Files

Abstract

Cyclosporin is a cyclic undecapeptide useful as drug for organ transplantation due to its immunosuppressive properties. Ten years ago, an anti-viral activity was discovered, incompatible with immunosuppression. These two activities originate in the presence of two receptors, i.e. cyclophiline A, which interferes in two processes and calcineurine, regulating immunosuppression. Consequently, the synthesis of modified analogues at different positions represents a research domain of high importance for elucidating structure-activity relationships of these receptors. Position 2 of cyclosporin is in close contact to cyclophiline and, consequently, of particular interest. cyclosporin C (CsC) differs from the well-known immunosuppressive analogue cyclosporin A (CsA) by the presence of a trifunctional amino acid at position 2, i.e. a threonine residue replacing 2-amino butyric acid (Abu2) in CsA. While the biological and pharmacokinetic properties of CsC and CsA are very similar, the presence of the threonine makes CsC a most attractive candidate for structure-activity relationship studies in applying the pseudo-proline concept (ΨPro) developed in the group of Prof. M. Mutter. The advantage of this concept relies in the direct incorporation of the pseudoproline moiety into CsC by cyclocondensation of ketals with yields reaching up to 60%, depending on the substituents R1 et R2 (figure 1). Figure 1: Direct insertion of the pseudo-proline system into CsC NMR analysis showed that monosubstitutions at C2 strongly effect the conformational properties. For example, unique conformations were found in weakly polar (CDCl3) and polar solvents (DMSO), depending on R1/R2. In contrast, the simultaneous presence of R1 and R2 result in intermediate effects. The drastic conformational impact on the Cs backbone by the invertion of ΨPro-systems is manifested by the loss of biological activity. Based on these results, the ΨPro concept was applied for the synthesis of so-called soft prodrugs of CsC. As prototype, a derivative containing a phosphate group (R1 = 3,5-Dimethoxy-4-phosphonooxyphenyle; R2 = H) improved the water solubility of cyclosporin by a factor higher than 500. The developed strategies can be applied to any peptide featuring a threonine or serine in its primary sequence, paving the way to a new class of prodrugs. With the goal to obtain analogues of higher receptor selectivity, the second part of the thesis focuses on the modification of position 8 of cyclosporin. Most notably, located in the effector domain of calcineurin, modifications of residue D-Ala8 should prevent the binding of the dimeric complex Cs-cyclophiline A to calcineurin, a prerequisite for antiviral and non-immunosuppressive activity. Based on experiments on the alkylation of CsA at D-NAla8, a selective ring-opening procedure of CsA between residues Ala7 and D-Ala8 was elaborated in three major steps: Selective alkylation of the nitrogen of D-Ala8 in 43% yield (5 steps), N —> O acyl transfer of Ala7 (>70%) and a solvolysis followed by Edman degradation to the linear decapeptide in 24.5% yield. Starting from CsA, the linear decapeptide was obtained on 10.5% overall yield for 10 steps (figure 2). Figure 2: Retrosynthesis of the 7-8 ring-opening of CsA While the reported ring opening procedures allow the study of the influence of modifications on positions 11 to 5 of Cs, the elaborated strategy gives access to the efficient modification of key position 8, opening interesting perspectives for structureactivity relationship studies of high therapeutical potential.

Details

Actions