Novel Peptidyl α-Keto Amide Inhibitors of Calpains and Other Cysteine Proteases

A series of new dipeptidyl α-keto amides of the general structure R1-l-Leu-d,l-AA-CONH-R2 were synthesized and evaluated as inhibitors for the cysteine proteases calpain I, calpain II, and cathepsin B. They combine 10 different N-protecting groups (R1), 3 amino acids residues in P1 (AA), and 44 distinct substituents on the α-keto amide nitrogen (R2). In general, calpain II was more sensitive to these inhibitors than calpain I, with a large number of inhibitors displaying dissociation constants (K i) in the 10−100 nM range. Calpain I was also effectively inhibited, but very low K i values were observed with a smaller number of inhibitors than with calpain II. Cathepsin B was weakly inhibited by most compounds in this study. The best inhibitors for calpain II were Z-Leu-Abu-CONH-CH2-CHOH-C6H5 (K i = 15 nM), Z-Leu-Abu-CONH-CH2-2-pyridyl (K i = 17 nM), and Z-Leu-Abu-CONH-CH2-C6H3(3,5(OMe)2) (K i = 22 nM). The best calpain I inhibitor in this study was Z-Leu-Nva-CONH-CH2-2-pyridyl (K i = 19 nM). The peptide α-keto amide Z-Leu-Abu-CONH-(CH2)2-3-indolyl was the best inhibitor for cathepsin B (K i = 31 nM). Some compounds acted as specific calpain inhibitors, with comparable activity on both calpains I and II and a lack of activity on cathepsin B (e.g., 40, 42, 48, 70). Others were specific inhibitors for calpain I (e.g., 73) or calpain II (e.g., 18, 19, 33, 35, 56). Such inhibitors may be useful in elucidating the physiological and pathological events involving these proteases and may become possible therapeutic agents.