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 characteristics of JNS4[1] Given the excellent uric acid-lowering effect of JNS4 in vivo, this study evaluated the pharmacokinetic characteristics of JNS4 and BM in SD rats. The results are summarized in Table 3 and Figure 8. After intravenous injection of 5 mg/kg JNS4, its half-life (t1/2), time to peak concentration (Tmax), maximum concentration (Cmax), and mean residence time (MRT) were 6.80 h, 0.52 h, 18428.57 ng/mL, and 9.85 h, respectively. At a dose of 5 mg/kg (oral), JNS4 was rapidly absorbed, with a time to peak concentration (Tmax) of 0.29 h, a half-life (t1/2) of 4.61 h, a mean residence time (MRT) of 3.48 h, a peak plasma concentration (Cmax) of 6833.07 ng/mL, and an area under the curve (AUC) of 35278.21 ng/mL·h. Notably, JNS4 boasts an oral bioavailability of 55.28%, significantly superior to BM (36.11%), sufficient to meet the requirements for oral drug candidates. These data may explain JNS4's superior uric acid-lowering effect in vivo.

In vitro and in vivo toxicity assessment of JNS4 [1]
Currently available anti-gout drugs can cause liver and kidney damage, especially benzbromarone (BM), which has been reported to impair mitochondrial function in HepG2 cells, leading to fulminant hepatitis. Therefore, assessing the toxicity of JNS4 is crucial. We first assessed the cytotoxicity of JNS4 on HepG2 and HK2 cells by the MTT assay. As shown in Figures 9A and 9B, neither JNS4 nor BM (0–400 μM) showed significant cytotoxicity at 24 hours. However, after 72 hours of cell incubation, BM began to produce cytotoxicity in HepG2 cells at 100 μM, with an inhibition rate of 65.71% at 400 μM (Figure 9C). In contrast, JNS4 had an inhibition rate of 34.75% at 400 μM, which was significantly lower than that of BM (p < 0.001). In HK2 cells, both JNS4 and BM exhibited similar and weak inhibitory effects, with inhibition rates of approximately 35.9% and 40.12% at 400 μM (Figure 9D). Notably, the concentrations of 400 μM and 72 hours significantly exceeded the usual plasma exposure dose and duration. Regarding in vivo toxicity, no death or abnormal behavior (somnolence, clonic seizures, anorexia, or ruffled fur) was observed in mice after 14 consecutive days of oral administration of 50 mg/kg JNS4 and BM. Furthermore, the weight gain in each experimental group was the same as in the control group (Figure 10A). Next, we assessed the hepatotoxicity of JNS4 and BM by measuring ALT/AST levels in mice. The hepatotoxicity of BM is associated with its mitochondrial toxicity. Compared with the control and JNS4-treated groups, BM administration significantly increased serum AST activity. Pretreatment with sulfoxide (BSO, the rate-limiting enzyme in glutathione biosynthesis) enhanced the JNS4 and BM-induced increases in ALT and AST levels. However, BM-induced ALT and AST levels were higher than those induced by JNS4 (Fig. 10B). Furthermore, we assessed the nephrotoxicity of JNS4 and BM by detecting serum creatinine (CR) and blood urea nitrogen (BUN) levels. As shown in Fig. 10C and D, no significant kidney damage was detected in either the JNS4 or BM groups compared to the control group. In summary, the above results indicate that JNS4 does not cause nephrotoxicity and has lower hepatotoxicity than BM.

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JNS4 and BM-induced GSH depletion[1]
Glutathione (GSH) has a protective effect against protein covalent binding and the resulting hepatotoxicity of xenobiotic metabolites. GSH depletion is a biomarker of the production of active metabolites. Therefore, we examined the serum GSH levels in mice treated with hepatotoxic doses (50 mg/kg) of JNS4 and BM. As shown in Figure 11, BM significantly reduced serum GSH levels by 74.29% (from 41.68 μM to 10.72 μM) within the first hour and recovered slowly within 12 hours. Although JNS4 also caused a decrease in serum GSH levels within the first 30 minutes (a decrease of 48.9%, from 42.23 μM to 20.39 μM), its GSH consumption was much lower than that of BM. Benzbromarone has been reported to have multiple possible metabolic pathways. The benzofuran ring and phenolic hydroxyl group are crucial for the occurrence of benzbromarone-induced hepatotoxicity. Due to the presence of phenolic hydroxyl group in JNS4, its metabolites may also reduce serum GSH levels, but as mentioned above, its GSH consumption was much lower than that of BM. However, the exact difference between the metabolic pathways of JNS4 and benzbromarone remains to be studied, which is beyond the scope of this study. These results suggest that JNS4 is less likely to cause hepatotoxicity due to GSH consumption than BM.

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Hepatic and renal histopathological analysis[1]
The metabolism of BM usually occurs in the liver. Mice were sacrificed 14 days after treatment with JNS4 and BM, and their livers and kidneys were removed for pathological examination. Hematoxylin and eosin (HE) staining of the liver and kidney tissues was performed (Figure 12). Compared with normal mice, the liver tissue of mice in the BM group showed mild inflammatory cell infiltration (black arrows) and vacuolar expansion, without significant cell disorganization or fatty degeneration. In contrast, the liver tissue of mice in the JNS4 group showed normal cell arrangement and no significant inflammatory cell infiltration. After a single dose, the renal excretion of BM was typically 8%–9% of the ingested dose, reaching 15%–20% after long-term administration (5 days and 14 days). Compared with the control group, the kidney tissues of both the BM and JNS4 groups showed normal renal tubular structure and glomerular cells, with no significant inflammatory cell infiltration observed. However, mild expansion was observed in the kidney tissue of the BM group. In summary, these data indicate that JNS4 (50 mg/kg) did not cause liver and/or kidney damage compared with 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 inhibitory efficacy against URAT1, with an IC50 value of 0.80 μM, comparable to BM (IC50 = 0.53 μM). Since BM is a non-selective URAT1 inhibitor, it also inhibits uric acid secretion transporters such as OAT1 and ABCG2. Therefore, we investigated the effects of JNS4 on these transporters. Encouragingly, JNS4 showed weak inhibition of OAT1 and ABCG2, with IC50 values of 4.04 μM and 10.16 μM, respectively, significantly lower than BM (IC50 values of 2.12 μM and 0.34 μM, respectively), indicating that JNS4 is unlikely to cause OAT1/ABCG2-mediated drug interactions and/or anti-uric acid excretion effects. Notably, in a mouse model of hyperuricemia, JNS4 demonstrated superior in vivo uric acid-lowering efficacy at doses of 1–4 mg/kg compared to BM and retinoic acid. In addition, JNS4 has good pharmacokinetic properties and an oral bioavailability of 55.28%, which is significantly higher than that of BM (36.11%). The efficient in vivo uric acid-lowering effect of JNS4 may be attributed to its selective inhibition of URAT1 and its superior oral bioavailability compared to BM. Furthermore, JNS4 has good toxicity characteristics (no cytotoxicity to HepG2 and HK2 cells at a concentration of 400 μM; no in vivo hepatotoxicity or nephrotoxicity observed at a dose of 50 mg/kg), while the same dose of BM caused mild to moderate hepatotoxicity and nephrotoxicity. In conclusion, JNS4 is a novel, safe, and selective URAT1 inhibitor with good drug-like properties and deserves further study as an anti-hyperuricemia drug. [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.)