KPH2f

URAT1; GLUT9

化合物KPH2f表现出强大的URAT1抑制活性，IC50为0.24 mM，与维瑞脲相当（IC50¼0.17 mM）。KPH2f还抑制GLUT9，IC50值为9.37±7.10 mM，表明具有双重URAT1/GLUT9靶向能力。此外，KPH2f对OAT1和ABCG2的影响很小，因此不太可能引起OAT1/ABCG2介导的药物相互作用和/或中和URAT1/GLUT9抑制剂的促尿酸作用[1]。

KPH2f（10mg/kg）在降低血尿酸水平方面同样有效，并且在小鼠高尿酸血症模型中表现出更高的促尿酸作用，与维瑞脲（10mg/kg的）相比。此外，KPH2f表现出良好的药代动力学特性，口服生物利用度为30.13%，明显优于韦尼拉德（21.47%）。此外，KPH2f表现出良好的安全性，不会引起hERG毒性、体外细胞毒性（低于维瑞脲）和体内肾损伤。总的来说，这些结果表明，KPH2f代表了一种新型、安全有效的双重URAT1/GLUT9抑制剂，具有更好的药物耐受性，值得作为抗高尿酸血症候选药物进行进一步研究[1]。

14C-尿酸摄取抑制试验[1]
将HEK293细胞以1105/孔的密度接种到聚-D-赖氨酸（PDL）涂覆的96孔板中。使用脂质体3000将URAT1质粒（100ng/孔）瞬时转染入HEK293细胞。转染24小时后，将细胞与含有或不含有不同浓度受试化合物的尿酸摄取缓冲液一起孵育30分钟。通过加入终浓度为25mM的14C-尿酸15分钟来启动摄取。然后用冰冷的DPBS洗涤细胞三次以终止反应。通过加入100ml 0.1M氢氧化钠获得细胞裂解物。在加入0.5mL闪烁液后，使用液体闪烁计数器测定细胞内放射性。实验一式三份。受试化合物的抑制率计算如下：比抑制率¼[1-（CPMtest-CPM0）/（CPMcon-CPM0）]100%其中CPMt是受试组的放射性，CPMcon是对照组的洗脱液内放射性。CPM0是没有hURAT1的空载体细胞的放射性。

---

化合物对ABCG2、OAT1和GLUT9的抑制作用[1]
对受试化合物对尿酸转运相关转运蛋白（包括GLUT9、ABCG2和OAT1）的抑制作用进行了研究，以评估受试化合物的选择性。GLUT9:500 ng pcDNA3.1（"）-GLUT9质粒用脂质体3000瞬时转染到24孔板中的HEK293细胞中。24小时后，使用全细胞膜片钳技术通过电生理记录电流在HEK293-GLUT9细胞中测定化合物的GLUT9抑制作用。灌注装置持续灌注含有或不含化合物的溶液，实现了快速的溶液交换。如我们之前报道的，电流是用MultiClamp 700B膜片钳放大器/Digidata 1550B数字化仪测量的，并由pClamp 10软件计算。

细胞活力测定（MTT法）[1]
人肾（HK2）细胞用于检测化合物诱导的细胞毒性和细胞存活率。将HK2细胞以5000/孔的密度镀入96孔板中。当细胞融合达到70%时，用一系列浓度的化合物（0e200 mM）处理细胞24小时孵育。向每个孔中加入0.5mg/mL MTT溶液，并将平板在37℃下进一步孵育2小时。此后，取出培养基，向每个孔加入100ml DMSO。将板以250rpm摇动30分钟，溶解甲氮晶体后，在570nm[1]处测定吸光度。

Evaluation of urate lowering effects of compounds in vivo [1]
Mice were feed one week to adapt to the environment before experiments. Mice then were randomly divided into 6 groups. Control group (n ¼ 8), model group (n ¼ 12), 3 compound groups (SG1C, KP, KPH2F) with 10 mg/kg treatment (n ¼ 8) and positive group (verinurad) with 10 mg/kg treatment (n ¼ 8). The method of hyperuricemia induction was conducted as we previous reported. Potassium oxonate (PO), a uricase inhibitor, was subcutaneous injected into mice at 400 mg/kg in 0.5% CMC-Na and 600 mg/kg of hypoxanthine was oral gavage in model and compound groups, 0.5 h after PO injection, the mice received 10 mg/kg compounds by oral gavage, while the control group was treatmented with 0.5% CMC. 3 h after the drug administration, blood samples were obtained from the orbital vein. The samples were then centrifuged (3000g, 10 min) to obtain serum for further analysis. Urine samples were collected after drug administration for 24 h in metabolic cages. The serum and urine uric acid level were determined by uric acid assay kit.

---

In vivo pharmacokinetic study [1]
The male Sprague-Dawley rats (300 ± 20 g) were provided by Laboratory Animal Center of Southern Medical University. 24 male rats were divided into 4 groups for intravenous (5 mg/kg) and oral administration (5 mg/kg) of KPH2F and verinurad. KPH2F and verinurad were dissolved with normal saline for intragastric and intravenous administration. Blood samples (0.5 mL) were collected into heparinized tubes from the orbital vein at 2min, 5 min, 15 min, 30min, 1 h, 1.5 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h. All the samples were immediately centrifuged at 8000 rpm for 5 min, and then the plasma were stored at 80 C until analysis. The samples were quantified with the LC-MS/MS system. 100 mL of plasma samples were spiked with 10 mL of the internal standard solution (testosterone, 1 mg/mL). The mixture was extracted with 600 mL of ethyl acetate. The supernatant was combined twice in succession and evaporated to dryness at 40e50 C. The residue was reconstituted in 400 mL of mobile phase (50% methanol) and then centrifuged at 14000 rpm for 15 min. A 300 mL aliquot of the resulting solution was injected into the LC-MS/MS system for analysis. Related pharmacokinetic parameters were calculated using DAS 2.0 software. The pharmacokinetic parameters determined included the elimination half-life (t1/2), time of peak plasma concentration (Tmax), maximum plasma concentration (Cmax), area under the concentrationetime curve (AUC), mean residence time (MRT) and bioavailability (F).

The pharmacokinetic properties of KPH2f and verinurad were assessed in SD rats. The results are summarized in Table 5 and Fig. 5. After a single 5 mg/kg i.v. dose of KPH2f, the half-life (t1/2), time-tomaximum- concentration (Tmax), maximum concentration (Cmax) and mean residence time (MRT) values were 4.19 h, 0.17 h, 11093.32 ng/mL and 4.80 h respectively. At the dose of 5 mg/kg (oral), KPH2f was rapidly absorbed with a Tmax of 0.50 h, a t1/2 of 5.14 h, an MRTof 3.17 h, a Cmax of 7649.04 ng/mL, and an area under curve (AUC) of 5656.03 ng/mLh. Notably, KPH2f exhibited a reasonable oral bioavailability of 30.13%, which is clearly better than that of verinurad (21.47%) and is sufficient for an oral drug candidate. Overall, KPH2f demonstrated more favorable drug-like properties when compare to verinurad. [1]

In vitro cytotoxicity assay [1]
KPH2f was selected for evaluating the in vitro cytotoxicity to normal cells by a well-established MTT assay. Human kidney HK2 cells were incubated with various concentrations of KPH2f for 24 h, and cell viabilities were measured. As shown in Fig. 6, KPH2f exhibited little or essentially no cytotoxicity to the HK2 cells with IC50 values of 207.56 mM and 167.24 mM for 24 h and 48 h incubation, respectively. While the cytotoxicity of verinurad was stronger (IC50 ¼ 197.45 mM and 108.78 mM for 24 h and 48 h incubation, respectively) than KPH2f. These results suggest that KPH2f has a benign cytotoxicity profile and is unlikely to cause toxicity at therapeutic concentrations (e.g. <50 mM).

---

Determination of hERG-inhibitory activity [1]
Compounds that bind to hERG potassium channels with high affinities may induce QT prolongation, thus causing severe cardiotoxicity. Therefore, we tested the in vitro hERG inhibitory activity of KPH2f using a manual patch-clamp method. As shown in Fig. 7, KPH2f showed no inhibitory effects on hERG potassium channel at 50 mM, while the positive control cisapride inhibited the potassium channel completely at a concentration of 1 mM (100% reduction of the current), which is consistent with previous reports.

[1]. Discovery of novel verinurad analogs as dual inhibitors of URAT1 and GLUT9 with improved Druggability for the treatment of hyperuricemia. Eur J Med Chem. 2022;229:114092.

Among them, compound KPH2f exhibited the highest URAT1-inhibitory activity with IC50 value of 0.24 mM, comparable to that of verinurad (IC50 ¼ 0.17 mM). Mechanism study demonstrated that verinurad at 10 mM had no effects on GLUT9, while KPH2f inhibited GLUT9 effectively with an IC50 value of 9.37 ± 7.10 mM, suggesting a dual URAT1/GLUT9 targeting mechanism. In addition, KPH2f showed little inhibitory effects against OAT1 and ABCG2, two important transporters involved in urate secretion and the excretion of various drug molecules. Thus, it is unlikely for KPH2f to cause OAT1/ABCG2-mediated drug-drug interactions and/or to neutralize the uricosuric effects of URAT1/GLUT9 inhibitors. Importantly, KPH2f (10 mg/kg) was equally effective in reducing serum uric acid levels and exhibited higher uricosuric effects in a mice hyperuricemia model, as compared to verinurad (10 mg/kg). The higher uricosuric effects of KPH2f may be attributed to the dual URAT1/GLUT9 targeting capabilities, as compared to verinurad which is a single URAT1-targeting agent. Furthermore, KPH2f demonstrated favorable pharmacokinetic properties with an oral bioavailability of 30.13%, clearly better than that of verinurad (21.47%). Moreover, KPH2f presented benign safety profiles without causing hERG toxicity, cytotoxicity in vitro (lower than verinurad), and renal damage in vivo. Finally, molecular docking analysis using a homology model indicated that KPH2f and verinurad bound similarly to URAT1, and the flexible NH-linker contributed to the binding affinity of KPH2f to URAT1. Site directed mutagenesis study further confirmed the binding interactions between KPH2f and URAT1, and the residues F358 and R487 are unique for the binding of KPH2f to URAT1. Collectively, these results suggest that KPH2f represents a novel, safe and effective dual URAT1/GLUT9 inhibitor with improved druggabilities and is worthy of further investigation as an anti-hyperuricemic drug candidate.[1]

C24H17N3NAO2S

434.47

433.086

2760615-09-4

NA for KPH2f

163196377

Light yellow to light brown solid powder

114

1

6

6

31

642

0

N(C1=CN=CC=C1SCC1C=CC=CC=1C(=O)O)C1=CC=C(C2C=CC=CC1=2)C#N.[Na]

VFQCYGPPRZHSKX-UHFFFAOYSA-M

InChI=1S/C24H17N3O2S.Na/c25-13-16-9-10-21(20-8-4-3-6-18(16)20)27-22-14-26-12-11-23(22)30-15-17-5-1-2-7-19(17)24(28)29;/h1-12,14,27H,15H2,(H,28,29);/q;+1/p-1

sodium;2-[[3-[(4-cyanonaphthalen-1-yl)amino]pyridin-4-yl]sulfanylmethyl]benzoate

KPH2f; 2760615-09-4; CHEMBL5202967; URAT1/GLUT9 inhibitor; Sodium 2-(((3-((4-cyanonaphthalen-1-yl)amino)pyridin-4-yl)thio)methyl)benzoate

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

---

注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

View More

注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

---

口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

View More

口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

---

注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

---

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;
5、为保证最佳实验结果，工作液请现配现用！
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。