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.

Cyclic nucleotide phosphodiesterase PDE1C1 in human cardiac myocytes.

Isoforms in the PDE1 family of cyclic nucleotide phosphodiesterases were recently found to comprise a significant portion of the cGMP-inhibited cAMP hydrolytic activity in human hearts. We examined the expression of PDE1 isoforms in human myocardium, characterized their catalytic activity, and quantified their contribution to cAMP hydrolytic and cGMP hydrolytic activity in subcellular fractions of this tissue. Western blotting with isoform-selective anti-PDE1 monoclonal antibodies showed PDE1C1 to be the principal isoform expressed in human myocardium. Immunohistochemical analysis showed that PDE1C1 is distributed along the Z-lines and M-lines of cardiac myocytes in a striated pattern that differs from that of the other major dual-specificity cyclic nucleotide phosphodiesterase in human myocardium, PDE3A. Most of the PDE1C1 activity was recovered in soluble fractions of human myocardium. It binds both cAMP and cGMP with K(m) values of approximately 1 microm and hydrolyzes both substrates with similar catalytic rates. PDE1C1 activity in subcellular fractions was quantified using a new PDE1-selective inhibitor, IC295. At substrate concentrations of 0.1 microm, PDE1C1 constitutes the great majority of cAMP hydrolytic and cGMP hydrolytic activity in soluble fractions and the majority of cGMP hydrolytic activity in microsomal fractions, whereas PDE3 constitutes the majority of cAMP hydrolytic activity in microsomal fractions. These results indicate that PDE1C1 is expressed at high levels in human cardiac myocytes with an intracellular distribution distinct from that of PDE3A and that it may have a role in the integration of cGMP-, cAMP- and Ca(2+)-mediated signaling in these cells.