beneficial biological activity as antibacterial and antifungal, trypanocidal, anticancer and antiviral. The mechanism of action of pyran naphthoquinones is not entirely elucidated despite the broad range of biological activities of these molecules. Some studies suggest that they are active at the level of the nuclear enzymes topoisomerases I and II, which are essential for chromosome structure, DNA transcription, and replication. Other authors point out that the biological profiles of these substances are due to their ortho or para-quinonoid moiety that can accept one and/or two electrons creating a redox cycling, which generates radical anion, superoxide anion radical, or dianion species, leading to an intracellular deleterious hypoxic condition. In this study, we demonstrated that 1,4-pyran naphthoquinones are potent inhibitors of DENV-2 replication in cells and impact on the in vitro ATPase activity of NS3. The methodology for synthesis of the pyran naphthquinones used in this study has been reported elsewhere. Briefly, the compounds were obtained by reacting of lawsone with an appropriate aldehyde that generate in situ an oquinone methide FRAX1036 intermediate on o-quinone methide intermediate followed by dehydration. The plasmid pRS424-FLDEN2-NG-CDNA, containing the full-length DENV-2 genome from the New Guinea strain, was used as template for the generation of the full-length NS3 construct. The ATPase activity of NS3 was determined by measuring the extension of hydrolysis of NTP to NDP and Pi. The amount of free inorganic phosphate released was calculated by the hydrolysis of ATP using a standard curve with known Pi concentrations, and the reaction was 852808-04-9 measured at 660 nm using SpectraMax M5 spectrophotometer. Compounds were diluted from 20 mM or 40 mM stock solution prepared in 100% DMSO. These compounds were diluted to a final concentration of 10% DMSO in the assay buffer. Since the compounds 9b and 9c demonstrated specific inhibitory activity in DENV-2 replication in Vero cells, we further investigated their efficacy against NS3 ATPase and helicase activities by performing a dose response analysis. Due to the low solubility of the naphthoquinone compounds in
