Discovery of novel curcumin derivatives targeting xanthine oxidase and urate transporter 1 as anti-hyperuricemic agents

Article abstract of DOI:10.1016/j.bmc.2016.10.022

A series of curcumin derivatives as potent dual inhibitors of xanthine oxidase (XOD) and urate transporter 1 (URAT1) was discovered as anti-hyperuricemic agents. These compounds proved efficient effects on anti-hyperuricemic activity and uricosuric activity in vivo. More importantly, some of them exhibited proved efficient effects on inhibiting XOD activity and suppressing uptake of uric acid via URAT1 in vitro. Especially, the treatment of 4d was demonstrated to improve uric acid over-production and under-excretion in oxonate-induced hyperuricemic mice through regulating XOD activity and URAT1 expression. Docking study was performed to elucidate the potent XOD inhibition of 4d. Compound 4d may serve as a tool compound for further design of anti-hyperuricemic drugs targeting both XOD and URAT1.

Full text of DOI:10.1016/j.bmc.2016.10.022

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel curcumin derivatives targeting xanthine oxidase
and urate transporter 1 as anti-hyperuricemic agents
Gui-Zhen Ao a, Meng-Ze Zhou b, Yu-Yao Li a, Si-Ning Li a, Hua-Nian Wang a, Qian-Wen Wan a,
Huan-Qiu Li a, , Qing-Hua Hu b,
⇑
⇑
a Department of Medicinal Chemistry, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, PR China
b State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of curcumin derivatives as potent dual inhibitors of xanthine oxidase (XOD) and urate trans-
porter 1 (URAT1) was discovered as anti-hyperuricemic agents. These compounds proved efficient effects
on anti-hyperuricemic activity and uricosuric activity in vivo. More importantly, some of them exhibited
proved efficient effects on inhibiting XOD activity and suppressing uptake of uric acid via URAT1 in vitro.
Especially, the treatment of 4d was demonstrated to improve uric acid over-production and under-excre-
tion in oxonate-induced hyperuricemic mice through regulating XOD activity and URAT1 expression.
Docking study was performed to elucidate the potent XOD inhibition of 4d. Compound 4d may serve
as a tool compound for further design of anti-hyperuricemic drugs targeting both XOD and URAT1.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 5 September 2016
Revised 17 October 2016
Accepted 18 October 2016
Available online xxxx
Keywords:
Xanthine oxidase
Urate transporter 1
Anti-hyperuricemic
Uricosuric
Curcumin derivatives
1. Introduction
Stevens Johnson syndrome, which may be fatal.10 In addition,
febuxostat has been associated with cardiovascular complications
Uric acid is the end product of purine metabolism in human
beings because a gene encoding uricase has undergone mutational
silencing during hominoid evolution.1–3 Hyperuricemia, character-
ized by the high level of serum uric acid, is a pathological condition
which may originate from excessive production and/or impaired
excretion of uric acid.4 Persistent hyperuricemia is widely accepted
as the primary risk factor for urate deposition diseases, such as
gout and renal damage.5,6 Reduction of the uric acid would play
a causal role in the treatment of urate-related disease. Therefore,
development of anti-hyperuricemic drugs has become increasingly
important.
Currently, there are several drug strategies to control urate
levels (Fig. 1). For example, the xanthine oxidase inhibitor allopuri-
nol and febuxostat have been the most commonly used urate-low-
ering drug. Xanthine oxidase (XOD) in liver is the key enzyme to
catalyze uric acid production, which catalyzes the oxidation of
hypoxanthine to xanthine and further catalyze the oxidation of
xanthine to uric acid.7 Inhibitors of xanthine oxidase block conver-
sion of xanthine to uric acid, exhibiting potentially effects on hype-
ruricemia.8,9 Nevertheless, only about 40% of patients are able to
meet treatment goals via allopurinol, and it occasionally causes
causing the Food and Drug Administration (FDA) to require a cau-
tionary statement on the drug insert.11 Thus, it requires more
diversity of treatment option for hyperuricemia.
On the other hand, benzbromarone, a drug with potent urico-
suric activity, effectively reduces serum urate levels through tar-
geting urate transporter 1 (URAT1), with most people achieving
normal uric acid values.12–14 URAT1 (SLC22A12) at the luminal
membrane, is the main renal-specific transporter involved in urate
reabsorption in kidneys, which has been considered as a efficient
target for treating hyperuricemia.15 Hypouricemia was induced
by an increase in urate urinary excretion and modest inhibition
of XO. Urate urinary excretion was observed to be due to inhibition
of URAT1 but not GLUT9. KUX-1151 is a potential XO and URAT1
dual inhibitors currently undergoing phase II trials (Kissei Pharma-
ceutical CO).16 Given these considerations, the development of
novel compounds that acted by dual inhibition of XOD and URAT1
could be a promising approach for treating hyperuricemia.
Curcumin, a polyphenolic compound derived from dietary spice
turmeric, possesses diverse pharmacologic effects including anti-
inflammatory, antioxidant17–19, antiproliferative20 and uricosuric
activities.21,22 In our previous publication23, a series of a,b-unsatu-
rated curcumin cyclohexanone analogous their antiproliferative
activities have been studied. Recently, the uricosuric activity of
curcumin has been investigated; it is potentially useful for treat-
⇑
Corresponding authors.
E-mail addresses: huanqiuli@suda.edu.cn (H.-Q. Li), huqh@cpu.edu.cn (Q.-H. Hu).
http://dx.doi.org/10.1016/j.bmc.2016.10.022
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ao, G.-Z.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.022

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G.-Z. Ao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3. Results and discussion
N
N
H
N
COOH
N
3.1. Anti-hyperuricemia activity in vivo
S
N
N
O
In order to monitor the efficacy of different synthesized
a,b-
OH
unsaturated cyclohexanone and cyclopentanone analogous of cur-
cumin, serum uric acid levels were determined using hyper-
uricemic mice models induced by oxonate treatment, allopurinol
and benzbromarone, reported to inhibit xanthine oxidase and
URAT1, respectively, were also screened under identical conditions
for comparison. The inhibition ratios exhibited the anti-hyper-
uricemia activities of the synthesized curcumin derivatives and
the results were summarized in Table 1. As expected, these cur-
Allopurinol
Febuxostat
Br
HO
Br
O
O
O
O
O
HO
OH
cumin
tives
a
,b-unsaturated cyclohexanone and cyclopentanone deriva-
O
exhibited remarkable anti-hyperuricemia activity
comparable to the positive control.
Curcumin
Benzbromarone
Preliminary SAR (Structure Activity Relationship) studies were
performed to deduce how the structure variation and modification
could affect the anti-hyperuricemia activity. Generally, the results
in Table 1 showed that those cyclohexanone derivatives would
perform better than cyclopentanone analogous. As to cyclohex-
anone derivatives 4a–4j, the electron withdrawing substituents
on the phenyl ring at R1 position input substantial effects on the
antiproliferative capability of the compounds. Compounds 4a–4e
and 4j exhibited strong uric acid lowering activity (32.1–92.4%),
which is slightly more potent compared to other compounds with
electron-donating group at R1 position (4f–4i). 4f and 4g with
strong electron-donating hydroxyl substitution on R-phenyl ring,
showed low in vivo efficacy (7.7% and 11.8%, respectively).
Secondly, as for compounds with halogen substitution on R-
phenyl ring, we could perceive the tendency that Br>Cl in the ser-
ies. Compounds with R1 substitution at the para position (4d, 4i,
7d and 7l) showed better activities than those with substitution
at the meta position (4c, 4e, 7c and 7a). Especially, compounds
4d showed the most potent inhibitory activity (uric acid lowering
activity 92.4%), and comparable to the positive control allopurinol
(148.32%) and benzbromarone (99.7%).
Figure 1. Chemical structures for allopurinol, febuxostat, benzbromarone and
curcumin.
ment of hyperuricemia or gout as a new URAT1 inhibitor.21 There-
fore, in continuation of our earlier studies focus on the diverse bio-
logical activity of curcumin analogs, the a,b-unsaturated curcumin
cyclohexanone analogous had been screened for their anti- hyper-
uricemia activity and some compounds showed potent anti-hype-
ruricemia activity. Thus, in this manuscript, we discovered a series
of
a,b-unsaturated cyclohexanone and cyclopentanone analogous
as anti-hyperuricemia agents. Notably, compound 4d is the XOD/
URAT1 dual inhibitors with excellent in vitro and in vivo anti-
hyperuricemia potency.
2. Chemistry
Twenty-two
tanone analogous of curcumin were synthesized to be screened
for the anti-hyperuricemia. The preparation of the ,b-unsaturated
a,b-unsaturated cyclohexanone and cyclopen-
a
cyclohexanone analogous have been reported in our previous pub-
lication.23 As show in Scheme 1, the intermediates 3 or 6 was pre-
pared by the Stork reaction. Enamines 2 or 5 were synthesized by
cyclohexanone/cyclopentanone and morpholine in benzene. 3 or 6
were subsequently obtained by hydrolysis of 3,5-dimethoxyben-
3.2. Uricosuric activity in vivo
To confirm whether the anti-hyperuricemia activities of synthe-
sized curcumin derivatives were attributed to their uricosuric
effects, we detected uric acid excretion in 24 h of hyperuricemic
mice with or without drug treatment. The results were summa-
rized in Table 1. Taken together, the designed and synthesized cur-
zaldehyde and 2 or 5. Then the
a,b-unsaturated cyclohexanone
and cyclopentanone analogous were synthesized by Claisen-Sch
midt reaction, various substitute benzaldehydes and 3 or 6 were
dissolved in 10% NaOH ethanol solution at room temperature to
give the target compounds 4a–4j and 7a–7l. All compounds were
purified by silicagel column and identified by elemental H and C
NMR and HRMS.
cumin a,b-unsaturated cyclohexanone analogous did demonstrate
fairly potent uricosuric activity in vivo. Compounds 4c, 4d and 4j
displayed the most potent uricosuric in the in vivo assay, with uric
acid excretion in 24 h elevation of 44.7%, 75.7%, and 36.4%, respec-
O
O
O
R
CHO
H3CO
1. 3,5-OCH3PhCHO
H3CO
O
N
Morpholine
Morpholine
R
R
2.HCl
NaOH or HCl
OCH3
OCH3
3
4a-4j
2
1
O
O
O
R
CHO
H3CO
H3CO
O
1. 3,5-OCH3PhCHO
N
2.HCl
NaOH or HCl
OCH3
OCH3
6
7a-7l
5
1
Scheme 1. Synthesis of target compounds.
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G.-Z. Ao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Table 1
In vivo anti-hyperuricemia activity and uricosuric activity of the synthesized compounds. (% at 10 mg/kg)
Compd.
R
Serum uric acid
level inhibition
Uric acid excretion
inhibition in 24 h
elevation
Compd.
R
Serum uric acid level
inhibition (% at 10 mg/kg) inhibition in 24 h
elevation
Uric acid excretion
4a
4b
4c
4d
4e
3-ClPh
2-ClPh
3-BrPh
4-BrPh
3-OCH3Ph
32.15%
36.52%
47.33%
92.45%
15.82%
22.17%
38.68%
44.71%
75.77%
16.93%
7a
7b
7c
7d
7e
3-ClPh
2-ClPh
3-BrPh
4-BrPh
3-
40.17%
16.82%
25.20%
44.52%
12.95%
26.40%
17.52%
23.48%
25.79%
8.60%
OCH3Ph
3,4-
4f
3,5-
7.74%
19.35%
7f
11.78%
12.90%
diisopropyPhenol
4-OHPh
4-N(CH3)2Ph
diOHPh
4-OHPh
4-N
4g
4h
11.88%
20.06%
15.82%
10.74%
7g
7h
9.36%
15.10%
10.16%
9.64%
(CH3)2Ph
4-
OCH3Ph
4-
4i
4j
4-OCH3Ph
25.21%
50.78%
7.35%
7i
7j
18.82%
54.39%
11.52%
40.55%
4-SO2CH3Ph
36.47%
SO2CH3Ph
2-BrPh
4-ClPh
Allopurinol
Benzbromarone
118.32%
99.74%
88.60%
37.58%
7k
7l
14.09%
30.62%
9.90%
7.35%
tively, and comparable to the positive control benzbromarone
(88.6%). Impaired renal excretion of uric acid was considered to
be the major cause of hyperuricemia, increased uric acid excretion
played an important role in lowering serum uric acid levels. Given
these considerations, these series of curcumin derivatives dis-
played potent uricosuric activities, contributing to their beneficial
effects on hyperuricemia. Nevertheless, the results also reflected
that the effects of curcumin derivatives on hyperuricemia were
not completely parallel to their uricosuric effects, which suggested
that the curcumin derivatives might affect the production of uric
acid.
4d (ꢀ8.19 kcal/mol) was less than score of febuxostat
(ꢀ11.14 kcal/mol). Compound 4d shows important interactions
between hydroxybenzyl ring and the residues known to be impor-
tant for binding of endogenous substrate of XOD, GLU802, and
Thr1010, the cyano groups of 4d interacts with GLU802 and
Thr1010 via hydrogen bond interactions. The modeling also sug-
gested that there is a
p-cation interaction between 4-BrPh ring of
compound 4d and the side chain of LYS771,
p-cation interaction
energies are of the same order of magnitude as hydrogen bonds
or salt bridges and play an important role in stabilizing the three
dimensional structure of a protein.
3.3. In vitro xanthine oxidase inhibition activity and molecular
docking
3.4. In vitro URAT1 inhibition activity
Since the URAT1 is the main renal-specific transporter involved
in urate reabsorption in kidneys, we examined URAT1 inhibitory
activities of curcumin derivatives 4a–4d, 4j, 7a–7c and 7j, which
exhibited their potent capacities on uric acid under-excretion. This
result was well consistent with that from their uricosuric activity.
As shown in Table 3, compounds 4d displayed the most potent
inhibitory activity (38.2%), less comparable to the positive control
Since the xanthine oxidase (XOD) was the rate-limiting enzyme
involved in uric acid production, we examined XOD inhibitory
activities of curcumin derivatives 4a–4d, 4j, 7a–7c and 7j, which
exhibited their potent hypouricemic capacities. This result corre-
lates well with that from the anti-hyperuricemia activity. As
shown in Table 2, compounds 4d and 4j displayed the most potent
inhibitory activity (IC50 = 5.7 nM and 10.8 nM), less comparable to
the positive control allopurinol (IC50 = 3.6 nM). This biological
benzbromarone (42.5%). This biological assay indicated that
a,b-
unsaturated cyclohexanone analogous 4d are potential small-
assay indicated that
a,b-unsaturated cyclohexanone analogous
molecule XOD inhibitors as anti-hyperuricemia agents.
4d and 4j are potential small-molecule XOD inhibitors as anti-
hyperuricemia agents.
3.5. Anti-hyperuricemia effect of 4d and its possible
mechanisms
In order to explore probable interaction model of inhibitors and
enzyme active site, molecular docking of the most potent inhibitor
4d into the substrate binding pocket of XOD was performed on the
binding model based on the XOD complex structure (1N5X.pdb).
Docking of 4d was performed using GLIDE (2015, Schrodinger
suite), and the binding model was illustrated by Discovery Studio
3.5 client. Figure 2 shows the docking conformations of the hit
compound at the bindingsite of XOD. Docking score of compound
As shown in Tables 2 and 3, we found that 4d could both inhibit
XOD activity and uric acid transportation via URAT1, exhibiting
most potent ability in lower serum uric acid levels and elevate uric
acid excretion among all the synthesized curcumin derivatives.
Therefore, we treated hyperuricemic mice with 4d at different
doses to further verify the anti-hyperuricemia activity of 4d, as
well as its possible mechanisms via URAT1 and XOD.
As shown in Figure 3A and B, serum and hepatic uric acid levels
were significantly increased in mice of oxonate + vehicle group,
which could be lowered by treatment of 4d at all the doses in a
dose-dependent manner, as well as allopurinol and benzbro-
marone at 10 mg/kg. On the other hand, 4d dose-dependently
recovered urinary uric acid excretion in hyperuricemic mice orally
administrated by oxonate for 7 consecutive days. Moreover, allop-
urinol and benzbromarone at 10 mg/kg also exhibited beneficial
effects on decrease of urinary uric acid excretion (Fig. 3C). More
Table 2
In vitro xanthine oxidase inhibition activity of the selected compounds
Compd.
In vitro IC50 (nM)
Compd.
In vitro IC50 (nM)
4a
4b
4c
4d
4j
49.4 1.2
24.5 0.9
18.9 1.1
5.7 0.8
7a
7b
7c
7j
29.3 2.1
37.0 1.4
35.2 1.7
17.7 0.6
3.6 0.7
10.8 0.6
Allopurinol
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G.-Z. Ao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 2. (A) The 3D model structure of compound 4d binding model with XOD complex structure (1N5X.pdb). (B) Ligand interaction diagram of compound 4d with XOD
complex structure.
Table 3
porter 1 dual inhibitors, the treatment of 4d led to the XOD and
URAT1 expression in hyperuricemic mice with excellent in vivo
anti-hyperuricemic activity.
In vitro URAT1 inhibition activity of the selected compounds
Compd.
[
14C] uric acid re-
absorption inhibition
Compd.
[
14C] uric acid re-
absorption inhibition
(% at 100
lM)
(% at 100 lM)
5. Experimental section
5.1. General chemistry
4a
4b
4c
4d
4j
15.1%
12.4%
20.7%
38.2%
18.9%
7a
7b
7c
7j
11.0%
5.8%
5.2%
23.5%
All chemicals (reagent grade) used were purchased from
Sigma–Aldrich (USA) and Sinopharm Chemical Reagent Co. Ltd.
(China). 1H NMR spectra were measured on Varian Unity Inova
300/400 MHz NMR Spectrometer at 25 °C and referenced to TMS.
Chemical shifts are reported in ppm (d) using the residual solvent
line as internal standard. Splitting patterns are designed as s, sin-
glet; d, doublet; t, triplet; m, multiplet. HRMS spectra were
acquired on Bruker Esquire Liquid Chromatography-Ion Trap Mass
Spectrometer. Analytical thin-layer chromatography (TLC) was
performed on the glass-backed silica gel sheets (silica gel 60 Å
GF254). All compounds were detected using UV light (254 or
365 nm). Analytical HPLC was conducted on SHIMADZU LC-20AD.
Prior to biological evaluation, all compounds were determined to
be >95% pure using appropriate analytical methods (MeOH/H2O
80% v/v, MeOH/H2O 75% v/v, MeOH/H2O 60% v/v) based on the
peak area percentage.
Benzbromarone 42.5%
interestingly, although 4d restored hepatic uric acid and urinary
uric acid excretion to normal levels, the effect of 4d on hepatic uric
acid levels seemed to be less potent than that of allopurinol, nev-
ertheless, 4d was more efficient in promoting uric acid excretion.
As shown in Figure 4, compared with oxonate + vehicle group,
5, 10, 20 mg/kg 4d notably inhibited hepatic XOD activity by
58.6%, 84.5% and 95.9%, respectively. In addition, allopurinol at
10 mg/kg significantly reduced XOD activity in livers of hyper-
uricemic mice, while benzbromarone did not affect hepatic XOD
activity. More interestingly, the XOD inhibitory action of 4d
seemed to be less potent than that of allopurinol, nevertheless,
they had similar anti-hyperuricemia effects, indicating that the
effect of 4d on hyperuricemia might be partly rather than com-
pletely attributed to its ability in inhibiting XOD activity. There-
fore, we detected the protein expression of URAT1 in the kidneys
of mice, which was considered to be the most primary urate trans-
porter mediating uric acid reabsorption in the kidney. As shown in
Figure 5, potassium oxonate treatment significantly increased
expressions of renal URAT1, which could be recovered by 4d at
all the three doses and benzbromarone at 10 mg/kg. Allopurinol,
as typical XOD inhibitor, also weakly decreased renal URAT1 pro-
tein level in hyperuricemic mice with no significance. These obser-
vations suggested that treatment of 4d led to recovery of renal
URAT1 expression, resulting in urate reabsorption reduction and
urate excretion enhancement in hyperuricemic mice.
5.2. General procedure for the preparation of compounds
Cyclohexanone or cyclopentane (0.11 mmol) and morpholine
(0.12 mmol) were dissolved in benzene (20 ml). The mixture was
heated under reflux for 3 h. After cooling to room temperature,
the reaction mixture was concentrated under reduced pressure
to give compound 2 or 5 (0.043 mmol). Next, compound 2
(0.043 mmol), 3, 5-dimethoxy benzaldehyde (0.033 mmol) were
dissolved in benzene (20 ml), and the mixture was heated under
reflux for 8 h. After cooling to room temperature, 20 ml HCl
(6 mg/L) was added dropwise, and the resulting solution was stir-
red at rt for 2 h. The reaction mixture was partitioned between H2O
(20 mL) and benzene (20 ml). The organic layers were combined,
dried with MgSO4, filtered and then concentrated under reduced
pressure. The residue was recrystallized from toluene to give the
intermediates 3 or 6.
4. Conclusion
The current study prepared a series of
hexanone and cyclopentanone analogous as anti-hyperuricemic
agents. As expected, these curcumin ,b-unsaturated cyclohex-
anone and cyclopentanone derivatives exhibited remarkable anti-
hyperuricemia activity and uricosuric activity in vivo. Notably,
compound 4d is a first-in-class xanthine oxidase and urate trans-
a,b-unsaturated cyclo-
Various substitute benzaldehydes (1.22 mmol) and 3 or 6
(1.22 mmol) were dissolved in 10% NaOH ethanol solution
(10 ml), and stirring at rt for 30 min. Then water (50 ml) was
added, the product extraction was carried out with EtOAc. The
a
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G.-Z. Ao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
A
B
Oxonate-treated
Oxonate-treated
8
4
0
0.8
0.4
0.0
###
###
*
*
**
***
***
***
**
***
***
***
(mg/kg)
(mg/kg)
Normal Vehicle
5
10
4d
20
10
10
Normal Vehicle
5
10
4d
20
10
10
AP
AP
BM
BM
C
Oxonate-treated
0.8
0.4
0.0
***
***
***
***
*
*
###
(mg/kg)
Normal Vehicle
5
10
4d
20
10
10
AP
BM
Figure 3. Effects of 4d, allopurinol (AP) and benzbromarone (BM) on serum uric acid levels (A), hepatic uric acid levels (B), urinary uric acid excretion in 24 h (C) and in
hyperuricemic mice. Data were displayed as mean S.E.M (n = 8). ###P <0.001, compared with normal vehicle group; *P <0.05, **P <0.01, ***P <0.001, compared with oxonate-
treated vehicle group.
Oxonate-treated
2.5
11.3%
43.4%
31.1%
2.0
27.5%
19.0%
1.5
###
*
1.0
0.5
0.0
***
***
***
(mg/kg)
Normal
Vehicle
5
10
4d
20
10
10
AP
BM
Figure 4. Effects of 4d, allopurinol (AP) and benzbromarone (BM) on XOD activity in hyperuricemic mice. Data were displayed as mean S.E.M (n = 8). ###P <0.001, compared
with normal vehicle group; *P <0.05, ***P <0.001, compared with oxonate-treated vehicle group.
organic layer was dried over Na2SO4, and after solvent evaporation
the residue was purified by flash column chromatography (silica
gel 60, 35–70 mesh) using CHCl3 as the eluent to give compounds
4a–4j and 7a–7l.
5.2.1. (2E,6E)-2-(3-Chlorobenzylidene)-6-(3,5-dimethoxybenzy-
lidene)cyclohexanone (4a)
Mp: 107.5–108.4 °C. 1H NMR(400 MHz, CDCl3), d(ppm): 7.72(s,
1H, @CH), 7.70(s, 1H, @CH), 7.43(s, 1H, ArH), 7.33(m, 3H, ArH),
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G.-Z. Ao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Oxonate-treate
4d
10
AP
10
BM
10 (mg/kg)
Normal Vehicle
5
20
URAT1
70 kDa
55 kDa
α-Tubulin
4
2
0
###
*
***
***
***
Figure 5. Effects of 4d, allopurinol (AP) and benzbromarone (BM) on renal URAT1 protein expressions in hyperuricemic mice. Data were expressed as the mean S.E.M. for 4
mice. ###P <0.001, compared with normal vehicle group; *P <0.05, ***P <0.001, compared with oxonate-treated vehicle group.
6.61(d, 2H, J = 2.1 Hz, ArH), 6.47(t, 1H, J = 2.1 Hz, ArH), 3.82(s, 6H,
OCH3), 2.92(m, 4H, CH2), 1.81(quint, 2H, J = 6.5 Hz, CH2). 13C NMR
(400 MHz, CDCl3), d(ppm): 190.257, 160.841, 137.985, 137.876,
137.577, 137.541, 136.631, 135.558, 134.564, 130.172, 129.936,
128.795, 108.625, 101.106, 55.707, 28.762, 28.684, 23.117. HR-
MS: Calcd. For C22H21ClO3 [M+H]+: 369.1252, Found: 369.1257.
3.82(s, 6H, OCH3), 2.93(t, 2H, J = 5.4 Hz, CH2), 2.88(t, 2H,
J = 5.4 Hz, CH2), 1.79(quint, 2H, J = 6.4 Hz, CH2). 13C NMR
(400 MHz, CDCl3), d(ppm): 190.323, 160.845, 137.912, 137.454,
136.967, 136.685, 135.922, 135.069, 132.115, 131.905, 123.159,
108.617, 101.084, 55.718, 28.755, 23.137. HR-MS: Calcd. For C22
-
H
21BrO3 [M+H]+: 413.0747, Found: 413.0761.
5.2.2. (2E,6E)-2-(2-Chlorobenzylidene)-6-(3,5-dimethoxybenzy-
lidene)cyclohexanone (4b)
5.2.5. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(3-methoxyben-
zylidene)cyclohexanone (4e)
Mp: 104.0–104.7 °C. 1H NMR(400 MHz, CDCl3), d(ppm): 7.88(s,
1H, @CH), 7.74(s, 1H, @CH), 7.44(m, 1H, ArH), 7.33(m, 1H, ArH),
7.28(m, 2H, ArH), 6.61(d, 2H, J = 2.0 Hz, ArH), 6.47(t, 1H,
J = 2.1 Hz, ArH), 3.82(s, 6H, OCH3), 2.94(t, 2H, J = 5.6 Hz, CH2),
2.76(t, 2H, J = 5.5 Hz, CH2), 1.77(quint, 2H, J = 6.2 Hz, CH2). 13C
NMR (400 MHz, CDCl3), d(ppm): 190.280, 160.837, 138.169,
137.948, 137.752, 136.739, 135.260, 134.694, 133.913, 130.818,
130.009, 129.827, 126.555, 108.627, 101.090, 55.706, 28.983,
28.483, 23.363.HR-MS: Calcd. For C22H21ClO3 [M+H]+: 369.1252,
Found: 369.1251.
Mp: 92.0–92.6 °C. 1H NMR d(ppm): 7.76(s, 1H, @CH), 7.71(s, 1H,
@CH), 7.32(t, 1H, J = 7.9 Hz, ArH), 7.06(d, 1H, J = 7.7 Hz, ArH), 7.00
(s, 1H, ArH), 6.90(d, 1H, J = 8.2 Hz, ArH), 6.60(d, 2H, J = 1.6 Hz,
ArH), 6.50(s, 1H, ArH), 3.84(s, 3H, CH3), 3.82(s, 6H, OCH3), 2.90(t,
4H, J = 5.2 Hz, CH2), 1.80(quint, 2H, J = 6.4 Hz, CH2). 13C NMR d
(ppm): 190.429, 160.792, 159.652, 137.955, 137.471, 137.107,
136.832, 136.619, 129.600, 123.085, 116.005, 114.455, 108.548,
100.994, 55.630, 55.507, 28.755, 28.728, 23.156. HR-MS: Calcd.
For C23H24O4[M+H]+: 365.1747, Found: 365.1747.
5.2.6. (2E,6E)-2-(3,5-Di-tert-butyl-4-hydroxybenzylidene)-6-(3,
5-dimethoxybenzylidene)cyclohexanone (4f)
5.2.3. (2E,6E)-2-(3-Bromobenzylidene)-6-(3,5-dimethoxybenzy-
lidene)cyclohexanone (4c)
Mp: 148.2–149.9 °C. 1H NMR d(ppm): 7.79(s, 1H, @CH), 7.70(s,
1H, @CH), 7.37(s, 2H, ArH), 6.60(s, 2H, ArH), 6.45(s, 1H, ArH),
5.48(s, 1H, OH), 3.82(s, 6H, OCH3), 2.903(m, 4H, CH2), 1.80(quint,
2H, J = 5.8 Hz, CH2), 1.46(s, 18H, CH3). 13C NMR d(ppm): 190.493,
160.829, 155.078, 139.015, 138.272, 137.232, 136.481, 136.151,
133.471, 128.514, 127.638, 108.539, 100.878, 55.704, 34.704,
30.539, 28.925, 28.785, 23.435. HR-MS: Calcd. For C30H38O4[M
+H]+: 463.2843, Found: 463.2829.
Mp: 111.9–112.7 °C. 1H NMR(400 MHz, CDCl3), d(ppm): 7.72(s,
1H, @CH), 7.69(s, 1H, @CH), 7.59(s, 1H, ArH), 7.46(d, 1H,
J = 7.7 Hz, ArH), 7.36(d, 1H, J = 7.6 Hz, ArH), 7.28(t, 1H, J = 7.9 Hz,
ArH), 6.60(s, 2H, ArH), 6.47(s, 1H, ArH), 3.82(s, 6H, OCH3), 2.91
(m, 4H, CH2), 1.79(quint, 2H, J = 6.3 Hz, CH2). 13C NMR(400 MHz,
CDCl3), d(ppm): 190.151, 160.825, 138.269, 137.846, 137.558,
136.593, 135.402, 133.047, 131.667, 130.167, 129.157, 122.721,
108.617, 101.105, 55.676, 28.728, 28.622, 23.097. HR-MS: Calcd.
For C22H21BrO3 [M+H]+: 413.0747, Found: 413.0747.
5.2.7. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(4-hydroxyben-
zylidene)cyclohexanone (4g)
5.2.4. (2E,6E)-2-(4-Bromobenzylidene)-6-(3,5-dimethoxybenzy-
lidene)cyclohexanone (4d)
Mp: 155.0–155.6 °C. 1H NMR d(ppm): 7.76(s, 1H, @CH), 7.72(s,
1H, @CH), 7.39(d, 2H, J = 8.3 Hz, ArH), 6.90(d, 2H, J = 8.4 Hz, ArH),
6.59(s, 2H, ArH), 6.46(s, 1H, ArH), 6.27(s, 1H, OH), 3.81(s, 6H,
OCH3), 2.90(m, 4H, CH2), 1.79(m, 2H, CH2). 13C NMR d(ppm):
191.373, 160.838, 157.381, 138.314, 138.045, 137.259, 137.102,
Mp: 126.4–128.6 °C. 1H NMR(400 MHz, CDCl3), d(ppm): 7.71(s,
1H, @CH), 7.70(s, 1H, @CH), 7.53(d, 2H, J = 8.3 Hz, ArH), 7.32(d,
2H, J = 8.3 Hz, ArH), 6.60(d, 2H, J = 1.8 Hz, ArH), 6.47(s, 1H, ArH),
Please cite this article in press as: Ao, G.-Z.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.022

G.-Z. Ao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
134.065, 133.039, 128.507, 115.982, 108.648, 101.149, 55.748,
28.860, 28.718, 23.219. HR-MS: Calcd. For C22H22O4 [M+H]+:
351.1591, Found: 351.1590.
ArH), 6.75(d, 2H, J = 1.7 Hz, ArH), 6.52(s, 1H, ArH), 3.84(s, 6H,
AOCH3), 3.11–3.13(m, 4H, ACH2). 13C NMR(400 MHz, CDCl3), d
(ppm): 196.237, 161.097, 138.830, 138.117, 137.668, 137.584,
134.639, 133.345, 132.418, 132.305, 130.519, 129.558, 123.137,
5.2.8. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(4-(dimethylam-
ino)benzylidene)cyclohexanone (4h)
109.041, 102.069, 55.700, 26.755, 26.693. HR-MS: Calcd. For C21
H
-
19BrO3[M+H]+: 399.0590, Found: 399.0573.
Mp: 109.6–110.3 °C. 1H NMR d(ppm): 7.78(s, 1H, @CH), 7.70(s,
1H, @CH), 7.46(d, 2H, J = 8.8 Hz, ArH), 6.72(d, 2H, J = 8.8 Hz, ArH),
6.60(d, 2H, J = 2.0 Hz, ArH), 6.44(s, 1H, ArH), 3.81(s, 6H, OCH3),
3.03(s, 6H, NCH3), 2.95(t, 2H, J = 6.3 Hz, CH2), 2.90(t, 2H,
J = 5.6 Hz), 1.80(quint, 2H, J = 6.2 Hz, CH2). 13C NMR d(ppm):
190.267, 160.794, 150.851, 138.751, 138.411, 137.439, 135.885,
133.004, 131.900, 124.155, 111.895, 108.468, 100.776, 55.675,
40.389, 29.099, 28.733, 23.333. HR-MS: Calcd. For C24H27NO3[M
+H]+: 378.2064, Found: 378.2060.
5.2.14. (2E,5E)-2-(4-Bromobenzylidene)-5-(3,5-dimethoxybenz-
ylidene)cyclopentanone (7d)
Mp: 182.0–183.3 °C. 1H NMR(400 MHz, CDCl3), d(ppm): 7.57(d,
2H, J = 8.4 Hz, ArH), 7.52(s, 2H, @CH), 7.45(d, 2H, J = 8.4 Hz, ArH),
6.74(s, 2H, ArH), 6.51(s, 1H, ArH), 3.84(s, 6H, AOCH3), 3.04–3.14
(m, 4H, ACH2). 13C NMR(400 MHz, CDCl3), d(ppm): 196.373,
161.116, 138.169, 137.733, 134.952, 134.503, 132.803, 132.335,
132.318, 124.064, 109.053, 102.019, 55.736, 26.775. HR-MS: Calcd.
For C21H19BrO3[M+H]+: 399.0590, Found: 399.0588.
5.2.9. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(4-methoxyben-
zylidene)cyclohexanone (4i)
5.2.15. (2E,5E)-2-(3,5-Dimethoxybenzylidene)-5-(3-methoxybe-
nzylidene)cyclopentanone (7e)
Mp: 97.7–98.1 °C. 1H NMR d(ppm): 7.77(s, 1H, @CH), 7.71(s, 1H,
@CH), 7.46(d, 2H, J = 8.7 Hz, ArH), 6.94(d, 2H, J = 8.7 Hz, ArH), 6.60
(d, 2H, J = 1.9 Hz, ArH), 6.46(s, 1H, ArH), 3.85(s, 3H, CH3), 3.82(s, 6H,
OCH3), 2.92(t, 4H, J = 5.9 Hz, CH2), 1.80 (quint, 2H, J = 6.4 Hz, CH2).
13C NMR d(ppm): 190.280, 160.745, 160.225, 138.045, 137.273,
136.970, 136.560, 134.257, 132.562, 128.771, 114.127, 108.460,
100.849, 55.573, 28.774, 28.657, 23.150. HR-MS: Calcd. For
Mp: 125.0–126.2 °C. 1H NMR(400 MHz, CDCl3), d(ppm): 7.56(s,
1H, @CH), 7.52(s, 1H, @CH), 7.36(t, 1H, J = 7.8 Hz, ArH), 7.20(d,
1H, J = 7.7 Hz, ArH), 7.12(s, 1H, ArH), 6.94(dd, 1H, J = 1.8 Hz,
8.3 Hz, ArH), 6.75(d, 2H, J = 1.8 Hz, ArH), 6.51(s, 1H, ArH), 3.85(s,
3H, AOCH3), 3.83(s, 6H, AOCH3), 3.11(s, 4H, ACH2). 13C NMR
(400 MHz, CDCl3), d(ppm): 196.505, 161.073, 159.964, 137.969,
137.809, 137.769, 137.358, 134.151, 134.101, 129.992, 123.588,
116.266, 115.380, 108.979, 101.927, 55.690, 55.570, 26.798. HR-
MS: Calcd. For C22H22O4[M+H]+: 351.1591, Found: 351.1578.
C
23H24O4[M+H]+: 365.1747, Found: 365.1747.
5.2.10. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(4-(methylsulf-
onyl)benzylidene)cyclohexanone (4j)
Mp: 162.0–163.5 °C. 1H NMR d(ppm): 7.79(d, 2H, J = 8.2 Hz,
ArH), 7.76(s, 1H, @CH), 7.73(s, 1H, @CH), 7.61(d, 2H, J = 8.2 Hz,
ArH), 6.61(s, 2H, ArH), 6.48(s, 1H, ArH), 3.82(s, 6H, OCH3), 3.09(s,
3H, SO2CH3), 2.95(t, 2H, J = 5.1 Hz, CH2), 2.89(t, 2H, J = 5.3 Hz,
CH2), 1.80(quint, 2H, J = 5.6 Hz,CH2). 13C NMRd(ppm): 189.744,
160.713, 141.522, 139.929, 139.108, 137.843, 137.523, 136.255,
134.311, 130.899, 127.501, 108.540, 101.071, 55.543, 44.536,
5.2.16. (2E,5E)-2-(3,4-Dihydroxybenzylidene)-5-(3,5-dimethox-
ybenzylidene)cyclopentanone (7f)
Mp: 157.6–158.7 °C. 1H NMR(400 MHz, CDCl3), d(ppm): 7.79(s,
1H, @CH), 7.71(s, 1H, @CH), 7.19(s, 1H, ArH), 7.04(d, 1H,
J = 8.3 Hz, ArH), 6.93(d, 1H, J = 8.2 Hz, ArH), 6.60(s, 2H, ArH), 6.47
(d, 1H, J = 1.6 Hz, ArH), 3.82(d, 6H, J = 1.6 Hz, AOCH3), 2.86–2.94
(m, 4H, ACH2). 13C NMR(400 MHz, CDCl3), d(ppm): 189.808,
160.872, 147.521, 145.617, 137.848, 137.753, 137.630, 135.768,
133.608, 127.474, 124.263, 118.335, 116.360, 108.601, 101.240,
28.541, 28.499, 22.860. HR-MS: Calcd. For
413.1417, Found: 413.1396.
C
23H24O5S[M+H]+:
55.840, 28.557, 28.367. HR-MS: Calcd. For
353.1384, Found: 352.1385.
C
21H20O5[M+H]+:
5.2.11. (2E,5E)-2-(3-Chlorobenzylidene)-5-(3,5-dimethoxybenz-
ylidene)cyclopentanon (7a)
Mp: 145.8–147.6 °C. 1H NMR(400 MHz, CDCl3), d(ppm): 7.90(s,
1H, @CH), 7.56–7.58(m, 1H, ArH), 7.53(s, 1H, @CH), 7.45–7.47(m,
1H, ArH), 7.29–7.32(m, 2H, ArH), 6.73(d, 2H, J = 1.7 Hz, ArH), 6.51
(s, 1H, ArH), 3.83(s, 6H, AOCH3), 3.00–3.10(m, 4H, ACH2). 13C
NMR(400 MHz, CDCl3), d(ppm): 196.050, 161.048, 139.710,
137.664, 136.288, 134.494, 134.061, 130.403, 130.333, 130.290,
129.769, 126.898, 108.956, 101.960, 55.654, 26.900, 26.564. HR-
MS: Calcd. For C21H19ClO3[M+H]+: 355.1095, Found: 355.1089.
5.2.17. (2E,5E)-2-(3,5-Dimethoxybenzylidene)-5-(4-hydroxybe-
nzylidene)cyclopentanone (7g)
Mp: 253.4–255.3 °C. 1H NMR(400 MHz, d6-DMSO), d(ppm):
10.10(s, 1H, AOH), 7.52(d, 2H, J = 8.3 Hz, ArH), 7.36(s, 1H, @CH),
7.30(s, 1H, @CH), 6.86(d, 2H, J = 8.3 Hz, ArH), 6.80(s, 2H, ArH),
6.55(s, 1H, ArH), 3.76(s, 6H, AOCH3), 2.94–3.10(m, 4H, ACH2). 13C
NMR(400 MHz, CDCl3), d(ppm): 195.023, 160.557, 159.245,
138.635, 137.340, 134.084, 133.392, 132.957, 131.854, 126.519,
115.998, 108.393, 101.552, 55.361, 55.281, 25.966. HR-MS: Calcd.
For C21H20O4[M+H]+: 337.1434, Found: 337.1452.
5.2.12. (2E,5E)-2-(2-Chlorobenzylidene)-5-(3,5-dimethoxybenz-
ylidene)cyclopentanone (7b)
Mp: 162.4–162.9 °C. 1H NMR(400 MHz, CDCl3), d(ppm): 7.91(s,
1H, @CH), 7.57(d, 1H, J = 6.7 Hz, ArH), 7.53(s, 1H, @CH), 7.46(d,
1H, J = 7.6 Hz, ArH), 7.30–7.32(m, 2H, ArH), 6.74(s, 2H, ArH), 6.51
(s, 1H, ArH), 3.83(s, 6H, AOCH3), 3.01–3.11(m, 4H, ACH2). 13C
NMR(400 MHz, CDCl3), d(ppm): 196.121, 161.080, 139.751,
137.709, 136.326, 134.542, 134.119, 130.431, 130.377, 130.320,
129.863, 126.914, 108.987, 101.982, 55.705, 26.956, 26.613. HR-
MS: Calcd. For C21H19ClO3[M+H]+: 355.1095, Found: 355.1086.
5.2.18. (2E,5E)-2-(3,5-Dimethoxybenzylidene)-5-(4-(dimethyl-
amino)benzylidene)cyclopentanone (7h)
Mp: 194.4–196.4 °C. 1H NMR(400 MHz, CDCl3), d(ppm): 7.58(s,
1H, @CH), 7.53(d, 2H, J = 8.9 Hz, ArH), 7.46(s, 1H, @CH), 6.75(d,
2H, J = 2.1 Hz, ArH), 6.73(d, 2H, J = 9.1 Hz, ArH), 6.49(t, 1H,
J = 2.1 Hz, ArH), 3.83(s, 6H, AOCH3), 3.07–3.10(m, 4H, ACH2), 3.05
(s, 6H, ACH3). 13C NMR(400 MHz, CDCl3), d(ppm): 196.318,
161.069, 151.343, 139.173, 138.343, 135.583, 133.223, 132.644,
132.564, 124.053, 112.171, 108.834, 101.583, 55.731, 40.402,
26.938, 26.896. HR-MS: Calcd. For C23H26NO3[M+H]+: 364.1907,
Found: 364.1906.
5.2.13. (2E,5E)-2-(3-Bromobenzylidene)-5-(3,5-dimethoxybenz-
ylidene)cyclopentanone (7c)
Mp: 141.8–142.7 °C. 1H NMR(400 MHz, CDCl3), d(ppm): 7.73(s,
1H, @CH), 7.50–7.53(m, 4H, ArH, @CH), 7.32(t, 1H, J = 7.7 Hz,
Please cite this article in press as: Ao, G.-Z.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.022

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G.-Z. Ao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5.2.19. (2E,5E)-2-(3,5-Dimethoxybenzylidene)-5-(4-methoxybe-
nzylidene)cyclopentanone (7i)
mals 1 h prior to the administration. Briefly, mice were adminis-
tered once daily with oxonate (250 mg/kg) or 0.5% CMC-Na
(vehicle) at 8:00 a.m. followed by test compounds, allopurinol
and benzbromarone received at 9:00 a.m. for seven consecutive
days in a volume of 15 mL/kg via oral gavage.
On the sixth day, mice were transferred to metabolic cages to
collect 24 h urine samples for each mouse. After filtering, urine
samples were used to perform uric acid analysis within 24 h.
Whole blood samples were collected 1 h after final administration
on the seventh day and centrifuged at 8000g for 5 min to obtain
serum for uric acid assays. Phosphotungstic acid method was
adopted to estimate uric acid concentrations in serum (Sur) and
urine (Uur).
Mp: 138.9–140.0 °C. 1H NMR(400 MHz, CDCl3), d(ppm): 7.55–
7.58(m, 3H, @CH, ArH), 7.49(s, 1H, @CH), 6.97(d, 2H, J = 8.6 Hz,
ArH), 6.75(d, 2H, J = 1.7 Hz, ArH), 6.50(s, 1H, ArH), 3.86(s, 3H,
AOCH3), 3.83(s, 6H, AOCH3), 3.06–3.10(m, 4H, ACH2). 13C NMR
(400 MHz, CDCl3), d(ppm): 196.433, 161.030, 160.921, 138.324,
137.951, 135.142, 134.163, 133.504, 132.896, 128.834, 114.582,
108.891, 101.747, 55.663, 26.773, 26.705. HR-MS: Calcd. For
C
22H22O4[M+H]+: 351.1591, Found: 351.1578.
5.2.20. (2E,5E)-2-(3,5-Dimethoxybenzylidene)-5-(4-(methylsul-
fonyl)benzylidene)cyclopentanone (7j)
1H NMR(400 MHz, CDCl3), d(ppm): 1H NMR(400 MHz, CDCl3), d
(ppm): 8.08(d, 2H, J = 8.3 Hz, ArH), 7.75(d, 2H, J = 8.4 Hz, ArH), 7.58
(s, 1H, @CH), 7.56(s, 1H, @CH), 6.75(d, 2H, J = 2.0 Hz, ArH), 6.53(t,
1H, J = 2.0 Hz, ArH), 3.84(s, 6H, AOCH3), 3.14–3.16(m, 4H, ACH2),
3.09(s, 3H, ASO2CH3). 13C NMR(400 MHz, CDCl3), d(ppm):
161.186, 141.402, 141.069, 140.606, 137.534, 137.265, 135.393,
131.394, 131.334, 128.090, 109.184, 102.235, 55.780, 44.805,
26.886, 26.791. HR-MS: Calcd. For C22H32O5S[M+H]+: 399.1261,
Found: 399.1262.
5.4. Docking study
Docking of 4d was performed using GLIDE (2015, Schrodinger
suite), and the binding model was illustrated by Discovery Studio
3.5 client. The crystal structures of XOD complex (1N5X.pdb) was
retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/
pdb/home/home.do). All bound waters and ligands were elimi-
nated from the protein and the polar hydrogen was added to the
proteins. Molecular docking of all twenty compounds was then
carried out using the Discovery Studio (version 3.1) as imple-
mented through the graphical user interface CDocker protocol.
5.2.21. (2E,5E)-2-(2-Bromobenzylidene)-5-(3,5-dimethoxyben-
zylidene)cyclopentanone (7k)
Mp: 159.0–160.3 °C. 1H NMR(400 MHz, CDCl3), d(ppm): 7.81(s,
1H, @CH), 7.59(t, 1H, J = 7.7 Hz, ArH), 7.53(s, 1H, @CH), 7.34–7.39
(m, 1H, ArH), 7.21(t, 1H, J = 7.6 Hz, ArH), 7.14(t, 1H, J = 9.6 Hz,
ArH), 6.75(d, 2H, J = 1.1 Hz, ArH), 6.51(s, 1H, ArH), 3.84(s, 6H,
AOCH3), 3.04–3.13(m, 4H, ACH2). 13C NMR(400 MHz, CDCl3), d
(ppm): 196.063, 161.071, 139.772, 137.720, 137.691, 135.811,
134.545, 133.653, 132.434, 130.562, 130.394, 127.520, 126.720,
5.5. Determination of XOD inhibitory activity
Assay of xanthine oxidase inhibitory activity was performed as
follows. 1092
lL of 0.1 unit of XOD in buffer (200 mM sodium
pyrophosphate, pH 7.5) and 2
lL (2.5, 5, 10, 20, 40 M) of the test
l
compounds or allopurinol in DMSO were mixed at 37 °C for 5 min.
108.976, 101.979, 55.703, 26.957, 26.473. HR-MS: Calcd. For C21
H
-
The control group did not contain the test compounds or allopuri-
nol. Immediately after 200 lL 0.6 mM xanthine in doubly distilled
19BrO3[M+H]+: 399.0590, Found: 399.0584.
water was added to the mixture, the absorption increments at
295 nm indicating the formation of uric acid were determined
every minute up to 8 min. Three replicates were made for each test
sample. The percent inhibition ratio (%) was calculated according
to the following equation, thereby the agent concentration needed
for 50% inhibition was calculated as IC50 values.
5.2.22. (2E,5E)-2-(4-Chlorobenzylidene)-5-(3,5-dimethoxyben-
zylidene)cyclopentanone (4l)
Mp: 178.5–179.2 °C. 1H NMR(400 MHz, CDCl3), d(ppm): 7.53(s,
1H, @CH), 7.52(d, 2H, J = 8.2 Hz, ArH), 7.51(s, 1H, @CH), 7.41(d,
2H, J = 8.4 Hz, ArH), 6.74(d, 2H, J = 1.6 Hz, ArH), 6.51(s, 1H, ArH),
3.84(s, 6H, AOCH3), 3.05–3.14(m, 4H, ACH2). 13C NMR(400 MHz,
CDCl3), d(ppm): 196.362, 161.115, 137.996, 137.749, 135.650,
134.542, 134.454, 132.750, 132.135, 129.352, 109.048, 102.006,
55.734, 26.790, 26.753. HR-MS: Calcd. For C21H19ClO3[M+H]+:
355.1095, Found: 355.1081.
% inhibition ¼ ½ðrate of control reaction
ꢀ rate of sample reactionÞ=rate of control reactionꢂ
ꢁ100
5.3. Determination of anti-hyperuricemic and uricosuric
activity in vivo
5.6. Determination of uric acid uptake through URAT1 in
HEK293 cells
Potassium oxonate, a potent uricase inhibitor, was used to
induce hyperuricemia in mice.24 Male Kun-Ming strain of mice,
weighing 18–22 g, were purchased from the Central Institute for
Experimental Animals of Zhejiang and were allowed one week to
adapt to the laboratory environment before used for experiments.
Animals were housed 5 per cage (320 ꢁ 180 ꢁ 160 mm) with free
access to food and water on standard laboratory conditions of tem-
perature 24 2 °C, relative humidity 55 5% and a 12-h light/12-h
dark cycle for the duration of the study. Mice were randomly
divided into different groups: normal + vehicle, oxonate + vehicle,
Human embryonic kidney (HEK)293 cells (American Type Cul-
ture Collection, Manassas, VA) were cultured in complete medium
consisting of Dulbecco’s modified Eagle’s medium with 10% fetal
calf serum, which were set to temperatures of 37 °C and supple-
mented with 5% CO2. The cell culture and transfection were per-
formed as described in Ahn’s study (Ahn et al., 2016). Cells were
transfected with URAT1 plasmid or control vector [pcDNA3.1(+)],
using Lipofectamine 2000 transfection reagent (Life Technologies/
Thermo Fisher Scientific) according to the manufacturer’s instruc-
tions. After 6 h of transfection, cells were subcultured in poly-D-
lysine-coated 24-well plates at a density of 2 ꢁ 105 cells/well. At
48 h after transfection, the cells were used for uptake experiments.
Cells expressing URAT1 or control vector were used to measure
the amount of uric acid taken up at various concentrations of cur-
cumin derivatives or benzbromarone. The composition of the incu-
bation medium was as follows: 140 mM sodium gluconate, 2.7 mM
oxonate + test
compounds
(10 mg/kg),
oxonate + 10 mg/kg
allopurinol, oxonate + 10 mg/kg benzbromarone. Oxonate, test
compounds, allopurinol and benzbromarone at various concentra-
tions were dissolved or suspended in 0.5% CMC-Na, and the
dosages of all the agents were selected based on our preliminary
experiments. Food, but not water, was withdrawn from the ani-
Please cite this article in press as: Ao, G.-Z.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.022

G.-Z. Ao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
potassium gluconate, 1.8 mM KH2PO4, and 10 mM Na2HPO4. The
Acknowledgments
cells were incubated with incubation medium containing cur-
cumin derivatives or benzbromarone for 30 min. A certain amount
medium containing [14C]uric acid was added to initiate the uptake.
The medium was aspirated at the end of the incubation period, and
the monolayers were rapidly washed twice with 1 ml of ice-cold
incubation medium. The cells were solubilized in 0.4 ml of 0.5 N
NaOH, and then the radioactivity in the aliquots was determined
by liquid scintillation counting.
The work was supported by National Natural Science Founda-
tion of China (Grant No. 81202573); the Suzhou Science & Technol-
ogy Foundation (Grant No. SYS201665) and PAPD (A Project
Funded by the Priority Academic Program Development of Jiangsu
Higher Education Institutions).
Supplementary data
5.7. Effects of 4d on XOD activity and URAT1 expression in vivo
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.10.022.
Animal model establishment and drug administration were fol-
lowed as 5.2. Mice were randomly divided into 7 groups: normal
+ vehicle, oxonate + vehicle, oxonate + 4d (5 mg/kg, 10 mg/kg,
20 mg/kg), oxonate + 10 mg/kg allopurinol, oxonate + 10 mg/kg
benzbromarone. Beside urine and serum samples, liver tissues
and kidney cortex tissues of mice were rapidly separated on ice
plate, frozen in liquid nitrogen, and stored at ꢀ70 °C.
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Please cite this article in press as: Ao, G.-Z.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.022