Application of the Phosphoramidate ProTide Approach to 4‘-Azidouridine Confers Sub-micromolar Potency versus Hepatitis C Virus on an Inactive Nucleoside

Abstract

We report the application of our phosphoramidate ProTide technology to the ribonucleoside analogue 4'-azidouridine to generate novel antiviral agents for the inhibition of hepatitis C virus (HCV). 4'-Azidouridine did not inhibit HCV, although 4'-azidocytidine was a potent inhibitor of HCV replication under similar assay conditions. However 4'-azidouridine triphosphate was a potent inhibitor of RNA synthesis by HCV polymerase, raising the question as to whether our phosphoramidate ProTide approach could effectively deliver 4'-azidouridine monophosphate to HCV replicon cells and unleash the antiviral potential of the triphosphate. Twenty-two phosphoramidates were prepared, including variations in the aryl, ester, and amino acid regions. A number of compounds showed sub-micromolar inhibition of HCV in cell culture without detectable cytotoxicity. These results confirm that phosphoramidate ProTides can deliver monophosphates of ribonucleoside analogues and suggest a potential path to the generation of novel antiviral agents against HCV infection. The generic message is that ProTide synthesis from inactive parent nucleosides may be a warranted drug discovery strategy.

Inhibition of Dengue Virus RNA Synthesis by an Adenosine Nucleoside

“…For nucleosides that could not be efficiently converted to their monophosphate forms, one could synthesize the monophosphate or phosphonate prodrugs. The phosphoramidate approach was successfully used to convert inactive nucleoside analogs to potent inhibitors of HCV (23,24). Other examples of nucleotide phosphonates are adefovir disoproxil and tenofovir disoproxil, which are in clinical use for treatment of hepatitis B virus and HIV, respectively.…”

2′-Deoxy-4′-azido Nucleoside Analogs Are Highly Potent Inhibitors of Hepatitis C Virus Replication Despite the Lack of 2′-α-Hydroxyl Groups

“…Options to further increase the antiviral potency of nucleoside analogs as inhibitors of HCV replication include optimization of nucleotide analog incorporation efficiency by HCV NS5B, or optimization of phosphorylation efficiency to increase the intracellular concentration of the nucleoside triphosphate analog. In particular, the first step in the pathway to the triphosphate, the formation of nucleoside monophosphate, appears to be a limiting step for a majority of nucleoside analogs (9,26,27).…”

“…Antiviral potency of nucleosides is often limited by phosphorylation efficiency, as modified analogs can be poor substrates for human nucleoside kinases (9). To identify novel nucleosides with increased antiviral potency, the focus of optimization was therefore on increasing phosphorylation efficiency by human kinases as well as on increasing intrinsic incorporation efficiency by the recombinant NS5B enzyme.…”

“…However, the incorporation efficiency of ara-CTP was 140-fold lower as compared with CTP, in line with an important interaction of the 2Ј-␣-hydroxy group. Earlier, we discovered that 4Ј-substituents on ribonucleoside analogs may pick up additional binding interactions in the HCV polymerase active site, with 4Ј-azido being the most effective to allow productive binding to both HCV polymerase and human nucleoside kinases (6,7,9). The addition of a 4Ј-azido moiety to the ara-CTP molecule resulted in an unexpected 15-30-fold increase in inhibitory potency of 2Ј-␣-deoxy-2Ј-␤-hydroxy-4Ј-azido-CTP (RO-9187-TP) as compared with 2Ј-␣-deoxy-ara-CTP.…”

Characterization of the Metabolic Activation of Hepatitis C Virus Nucleoside Inhibitor β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) and Identification of a Novel Active 5′-Triphosphate Species

“…Similarly, the uridine analog of the HCV replication inhibitor R1479 (4Ј-azidocytidine) was inactive in the replicon system. However, when delivered as a monophosphate prodrug, 4Ј-azidouridine could be converted into a potent inhibitor of HCV replication, demonstrating that a block of monophosphate formation resulted in lack of antiviral activity of 4Ј-azidouridine (21). Assuming a likely block of RO2433 phosphorylation to its monophosphate, RO2433-MP in human hepatocytes was most likely formed through the deamination of PSI-6130-MP by the cellular dCMP deaminase and subsequently further phosphorylated to RO2433-DP and -TP by uridine/cytidine monophosphate kinase and possibly nucleoside diphosphate kinase (22)(23)(24).…”