Chem. Res. Toxicol. 1992,5,89-94 an aid to drug development. B o g . Drug Res. 31,427-479. (17) Dixit, R.,Seth, P. K., and Mukhtar, H. (1982) Mercapturates from acrylamide. Drug Metab. Dispos. 10,196-197. (18) Petersen, D. W.,and Lech, J. J. (1987) Hepatic effects of acrylamide in rainbow trout. Toxicol. Appl. Phormacol. 89, 249-255.
89
(19) Hashimoto, K.,and Tanij, H. (1985) Mutagenicity of acrylamide and ita analogues in Salmonella typhimurium. Mutat. Res. 158,129-133. (20) Barfknecht, T. R.,Mecca, D. J., and Naismith, R. W. (1988) The genotoxic activity of acrylamide. Enuiron. Mol. Mutagen. 11, Suppl. 11.
Synthesis and Neurotoxicological Evaluation of Putative Metabolites of the Serotonergic Neurotoxin 2-( Methylamino)-1-[3,4-( methylenedioxy)phenyllpropane [(Methylenedioxy)methamphetamine] Zhiyang Zhao and Neal Castagnoli, Jr.* Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061
George A. Ricaurte, Thomas Steele, and Mary Martello Department of Neurology, Johns Hopkins University School of Medicine, Francis Scott K e y Medical Center, Baltimore, Maryland 21224 Received August 9,1991 Theoretical considerations and recent experimental data have prompted an investigation of the neurotoxicologikal prooerties of the 6-hydroxydopamine analogue 2-(methylamino)-1(2,4,5trihydroxyphenyl)propane( 5 ) and its possible precursor 1-[2-hydroxy-4,5-(methylenedioxy)phenyl]-2-(methylamino)propane(4), potential metabolites of the serotonergic neurotoxin MDMA. Systemic, intracerebroventricular,and intraparenchymal (intrastriatal and intracortical) administration of 4 led to no detectable alterations of hippocampal or cortical serotonin or striatal dopamine levels in the rat under conditions that caused significant biogenic amine depletions by established neurotoxins. By contrast, intraparenchymal administration of 5 caused profound depletions of dopamine and serotonin, with the former being more severely depleted than the latter. Although not conclusive, these data suggest a possible role for 5 in the mediation of MDMA’s neurotoxic actions.
Introduction The methamphetamine derivative 2-(methylamino)-1[3,4-(methy1enedioxy)phenyllpropane [(methylenedioxy)methamphetamine, MDMAl (1);see Chart I], a commonly abused substance (l),has been promoted by some practitioners as a useful adjuvant psychotherapeutic agent (2). These human exposures are of some concern since studies in rats (3-5)and primates (6)have documented that MDMA is a neurotoxin with selectivity for serotonergic neurons. The molecular events responsible for the neurotoxic effects of MDMA and structurally related amphetaminetype neurotoxins remain unknown (7). Serotonin (5-HT) uptake inhibitors protect against the neurotoxicity of MDMA (8). However, since MDMA itself does not appear to be a substrate of the 5-HT transporter (9),it has been postulated that one or more metabolites of MDMA may mediate the observed neurotoxic effects of the parent drug (10).Consistent with this metabolic theory is the report Abbreviations: MDMA, (methy1enedioxy)methamphetamine [2(methylamino)-l-[3,4-(methylenedioxy)phenyl]propane];5-HT, 5hydroxytryptamine (serotonin); 6-OHDA, 6-hydroxydopamine [ 2-(2,4,5trihydroxypheny1)ethylaminel;DA, dopamine; ppm, parts per million; TMS, tetramethylsilane; HP, Hewlett Packard; DIPEI, direct insertion probe electron ionization; IR, infrared; UV, ultraviolet; TLC, thin-layer chromatography; THF, tetrahydrofuran; GC/EIMS, gas chromatography/electron ionization mass spectra; ip, intraperitoneal; 5,7-DHT, 5,7dihydroxytryptamine; EC, electrochemical;DIPCI, direct insertion probe chemical ionization; EDTA, ethylenediaminetetraaceticacid.
that intraventricular administration of MDMA does not lead to alterations in brain 5-HT levels (11). These considerations have led to a series of metabolic studies in rats (12)and humans (13)designed to evaluate the possible conversion of MDMA to neurotoxic metabolites. Of particular interest is the ability of rat liver and brain enzymes to catalyze the oxidative cleavage of the 3,4-(methylenedioxy)group present in MDMA to generate the corresponding catechol 2 (12).This dopamine analogue is reported to be oxidized rapidly to the corresponding electrophilic o-quinone (3), a potential neurotoxic alkylating agent (14). An alternative bioactivation pathway would involve initial C-2 oxidation of the aromatic ring, a type of biotransformation known to be catalyzed by liver (15)and brain (16)cytochrome P-450 monooxygenases, to yield the species 4. corresponding 2-hydroxy-4,5-(methylenedioxy) Subsequent oxidative cleavage of the methylenedioxy group in the brain would generate the 2,4,5-trihydroxy compound 5, a close structural analogue of the potent neurotoxin “&hydroxydopamine”(6-OHDA,6) (17). Since the a-methyl analogue 7 of 6-OHDA (18)and the structurally related hydroquinone 8 (19) display neurotoxic propertiea similar to those of 6-OHDA, the a-methyl group present in 6 is unlikely to affect the toxic potential of this system. The neurotoxic properties of the corresponding N-methylated analogue of 6-OHDA have not been reported. In an effort to evaluate the possible toxicological significance of this putative metabolic sequence, we have
0893-228~/92/2705-0089$03.00/00 1992 American Chemical Society
Zhao et al.
90 Chem. Res. Toxicol., Vol. 5, No. 1, 1992 Chart I NHCH3 m 1: R = H 4: R = O H
2: R = H 5: R = O H
synthesized compounds 4 and 5 as racemic mixtures and have estimated their neurotoxic potentials in the r a t by measuring their 5-HT and dopamine (DA) depleting properties following systemic (compound 4) a n d intracerebral (compounds 4 and 5) administration.
Experimental Section Chemistry. Commercial chemicals and solvents were purchased from the Aldrich Chemical Co., Milwaukee, WI, and Fisher Scientific Products, Pittsburgh, PA. Melting points were determined on a Thomas-Hoover melting point apparatus and are uncorrected. 'H and 13CNMR spectra were taken on a Bruker WP27OSY 270-MHz spectrometer. Chemical shifts (6) are reported in parts per million (ppm) relative to TMS as internal standard, and spin multiplicities are given as s (singlet), d (doublet), t (triplet), q (quartet), or m (multiplet). Gas chromatography/electron ionization mass spectra (GC/EIMS) were obtained with a temperature-programmedHewlett-Packard (HP) capillary column (HP-1,12 m x 0.2 mm) on an HP 5890 GC linked to an H P 5970B quadrupole mass spectrometer. The oven temperature was held at 125 "C for 1 min after injection and then was temperature-programmed to 275 "C at 25 "C/min. Direct insertion probe electron ionization (DIPEI) and CI (chemical ionization) mass spectra were obtained on a VG 7070E-HF mass spectrometer. Infrared (IR)spectra were performed on a Perkin Elmer 710 B IR spectrophotometer, and ultraviolet (UV) spectra were recorded on a Beckman DU 50 UV spectrophotometer. Analytical thin-layer chromatography (TLC) was carried out on 0%" silica gel 60 FZMplates. Flash chromatography was performed by using silica gel 60 (230-400 mesh), and column shes were varied on the basis of amount of sample.
(A)2-Hydroxy-4,5-(methylenedioxy)benzaldehyde(10)and Tris[2-hydroxy-3,4-(methylenedioxy)phenyl]methane( 11). A mixture of phosphorous oxychloride (16.45 g, 10 mL, 0.11 mol) and N-methylformanilide (14.24 g, 13 mL, 0.11 mol) was stirred for 90 min at room temperature under Nz to give the solid formamidinium chloride. To a suspension of this solid in CHzClz (50 mL) was added dropwise over a 30-min period 3,4-(methy1enedioxy)phenol (9, 10 g, 0.07 mol) in 40 mL of CHzClz (20). Following an additional 2 h the reaction mixture was extracted with ice-water (3 X 25 mL) and then with 10% NaOH. The combined aqueous extracts were acidified to pH 2 with concentrated HCl, and the resulting solution was extracted with diethyl ether (4 X 40 mL). The combined diethyl ether extracts with dried over MgS04, filtered, and concentrated to give a dark yellow residue which upon treatment with 40 mL of CHzClzyielded an off-white solid (6.0 g, see below) and a yellow solution. After filtering off the solid, the desired benzaldehyde derivative 10 (3.19 g, 27.4%)was isolated from the CHzCl2fdtrate by silica gel column chromatography with CHzC12as eluent. The pure product was obtained as yellow crystals: mp 122-124 "C; UV (MeOH) ,A, 348 (82001,278 (7100), and 242 nm (17000); IR (KBr) 1660,1500, and 240 cm-'; 'H NMR (CDC13)6 11.78 (s, 1 H, CHO), 9.59 (s, 1 H, OH, exchanges with DzO), 6.84 (8, 1 H, ArH-6), 6.44 (s, 1 H, ArH-3), 5.99 (8, 2 H, OCHzO); 13C NMR (DMSO-&) 6 192 (carbonyl carbon), 160 (C-2), 155 (C-4), 142 (C-5), 116 (C-l), 107 (C-6), 103 (C-3), 98 (CH,); GC/EIMS, retention time = 4.26 min [m/z (assignment, %)I, 166 ( M + ,loo), 165 (96),148 (6), 137 (12), 120 (lo), 107 (15), 79 (151, 53 (25). Anal. Calcd for C8HS04:C, 57.84; H, 3.64. Found: C, 57.74; H, 3.69. (B)Compounds 12 and 13. The off-white solid which separated when the crude isolate was treated with CHZCl2was recrystallized from acetonewater to give 3.1 g (31%)of the trimer 11: mp 195-197 "C dec; 'H NMR (DMSO-d,) 6 8.90 (s, 1 H, OH, exchanges with DzO), 6.39 (s, 3 H, ArH-6), 6.14 (s, 3 H, ArH-3),
H
3
3
6: R 1 = H, Rp = OH 7: R 1 = CH3, R2 = OH 8: R 1 = R * = C k $
5.94 ( s , l H, CH), and 5.86 ( ~ , H, 6 OCHZO); 13CN M R (DMSO-de) 6 149.0 (ArC-2),146.5 (ArC-4),139.5 (ArC-5),123.0 ( M - 1 1 , 108.5 ( M - 6 ) , 101.5 ( M - 3 ) , 98 (CH,), 37.5 (CHI; DIPEI mass spedrum [m/z (assignment, %)] 424 (M'*, l),406 (M' - 18,3), 269 (70), C 61.40, H 4.00. 138 (100). Anal. Calcd for C22H160~-1/3H20: Found: C 61.57, H 3.71. The trimer 11 (20 mg, 0.047 "01) was treated with chlorotrimethylsilane (0.11 mL, 0.89 mmol) and triethylamine (0.1 mL, 0.74 mmol) in 15 mL of dry THF under N2 (21). The resulting cloudy reaction mixture was allowed to stir at room temperature overnight. GC/EI mass spectral analyak of the reaction mixture displayed a single peak (10.56 min), and a mass spectrum corresponded to tris[2-[(trimethylsily1)oxy]-4,5-(methylenedioxy)phenyl]methane (12): [m/z (assignment, %)] 640 ( M + , 95), 625 (M" - CH3, lo), 551 (M'+OSiMe3,20), 521 (20), 431 (40), 357 (15), 341 (lo), 269 (15), 233 (15), 73 (100). The preparation of tris[3,4-(methylenedioxy)-2-methoxyphenyl]methane(13)was achieved by an overnight reaction between the trimer 11 (43 mg, 0.1 mmol) in 15 mL of CH30H and diazomethane (1.52 mmol) in 4 mL of ether (22). The excess diazomethane was destroyed by adding a few drop of acetic acid, and the solvent was evaporated under vacuum. A diethyl ether solution of the residue was washed with aqueous NaHC03 followed by water, dried over anhydrous MgS04, and evaporated to dryness to yield 30 mg (70%) of an off-white solid mp 202-204 "C dec; GC/EIMS, retention time 11.19 min [m/z (assignment, %)I, 466 (M+,loo),435 ( M + - OCH3, 851,405 (15), 315 (E),269 (lo), 165 (50), 135 (30); 'H NMR (CDC13)6 6.51 (8, 3 H, ArH-6), 6.33 ( ~ , H, 3 ArH-3), 6.15 ( ~ ,H,l CH), 5.86 ( ~ , H, 6 OCHzO),3.65 ( s , 9 H, OCH3). Anal. Calcd for Cz5HzzO9.2H20: C, 59.76; H, 5.22. Found: C, 59.97; H 4.90.
(C)2-(Benzyloxy)-4,5-(methylenedioxy)benzaldehyde(14). A solution of 2-hydroxy-3,4-(methylenedioxy)benzaldehyde(10, 2 g, 2.05 mmol) in 25 mL of dry THF containing NaH (0.31 mg, 13 mmol), tetrabutylammonium iodide (90mg, 0.24 mmol), and (bromomethy1)benzene(6.15 g, 4.28 mL, 36 mmol) was stirred at room temperature under an atmosphere of Nz for 20 h (23). The solvent was removed, and a diethyl ether solution (50 mL) of the residue was washed with water, dried over MgS04, and concentrated to yield the crude product which was purified by silica gel column chromatography. Elution with hexane-dichloromethane (1:l) and CHzClzprovided 2.67 g (90%) of 14 as light yellow crystals: mp 94-95 "C; 'H NMR (CDC13)6 10.38 (s, 1 H, CHO), 7.45-7.32 (m, 5 H, C&), 7.28 (8, 1 H, ArH-6), 6.60 (s, 1 H, ArH-3), 6.00 (s, 2 H, OCHzO), 5.13 (s, 2 H, CHZPh); GC/EIMS, retention time = 7.89 min [m/z (assignment, %)I, 256 (M'+, 20), 227 (lo),164 (E),91 (loo),65 (15). Anal. Calcd for C15H1204: C, 70.31; H, 4.72. Found: C, 70.05; H, 4.76. (D)1- [2-(Benzyloxy)-4,5-(methylenedioxy) p henyll-2nitropropene (15). A solution of 14 (2.3 g, 8.98 mmol) and ammonium acetate hydrate (6.90 mg, 9.0 mmol) in 20 mL of nitroethane was heated under reflux for 9 h. The residue obtained after removing the nitroethane in vacuo was purified by silica gel column chromatography with CHC13 to yield the desired nitropropene 15 (2.11 g, 80%) as a yellow crystalline solid mp 90-92 "C; 'H NMR (CDC13)6 8.35 (8, 1 H, vinyl H), 7.45-7.32 (m, 5 H, CSH.~), 6.85 ( ~ H, , lArH-6), 6.62 (9, 1 H, ArH-3), 5.98 (s,OCH,O), 5.10 (s, CH2Ph),2.38 (s,3 H, CCH,); DIPEI mass spectrum [m/z (assignment, %)] 313 ( M + ,3), 176 (40), 91 (loo),65 (20). Anal. Calcd for CI7Hl5NO5:C, 65.17;H, 4.83; N, 4.47. Found C, 65.12; H, 4.84; N, 4.45. (E) l-[2-(Benzyloxy)-4,5-(methylenedioxy)phenyl]-2aminopropane Hydrochloride (16.HC1). A suspension of LiAlH4(971 mg,25.56 "01) and nitropropene 15 (2 g, 6.39 m o l ) in 30 mL of dry THF was stirred overnight under an atmosphere of NP. After decomposing the excess hydride with water, the reaction mixture was filtered with the aid of an additional 30 mL
Putative Metabolites of MDMA of THF. The filtrate was dried with KzCO3, filtered, and concentrated to yield 1.12 g (61.5%) of amine 16 as a yellow oil: 'H NMR (CDC1,) 6 7.45-7.32 (m, 5 H, CsH5),6.65 (8, 1H, ArH-6), 6.56 ( ~ ,H, 1 ArH-3), 5.90 (8,2 H, OCHZO), 5.02 ( ~ $H, 2 CHZPh), 3.20 (m, 1 H, CH), 2.62 (dd, 2 H, CHZ), 1.12 (d, 3 H, CH3); GC/EIMS, retention time = 6.37 min [ m / z (assignment, %)I, 285 (M'+, 3), 242 (62), 151 (42), 121 (lo), 91 (loo), 65 (20). The HCl salt was prepared in anhydrous ethereal HCl and recrystallized from methanol-ethyl ether (2:8) to yield 1.06 g (52%) of a light yellow crystalline product: mp 183-185 "C; 'H NMR (CDCl,) 6 7.45-7.32 (m, 5 H, C6H5),6.78 (8, 1 H, ArH-61, 6.58 (8, 1 H, ArH-3), 5.84 (8, 2 H, OCH,O), 5.04 (4, 2 H, CH2Ph), 3.62 (m, 1 H, CH), 2.98 (m, 2 H, CH2), 1.38 (d, 3 H, CH,). Anal. Calcd for C17H&J03*HCl:C, 63.51; H, 6.27; N, 4.36. Found C, 63.23; H, 6.30; N, 4.31. (F) 1- [2- (Benzyloxy)-4,5-(met hylenedioxy)phenyl] -2formamidopropane (17). The free base obtained from the above HC1 salt (2.5 g, 7.78 mmol) was heated under reflux and an atmosphere of Nz in 150 mL of ethyl formate for 6 h. The crude yellow product (2.45 g) obtained after removing the excess ethyl formate was dissolved in CH2C12,and the resulting solution was washed with saturated aqueous NaHC03 followed by 1N HC1. The residue isolated from the CHzClzlayer was purified by silica gel column chromatography with dichloromethane-ethyl acetate (1:l)as the eluent. The resulting solid was recrystallized from diethyl ether to yield 1.6 g (66%)of the analytically pure product: mp 96-97 "C;IR (KBr) 1658 cm-' (amide carbonyl); 'H NMR 6 7.90 and 7.72 (8 and d, respectively, 1 H, 2 CHO rotamers), 7.48-7.28 (m, 5 H, C$15), 6.62 (s, 1H, ArH-6),6.58 (8, 1H, ArH-3), 5.90 (s, 2 H, OCHZO), 5.00 (8, 2 H, PhCHZO), 3.75 and 4.18 (both m, 1H, NCHCH3rotamers), 2.68 (m, 2 H, CHCHJ, 1.14 and 1.17 (both d, 3 H, NCHCH, rotamers); GC/EIMS, retention time = 7.51 min [m/z (assignment, %)I, 313 (M', 40),268 (80),250 (15), 211 (25), 177 (40), 151 (50), 91 (loo), 65 (15). Anal. Calcd for ClBHia04: C, 69.08; H, 6.12; N, 4.48. Found C, 68.92; H, 6.16; N, 4.42. (G) 1-[2- (Benzyloxy)-4,5-(methylenedioxy) phenyl]-2(methy1amino)propane Hydrochloride (18.HCl). A stirred suspension of LiAlI& (1.5g, 38.34 "01) and the above formamide (2.0 g, 6.39 "01) in 70 mL of dry THF was heated under reflux and an atmosphere of N2 for 8 h. The excess hydride was decomposed by the careful addition of water, and the resulting mixture was filtered free of solids and dried over K2C03and the solvent removed to yield 1.4 g (73%) of a yellow crystalline solid. The HCl salt obtained from this free base with ethereal HCl was recrystallizedfrom acetonitrile: mp 144-146 "C; 'H NMR (CDCl,) 6 7.3-7.5 (m, 5 H, C6H5),6.7 (s, 1 H, ArH-6), 6.6 (s, 1 H, ArH-3), 5.95 (8, 2 H, OCH,O), 5.0 (s, 2 H, CH,Ph), 3.35 (m, 1H, CH), 3.35 and 2.70 (m's, 2 H, CHCH,), 2.48 (t, 3 H, NCHJ, 1.45 (d, 3 H, CCH,); GC/EIMS, retention time = 8.16 min [m/z (assignment, %)I, 299 ( M + , 2), 268 (2), 242 (40), 151 (lo), 91 (40), 58 (100). Anal. Calcd for ClJ-Iz1NO3.HC1:C, 64.34;H, 6.61; N, 4.18. Found C, 63.92; H, 6.62; N, 4.29. (H)1- [2-Hydroxy-4,5-(met hylenedioxy ) p hen yll-2- (methy1amino)propane Hydrochloride (4-HC1). Hydrogenolysis (23)of the above benzyl ether (800 mg, 2.39 mmol) in 100 mL of EtOH containing 40 mg of 10% Pd/C at room temperature under 1atm of Hz for 5 h gave 566 mg (96%)of an off-whitesolid which was crystallized from acetonitrile: mp 133-135 "C; 'H N M R (CDC13)6 6.68 (8, 1 H, A&), 6.45 (8, 1 H, ArH,), 5.85 (8, 2 H, OCH,O), 3.38 (m, 1 H, CH), 3.15 and 2.75 (dd's, 2 H, CHCH,), 2.65 (s,3 H, NCH,), 1.45 (d, 3 H, CH,); GC/EIMS, retention time = 3.75 min [m/z (assignment, %)I, 209 (M', E),58 (100). Anal. Calcd for C11H15N03-HC1:C, 53.81; H, 6.57; N, 5.71. Found: C, 53.88; H, 6.58; N, 5.75. (I) 1-(2,4,5-Trihydroxyphenyl)-2-(iV-methylamino)propane Hydrochloride (5.HC1). To a solution of 18-HC1(700 mg, 2.1 "01) in 25 mL of CHCl, cooled to 0 "C was added dropwise BBr3 1M solution in CH,Cl,) under N2 (24).The (10.5 mL, 10.5 "01, resulting cloudy solution was stirred overnight at room temperature under an atmosphere of NZ. After cooling, the reaction was quenched by the dropwise addition of MeOH. The brown, solid residue obtained after evaporation of the solvent was dissolved in a small volume of water and chromatographed on a cationic exchange column (15 mL of Dowex AG 50W-X4,50-100 mesh, washed with 30 mL of 6 N HCl followed by distilled water with
Chem. Res. Toxicol., Vol. 5, No. 1, 1992 91 neutral). The column was washed with water until the eluent was free of halide (silver nitrate test). The product was eluted with 6 N HC1, and the eluent was lyophilized to give 300 mg (61.5%) of an off-white crystalline solid mp 202-204 "C dec; 'H NMR (CD,OD) 6 6.49 (8, 1H, ArH-6), 6.33 (8, 1 H, ArH-3),3.34 (m, 1 H, CH), 2.83 (dd) and 2.63 (partially obscured dd) (2 H, CH2),2.60 (s,3 H, NCH,), 1.18 (d, 3 H, CCH,). Anal. Calcd for C10H15N03'HCk C, 51.43; H, 6.91; N, 5.99. Found: C, 51.29; H, 6.90; N, 5.97. Biological Studies. (A) Animals. Male albino SpragueDawley rata (Harlan, Indianapolis, IN) weighing 180-200 g were housed individually in wire mesh cages with free access to food and water. (B) Drug Administration. The neurotoxic effects of MDMA were compared with those of its "&hydroxy" analogue 4 following intraperitoneal (ip) administration (Table I). The neurotoxic effects of 4 (Tables I1 and III; 25)and 5 (Tables IV and V) were compared with those of the established neurotoxins 5,7-dihydroxytryptamine (5,7-DHT) and/or 6-OHDA following intracerebroventricular and intrastriatal administration. The neurotoxicity of compound 5 also was compared to that of 5,7-DHT following intracortical administration (Table VI). Intrastriatal and intracortical injections were performed as follows: Rata were anesthetized with chloral hydrate (300 mg/kg, ip) and a midsagittal incision was made to expose the skull. Small burr holes were made with a 26 guage needle 3 mm lateral to the bregma and approximately 0.5 mm posterior to the coronal suture. Compounds were injected over a 60-8 interval using a 10-pL Hamilton syringe equipped with cannulae of fixed lengths to protrude 0.2 and 0.6 mm below the surface of the skull for intracortical and intrastriatal injections, respectively. Test compounds were always administered into the right hemisphere; an equivalent volume of vehicle was delivered into either cerebral hemisphere (or striatum) of a different animal. Drugs were dissolved in a sterile saline solution containing 0.1% ascorbic acid to minimize oxidation. Volumes were 5 p L for intrastriatal and intracortical injections and 10 pL for intracerebroventricular injections. (C)Brain Dissection. One week after drug administration, rata were killed by decapitation and the striatum, hippocampus, and cerebral cortex were dissected free as described previously (26). Immediately after dissection, tissue parts were wrapped in aluminum foil and stored in liquid N2 until assayed. (D) HPLC-EC Determination of Brain 5-HT a n d DA. Concentrations of 5-HT and DA in brain tissue were determined by reverse-phase liquid chromatography coupled with electrochemical (EC) detection using the method of Kotake et al. (27) with minor modifications. Briefly, frozen tissue was weighed, placed in 10 parts of ice-cold 0.4% HC104,and then homogenized for 15 s using a Polytron homogenizer (setting = 5). The homogenate was centrifuged for 10 min at 15OOO rpm in a refrigerated Sorvall RC2B centrifuge. Fractions of the supernatant were transferred to polypropylene tubes which were then stored in liquid NZ. Separation of monoamines and their metabolites was accomplished using a Brownlee Spheri-5RP-18 250 x 4.6 mm column (5 pm particle size). For separation of 5-HT and DA, a mobile phase consisting of 98 parts of an aqueous phase (125 mM citric acid, 125 mM sodium phosphate, 0.27 mM EDTA, and 0.12 mM sodium octyl sulfate) and 2 parts MeOH with a pH of 2.5 was used. The flow rate of the mobile phase was approximately 1.0 mL/min. The column was maintained in a CTO-6A column oven module (Shimadzu, Concord, CA) at 40 "C. Detection was achieved by means of an amperometric L-ECD-6A detector (Shimadzu),with a glassy carbon working electrode and a Ag/AgCl reference electrode. The fixed potential difference between the reference and working electrodes was +0.70 V. The EC response was quantified using a Shimadzu Chromatopac C-R4A data processor equipped to measure the area under the curve for a given sample and to compare the result with areas obtained from standards processed in an identical manner. (E) Statistics. Differences between group means were d using analysis of variance followed by Duncan's multiple range test where appropriate. Student's t test also was used when multiple comparisons were not involved. Differences were considered significant when a P value of less than 0.05 was obtained. Version 4.1 of the computer progam STATPAK (Northwest Ana-
Zhao et al.
92 Chem. Res. Toxicol., Vol. 5, No. 1, 1992
a - ?J-JcH:mo7(mHy"'
Scheme I. Synthetic Pathway to Proposed 2-Hydroxylated Derivative (4) of MDMA and 6-OHDA Analogue 5 OR
OH
OBn
10: R = H 14: R = B n
9
OBn
15
16
Y
(mfsHOBn
a H ; c H 3 < 4
17
5
18
Table I. Regional Brain Levels of 5-HT and DA after ip Administration of Compound 4 hippocampal 5-HT, pg/f % of control cortical 5-HT, f i g / $ % of control striatal DA, rrgla" 0.44 f 0.02 100 0.41 f 0.03 100 10.6 f 0.2
treatment saline compound 4 95 0.38 f 0.03 0.42 f 0.03 10 w / k g 40 mg/kg 0.43 f 0.02 98 0.44 f 0.08 MDMA 0.17 f 0.02' 39 0.15 f 0.02' 10 w / k g aAssays were performed 1 week after ip treatment (n = 6). Drug or saline was injected cantly different from control (p < 0.05; ANOVA followed by Duncan's multiple range test).
93 107 37
NA' 10.7 f 0.5
% of control
100 101
NA
four times at 2-h intervals. 'Designates signifi'NA = not assayed.
Table 11. Regional Brain Levels of 5-HT and DA after Intracerebroventricular Administration of Compound 4 % of treatment hippocampal 5-HT, pg/g control cortical 5-HT, pg/g % of control striatal DA, pg/g % of control vehicle 0.34 f 0.02 100 0.26 f 0.01 100 10.6 f 0.2 100 compound 4 100 rg 0.33 f 0.01 97 0.28 f 0.01 108 NA' 94 0.27 f 0.02 104 9.5 f 0.5 90 400 rg 0.32 f 0.02 5,7-DHT loo rg 0.02 f 0.01' 6 0.05 f 0.02' 19 NA 6-OHDA 100 rg NA NA 3.7 f 0.56 35
"Assays were performed 1 week after treatment (n = 6 unless otherwise noted). Drug or vehicle (5 or 10 pL) was injected into the appropriate brain region. See Table I footnotes. lytical Inc., Portland, OR) was used.
Results and Dlscusslon Synthetic Studies. The synthetic pathway to the target compounds 4 and 5 (Scheme I) follows well-established literature procedures (20). The formylation of commercially available 3,4-(methy1enedioxy)phenol (9) yielded the expected ortho substitution product 10 which, as the corresponding protected benzyloxy derivative 14, was converted to the nitrostyrene intermediate 15. LiAlH4 reduction of 15 yielded primary amine 16. Treatment of this amine with ethyl formate gave the corresponding N-formyl derivative 17 which was characterized by 'H NMR analysis as a mixture of rotomers (see Experimental Section). LiAlH4 reduction of 17 yielded the desired protected N-methyl product 18 that was converted to the final amine 4 by hydrogenolysis. Treatment of 18 with BBr3 followed by ion-exchange chromatography provided the trihydroxy compound 5 which, while reasonably stable as its solid HC1 salt, rapidly decomposed in pH 7.4 buffer. The above synthetic sequence proceeded as expected except that the formylation of 9 gave a major, acidic (phenolic) side product. The GC/EI mass spectrum displayed a base peak at m / z 269 and a strong (90%) peak at m / z 270. The 'H NMR spectrum was similar to that of 10 but lacked an aldehyde proton signal and displayed in addition a weak singlet at 5.94 ppm. The two singlet aromatic proton signals suggested a 1,2,4,5-tetrasubstituted product. Although the 9-hydroxyxanthene derivative 19 is consistent with these NMR spectral data and would be expected to generate the fragment ion 20 (m/z 269) under
Table 111. Striatal 5-HT and DA Levels after Intrastriatal Drug Administration % of % of treatment 5-HT, rg/@ control DA, pg/$ control vehicle 0.22 f 0.02 100 7.8 k 0.5 100 100 7.9 f 0.7 101 compound 4 0.22 f 0.02
See Table I1 footnote.
GC/EIMS conditions, the 13CNMR spectrum of the unknown ruled this structure out since the only unassigned signal at 38.5 ppm is too far upfield to accommodate the C-9 carbinol atom of 19. The GC/EI mass spectra of the corresponding pertrimethylsilylated and permethylated derivatives displayed intense molecular ions at m/z 640 and 466,respectively. All of these data, together with the elemental analyses, are consistent with the trimer structure 11 in which case the tris-0-(trimethylsilyl) and tris-0methyl derivatives would be 12 and 13, respectively. The formation of 11 presumably occurs via the pathway summarized in Scheme 11, in which the initial Vilsmeier adduct 21 is attacked by the nucleophilic (methy1enedioxy)phenol 9 to generate a dimeric product 22 that undergoes an additional reaction with 9 to yield the trimer 11.
19
20 (miz 269)
Putative Metabolites of MDMA
Chem. Res. Toxicol., Vol. 5, No. 1, 1992 93 Scheme 11. Proposed Pathway Leading to Trimer 11 r
p 3H
21
Table IV. Striatal 5-HT and DA Levels after Intracerebroventricular Administration of Comeound 5 % of % of control control DA, p g / g treatment 5-HT, p g / g 100 100 10.5 f 0.6 vehicle 0.36 f 0.02 compound 5 92 7.8 f 0.5b 72 100 pg 0.33 f 0.03 100 10.9 f 0.1 vehicle NA' 6-OHDA 3.7 f 0.9b 34 100pg NA 100 NA vehicle 0.34 k 0.02 5,7-DHT 6 NA 100 pg 0.02 f 0.01b ~~
b,c See Table
Table V. Striatal 5-HT and DA Levels after Intrastriatal Administration of Compound 5 9% of % of treatment n 5-HT, p g / g control DA, p g / g control vehicle 9 0.25 f 0.01 100 8.7 f 0.4 100 compound 5 50 pg 5 0.18 6 O.Olb 72 3.7 f 0.6b 43 100 pg 4 0.13 f O.Olb 52 1.0 A 0.6b 15 vehicle 3 0.24 f 0.02 100 10.1 f 0.3 100 6-OHDA 50 pg 3 0.27 f 0.04 113 6.4 f 0.Bb 63 (I
See Table I1 footnote. See Table I footnote. Table VI. Cortical 5-HT Levels after Intracortical Administration of Compound 5 treatment 5-HT. ~ / e " % of control vehicle 0.18 f 0.02 100 compound 5 100 0.14 f 0.01 78 400 Pg 0.08 0.02* 45 vehicle 0.15 f 0.01 100 5,7-DHT 100 Pg 0.08 f 0.02b 53
I footnotes.
Neurotoxicological Studies. Compound 4 failed to deplete 5-HT or DA on a long-term basis in the rat brain regardless of whether it was given systemically (Table I), intracerebroventricularly (Table 11), or intrastriatally (Table 111). MDMA given systemically produced a large depletion of regional brain 5-HT (Table I), and 6-OHDA and 5,7-DHT given intracerebroventricularly produced large depletions of DA and 5-HT, respectively (Table 11). The inability of compound 4 to deplete rat brain DA or 5-HT did not appear to be related to administration of insufficient dosage since lower doses of the established neurotoxins had clear depleting effects. For example, the 10 mg/kg regimen of MDMA given systemicallyproduced a 61% depletion of 5-HT while a 4-fold higher dose (40 mg/kg) of compound 4 did not cause significant toxic effects toward dopaminergic or serotonergic neurons. On the basis of these results, it is unlikely that compound 4 plays a direct role in the neurotoxic action of MDMA. The long-term effects on brain 5-HT and DA of the 6-OHDA analogue 5 were examined following intracerebral administration only since it is unlikely to cross the blood-brain barrier. Intracerebroventricular administration of compound 5 produced a moderate (28%)depletion of striatal DA but had no effect on striatal 5-HT (Table IV). This behavior of compound 5 is similar to that observed with 6-OHDA which, however, was more potent (66% DA depletion). The ability of this preparation to demonstrate a toxic effect on serotonergic neurons was confirmed with 5,7-DHT which caused a profound depletion of striatal 5-HT. Intrastriatal administration of compound 5 produced a more severe depletion of DA than that produced by an equivalent dose given intracerebroventricularly (85% versus 28%). Notably, when given intrastriatally, compound 5 had greater DA-depleting effects than 6-OHDA (57% versus 37%). The larger DA-depleting effects of compound 5 when given directly into the striatum (rather than the ventricle) did not appear to be related to the method of drug delivery since injections of the vehicle alone were without effect (Table V). Intrastriatal administration of 5 also produced a lasting depletion of striatal 5-HT. This depletion, while smaller than the depletion of DA, was still substantial and dose-related (48% depletion after the 1OO-Mg dose). These results
-I 11: R = H 12: R = Si(CH,), 13: R=CH,
22
-
See Table I1 footnote.
H?
~
(I
See Table I1 footnote. See Table I footnote.
highlight the fact that site of injection is an important determinant of the monoamine-depleting action of compound 5. We also have evaluated the effects of compound 5 in the motor cortex to determine if its 5-HT-depleting properties of 5 are dependent on DA. As before, the effects of compound 5 were compared to those of a documented serotonergic neurotoxin (5,7-DHT). When given intracortically, compound 5 again produced a dose-related depletion of brain 5-HT which was approximately one-half of that produced by 5,7-DHT (Table VI). These findings,coupled with those in the striatum, indicate that compound 5 possesses significant 5-HT-depleting activity and that the prolonged effects of compound 5 on 5-HT levels are not dependent on dopaminergic innervation. The ability of compound 5 to cause a long-lasting depletion of brain 5-HT is consistent with the view that this potential metabolite of MDMA may mediate the neurotoxic properties of the parent drug. At seeming odds with this view is the fact that MDMA is highly selective for 5-HT neurons (28) whereas compound 5 depletes both brain 5-HT and DA. Although this lack of selectivity of compound 5 argues against its possible role in mediating MDMA neurotoxicity, some factors should be considered which may contribute to this divergence in the neurotoxic profiles of MDMA and compound 5. First, the intraparenchymal route of administration could engender levels of compound 5 within dopaminergic neurons which do not parallel those which develop after systemic administration of MDMA. This specificity may be due to selective transport of MDMA. Alternatively, the enzymes responsible for the generation of compound 5 from its precursor may be present exclusively in serotonergic neurons. Finally, it is important to recall that when given at high dosages, MDMA, like compound 5, depletes both brain DA
94 Chem. Res. Tonicol., Vol. 5, No. 1, 1992
and 5-HT (28). Taken together, these considerations suggest that it would be premature to exclude compound 5 from consideration as a mediator of MDMA neurotoxicity. Biochemical studies are in progress to assess the possible role(s) of the aforementioned factors in the neurotoxicity of MDMA and its metabolites. Acknowledgment. Supported by NIDA Grant DA 06275 and the Harvey W. Peters Research Center for
Parkinson’s Disease and Disorders of the Central Nervous System.
References (1) Peroutka, S. J. (1987) Incidence of recreational w e of 3,4-
methylenedioxymethamphetamine (MDMA, “Ectasy”) on an undergraduate campus. N. Engl. J. Med. 317, 1542-1543. (2) Downing, J. (1986) The psychological and physiological effects of MDMA on normal volunteers. J. Psychoact. Drugs 18, 335-340. (3) Ricaurte, G., Bryan, G., Straws, L., Seiden, L., and Schuater, C. (1985) Hallucinogenic amphetamine selectively destroys brain serotonin nerve terminals. Science 229,986-988. (4) Schmidt, C. J., Wu, L., and Lovenberg, W. (1986) Methylenedioxymethamphetamine: a potentially neurotoxic amphetamine analogue. Eur. J. Pharmacol. 124, 175-178. (5) Schmidt, C. J., and Taylor, V. L. (1989) Neurochemical effecta of methylenedioxymethamphetamine in the rat: acute versus long-term changes. In MDMA: “Ecstasy” and/or human neurotoxin (Peroutka, S. J., Ed.) pp 151-169, Kluwer Academic Publishers, Norwell, MA. (6) Ricaurte, G. A., DeLanney, L. E., Irwin, L., and Langston, J. W. (1988) Toxic effect of MDMA on central serotonergic neurons in the primate: Importance of route and frequency of drug administration. Brain Res. 446, 165-168. (7) McKenna, D. J., and Peroutka, S. J. (1990) Neurochemistry and neurotoxicity of 3,4-methylenedioxymethamphetamine(MDMA, “Ecstasy”). J. Neurochem. 54, 14-22. (8) Schmidt, C. J. (1987) Neurotoxicity of the psychedelic amphetamine, methylenedioxymethamphetamine. J. Pharmacol. Exp. Ther. 240, 1-7. (9) Wang, S. S., Ricaurte, G. A., and Peroutka, S. J. (1987) [HI3,4-Methylenedioxymethamphetamine(MDMA) interactions with brain membranes and glass fiber filter paper. Eur. J. Pharmacol. 138,439-443. (10) Slikker, W., Jr., Gollamude, R., Ali, S. F., Kolta, M., Kipe, G., Webb, P., Lopez, M., and Leakey, J. (1989) The effect of metabolic induction and inhibition of methylenedioxymethamphetamine (MDMA) induced neurochemical alterations. FASEB J. 3, A1036. (11) Molliver, M. E., O’Hearn, E., Battaglia, G., and De Souza, E. B. (1986)Direct intracerebral administration of MDA and MDMA does not produce serotonin neurotoxicity. SOC. Neurosci. Abstr. 12, 1234. (12) Lim, H. K., and Foltz, R. L. (1988) In vivo and in vitro metabolism of 3,4-methylenedioxymethamphetaminein the rat:
Zhao et al. Identification of metabolites using an ion trap detector. Chem. Res. Toxicol. 1, 370-378. (13) Lim, H. K., and Foltz, R. L. (1989) Identification of metabolites of 3,4-(methy1enedioxy)methamphetamine in human urine. Chem. Res. Toricol. 2, 142-143. (14) Miramatsu, M., Kumagal, Y., Unger, S. E., and Cho., A. K. (1990) Metabolism of methylenedioxymethamphetamine: Formation of dihydroxymethamphetamine and a quinone identified as ita glutathione adduct. J. Phurmacol. Exp. Ther. 254,521-526. (15) Dougan, D., Wade, D., and Duffield, P. (1987) How metabolites may augment some psychostimulant actions of amphetamine. Trends Pharmacol. Sci. 8, 277-280. (16) Paul, S., Axelrol, J., and Diliberto, E. (1977) Catechol estrogen-forming enzyme of brain: Demonstration of a cytochrome P-450 monooxygenase. Endocrinology 101,1604-1610. (17) Biel, J. H., and Abood, L. G. (1971) Biogenic amines and physiological membranes in drug therapy, Vol. 5, Part B, pp 390-391, Marcel Dekker, New York. (18) Rotman, A., Daly, J. W., and Creveling, C. R. (1976) Oxygendependent reaction of 6-hydroxydopamine, 5,6-dihydroxytryptamine, and related compoundswith proteins in vitro: a model for cytotoxicity. Mol. Pharmacol. 12, 887-899. (19) Butcher, L. L. (1975) Degenerative processes after punctate intracerebral administration of 6-hydroxydopamine. J. Neural Tranam. 37,189-208. (20) Zweig, J. S., and Castagnoli, N., Jr. (1977) In uitro O-demethylation of the psychomimetic amine, 1-(2,5-dimethyloxy-4methylphenyl)-2-aminopropane.J. Med. Chem. 20,414-421. (21) Greene, T. W. (1981) Protectiue groups in organic synthesis, p 40, John Wiley & Sons, New York. (22) Neeman, M., and Hashimoto,Y. (1962)The structure of estriol monoglucoeiduronic acid from human pregnancy urine. J. Am. Chem. SOC. 84, 2972-2978. (23) Heathcock, C. H., and Ratcliffe, R. (1971) A stereoselectivetotal synthesis of guaiazulenic sesquiterpenoids a-bulnesene and bulnesol. J. Am. Chem. SOC. 93,1746-1757. (24) Cheng, A. C., and Castagnoli, N., Jr. (1984) Synthesis and physicochemical and neurotoxicity studies of l-(4-substituted2,5-dihydroxyphenyl)-2-aminoethaneanalogues of 6-hydroxydopamine. J. Med. Chem. 27,513-520. (25) McCann, U., and Ricaurte, G. A. (1991) Major metabolites of 3,4methylenedioxyamphetamine(MDA) do not mediate its toxic effecta on brain serotonin neurons. Brain Res. 545, 279-282. (26) Ricaurte, G. A., Fuller, R. W., Perry, K. W., and Seiden, L. S. (1983) Fluoxetine increases long-lasting neostriatal dopamine depletion after administration of d-methamphetamine and damphetamine. Neuropharmacology 22, 1165-1169. (27) Kotake, C., Heffner, T., Vosmer, G., and Seiden, L. (1985) Determination of dopamine, norepinephrine, serotonin and their major metabolic products in rat brain by reverse-phase ion-pair high performance liquid chromatography with electrochemical detection. Pharmacol., Biochem. Behav. 22, 85-90. (28) Commins, D. L., Vosmer, G., Virus, R. M., Woolverton, W. L., Schwter, C. R., and Seiden, L. S. (1987) Biochemical and histological evidence that methylenedioxymethamphetamine (MDMA) is toxic to neurons in the rat brain. J. Phurmucol. Erp. Ther. 241, 338-345.
