e preclinical antimalarial drugs. N-89 is a 14192894 chemically synthesized endoperoxide compound with potent antimalarial activity against Plasmodium falciparum in vitro and Plasmodium berghei in vivo, and it shows low levels of cytotoxicity in mice and rats . N-251 hexan-1-ol), which bears a functional side chain hydroxyl group that allows derivatization, is synthesized by replacing the hydrogen at C-4 of N-89 with hexanol, and it is as potent as N-89 against malaria parasites. We first evaluated the anti-HCV activities of N-89 and N-251 using HuH-7-derived OR6 and Li23-derived ORL8 and ORL11 assay systems. The results revealed that both N-89 and N-251 possessed strong anti-HCV activities. The EC50 and SI values of N-89 in each assay were calculated , and the anti-HCV activity of N-251 was found to be as potent as that of N-89. The anti-HCV activities of N-89 and N-251 were confirmed by Western blot analysis of HCV Core. To further evaluate the activities of N-89 and N-251, as additional assay systems, we used HuH-7-derived 1B-4R and AH1R , and Li23derived 1B-4RL and KAH5RL . These assays also showed that N-89 and N-251 possessed potent anti-HCV activities. It was noteworthy that N-89 exhibited the strongest antiHCV activity in the AH1R assay. These results suggest that the anti-HCV activity of N-89 or N-251 is not influenced by the cell line or HCV strain. We next examined the activities of N-89 and N-251 using polyclonal cell-based assay systems that facilitate the monitoring replication of HCV subgenomic replicon RNA. These assays also showed that N-89 and N-251 possessed anti-HCV activity with EC50 values of less than 1 mM. Taken together, these results indicate that the anti-HCV activities of N-89 and N-251 are not dependent on the specific cloned cell line or HCV structural proteins. N-89 and N-251 Inhibited Authentic HCV-RNA Replication The genome-length HCV-RNA used in the assay systems described above contains three non-natural elements: 17636045 RL, neomycin AEB-071 biological activity phosphotransferase, and an internal ribosomal entry site of encephalomyocarditis virus. To exclude the possibility that the anti-HCV activity of N-89 or N-251 was due to the inhibition of these three exogenous elements, we examined the anti-HCV activities of N-89 and N-251 using the authentic 9.6 kb HCVRNA-replicating HCV-O/RLGE cells, which were developed 
by the introduction of in vitro synthesized HCV-O/RLGE RNA into OR6c cured cells. We could demonstrate by quantitative RT-PCR and Western blot analyses that N-89 and N251 at the expected concentrations efficiently prevented HCVRNA replication and HCV Core expression in HCV-O/RLGE cells in a dose-dependent manner, respectively. The EC50 and SI values of N-89 and N-251 in this assay were calculated as follows each: EC50 2.0 mM and SI.5.0 in N-89; EC50 1.6 mM and SI 2.8 in N-251. To further confirm that N-89 or N-251 does not inhibit the RL activity, we examined the direct effect of each reagent by adding it along with substrate to the cell lysate in the RL assay. No suppressive effects by N-89 and N-251 were observed in either the OR6 assay or the ORL8 assay. These results indicate that the anti-HCV activities of N89 and N-251 were due to the inhibition of HCV-RNA itself, but not to exogenous elements contained in the genome-length HCVRNA. N-89 and N-251 did not Inhibit RNA Replication of HCVJFH-1 Strain We next examined whether N-89 and N-251 worked in an HCV production system using HCV-JFH-1 strain. Unexpectedly, the results using the J