HCV NS5B 1a (IC50 = 0.94 μM); HCV NS5B 1b (IC50 = 1.2 μM)

体外活性：VX-222 与 HCV RNA 依赖性 RNA 聚合酶的拇指 II 变构袋结合。 VX-222 对基因型 1a 和 1b 的 HCV NS5B 表现出非竞争性和选择性抑制，IC50 分别为 0.94 和 1.2 μM。 VX-222 选择性抑制亚基因组 HCV 基因型 1a 和 1b 的复制，EC50 分别为 22.3 和 11.2 nM。同样，最近的一项研究表明，VX-222 抑制 1b/Con1 HCV 亚基因组复制子，EC50 为 5 nM。 VX-222 优先抑制引物依赖性 RNA 合成，对从头启动的 RNA 合成仅显示出适度的影响或没有影响。激酶测定：VX-222 对 HCV NS5B 活性的抑制作用是通过使用均聚物 RNA 模板/引物（即聚 rA）评估新合成的 RNA 中酶的 C 末端 Δ21 截短形式掺入的放射性标记 UTP 的量来测量的。 / 寡聚 dT。使用液体闪烁计数器对掺入的放射性进行定量检测。 VX-222 抑制基因型 1b 菌株 BK 的 HCV NS5B 的体外动力学是使用 NS5B 的 C 末端 Δ21 截短版本确定的。 VX-222（1 至 1.5 μM）在 10 至 75 μM 非放射性 UTP 与 0.89 至 6.70 μCi 的 [α-33P] 标记 UTP 混合的情况下进行测试。 RNA 依赖性 RNA 聚合酶反应可在 22 °C 下进行 18 分钟。细胞测定：将含有 HCV RNA 复制子的 Huh7.5 细胞用胰蛋白酶处理，并以 4 × 104 个细胞/孔的浓度接种到 48 孔板中。第二天更换培养基，并将 VX-222 添加到 200 μL 完全培养基中。 48 小时后，提取总 RNA，并通过实时逆转录 PCR (RT-PCR) 定量病毒 RNA。通过对数曲线拟合的非线性回归分析计算使 HCV RNA 复制子水平降低 50% 的有效药物浓度 (EC50)。

在大鼠和狗中，VCH-222 显示出良好的药代动力学特征，包括较低的全身清除率和出色的口服生物利用度（大于 30%）以及良好的 ADME 特性。 VCH-222 可通过多种酶（CYP1A1、2A6、2B6、2C8、CYP 3A4、UGT1A3）进行生物转化，预计会在肝脏中主动转运，并在胆汁中或以葡萄糖醛酸加合物的形式主要完整地排泄。

VX-222 对 HCV NS5B 活性的抑制作用是通过使用称为聚 rA / 寡聚 dT 的均聚物 RNA 模板/引物测量新鲜合成的 RNA 中酶的 C 末端 Δ21 截短形式掺入的放射性标记 UTP 的量来量化的。液体闪烁计数器用于定量检测掺入的放射性。通过使用 NS5B 的 C 末端 Δ21 截短形式，确定了 VX-222 诱导的对基因型 1b 菌株 BK 的 HCV NS5B 抑制的体外动力学。当VX-222（1至1.5μM）与0.89至6.70μCi的[α-33P]标记UTP和10至75μM非放射性UTP组合时，进行测试。 RNA 依赖性 RNA 聚合酶反应在 22 °C 下进行 18 分钟。

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大鼠和人肝微粒体的稳定性研究：[2]
孵育混合物中含有最终蛋白浓度为0.5 mg/mL的肝微粒体，1µM的试验品和2 mM的NADPH， 0.1 M的磷酸钾缓冲液（pH 7.4）含有1 mM的EDTA。在0、5、10、15和30分钟收集等分，通过加入含有内标的乙腈沉淀蛋白质来终止反应。采用LC/MS/MS对上清液进行定量分析。

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CYP抑制试验：[2]
对实验品抑制人肝微粒体中各种CYP酶的潜力进行了评价。含0.25 mg/mL混合性别人肝微粒体蛋白和2 mM NADPH的孵育混合物，在0.1 M磷酸钾缓冲液中加入1.0 mM EDTA, pH 7.4。测试品与酶特异性探针底物在0至75µM的浓度范围内孵育。在37°C摇晃培养箱中进行3次10分钟的培养。通过LC/MS/MS监测每个探针特异性代谢物的外观，并相对于载体对照测定其形成的抑制百分率，以确定IC50值。

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PXR试验（CYP诱导电位）：[2]
利用稳定转染PXR和荧光素酶报告基因的DPX2人肝癌细胞系，评估PXR受体的激活。底物浓度范围（0.1-100µM）与细胞孵育24小时，绘制PXR激活相对于载体对照的倍数，以确定EC50和Emax值。利福平作为阳性对照。

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快速平衡透析（RED）评估血浆蛋白结合：[2]
将试验品以终浓度为1和10 μ M（1%有机）加入血浆中。将血浆样品装入RED装置的血浆室，并将磷酸盐缓冲液装入缓冲室。在37℃、饱和湿度的CO2培养箱（5% CO2）中，密封并在振动平台上搅拌6小时。孵育后，将装置两侧的等分液与200µL内标溶液在乙腈中涡流混合，沉淀蛋白质。采用LC/MS/MS对上清液进行分析。通过比较缓冲液样品与等离子体样品的峰面积比来确定未结合的部分。

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表面等离子体共振。[1]
所有分析物结合实验均在Biacore T100仪器上进行，使用n -羟基琥珀酰亚胺酯和1-乙基-3（3-二氨基丙基）盐酸碳二亚胺活化的预处理CM5传感器芯片。1bΔ21 NS5B蛋白及其变体在注射5分钟后固定到约9,000反应单位的标称密度。将化合物注入含有25 mM HEPES （pH 7.4）、10 mM MgCl2、150 mM NaCl、0.01% Tween 20、0.05% β-巯基乙醇和5% DMSO的缓冲液中。所有化合物均表现出1:1的饱和结合行为。为了竞争结合，实验设计包括注射饱和浓度的第一种分析物（160 nM非布韦），然后立即注射等摩尔比例的分析物混合物（160 nM非布韦加160 nM VX-222或ANA-598）。

将含有 HCV RNA 复制子的胰蛋白酶处理的 Huh7.5 细胞以每孔 4 × 10⁴ 细胞的密度接种到 48 孔板中。第二天，将 200 μL 完整培养基与 VX-222 一起添加到新培养基中。 48 小时后，提取总 RNA，并使用实时逆转录 PCR (RT-PCR) 定量病毒 RNA。通过使用非线性回归分析和对数曲线拟合，确定了使 HCV RNA 复制子水平降低 50% 的有效药物浓度 (EC50)。

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肝细胞主动摄取：[2]
在37℃和4℃孵育15分钟的时间过程中，通过比较肝细胞中底物与培养基中底物的比例来评估试验品对人肝细胞的积极吸收。匹伐他汀作为阳性对照。用试验化合物孵育后，通过油层离心，在含内标的甲醇中裂解，将肝细胞从培养液中分离出来。采用LC/MS/MS对上清液进行分析，并在37℃和4℃下计算肝细胞中化合物与培养液中化合物的峰值比，以评估活性摄取。

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Caco2渗透性测定：[2]
简单地说，Caco-2细胞单层在12孔Costar Transwell®板上生长，融合在胶原包被的微孔聚碳酸酯膜上。渗透性试验缓冲液为Hank 's平衡盐溶液，含有10 mM HEPES和15 mM葡萄糖，pH值为7.4。给药液浓度为5µM。细胞在根尖侧（a -to- b）或基底侧（B-to-A）给药，在37℃、5% CO2的湿室中孵育。试验一式两份。在每个时间点（1和2小时），从接收腔中取出200µL的等分液，并用新鲜的实验缓冲液替换。通过无细胞（空白）膜进行渗透，以解释与设备的非特异性结合和测试品通过设备的自由扩散。经上皮电阻作为细胞单层完整性的定性测量。在测试品存在的情况下，通过每个单层测量路西法黄通量，以确保在分析过程中细胞的完整性。心得安作为阳性对照。定量分析采用LC-MS/MS，采用4点标准曲线。视渗透性（Papp）作为受试物在井内累积浓度随时间变化的函数计算。

Rats or dogs
5 mg/kg for rats or 10 mg/kg for dogs
By oral gavage

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In Vivo Studies [2]
Rat IV formulations were prepared as solutions in 10% DMI/15% EtOH/35% PPG/40% dextrose/5% in water. IV formulations for dogs and monkeys were prepared as solutions in saline. PO formulations for rats were prepared as suspensions in 0.5% MC/0.1% SLS/99.4% water. For the PO studies, male Sprague-Dawley rats were instrumented with either a carotid artery cannula to facilitate blood collection and/or a single bile duct cannula to facilitate bile collection. For IV studies, male Sprague-Dawley rats were additionally fitted with a jugular vein catheter for dose administration. Blood samples were collected at intervals to 72 hours post dose. EDTA was used as anticoagulant and plasma was prepared by centrifugation. Bile duct cannulated rats received a 15mg/kg PO dose; bile, urine, and feces were collected at intervals to 72 hours. In the tissue distribution study, male Sprague-Dawley rats received a 10 mg/kg PO dose and specified tissues were collected following euthanasia at 1, 2, 4, 7 and 24 hours post dose.

[1]. Biochemical study of the comparative inhibition of hepatitis C virus RNA polymerase by VX-222 and filibuvir. Antimicrob Agents Chemother. 2012;56(2):830-837.

[2]. Discovery of Novel Allosteric HCV NS5B Inhibitors. 2. Lactam-Containing Thiophene Carboxylates. ACS Med Chem Lett. 2017;8(2):251-255.

5-(3,3-dimethylbut-1-ynyl)-3-[(4-hydroxycyclohexyl)-[(4-methylcyclohexyl)-oxomethyl]amino]-2-thiophenecarboxylic acid is a thiophenecarboxylic acid.
Lomibuvir has been used in trials studying the treatment of Chronic Hepatitis C Virus and Chronic Hepatitis C Virus Infection.

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Filibuvir and VX-222 are nonnucleoside inhibitors (NNIs) that bind to the thumb II allosteric pocket of the hepatitis C virus (HCV) RNA-dependent RNA polymerase. Both compounds have shown significant promise in clinical trials and, therefore, it is relevant to better understand their mechanisms of inhibition. In our study, filibuvir and VX-222 inhibited the 1b/Con1 HCV subgenomic replicon, with 50% effective concentrations (EC(50)s) of 70 nM and 5 nM, respectively. Using several RNA templates in biochemical assays, we found that both compounds preferentially inhibited primer-dependent RNA synthesis but had either no or only modest effects on de novo-initiated RNA synthesis. Filibuvir and VX-222 bind to the HCV polymerase with dissociation constants of 29 and 17 nM, respectively. Three potential resistance mutations in the thumb II pocket were analyzed for effects on inhibition by the two compounds. The M423T substitution in the RNA polymerase was at least 100-fold more resistant to filibuvir in the subgenomic replicon and in the enzymatic assays. This resistance was the result of a 250-fold loss in the binding affinity (K(d)) of the mutated enzyme to filibuvir. In contrast, the inhibitory activity of VX-222 was only modestly affected by the M423T substitution but more significantly affected by an I482L substitution.[1]

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Lomibuvir (1) is a non-nucleoside, allosteric inhibitor of the hepatitis C virus NS5B polymerase with demonstrated clinical efficacy. Further development efforts within this class of inhibitor focused on improving the antiviral activity and physicochemical and pharmacokinetic properties. Recently, we reported the development of this series, leading to compound 2, a molecule with comparable potency and an improved physicochemical profile relative to 1. Further exploration of the amino amide-derived side chain led to a series of lactam derivatives, inspired by the X-ray crystal structure of related thiophene carboxylate inhibitors. This series, exemplified by 12f, provided 3-5-fold improvement in potency against HCV replication, as measured by replicon assays. The synthesis, structure-activity relationships, in vitro ADME characterization, and in vivo evaluation of this novel series are discussed.[2]

C25H35NO4S

445.61

445.228

C, 67.38; H, 7.92; N, 3.14; O, 14.36; S, 7.19

1026785-55-6

Lomibuvir;1026785-55-6; 1026785-59-0

24798764

White to off-white solid powder

1.2±0.1 g/cm3

640.5±55.0 °C at 760 mmHg

341.2±31.5 °C

0.0±2.0 mmHg at 25°C

1.589

5.15

106.08

2

5

6

31

717

0

O=C(C1=C(N([C@H]2CC[C@H](O)CC2)C([C@H]3CC[C@H](C)CC3)=O)C=C(C#CC(C)(C)C)S1)O

WPMJNLCLKAKMLA-UHFFFAOYSA-N

InChI=1S/C25H35NO4S/c1-16-5-7-17(8-6-16)23(28)26(18-9-11-19(27)12-10-18)21-15-20(13-14-25(2,3)4)31-22(21)24(29)30/h15-19,27H,5-12H2,1-4H3,(H,29,30)

5-(3,3-dimethylbut-1-ynyl)-3-[(4-hydroxycyclohexyl)-(4-methylcyclohexanecarbonyl)amino]thiophene-2-carboxylic acid

TD-4208; TD4208; GSK-1160724; GSK-1160724; Lomibuvir; VX-222; 1026785-59-0; 1026785-55-6; VCH-222; VX-222 (VCH-222, Lomibuvir); cis-Lomibuvir; Lomibuvir (VX-222); TD 4208; GSK1160724; trade name: Yupelri; TD-4208; GSK 1160724

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)