Thiol-based Inhibitors of Mammalian Collagenase: Substituted Amide and Peptide Derivatives of the Leucine Analogue.

To define the inhibitory requirements of mammalian collagenase,several N-substituted amide and peptide derivatives of the mercaptomethyl analogue of leucine,2-[(R,S)mercaptomethyl]-4-methylpentanoic acid (H psi[SCH2]-DL-leucine),were synthesized and tested as inhibitors of pig synovial collagenase with soluble type I collagen as substrate. H psi[SCH2]-DL-leucine (IC50 = 320 microM) was about 10 times more potent than the beta-mercaptomethyl compound,N-acetylcysteine. The amide of H psi[SCH2]-DL-leucine was six times more potent than the parent thiol acid. Aliphatic N-substituted amides were less potent than the unsubstituted amide,whereas the N-benzyl amide was slightly more potent. Dipeptides,particularly those with an aromatic group at P2',were up to 20-fold more potent,while tripeptides with an aromatic L-amino acid at P2' and Ala-NH2 at P3' were up to 2200 times more potent than H psi[SCH2]-DL-leucine. The resolved diastereomers of H psi[SCH2]-DL-Leu-Phe-Ala-NH2 inhibited by 50% at 0.3 and 0.04 microM,respectively. The most potent inhibitor synthesized,an isomer of H psi[SCH2]-DL-Leu-L-3-(2'-naphthyl)alanyl-Ala-NH2,exhibited an IC50 of 0.014 microM,a value about 300 times less than similar thiol-based analogues of the P'-cleavage sequence of type I collagen,H psi[SCH2]-DL-Leu-Ala-Gly-Gln-. These structure-function studies establish within the present series of compounds that the most effective inhibitors of mammalian collagenase are not closely related to the P2'-P3' elements of the cleavage site of the natural substrate but rather have an aromatic group at the P2' position and Ala-NH2 at the P3' position.