Rational Design of True Hirudin Mimetics: Synthesis and Characterization of Multisite-Directed r-Thrombin Inhibitors1

We describe here the design, synthesis, and activity of a novel class of R-thrombin inhibitors named hirunorms. They were rationally designed to interact through their N-terminal end with the R-thrombin active site in a nonsubstrate mode and to specifically bind the fibrinogen recognition exosite. An appropriate spacer that is able to properly orient the N-terminal end in the active site was also selected. This spacer allowed the size of the inhibitors to be reduced to about one-third of the amino acid residues in the hirudin sequence. Hirunorms specifically inhibit the amidolytic action of human R-thrombin toward a small chromogenic substrate. The most active compounds of the series, hirunorms IV and V, inhibit R-thrombin catalyzed hydrolysis of Tos-Gly-Pro-Arg-p-nitroanilide with Ki ) 0.09 and Ki ) 0.21 nM, respectively. Comparison of the anticoagulant properties of hirunorms, natural hirudin from the European leech Hirudo medicinalis, and the synthetic analog hirulog-1 revealed that hirunorm IV is about 10-fold and 3-fold more active, on a molar base, than hirudin and hirulog-1 in increasing the aPTT, PT, and TT of normal human plasma. The peculiar structure of hirunorms makes them stable to the amidolytic action of thrombin without the introduction of any peptide bond modification. These molecules display long-lasting activity in human plasma, due to the presence of several unnatural amino acids in susceptible positions. Hirunorms are potential candidates for injectable anticoagulants, due to their potency, specificity of action, long-lasting activity, and safety profiles.