Serotonergic neurotoxic thioether metabolites of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"): synthesis, isolation, and characterization of diastereoisomers

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a synthetic recreational drug of abuse that produces long-term toxicity associated with the degeneration of serotonergic nerve terminals. In various animal models, direct administration of MDMA into the brain fails to reproduce the serotonergic neurotoxicity, implying a requirement for the systemic metabolism and bioactivation of MDMA. Catechol-thioether metabolites of MDMA, formed via oxidation of 3,4-dihydroxymethamphetamine and 3,4-dihydroxyamphetamine (HHMA and HHA) and subsequent conjugation with glutathione (GSH), are selective serotonergic neurotoxicants when administered directly into brain. Moreover, following systemic administration of MDMA, the thioether adducts are present in rat brain dialysate. MDMA contains a stereogenic center and is consumed as a racemate. Interestingly, different pharmacological properties have been attributed to the two enantiomers, (S)-MDMA being the most active in the central nervous system and responsible for the entactogenic effects, and most likely also for the neurodegeneration. The present study focused on the synthesis and stereochemical analysis of the neurotoxic MDMA thioether metabolites, 5-(glutathion-S-yl)-HHMA, 5-(N-acetylcystein-S-yl)-HHMA, 2,5-bis-(glutathion-S-yl)-HHMA, and 2,5-bis-(N-acetylcystein-S-yl)-HHMA. Both enzymatic and electrochemical syntheses were explored, and methodologies for analytical and semipreparative diastereoisomeric separation of MDMA thioether conjugates by HPLC-CEAS and HPLC-UV, respectively, were developed. Synthesis, diastereoisomeric separation, and unequivocal identification of the thioether conjugates of MDMA provide the chemical tools necessary for appropriate toxicological and metabolic studies on MDMA metabolites contributing to its neurotoxicity.This work was supported by a post-doctoral grant financed by the Secretaria de Estado de Universidades e Investigación del Ministerio de Educación y Ciencia, Spain; NIDA Grant 5R01BA017987-01; Ministerio de Educación y Ciencia (Spain) SAF2005-0189; and Generalitat de Catalunya (Spain) 2005SGR00032. This work was supported by a post-doctoral grant financed by the Secretaria de Estado de Universidades e Investigación del Ministerio de Educación y Ciencia, Spain; NIDA Grant 5R01BA017987-01; Ministerio de Educación y Ciencia (Spain) SAF2005-0189; and Generalitat de Catalunya (Spain) 2005SGR00032. The authors acknowledge assistance from the NIEHS supported Southwest Environmental Health Sciences Center (SWEHSC) Grant P30 ES06694, at the University of Arizona. In particular, we thank Drs. M. Ahad Ali and Eugene A. Mash (SWEHSC Synthetic Chemistry Facility Core) for synthesizing HHMA·HBr; Yelena Feinstein (SWEHSC Proteomics Facility Core) for the HPLC-MS and HPLC-MS/MS analyses; and Dr. Neil Jacobsen (NMR facility, Department of Chemistry, University of Arizona) for the NMR analyse