NOVEL AMIDINE-CONTAINING PEPTIDYL PHOSPHONATES AS IRREVERSIBLE INHIBITORS FOR BLOOD-COAGULATION AND RELATED SERINE PROTEASES

A series of new peptidyl (alpha-aminoalkyl)phosphonate diphenyl esters containing the 4-amidinophenyl group were synthesized and tested as i rreversible inhibitors,for thrombin- and other trypsin-like enzymes. T hese phosphonates irreversibly inhibited several coagulation enzymes a nd trypsin. Boc-D-Phe-Pro-(4-AmPhGly)(P)(OPh)(2) is the best human thr ombin inhibitor in the series with a k(obs)/[I] value of 11 000 M(-1) s(-1), and it inhibits thrombin more than 5-fold more effectively than the other enzymes tested. Z-(4-AmPhGly)(P)(OPh)(2) is the best inhibi tor for plasma kallikrein with a k(obs)/[I] value of 18 000 M(-1) s(-1 ). Generally, the (4-AmPhGly)(P)(OPh)(2) derivatives are better inhibi tors of thrombin and trypsin than the corresponding (4-AmPhe)P(OPh)2 d erivatives which contain an extra CH2 separating the amidinophenyl gro up from the peptide backbone. The amidino phosphonates did not inhibit acetylcholinesterase and were chemically stable in neutral buffers. I n addition, the inhibited trypsin derivative did not regain any enzyme activity after removal of excess inhibitor and incubation in a pH 7.5 buffer for 1 day. Boc-D-Phe-Pro-(4-AmPhGly)(P)(OPh)(2) and D-Phe-Pro- (4-AmPhe)(P)(OPh)(2) prolonged the prothrombin time ca. 2-fold and pro longed the activated partial thromboplastin time ca. 3-4-fold in human plasma at concentrations of 63 and 125 mu M, respectively. The novel amidine-containing peptidyl phosphonates reported here are thus effect ive anticoagulants in vitro, and they may have utility for use in vivo .