Structure-based studies led to the identification of a constrained derivative of S-trityl-l-cysteine (STLC) scaffold as a candidate inhibitor of hepatitis C virus (HCV) NS5B polymerase. A panel of STLC derivatives were synthesized and investigated for their activity against HCV NS5B. Three STLC derivatives, 9, F-3070, and F-3065, were identified as modest HCV NS5B inhibitors with IC 50 values between 22.3 and 39.7 μM. F-3070 and F-3065 displayed potent inhibition of intracellular NS5B activity in the BHK-NS5B-FRLuc reporter and also inhibited HCV RNA replication in the Huh7/Rep-Feo1b reporter system. Binding mode investigations suggested that the STLC scaffold can be used to develop new NS5B inhibitors by further chemical modification at one of the trityl phenyl group. © 2012 Elsevier Masson SAS. All rights reserved.