N-[2,2-Dimethyl-3-(N-(4-cyanobenzoyl)amino)nonanoyl]-l-phenylalanine Ethyl Ester as a Stable Ester-Type Inhibitor of Chymotrypsin-like Serine Proteases: Structural Requirements for Potent Inhibition of α-Chymotrypsin

We introduce a new potent inhibitor, N-[2,2-dimethyl-3-(N-(4-cyanobenzoyl)amino)nonanoyl]-l-phenylalanine ethyl ester (3), which preferentially inhibits serine proteases belonging to a chymotrypsin superfamily. This inhibitor, despite consisting of a stable ethyl ester structure, showed strong inhibitory activities toward bovine α-chymotrypsin, human cathepsin G, and porcine elastase by acting as an acylating agent. The calculated inactivation rate constant (kinact) and enzyme−inhibitor dissociation constant (Ki) against α-chymotrypsin were 0.0028 s-1 and 0.0045 μM, respectively (kinact/Ki = 630 000 M-1 s-1). These kinetic parameters indicate that this inhibitor is one of the most powerful α-chymotrypsin inactivators ever reported. On the basis of structure−activity relationship (SAR) and structure−stability relationship studies of analogues of 3, which were modified in three parts of the molecule, i.e., the 4-cyanophenyl group, β-substituent at the β-amino acid residue, and ester structure, we suggest th...