Optimization of Cyclic Plasmin Inhibitors: From Benzamidines to Benzylamines

Abstract

New macrocyclic plasmin inhibitors based on our previously optimized P2-P3 core segment have been developed. In the first series, the P4 residue was modified, whereas the 4-amidinobenzylamide in P1 position was maintained. The originally used P4 benzylsulfonyl residue could be replaced by various sulfonyl- or urethane-like protecting groups. In the second series, the P1 benzamidine was modified and a strong potency and excellent selectivity was retained by incorporation of p-xylenediamine. Several analogues inhibit plasmin in the subnanomolar range, and their potency against related trypsin-like serine proteases including trypsin itself could be further reduced. Selected derivatives have been tested in a plasma fibrinolysis assay and are more effective than the reference inhibitor aprotinin. The crystal structure of one inhibitor was determined in complex with trypsin. The binding mode reveals a sterical clash of the inhibitor's linker segment with the 99-hairpin loop of trypsin, which is absent in plasmin.

Optimization of Substrate‐Analogue Furin Inhibitors

“…Most of the used free or protected P1 residues were prepared according to known published procedures, their synthesis and analytical data are provided in the Supporting Information, e.g., N ‐(4‐(aminomethyl)benzyl)‐2,2,2‐trifluoroacetamide⋅HCl ( 32 ) used for inhibitors 13 and 19 , tert ‐butyl (5‐(aminomethyl)pyridin‐2‐yl)carbamate⋅CH 3 COOH ( 33 ) used for inhibitors 10 , 15 , and 20 , 4‐(aminomethyl)pyridin‐2‐amine ( 34 ) used for inhibitors 11 and 16 , (9 H ‐fluoren‐9‐yl)methyl 4‐aminobenzylcarbamate ( 45 ) used for inhibitors 6 and 7 , and (9 H ‐fluoren‐9‐yl)methyl 3‐aminobenzylcarbamate ( 46 ) used for inhibitor 8 , 9 , and 14 . The synthesis of 4‐(aminomethyl)benzohydrazonamide ( 37 ) used as a P1 residue in inhibitors 12 and 17 started from p ‐cyanobenzylamine⋅HCl according to Scheme .…”

Deracemization and Stereoinversion of α‐Amino Acids by l‐Amino Acid Deaminase

“…The generation of d ‐4‐Npa (the starting point for the synthesis of plasmin inhibitors and anti‐fibrinolytic drug production), from the corresponding l ‐enantiomer was established by coupling the deamination of the l ‐amino acid to the corresponding achiral imino acid and its nonselective in situ reduction by the borane tert ‐butylamine complex (Scheme B). A 12.5 mM l ‐4‐Npa solution was incubated with PmaLAAD under standard conditions and then a 5‐fold molar excess of the reducing agent was added.…”

Design, Synthesis, and Characterization of Macrocyclic Inhibitors of the Proprotein Convertase Furin

“…After N-terminal Boc protection the side chain Dde groups on the P2 and P6 Lysr esidues were removed.T he peptides were cyclized on-resin using the protonatable3 ,3'-(piperazine-1,4-diyl)dipropanoic acid, which was also previously used for the cyclization of numerous plasmini nhibitors. [8,9] The following steps were performed as described before. Notably,t he synthesis of inhibitor 5 was achieved only with poor yield.…”

“…An even stronger inhibition wasd etermined for most of the type 4i nhibitors (9)(10)(11)(12)(13)(14), where the furin inhibitor segment was fused to the cyclic CPP sequence. Thei nhibition constants of compounds 10, 11,a nd 13 (< 150 pm)w ere determined under tight-binding conditions, as shown in Figure 2f or the most potent analogue 10.T he equations used for K i value calculations are described in the Supporting Information.…”

“…Many other examples of potent and selective experimental macrocyclic serine protease inhibitors have been described, including inhibitors of the clottinge nzymes thrombin, [3] factor VIIa [4][5][6] or factor XIa, [7] and of the fibrinolytic protease plasmin. [8,9] These reports encouraged us to test various macrocyclizations for the design of new furin inhibitors.…”