INHIBITION OF THROMBIN BY ARGININE-CONTAINING PEPTIDE CHLOROMETHYL KETONES AND BIS CHLOROMETHYL KETONE-ALBUMIN CONJUGATES

Arg-containing peptide chloromethyl ketones including D-Phe-Pro-Arg-CH 2Cl derivatives have been synthesized and tested as inhibitors for thr ombin and several blood coagulation enzymes. The parent compound, D-Ph e-Pro-Arg-CH2Cl is still the best thrombin inhibitor in the series wit h k(obs)/[I] value of 10(7) M(-1)s(-1). Extension by one amino acid (P he or Gly), or a peptide moiety (ClCH2-Arg<-Pro<-D-Phe<-CO-CO-, ClCH2- Arg<-Pro<-D-Phe<-CO-(CH2)(3)-CO-, where <- indicates a reversed amino acid residue, -CO-CHR-NH-) on the N-terminus of D-Phe-Pro-Arg-CH2Cl re duces the inhibition constant by 1-2 orders of magnitude, which indica tes the importance of a free amino group at the N-terminus. The tripep tide D-Phe-Pro-Arg-CH2Cl and related tetrapeptide inhibitors inhibit t hrombin more potently than factor IXa and plasma kallikrein by 2-5 ord ers of magnitude. Z-Arg-CH2Cl and Phe-Phe-Arg-CH2Cl which contain a la rge hydrophobic group at the P-2 site inhibit thrombin poorly. All the peptide chloromethyl ketones inhibit plasma kallikrein moderately wit h k(obs)/[I] values of 10(2)-10(3) M(-1)s(-1) but inhibit factor IXa p oorly (k(obs)/[I] < 20 M(-1)s(-1)). Conjugates of albumin with the bis chloromethyl ketones [(CO-D-Phe-Pro-Arg-CH2Cl)(2) (CH2)(3)-(CO-D-Phe- Pro-Arg-CH2Cl)(2)] were prepared and are potent thrombin inhibitors. T hese conjugates are model compounds for developing specific thrombus-b ound thrombin inhibitors which may have therapeutic application in the treatment of coagulation disorders.