Synthesis and neurotoxicity profile of 2,4,5-trihydroxymethamphetamine and its 6-(N-acetylcystein-S-yl) conjugate

Article abstract of DOI:10.1021/tx2001459

The purpose of the present study was to determine if trihydroxymethamphetamine (THMA), a metabolite of methylenedioxymethamphetamine (MDMA, "ecstasy"), or its thioether conjugate, 6-(N-acetylcystein-S- yl)-2,4,5-trihydroxymethamphetamine (6-NAC-THMA), pla

Full text of DOI:10.1021/tx2001459

ARTICLE
pubs.acs.org/crt
Synthesis and Neurotoxicity Profile of 2,4,5-Trihydroxymethamphetamine
and Its 6-(N-Acetylcystein-S-yl) Conjugate
Anne Neud€orffer,† Melanie Mueller,‡ Claire-Marie Martinez,† Annis Mechan,‡ Una McCann,§
George A. Ricaurte,*,‡ and Martine Largeron*,†
†UMR 8638 CNRS-Universitꢀe Paris Descartes, Synthꢁese et Structure de Molꢀecules d'Intꢀer^et Pharmacologique,
Facultꢀe des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l'Observatoire, 75270 Paris cedex 06, France
‡Department of Neurology and §Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine,
5501 Hopkins Bayview Circle, Baltimore, Maryland 21224, United States
S Supporting Information
b
ABSTRACT: The purpose of the present study was to deter-
mine if trihydroxymethamphetamine (THMA), a metabolite of
methylenedioxymethamphetamine (MDMA, “ecstasy”), or its
thioether conjugate, 6-(N-acetylcystein-S-yl)-2,4,5-trihydroxy-
methamphetamine (6-NAC-THMA), play a role in the lasting
effects of MDMA on brain serotonin (5-HT) neurons. To this
end, novel high-yield syntheses of THMA and 6-NAC-THMA
were developed. Lasting effects of both compounds on brain
serotonin (5-HT) neuronal markers were then examined. A
single intraventricular injection of THMA produced a significant lasting depletion of regional rat brain 5-HT and 5-hydroxyindo-
leacetic acid (5-HIAA), consistent with previous reports that THMA harbors 5-HT neurotoxic potential. The lasting effect of THMA
on brain 5-HT markers was blocked by the 5-HT uptake inhibitor fluoxetine, indicating that persistent effects of THMA on 5-HT
markers, like those of MDMA, are dependent on intact 5-HT transporter function. Efforts to identify THMA in the brains of animals
treated with a high, neurotoxic dose (80 mg/kg) of MDMA were unsuccessful. Inability to identify THMA in the brains of these
animals was not related to the unstable nature of the THMA molecule because exogenous THMA administered intracerebroven-
tricularly could be readily detected in the rat brain for several hours. The thioether conjugate of THMA, 6-NAC-THMA, led to no
detectable lasting alterations of cortical 5-HT or 5-HIAA levels, indicating that it lacks significant 5-HT neurotoxic activity. The
present results cast doubt on the role of either THMA or 6-NAC-THMA in the lasting serotonergic effects of MDMA. The possibility
remains that different conjugated forms of THMA or oxidized cyclic forms (e.g., the indole of THMA) play a role in MDMA-induced
5-HT neurotoxicity in vivo.
’ INTRODUCTION
MDMA’s neurotoxic properties for more than two decades,9
the mechanisms by which MDMA damages brain axons and
axon terminals are not known.2,10
3,4-Methylenedioxymethamphetamine (MDMA, “ecstasy”)
is a drug of abuse that produces lasting decreases in various
presynaptic serotonergic neuronal markers including 5-HT, its
major metabolite, 5-hydroxyindoleacetic acid (5-HIAA), its
biosynthetic enzyme, tryptophan hydroxylase (TPH), and its
membrane reuptake site, the 5-HT transporter (SERT).1,2
These decrements in 5-HT axonal markers are accompanied
by anatomical signs of 5-HT axonal damage3,4 and a lasting
decrease in anterograde [3H]proline transport from the dorsal
raphe nucleus to the forebrain.5 Collectively, these lasting
chemical and anatomical alterations after MDMA exposure
have been widely interpreted as being indicative of a toxic
effect of MDMA on brain 5-HT axon terminals.1,2,6 Such
neurotoxic effects of MDMA have been documented in a variety
of species, including nonhuman primates. In most species
(including primates), MDMA selectively damages brain 5-HT
neurons.1,2,7 For uncertain reasons, in mice, MDMA selectively
damages brain dopamine (DA) neurons.8 Despite knowledge of
Research demonstrating that central administration of
MDMA fails to produce 5-HT neurotoxicity11ꢀ14 has led to
the suspicion that systemic metabolism of peripherally admi-
nistered MDMA is required for the expression of neurotoxicity.
This suspicion has generally been taken to suggest that a toxic
metabolite is responsible for the lasting effects of MDMA on
brain 5-HT neurons. In this regard, thioether conjugates of
MDMA metabolites have drawn particular attention.15ꢀ18
Of the various potentially toxic metabolites of MDMA
(Scheme 1), recent evidence indicates that several metabolites
do not appear to be directly involved. These include 3,4-
14,19,20
dihydroxymethamphetamine (HHMA)
and 4-hydro-
xy-3-methoxymethamphetamine (HMMA),20 both of which
Received: April 7, 2011
Published: May 10, 2011
r
2011 American Chemical Society
968
dx.doi.org/10.1021/tx2001459 Chem. Res. Toxicol. 2011, 24, 968–978
|

Chemical Research in Toxicology
ARTICLE
Scheme 1. Metabolic Pathways of MDMA along with the Associated Microsomal Enzymes Including the Postulated Conjugate
Formationa
a The numbered compounds correspond to the postulated metabolites synthesized and used for the biological experiments.
are phase I and phase II metabolites generated through O-
demethylenation, and 3,4-dihydroxyamphetamine (HHA),21
which is produced via N-demethylation followed by O-de-
methylenation. In particular, exposure to these metabolites
does not result in lasting 5-HT deficits. More recently, the
thioether conjugate of HHMA, 5-(N-acetylcystein-S-yl)-
HHMA (5-NAC-HHMA) has been implicated in MDMA
neurotoxicity,18 but efforts to replicate these findings have
been unsuccessful.20
One metabolite of MDMA that has not yet been excluded
as the mediator of MDMA neurotoxicity is 2,4,5-trihydroxy-
methamphetamine (THMA). This metabolite, arising from the
ring hydroxylation biotransformation step (Scheme 1), is a
particularly intriguing candidate because it has structural simi-
larity to the well-known DA neurotoxin, 6-hydroxydopamine
(6-OHDA), has been reported to be formed endogenously in the
liver,22 and has already been shown to produce lasting 5-HT
deficits when administered centrally. Of note, however, THMA
has never been detected in the brains of animals treated
with MDMA.
The goal of the present investigation was to accomplish high-
yield syntheses of THMA 4 and its thioether conjugate, 6-(N-
acetylcystein-S-yl)-2,4,5-trihydroxymethamphetamine (6-NAC-
THMA) 5 (Scheme 1), and to determine if either of these
MDMA metabolites plays a role in MDMA neurotoxicity. In
particular, after confirming earlier findings that centrally admi-
nistered THMA produces lasting effects on brain 5-HT
neurons,23,24 we sought to (1) determine if the lasting effect of
THMA on 5-HT can be blocked with a 5-HT transporter
blocker, fluoxetine, which is known to protect against MDMA
neurotoxicity;30 (2) employ newly developed liquid chromato-
graphic-mass spectrometric (LC-MS) methods to ascertain if
THMA can be detected in the brains of rats treated with
peripherally administered neurotoxic dose of MDMA; (3) test
if the inherent instability of the THMA molecule impacts its
detectability in brain tissue; and (4) determine if the thioether
conjugate of THMA, 6-NAC-THMA 5, produces lasting effects
on brain 5-HT neuronal markers. The present results cast doubt
on the role of THMA and its thioether conjugate, 6-NAC-
THMA, in MDMA neurotoxicity.
Although extensive work has been done with thioether con-
jugates of HHMA,18,20,25ꢀ28 there are no published reports on
thioether conjugates of THMA. Notably, thioether conjugates of
6-OHDA have been detected in the brains of rats and mice given
6-OHDA centrally,29 suggesting that similar processes may occur
with THMA. To our knowledge, synthesis of the thioether
conjugate of THMA has yet to be reported.
’ EXPERIMENTAL PROCEDURES
Chemicals. All reagents and solvents (HPLC grade) were commer-
cial products of the highest available purity and were used as supplied.
Analytical thin-layer chromatography was carried out on silica gel
Macherey-Nagel Polygram SIL G/UV 254 (0.25 mm). Column
969
dx.doi.org/10.1021/tx2001459 |Chem. Res. Toxicol. 2011, 24, 968–978

Chemical Research in Toxicology
ARTICLE
Scheme 2. Four-Step Synthesis of THMA 4
chromatography was performed on Macherey-Nagel Si 60 M silica gel
(40ꢀ63 μm). Melting points were measured on a K€ofler apparatus.
HPLC was carried out using a Waters system consisting of a 600E
multisolvent delivery system, a Rheodyne-type loop injector, and a 2487
dual-channels UVꢀvisible detector set at 254 and 278 nm. A mixture of
two solvents A and B constituted the mobile phase. Solvent A was
prepared by adding 1% concentrated trifluoroacetic acid (TFA) to
deionized water. Solvent B was prepared by adding 0.5% TFA to a 1:1
(v/v) mixture of methanol and deionized water. Semipreparative
reversed-phase HPLC was performed using a 250 ꢁ 20 mm, 5 μm
Kromasil C18 column and a 2 mL loop injector, whereas for the
analytical reversed-phase HPLC, a 250 ꢁ 4.6 mm, 5 μm Kromasil
C18 column, together with a 50 μL loop injector, was used.
residue was washed with ethyl acetate (120 mL). The resulting organic
phase was washed with 50 mL of water, dried over MgSO4, and filtered
off, and the solvent was evaporated. After column chromatography
(toluene/acetone 92.5/7.5), the crude pale yellow solid was recrystal-
lized from diethyl ether affording compound 2 (1.08 g, 4.8 mmol) in
1
80.5% yield; mp 55ꢀ57 °C. H NMR (CDCl3) δ 2.15 (s, 3H, CH3),
3.63 (s, 2H, CH2), 3.81 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.90 (s, 3H,
OCH3), 6.55 (s, 1H, H3), 6.68 (s, 1H, H6). 13C NMR (CDCl3) δ 29.1,
44.8, 56.0, 56.1, 56.5, 97.4, 114.6, 114.8, 142.8, 148.8, 151.5, 207.3.
HRMS (ESI) m/z calcd for [M þ Na]þ 247.0946; found, 247.0942.
2,4,5-Trimethoxymethamphetamine (3). To a stirred solu-
tion of compound 2 (1.0 g, 4.5 mmol) in dry dichloromethane (10 mL)
cooled at ꢀ5 °C was added a 2 M solution of methylamine in THF
(10.1 mL, 20.25 mmol, 4.5 equiv). After the reaction mixture was stirred
at room temperature for 1 h, 1.15 mL of glacial acetic acid (4.5 equiv)
was added, followed by sodium tri-acetoxyborohydride (Na(AcO)3BH)
(1.43 g, 6.75 mmol, 1.5 equiv) in small portions. Then, the reaction
mixture was stirred at room temperature for 3 h and quenched with
2.5 M sodium hydroxide aqueous solution (5 mL). The aqueous layer
was extracted with diethyl ether (3 ꢁ 100 mL), and the combined ether
extracts were washed with 2.5 M sodium hydroxide aqueous solution
(5 mL) dried over anhydrous MgS04 and evaporated to dryness to give
compound 3 as a white solid (1.07 g, 4.5 mmol) in quantitative yield; mp
74ꢀ76 °C. 1H NMR (CDCl3) δ 1.01 (d, J = 6.0 Hz, 3H), 1.55 (broad s,
1H), 2.36 (s, 3H), 2.52 (m, J = 13.0 Hz, 1H), 2.64 (m, J = 13.0 Hz, 1H),
2.73 (m, 1H), 3.76 (s, 3H), 3.80 (s, 3H), 3.85 (s, 3H), 6.49 (s, 1H), 6.66
(s, 1H). 13C NMR (CDCl3) δ 19.8, 34.0, 37.5, 55.3, 56.1, 56.3, 56.6,
97.7, 115.1, 119.4, 142.7, 147.9, 151.9. HRMS (ESI) m/z calcd for [M þ
H]þ 240.1600; found, 240.1594.
1H NMR and 13C NMR spectra were performed on a Br€uker AC-300
spectrometer operating at 300 and 75 MHz, respectively. Chemical shifts
are expressed as δ units (parts per million) downfield from TMS
(tetramethylsilane). The measurements were carried out using the
standard pulse sequences. The carbon type (methyl, methylene,
1
methine, or quaternary) was determined by DEPT experiments. H
and 13C NMR spectra of all compounds are included in the Supporting
Information as a proof of their identity. High-resolution mass spectra
(HRMS) were recorded on a LTQ-Orbitrap spectrometer operating in
positive ion mode. UV/vis spectra were recorded on a VARIAN Cary
100 spectrophotometer.
(()-2,4,5-Trihydroxymethamphetamine hydrobromide (THMA,
HBr) was synthesized in four steps (Scheme 2) from commercially
available 2,4,5-trimethoxybenzaldehyde and nitroethane, through a
procedure close to those previously reported for the synthesis of (()-
3,4-dihydroxymethamphetamine hydrobromide (HHMA, HBr) with
some modifications.31ꢀ34
(()-2,4,5-Trihydroxymethamphetamine Hydrobromide
(THMA, HBr) (4). THMA was previously described as the hydrochloric
salt.23 A 48% aqueous solution of HBr (5.2 mL) (46 mmol) was added to
compound 3 (954 mg, 4.0 mmol). The resulting solution was heated to
reflux for 2.0 h, under inert atmosphere. After evaporation under
reduced pressure, the brown solid was recrystallized successively in
dichloromethane-MeOH 90:10 v/v, and chloroformꢀisopropanol
85:15 v/v mixture, affording THMA, HBr 4 as a white solid (845 mg,
3.04 mmol) in 76% yield. The degree of purity for compound 4 (99%)
was determined by analytical HPLC (eluent, solvent A/solvent B 93/7;
flow rate, 0.6 mL minꢀ1); mp 172ꢀ174 °C. UV absorption (0.2 M
aqueous HCl) λmax = 292 nm, ε = 4300 molꢀ1 L cmꢀ1. 1H NMR (D2O)
δ 1.14 (d, J = 6,5 Hz, 3H), 2.57 (s, 3H), 2.69 (m, 2H), 3.37 (m, 1H), 6.38
(s, 1H), 6.60 (s, 1H). 13C NMR (D2O) δ 14.8, 30.1, 32.9, 55.8, 104.2,
113.9, 118.9, 137.1, 143.9, 147.7. Note, when the 1H NMR spectra of
THMA was performed in DMSO D6, three additional signals were
recorded at 8.09 (1H, OH, D2O exchanged), 8.38 (2H, OH, D2O
2,4,5-Trimethoxy-β-methyl-β-nitrostyrene (1). A solution of
2,4,5-trimethoxybenzaldehyde (2.94 g, 15.0 mmol) and of ammonium
acetate (1.18 g, 15.3 mmol) in 60 mL of nitroethane was heated to reflux
for 45 min. After evaporation under reduced pressure, H2O (50 mL) was
added to the resulting orange oil, and the reaction mixture was extracted
with ethyl acetate (2 ꢁ 100 mL). The organic phase was dried over
MgSO4 and filtered off. Evaporation of the solvent under reduced
pressure afforded 1 in 99.5% (3.77 g, 14.90 mmol) as an orange solid;
mp, 94ꢀ96 °C; (literature34 mp, 93ꢀ95 °C). Spectroscopic data were
identical to those previously reported.34
2,4,5-Trimethoxyphenylacetone (2). Compound 1 (1.52 g,
6.0 mmol) was dissolved at 50 °C in 4.8 mL of toluene. Then, 2.0 g of Fe
(36.0 mmol, 6 equiv) (electrolytic powder), 81.1 mg of ferric chloride
(0.3 mmol, 0.05 equiv), 19.2 mL of water, and 2.1 mL of 35% aqueous
solution of HCl (2 equiv) were successively added, and the resulting
mixture was heated to reflux for 6 h. After filtration through Celite, the
970
dx.doi.org/10.1021/tx2001459 |Chem. Res. Toxicol. 2011, 24, 968–978

Chemical Research in Toxicology
ARTICLE
exchanged), and 8.74 (2H, MeNH2þ, D2O exchanged) that confirmed
the presence of the three hydroxyl groups at C2, C4, and C5 and that of
the protonated secondary amino group on the side chain. As reported
earlier, HRMS did not allow us to obtain the exact mass of THMA but
that of the 5,6-dihydroxyindole species. Only derivatization with methyl
chloroformate provided a means of converting the highly reactive
THMA to a stable derivative, then avoiding the cyclization into indole.22
HRMS (ESI) m/z calcd for [M þ Na]þ 200.06822; found, 200.06823
(5,6-dihydroxyindole species). However, it was possible to detect
THMA by LC-MS (see Figure 5 below).
Electrochemistry. Controlled-potential electrolyses were carried
out in a cylindrical three-electrode divided cell (9 cm diameter), using a
Voltalab 32 electrochemical analyzer (Radiometer, Copenhagen). In the
main compartment, a cylindrical platinum grid (area = 60 cm2) served as
the anode (working electrode). A platinum sheet was placed in the
concentric cathodic compartment (counter-electrode), which was sepa-
rated from the main compartment with a glass frit. The reference
electrode was an Ag/AgCl electrode, to which all potentials quoted
are referred.
reversed-phase HPLC was performed using the following mobile phase
gradient (flow rate: 5.0 mL minꢀ1): 0ꢀ10 min, mixture of solvent
A/solvent B 80/20; 10ꢀ70 min, mixture of solvent A/solvent B from
80/20 to 100% solvent B. Fractions containing 6-(N-acetylcystein-S-yl)-
2,4,5-trihydroxymethamphetamine (5) were collected, immediately
frozen at ꢀ80 °C, and then freeze-dried. Compound 5 was isolated in
27% yield (24.0 mg, 0.07 mmol). Twenty- one percent of the starting
THMA (10.5 mg, 0.05 mmol) were also recovered because the electro-
lysis was stopped before the end of the oxidation (after the consumption
of 1.75 electron per molecule) since, in pH 7.4 buffer, compound 5
underwent oxidative cyclization into an indole species which rapidly
polymerized as do other indoles of this nature.29
Enzymatic Oxidation. Mushroom tyrosinase (12500 units) was
added to a solution of THMA, HBr (4) (69.5 mg, 0.25 mmol) contain-
ing 5 equiv of N-acetylcysteine (208 mg, 1.25 mmol) in 10ꢀ2 M sodium
phosphate buffer (250 mL, pH 7.4) at 37 °C. The solution became
immediately red. After 10 min of reaction at 37 °C, the solution was
acidified to pH 2.0 with concentrated HCl, frozen at ꢀ80 °C, and then
freeze-dried. Semipreparative reversed-phase HPLC was performed
using the following mobile phase gradient (flow rate: 5.0 mL minꢀ1):
0ꢀ10 min, mixture of solvent A/solvent B 80/20; 10ꢀ80 min, mixture
of solvent A/solvent B from 80/20 to 100% solvent B. Fractions
containing 6-(N-acetylcystein-S-yl)-2,4,5-trihydroxymethamphetamine
(5) were collected, immediately frozen at ꢀ80 °C, and then freeze-dried.
Compound 5 was isolated in 21.5% yield (19.2 mg, 0.05 mmol). Note
14.5% of THMA (7.5 mg, 0.04 mmol) was also recovered.
Biological Studies. Male albino SpragueꢀDawley rats weighing
approximately 300 g were used. Animals were housed in a colony room
maintainedat22 ( 2 °C with freeaccessto food and water. Lighting inthe
room was automatically regulated on a 12:12 h light/dark cycle. Animals
were single housed throughout. The facilities used are accredited by the
American Association for the Assessment and Accreditation of Laboratory
Animal Care. All experimental procedures were approved by the Institu-
tional Animal Care and Use Committee at the Johns Hopkins University
School of Medicine and were in accordance with the National Institutes of
Health Guide for the Care and Use of Laboratory Animals Housing.
To evaluate of the lasting effects of THMA and 6-NAC-THMA on
brain 5-HT axonal markers, five experiments were performed. The
purpose and design of each of these experiments were as follows.
Experiment 1 assessed regional brain 5-HT and 5-HIAA levels one
week after the administration of newly synthesized THMA. THMA, as
the hydrobromide salt, was administered into the right lateral ventricle of
rats at two different doses (250 and 1000 nmol). Doses refer to the base
form. These doses were selected on the basis of previous studies.23,24
Rats received either 10 μL of artificial cerebrospinal fluid (aCSF)
(control group, N = 5), 250 nmol of THMA (N = 6), or 1000 nmol
of THMA (N = 7).
Experiment 2 determined if the lasting effect of THMA on central
5-HT neuronal markers could be blocked with a 5-HT transporter
blocker, fluoxetine. There were four treatment groups (n = 5ꢀ7 per
group): (1) SAL/aCSF; (2) Fluox/aCSF; (3) SAL/THMA; and (4)
Fluox/THMA. Rats received saline or fluoxetine (10 mg/kg, i.p.) 65 and
5 min prior to injection of THMA (150 μg) into the lateral ventricle.
THMA was dissolved in aCSF immediately prior to intraventricular
injection. One week after treatment, rats were sacrificed for the
determination of brain 5-HT and 5-HIAA levels.
6-(N-Acetylcystein-S-yl)-2,4,5-trihydroxymethampheta-
mine (5). Procedure A. A solution of THMA, HBr (69.5 mg, 0.25
mmol), in 0.2 M HCl (250 mL), was oxidized under nitrogen at 10 °C at
a platinum grid whose potential was fixed at þ1.0 V versus Ag/AgCl.
After the consumption of 2 electrons per molecule, 2 equiv of
N-acetylcysteine (83 mg, 0.50 mmol) was added to the pale orange
solution, which slowly turned yellow. After 50 min, the reaction mixture
was frozen at ꢀ80 °C and then freeze-dried. The residue was subdivided
into fractions of about 25 mg. Each fraction was dissolved in 2 mL of
water and then purified by semipreparative reversed-phase HPLC, using
a mixture of solvent A/solvent B 78/22 as the eluent (flow rate, 4.5 mL
minꢀ1; detection, 292 and 264 nm). Fractions containing 6-(N-acet-
ylcystein-S-yl)-2,4,5-trihydroxy-methamphetamine (5) were collected,
immediately frozen at ꢀ80 °C, and then freeze-dried. Compound
5 (diastereomeric mixture) was isolated as a pale pink solid (64 mg,
0.18 mmol) in 71% yield. Its degree of purity (96.5%) was determined by
analytical HPLC (eluent, solvent A/B 90/10; flow rate, 0.65 mL minꢀ1);
mp >110 °C (dec.). UV absorption (0.2 M aqueous HCl) λmax = 257
(ε = 2420 molꢀ1 L cmꢀ1) and 307 nm (ε = 3118 molꢀ1 L cmꢀ1).
1H NMR (D2O) δ 1.30 (d, J = 6.5 Hz, 3H), 1.76 (s, 3H), 2.56 (s, 3H),
3.03 (m, 3H), 3.27 (m, 1H), 3.29 (m, 1H), 4.23 (m, 1H), 6.44 (s, 1H).
13C (CDCl3) Diastereoisomer A: δ 14.8, 21.4, 30.2, 31.1, 34.5, 53.2, 56.4,
105.2, 117.6, 120.3, 139.9, 143.9, 147.9, 173.8. Diastereoisomer B: δ 14.8,
21.4, 30.2, 31.2, 34.5, 53.4, 56.4, 105.2, 117.7, 120.4, 139.9, 143.9, 147.9,
1
173.8. Note, when the H NMR spectra of 6-NAC-THMA was per-
formed in DMSO D6, four additional signals were recorded at 8.26
(1H, NHCOMe, D2O exchanged), 8.38 (2H, OH, D2O exchanged), 9.04
(1H, OH, D2O exchanged), and 9.42 (1H, CO2H, D2O exchanged) that
confirmed the presence of the three hydroxyl groups at C2, C4, and C5,
that of the acetylated amino group, and that of the carboxylic acid
function. As mentioned in the case of THMA, HRMS did not allow us to
obtain the exact mass of 6-NAC-THMA but that of its corresponding
5,6-dihydroxyindole species. HRMS (ESI) m/z calcd for [M þ Na]þ
361.08282; found, 361.08286.
For biological evaluation, a second purification was performed by
semipreparative reversed-phase HPLC, using a mixture of solvent A/solvent
B 92/8 as the eluent (flow rate: 10.5 mL minꢀ1). Then, compound 5 could
be isolated with 99.5% purity grade (see the Supporting Information).
Procedure B. A solution of THMA, HBr (4) (69.5 mg, 0.25 mmol) in
10ꢀ2 M sodium phosphate buffer (250 mL, pH 7.4) containing 5 equiv
of N-acetylcysteine (208 mg, 1.25 mmol) was oxidized under nitrogen at
10 °C at a platinum grid whose potential was fixed at þ0.4 V versus Ag/
AgCl. After the consumption of 1.75 electron per molecule, the resulting
red solution was acidified to pH 2.0 with 0.2 M HCl. The resulting
solution was frozen at ꢀ80 °C and then freeze-dried. Semipreparative
Experiment 3 tested for the presence of THMA in the brain of animals
treated with a high neurotoxic dose of MDMA (80 mg/kg; s.c.). Six rats
were treated with MDMA: 3 were examined 1.5 h after MDMA
administration; the other 3 were examined 3 h after MDMA administra-
tion. Cortical and hippocampal tissues from these animals were analyzed
for THMA, MDMA, and various MDMA metabolites, as described below.
These times for analysis were selected on the basis of prior pharmacoki-
netic studiesofMDMAin theratindicatingthatthese timescorrespond to
971
dx.doi.org/10.1021/tx2001459 |Chem. Res. Toxicol. 2011, 24, 968–978

Chemical Research in Toxicology
ARTICLE
Scheme 3. Oxidative Polymerization of THMA 4 in pH 7.4 Phosphate Buffered Aqueous Solution under Atmospheric Conditions
(Path a) and Two-Step One-Pot Electrochemical Synthesis of 6-NAC-THMA 5 under Acidic Conditions (Path b)
then divided into subgroups (N = 3 at each time point) and sacrificed at
various time points (0.5, 1, 2, and 4 h) after treatment. Regional brain
levels of THMA were measured, as described below.
Experiment 5 assessed regional brain 5-HT and 5-HIAA levels two
weeks after the administration of 6-NAC-THMA, the thioether con-
jugate of THMA. 6-NAC-THMA was administered directly into the
right frontal cortex. Rats received either 1 μL of aCSF (control group,
N = 6) or 42 nmol of 6-NAC-THMA (N = 8), or 52 nmol of
5,7-dihydroxytryptamine (5,7-DHT) (positive control group, N = 6,
single dose). Animals treated with aCSF and 6-NAC-THMA received
four consecutive doses, with each dose administered 12 h apart. The
neurotoxin 5,7-DHT was administered in a single dose. The dose and
frequency chosen are the same as those that were used in previous
studies to test for the neurotoxic potential of various thioether con-
jugates derived from HHMA and/or HHA.15,16,18,20
Intracerebroventricular Administration. Animals were an-
esthetized with sodium pentobarbital (60 mg/kg, i.p.), and the head
was shaved and placed into a stereotaxic apparatus. Using a surgical
scalpel, a midsagittal incision was made to expose the skull. A small burr
hole was made with a hand drill: (ꢀ) 0.92 mm from bregma and (ꢀ)
1.4 mm lateral to the midline. A 10 μL Hamilton 7000 series glass syringe
(Hamilton Co., Reno, NV) containing the various injection solutions
was inserted (ꢀ) 3.5 mm dorsoventrally.35 Artificial CSF serving as a
vehicle control was prepared as described previously by Miller et al.15
Ten microliters of the drug solution was injected manually into the right
ventricle at a rate of 10 μL over 2 min. After the injection was completed,
the needle was left in the ventricle for an additional 2 min.
Intracortical Administration. Animals were anesthetized with
Figure 1. Spectrophotometric changes accompanying the electrosynth-
esis of 6-NAC-THMA 5. (Spectra aꢀe): electrochemical oxidation of
THMA(1 mM), at a platinum anode (E = þ1.0 V versus Ag/AgCl), in
deaerated 0.2 M HCl aqueous solution; (a) 0 (before electrolysis),
(b) 0.5, (c) 1.0, (d) 1.5, and (e) 2.0 mol of electrons. (Spectra fꢀi):
evolution after the addition of 2 equiv of N-acetylcysteine to the
two electron-oxidized solution; (f) 10 min after addition, (g) 20 min,
(h), 40 min, and (i) 50 min (end of the process). Cell thickness is 0.1 cm.
xylazine (25 mg/kg, i.p.) and ketamine (35 mg/kg i.p.). Guide cannulae
(20 gauge; Plastic One, Roanoke, VA) were surgically implanted into the
right frontal cortex [anteroanterior, 3.0 mm; mediolateral, 2.0 mm;
dorsoventral, 2.0 mm35]. Cannulae were fixed to the skull with dental
acrylic (Ortho-Jet, Lang Dental, Wheeling, IL) and two stainless steel
screws. Dummy cannulae were placed in the guide cannulae, and animals
were individually housed and allowed a 7-day recovery period. The
dummy cannulae were replaced with internal cannulae (24 gauge; Plastic
One) connected to PE 20 tubing that in turn was connected to a 1 μL
Hamilton 7000 series glass syringe (Hamilton Co., Reno, NV) containing
the various injection solutions. Artificial CSF served as a vehicle control
and was prepared as described previously by Miller et al.20 One microliter
of the drug solution was injected manually into the frontal cortex at a rate
of 0.2 μL over 5 min for a total of four consecutive doses, with each dose
administered 12 h apart. After the injection was completed, the internal
cannulae were left in the cortex for an additional 2 min. Animals were
awake but gently restrained during the injections. After injection, the
dummy cannulae were replaced.
peak concentrations of MDMA [i.e., times when maximum concentra-
tions of the precursor would be available for potential conversion into
THMA20]. Also, after intracerebroventricular administration, the highest
levels of THMA were evident 30 to 120 min later (see Figure 6).
Experiment 4 determined if THMA, an inherently unstable molecule,
could be reliably measured in brain tissue after central doses that
produce a lasting effect on brain 5-HT and 5-HIAA, namely, 250 and
1000 nmol (see Figure 2). THMA (250 or 1000 nmol) was infused into
the right lateral ventricle of rats (N = 12 for each dose). Animals were
972
dx.doi.org/10.1021/tx2001459 |Chem. Res. Toxicol. 2011, 24, 968–978

Chemical Research in Toxicology
ARTICLE
Figure 2. 5-HT (left panels) and 5-HIAA levels (right panels) in the hippocampus (a), cerebral cortex (b), and striatum (c) of rats one week after
intracerebroventricular administration of 250 or 1000 nmol THMA. Control animals received unilateral intracerebroventricular injections of an
equivalent volume of aCSF. Values represent the mean ( SD (N = 5ꢀ7) side ipsilateral to injections. A one-way ANOVA followed by Tukey’s multiple
comparison test was performed and differences regarded as significant when p < 0.05. * Indicates significant differences from the control group.
Brain Dissection. Rats were euthanized by decapitation, and the
striatum, hippocampus, and cerebral cortex were dissected free as
previously described.36 Immediately after dissection, tissue parts were
wrapped in aluminum foil and stored in liquid nitrogen until assayed.
Regional Brain 5-HT and 5-HIAA Determination. Tissue
samples were analyzed for their content of 5-HT and 5-HIAA 1 or 2
weeks after drug treatment by means of high performance liquid
chromatography coupled with electrochemical detection (HPLC-EC)
as previously described.7
Determination of Brain THMA and MDMA Metabolite
Concentrations. For the determination of brain concentrations of
THMA, samples were prepared and analyzed using LC-MS methods. In
particular, aliquots of rat cortex (approximately 100 mg) and hippo-
campus (approximately 50 mg) were weighed and for each microgram of
tissue, and 10 μL of internal standard solution (pholedrine) was added.
After homogenization in 0.01 M HCl (preserved with 6% of each
250 mmol SMBS and 250 mmol EDTA) with a Polytron homogeniza-
tion unit (model PT 10-35, 15 s, setting 6; Kinematica Inc., Bohemia,
NY), the samples were centrifuged (16,000g for 10 min), and the
supernatant was transferred to autosampler vials. Aliquots (5 μL) were
injected into the LC-MS system. All samples were analyzed using an
Agilent Technologies (AT, Waldbronn, Germany) AT Series 1100 LC/
MSD, VL version, using electrospray ionization (ESI) in positive ioniza-
tion mode, including an AT 1100 Series HPLC system which consisted of
a degasser, a quaternary pump, a column thermostat, and an autosampler.
Isocratic elution was performed on a Zorbax 300-SCX column (Narrow-
Bore 2.1 ꢁ 150 mm, 5 μm) and a Zorbax SCX guard column (4.6 ꢁ 12.5
mm, 5 μm). The mobile phase consisted of 5 mM aqueous ammonium
formate adjusted to pH 3 with formic acid (eluent A) and acetonitrile
(eluent B). Samples were analyzed in positive (selected ion monitoring)
SIM mode with the following ions: m/z 198 (target ion), 167 for THMA,
m/z 196 (t), 165 for the cyclic indoline form of THMA, and m/z 166 (t),
135 for pholedrine (IS). Fragmentor voltage was 100 V. The linear range
was 5 to 100 μg/g for THMA as well as for the indoline species. The
lowest point of the calibration curve was defined as the limits of
quantitation of the method (5 μg/g). For the determination of MDMA
and its metabolites HHMA, HMMA, and MDA, samples were prepared
and analyzed using previously described LC-MS methods.37
Statistics. The significance of differences between means was
determined using one-way analysis of variance (ANOVA) followed by
Tukey’s multiple comparison test. Statistical analyses were performed
using Prism, version 3.02 (GraphPad Software, Inc. San Diego, CA,
USA). Differences were considered significant if p < 0.05.
’ RESULTS AND DISCUSSION
Synthesis of THMA. THMA was synthesized in a straightfor-
ward manner (Scheme 2), starting from commercially available
2,4,5-trimethoxybenzaldehyde. Briefly, the previously reported
Knoevenagel condensation beetween 2,4,5-trimethoxybenzaldehyde
973
dx.doi.org/10.1021/tx2001459 |Chem. Res. Toxicol. 2011, 24, 968–978

Chemical Research in Toxicology
ARTICLE
Figure 3. DA (upper panel) and DOPAC (lower panel) levels in the
striatum of rats one week after intracerebroventricular administration of
THMA (1000 nmol). Control animals received unilateral intracerebro-
ventricular injections of an equivalent volume of aCSF. Values represent
the mean ( SD. A one-way ANOVA followed by Tukey’s multiple
comparison test was performed and differences regarded as significant
when p < 0.05. * Indicates significant differences from the control group.
Figure 4. Effect of fluoxetine on lasting deficits of 5HT and 5-HIAA
after THMA. Shown are the concentrations of 5-HT and 5-HIAA in the
hippocampus of rats 1 week after drug administration in four different
groups of animals. Rats were administered either aCSF or THMA (150
μg) into the lateral ventricle. Rats were pretreated with either saline or
fluoxetine (10 mg/kg, i.p.) 65 and 5 min prior to the intracerebroven-
tricular THMA injection. A one-way ANOVA followed by Tukey’s test
for multiple comparisons was used to test for significant differences
among treatment groups. Differences were considered significant when
p < 0.05. * Indicates significant differences from the saline/aCSF group;
** indicates significant differences between the saline/THMA and
fluoxetine/THMA group.
and nitroethane34 quantitatively led to β-methyl-β-nitrostyrene (1),
which was converted, after the reduction of the nitro group and
hydrolysis of the imine function, into the corresponding 2,4,5-
trimethoxyphenylacetone (2) in 80.5% yield. Then, acid-catalyzed
reductive amination by sodium triacetoxyborohydride38 quantita-
tively yielded 2,4,5-trimethoxymethamphetamine (3). Finally, com-
plete demethylation of 3 using hydrobromic acid heated at reflux,
afforded THMA 4 in 76% yield. Interestingly, this four-step reaction
sequence produced THMA in a markedly improved overall yield
(60%) when compared with that of the previously described
procedure, starting from commercially available 3,4-(methyl-
enedioxy)phenol, for which the overall yield did not exceed 5% after
seven steps.23 Although it was previously reported that THMA could
be synthesized from 2,4,5-trimethoxybenzaldehyde by modification
of the procedure for 4-hydroxy-3-methoxymethamphetamine, no
details of the experiments were provided.22 Therefore, the experi-
ments and the spectroscopic characterizations are thoroughly de-
tailed in this work.
insoluble melanin-like polymers (Scheme 3, path a). To over-
come this problem, we decided to adapt our electrochemical
procedure, previously described for the preparation of thioether
conjugates of HHMA,40,41 to the synthesis of 6-NAC-THMA 5.
Therefore, the anodic oxidation of THMA 4 was conducted
under acidic conditions (Scheme 3, path b), in the absence
of N-acetylcysteine (NAC) because 6-NAC-THMA 5 was also
electroactive at the applied potential, giving no desired additional
oxidation products.
Controlled potential electrolysis was used as a preparative
method for the isolation of 6-NAC-THMA 5. When the con-
trolled potential of the platinum anode was fixed at þ1.0 V versus
Ag/AgCl, which is at a potential at which THMA could be
oxidized to the p-quinone species, a coulometric value of 2.0 (
0.1 was found for the number of electrons involved in the
oxidation of one molecule of THMA into the transient p-quinone
species. The latter was rather stable in aqueous 0.2 M HCl as
shown by monitoring the UV absorption spectrum in the course
of the electrolysis (Figure 1). After the application of the
Electrosynthesis of 6-NAC-THMA 5. As previously described
in the case of 6-hydroxydopamine,39 2,4,5-trihydroxymetham-
phetamine was poorly stable in potassium phosphate buffer (pH
7.4) and rapidly air oxidized to a putative p-quinone species.
At pH 7.4, the ionization of the phenol group at the 4 position
[pKa = 4ꢀ5, as reported for 6-OHDA39] facilitated the deproto-
nation of the secondary amine function at the origin of the
intramolecular cyclization, leading to an indoline intermediate.
This was converted into a redox active 5,6-dihydroxyindole
species which, after subsequent oxidation reaction, afforded
974
dx.doi.org/10.1021/tx2001459 |Chem. Res. Toxicol. 2011, 24, 968–978

Chemical Research in Toxicology
ARTICLE
Figure 5. Mass chromatograms of the target ions of THMA (a), HHMA (b), and HMMA (c). Top chromatograms of each part (aꢀc) show given ions in
rat brain tissue spiked with THMA, HHMA, and HMMA, respectively. Final tissue concentration of each spiked analyte was 5 μg/g. The bottom
chromatograms of each part (aꢀc) show the given ions in authentic brain tissue samples taken from rats (N = 3) treated with a high neurotoxic dose of
MDMA (80 mg/kg; SC) 3 h previously. Similar results were obtained in rats treated identically but sacrificed 1.5 (instead of 3) h after drug administration.
potential, a decrease in the UV absorption band shown by
THMA at 292 nm (ε = 4300 molꢀ1 L cmꢀ1) was observed,
while two new bands at 264 and 389 nm developed. Spectral
changes showed three isosbestic points at 230, 283, and 309 nm,
indicating that a simple equilibrium between two species was
shifted (Figure 1, spectra aꢀe). Subsequent addition of 2 equiv of
N-acetylcysteine resulted in the slow discoloration of the pale
orange solution due to the formation of the catecholꢀ-thioether
conjugate 5, which was identified by the change in the UV
absorption spectrum (Figure 1, spectra eꢀi), showing new
absorption maxima at 261 and 298 nm. Treatment of the
electrolysis solution afforded, after semipreparative reversed-
phase HPLC (see the Experimental Procedures section),
6-NAC-THMA 5 in 71% yield.
In a second experiment aimed at comparing the results of
anodic oxidation with that of the enzymatic procedure reported
below, electrolysis was performed in potassium phosphate buffer
(pH 7.4), in the presence of NAC, in other words, under the
experimental conditions required for enzymatic oxidation. After
the application of the potential, the electrolysis solution rapidly
became red, in agreement with the formation of quinonoid
species. This was substantiated by monitoring the UV absorption
spectrum in the course of the electrolysis. A decrease in the UV
absorption band at 292 nm was recorded while new bands at 278
and 489 nm developed. Contrary to what has been observed
under acidic conditions, spectral changes did not show isosbestic
points. Consequently, to limit the decomposition of the electro-
generated catecholꢀthioether conjugate
5
which was
975
dx.doi.org/10.1021/tx2001459 |Chem. Res. Toxicol. 2011, 24, 968–978

Chemical Research in Toxicology
ARTICLE
electroactive at the applied potential, the electrolysis was stopped
after the consumption of 1.75 electron per molecule. Treatment
of the electrolysis solution afforded, after semipreparative re-
versed-phase HPLC, 6-NAC-THMA 5 in 27% yield.
Enzymatic Procedure for the Synthesis of 6-NAC-THMA 5.
To ensure that 6-NAC-THMA 5 could be formed enzymatically,
THMA was oxidized with mushroom tyrosinase in the presence
of 5 equiv of NAC (see the Experimental Procedures section), a
method previously reported for the synthesis of thioether con-
jugates of HHMA.18,25,42 Because of the rapid degradation of the
solution at 37 °C, the reaction was stopped after only 10 min by
acidification of the reaction mixture to pH 2.0 with concentrated
HCl. After semipreparative reversed-phase HPLC, 6-NAC-
THMA 5 was isolated in 21% yield. Although 6-NAC-THMA
5 can be prepared enzymatically, this procedure was not adapted
for routine synthesis because it was too expensive and not
suitable for yielding substantial amounts of thioether conjugates.
In this respect, the electrosynthesis performed under acidic
conditions proved to be particularly attractive for routine synth-
esis, leading to 6-NAC-THMA 5 in high yield (71%).
Biological Studies of THMA 4. THMA 4 administered into
the right lateral ventricle of rats produced a significant lasting
depletion of 5-HT and 5-HIAA in the cortex and hippocampus
(Figure 2). 5-HT and 5-HIAA levels were also reduced in the
striatum, but the reductions did not achieve statistical signifi-
cance. In contrast, significant effects on DA were evident in the
striatum (Figure 3). Taken together, these results confirm
previous reports that THMA has the potential to produce lasting
effects on brain 5-HT and DA neurons.23,24
The lasting effect of THMA on brain 5-HT neurons could be
blocked with the 5-HT uptakeinhibitor fluoxetine (Figure4). This
observation indicates that the lasting effect of THMA on brain
5-HT neurons, like that of MDMA, is dependent on intact 5-HT
transporter function.30,43 Further, it suggests that THMA could
play a role in MDMA’s long-term effects on brain 5-HT neurons.
If THMA is, in fact, the metabolite that mediates MDMA
neurotoxicity, its presence in the brain would be anticipated. We
therefore sought to identify THMA in the brain of MDMA-
treated animals. While other O-demethylenated MDMA meta-
bolites of MDMA (HHMA and HMMA) could be readily and
reliably detected in the brains of rats treated with a high
neurotoxic dose of MDMA (80 mg/kg), THMA could not be
detected in either the cortex or hippocampus, at either 1.5 or 3 h
after MDMA administration (Figure 5).
Recognizing that our inability to detect THMA in the brain of
MDMA-treated rats might be due to the inherent instability of
the THMA molecule, we next carried out studies to determine
the stability of THMA in vivo. In particular, we administered
THMA to rats intracerebroventricularly, then attempted to
measure THMA in various rat brain regions at several times
after THMA administration. Figure 6 shows timeꢀconcentra-
tion profiles of THMA in the cortex, hippocampus, and striatum
of rats treated in manner identical to those whose lasting 5-HT
and 5-HIAA depletions are shown in Figure 2. THMA levels
peaked approximately 0.5ꢀ1 h after THMA administration and
could be measured up to 4 h. These results demonstrate that it is
feasible to detect and reliably measure THMA (and its indoline
formed via cyclization; not shown) in rat brain tissue for several
hours after its intracerebroventricular administration. Thus, it
appears that our inability to identify THMA in the rat brain after
peripheral MDMA administration is not related to instability of
the THMA molecule. The absence of detectable levels of THMA
Figure 6. Concentrations of THMA in the ipsilateral hippocampus (a),
striatum (b), and cerebral cortex (c) at various time points after
intracerebroventricular administration of 250 or 1000 nmol of THMA.
Values represent the mean ( SD (N = 3 rats at each time point).
after MDMA administration casts doubt on the view that
THMA, at least in its free (unconjugated) form, mediates
MDMA’s lasting effects on brain 5-HT neurons. At this time, it
cannot be stated with absolute certainty that other conjugated
forms of THMA, such as sulfate or glucuronic conjugates, are not
present in the brains of MDMA-treated rats because the present
THMA analyses were done without performing conjugate clea-
vage (because the cleavage procedure results in THMA degrada-
tion and disappearance).
Notably, inspection of Figures 2 and 6 shows that there was no
correlation between THMA concentrations and lasting 5-HT
deficits in various rat brain regions. That is, higher concentrations
of THMA were not associated with greater 5-HT deficits. This
may be yet another indication that THMA does not mediate the
long-term effects of MDMA on brain 5-HT neurons because, in
general, there is an excellent correlation between brain MDMA
concentrations and lasting 5-HT deficits.20 In considering the
lack of correlation between THMA concentrations and regional
brain 5-HT deficits, it is also important to take into account that
5-HT terminals in different brain regions may differ in their
susceptibility to various neurotoxicants, including THMA.
Further, it is possible that 5-HT terminals in the striatum are
less affected because some THMA in this brain region may be
sequestered within DA terminals, effectively reducing the
amount of THMA to which 5-HT terminals are exposed.
Biological Studies of 6-NAC-THMA 5. Given reports impli-
cating thioether conjugates of O-demethylenated metabolites of
976
dx.doi.org/10.1021/tx2001459 |Chem. Res. Toxicol. 2011, 24, 968–978

Chemical Research in Toxicology
ARTICLE
THMA, 6-NAC-THMA, lacks 5-HT neurotoxic potential, as
evidenced by the fact that it fails to produce lasting effects on
brain 5-HT axonal markers. Taken together, these observations
suggest that neither THMA 4 nor 6-NAC-THMA 5 is directly
responsible for MDMA neurotoxicity but leave open the possibi-
lity that forms of THMA different from those measured here (e.g.,
different THMA conjugates or oxidized cyclic forms) could be
involved. Additional research is required to test these possibilities.
’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR spectra of
S
b
THMA and its synthetic intermediates, together with 6-(N-
acetylcystein-S-yl)-THMA; analytical HPLC chromatograms of
THMA and of 6-(N-acetylcystein-S-yl)-THMA. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: (þ) 33 1 53 73 96 46. Fax: (þ) 33 1 44 07 35 88. E-mail:
ricaurte@jhmi.edu (G.A.R.); martine.largeron@parisdescartes.fr
(M.L.).
Funding Sources
C.M.M. is a Ph.D. student funded by the French MENRT. This
Research was supported by the joint grants of the “Mission
Interministꢀerielle de Lutte contre la Drogue et la Toxicomanie”
(MILDT) and the “Institut National de la Santꢀe et de la
Recherche Mꢀedicale” (INSERM).(Appel ꢁa projets commun
2007 MILDT-INSERM “Recherche sur les drogues et la
toxicomanie”) (to M.L.) as well as by NIH grants DA 05707
and DA 01796401 (to G.A.R.).
Figure 7. 5-HT levels and 5-HIAA levels in the ipsilateral frontal cortex
of rats two weeks after cortical administration of 42 nmol of 6-NAC-
THMA. The thioether conjugate was injected four times, with a 12-h
interval between each injection. 6-NAC-THMA was dissolved in aCSF
shortly before each injection. Control animals received unilateral
intraparenchymal injections of an equivalent volume of aCSF. A positive
control group consisted of animals that received a single intraparench-
ymal injection of 52 nmol of 5,7-DHT. Values represent the mean ( SD
(N = 8 in 6-NAC-THMA groups, N = 6 in positive control groups, and
N = 6 in control groups). One-way ANOVA followed by Tukey’s multiple
comparison test was performed and differences regarded as significant
when p < 0.05. * Indicates significant differences from the control group.
’ REFERENCES
(1) Green, A. R., Mechan, A. O., Elliott, J. M., O’Shea, E., and
Colado, M. I. (2003) The pharmacology and clinical pharmacology of
3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”). Pharmacol.
Rev. 55, 463–508.
MDMA as mediators of MDMA neurotoxicity,10,15ꢀ18,20,27,28 we
next assessed the neurotoxic potential of 6-NAC-THMA 5, a
thioether conjugate of THMA. These studies involved a direct
injection of 6-NAC-THMA 5 into cortical tissue, using the
known 5-HT neurotoxin, 5,7-DHT, as a positive control. As
expected, 5,7-DHT produced lasting depletions of both 5-HT
and 5-HIAA in the brain (Figure 7). In contrast, 6-NAC-THMA
5 was without long-term effects on brain 5-HT neuronal markers.
These results suggest that 6-NAC-THMA 5 lacks significant
5-HT neurotoxic potential and do not support the view that this
particular thioether conjugate of THMA is responsible for
mediating MDMA-induced 5-HT neurotoxicity.
(2) Sakar, S., and Schmued, L. (2010) Neurotoxicity of ecstasy
(MDMA): an overview. Curr. Pharm. Biotechnol. 11, 460–469.
(3) Molliver, M. E., Berger, U. V., Mamounas, L. A., Molliver, D. C.,
O’Hearn, E., and Wilson, M. A. (1990) Neurotoxicity of MDMA and
related compounds: anatomic studies. Ann. N.Y. Acad. Sci. 600, 649–661.
(4) Commins, D. L., Vosmer, G., Virus, R. M., Woolverton, W. L.,
Schuster, C. R., and Seiden, L. S. (1987) Biochemical and histological
evidence that methylenedioxymethylamphetamine (MDMA) is toxic to
neurons in the rat brain. J. Pharmacol. Exp. Ther. 241, 338–345.
(5) Callahan, B. T., Cord, B. J., and Ricaurte, G. A. (2001) Long-term
impairment of anterograde axonal transport along fiber projections
originating in the rostral raphe nuclei after treatment with fenfluramine
or methylenedioxymethamphetamine. Synapse 40, 113–121.
(6) Yamamoto, B. K., Moszczynska, A., and Gudelsky, G. A. (2010)
Amphetamine toxicities: classical and emerging mechanisms. Ann. N.Y.
Acad. Sci. 1187, 101–121.
(7) Mechan, A., Yuan, J., Hatzidimitriou, G., Irvine, R. J., McCann,
U. D., and Ricaurte, G. A. (2006) Pharmacokinetic profile of single and
repeated oral doses of MDMA in squirrel monkeys: relationship to lasting
effects on brain serotonin neurons. Neuropsychopharmacology 31, 339–350.
(8) Easton, N., and Marsden, C. A. (2006) Ecstasy: are animal data
consistent between species and can they translate to humans?
J Psychopharmacol. 20, 194–210.
’ CONCLUSIONS
THMA produces lasting effects on brain 5-HT neurons. The
lasting effect of THMA on brain 5-HT neurons, like that of
MDMA, is dependent upon intact 5-HT transporter function. In
contrast to other O-demethylenated MDMA metabolites
(HHMA and HMMA), THMA was not detected in the brain
tissue of rats treated peripherally with a high neurotoxic dose of
MDMA. Inability to detect THMA in the brain after peripheral
MDMA administration was not due to the instability of the
THMA molecule because exogenous THMA administered cen-
trally could be readily detected in rat brains for several hours after
intracerebroventricular administration. The thioether conjugate of
(9) Schmidt, C. J., Wu, L., and Lovenberg, W. (1986) Methylene-
dioxymethamphetamine: a potentially neurotoxic amphetamine analo-
gue. Eur. J. Pharmacol. 124, 175–178.
977
dx.doi.org/10.1021/tx2001459 |Chem. Res. Toxicol. 2011, 24, 968–978

Chemical Research in Toxicology
ARTICLE
(10) Capela, J. P., Carmo, H., Remiao, F., Lourdes Bastos, M.,
Meisel, A., and Carvalho, F. (2009) Molecular and cellular mechanisms
of ecstasy-induced neurotoxicity: an overview. Mol. Neurobiol. 39,
210–271.
(11) Schmidt, C. J., and Taylor, V. L. (1988) Direct central effects of
acute methylenedioxy-methamphetamine on serotonergic neurons. Eur.
J. Pharmacol. 156, 121–131.
(12) Paris, J. M., and Cunningham, K. A. (1992) Lack of serotonin
neurotoxicity after intraraphe microinjection of (()-3,4-methylenediox-
ymethamphetamine (MDMA). Brain Res. Bull. 28, 115–119.
(13) Esteban, B., O’Shea, E., Camarero, J., Sanchez, V., Green, A. R.,
and Colado, M. I. (2001) 3,4-Methylenedioxymethamphetamine in-
duces monoamine release, but not toxicity, when administered centrally
at a concentration occurring following a peripherally injected neurotoxic
dose. Psychopharmacology (Berlin) 154, 251–260.
metabolites of 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”):
synthesis, isolation and characterization of diastereoisomers. Chem. Res.
Toxicol. 21, 2272–2279.
(27) Perfetti, X., O’Mathuna, B., Pizarro, N., Cuyas, E., Khymenets,
O., Almeida, B., Pellegrini, M., Pichini, S., Lau, S. S., Monks, T. J., Farrꢀe,
M., Pascual, A., Joglar, J., and de La Torre, R. (2009) Neurotoxic
thioether adducts of MDMA identified in human urine after ecstasy
ingestion. Drug Metab. Dispos. 37, 1448–1455.
(28) Capela, J. P., Macedo, C., Branco, P. S., Ferreira, L. M., Lobo,
A. M., Fernandes, E., Remiao, F., Bastos, M. L., Dirnagl, U., Meisel, A.,
and Carvalho, F. (2007) Neurotoxicity mechanisms of thioether ecstasy
metabolites. Neuroscience 146, 1743–1757.
(29) Liang, Y. -O., Plotsky, P. M., and Adams, R. N. (1977) Isolation
and identification of an in vivo reaction product of 6-hydroxydopamine.
J. Med. Chem. 20, 581–583.
(14) Escobedo, I., O’Shea, E., Orio, L., Sanchez, V., Segura, M., de la
Torre, R., Farre, M., Green, A. R., and Colado, M. I. (2005) A
comparative study on the acute and long-term effects of MDMA and
3,4-dihydroxymethamphetamine (HHMA) on brain monoamine levels
after i.p. or striatal administration in mice. Br. J. Pharmacol. 144,
231–241.
(15) Miller, R. T., Lau, S. S., and Monks, T. J. (1997) 2,5-Bis-(glu-
tathion-S-yl)-alpha-methyldopamine, a putative metabolite of (()-3,4-
methylenedioxyamphetamine, decreases brain serotonin concentra-
tions. Eur. J. Pharmacol. 323, 173–180.
(16) Bai, F., Lau, S. S., and Monks, T. J. (1999) Glutathione and
N-acetylcysteine conjugates of R-methyldopamine produce serotoner-
gic neurotoxicity: possible role in methylenedioxyamphetamine-
mediated neurotoxicity. Chem. Res. Toxicol. 12, 1150–1157.
(17) Monks, T. J., Jones, D. C., Bai, F., and Lau, S. S. (2004) The role
of metabolism in 3,4-(þ)-methylenedioxyamphetamine and 3,4-(þ)-
methylenedioxymethamphetamine (ecstasy) toxicity. Ther. Drug Monit.
26, 132–136.
(30) Schmidt, C. J. (1987) Neurotoxicity of the psychedelic ampheta-
mine, methylenedioxymethamphetamine. J. Pharmacol. Exp. Ther. 240, 1–7.
(31) Borgman, R. J., Baylor, M. R., McPhillips, J. J., and Stitzel, R. E.
(1974) R-Methyldopamine derivatives. Synthesis and pharmacology.
J. Med. Chem. 17, 427–430.
(32) Morgan, P. H., and Beckett, A. H. (1975) Synthesis of some
N-oxygenated products of 3,4-dimethoxyamphetamine and its N-alkyl
derivatives. Tetrahedron 31, 2595–2601.
(33) Cannon, J. G., Perez, Z., Long, J. P., Rusterholtz, D. B., Flynn,
J. R., Costall, B., Fortune, D. H., and Naylor, R. J. (1979) N-alkyl
derivatives of (()-R-methyldopamine. J. Med. Chem. 22, 901–907.
(34) Milhazes, N., Cunha-Oliveira, T., Martins, P., Garrido, J.,
Oliveira, C., Rego, A. C., and Borges, F. (2006) Synthesis and cytotoxi-
city profile of 3,4-methylenedioxymethamphetamine (“ecstasy”) and its
metabolites on undifferentiated PC12 cells: a putative structure-toxicity
relation ship. Chem. Res. Toxicol. 19, 1294–1304.
(35) Paxinos, G., and Watson, C. (1986) The Rat Brain in Stereotaxic
Coordinates, Academic Press, Inc., New York.
(18) Jones, D. C., Duvauchelle, C., Ikegami, A., Olsen, C. M., Lau,
S. S., de la Torre, R., and Monks, T. J. (2005) Serotonergic neurotoxic
metabolites of ecstasy identified in rat brain. J. Pharmacol. Exp. Ther.
313, 422–431.
(19) Steele, T. D., Brewster, W. K., Johnson, M. P., Nichols, D. E.,
and Yim, G. K. W. (1991) Assessment of the role of alpha-methylepinine
in the neurotoxicity of MDMA. Pharmacol., Biochem. Behav.
38, 345–351.
(20) Mueller, M., Yuan, J., Felim, A., Neud€orffer, A., Peters, F. T.,
Maurer, H. H., McCann, U. D., Largeron, M., and Ricaurte, G. A. (2009)
Further studies on the role of metabolites in (()-3,4-methylenediox-
ymethamphetamine-induced serotonergic neurotoxicity. Drug Metab.
Dispos. 37, 2079–2086.
(21) McCann, U. D., and Ricaurte, G. A. (1991) Major metabolites
of (()-3,4-methylenedioxyamphetamine (MDA) do not mediate its
toxic effects on brain serotonin neurons. Brain Res. 545, 279–282.
(22) Lim, H. K., and Foltz, R. L. (1991) Ion trap tandem mass
spectrometric evidence for the metabolism of 3,4-(methylenedioxy)-
methamphetamine to the potent neurotoxins 2,4,5-trihydroxymethamphe-
tamine and 2,4,5-trihydroxyamphetamine. Chem. Res. Toxicol. 4, 626–632.
(23) Zhao, Z., Castagnoli, N., Ricaurte, G. A., Steele, T., and Martello, M.
(1992) Synthesis and neurotoxicological evaluation of putative metabolites of
the serotonergic neurotoxin 2-(methylamino)-1-[3,4-(methylenedioxy)-
phenyl]propane[(Methylenedioxy)methamphetamine]. Chem. Res. Toxicol.
5, 89–94.
(24) Johnson, M., Elayan, I., Hanson, G. R., Foltz, R. L., Gibb, J. W.,
and Lim, H. K. (1992) Effects of 3,4-dihydroxymethamphetamine and
2,4,5-trihydroxymethamphetamine, two metabolites of 3,4-methylene-
dioxymethamphetamine, on central serotonergic and dopaminergic
systems. J. Pharmacol. Exp. Ther. 261, 447453.
(25) Erives, G. V., Lau, S. S., and Monks, T. J. (2008) Accumulation
of neurotoxic thioether metabolites of 3,4-(()-methylenedioxy-
methamphetamine in rat brain. J. Pharmacol. Exp. Ther. 324, 284–291.
(26) Pizarro, N., de la Torre, R., Joglar, J., Okumura, N., Perfetti, X.,
Lau, S. S., and Monks, T. J. (2008) Serotonergic neurotoxic thioether
(36) Heffner, T. G., Hartman, J. A., and Seiden, L. S. (1980) A rapid
method for the regional dissection of the rat brain. Pharmacol., Biochem.
Behav. 13, 453–456.
(37) Mueller, M., Peters, F. T., Ricaurte, G. A., and Maurer, H. H.
(2008) Liquid chromatographic-electrospray ionization mass spectro-
metric assay for simultaneous determination of 3,4-methylenedioxy-
methamphetamine and its metabolites 3,4-methylenedioxyamphetamine,
3,4-dihydroxymethamphetamine, and 4-hydroxy-3-methoxymethamphe-
tamine in rat brain. J. Chromatogr., B 874, 119–124.
(38) Jozwiak, K., Yiu-Ho Woo, A., Tanga, M. J., Toll, L., Jimenez, L.,
Kozocas, J. A., Plazinska, A., Xiao, R.-P., and Wainer, I. W. (2010)
Comparative molecular field analysis of fenoterol derivatives: a platform
towards highly selective and effective β2-adrenergic receptor agonists.
Bioorg. Med. Chem. 18, 728–736.
(39) Blank, C. L., Kissinger, P. T., and Adams, R. N. (1972) 5,6-
dihydroxyindole formation from oxidized 6-hydroxydopamine. Eur. J.
Pharmacol. 19, 391–394.
(40) Felim, A., Urios, A., Neud€orffer, A., Herrera, G., Blanco, M., and
Largeron, M. (2007) Bacterial plate assays and electrochemical meth-
ods: an efficient tandem for evaluating the ability of catechol-thioether
metabolites of MDMA (“ecstasy”) to induce toxic effects through redox-
cycling. Chem. Res. Toxicol. 20, 685–693.
(41) Felim, A., Neud€orffer, A., Monnet, F. P., and Largeron, M.
(2008) Environmentally friendy expeditious one-pot electrochemical
synthesis of bis-catechol-thioether metabolites of ecstasy: in vitro neuro-
toxic effects in the rat hippocampus. Int. J. Electrochem. Sci. 3, 266–281.
(42) Macedo, C., Branco, P. S., Ferreira, L. M., Lobo, A. M., Capela,
J. P., Fernandes, E., de Lourdes Bastos, M., and Carvalho, F. (2007)
Synthesis and cyclic voltammetry studies of 3,4-methylenedioxymetham-
phetamine (MDMA) human metabolites. J. Health Sci. 53, 31–42.
(43) Sanchez, V., Camarero, J., Esteban, B., Peter, M. J., Green, A. R., and
Colado, M. I. (2001) The mechanisms involved in the long-lasting neuro-
protective effect of fluoxetine against MDMA (“ecstasy”)-induced degenera-
tion of 5-HT nerve endings in rat brain. Br. J. Pharmacol. 134, 46–57.
978
dx.doi.org/10.1021/tx2001459 |Chem. Res. Toxicol. 2011, 24, 968–978