P85: Synthesis and anti-cancer effects of CpdA enantiomers, non-steroidal glucocorticoid receptor ligands

Abstract

Glucocorticoids (GCs) are the most important component of therapy for a number of diseases including combined chemotherapy of hematological malignancies. However, their application is strongly restricted by the development of serious side effects as well as glucocorticoid resistance. Both side effects and resistance affect child cancer patients more intensively. Adverse side-effects of glucocorticoid treatment are the result of glucocorticoid receptor (GR)-mediated gene activation, while the beneficial anti-inflammatory effects result from GR-mediated ‘transrepression’. Difference in mechanisms of therapeutic and side effects of GCs became obvious and selective glucocorticoid agonists (SEGRA) were developed aiming to separate transactivation from transrepression. Recently we have demonstrated that one of the modern SEGRAs, 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride or CpdA, selectively induces transrepression in lymphoma cells and reveals GR-dependent anti-lymphoma activity in vitro and in vivo. However, CpdA is a chiral molecule and exists as a racemic mixture of two enantiomers. Enantiomers often possess the same physical and chemical properties but their biological effects may differ drastically. They differently interact with cell receptors and it can lead to the essential diversity in pharmacokinetics and pharmacodynamics. The notorious example of such a molecule is Thalidomide which was originally designed and used as a sedative drug during the pregnancy, but it was withdrawn chiefly because of its severe teratogenicity. As it was demonstrated, only (R)-thalidomide exhibited significant sedative effects, while (S)-thalidomide revealed the teratogenic effects. The application of enantiopure compounds may lead to reduction of metabolism variability in patients and to decrease of drug effective dose. Thereby, the synthesis of enantiopure isomers of CpdA and the study of their anti-cancer activity are of immediate interest in cancer research. We synthesized for the first time enantiomers of the CpdA based on literature precedent Sharpless asymmetric dihydroxylation with AD-mix-alpha or AD-mix-beta. (S)- and (R)-enantiomers with enantiopure excess of 98% were obtained. Then we evaluated the cytotoxic effects on acute lymphoblastic leukemia cells CEM and mantle cell lymphoma cells Granta by direct cell counting and found that cytotoxic activities of both enantiomers were comparable with the effect of racemic mixture on cell growth and survival. Glucocorticoids modulate the expression of genes through transrepression and transactivation mechanisms realized in equal measure. In present work we estimated the potential ability of newly synthesized enantiomers to activate these mechanisms. Transactivation realizes through direct binding of receptor homodimers to specific sequences (glucocorticoid responsive elements (GREs)) in promoter or enhancer regions of GR target genes. Therefore, we studied the level of transactivation as the expression of GR-regulated genes, immunophiline FKBP51 and glucocorticoid-induced leucine zipper GILZ. As shown by Q-RT-PCR, the expression of FKBP51 and GILZ was unaffected after cell treatment by both CpdA enantiomers; hence, these compounds did not induce GR transactivation. GR-transrepression is chiefly mediated by protein–protein interaction between GR and other transcription factors like NF-kB and AP1, followed by inhibition of their transcriptional activity. We evaluated the transrepression by expression of NF-kB-depended genes, cyclines D1 and D2 (CCND and CCND2). (S)-enantiomer of CpdA down-regulated the expression of CCND1 and CCND2 to the level compared with Dex while (R)-enantiomer surprisingly increased level of CCND1 and CCND2 expression. This fact demonstrated that (S)-CpdA is perspective as antiproliferative and cell growth inhibiting agent. Overall, our data provide the potential for further studies of CpdA optical isomers, especially (S)-enantiomer, as GR-dependent anti-cancer agents.