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NHC-diphosphate is an active phosphorylated intracellular metabolite of β-d-N4-Hydroxycytidine (NHC) as a diphosphate form[1]. NHC is a pyrimidine ribonucleoside and behaves as a potent anti-virus agent. NHC effectively inhibits the replication of venezuelan equine encephalitis virus (VEEV), Chikungunya virus (CHIKV) and hepatitis C virus (HCV)[1].

Huh-7 cells are incubated with (10-50 μM; 4 h) NHC or a McGuigan phosphoramidate prodrug of NHC.Intracellular levels of the parental compounds and phosphorylated metabolites are measured using LC-MS/MS. Small amounts of NHC-monophosphate (MP) and NHC-diphosphate can be observed, while NHC-triphosphate remains the most abundant metabolite.[1].

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