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

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Abstract
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 that the potent inhibitory activity of 3 is due to the following structural features: (1) the ethyl ester which enforces specific acyl-enzyme formation, (2) the n-hexyl group at the β-position and 4-cyanophenyl group which stabilize the acyl-enzyme, and (3) the phenylalanine residue which functions for the specific recognition of S1 site in the enzyme. In particular, the action of 3 as a potent inhibitor, but poor substrate, can be ascribed largely to the very slow deacylation rate depending on the structure factors cited in feature 2. The results of inhibition by 3 and its analogues against different serine proteases such as chymase, cathepsin G, and elastase suggest that these compounds recognize common parts in the active sites among these chymotrypsin-like serine proteases, and 3 is one of the most suitable structures to recognize those common parts. Our results provide an intriguing basis for further developments in the design of a stable ester-based selective serine protease inhibitor.