The naturally occurring nucleotide 3′-deoxy-3′,4′-didehydro-cytidine-5′-triphosphate (ddhCTP) was recently found to exert potent and broad-spectrum antiviral activity. However, nucleoside 5′-triphosphates in general are not cell-permeable, which precludes the direct use of ddhCTP as a therapeutic. To harness the therapeutic potential of this endogenous antiviral nucleotide, we synthesized phosphoramidate prodrug HLB-0532247 (1) and found it to result in dramatically elevated levels of ddhCTP in cells. We compared 1 and 3′-deoxy-3′,4′-didehydro-cytidine (ddhC) and found that 1 more effectively reduces titers of Zika and West Nile viruses in cell culture with minimal nonspecific toxicity to host cells. We conclude that 1 is a promising antiviral agent based on a novel strategy of facilitating elevated levels of the endogenous ddhCTP antiviral nucleotide
