Background: Standard treatment of chronic hepatitis C with pegylated interferon and ribavirin is associated with suboptimal virological response rates and substantial side effects. This study describes the in vitro and in vivo development of JTK- 652, a novel pyrrolopyridazin-derived HCV infection inhibitor. Methods: JTK-652 was evaluated in multiple cell lines using an in vitro HCV infection model consisting of HCV pseudotype vesicular stomatitis virus bearing HCV E1/E2 envelope proteins. Safety, tolerability, pharmacokinetics and efficacy of JTK-652 were tested in a randomized double-blind and placebo-controlled study in healthy male volunteers (n=36) and chronic hepatitis C patients. A total of 10 HCV genotype-1-infected patients (treatment-naive [n=2] and treatment-experienced [n=8]) with HCV RNA>1x10(5) IU/ml received an oral dose of 100 mg JTK-652 three times daily or placebo (8:2 ratio) for 4 weeks. Results: JTK-652 showed potent inhibitory activity against HCV genotype 1a and 1b pseudotype viruses bearing HCV E1/E2 envelope proteins in HepG2 cells and in human primary hepatocytes. No significant clinical laboratory, vital sign, ECG or physical examination abnormalities were observed during the Phase I trial. JTK-652 was found to be well tolerated. No significant changes in HCV RNA levels compared with baseline were observed at the end of treatment. Conclusions: Although results from the preclinical studies indicated that JTK-652 has well-established antiviral properties and a Phase I clinical trial has showed that JTK-652 was safe and well tolerated at a 100 mg three times daily dose level, plasma HCV RNA levels in chronically HCV-infected patients did not decrease during 28 days of dosing at a 100 mg three times daily dose level.