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-CH2Cl derivatives have been synthesized and tested as inhibitors for thrombin and several blood coagulation enzymes. The parent compound, D-Phe-Pro-Arg-CH2Cl is still the best thrombin inhibitor in the series with kobs/[I] value of 10(7) M-1s-1. Extension by one amino acid (Phe 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 reduces the inhibition constant by 1-2 orders of magnitude, which indicates the importance of a free amino group at the N-terminus. The tripeptide D-Phe-Pro-Arg-CH2Cl and related tetrapeptide inhibitors inhibit thrombin more potently than factor IXa and plasma kallikrein by 2-5 orders of magnitude. Z-Arg-CH2Cl and Phe-Phe-Arg-CH2Cl which contain a large hydrophobic group at the P2 site inhibit thrombin poorly. All the peptide chloromethyl ketones inhibit plasma kallikrein moderately with kobs/[I] values of 10(2)-10(3) M-1s-1 but inhibit factor IXa poorly (kobs/[I] < 20 M-1s-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. These conjugates are model compounds for developing specific thrombus-bound thrombin inhibitors which may have therapeutic application in the treatment of coagulation disorders.