NHC-triphosphate triammonium is an active phosphorylated intracellular metabolite of β-d-N4-Hydroxycytidine (NHC) as a triphosphate form[1]. NHC-triphosphate triammonium is a weak alternative substrate for the viral polymerase and can be incorporated into HCV replicon RNA[1][2].

In an intracellular metabolism assay conducted on HCV replicon cells exposed to 10 μM 3H-labeled NHC, the transformation of NHC into its mono-, di-, and triphosphate forms is documented, exhibiting a significant increase in the NHC-triphosphate (NHC-TP) concentration up to 71.12 pM after 8 hours. The absence of NHC-TP at concentrations ranging from 5-40 μM results in complete polymerization, serving as a modest alternate substrate. Moreover, substituting NHC-TP for CTP during polymerization increases the weight of the resultant product by 16 units (attributable to an additional oxygen molecule), causing a noticeable electrophoretic shift in cell-free HCV NS5B polymerization reactions. In experiments utilizing Huh-7 cells treated with 10-50 μM NHC or a McGuigan phosphoramidate prodrug of NHC for 4 hours, analysis through LC-MS/MS reveals minor levels of NHC-mono- and diphosphates, with NHC-TP remaining the predominant metabolite. The NHC-TP metabolite is hypothesized to directly interact with the viral polymerase, acting as a potential nonobligate chain terminator and playing a crucial role in inhibiting the synthesis of early negative-strand RNA. This inhibition could lead to the disruption of proper replicase complex formation, either through chain termination or mutagenesis.