Phosphoramidate ProTides of 2′-C-Methylguanosine as Highly Potent Inhibitors of Hepatitis C Virus. Study of Their in Vitro and in Vivo Properties

Abstract

Hepatitis C virus infection constitutes a serious health problem in need of more effective therapies. Nucleoside analogues with improved exposure, efficacy, and selectivity are recognized as likely key components of future HCV therapy. 2'-C-Methylguanosine triphosphate has been known as a potent inhibitor of HCV RNA polymerase for some time, but the parent nucleoside is only moderately active due to poor intracellular phosphorylation. We herein report the application of phosphoramidate ProTide technology to bypass the rate-limiting initial phosphorylation of this nucleoside. Over 30 novel ProTides are reported, with variations in the aryl, ester, and amino acid regions. l-Alanine compounds are recognized as potent and selective inhibitors of HCV in replicon assay but lack rodent plasma stability despite considerable ester variation. Amino acid variation retaining the lead benzyl ester moiety gives an increase in rodent stability but at the cost of potency. Finally l-valine esters with ester variation lead to potent, stable compounds. Pharmacokinetic studies on these agents in the mouse reveal liver exposure to the bioactive triphosphate species following single oral dosing. Systemic exposure of the ProTide and parent nucleoside are low, indicating possible low toxicity in vivo, while liver concentrations of the active species may be predictive of efficacy in the clinic. This represents one of the most thorough cross-species studies of ProTides to date.

INX-08189, a Phosphoramidate Prodrug of 6- O -Methyl-2′- C -Methyl Guanosine, Is a Potent Inhibitor of Hepatitis C Virus Replication with Excellent Pharmacokinetic and Pharmacodynamic Properties

“…In the case of 2Ј-C-MeG, a striking improvement in cell-based potency over that of the parent nucleoside can be achieved (Ͼ80 fold) with phosphoramidates incorporating 1-naphthyl as the aryl-leaving group (27). These prodrugs of 2Ј-C-MeG also have been shown to generate high concentrations of the active 2Ј-C-MeG triphosphate in liver tissue following oral administration in mice (24). To further improve the performance of these compounds, alterations at the C-2 and C-6 positions of the base were investigated.…”

“…For HCV genotype 2a (J6/JFH), Huh-7 cells were transfected transiently with a full-length, monocistronic HCV genome construct expressing Renilla luciferase. The inhibitory effect on HCV RNA replication using the luciferase assay and the cytotoxic effect of compounds were performed as previously described (24). For HCV genotype 1a (H77), a stable, full-length, bicistronic replicon cell line was used (3).…”

“…The concentration of 2Ј-C-MeGTP in liver samples and the concentration of 2Ј-C-MeG in plasma samples from rats and primates was performed by LC-MS/MS as described previously (24). The assay measuring 2Ј-C-MeGTP in rat or primate liver was linear (r 2 Ն 0.99) in the concentration range of 100 to 4, 000 ng per g of tissue, with Ն85% accuracy and Յ2% CV.…”

“…This approach is designed to bypass the rate-limiting initial phosphorylation step of activation by delivering the monophosphate form of the nucleoside analog to the liver, where it can be efficiently converted to the active triphosphate (24). INX-08189 is a phosphoramidate of O-6-methyl-2Ј-C-methyl guanosine ( Fig.…”

Overcoming Stability Challenges in the Quantification of Tissue Nucleotides: Determination of 2′-<i>C</i>-Methylguanosine Triphosphate Concentration in Mouse Liver

“…Previously a method for analyzing 2′-MeGTP in mouse liver was reported using a chelating reagent to stabilize the triphosphate homogenization step, which lacks sufficient detailed descriptions, and included a laborious pulverizing step on dry ice. 16) Other poorly attractive extraction procedures and LC-MS methods for quantification of different nucleotides in tissues have been developed and reported. [17][18][19][20][21][22] A limitation in the measurement of 2′-MeGTP is that it undergoes rapid ischemic degradation in collected liver tissue.…”