URAT1 inhibitor 3   中文名称: URAT1 抑制剂 3

URAT1 (IC50 = 0.8 nM)[1]

在高浓度下，URAT1 抑制剂 3（0-400 μM；24 和 72 小时；HepG2 和 HK2 细胞）会降低细胞活力，且毒性最小[1]。尿酸盐排泄转运蛋白受 URAT1 抑制剂 3 (0.01-100 μM) 的抑制较小，ABCG2 的 IC50 值为 4.04 μM，OAT1 的 IC50 值为 10.16 μM[1]。

在高尿酸血症小鼠模型中，URAT1 抑制剂 3（1-4 mg/kg；ig；一次）具有降低尿酸的功效[1]。在小鼠中，URAT1 抑制剂 3（50 mg/kg；ig；每日一次，持续 14 天）对肝脏或肾脏没有不良影响[1]。高尿酸血症模型昆明小鼠接受URAT1抑制剂3（50mg/kg；ig；一次）诱导的GSH消耗。

HEK293-ABCG2囊泡中14C-尿酸摄取测定[1]
将20μg pcDNA3.1（+）-ABCG2质粒瞬时转染到100 mm培养皿中的HEK293细胞中，用脂质体3000表达ABCG2。24小时后，通过超速离心从HEK293-ABCG2细胞制备膜囊泡。正如我们之前报道的那样，试验化合物的ABCG2抑制作用是通过14C尿酸测定法测定的。

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通过电生理记录检测GLUT9抑制作用[1]
用脂质体3000将PcDNA3.1（+）-GLUT9质粒瞬时转染到24孔板中的HEK293细胞中。通过使用我们之前报道的全细胞膜片钳技术的电生理记录电流，在HEK293-GLUT9细胞中确定了化合物的GLUT9抑制作用。

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通过6-CFL摄取检测OAT1抑制作用[1]
将100ng/孔的pcDNA3.1（+）-OAT1质粒瞬时转染到PDL包被的96孔板中的HEK293细胞中。细胞生长24小时后，将HEK293-OAT1细胞与受试化合物预孵育30分钟，然后摄取6-CFL 15分钟。之后，用冰冷的DPBS洗涤细胞。在微孔板中测定细胞裂解物的荧光强度。

细胞活力测定[1]
细胞类型： HepG2 和 HK2 细胞
测试浓度： 0、100、200、300 和 400 μM
孵育时间：24小时和72小时
实验结果：24小时几乎没有细胞毒性，对HepG2和HK2细胞的抑制率分别为34.75%和35.9% ， 分别。

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

55.1

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)

配方 1 中的溶解度: ≥ 10 mg/mL (32.56 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 100.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
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6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。