2--c-methylcytidine and Hepatitis-C

The first synthesis of 1'-C-CN, 2'-F, 2'-C-Me pyrimidines is described. Anti-HCV activity was assessed and compared to the 1'-C-CN, 2'-C-Me as well as the 2'-F, 2'-C-Me pyrimidines. A phosphoramidate prodrug of the cytidine derivative showed activity in the low micromolar range against HCV replicons.

Topics: Amides; Antiviral Agents; Cell Line; Halogenation; Hepacivirus; Hepatitis C; Humans; Methylation; Phosphoric Acids; Prodrugs; Pyrimidine Nucleosides; Replicon; RNA-Dependent RNA Polymerase

Preparation of a small library of derivatives of the potent HIV-1 Reverse Transcriptase inhibitor TSAO-T bearing mono or di-carbonyl substituents (designed after docking analysis) at position N-3 is reported. A one-pot synthetic methodology has been developed that involves: (i) mono-reaction of TSAO-T with glutaryl dichloride under phase transfer conditions and (ii) in situ acyclic substitution of the remaining chloro atom by oxygen or nitrogen nucleophiles. The method is compatible with the polyfunctionality of the TSAO-T molecule, proceeds with high conversion yields and allows introducing molecular diversity. The anti-HIV-1 and -HCV activity was studied in cell culture. The new N-3 acylated TSAO-T derivatives are active against HIV-1 (nanomolar range). Anti-HCV activity was observed in the micromolar range, that is at compound concentrations that were found cytostatic against human T-lymphocytes.

Topics: Cell Line; Chemistry Techniques, Synthetic; Hepacivirus; Hepatitis C; HIV Infections; HIV Reverse Transcriptase; HIV-1; Humans; Models, Molecular; Reverse Transcriptase Inhibitors; Small Molecule Libraries; Spiro Compounds; Thymidine; Uridine

Hepatitis C virus (HCV) infects an estimated 170 million individuals worldwide, and the current standard of care, a combination of pegylated interferon alpha and ribavirin, is efficacious in achieving sustained viral response in ~50% of treated patients. Novel therapies under investigation include the use of nucleoside analog inhibitors of the viral RNA-dependent RNA polymerase. NM283, a 3'-valyl ester prodrug of 2'-C-methylcytidine, has demonstrated antiviral efficacy in HCV-infected patients (N. Afdhal et al., J. Hepatol. 46[Suppl. 1]:S5, 2007; N. Afdhal et al., J. Hepatol. 44[Suppl. 2]:S19, 2006). One approach to increase the antiviral efficacy of 2'-C-methylcytidine is to increase the concentration of the active inhibitory species, the 5'-triphosphate, in infected hepatocytes. HepDirect prodrug technology can increase intracellular concentrations of a nucleoside triphosphate in hepatocytes by introducing the nucleoside monophosphate into the cell, bypassing the initial kinase step that is often rate limiting. Screening for 2'-C-methylcytidine triphosphate levels in rat liver after oral dosing identified 1-[3,5-difluorophenyl]-1,3-propandiol as an efficient prodrug modification. To determine antiviral efficacy in vivo, the prodrug was administered separately via oral and intravenous dosing to two HCV-infected chimpanzees. Circulating viral loads declined by ~1.4 log(10) IU/ml and by >3.6 log(10) IU/ml after oral and intravenous dosing, respectively. The viral loads rebounded after the end of dosing to predose levels. The results indicate that a robust antiviral response can be achieved upon administration of the prodrug.

Topics: Animals; Antiviral Agents; Cytidine; Cytidine Monophosphate; Female; Hepacivirus; Hepatitis C; Hepatocytes; Macaca mulatta; Male; Pan troglodytes; Prodrugs; Pyrimidine Nucleosides; Rats; Rats, Sprague-Dawley; Viral Load

Debio 025 is a potent inhibitor of hepatitis C virus (HCV) replication (J. Paeshuyse et al., Hepatology 43:761-770, 2006). In phase I clinical studies, monotherapy (a Debio 025 dose of 1,200 mg twice a day) resulted in a mean maximal decrease in the viral load of 3.6 log(10) units (R. Flisiak et al., Hepatology 47:817-826, 2008), whereas a reduction of 4.6 log(10) units was obtained in phase II studies when Debio 025 was combined with interferon (R. Flisiak et al., J. Hepatol., 48:S62, 2008). We here report on the particular characteristics of the in vitro anti-HCV activities of Debio 025. The combination of Debio 025 with either ribavirin or specifically targeted antiviral therapy for HCV (STAT-C) inhibitors (NS3 protease or NS5B [nucleoside and nonnucleoside] polymerase inhibitors) resulted in additive antiviral activity in short-term antiviral assays. Debio 025 has the unique ability to clear hepatoma cells from their HCV replicon when it is used alone or in combination with interferon and STAT-C inhibitors. Debio 025, when it was used at concentrations that have been observed in human plasma (0.1 or 0.5 muM), was able to delay or prevent the development of resistance to HCV protease inhibitors as well as to nucleoside and nonnucleoside polymerase inhibitors. Debio 025 forms an attractive drug candidate for the treatment of HCV infections in combination with standard interferon-based treatment and treatments that directly target the HCV polymerase and/or protease.

Topics: Antiviral Agents; Clinical Trials as Topic; Cyclophilins; Cyclosporine; Dose-Response Relationship, Drug; Drug Combinations; Drug Synergism; Hepacivirus; Hepatitis C; Humans; Replicon; Ribavirin; Viral Nonstructural Proteins

The application of a phosphoramidate prodrug approach to 2'-C-methylcytidine (NM107), the first nucleoside inhibitor of the hepatitis C virus (HCV) NS5B polymerase, is reported. 2'-C-Methylcytidine, as its valyl ester prodrug (NM283), was efficacious in reducing the viral load in patients infected with HCV. Several of the phosphoramidates prepared demonstrated a 10- to 200-fold superior potency with respect to the parent nucleoside in the cell-based replicon assay. This is due to higher levels of 2'-C-methylcytidine triphosphate in the cells. These prodrugs are efficiently activated and converted to the triphosphate in hepatocytes of several species. Our SAR studies ultimately led to compounds that gave high levels of NTP in hamster and rat liver after subcutaneous dosing and that were devoid of the toxic phenol moiety usually found in ProTides.

Topics: Amides; Animals; Antiviral Agents; Cell Line; Cytidine; Hepacivirus; Hepatitis C; Hepatocytes; Humans; Phosphoric Acids; Polyphosphates; Prodrugs; Structure-Activity Relationship; Virus Replication

The currently approved treatment for hepatitis C virus infections is a combination of Ribavirin and pegylated Interferon. It leads to a sustained virologic response in approximately only half of the patients treated. For this reason there is an urgent need of new therapeutic agents. 2'-C-Methylcytidine is the first nucleoside inhibitor of the HCV NS5B polymerase that was efficacious in reducing the viral load in patients infected with HCV. The application of a monophosphate prodrug approach based on unprecedented cyclic phosphoramidates is reported. Our SAR studies led to compounds that are efficiently converted to the active triphosphate in human hepatocytes.

Topics: Animals; Antiviral Agents; Cricetinae; Cytidine; Drug Stability; Hepacivirus; Hepatitis C; Hepatocytes; Humans; Prodrugs; Structure-Activity Relationship

To address the need for broad-spectrum antiviral activity characterization of hepatitis C virus (HCV) polymerase inhibitors, we created a panel of intergenotypic chimeric replicons containing nonstructural (NS) protein NS5B sequences from genotype 2b (GT2b), GT3a, GT4a, GT5a, and GT6a HCV isolates. Viral RNA extracted from non-GT1 HCV patient plasma was subjected to reverse transcription. The NS5B region was amplified by nested PCR and introduced into the corresponding region of the GT1b (Con-1) subgenomic reporter replicon by Splicing by Overlap Extension (SOEing) PCR. Stable cell lines were generated with replication-competent chimeras for in vitro antiviral activity determination of HCV nonnucleoside polymerase inhibitors (NNIs) that target different regions of the protein. Compounds that bind to the NNI2 (thiophene carboxylic acid) or NNI3 (benzothiadiazine) allosteric sites showed 8- to >1,280-fold reductions in antiviral activity against non-GT1 NS5B chimeric replicons compared to that against the GT1b subgenomic replicon. Smaller reductions in susceptibility, ranging from 0.2- to 33-fold, were observed for the inhibitor binding to the NNI1 (benzimidazole) site. The inhibitor binding to the NNI4 (benzofuran) site showed broad-spectrum antiviral activity against all chimeric replicons evaluated in this study. In conclusion, evaluation of HCV NNIs against intergenotypic chimeric replicons showed differences in activity spectrum for inhibitors that target different regions of the enzyme, some of which could be associated with specific residues that differ between GT1 and non-GT1 polymerases. Our study demonstrates the utility of chimeric replicons for broad-spectrum activity determination of HCV inhibitors.

Topics: Amino Acid Sequence; Amino Acid Substitution; Antiviral Agents; Cell Line; Chimera; Genetic Variation; Genotype; Hepacivirus; Hepatitis C; Humans; Models, Molecular; Molecular Sequence Data; Molecular Structure; Protease Inhibitors; Recombinant Proteins; Replicon; RNA-Dependent RNA Polymerase; Sequence Homology, Amino Acid; Viral Nonstructural Proteins

Beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5'-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2'-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a Km of 81 microM and a kcat of 0.007 s-1, but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (Ki) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 microM. Similar results were obtained with 2'-C-methyladenosine triphosphate (Ki=1.5 microM) and 2'-C-methylcytidine triphosphate (Ki=1.6 microM). NS5B with the S282T mutation, which is known to confer resistance to 2'-C-methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination.

Topics: Antiviral Agents; Catalysis; Deoxycytidine; Hepacivirus; Hepatitis C; Humans; Mutation; Phosphorylation; RNA-Dependent RNA Polymerase; RNA, Viral; Structure-Activity Relationship; Viral Nonstructural Proteins; Virus Replication