HCV NS5B 1a (IC50 = 0.94 μM); HCV NS5B 1b (IC50 = 1.2 μM)
Lomibuvir (VX-222) is a potent inhibitor of hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (the viral enzyme responsible for viral genome replication), with an IC50 of 16 nM for HCV genotype 1a NS5B polymerase and 12 nM for genotype 1b NS5B polymerase in cell-free enzyme assays [1]
- Lomibuvir acts as a non-nucleoside inhibitor (NNI) of HCV NS5B, binding to the thumb domain of the enzyme; it shows no significant inhibition of human RNA polymerases (e.g., Pol I, Pol II) at concentrations up to 10 μM [1]

VX-222 attaches itself to the HCV RNA-dependent RNA polymerase's thumb II allosteric pocket. With an IC50 of 0.94 and 1.2 μM, respectively, VX-222 shows non-competitive and selective inhibition in HCV NS5B of genotypes 1a and 1b. Subgenomic HCV genotypes 1a and 1b are specifically inhibited by VX-222, with EC50 values of 22.3 and 11.2 nM, respectively. (Source: ) Similarly, VX-222 inhibits the 1b/Con1 HCV subgenomic replicon with an EC50 of 5 nM, according to a recent study. While having little to no effect on de novo-initiated RNA synthesis, VX-222 preferentially inhibits primer-dependent RNA synthesis.

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In cell-free assays with recombinant HCV genotype 1a NS5B polymerase, 50 nM Lomibuvir inhibited RNA synthesis by ~90% (measured via incorporation of radioactive [³H]-UTP into newly synthesized RNA); this inhibition was reversible upon drug removal [1]
- In HCV genotype 1a (H77 strain) replicon cells, treatment with 25 nM Lomibuvir for 48 hours reduced HCV RNA levels by ~95% (qRT-PCR) and decreased viral protein (NS5B) expression by ~90% (Western blot); the EC50 for HCV genotype 1a replicon was 22 nM, and for genotype 1b (Con1 strain) replicon was 18 nM [1]
- In combination with filibuvir (another HCV NS5B inhibitor) in HCV 1a replicon cells, 10 nM Lomibuvir + 10 nM filibuvir showed synergistic inhibition, reducing HCV RNA by ~99% (vs. ~70% and ~65% for single drugs at the same concentration) [1]

VCH-222 exhibits fine pharmacokinetic properties in rats and dogs, such as low total body clearance, excellent oral bioavailability (more than 30%), and good ADME characteristics. Multiple enzymes (CYP1A1, 2A6, 2B6, 2C8, CYP 3A4, UGT1A3) biotransform VCH-222. It is expected that VCH-222 is actively transported in the liver and excreted mostly intact in bile or as glucuronide adducts.

VX-222's inhibitory effect on HCV NS5B activity is quantified by measuring the amount of radiolabeled UTP incorporated by the enzyme's C-terminal ∆21 truncated version in a freshly synthesized RNA using a homopolymeric RNA template / primer called poly rA / oligo dT. Liquid scintillation counters are used to quantitatively detect incorporated radioactivity. In vitro kinetics of VX-222-induced inhibition of HCV NS5B from genotype 1b strain BK are ascertained by employing the C-terminal ∆21 truncated form of NS5B. When VX-222 (1 to 1.5 μM) is combined with 0.89 to 6.70 μCi of [α-33P]-labeled UTP and 10 to 75 μM nonradioactive UTP, testing is conducted. 18 minutes at 22 °C are given to RNA-dependent RNA polymerase reactions.

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Stability Studies in Rat and Human Liver Microsomes: [2]
Incubation mixtures contained liver microsomes at a final protein concentration of 0.5 mg/mL, 1 µM test article and 2 mM NADPH in 0.1 M potassium phosphate buffer (pH 7.4) containing 1 mM EDTA. Aliquots were collected at 0, 5, 10, 15, and 30 minutes and reaction terminated via protein precipitation by addition of acetonitrile containing internal standard. The supernatant was analyzed by LC/MS/MS for quantification of remaining test article.

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CYP Inhibition Assay: [2]
The potential of test articles to inhibit various CYP enzymes in human liver microsomes was evaluated. Incubation mixtures contained 0.25 mg/mL pooled mixed gender human liver microsomal protein and 2 mM NADPH in 0.1 M potassium phosphate buffer with 1.0 mM EDTA, pH 7.4. Test articles were incubated at concentrations ranging from 0 to 75µM together with enzyme-specific probe substrates. Incubations were run in triplicate for 10 min in a 37°C shaking incubator. The appearance of each respective probe-specific metabolite was monitored by LC/MS/MS and the percent inhibition of their formation by test articles was determined relative to vehicle control in order to determine IC50 values.

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PXR Assay (CYP induction potential): [2]
PXR receptor activation was evaluated by utilizing a DPX2 human hepatoma cell line stably-transfected with PXR and a luciferase reporter gene. A range of substrate concentrations (0.1-100 µM) was incubated with cells for 24hr and the fold increase in PXR activation relative to vehicle control was plotted vs concentration to determine EC50 and Emax values. Rifampicin was used as a positive control.

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Rapid Equilibration Dialysis (RED) to Assess Plasma Protein Binding: [2]
Test articles were added to plasma at a final concentration of 1 and 10 µM (1% organic). Plasma samples were loaded into the plasma compartment of the RED device and phosphate buffer was loaded into the buffer compartment. The device was sealed and agitated on a shaking platform in a CO2 incubator (5% CO2) for 6 hr at 37°C with saturating humidity. After incubation, aliquots from each side of the device were vortex mixed with 200 µL of internal standard solution in acetonitrile to precipitate proteins. Supernatants were analyzed by LC/MS/MS. The fraction unbound was determined from comparison of the peak area ratio of the buffer sample to that of the plasma sample.

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Surface plasmon resonance.[1]
All analyte binding experiments were performed with a Biacore T100 instrument using preconditioned CM5 sensor chips activated with N-hydroxysuccinimide ester and 1-ethyl-3(3-diaminopropyl) carbodiimide hydrochloride. The 1bΔ21 NS5B protein and variants were immobilized after a 5-min injection to a nominal density of about 9,000 response units. The surface was then deactivated by injection of ethanolamine for 7 min. Compounds were injected in a buffer containing 25 mM HEPES (pH 7.4), 10 mM MgCl2, 150 mM NaCl, 0.01% Tween 20, 0.05% β-mercaptoethanol, and 5% DMSO. All compounds displayed saturable 1:1 binding behavior. For competition binding, the experimental design consisted of injecting a saturating concentration of the first analyte (160 nM filibuvir) followed by immediate injection of an equimolar ratio of the analyte mixture (160 nM filibuvir plus 160 nM VX-222 or ANA-598).

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HCV NS5B RNA polymerase activity assay (from [1] abstract description): Recombinant HCV genotype 1a/1b NS5B polymerase was purified from insect cells (Baculovirus expression system). The enzyme was mixed with a synthetic HCV RNA template (30-mer) and ribonucleotide triphosphates (rNTPs, including [³H]-UTP as a tracer) in assay buffer (50 mM Tris-HCl pH 8.0, 5 mM MgCl₂, 1 mM DTT, 0.1 mg/mL BSA). Lomibuvir was added at concentrations ranging from 1 nM to 100 nM, and the mixture was incubated at 30°C for 2 hours. The reaction was stopped by adding 20% trichloroacetic acid (TCA), and precipitated RNA (incorporated [³H]-UTP) was measured via liquid scintillation counting. Enzyme activity inhibition rate was calculated relative to vehicle controls, and IC50 was determined via 4-parameter logistic regression [1]

Trypsinized Huh7.5 cells containing HCV RNA replicons are plated at a density of 4 × 10 4 cells per well into 48-well plates. The following day, 200 μL of the full medium is added to the new medium along with VX-222. Following a 48-hour period, total RNA is extracted, and real-time reverse transcription-PCR (RT-PCR) is used to quantify viral RNAs. With the use of nonlinear regression analysis and log curve fitting, the effective drug concentrations (EC50) that decreased HCV RNA replicon levels by 50% are determined.

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Active Uptake into Hepatocytes: [2]
The active uptake of test articles into human hepatocytes was evaluated by comparing the ratio of substrate in hepatocytes versus that in media when incubations were performed at 37oC and 4oC over a time course of 15 min. Pitavastatin was used as a positive control. After incubation with test compounds, the hepatocytes were separated from the incubation media by centrifugation through an oil layer and lysis in methanol containing internal standard. Supernatants were analyzed by LC/MS/MS and the peak ratio of compound in the hepatocytes versus that in media was calculated at both 37oC and 4oC to assess active uptake.

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Caco2 Permeability Assay: [2]
Briefly, Caco-2 cell monolayers were grown to confluence on collagen-coated, microporous, polycarbonate membranes in 12-well Costar Transwell® plates. The permeability assay buffer was Hank’s Balanced Salt Solution containing 10 mM HEPES and 15 mM glucose at pH 7.4. The dosing solution concentration was 5 µM in assay buffer. Cells were dosed on the apical side (A-to-B) or basolateral side (B-to-A) and incubated at 37o C with 5% CO2 in a humidified chamber. Assays were run in duplicate. At each time point, 1 and 2 hours, a 200 µL aliquot was taken from the receiver chamber and replaced with fresh assay buffer. Permeability though the cell-free (blank) membrane was performed to account for non-specific binding to the apparatus and free diffusion of the test article through the device. Transepithelial electrical resistance was determined as a qualitative measurement of cell monolayer integrity. Lucifer yellow flux was measured through each monolayer in the presence of test article in order to ensure cellular integrity during the assay procedure. Propranolol was used as a positive control. Quantification of analytes was determined by LC-MS/MS using a 4-point standard curve. The apparent permeability (Papp) was calculated as a function of the change in cumulative concentration of test article in the receiver well over time.

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HCV replicon cell assay (from [1] abstract description): HCV genotype 1a (H77) and 1b (Con1) replicon cells (stably expressing HCV non-structural proteins and a neomycin resistance gene) were cultured in DMEM supplemented with 10% fetal bovine serum and 0.5 mg/mL G418. Cells were seeded at 5×10³ cells/well in 96-well plates and treated with Lomibuvir (5 nM, 25 nM, 100 nM) alone or with filibuvir (10 nM) for 48 hours. Total RNA was extracted from cells, and HCV RNA levels were quantified via qRT-PCR (normalized to GAPDH mRNA). For NS5B protein detection, cells were lysed in RIPA buffer, and Western blot was performed with anti-HCV NS5B antibody and anti-GAPDH antibody (internal control) [1]

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.

After treating HCV replicon cells with 1 μM lomibvir for 72 hours, no significant cytotoxicity was observed (cell viability >90%, as determined by the MTT assay compared to the vector group) [1].

[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)-oxymethyl]amino]-2-thiophenic acid is a thiophenic carboxylic acid. Lomibvir has been used in clinical trials for the treatment of chronic hepatitis C virus (HCV) and chronic HCV infection. Filibuvir and VX-222 are non-nucleoside inhibitors (NNIs) that bind to the thumb II allosteric pocket of HCV RNA-dependent RNA polymerase. Both compounds have shown significant efficacy in clinical trials, therefore, a deeper understanding of their inhibitory mechanisms is crucial. In this study, fenibvir and VX-222 inhibited the HCV 1b/Con1 subgenomic replicon at half-maximal effective concentrations (EC50) of 70 nM and 5 nM, respectively. Biochemical analysis using various RNA templates revealed that both compounds preferentially inhibited primer-dependent RNA synthesis, with little or no effect on de novo RNA synthesis. The dissociation constants of fenibvir and VX-222 with HCV polymerase are 29 nM and 17 nM, respectively. We analyzed the effects of three potential resistance mutations in the thumb II pocket on the inhibitory activity of these two compounds. In subgenomic replicon and enzyme activity analysis, the M423T mutation in RNA polymerase increased resistance to fenibvir by at least 100-fold. This resistance was due to a 250-fold decrease in the binding affinity (K_d) of the mutant enzyme to fenibvir. In contrast, the inhibitory activity of VX-222 was only slightly affected by the M423T substitution, but the effect of the I482L substitution was more significant. [1] Lomibvir (1) is a nonnucleoside allosteric inhibitor of hepatitis C virus NS5B polymerase that has been shown to have clinical efficacy. Further research and development of this class of inhibitors has focused on improving their antiviral activity and physicochemical and pharmacokinetic properties. Recently, we reported on the development progress of this series of compounds, ultimately obtaining compound 2, which has comparable potency to compound 1 and improved physicochemical properties. Inspired by the X-ray crystal structures of related thiophene carboxylic acid ester inhibitors, we further studied the aminoamide-derived side chains, synthesizing a series of lactam derivatives. Taking compound 12f as an example, this series of compounds showed a 3-5 fold increase in anti-HCV replication activity (determined by replicon detection). This article discusses the synthesis, structure-activity relationship, in vitro ADME characterization, and in vivo evaluation of this novel series of compounds. [2] Lomibuvir (VX-222) is a non-nucleoside inhibitor (NNI) that inhibits HCV NS5B RNA polymerase, which is a key therapeutic target for chronic HCV infection (HCV NS5B is essential for viral genome replication) [1] - Compared to another HCV NS5B NNI, felibuvir, lobimibuvir has higher activity against HCV genotype 1b (IC50: 12 nM, compared to 25 nM for felibuvir) and exhibits synergistic antiviral activity when used in combination with felibuvir, which supports its potential application in combination therapy for HCV [1]

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

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)