Pyrrolopyrimidine nucleoside 1 was designed and synthesized as a potential mutagen for HCV. An in vitro HCV NS5B enzymatic assay indicated that pyrrolopyrimidine triphosphate acts as a CTP analog rather than a UTP analog. The SATE-prodrug of pyrrolopyrimidine monophosphate showed a weak inhibitory activity in an HCV replicon system (EC 50 =60μM) and did not exhibit cytotoxicity (CC 50 >100μM). Investigation of phosphorylation events using nucleoside kinases and LC–MS analysis revealed that the second phosphorylation step, from monophosphate ester to diphosphate ester, is unfavorable.