Synthèse, structure et propriétés physicochimiques des cyclosporines

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.

Cyclosporin analogues

Arnaud Hamel

Cyclosporins are a family of hydrophobic cyclic undecapeptides produced by the fungus Tolypocladium niveum. The main metabolite cyclosporin A (CsA, see Scheme, R = R' = H) is the first line drug currently used to prevent rejection of organ transplants. Its strong immunosuppressive activity is achieved through the formation of a cyclosporin–cyclophilin A (CypA) complex inhibiting the calcium- and calmoduline-dependent protein phosphatase calcineurin (Cn). This trimeric complex prevents translocation of the transcription enhancer NFAT into the nucleus, inhibiting T-cell activation. It was also shown that, in the presence of CsA, chronically infected cells produce non-infectious HIV particles. By binding with the HIV capsid gag polyprotein pr55, CypA is recruited in stoichiometric amounts in the nascent HIV virion. The formation of the CsA-CypA complex disrupts this interaction at the maturation stage of the HIV infection and blocks the replication of HIV in cells. Based on SAR studies, the present thesis aims to develop novel analogues and prodrugs of CsA exhibiting antiviral, non-immunosuppressive activities. The first part of this work describes the regioselective synthesis and the pharmacological properties of a novel series of dimers of cyclosporin analogs modified at position 8 (see Scheme, in blue). Our findings demonstrate that the dimerization at position 8 of cyclosporin derivatives strongly increase their affinity to CypA and drastically alter their binding to Cn, thus representing the first derivatives of cyclosporin exhibiting non-immunosuppressive (loss of Cn binding), anti-viral (CypA binding) activities by the exclusive modification at position 8. By designing linkers of tailored length to permit the cooperative interaction with both immunophilin receptors, bivalent ligands have been developed. As indicated by receptor binding studies, these dimers represent the most potent non-immunosuppressive cyclophilin binders reported up to now, potential anti-HIV-1 inhibitors. The goal of the second part of this thesis was to use various concepts for prodrug design for modulating the pharmacokinetic properties and for increasing the low water-solubility of cyclosporin analogs that is responsible for undesirable pharmaceutical properties of cyclosporins such as erratic oral absorption profile, poor oral bioavailability and complications in formulating. A prodrug is a pharmacologically inactive derivative of a parent drug molecule that requires spontaneous or enzymatic transformation within the body in order to release the active drug, exhibiting improved delivery properties compared to the parent drug molecule. The elaborated chemical modifications of the cyclosporin derivatives allowed us (1) to mask the bioactive conformation of the cyclosporine, leading to the loss of binding to CypA and Cn, and consequently in a loss of the immunosuppressive activity and (2) to increase the solubility of the cyclosporins through the introduction of a solubilizing group (SG). As a first chemical modification, the hydroxyl group of the residue MeBmt-1 of cyclosporins was masked by a chemical unit that can be selectively removed enzymatically (Scheme, in green). The double prodrugs showed an increase in water-solubility of factors up to 1000 compared to their parent drug and a specific enzymatic controlled drug release. Alternatively, a novel strategy for the selective introduction of an acyl-function at different positions of hydroxyl-containing cyclosporins (Scheme, blue and red) was explored. The corresponding O-acyl isopeptides undergo spontaneous O,N-acyl migration (serving as chemical switch), resulting in the recovery of the parent compound under physiologically conditions (pH controlled drug release). The investigated derivatives showed high thermodynamic stability, differential conversion rates and strongly increased water solubility, offering novel chemoreversible prodrugs of cyclosporin analogs. In extending this concept, O-acyl iso-cyclosporins stabilized by a pro-moiety were developed, setting the stage for a novel class of double prodrugs exhibiting improved thermodynamic stability and tailored chemical and enzymatic conversion rates. With the increasing challenges in drug delivery of complex therapeutic agents, the elaborated systems provide versatile, generally applicable strategies for hydroxyl group containing drug candidates.

EPFL2005

Cyclosporin analogues

Arnaud Hamel

EPFL2005