Novel coumarinic derivatives were synthesized and tested for their inhibitory potency toward α-CT and HLE. Cycloalkyl esters and amides were found to be essentially inactive on both enzymes. On the opposite, aromatic esters strongly inactivated α-CT whereas HLE was less efficiently inhibited with dichlorophenyl ester derivatives (k(inact)/K(I)=4000M-1 s-1 for 36). Representative examples of amide, ester, thioester and ketone derivatives were prepared in order to evaluate the influence of the link between the coumarinic ring and the phenyl side chain. The irreversible inactivation of α-CT by 6-chloromethyl derivatives should be due to alkylation of a histidine residue as suggested by the amino acid analysis of the modified chymotrypsin. Conversely the inhibition of HLE was transient. Intrinsic reactivity of coumarins has been calculated using a model of a nucleophilic reaction between the ligand and the couple methanol-water. From this calculation, it appears that differences in the inhibitory potency expressed by these molecules cannot only be explained by differences in the reactivity of the lactonic carbonyl group toward the nucleophilic attack. Copyright (C) 2000 Elsevier Science Ltd.