Synthesis and Cyclic Voltammetry Studies of 3,4-Methylenedioxymethamphetamine (MDMA) Human Metabolites
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Journal of Health Science, 53(1) pp 31–42 (2007) 31 Synthesis and Cyclic Voltammetry Studies of 3,4-Methylenedioxymethamphetamine (MDMA) Human Metabolites Carla Macedo,a Paula Serio´ Branco,∗,a Lu´ısa Maria Ferreira,a Ana Maria Lobo,a Joao˜ Paulo Capela,b Eduarda Fernandes,c Maria de Lourdes Bastos,b and Felix Carvalhob aREQUIMTE/CQFB, Departamento de Qu´ımica, FCT, Universidade Nova de Lisboa, 2829–516 Caparica, Portugal, bREQUIMTE, Departamento de Toxicologia, and cREQUIMTE, Departamento de Qu´ımica-F´ısica, Faculdade de Farm´acia da Universidade do Porto, 4099–1030, Porto, Portugal (Received July 6, 2006; Accepted October 27, 2006) 3,4-Methylenedioxymethamphetamine (MDMA or “Ecstasy”) is a widely abused, psychoactive recreational drug. There are growing evidences that the MDMA neurotoxic profile may be highly dependent on its hepatic metabolism. MDMA metabolism leads to the production ofhighly reactive derivates, namely catechols, cate- chol thioethers, and quinones. In this study the electrochemical oxidation-reduction processes of MDMA human metabolites, obtained by chemical synthesis, were evaluated by cyclic voltammetry based on an electrochem- ical cell with a glassy carbon working electrode. The toxicity of α-methyldopamine (α-MeDA), N-methyl-α- methyldopamine (N-Me-α-MeDA) and 5-(glutathion-S-yl)-α-methyldopamine [5-(GSH)-α-MeDA] to rat cortical neurons was then correlated with their redox potential. The obtained datademonstratedthatthe lower oxidation potential observed for the catecholic thioether of α-MeDA correlatedwith the higher toxicity of this adduct. This accounts for the use of voltammetry data in predicting the toxicity of MDMA metabolites. Key words —— 3,4-Methylenedioxymethamphetamine, Neurotoxicity, Cyclic voltammetry, Metabolites, Rat cortical neurons INTRODUCTION in laboratory animals and humans have been demonstrating that the exposure to MDMA may Theresearch in the area of drug abuse is mainly elicit long-term changes in the neurochemistry and focused in its direct or indirect effects on receptors behavior. These changes result from the selective and transmitters1) and less attention has been given neurotoxicity to the 5-hydroxytryptamine (5-HT) to the putative chemical reactivity of their metabolic containing axon terminals.4) products. However, there are overwhelming evi- Systemic metabolism seems important for the dences indicating that metabolites are often crucial neurotoxic response since, in some animal stud- participants in the action of drugs and toxins. ies, direct injection of MDA or MDMA into the The ring-substituted amphetamines, 3,4- brain did not cause long-term 5-HT deficits.5–7) The Methylenedioxymethamphetamine (MDMA), 1 metabolism of MDMA and MDA starts with O- and methylenedioxyam-phetamine (MDA), 2 are demethylenation to N-methyl-α-methyldopamine , well established serotonergic neurotoxicants.2 3) (N-Me-α-MeDA), 3 and α-methyldopamine (α- MDMA, also known as “Ecstasy,” is a widely MeDA), 4 respectively, both of which are cate- abused psychoactive recreational drug associated cholamines that can undergo oxidation to the cor- with acute and long-term toxic effects. MDA, a responding ortho-quinones 5 and 6 (for detailed in- well characterized metabolite of MDMA, has also sights on MDMA metabolism see Fig. 1).8, 9) These been used as a drug of abuse. Various studies quinones are highly active redox molecules, which ∗ can enter redox cycles with their semiquinone radi- To whom correspondence should be addressed: REQUIMTE/ cals, leading to formation of reactive oxygen species CQFB, Departamento de Qu´ımica, FCT, Universidade Nova de (ROS) and reactive nitrogen species (RNS).10, 11) Lisboa, 2829–516 Caparica, Portugal. Tel.: +351-212948300; Thetoxicity of these metabolites has been corre- Fax: +351-212948550; E-mail: [email protected] 32 Vo l. 5 3 (2007) Fig. 1. Proposed Pathway for MDMA Metabolism Leading to Reactive Intermediates Capable of Alkylating Cellular Components lated with electron transfer (ET), ROS formation MeDA the most toxic. We have also previously and consequent oxidative stress (OS).12, 13) Acyclic demonstrated that the metabolism is important for voltammetry study on the oxidation-reduction be- MDMA-induced toxicity to peripheral organs.18–20) havior of bioactive catecholamines14, 15) showed the The underlying mechanisms of MDMA and straightforward formation of the corresponding o- MDA-induced neurotoxicity, namely the toxic ef- quinones via electro-oxidation (a two electron trans- fects mediated by its metabolite(s), remain to be fer process). These o-quinones can readily undergo completely elucidated. Herein we present cyclic reduction, indicating the potential for ET occur- voltammetry studies that provide evidence for a cor- rence in vivo.Alternatively, the reactive o-quinone relation between the toxicity of MDMA and MDA- intermediates are Michael acceptors, and cellular metabolites and their redox potential. damage can occur through alkylation of crucial cel- lular proteins and/or DNA. In the presence of other bionucleophiles, as GSH, o-quinones may conju- MATERIALS AND METHODS gate to form adducts. These conjugates remain re- dox active being in turn readily oxidized to the cor- Reagents and Apparatuses —— N-Acetylcysteine responding quinone-adducts, which are susceptible (NAC), GSH, mushroom tyrosinase (4400 to an addition of a second nucleophile molecule.16) units/mg) and other chemicals were obtained Our group haspreviouslyevaluated the toxi- from Sigma-Aldrich R (Steinheim, Germany). city of MDMA and three of its metabolites, ob- MDMA (HCl salt) was extracted and purified from tained by synthesis, namely N-Me-α-MeDA, α- high purity MDMA tablets that were provided by MeDA and 5-(glutathion-S-yl)-α-methyldopamine the Portuguese’s Criminal Police Department. The [5-(GSH)-α-MeDA], 7,inratcortical neuronal obtained salt was pure and fully characterized by ◦ serum free cultures under normal (36.5 C) and NMRand mass spectrometry methodologies. ◦ hyperthermic (40 C) conditions.17) It was shown Solvents were dried by standard methods21) and that these metabolites are more neurotoxic than distilled before use. Analytical thin-layer chro- the parent compound MDMA, being 5-(GSH)-α- matography was conducted on Merck (Darmstadt, No. 1 33 Germany) Kieselgel 60, F254 silica gel 0.2 mm HC). thick plates; column chromatography was per- 1-(3,4,5-Trimethoxyphenyl)-2-aminopro- formed on Merck Kieselgel 60 (240–400 nm) silica pane hydrochloride. To a solution of LiAlH4 gel or reverse-phase RP-18 modified silica. Melt- (0.4 g, 0.01 mol) in dry tetrahydrofuran (THF, ing points were recorded on a Reichert-Thermovar 10 ml) was added dropwise a solution of trans- hot stage apparatus and are reported uncorrected. 1-(3,4,5-trimethoxyphenyl)-2-nitropropene (1 g, InfRaRed (IR) spectra were recorded on a Perkin 3.97 × 10−3 mol) in THF (10 ml). The solution Elmer(Beaconsfield, UK) Spectrum 1000 as potas- was refluxed for 3 hr after which the excess of sium bromide (KBr) pellets or as film over sodium LiAlH4 was destroyed by slow addition of 2 ml of chloride (NaCl) windows. UV/vis spectra were water followed by 2 ml of aqueous 15% NaOH and recorded between 200 and 500 nm on a Thermo finally 5 ml of water. The mixture was filtered and Electron Helios γ.Proton and carbon nuclear mag- the residue washed with THF. The solution was 1 13 netic resonance spectra ( H- and C-NMR) were dried with anhydrous Na2SO4 and evaporated to recorded on a Bruker (Wissembourg, France) ARX dryness. The yellow oil was dissolved in hexane 400 spectrometer at 400 and 100.62 MHz respec- and the compound precipitated by freezing. The tively. Chemical shifts are expressed in ppm, down- product was obtained as its hydrochloride salt by field from trimethylsilyl (TMS, δ = 0) as an in- precipitation of the salt by slow addition of an HCl ternal standard; J-values are given in Hz. The ex- dry ethyl ether solution: mp 221–223◦C (lit.25) ◦ −1 act attribution of NMR signals was preformed using 216–217 C), IV (KBr, νmax cm ): 3400, 2938, two dimensional NMR experiments. Low resolu- 1587, 1514, 1321, 1246, 1136, 1092; 1H-NMR tion mass spectra were acquired with a Micromass (CDCl3, δ): 1.14 (3H, d, J = 6.2, CH3), 2.42 (1H, (Manchester, UK) GC-TOF Gas Chromatogra-phy dd, J = 13.0and8.4, CH2), 2.68 (1H, dd, J = 13.0 Time-of-fly (GCT) mass spectrometer. HPLC was and 4.8, CH2), 3.19–3.14 (1H, m, CH), 3.83 (3H, s, conducted on a Merck Hitachi (Tokyo, Japan) sys- OCH3), 3.85 (6H, s, OCH3), 6.41 (2H, s, ArH). tem consisting of an L-7100 pump, a Rheodyne 5-OH-α-MeDA (13). Asolution of 1-(3,4,5- type injector, a D-7000 interface, and an L7450A trimethoxyphenyl)-2-aminopropane hydrochloride diode array spectrometric detector. Cyclic voltam- (0.1 g, 3.82×10−4 mol) in 10 ml of 48% HBr was de- mograms were run at room temperature on an Auto- gassed for one hour, warmed to 125◦Cfor3hrand lab (Utrecht, Netherlands) PGSTAT12 potentiostat finally stirred 1 hr at room temperature. The prod- connected to a conventional three-electrode cell- uct purification was performed by reverse-phase assembly (saturated calomel electrode (SCE) as ref- RP-18 modified silica column chromatography with erence, Pt wire as counter electrode and glassy car- water. The solution was concentrated to dryness. bon as working electrode). The air sensitive 1-(3,4,5-trihydroxiphenyl)-2- aminopropane hydrobromide was obtained as a or- 1 Chemical Synthesis ange oil (0.06 g) in 60% yield. H-NMR (D2O, δ): Hydrobromide salts of N-methyl-α-methy- 1.16 (3H, d, J = 6.5, CH3), 2.58 (1H, dd, J = 13.9 ldopamine (3) and α-methyldopamine (4). N-Me- and 7.7, CH2), 2.66 (1H, dd, J = 13.9and6.7, α-MeDA.HBr and α-MeDA.HBr were prepared fol- CH2), 3.45–3.39 (1H, m, CH), 6.29 (2H, s, ArH); 22) 23) 13 lowing the procedures of Borgman and Pizarro C-RMN (D2O, δ): 17.6 (CH3), 39.6 (CH2), 49.1 starting from the corresponding benzaldehyde and (CH), 109.6 (CAr2+6 ), 128.5 (CAr1+3/5 ), 131.3 nitroethane.