EFFECT OF THE 7-AMINO SUBSTITUENT ON THE INHIBITORY POTENCY OF MECHANISM-BASED ISOCOUMARIN INHIBITORS FOR PORCINE PANCREATIC AND HUMAN NEUTROPHIL ELASTASES - A 1.85-A X-RAY STRUCTURE OF THE COMPLEX BETWEEN PORCINE PANCREATIC ELASTASE AND LPHENYLALANYL)AMINO]-4-CHLORO-3-METHOXYISOCOUMARIN

A series of new acyl, urea, and carbonate derivatives of 7-amino-4-chl oro-3-methoxyisocoumarin were synthesized and evaluated as irreversibl e inhibitors of human neutrophil elastase (HNE) and porcine pancreatic elastase (PPE). Inhibition of HNE is directly related to the hydropho bicity of the substituent on the 7-amino group. The N-Tos-Phe derivati ve (19) is the best HNE inhibitor with a second-order rate constant k( obs)/[I] = 200 000 M-1 s-1. The closest analogue in this series, the 3 ,3-diphenylpropionyl derivative 5, had a k(obs)/[I] = 130 000 M-1 s-1 with HNE. In contrast to the Tos-Phe derivative 19, phenylacetyl deriv ative 2 and carbonates 22 and 25 gave extremely stable enzyme-inhibito r complexes with deacylation half-lives longer than 48 h with both ela stases. N-Phenylurea derivative 25 was the best inhibitor for PPE with a second-order rate constant k(obs)/[I] = 7300 M-1 s-1. The crystal s tructure of a complex of PPE with N-tosyl-Phe derivative 19 was determ ined at 1.85-angstrom resolution and refined to a final R factor of 16 .9%. The isocoumarin forms an acyl enzyme with Ser-195, while His-57 i s near the inhibitor, but not covalently linked. The Tos-Phe makes a f ew hydrophobic contacts with the S' subsites of PPE, but appears to be interacting primarily with itself in the PPE structure. This region o f HNE is more hydrophobic and modeling indicates that the inhibitor wo uld probably make additional contacts with the enzyme.