P<0.05, **P<0.01, ***P<0.001, compared with hyperuricemia + vehicle group. Values
were mean SEM (n = 8)
The procedure for acute toxicity study followed similar protocol from the reported
study.44 After administration of the compounds 16 and 18 (5,000 mg/kg), mice were
monitored continuously for the first 4 h for any abnormal behavior and mortality
changes, intermittently for the next 24 h. During the experimental period, no acute
toxicity, such as mortality, or significant abnormal changes in water or food
consumption or body weight reduction were observed. This indicated that 16 and 18
were nontoxic and tolerated at doses up to 5,000 mg/kg.
In conclusion, this work reported the synthesis and evaluation of hydroxychalcone
derivatives as multifunctional XO inhibitors with antioxidant and reducing serum uric
acid potency in vitro and in vivo. Among them, compounds 16 and 18 emerged as the
most potent XO inhibitors and possessed balanced activities. Molecular docking and
kinetic studies revealed that representative compound 16 acted as a mixed-type
inhibitor for XO. In addition, compounds 16 and 18 showed good in vitro free radical
scavenging activity and significant in vivo serum uric acid reducing potency.
Moreover, the acute toxicity test indicated that 16 and 18 were nontoxic.
Consequently, compounds 16 and 18 appeared to be promising candidates for treating
hyperuricemia, gout, and other diseases involving XO.
Acknowledgements
This work was supported in part by the Guangxi basic ability promotion project of
young teachers (KY2016YB097 and KY2016YB091).
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Graphical Abstract:
16

Highlights
ꢀ A series of hydroxychalcone derivatives (1-18) were designed and synthesized.
ꢀ Some compounds exhibited XO inhibition, antioxidant and hypouricemic
properties.
ꢀ 16 and 18 can inhibit hepatic XO and reduced serum uric acid level of
hyperuricemic mice.
ꢀ 16 and 18 might be promising candidates for treating hyperuricemia and gout.
17