The antiviral efficacies and cytotoxicities of 2,3-and 4-substituted 2,3-didehydro-2,3-dideoxycytidine analogs were evaluated. All compounds were tested (i) against a wild-type human immunodeficiency virus type 1 (HIV-1) isolate (strain xxBRU) and lamivudine-resistant HIV-1 isolates, (ii) for their abilities to inhibit hepatitis B virus (HBV) production in the inducible HepAD38 cell line, and (

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a cytidine analogue with potent and selective anti-hepatitis C virus (HCV) activity in the subgenomic HCV replicon assay, 90% effective concentration (EC90)=4.6 +/- 2.0 microM. The spectrum of activity and cytotoxicity profile of PSI-6130 was evaluated against a diverse panel of viruses and cell types, and against two additional HCV-1b replicons. The S282T mutation, which confers resistance to 2'-C-methyl adenosine and other 2'-methylated nucleosides, showed only a 6.5-fold increase in EC90. When assayed for activity against bovine diarrhoea virus (BVDV), which is typically used as a surrogate assay to identify compounds active against HCV, PSI-6130 showed no anti-BVDV activity. Weak antiviral activity was noted against other flaviviruses, including West Nile virus, Dengue type 2, and yellow fever virus. These results indicate that PSI-6130 is a specific inhibitor of HCV. PSI-6130 showed little or no cytotoxicity against various cell types, including human peripheral blood mononuclear and human bone marrow progenitor cells. No mitochondrial toxicity was observed with PSI-6130. The reduced activity against the RdRp S282T mutant suggests that PSI-6130 is an inhibitor of replicon RNA synthesis. Finally, the no-effect dose for mice treated intraperitoneally with PSI-6130 for six consecutive days was > or =100 mg/kg per day.

Background: Hepatitis C virus (HCV) polymerase is an essential enzyme for HCV replication and has multiple inhibitor binding sites making it a major target for antiviral intervention. It is apparent that no single drug can inhibit HCV replication in humans. Hence, combinations of nucleoside analogues β-d-2'-C-methylcytidine (2'-C-MeC; NM-107) or β-d-2'-deoxy-2'-fluoro-2'-C-methylcytidine (2'-F-C-MeC; PSI-6130) with interferon-a2b (IFN-a2b) or triple combination with ribavirin (RBV) were evaluated. Methods: Huh-7 cells containing the self-replicating subgenomic HCV replicon (Clone B) were used for drug combination studies. After drug treatment for 5 days, total cellular RNA was then extracted and both ribosomal RNA and HCV replicon RNA were amplified in a singlestep multiplex real-time PCR assay. Drug interaction analyses were performed using the CalcuSyn program. The current standard of care for chronic hepatitis C virus (HCV) infection is a combination of pegylated interferon (IFN)-a and ribavirin (RBV), which results in a sustained virological response in about a half of infected individuals; however, this combined modality is associated with considerable side effects [1,2]. Several new HCV inhibitors have been identified and some are being evaluated in controlled clinical trials [3]. Recently, promising 2′-modified nucleoside analogues with activity against HCV have been reported; for instance, valopicitabine (NM-283), the 3′-valine ester of β-d-2′-Cmethylcytidine (2′-C-MeC; NM-107), is a prodrug nucleoside that after intracellular phosphorylation to its 5′-triphosphate metabolite, selectively inhibits HCV RNA-dependent RNA polymerase [4,5]. Although valopicitabine was recently evaluated in clinical trials and the combination of valopicitabine with IFN-a (plus RBV) has led to successful viral RNA suppression in a number of HCV-infected individuals, this drug was found to have gastrointestinal toxicity leading to its discontinuation in its present form [6]. Another novel cytidine analogue, β-d-2′-deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130, 2′-F-C-MeC has demonstrated potent and specific in vitro anti-HCV activity with no apparent cytotoxicity [7,8]. Recently, in a 14 day Phase I monotherapy study, a new prodrug of 2′-F-C-MeC named R7128 was shown to be highly potent at lowering levels of HCV RNA, with a 2.7-log reduction in the viral load of individuals infected with HCV genotype 1 who had failed prior IFN therapy [9,10]. These nucleoside analogues have demonstrated excellent anti-HCV activity in vitro in both antireplicon and infectious HCV systems [11]. Furthermore, nucleoside analogue prodrugs can be administered in combination with IFN, with or without Results: Double combinations of 2'-C-MeC or 2'-F-C-

This work is the first example of both inactive uridine and cytidine analogues of a nucleoside being converted to active anti-HCV nucleosides via 5'-monophosphate prodrugs.

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