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

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 b

Department of Medicinal Chemistry, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, PR China State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China

a r t i c l e

i n f o

Article history: 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

a b s t r a c t 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. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction 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, characterized 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 allopurinol and febuxostat have been the most commonly used urate-lowering 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 conversion of xanthine to uric acid, exhibiting potentially effects on hyperuricemia.8,9 Nevertheless, only about 40% of patients are able to meet treatment goals via allopurinol, and it occasionally causes ⇑ Corresponding authors. E-mail addresses: [email protected] (H.-Q. Li), [email protected] (Q.-H. Hu).

Stevens Johnson syndrome, which may be fatal.10 In addition, febuxostat has been associated with cardiovascular complications causing the Food and Drug Administration (FDA) to require a cautionary 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 uricosuric activity, effectively reduces serum urate levels through targeting 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 Pharmaceutical 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 antiinflammatory, antioxidant17–19, antiproliferative20 and uricosuric activities.21,22 In our previous publication23, a series of a,b-unsaturated curcumin cyclohexanone analogous their antiproliferative activities have been studied. Recently, the uricosuric activity of curcumin has been investigated; it is potentially useful for treat-

http://dx.doi.org/10.1016/j.bmc.2016.10.022 0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

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G.-Z. Ao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

N

H N

N

3. Results and discussion

N

N

COOH

S

N

3.1. Anti-hyperuricemia activity in vivo

O

OH Allopurinol

In order to monitor the efficacy of different synthesized a,bunsaturated cyclohexanone and cyclopentanone analogous of curcumin, serum uric acid levels were determined using hyperuricemic 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-hyperuricemia activities of the synthesized curcumin derivatives and the results were summarized in Table 1. As expected, these curcumin a,b-unsaturated cyclohexanone and cyclopentanone derivatives exhibited remarkable anti-hyperuricemia activity comparable to the positive control. 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 cyclohexanone 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 Rphenyl ring, we could perceive the tendency that Br>Cl in the series. 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%).

Febuxostat

Br HO Br

O

O

O

O

O

HO

OH

O Curcumin

Benzbromarone

Figure 1. Chemical structures for allopurinol, febuxostat, benzbromarone and curcumin.

ment of hyperuricemia or gout as a new URAT1 inhibitor.21 Therefore, in continuation of our earlier studies focus on the diverse biological activity of curcumin analogs, the a,b-unsaturated curcumin cyclohexanone analogous had been screened for their anti- hyperuricemia activity and some compounds showed potent anti-hyperuricemia 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 antihyperuricemia potency. 2. Chemistry Twenty-two a,b-unsaturated cyclohexanone and cyclopentanone analogous of curcumin were synthesized to be screened for the anti-hyperuricemia. The preparation of the a,b-unsaturated cyclohexanone analogous have been reported in our previous publication.23 As show in Scheme 1, the intermediates 3 or 6 was prepared 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-dimethoxybenzaldehyde 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.

3.2. Uricosuric activity in vivo To confirm whether the anti-hyperuricemia activities of synthesized 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 summarized in Table 1. Taken together, the designed and synthesized curcumin 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 Morpholine

N

O H3CO 1. 3,5-OCH 3PhCHO 2.HCl

Morpholine

N

1

OCH 3

3

4a-4j O

H 3CO 1. 3,5-OCH 3PhCHO 2.HCl

5

O H3CO

NaOH or HCl

OCH 3

O O

CHO

R

2

1

R

R

CHO

O H 3CO R

OCH3

NaOH or HCl

6

OCH3 7a-7l

Scheme 1. Synthesis of target compounds.

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G.-Z. Ao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx Table 1 In vivo anti-hyperuricemia activity and uricosuric activity of the synthesized compounds. (% at 10 mg/kg) Uric acid excretion inhibition in 24 h elevation

Compd.

R

Serum uric acid level inhibition (% at 10 mg/kg)

Uric acid excretion inhibition in 24 h elevation

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

40.17% 16.82% 25.20% 44.52% 12.95%

26.40% 17.52% 23.48% 25.79% 8.60%

7.74%

19.35%

7f

11.78%

12.90%

11.88% 20.06%

15.82% 10.74%

7g 7h

9.36% 15.10%

10.16% 9.64%

4-OCH3Ph

25.21%

7.35%

7i

18.82%

11.52%

4-SO2CH3Ph

50.78%

36.47%

7j

54.39%

40.55%

118.32% 99.74%

88.60% 37.58%

7k 7l

3-ClPh 2-ClPh 3-BrPh 4-BrPh 3OCH3Ph 3,4diOHPh 4-OHPh 4-N (CH3)2Ph 4OCH3Ph 4SO2CH3Ph 2-BrPh 4-ClPh

14.09% 30.62%

9.90% 7.35%

Compd.

R

4a 4b 4c 4d 4e

3-ClPh 2-ClPh 3-BrPh 4-BrPh 3-OCH3Ph

4f 4g 4h

3,5diisopropyPhenol 4-OHPh 4-N(CH3)2Ph

4i 4j Allopurinol Benzbromarone

Serum uric acid level inhibition

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 displayed 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 important 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 suggested 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 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 correlates 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 assay indicated that a,b-unsaturated cyclohexanone analogous 4d and 4j are potential small-molecule XOD inhibitors as antihyperuricemia agents. 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

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 10.8 ± 0.6

7a 7b 7c 7j Allopurinol

29.3 ± 2.1 37.0 ± 1.4 35.2 ± 1.7 17.7 ± 0.6 3.6 ± 0.7

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 benzbromarone (42.5%). This biological assay indicated that a,bunsaturated cyclohexanone analogous 4d are potential smallmolecule XOD inhibitors as anti-hyperuricemia agents. 3.5. Anti-hyperuricemia mechanisms

effect

of

4d

and

its

possible

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 benzbromarone 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, allopurinol and benzbromarone at 10 mg/kg also exhibited beneficial effects on decrease of urinary uric acid excretion (Fig. 3C). More

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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 In vitro URAT1 inhibition activity of the selected compounds Compd.

[14C] uric acid reabsorption inhibition (% at 100 lM)

Compd.

[14C] uric acid reabsorption inhibition (% at 100 lM)

4a 4b 4c 4d 4j

15.1% 12.4% 20.7% 38.2% 18.9%

7a 7b 7c 7j Benzbromarone

11.0% 5.8% 5.2% 23.5% 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, nevertheless, 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 hyperuricemic 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 completely attributed to its ability in inhibiting XOD activity. Therefore, we detected the protein expression of URAT1 in the kidneys of mice, which was considered to be the most primary urate transporter 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 protein level in hyperuricemic mice with no significance. These observations suggested that treatment of 4d led to recovery of renal URAT1 expression, resulting in urate reabsorption reduction and urate excretion enhancement in hyperuricemic mice. 4. Conclusion The current study prepared a series of a,b-unsaturated cyclohexanone and cyclopentanone analogous as anti-hyperuricemic agents. As expected, these curcumin a,b-unsaturated cyclohexanone and cyclopentanone derivatives exhibited remarkable antihyperuricemia activity and uricosuric activity in vivo. Notably, compound 4d is a first-in-class xanthine oxidase and urate trans-

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. 5. Experimental section 5.1. General chemistry 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, singlet; 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. 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 stirred 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. 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

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A

B Oxonate-treated

Oxonate-treated

0.8

8

### * ***

***

Hepatic uric acid levels (mg/g tissue)

Serum uric acid levels (mg/dL)

###

*** ***

4

0 Normal

Vehicle

5

10

20

4d

10 BM

** **

*** ***

0.4

0.0

(mg/kg)

10 AP

*

Normal

Vehicle

5

10

20

4d

C

10

10

AP

BM

(mg/kg)

Oxonate-treated

Urinary uric acid excretion in 24 h (mg)

0.8

***

***

*** *

***

*

20

10

10

AP

BM

###

0.4

0.0 Normal

Vehicle

5

10 4d

(mg/kg)

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 oxonatetreated vehicle group.

Oxonate-treated

Xanthine Oxidase activity

(nmol uric acid/min per mg protein)

2.5 11.3% 43.4% 31.1%

2.0 27.5% 19.0%

1.5 ###

*

1.0

***

*** ***

0.5 0.0 Normal

Vehicle

5

10 4d

20

10

10

AP

BM

(mg/kg)

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-dimethoxybenzylidene)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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Oxonate-treate

Normal Vehicle

5

4d 10

20

AP 10

BM 10 (mg/kg)

URAT1

70 kDa

α-Tubulin

55 kDa

URAT1 protein levels (normalized to 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. HRMS: 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 C22H21BrO3 [M+H]+: 413.0747, Found: 413.0761.

5.2.2. (2E,6E)-2-(2-Chlorobenzylidene)-6-(3,5-dimethoxybenzylidene)cyclohexanone (4b) 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.

5.2.5. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(3-methoxybenzylidene)cyclohexanone (4e) 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.

5.2.3. (2E,6E)-2-(3-Bromobenzylidene)-6-(3,5-dimethoxybenzylidene)cyclohexanone (4c) 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. 5.2.4. (2E,6E)-2-(4-Bromobenzylidene)-6-(3,5-dimethoxybenzylidene)cyclohexanone (4d) 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),

5.2.6. (2E,6E)-2-(3,5-Di-tert-butyl-4-hydroxybenzylidene)-6-(3, 5-dimethoxybenzylidene)cyclohexanone (4f) 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. 5.2.7. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(4-hydroxybenzylidene)cyclohexanone (4g) 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,

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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. 5.2.8. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(4-(dimethylamino)benzylidene)cyclohexanone (4h) 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. 5.2.9. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(4-methoxybenzylidene)cyclohexanone (4i) 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). 13 C 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 C23H24O4[M+H]+: 365.1747, Found: 365.1747. 5.2.10. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(4-(methylsulfonyl)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, 28.541, 28.499, 22.860. HR-MS: Calcd. For C23H24O5S[M+H]+: 413.1417, Found: 413.1396. 5.2.11. (2E,5E)-2-(3-Chlorobenzylidene)-5-(3,5-dimethoxybenzylidene)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. HRMS: Calcd. For C21H19ClO3[M+H]+: 355.1095, Found: 355.1089. 5.2.12. (2E,5E)-2-(2-Chlorobenzylidene)-5-(3,5-dimethoxybenzylidene)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. HRMS: Calcd. For C21H19ClO3[M+H]+: 355.1095, Found: 355.1086. 5.2.13. (2E,5E)-2-(3-Bromobenzylidene)-5-(3,5-dimethoxybenzylidene)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,

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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, 109.041, 102.069, 55.700, 26.755, 26.693. HR-MS: Calcd. For C21H19BrO3[M+H]+: 399.0590, Found: 399.0573. 5.2.14. (2E,5E)-2-(4-Bromobenzylidene)-5-(3,5-dimethoxybenzylidene)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. 5.2.15. (2E,5E)-2-(3,5-Dimethoxybenzylidene)-5-(3-methoxybenzylidene)cyclopentanone (7e) 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. HRMS: Calcd. For C22H22O4[M+H]+: 351.1591, Found: 351.1578. 5.2.16. (2E,5E)-2-(3,4-Dihydroxybenzylidene)-5-(3,5-dimethoxybenzylidene)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, 55.840, 28.557, 28.367. HR-MS: Calcd. For C21H20O5[M+H]+: 353.1384, Found: 352.1385. 5.2.17. (2E,5E)-2-(3,5-Dimethoxybenzylidene)-5-(4-hydroxybenzylidene)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. 5.2.18. (2E,5E)-2-(3,5-Dimethoxybenzylidene)-5-(4-(dimethylamino)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.

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5.2.19. (2E,5E)-2-(3,5-Dimethoxybenzylidene)-5-(4-methoxybenzylidene)cyclopentanone (7i) 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 C22H22O4[M+H]+: 351.1591, Found: 351.1578. 5.2.20. (2E,5E)-2-(3,5-Dimethoxybenzylidene)-5-(4-(methylsulfonyl)benzylidene)cyclopentanone (7j) 1 H 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. 5.2.21. (2E,5E)-2-(2-Bromobenzylidene)-5-(3,5-dimethoxybenzylidene)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, 108.976, 101.979, 55.703, 26.957, 26.473. HR-MS: Calcd. For C21H19BrO3[M+H]+: 399.0590, Found: 399.0584. 5.2.22. (2E,5E)-2-(4-Chlorobenzylidene)-5-(3,5-dimethoxybenzylidene)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.

mals 1 h prior to the administration. Briefly, mice were administered 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). 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 eliminated 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 implemented through the graphical user interface CDocker protocol. 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 lM) of the test compounds or allopurinol in DMSO were mixed at 37 °C for 5 min. The control group did not contain the test compounds or allopurinol. Immediately after 200 lL 0.6 mM xanthine in doubly distilled 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.

% inhibition ¼ ½ðrate of control reaction
rate of sample reactionÞ=rate of control reaction  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 temperature 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, oxonate + test compounds (10 mg/kg), oxonate + 10 mg/kg allopurinol, oxonate + 10 mg/kg benzbromarone. Oxonate, test compounds, allopurinol and benzbromarone at various concentrations 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-

Human embryonic kidney (HEK)293 cells (American Type Culture 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 supplemented with 5% CO2. The cell culture and transfection were performed 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 instructions. After 6 h of transfection, cells were subcultured in poly-Dlysine-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 curcumin derivatives or benzbromarone. The composition of the incubation medium was as follows: 140 mM sodium gluconate, 2.7 mM

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potassium gluconate, 1.8 mM KH2PO4, and 10 mM Na2HPO4. The cells were incubated with incubation medium containing curcumin 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.

Acknowledgments

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 followed 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. Phosphotungstic acid method was adopted to estimate uric acid concentrations in serum, urine and livers. For XOD activity assay, enzyme extraction of liver tissues has been performed as described in our previous study.24 XOD activity in extraction of mice livers was determined by the XOD activity detection kit. The experimental process was performed according to manufacturer’s instructions. The protein extractions of renal cortical brush-border membrane for Western blot analysis of UART1 (1:1000) (SaiChi Biotech, Beijing, P. R. China) and a-tubulin (1:2000) (Cell Signaling Technology, Boston, MA, USA) were prepared as our previous studies. The Protein concentrations of kidney cortex supernatant were measured by the BCA Protein Quantification Kit (Vazyme Biotech, Nanjing, China). Western blot analysis were performed as our previous studies. The levels of target proteins were determined using a gel imaging system (ChemiScope 2850, Clinx Science Instruments Co., Ltd., Shanghai, China), and normalized by the reference band from a-tubulin. 5.8. Statistical analysis Data is presented as means ± S.E.M, and statistically significant differences were assessed by one-way analysis of variance (ANOVA) followed by post-hoc analysis. All statistical analyses are processed with Statistical Analysis System (GraphPad Prism 5, GraphPad Software, Inc., San Diego, CA, USA), and the difference was considered statistically significant when P <0.05.

The work was supported by National Natural Science Foundation of China (Grant No. 81202573); the Suzhou Science & Technology Foundation (Grant No. SYS201665) and PAPD (A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions). Supplementary data

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