Discovery and structure activity relationship of glyoxamide derivatives as anti-hepatitis B virus agents.

Chronic hepatitis B viral infection is a significant health problem world-wide, and currently available antiviral agents suppress HBV infections, but rarely cure this disease. It is presumed that antiviral agents that target the viral nuclear reservoir of transcriptionally active cccDNA may eliminate HBV infection. Through a series of chemical optimization, we identified a new series of glyoxamide derivatives affecting HBV nucleocapsid formation and cccDNA maintenance at low nanomolar levels. Among all the compounds synthesized, GLP-26 displays a major effect on HBV DNA, HBeAg secretion and cccDNA amplification. In addition, GLP-26 shows a promising pre-clinical profile and long-term effect on viral loads in a humanized mouse model.

Nucleoside Analogs with Antiviral Activity against Yellow Fever Virus.

Yellow fever virus (YFV) is a human Flavivirus reemerging in parts of the world. While a vaccine is available, large outbreaks have recently occurred in Brazil and certain African countries. Development of an effective antiviral against YFV is crucial, as there is no available effective drug against YFV. We have identified several novel nucleoside analogs with potent antiviral activity against YFV with 50% effective concentration (EC50) values between 0.25 and 1 µM with selectivity indices over 100 in culture.

In silico design of a novel nucleotide antiviral agent by free energy perturbation.

Nucleoside analogs are the backbone of antiviral therapies. Drugs from this class undergo processing by host or viral kinases to form the active nucleoside triphosphate species that selectively inhibits the viral polymerase. It is the central hypothesis that the nucleoside triphosphate analog must be a favorable substrate for the viral polymerase and the nucleoside precursor must be a satisfactory substrate for the host kinases to inhibit viral replication. Herein, free energy perturbation (FEP) was used to predict substrate affinity for both host and viral enzymes. Several uridine 5'-monophosphate prodrug analogs known to inhibit hepatitis C virus (HCV) were utilized in this study to validate the use of FEP. Binding free energies to the host monophosphate kinase and viral RNA-dependent RNA polymerase (RdRp) were calculated for methyl-substituted uridine analogs. The 2'-C-methyl-uridine and 4'-C-methyl-uridine scaffolds delivered favorable substrate binding to the host kinase and HCV RdRp that were consistent with results from cellular antiviral activity in support of our new approach. In a prospective evaluation, FEP results suggest that 2'-C-dimethyl-uridine scaffold delivered favorable monophosphate and triphosphate substrates for both host kinase and HCV RdRp, respectively. Novel 2'-C-dimethyl-uridine monophosphate prodrug was synthesized and exhibited sub-micromolar inhibition of HCV replication. Using this novel approach, we demonstrated for the first time that nucleoside analogs can be rationally designed that meet the multi-target requirements for antiviral activity.

Discovery of a Series of 2'-α-Fluoro,2'-ß-bromo-ribonucleosides and Their Phosphoramidate Prodrugs as Potent Pan-Genotypic Inhibitors of Hepatitis C Virus.

Hepatitis C virus (HCV) nucleoside inhibitors display pan-genotypic activity, a high barrier to the selection of resistant virus, and are some of the most potent direct-acting agents with durable sustained virologic response in humans. Herein, we report, the discovery of ß-d-2'-Br,2'-F-uridine phosphoramidate diastereomers 27 and 28, as nontoxic pan-genotypic anti-HCV agents. Extensive profiling of these two phosphorous diastereomers was performed to select one for in-depth preclinical profiling. The 5'-triphosphate formed from these phosphoramidates selectively inhibited HCV NS5B polymerase with no inhibition of human polymerases and cellular mitochondrial RNA polymerase up to 100 µM. Both are nontoxic by a variety of measures and display good stability in human blood and favorable metabolism in human intestinal microsomes and liver microsomes. Ultimately, a preliminary oral pharmacokinetics study in male beagles showed that 28 is superior to 27 and is an attractive candidate for further studies to establish its potential value as a new clinical anti-HCV agent.

2'-Chloro,2'-fluoro Ribonucleotide Prodrugs with Potent Pan-genotypic Activity against Hepatitis C Virus Replication in Culture.

Pan-genotypic nucleoside HCV inhibitors display a high genetic barrier to drug resistance and are the preferred direct-acting agents to achieve complete sustained virologic response in humans. Herein, we report, the discovery of a ß-d-2'-Cl,2'-F-uridine phosphoramidate nucleotide 16, as a nontoxic pan-genotypic anti-HCV agent. Phosphoramidate 16 in its 5'-triphosphate form specifically inhibited HCV NS5B polymerase with no marked inhibition of human polymerases and cellular mitochondrial RNA polymerase. Studies on the intracellular half-life of phosphoramidate 16-TP in live cells demonstrated favorable half-life of 11.6 h, suggesting once-a-day dosing. Stability in human blood and favorable metabolism in human intestinal microsomes and liver microsomes make phosphoramidate 16 a prospective candidate for further studies to establish its potential value as a new anti-HCV agent.