Synthesis and evaluation of the biological activity of analogues of leupeptin

Abstract

Leupeptin, N-acetyl or N-propionyl-L-leucyl-L-leucyl-DL-argininal, is a potent inhibitor of trypsin-like enzymes; however, it is not selective within this proteolytic class. In an effort to increase selectivity among trypsin-like enzymes the synthesis of several tripeptide analogues of leupeptin containing C-terminal argininal, lysinal, and ornithinal units has been accomplished. The synthetic tripeptide analogues were tested as inhibitors of trypsin, kallikrein, plasmin and cathepsin B-like enzymes activity in vitro. Carbobenzyloxy-L-leucyl-L-leucyl-L-agininal (43a) was significantly less effective as an inhibitor than leupeptin. Carbobenzyloxy-L-leucyl-L-leucyl-L-lysinal (55a) and carbobenzyloxy-L-leucyl-L-leucyl-L-ornithinal (55e) display significantly different inhibition characteristics than carbobenzyloxy-L-leucyl-L-leucyl-L-argininal (43a). While carbobenzyloxy-L-leucyl-L-leucyl-L-argininal (43a) showed a moderate inhibition of trypsin, plasmin, kallikrein, and the cathepsins carbobenzyloxy-L-leucyl-L-leucyl-L-ornithinal (55e) showed no inhibition of trypsin or plasmin. Carbobenzyloxy-L-leucyl-L-leucyl-L-lysinal (55a) was effective as an inhibitor of trypsin and plasmin, and more effective as an inhibitor of the cathespin than carbobenzyloxy-L-leucyl-L-leucyl-L-argininal (43a). These results indicate that the nature of the N-terminal protecting group and the side chain of the C-terminal aldehyde are important for biological activity of the peptides. Variations made in the composition and sequence of the amino acid substituents also resulted in variations in the biological activity of the peptides. In general, plasmin showed a strong preference for the tripeptide inhibitors which contain a L-phenylalanyl-L-leucyl unit; and the cathespins showed a strong preference for the tripeptide inhibitors which contain a L-leucyl-L-valyl unit. These data suggest that the composition and sequence of amino acid units play a significant role in the selectivity within a mechanistic class.