Inhibition of Thrombin by Arginine-Containing Peptide Chloromethyl Ketones and Bis Chloromethyl Ketone-Albumin Conjugates

Abstract

Arg-containing peptide chloromethyl ketones including D-Phe-Pro-Arg-CH₂CI derivatives have been synthesized and tested as inhibitors for thrombin and several blood coagulation enzymes. The parent compound, D-Phe-Pro-Arg-CH₂CI is still the best thrombin inhibitor in the series with k_obs/[I] value of 10⁷ M⁻¹s⁻¹ Extension by one amino acid (Phe or Gly), or a peptide moiety (ClCH₂-Arg<-Pro<-D-Phe<-CO-CO-, CICH₂-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-CH₂CI reduces the inhibition constant by 1–2 orders of magnitude, which indicates the importance of a free amino group at the N-terminus. The tnpeptide D-Phe-Pro-Arg-CH₂CI and related tetrapeptide inhibitors inhibit thrombin more potently than factor IXa and plasma kallikrein by 2–5 orders of magnitude. Z-Arg-CH₂CI and Phe-Phe-Arg-CH₂CI which contain a large hydrophobic group at the P, site inhibit thrombin poorly. All the peptide chloromethyl ketones inhibit plasma kallikrein moderately with k_obs,/[I] values of 10²-10³ M⁻¹s⁻¹ but inhibit factor IXa poorly (k_obs/[I] < 20 M⁻¹s⁻¹). Conjugates of albumin with the bis chloromethyl ketones [(CO-D-Phe-Pro-Arg-CH₂CI)₂, (CH₂)₃-(CO-D-Phe-Pro-Arg-CH₂CI)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.

Key Words: :

• Thrombin inhibitors
• peptide chloromethyl ketones