New 2-oxo-2H-1-benzopyran derivatives were prepared to optimize 2a,b, initially developed as mechanism-based alpha-chymotrypsin (alpha-CT) inhibitors, into potent and selective thrombin (THR) inhibitors. From this study, 22, characterized by a 2-(N-ethyl-2'-oxoacetamide)-5'-chlorophenyl ester side chain, was shown to be a good THR inhibitor (ki/KI = 3455 M(-1) x s(-1)), displaying an excellent selectivity profile against other serine proteases such as factor Xa, trypsin, and alpha-CT. Docking analysis of this compound into the different protein structures revealed the molecular basis responsible for its potency and selectivity.