Barbara Pacini

Potent Inhibitors of Subgenomic Hepatitis C Virus RNA Replication through Optimization of Indole-N-Acetamide Allosteric Inhibitors of the Viral NS5B Polymerase

Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. Compounds that block replication of subgenomic HCV RNA in liver cells are of interest because of their demonstrated antiviral effect in the clinic. In followup to our recent report that indole-N-acetamides (e.g., 1) are potent allosteric inhibitors of the HCV NS5B polymerase enzyme, we describe here their optimization as cell-based inhibitors. The crystal structure of 1 bound to NS5B was a guide in the design of a two-dimensional compound array that highlighted that formally zwitterionic inhibitors have strong intracellular potency and that pregnane X receptor (PXR) activation (an undesired off-target activity) is linked to a structural feature of the inhibitor. Optimized analogues devoid of PXR activation (e.g., 55, EC(50) = 127 nM) retain strong cell-based efficacy under high serum conditions and show acceptable pharmacokinetics parameters in rat and dog.

Preclinical pharmacokinetics and metabolism of a potent non-nucleoside inhibitor of the hepatitis C virus NS5B polymerase.

The disposition of compound A, a potent inhibitor of the hepatitis C virus (HCV) NS5B polymerase, was characterized in animals in support of its selection for further development. Compound A exhibited marked species differences in pharmacokinetics. Plasma clearance was 44 ml min−1 kg−1 in rats, 9 ml min−1 kg−1 in dogs and 16 ml min−1 kg−1 in rhesus monkeys. Oral bioavailability was low in rats (10%) but significantly higher in dogs (52%) and monkeys (26%). Compound A was eliminated primarily by metabolism in rats, with biliary excretion accounting for 30% of its clearance. Metabolism was mainly mediated by cyclohexyl hydroxylation, with N-deethylation and acyl glucuronide formation constituting minor metabolic pathways. Qualitatively, the same metabolites were identified using in vitro systems from all species studied, including humans. The low oral bioavailability of compound A in rats was mostly due to poor intestinal absorption. This conclusion was borne out by the findings that hepatic extraction in the rat was only 30%, intraperitoneal bioavailability was good, and compound A was poorly absorbed from the rat isolated intestinal loop, with no detectable intestinal metabolism. Compound A was not an inhibitor of major human cytochrome P450 enzymes, indicating minimal potential for clinical drug–drug interactions. The metabolic clearance of compound A in rat, dog and monkey hepatocytes correlated with the systemic clearance observed in these species. Since compound A was very stable in human hepatocytes, the results suggest that it will be a low clearance drug in humans.

2-(3-Thienyl)-5,6-dihydroxypyrimidine-4-carboxylic acids as inhibitors of HCV NS5B RdRp

A series of 2-(3-thienyl)-5,6-dihydroxypyrimidine-4-carboxylic acid inhibitors of the hepatitis C virus (HCV) NS5B polymerase enzyme are reported. Sulfonyl urea substituted analogs in this series proved to be the most potent active site non-nucleoside inhibitors of NS5B reported to date. These compounds had low nanomolar enzyme inhibition across HCV genotypes 1-3 and showed single digit micromolar inhibition in the HCV replicon assay. This improved cell-based activity allowed the binding mode of these compounds to be probed by selection of resistant mutations against compound 21. The results generated are in broad agreement with the previously proposed binding model for this compound class.