Entwicklung, Synthese und Charakterisierung makrozyklischer Plasmininhibitoren

The current work is focused on the development of new potent and selective active site inhibitors of plasmin as potential antifibrinolytics. Antifibrinolytics are important drugs for the reduction of bleeding after severe traumata and in major surgical interventions. Currently, only few antifibrinolytic drugs are approved for clinical use, which possess limited efficacies and can cause severe side effects. The most important antifibrinolytic drug tranexamic acid has been developed at the beginning of the 1960’s and reduces the plasmin formation from its zymogen plasminogen, but lacks an inhibitory efficacy against already activated plasmin. This limits its therapeutic usability. Hemostasis includes the formation of fibrin clots in the injured vessel, which facilitates the accumulation of platelets and erythrocytes. During wound healing, the clot removal is mediated by plasmin, which degrades insoluble fibrin polymers into soluble fragments during the process of fibrinolysis. In hyperfibrinolytic states, an increased plasmin activity impedes hemostasis and can thereby cause severe bleeding complications. An antifibrinolytic effect can be achieved either via a direct plasmin inhibition or by reducing the plasmin formation from its zymogen plasminogen. In our group, a multitude of highly active and selective macrocyclic plasmin inhibitors has been developed before, which address the S4-S1-region of the catalytic center. The high plasmin selectivity could be achieved by their sterically demanding macrocycle, which causes a steric clash with the characteristic 99-loop that is missing in Plasmin, but is present in all other trypsin like serine proteases. Among these inhibitors, compound 40 exhibits the best combination of plasmin inhibition and selectivity. Its macrocyclic core segment consists of two p-substituted phenylalanine-residues in P3- and P2-position, respectively, whose sidechains are connected with a symmetric piperazinyldicarboxylic acid amide linker. In this thesis, such structures and their derivatives are named macrocyclic substrate analogue inhibitors. Within the scope of this work, several derivatives of compound 40 have been synthesized varying both the P4-position (series 1) and the length of the linker segment (series 2). For the inhibitor series 2, a new synthesis strategy has been established that allows the selective incorporation of asymmetric linkers. Furthermore, a new type of macrocycle has been synthesized, in which the P4-residue is connected to the P2-amino acid via the aforementioned linker segments (series 3). Inhibitory constants were determined for plasmin and six related trypsin-like serine proteases. Although some derivatives inhibit plasmin with subnanomolar Ki-values, none of the new inhibitors showed an improved overall profile in comparison to the reference compound 40. In cooperation with an Australian group, the binding mode of these inhibitors has been determined by X-ray crystallography for the first time. In a second project, a new inhibitor type was developed based on the known crystal structure of the acyclic compound 11 (YO-2) in μPlasmin. This reference inhibitor addresses the S1-S2’-region of the catalytic center and possesses a relatively weak plasmin selectivity. The concept of macrocyclization was adapted to synthesize sterically demanding YO-2-derivatives, which should reveal a similar binding mode and would ideally show an improved selectivity profile over related trypsin-like serine proteases. For this thesis, A large number of new macrocyclic inhibitors has been synthesized, which share a common Tranexamoyl (Txa)- residue in P1-position and are therefore called macrocyclic Txa-conjugated inhibitors (series 4 and 5). The best derivatives possess subnanomolar inhibition constants for plasmin. Several of these compounds also show a significant selectivity against a panel of related trypsin-like serine proteases. However, in comparison to the reference inhibitor 40, their selectivity profile is less pronounced. Together with the Australian group, the binding mode of the new macrocyclic Txa-conjugated inhibitors in μPlasmin was determined, which resembles the conformation of the reference inhibitor YO-2 in the S1-S2’-region. Both the macrocyclic substrate analogue inhibitors and the macrocyclic Txa-conjugated inhibitors could potentially be further developed as potent and selective antifibrinolytic drugs for the reduction of bleeding.