Design and evaluation of novel bivalent thrombin inhibitors based on amidinophenylalanines

Two bivalent thrombin inhibitors were synthesized, which consist of a benza midine-based active-site-blocking segment, a fibrinogen recognition exosite inhibitor and a peptidic linker connecting these fragments. BZA-1 hirulog contains an N-alpha-(2-naphthylsulfonyl)-S-3-amidinophenylalanyl-isonipecot ic acid residue connected via the carboxyl group to the linker segment. The active-site-directed moiety of BZA-2 hirulog [N-alpha-(2-naphthylsulfonyl- glutamyl)-R-4-amidinophenylalanyl-piperidide] was coupled to the linker via the side chain of the glutamic acid. Both BZA-hirulogs contain almost iden tical linker-exo site inhibitor parts, except for the substitution of a gly cine as the first linker residue in BZA-1 hirulog by a gamma-amino butyric acid in BZA-2 hirulog, thus increasing flexibility and linker length by two additional atoms. BZA-1 hirulog showed moderate potency (K-i = 0.50 +/- 0. 14 nM), while BZA-2 hirulog was characterized as a slow, tight binding inhi bitor of thrombin (K-i = 0.29 +/- 0.08 pM). The stability in human plasma o f both analogs was strongly improved compared with hirulog-1. For BZA-2 hir ulog a significantly reduced plasma clearance was observed after intravenou s injection in rats compared with BZA-1 hirulog and hirulog-1. The X-ray st ructure of the BZA-2 hirulog in complex with human alpha-thrombin was solve d and confirmed the expected bivalent binding mode.