URAT1 (IC50 = 0.8 nM)[1]

At high concentrations, URAT1 inhibitor 3 (0-400 μM; 24 and 72 h; HepG2 and HK2 cells) decreases cell viability with minimal toxicity[1]. The urate excretion transporters are less inhibited by URAT1 inhibitor 3 (0.01-100 μM), with IC50 values of 4.04 μM for ABCG2 and 10.16 μM for OAT1[1].

In a mouse model of hyperuricemia, URAT1 inhibitor 3 (1-4 mg/kg; ig; once) has the efficacy to decrease urate[1]. In mice, URAT1 inhibitor 3 (50 mg/kg; ig; daily, for 14 d) had no adverse effects on the liver or kidneys[1]. Kunming mice with a hyperuricemia model are subjected to GSH depletion induced by URAT1 inhibitor 3 (50 mg/kg; ig; once).

14C-uric acid uptake assay in HEK293-ABCG2 vesicles [1]
20 μg of pcDNA3.1(+)-ABCG2 plasmid was transiently transfected into HEK293 cells in 100 mm dishes to express ABCG2 with lipofectamine 3000. After 24 h, the membrane vesicles were prepared from HEK293-ABCG2 cells by ultracentrifuge. The ABCG2 inhibitory effects of test compounds were determined by a14C-uric acid assay as we previously reported.

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Detection of GLUT9 inhibitory effects by electrophysiological recordings [1]
PcDNA3.1 (+)-GLUT9 plasmid was transiently transfected into HEK293 cells in 24 well plates by lipofectamine 3000. The GLUT9 inhibitory effects of compounds were determined in HEK293-GLUT9 cells by the electrophysiological recordings currents using the wholecell patch-clamp techniques previously reported by us.

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Detection of OAT1 inhibitory effects by 6-CFL uptake [1]
100 ng/well of pcDNA3.1(+)-OAT1 plasmid was transient transfected into HEK293 cells in PDL coated 96 well plate. After 24 h cell growth, the HEK293-OAT1 cells were pre-incubated with tested compounds for 30 min, and then uptake of 6-CFL were performed for 15 min. After that, cells were washed with ice cold DPBS. The fluorescence intensity of cell lysates were determined in microplate.

Cell Viability Assay[1]
Cell Types: HepG2 and HK2 cells
Tested Concentrations: 0, 100, 200, 300, and 400 μM
Incubation Duration: 24 and 72 hrs (hours)
Experimental Results: Had little cytotoxicity at 24 h and inhibition rate of 34.75% and 35.9% of HepG2 and HK2 cells, respectively.

Animal/Disease Models: Male Kunming (KM) mice with hyperuricemia model[1]
Doses: 1, 2, and 4 mg/kg
Route of Administration: Oral gavage; once
Experimental Results: diminished the serum urate levels in a dose-dependent manner.

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Animal/Disease Models: Male Kunming mice[1]
Doses: 50 mg/kg
Route of Administration: Oral gavage; daily, for 14 days
Experimental Results: Did not cause renal toxicity.

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Animal/Disease Models: Male Kunming mice with hyperuricemia model[1]
Doses: 50 mg/kg
Route of Administration: Oral gavage; once
Experimental Results: diminished the serum GSH levels from 42.23 μM to 20.39 μM.

Pharmacokinetic properties of JNS4 [1]
Considering the excellent urate lowering effect of JNS4 in vivo, the pharmacokinetic properties of JNS4 and BM were assessed in SD rats. The results were summarized in Table 3 and Fig. 8. After intravenous injection of 5 mg/kg JNS4, the half-life (t1/2), time-to-maximumconcentration (Tmax), maximum concentration (Cmax) and mean residence time (MRT) values were 6.80 h, 0.52 h, 18428.57 ng/mL and 9.85 h respectively. At the dose of 5 mg/kg (oral), JNS4 was rapidly absorbed with a Tmax of 0.29 h, a t1/2 of 4.61 h, an MRT of 3.48 h, a Cmax of 6833.07 ng/mL, and an area under curve (AUC) of 35278.21 ng/mL•h. Notably, JNS4 exhibited a high oral bioavailability of 55.28%, which was significantly better than that of BM (36.11%) and is sufficient for an oral drug candidate. These data may explain the better urate lowering effects of JNS4 in vivo.

Toxicity assessment of JNS4 in vitro and in vivo [1]
Currently available anti-gout drugs can cause liver and kidney damage, especially, Benzbromarone (BM) was reported to impair mitochondrial function in HepG2 cells, leading to fulminant hepatitis. Therefore, it is essential to evaluate the toxicity of JNS4. We first assessed the cytotoxicity of JNS4 against HepG2 and HK2 cells by an MTT assay. As shown in Fig. 9A and B, both JNS4 and BM (at 0–400 μM) showed little cytotoxicity at 24 h. However, when cells were incubated for 72 h, BM started to cause cytotoxicity against HepG2 cells at 100 μM, and the inhibition rate was 65.71% at 400 μM (Fig. 9C). In contrast, JNS4 exhibited an inhibition rate of 34.75% at 400 μM, significantly lower than that of BM (p < 0.001). In HK2 cells, both JNS4 and BM showed similar and mild inhibitory effects, with an inhibition rate of about 35.9% and 40.12% at 400 μM for JNS4 and BM, respectively (Fig. 9D). It is worthy of note that 400 μM and 72 h were far exceeding regular plasma exposed dose and time. As for the in vivo toxicity, no death and/or abnormal behaviors (lethargy, clonic convulsion, anorexia, or ruffled fur) were observed for mice treated with 50 mg/kg of JNS4 and BM (p.o.) for 14 days. Furthermore, the body weight gain in test groups was the same as in the control group (Fig. 10A). Next, we evaluated the hepatotoxicity of JNS4 and BM by measuring ALT/AST levels in mice. The hepatotoxicity of BM is related to its mitochondrial toxicity. Administration of BM significantly increased the serum AST activity compared with control and JNS4 treated group. Pretreatment with buthionine sulfoximine (BSO, a rate limiting enzyme in the biosynthesis of GSH) could potentiate JNS4 and BM induced elevation of ALT and AST levels. However, the ALT and AST levels induced by BM were higher than those by JNS4 (Fig. 10B). In addition, we evaluated the renal toxicities of JNS4 and BM by examining the serum CR and BUN levels. As shown in Fig. 10C, D, no obvious renal injury was detected in JNS4 and BM groups, when compared to the control group. In conclusion, the above results suggested that JNS4 did not cause renal toxicity, and showed less hepatotoxicity than BM.

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GSH depletion induced by JNS4 and BM [1]
Glutathione (GSH) plays a protective role for protein covalent binding and subsequent hepatotoxicity caused by xenobiotic metabolites. Depletion of GSH is a biomarker for the production of reactive metabolites. Therefore, we measured the serum GSH levels in mice treated with JNS4 and BM at a hepatotoxic dose (50 mg/kg). As shown in Fig. 11, BM significantly reduced the serum GSH levels by 74.29% (from 41.68 μM to 10.72 μM) during the first 1 h, and GSH was slowly recovered within 12 h. While the serum GSH levels were also decreased by JNS4 during the first 30 min (by 48.9%, from 42.23 μM to 20.39 μM), the degree of GSH depletion was far lesser than that of BM. It has been reported that there are multiple possible metabolic pathways for benzbromarone. Both the benzofuran ring and the phenolic hydroxyl group are critical for the development of benzbromarone induced hepatotoxicities. Due to the presence of the phenolic hydroxyl group in JNS4, it is the possible that the metabolic products of JNS4 may also decrease serum GSH levels, but the degree of GSH depletion was far lesser than that of BM as mentioned above. However, the exact difference between the metabolic pathways of JNS4 and benzbromarone remains to be investigated, which is beyond the scope of this work. These results indicate that JNS4 is less likely to cause hepatotoxicity than BM due to the depletion of GSH.

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Pathological analysis of liver and kidney tissues [1]
The metabolism of BM usually occurs in liver. After 14 days treatment of JNS4 and BM, mice were sacrificed; the livers and kidneys were removed for the purpose of pathological examination. HE staining of liver and kidney was performed (Fig. 12). Compared to normal mice, the liver tissues of BM group showed mild inflammatory infiltration (black arrow) and dilated vacuolization, no obvious irregular cell arrangements and steatosis were observed. In contrary to the BM group, the liver tissues of JNS4 group exhibited normal cell arrangements, and no obvious inflammatory infiltration was observed. The renal excretion of BM usually amounted to 8–9% of the dose ingested after a single dose, and reached 15–20% after chronic treatment (5 and 14 days). The renal tissues of both BM and JNS4 group had normal tubular structure and glomerulus cells compared with control group, and no obvious inflammatory infiltration was observed. However, a slight dilatation was observed in the kidney tissues of BM group. Collectively, these data suggested that JNS4 (50 mg/kg) did not cause hepatic and/or renal damage as compared to BM.

[1]. Discovery of novel benzbromarone analogs with improved pharmacokinetics and benign toxicity profiles as antihyperuricemic agents. Eur J Med Chem. 2022 Nov 15;242:114682.

JNS4 exhibited the highest URAT1 inhibitory potency with an IC50 of 0.80 μM, comparable to that of BM (IC50 = 0.53 μM). As BM is a nonselective URAT1 inhibitor, which also inhibits urate secretion transporters such as OAT1 and ABCG2. Thus, we investigated the effects of JNS4 on these transporters. To our delight, JNS4 showed little inhibitory effects against OAT1 and ABCG2 with IC50 of 4.04 μM and 10.16 μM, respectively, much weaker than that of BM (IC50 = 2.12 μM and 0.34 μM), indicating that JNS4 is less likely to cause OAT1/ABCG2-mediated drug-drug interactions and/or anti-uricosuric effects. Importantly, JNS4 demonstrated higher in vivo urate-lowering efficacy at doses of 1–4 mg/kg in a mouse model of hyperuricemia, as compared to BM and lesinurad. Furthermore, JNS4 exhibited favorable pharmacokinetic properties with an oral bioavailability of 55.28%, significantly higher than that of BM (36.11%). The high in vivo antihyperuricemic effects of JNS4 were probably due to selective inhibition of URAT1, as well as the excellent oral bioavailability when compared to BM. Moreover, JNS4 possessed benign toxicity profiles (no cytotoxicity against HepG2 and HK2 cells at 400 μM; no in vivo hepatic and renal toxicities observed at 50 mg/kg), while BM at the same dose caused mild to moderate damages to liver and kidney. In conclusion, JNS4 represents a novel, safe and selective URAT1 inhibitor with excellent druggabilities and is worthy of further investigation as an anti-hyperuricemic agent. [1]

C14H8CL2N2O2

307.131521224976

305.996

2850331-30-3

165412792

Off-white to light yellow solid powder

3.7

1

3

1

20

370

0

HVYNCNZJQZOKFM-UHFFFAOYSA-N

InChI=1S/C14H8Cl2N2O2/c15-10-6-9(7-11(16)12(10)19)14(20)18-5-3-8-2-1-4-17-13(8)18/h1-7,19H

(3,5-dichloro-4-hydroxyphenyl)-pyrrolo[2,3-b]pyridin-1-ylmethanone

URAT1 inhibitor 3; 2850331-30-3; CHEMBL5439993;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 100 mg/mL (325.60 mM)

Solubility in Formulation 1: ≥ 10 mg/mL (32.56 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 100.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)