Hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma. Although various classes of anti-HCV agents have been under clinical development, most of these agents target RNA replication in the HCV life cycle. To achieve a more effective multidrug treatment, the development of new, less expensive anti-HCV agents that target a different step in the HCV life cycle is needed. We prepared an in-house natural product library consisting of compounds derived from fungal strains isolated from seaweeds, mosses, and other plants. A cell-based functional screening of the library identified sulochrin as a compound that decreased HCV infectivity in a multi-round HCV infection assay. Sulochrin inhibited HCV infection in a dose-dependent manner without any apparent cytotoxicity up to 50 mu M. HCV pseudoparticle and trans-complemented particle assays suggested that this compound inhibited the entry step in the HCV life cycle. Sulochrin showed anti-HCV activities to multiple HCV genotypes la, 1b, and 2a. Co-treatment of sulochrin with interferon or a protease inhibitor telaprevir synergistically augmented their anti-HCV effects. Derivative analysis revealed anti-HCV compounds with higher potencies (IC50 < 5 mu M). This is the first report showing an antiviral activity of methoxybenzoate derivatives. Thus, sulochrin derivatives are anti-HCV lead compounds with a new mode of action. (C) 2013 Elsevier Inc. All rights reserved.