Synthesis of Novel Inhibitors of IdeS, a Bacterial Cysteine Protease Including Studies of Stereoselective Reductive Aminations

摘要

AbstractThe cysteine protease IdeS is an IgG degrading enzyme secreted by the bacterium Streptococcuspyogenes to evade the human immune system. In this thesis several inhibitors of IdeS have beensynthesized and evaluated. Such inhibitors should be highly useful when elucidating the detailedmechanism of IdeS action. They might also have a potential as treatment of acute and severe infectionscaused by the bacteria. Further, IdeS has a therapeutic application of its own due to the proteolytic abilityand an IdeS inhibitor might contribute during the development.Only irreversible, unselective inhibitors of IdeS were known five years ago. In this thesis, three strategieswith the aim to synthesize and identify more inhibitors have been undertaken. Focus was first set oncompounds with a substructure resembling the known inhibitors but with reversible warheads, i.e. nitrile,azide and aldehyde functions. The aldehyde derivatives were found to provide the first reversibleinhibitors of IdeS.Then, to avoid covalent interactions and obtain more selective inhibitors, a substrate based strategy wasundertaken. A 3-aminopiperidine fragment was used as replacement of either of the two residues adjacentto the scissile bond in IgG. Such fragments can be synthesized from N-protected 3-aminopiperidone andamino acid esters in reductive aminations in which a stereogenic center is formed. A series of di-, tri- andtetrapeptide analogues, together with eight peptides covering the cleavage site of IgG, were screened fortheir capacity to inhibit the cysteine proteases IdeS, SpeB and papain. Several analogues showedinhibition capacity, two compounds showed also high selectivity for IdeS. In contrast, none of the testedpeptides showed any inhibition. Computational docking studies indicate that the identified IdeS peptideanalogues and the non-active peptides do not share the same binding site in IdeS. Probably, the piperidinemoiety hinders the inhibitor to enter the catalytic site.A more detailed study of the stereoselectivity in the reductive aminations affording the 3-aminopiperidinefragment showed that a large protecting group (trityl) together with a large reducing agent (NaBH(O-2-ethylhexanoyl)3) gave the highest diastereomeric ratio. The highest ratio obtained was 21:79 when Lprolinemethyl ester was used. The newly formed stereogenic center had the R-configuration, determinedby chemical correlation. Computer based conformational analysis combined with Boltzmann distributioncalculations implies an axial attack by the reducing reagent on the intermediary imine.To improve the potency of the two identified di- and tripeptide analogues synthetic routes toconformationally restricted N-containing bicyclic derivatives was undertaken in a third strategy. Fivecompounds with different bicyclic scaffolds were screened for their inhibition capacity towards IdeS andpapain. One of the compounds was able to inhibit the first step of proteolytic cleavage of IgG by IdeS, aprocess usually completed in seconds.