Synthesis and in vitro cytotoxicity profile of the R-enantiomer of 3,4-dihydroxymethamphetamine (R-(-)-HHMA): Comparison with related catecholamines

Article abstract of DOI:10.1021/tx9003374

(±)-3,4-Methylenedioxymethamphetamine (MDMA, also known as "ecstasy") is a chiral drug that is essentially metabolized in humans through O-demethylenation into 3,4-dihydroxymethamphetamine (HHMA). There has recently been a resurgence of interest in the po

Full text of DOI:10.1021/tx9003374

Chem. Res. Toxicol. 2010, 23, 211–219
211
Synthesis and in Vitro Cytotoxicity Profile of the R-Enantiomer of
3,4-Dihydroxymethamphetamine (R-(-)-HHMA): Comparison with
Related Catecholamines
Anne Felim,† Guadalupe Herrera,‡ Anne Neudo¨rffer,† Manuel Blanco,‡
Jose´-Enrique O’Connor,*,‡ and Martine Largeron*,†
UMR 8638 CNRS, UniVersite´ Paris Descartes, Synthe`se et Structure de Mole´cules d’Inte´reˆt Pharmacologique, Faculte´
des Sciences Pharmaceutiques et Biologiques, 4 AVenue de l’ObserVatoire, 75270 Paris cedex 06, France, and
Laboratorio de Cito´mica, Unidad Mixta de InVestigacio´n CIPF-UVEG, Centro de InVestigacio´n Principe Felipe,
AVenida Autopista del Saler 16, 46012 Valencia, Spain
ReceiVed September 16, 2009
(()-3,4-Methylenedioxymethamphetamine (MDMA, also known as “ecstasy”) is a chiral drug that is
essentially metabolized in humans through O-demethylenation into 3,4-dihydroxymethamphetamine
(HHMA). There has recently been a resurgence of interest in the possibility that MDMA metabolites,
especially 5-(N-acetylcystein-S-yl)-N-methyl-R-methyldopamine (designated as 5-NAC-HHMA), might
play a role in MDMA neurotoxicity. However, the chirality of MDMA was not considered in previously
reported in vivo studies because HHMA, the precursor of the 5-NAC-HHMA metabolite, was used as
the racemate. Since the stereochemistry of this chiral drug needs to be considered, the first total synthesis
of R-(-)-HHMA is reported. Using L-DOPA as the chiral source, the preparation of R-(-)-HHMA is
achieved through seven steps, in 30% overall yield and 99.5% enantiomeric excess. The cytotoxicity of
R-(-)-HHMA and related catecholamines has been further determined by flow cytometric analysis of
propidium iodide uptake in human dopaminergic neuroblastoma SH-SY5Y cells and by an Escherichia
coli plate assay, specific for the detection of oxidative toxicity. The good correlation between the toxicities
observed in both systems suggests that SH-SY5Y cells are sensitive to oxidative toxicity and that cell
death (necrosis) would be mediated by reactive oxygen species mainly generated from redox active
quinonoid centers. In contrast, apoptosis was detected for 3,4-dimethoxymethamphetamine (MMMA),
the synthetic precursor of HHMA possessing a protected catechol group. MMMA was not toxic in the
bacterial assay, indicating that its toxicity is not related to increased oxidative stress. Finally, we can
conclude that there is a need to distinguish the toxicity ascribed to MDMA itself, also bearing a protected
catechol moiety, from that depending on MDMA biotransformation leading to catechol metabolites such
as HHMA and the thioether conjugates.
In humans, MDMA is essentially metabolized through
O-demethylenation into 3,4-dihydroxymethamphetamine (HHMA)
(16). Because of its catechol moiety, HHMA can be easily
oxidized to the corresponding o-quinone species, which exhibits
a double reactivity. First, it can undergo redox-cycling which
produces semiquinone radical and leads to the generation of
reactive oxygen species (ROS). Second, it behaves as a highly
electrophilic compound which can be conjugated with thiol
nucleophiles such as glutathione or N-acetylcysteine (NAC) to
form redox active catechol-thioether mono- and bis-conjugates
(17) able to induce cytotoxicity mediated by ROS (18).
Introduction
The alarming increase in the recreational use of (()-3,4-
methylenedioxymethamphetamine (MDMA, also known as
“ecstasy”) and the multitude of adverse effects resulting from
its misuse (1-5) require a complete understanding of the
pharmacology and toxicology of this synthetic psychoactive
drug. However, the precise mechanisms by which MDMA
induces neurotoxic effects, both in laboratory animals (rodents,
nonhuman primates) and humans, remain to be fully elucidated
(6-11). MDMA-mediated neurotoxicity could be related to
some of the adverse effects such as hyperthermia, the serotonin
(5-HT) transporter action, 5-HT2A agonism, glutamate excito-
toxicity, the monoamine oxidase metabolism of dopamine and
serotonin, and nitric oxide together with the formation of
damaging peroxynitrite, and, importantly, the formation of
MDMA neurotoxic metabolites (12). Among these factors,
which may act collectively in a synergistic manner conducive
to neurotoxicity, systemic metabolism of MDMA seems to be
a crucial step (13-15).
The potential role of metabolites in MDMA neurotoxicity
has been a topic of recent interest (12, 17, 19). Among them,
the catechol-thioether metabolite 5-(N-acetylcystein-S-yl)-N-
methyl-R-methyldopamine (here designated as 5-NAC-HHMA)
has been the most strongly implicated in MDMA neurotoxicity
(20, 21). Studies have been recently performed to further assess
the 5-HT neurotoxic potential of 5-NAC-HHMA, but the results
are at odds with those previously reported and argue against a
pivotal role for 5-NAC-HHMA in MDMA-induced 5-HT
neurotoxicity (19). The reasons for this discrepancy is not clear,
but it is important to note that 5-NAC-HHMA used in these
studies was prepared through a biomimetic electrochemical
oxidation process (18), whereas the procedure previously utilized
* Corresponding author. Tel: (+) 33 1 53 73 96 46. Fax: (+) 33 1 44 07
35 88. E-mail: eoconnor@ochoa.fib.es (J.E.O’C.); martine.largeron@
parisdescartes.fr (M.L.).
† Universite´ Paris Descartes.
‡ Centro de Investigacio´n Principe Felipe.
10.1021/tx9003374  2010 American Chemical Society
Published on Web 12/17/2009

212 Chem. Res. Toxicol., Vol. 23, No. 1, 2010
Felim et al.
Figure 2. Chemical structures of studied compounds.
Figure 1. Stereochemistry of the hepatic metabolism leading to R-(-)-
HHMA and S-(+)-HHMA.
In this article, we report the first total synthesis of R-(-)-
HHMA in 30% overall yield and 99.5% enantiomeric excess
(ee), using L-DOPA as the inducer of chirality. To the best of
our knowledge, a sole synthesis of enantiomerically enriched
S-(+)-HHMA (80% ee) is known (33). Consequently, as long
as the synthesis of optically pure S-enantiomer is not reported,
the enantioselective toxicity of both enantiomers of HHMA
cannot be addressed. Nevertheless, we have decided to examine
the in vitro toxicity profile of R-(-)-HHMA in a broader context,
using different assays which could be predictive of the
toxicological effects associated with catecholamine-type struc-
tures. The cytotoxicity of R-(-)-HHMA and related catechola-
mines (Figure 2) has been assessed in human dopaminergic
neuroblastoma SH-SY5Y cells. Flow cytometry has been used
to discriminate between live, apoptotic, and necrotic cells and
to measure the activity of caspase-3, an effector of apoptosis.
In addition, the cytotoxicity observed in SH-SY5Y cells has
been compared with that evaluated in an Escherichia coli plate
assay which is specific for the detection of oxidative toxicity
(34).
for synthesizing this metabolite involved the oxidation of the
catechol with mushroom tyrosinase (20), which yields a different
ratio of 5-NAC-HHMA diastereoisomers (22). Consequently,
for a comprehensive study on MDMA-induced neurotoxicity,
the stereochemistry of the metabolism of this chiral drug needs
to be considered (23).
MDMA is consumed as a racemate that is a 1:1 mixture of
R-(-)-MDMA and S-(+)-MDMA enantiomers (Figure 1). Both
enantiomers show different pharmacological and pharmacoki-
netic profiles (24-30). For most of the known psychomimetic
agents having at least one chiral center, the most potent isomer
exhibits the absolute R configuration (24). In the case of
MDMA, the potency assignment is reversed since S-(+)-MDMA
has been shown to display effective psychomimetic properties
in humans. Furthermore, although R-(-)-MDMA has the higher
affinity for the serotonin receptor 5-HT2, the S-(+)-MDMA
isomer produces the most lasting 5-HT deficits that probably
reflects its neurotoxicity (25). Metabolism could also contribute
to differences between both enantiomers. Especially, the dif-
ference in their pharmacokinetic properties may be caused by
enantioselective metabolism by cytochrome P450 isoenzymes,
in particular CYP2D6 and CYP2C19 (23). Therefore, HHMA,
which is a major MDMA metabolite in humans, exists also as
a pair of enantiomers (30) (Figure 1). HHMA being the
precursor of the thioether metabolites, these conjugates exist
as a mixture of diastereoisomers as they possess several chiral
centers: that of HHMA present in the alkylamino side chain
(carbon atom R to the amine function), together with those
present in the thiol moieties. Because MDMA enantiomers have
different biological activities, it is of a great interest to study
whether the catechol-thioether diastereoisomers of HHMA also
exhibit dissimilar biological properties. The preparation of such
diastereoisomers requires the previous synthesis of enantio-
merically pure precursors R-(-)-HHMA and S-(+)-HHMA,
which are more difficult to synthesize than the racemate. To
circumvent this problem, analytical and semipreparative meth-
odologies for the diastereoisomeric separation of MDMA
thioether conjugates have been recently reported, but this
separation method only furnished small quantities of both
diastereoisomers (22).
Experimental Procedures
Chemistry. All reagents and solvents (HPLC grade) were
commercial products of the highest available purity and were used
as supplied. Dopamine hydrochloride (DA, HCl) was purchased
from Aldrich.
Analytical thin-layer chromatography was carried out on silica
gel Macherey-Nagel Polygram SIL G/UV 254 (0.25 mm). Column
chromatography was performed on Macherey-Nagel Si 60 M silica
gel (40-63 µm). Optical rotations were measured at 25 °C using
a Perkin-Elmer 341 polarimeter. Melting points were measured on
a Ko¨fler 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 UV-visible detector set at 254 and 278
nm. A mixture of two solvents constituted the mobile phase:
acetonitrile (MeCN) and solvent A. Solvent A was prepared by
adding 1‰ concentrated trifluoroacetic acid (TFA) to 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, were used.
1H NMR and 13C NMR spectra were performed on a Bruker
AC-300 spectrometer operating at 300 and 75 MHz, respectively.
Chemical shifts are expressed as δ units (part per million) downfield
from TMS (tetramethylsilane). The measurements were carried out
using the standard pulse sequences. The carbon type (methyl,
methylene, methine, or quaternary) was determined by DEPT
experiments. 1H 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. (()-3,4-
This prompted us to envision the total synthesis of enantio-
merically pure precursors, R-(-)-HHMA and S-(+)-HHMA.
Starting from these compounds, it would be then easy to prepare
the corresponding diastereomerically pure forms of thioether
metabolites, either through the straightforward one-pot electro-
chemical synthesis we have recently reported for preparing
diverse catechol-thioether mono- and bis-conjugates starting
from (()-HHMA racemate (18, 31) or by using the enzymatic
procedure described by other investigators (20, 21, 32).

Synthesis and Cytotoxicity Profile of R-(-)-HHMA
Chem. Res. Toxicol., Vol. 23, No. 1, 2010 213
dihydroxymethamphetamine hydrobromide (HHMA, HBr) was
synthesized in two steps from commercially available 3,4-
dimethoxyphenylacetone using an adaptation of previously reported
procedures with slight modifications (35-38).
in 99% yield. mp 78-79 °C; (literature (39) mp 77-78 °C); [R]25
D
+ 4.6 (c 0.05, MeOH); literature (39) [R]23D +6.0 (c 1.0, MeOH).
Spectroscopic data were identical to those previously reported
(39, 42, 43).
(()-3,4-Dimethoxymethamphetamine [(()-MMMA]. (()-
MMMA was previously described as the hydrochloride salt (37, 38);
6.45 mL of a 40% aqueous solution of methylamine (8 equiv) were
added dropwise to a solution of 3,4-dimethoxyphenylacetone (3.0
g, 15.45 mmol) in MeOH (50 mL) at 0 °C. The resulting solution
was stirred under nitrogen at 0 °C for 30 min, and sodium
borohydride (NaBH4) (632 mg, 16.7 mmol) was added in small
portions for 30 min. Then, the reaction mixture was allowed to
warm at room temperature and stirred for 1 h. After the addition
of K2CO3 (2.2 g), the solvent was evaporated under reduced
pressure. H2O (15 mL) was added to the residue, and the reaction
mixture was extracted with diethyl ether (40 mL). The organic phase
was then washed with 1 N HCl (15 mL). The resulting acidic
aqueous phase was alkalinized with K2CO3 and extracted with
diethyl ether (50 mL). The organic phase was then dried over
MgSO4 and filtered off, and the solvent was evaporated. Column
chromatography (dichloromethane-MeOH 80/20 v/v) afforded
(()-MMMA as a yellow oil (2.23 g, 10.65 mmol) in 69% yield.
1H NMR (CDCl3) δ 1.09 (d, J ) 6.5 Hz, 3H), 1.44 (broad s, 1H),
2.41 (s, 3H), 2.62 (dd, J ) 13.4 Hz, 2H), 2.77 (m, 1H), 3.88 (s,
3H), 3.89 (s, 3H), 6.75 (m, 2H), 6.83 (d, J ) 8.1 Hz, 1H). 13C
NMR (CDCl3) δ 15.2, 30.4, 39.2, 55.8, 55.9, 57.0, 111.4, 112.1,
121.3, 128.0, 148.0, 149.2.
tert-Butyl [(S)-2-(3,4-Dimethoxyphenyl)-1-(hydroxymethyl)ethyl]-
carbamate (4). Lithium aluminum hydride (LiAlH4) solution in THF
(1 M, 35.4 mL) was added dropwise to a solution of compound 3
(3 g, 8.84 mmol) in dry THF (40 mL). The reaction mixture was
stirred under nitrogen at room temperature for 1 h. Then, 0.5 mL
of ice-water and 0.5 mL of 0.5 M NaOH were successively added.
The solid Al(OH)3 was filtered off and washed with dichlo-
romethane (2 × 40 mL). The filtrate was concentrated to dryness,
and the residue was dissolved in dichloromethane (40 mL). The
resulting organic phase was then washed with water (20 mL), dried
over MgSO4, filtered off, and then concentrated. Column chroma-
tography (petroleum ether/ethyl acetate 60/40 v/v) afforded N-
protected R-aminoalcohol 4 as a white solid (2.5 g, 8.0 mmol) in
90% yield. mp 94-95 °C; (literature (39) mp 91-92 °C); [R]25
D
-19.0 (c 0.01, MeOH); literature (39) [R]23D -19.6 (c 0.3, MeOH).
Spectroscopic data were identical to those previously reported
(39, 42, 43).
(S)-2-(tert-Butoxycarbonylamino)-3-(3,4-dimethoxyphenyl)-1-(4-
methylbenzenesulfonyloxy) Propane (5). To a solution of R-ami-
noalcohol (4) (2.34 g,7.51 mmol) in dichloromethane (15.5 mL)
dried over CaCl2 were added triethylamine (1.57 mL, 11.27 mmol),
p-toluenesulfonyl chloride (1.72 g, 9.01 mmol), and dimethylami-
nopyridine (DMAP) (918 mg, 0.750 mmol). After stirring for 18 h
at room temperature, under nitrogen, the reaction mixture was
diluted with dichloromethane (40 mL). The organic phase was then
washed with water (20 mL), dried over MgSO4, and filtered off,
and the solvent was evaporated under reduced pressure. The crude
residue was recrystallized from the diethyl oxide/dichloromethane
mixture affording compound 5 as a white solid (2.94 g, 6.3 mmol)
in 84% yield. mp 155-156 °C; (literature (44) mp 146-148 °C);
[R]25D -16.2 (c 0.09, MeOH); literature (44) [R]25D -15.4 (c 0.36,
(()-3,4-Dihydroxymethamphetamine hydrobromide (HHMA,
HBr). A 48% aqueous solution of HBr (3.3 mL) (18.7 mmol) was
added to (()-MMMA (523 mg, 2.5 mmol). The resulting solution
was heated to reflux for 1.5 h, under inert atmosphere. After the
addition of 0.5 mL of methanol and evaporation under reduced
pressure, the purple oil was purified by column chromatography
(dichloromethane-MeOH 90:10 v/v) affording HHMA, HBr as a
colorless oil (524 mg, 2.0 mmol) in 80% yield. Spectroscopic data
were identical to those previously reported (38).
1
CHCl3). H NMR (CDCl3) δ 1.41 (s, 9H), 2.47 (s, 3H), 2.80 (m,
2H), 3.87 (s, 6H), 3.95 (m, 2H), 4.73 (d, J ) 7.0 Hz, 1H), 6.64 (d,
J ) 7.9 Hz, 1H), 6.71 (s, 1H), 6.74 (d, J ) 7.9 Hz, 1H), 7.36 (d,
J ) 8.2 Hz, 2H), 7,78 (d, J ) 8.2 Hz, 2H). 13C NMR (CDCl3) δ
21.7, 28.3, 36.7, 50.8, 55.9 (×2), 69.9, 79.8, 111.2, 112.3, 121.3,
128.0, 129.1, 130.0, 132.5, 145.1, 147.8, 149.0, 155.0.
Methyl (S)-2-Amino-3-(3,4-dihydroxyphenyl)propanoate Hy-
drochloride (1). Thionyl chloride (6.57 mL, 90 mmol) was added
dropwise to a solution of L-DOPA (1.97 g, 10 mmol) in MeOH
(100 mL) at 0 °C. After 18 h at 20 °C, the solvent was evaporated
giving amine chlorohydrate 1 in 100% yield (2.48 g) as a white
solid; mp 95-96 °C (surprisingly, this value markedly differs from
that previously reported in the literature (39) mp 174-175 °C);
[R]25 + 9.4 (c 0.03, MeOH); literature (39) [R]23 +7.9 (c 1.0,
(R)-3,4-Dimethoxymethamphetamine (6) and (S)-3-(3,4-dimeth-
oxyphenyl)-2-(N-methylamino)-propan-1-ol (7). LiAlH4 solution in
THF (1 M, 4.45 mL) was added dropwise to a solution of compound
5 (501 mg, 1.08 mmol) in dry dichloroethane (13.5 mL) at 0 °C.
The resulting solution was allowed to warm to room temperature,
under nitrogen, and stirred for 45 min. Then, the reaction mixture
was heated to reflux for 1.5 h. After hydrolysis with 0.5 mL of
ice-water and 0.5 mL of 0.5 M NaOH, the solid Al(OH)3 was
filtered off and washed with dichloromethane (2 × 40 mL). The
filtrate was concentrated to dryness. Column chromatography of
the residue (dichloromethane/methanol 95/5 v/v with 1.5% triethy-
lamine) afforded compounds 6 (134.5 mg, 0.64 mmol) and 7 (31
mg, 0.15 mmol) as colorless oils in 60% and 13% yields,
respectively.
D
D
MeOH). Spectroscopic data were identical to those previously
reported (39, 40).
Methyl (R)-2-(tert-Butoxycarbonylamino)-3-(3,4-dihydroxyphe-
nyl)-propanoate (2). Satured aqueous NaHCO3 (7.5 mL) and di-
tert-butyl dicarbonate (Boc2O) (1.25 g, 5.41 mmol) in THF (5.5
mL) were added to ester 1 (0.96 g, 3.87 mmol) in THF (7.5 mL),
at 0 °C, under nitrogen. The resulting solution was allowed to warm
to room temperature and stirred for 1 h. After acidification with 1
N HCl aqueous solution (12 mL), the solvent was evaporated, and
the aqueous phase was extracted with ethyl acetate (40 mL). The
organic phase was then dried over MgSO4 and filtered off, and the
solvent was evaporated under reduced pressure. Column chroma-
tography (petroleum ether/ethyl acetate 60/40 v/v) afforded product
2 as a white solid (1.1 g, 3.53 mmol) in 92% yield. mp 138-139
°C; (literature (39) mp 134-135 °C); [R]25D + 6.0 (c 0.1, MeOH);
literature (39) [R]23D +6.9 (c 1.0, MeOH). Spectroscopic data were
identical to those previously reported (39, 41).
Methyl (S)-2-(tert-Butoxycarbonylamino)-3-(3,4-dimethoxyphe-
nyl)-propanoate (3). To compound 2 (1.3 g, 4.2 mmol) in acetone
(12 mL) were added MeI (2.1 mL, 33.6 mmol) and K2CO3 (5.8 g,
42.1 mmol). The reaction mixture was heated to reflux for 6 h.
After filtration of the precipitate, the solution was concentrated,
and the resulting residue was dissolved in diethyl ether (40 mL).
The organic phase was then washed with water (20 mL), dried over
MgSO4, filtered off, and the solvent evaporated under reduced
pressure. Column chromatography (petroleum ether/ethyl acetate
60/40 v/v) afforded product 3 as a white solid (1.41 g, 4.15 mmol)
1
Compound 6: H NMR (CDCl3) δ 1.02 (d, J ) 6.2 Hz, 3H),
2.02 (broad s, 1H), 2.34 (s, 3H), 2.57 (m, 2H), 2.70 (m, 1H), 3.81
(s, 3H), 3.82 (s, 3H), 6.69 (m, 2H), 6.76 (d, J ) 8.7 Hz, 1H). 13C
NMR (CDCl3) δ 19.5, 33.9, 43.0, 55.8 (×2), 56.3, 111.1, 112.3,
121.1, 131.9, 147.4, 148.7. HRMS (ESI) m/z calcd for [M + H]+
210.2955; found, 210.1489. [R]25 -1.9 (c 0.06, MeOH).
D
1
Compound 7: H NMR (CDCl3) δ 2.3 (m, 3H), 2.42 (s, 3H),
3.37 (dd, J ) 10.8 Hz, 1H), 3.63 (dd, J ) 10.8 Hz, 1H), 3.85 (s,
3H), 3.86 (s, 3H), 6.74 (m, 2H), 6.82 (d, J ) 8.1 Hz, 1H). 13C
NMR (CDCl3) δ 33.7, 37.1, 55.9 (×2), 61.9, 62.0, 111.3, 112.2,
121.1, 131.0, 147.5, 148.9. HRMS (ESI) m/z calcd for [M + H]+
226.2949; found, 226.1438. [R]25 + 10.1 (c 0.01, MeOH).
D
(R)-3,4-Dihydroxymethamphetamine Hydrobromide HHMA,
HBr (8). A 48% aqueous solution of HBr (2.6 mL) (15 mmol) was
added to compound 6 (420 mg, 2 mmol). The resulting solution
was heated to reflux for 1.5 h, under nitrogen. After the addition

214 Chem. Res. Toxicol., Vol. 23, No. 1, 2010
Felim et al.
Scheme 1. Two Step Synthesis of (()-HHMA
of 0.5 mL of methanol and evaporation under reduced pressure,
the orange oil was purified by column chromatography (dichlo-
romethane/methanol 80/20) affording compound 8 as a colorless
oil (376.5 mg, 1.44 mmol) in 72% yield. [R]25 -2.4 (c 0.05,
D
MeOH). 1H NMR (D2O) δ 1.16 (d, J ) 6.6 Hz, 3H), 2.57 (s, 3H),
2.68 (dd, J ) 13.9 Hz, 1H), 2.80 (dd, J ) 13.9 Hz, 1H), 3.35 (m,
1H), 6.62 (dd, J ) 1.9 and 8.1 Hz, 1H), 6.71 (s, J ) 1.9 Hz), 6.79
(d, 1H, J ) 8.1 Hz). 13C NMR (D2O) δ 14.8, 29.9, 37.9, 56.4,
116.2, 116.9, 121.7, 128.1, 143.0, 143.9. HRMS (ESI) m/z calcd
for [M + H]+ 182.2419; found, 182.1175.
(S)-3-(3,4-Dihydroxyphenyl)-2-(N-methylamino)-propan-1-ol Hy-
drobromide (9). The previous method, replacing compound 6 by
compound 7 (414 mg, 2 mmol), afforded after column chroma-
tography, compound 9 as a colorless oil (400.5 mg, 1.44 mmol) in
72% yield. [R]25D + 2.1 (c 0.016, MeOH). 1H NMR (D2O) δ 2.62
(s, 3H), 2.74 (dd, J ) 14.2 Hz, 1H), 2.83 (dd, J ) 14.2 Hz, 1H),
3.37 (m, 1H), 3.53 (dd, J ) 12.3 Hz, 1H), 3.73 (dd, J ) 13.6 Hz,
1H), 6.64 (dd, J ) 1.9 and 8.1 Hz, 1H), 6.73 (s, J ) 1.9 Hz, 1H),
6. 77 (d, J ) 8.1 Hz, 1H). 13C NMR (D2O) δ 30.0, 32.5, 57.8,
61.0, 116.4, 116.9, 121.7, 127.8, 143.1, 144.1. HRMS (ESI) m/z
calcd for [M + H]+ 198.2413; found, 198.1122.
Cell Culture. Human neuroblastoma SH-SY5Y cells were
obtained from the European Collection Cell Cultures (ECACC no.
94030304; Salisbury, UK). Cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) containing 15% fetal bovine
serum (FBS), 1% nonessential amino acids, 2 mM L-glutamine,
100 IU/mL penicillin, and 100 µg/mL streptomycin.
Flow Cytometry. For cell analysis, we used a Cytomics FC500
MPL multiwell-plate flow cytometer (Beckman-Coulter, Brea, CA),
equipped with an air-cooled argon-ion laser tuned at 488 nm (blue
light). To assay cell death, SH-SY5Y cells were seeded in plastic
96-well plates (final concentration in well 2.7 × 104 cells) and
incubated in 10% FBS containing medium for 24 h (50% conflu-
ence) in a CO2 incubator at 37 °C. Then, the indicated concentra-
tions of the compounds in 5% FBS containing medium were added
and the plates kept for further 24 h. Cells were then trypsinized
and resuspended in their original wells in 5% FBS containing
medium, in the presence of 2.5 µg/mL propidium iodide (PI). To
identify PI uptake by dead cells (PI positive) by flow cytometry,
cells were excited at 488 nm, and emission was detected at 625
nm. From dose-response curves of at least three experiments, TC50,
the molar concentration producing 50% of cell death, was calcu-
lated. SDs at doses in the vicinity of the TC50 values were lower
than 10%. The percentage of viable (PI negative), apoptotic (PI
intermediate), and necrotic (PI positive) cells was calculated by
means of a plot correlating cell size (forward-angle light scatter)
and PI uptake (45).
contained 6 g of Difco agar and 5 g of NaCl per liter of distilled
water. In the assays, 100 µL of a suitable dilution of the test
compound was added to 100 µL of the bacterial mix, containing
approximately 800 test cells (from IC5282 or IC5204 strains) and
3 × 107 filler cells (from IC2880 strain) unable to grow in minimal
E4 medium because of their requirement for tryptophan and used
to obtain a background lawn. Where indicated, mushroom tyrosinase
(EC 1.14.18.1; Sigma) was added to the assay (100 µL of a suitable
dilution from a starting solution in 0.1 M sodium phosphate at pH
7.0). After the addition of 2.5 mL of molten top agar, the mixture
was poured on plates containing solid E4 medium (about 35 mL),
which were incubated at 37 °C, for 24 or 48 h. Data were the mean
of at least three experiments. From the dose-response curves, the
TC50 value was calculated as the concentration (µmol/plate) needed
to reduce the survival to 50%. SDs at doses close to the TC50 values
were lower than 10%.
Results and Discussion
Chemistry. Synthesis of (()-HHMA. (()-HHMA was pre-
pared in a straightforward manner (Scheme 1), starting from
commercially available 3,4-dimethoxyphenylacetone by reduc-
tive amination with sodium borohydride leading to (()-MMMA
in 69% yield. Complete demethylation of (()-MMMA using
hydrobromic acid, heated at reflux, afforded (()-HHMA in 80%
yield. Interestingly, this two step reaction sequence produced
(()-HHMA in a markedly improved overall yield (55%) when
compared with that of earlier reported procedures starting from
3,4-dimethoxybenzaldehyde and nitroethane, for which the
overall yields did not exceed 20% (35-38).
Synthesis of R-(-)-HHMA. We prepared R-(-)-HHMA from
commercially available L-DOPA, chosen as the chiral source,
through a seven step sequence which involved protection and
deprotection reactions as reported in Scheme 2. This strategy
required the intermediary synthesis of N-protected R-amino
alcohol 4, which has been previously described in four steps
and 74.5% overall yield (39). Minor modifications of this
procedure (solvent especially) allowed us to improve the yield
in N-protected R-amino alcohol 4, which was prepared in 82%
overall yield. Quantitative esterification of L-DOPA in MeOH,
followed by protection of the resulting amino group by treatment
with di-tert-butyl-dicarbonate (Boc2O) in THF, gave access to
compound 2 in 92% yield. Methylation of the aromatic hydroxyl
groups in acetone led, in quantitative yield, to compound 3
whose ester function was finally reduced with LiAlH4 in THF,
affording N-protected R-amino alcohol 4 in 90% yield. Treat-
ment of 4 with para-toluenesulfonyl chloride (TsCl), in the
presence of dimethylaminopyridine (DMAP) as the catalyst,
provided the rather unstable tosylate 5 in 84% yield. Because 5
spontaneously converted through intramolecular cyclization
between its alcohol group and its carbamate function to
oxazolidinone on silica gel (39), tosylate 5 had to be purified
by recrystallization. Concomittant reduction of the tosyl and
To measure the mitochondrial generation of superoxide, trypsinized
cells were resuspended in their original wells in 5% FBS containing
medium in the presence of 1.25 µM MitoSOX Red probe (InVit-
rogen), a mitochondrial superoxide indicator having excitation/
emission maxima of approximately 510/580 nm. The mean
fluorescence intensity was calculated for each sample. The results
were expressed as Fluorescence Arbitrary Units (F.A.U). As a
positive control, we used the superoxide generator plumbagin (2-
methyl-5-hydroxy-1,4-naphthoquinone), inducing a mean intensity
of 20 FAU at the dose of 0.02 mM.
Caspase-3 activity was measured by flow cytometry, using
Cleaved Caspase-3 (Asp-175) Antibody (Alexa Fluor 488 Conju-
gate) in accordance with the protocol supplied by the manufacturer
(Cell Signaling Technology). As a positive control, we used
camptothecin inducing 54% of caspase-3 activity in SH-SY5Y cells
at the dose of 0.5 µM (24 h).
Escherichia coli Cytotoxicity Plate Assays. The E. coli strains
were IC5282 (OxyR+ strain), IC5204, deficient in the OxyR
function (OxyR- strain), and IC2880, a tryptophan-requiring strain
(18). Overnight cultures were grown in Oxoid Nutrient Broth No.
2. Solid minimal E4 medium contained 15 g of Difco agar and 4 g
of glucose per liter of Vogel-Bonner E buffer (containing MgSO4·7
H2O (10 g), citric acid monohydrate (100 g), K2HPO4 (500 g), and
NaHNH4PO4·7 H2O (175 g) per liter of distilled water). Top agar

Synthesis and Cytotoxicity Profile of R-(-)-HHMA
Chem. Res. Toxicol., Vol. 23, No. 1, 2010 215
Scheme 2. Synthetic Procedure for the Total Synthesis of R-(-)-HHMA
BOC groups was achieved using LiAlH4 in dichloroethane/THF
3/1 v/v, leading to R-(-)-MMMA 6 as the major product (60%),
with high optical purity (>99%) as determined by HPLC analysis
of the mixture of Mosher amides prepared from racemate (()-
MMMA and from enantiomerically pure R-(-)-MMMA 6
(Figures 3 and 4). (See the Supporting Information for the
synthesis of the Mosher amides.) Aminoalcohol 7 was also
obtained as the minor product in 13% yield. Obviously, this
reduction reaction proved to be the more difficult step of the
synthetic procedure because of the occurrence of both the
spontaneous cyclization of 5 to oxazolidinone and the partial
reduction of the tosyl group to aminoalcohol 7. Therefore, a
thorough control of temperature was necessary to obtain good
yields in R-(-)-MMMA 6. Finally, complete demethylation of
R-(-)-MMMA 6, using hydrobromic acid heated at reflux,
afforded R-(-)-HHMA 8 in 72% yield with 99.5% ee, whereas
that of aminoalcohol 7 led to the deprotected R-aminoalcohol
9 in 72% yield.
Toxicological Study. The in vitro toxicological study was
performed using the human dopaminergic neuroblastoma SH-
SY5Y cell line, which is commonly used for studying the
toxicity of catecholamine derivatives (46, 47). Loss of cell
viability was monitored by flow cytometric analysis of pro-
pidium iodide (PI) uptake in dead cells (PI positive cells) having
defective plasma membranes. In addition, as we previously
determined the cytotoxicity profile of (()-HHMA in Escherichia
coli assays, specific for the detection of oxidative toxicity
(18, 34), these bacterial tests were also included in the present
study.
that found for the control catecholamine dopamine (DA) (TC50
) 0.25 mM), whereas a lower toxicity (TC50 ) 1.0 mM) was
observed for aminoalcohol 9 isolated as a byproduct from the
synthesis of R-(-)-HHMA 8 (Scheme 2). For the (()-MMMA
racemate, an intermediate in the synthesis of (()-HHMA
possessing a protected catechol group (Scheme 1) utilized for
comparative purposes, toxicity was detected at high doses (TC50
) 11.3 mM). Similarly, MDMA metabolite 4-hydroxy-3-
methoxymethamphetamine [(()-HMMA] has been described
as considerably less toxic than catechol-containing metabolites
to PC12 cells, in the MTT reduction assay (38). Our results on
cytotoxicity obtained from the PI uptake assay directly correlated
with the extent of cell death evaluated by the MTT reduction
assay (data not shown).
E. coli based assays allow a mechanistic characterization of
toxicity so that they can be used as a model system. In these
assays, toxicity mediated by ROS (ROS-TOX) can be detected
in OxyR- cells deficient in OxyR function because they are
unable to exert antioxidant defenses (34, 48). In contrast, ROS-
TOX is inhibited in OxyR+ cells because of the elimination of
hydrogen peroxide by catalase and alkylhydroxyperoxide re-
ductase, both enzymes encoded by OxyR-regulated genes, so
that the toxicity arising from the binding of quinones with
cellular nucleophiles could then be shown (48, 49). The results
of the E. coli plate assays are shown in Table 1. R-(-)-HHMA
8 and (()-HHMA gave similar positive responses in the OxyR-
assay, with TC50 values of 0.8 and 0.75 µmol/plate, respectively,
indicating high ROS-TOX induction. Furthermore, an increase
in ROS-TOX (TC50 ) 0.6 and 0.5 µmol/plate, respectively) was
observed in the presence of tyrosinase, which catalyzed the
oxidation to o-quinone. For (()-MMMA, ROS-TOX induction
was abolished, whereas for aminoalcohol 9 and dopamine, it
was poor (TC50 ≈ 4 µmol/plate) but greatly increased following
catalytic oxidation by tyrosinase (TC50 ≈2 µmol/plate). The
The results of the flow cytometric analysis of acute (24 h)
cytotoxicity in SH-SY5Y cells are shown in Table 1. R-(-)-
HHMA enantiomer 8 induced a decrease in cell viability similar
to that resulting from exposure to the (()-HHMA racemate,
with a TC50 value of 0.30 mM. This decrease was also close to

216 Chem. Res. Toxicol., Vol. 23, No. 1, 2010
Felim et al.
Figure 3. HPLC chromatograms for the analytical resolution of Mosher
amide diastereoisomers prepared from (()-MMMA racemate [(H2O
+ 1 ‰TFA)/MeCN 53/47; flow rate, 0.9 mL.min-1]: (a) Mosher amide
of R-(-)-MMMA isolated after semipreparative HPLC; (b) Mosher
amide of S-(+)-MMMA isolated after semipreparative HPLC; (c)
mixture of the Mosher amides obtained from (()-MMMA racemate.
Figure 4. HPLC chromatograms for the analytical determination of
the enantiomeric excess of R-(-)-MMMA 6 [(H2O + 1 ‰TFA)/MeCN
53/47; flow rate, 0.9 mL.min-1]: (a) Mosher amide prepared from
enantiomerically pure R-(-)-MMMA 6; (b) mixture of the Mosher
amides obtained from (()-MMMA racemate. (c) Mosher amide
prepared from enantiomerically pure R-(-)-MMMA 6 together with
the mixture of Mosher amides obtained from (()-MMMA racemate.
enhancement of toxicity promoted by tyrosinase suggests that
the oxidation of these catecholamines to the corresponding
o-quinone opens the way for redox cycling, leading to the
formation of ROS (18). In contrast, the negative response in
the OxyR+ assay (Table 1), even in the presence of tyrosinase
(data not shown), was indicative of the absence of quinone-
promoted toxicity, which is resistant to inhibition by antioxidant
defenses (48, 49).
The good correlation found between the toxicity observed in
SH-SY5Y cells and in E. coli OxyR- cells would indicate a
susceptibility of the SH-SY5Y cell line to the oxidative toxicity
induced by the catecholamine derivatives. Consistent with this
possibility is the mitochondrial generation of superoxide anion
by these compounds in SH-SY5Y cells, detected by flow
cytometric analysis using the MitoSOX Red fluorochrome
(Table 2). Note, however, that HHMA and DA induced similar
toxicities in SH-SY5Y cells, whereas, converse to HHMA, DA
was found to be a poor inducer of ROS-TOX in E. coli OxyR-
cells (Table 1).
Flow cytometry offers the possibility of multiparametric
assays simultaneously providing several end points to character-
ize different modes of cell death at a single-cell level (45). In
addition to the assay of plasma membrane permeability (PI
uptake) for cell death evaluation (TC50 value), we used flow
cytometry to discriminate between live (PI negative), apoptotic
(PI intermediate), and necrotic (PI positive) subpopulations, by
means of a plot correlating cell size (forward-angle light scatter)
and PI uptake (Table 3). For R-(-)-HHMA, (()-HHMA,
aminoalcohol 9, and DA induction of necrotic cells but not that
of apoptotic cells was detected. In contrast, induction of
apoptotic cells (48% at 10 mM) was clearly observed for (()-

Synthesis and Cytotoxicity Profile of R-(-)-HHMA
Chem. Res. Toxicol., Vol. 23, No. 1, 2010 217
Table 1. In Vitro Cytotoxicity Profile of R-(-)-HHMA in
sequence order of events and the identity of specific neurotoxic
entities have yet to be determined. There has recently been a
resurgence of interest in the possibility that MDMA metabolites
(especially 5-NAC-HHMA) might play a role in MDMA
neurotoxicity. The discrepant findings between the recently
published work (19) with those previously reported (20, 21)
leave open the possibility that 5-NAC-HHMA may require the
presence of MDMA and/or elevated body temperature to be
toxic but also raise the question of the chirality of MDMA,
which was not considered in previously reported in vivo studies
because HHMA, the precursor of 5-NAC-HHMA metabolite,
was used as the racemic mixture. In vitro metabolism of MDMA
is enantioselective, with a preference for the S-enantiomer
caused in part by CYP2C19 and CYP2D6-mediated demeth-
ylenation (23). However, correlation with the in vivo situation
is not straightforward because in vivo catecholic phase I
metabolites such as HHMA are further metabolized by O-
methylation and/or glucuronidation/sulfation. Recent investiga-
tions have revealed that the S-enantiomer of catecholic phase I
metabolites was preferably O-methylated by catechol-O-meth-
yltransferase (COMT) (50), resulting in the accumulation of the
R-enantiomer. Therefore, the synthesis of R-(-)-HHMA, by
allowing the synthesis of noticeable amounts of the correspond-
ing diastereomerically pure form of 5-NAC-HHMA metabolite,
will permit a comprehensive examination of its participation in
the in vivo neurotoxic effects of ecstasy.
Comparison with Related Catecholamines
TC50 in Assay
SH-SY5Ya
PI
E. colib
OxyR-/Tyrc
compound
OxyR-
OxyR+
d
R-(-)-HHMA 8
(()-HHMA
aminoalcohol 9
(()-MMMA
DA
0.3
0.3
1.0
11.3
0.25
0.8
0.75
5
0.6
0.5
2
-
1.8
-
-
-
-
-
-
4
a SH-SY5Y
) human dopaminergic neuroblastoma cells. PI )
propidium iodide assay: live cells, PI negative; dead cells, PI positive.
TC50 in mM. b TC50 in µmol/plate. c Tyr ) 50 units of tyrosinase per
plate. d (-), survival >50% at the highest dose tested.
Table 2. Mitochondrial Generation of Superoxide Anion by
R-(-)-HHMA in Comparison with Related Catecholamines
compound
dose (mM)
F.A.U.a
_
_
8.0
R-(-)-HHMA 8
(()-HHMA
aminoalcohol 9
DA
0.1
0.5
0.1
0.5
0.25
0.5
0.1
0.31
8.0
14.8
8.6
13.3
11.5
13.3
9.1
10.1
a Mitochondrial generation of superoxide anion was determined by
The in vitro toxicological study, performed in human neu-
roblastoma SH-SY5Y cells, revealed similar toxicities for the
HHMA racemate and its R-enantiomer. The inclusion in this
work of an E. coli based plate assay, specific for the detection
of oxidative toxicity, showed a good correlation between the
toxic effects observed both in human and bacterial cells. This
correlation led us to conclude that SH-SY5Y cells exhibit a low
protection against oxidative toxicity and that the observed cell
death in these cells would be mediated by ROS, mainly
generated through the redox activity of the generated quinone
species. The toxicophore (i.e., the reactive chemical moiety)
would be essentially the catechol group, and therefore, the
problem of the toxicity of the phase I catecholic metabolites of
MDMA would be related to the toxicity induced by other
catecholamine compounds, as is the case with DA. Although
to a lesser extent, the nature of the alkylamino side chain of
catecholamine derivatives also plays a role. As a result, R-(-)-
HHMA and aminoalcohol 9, two enantiomeric pure forms,
exhibit different cytotoxicity in E. coli plate assays. Probably,
a physical parameter such as lipophilicity may contribute to the
exhaustive toxicity. Accordingly, R-(-)-HHMA is at once more
toxic (TC50 ) 0.75 µmol per plate) and more lipophilic (log P
) 0.90) than aminoalcohol 9, for which TC50 ) 5 µmol per
plate and log P ) 0.04.
flow cytometry using the MitoSOX Red fluorochrome. FAU
fluorescence arbitrary units.
)
Table 3. Flow Cytometric Characterization of Cell Death
and of Caspase-3 Activity in Treated SH-SY5Y Cells
dose
(mM)
apoptotic
cells (%)a
necrotic
caspase-3
compound
cells (%)b
activity (%)c
_
_
0.1
1
0.1
1
0.5
1
10
0.1
1
12
10
14
9
10
9
15
48
13
11
23
34
80
30
82
29
51
25
35
83
9
R-(-)-HHMA 8
36
37
42
37
15
27
18
16
22
(()-HHMA
aminoalcohol 9
(()-MMMA
DA
a PI intermediate cells. b PI positive cells. c Caspase-3 activity was
determined by flow cytometry using cleaved caspase-3 (Asp175)
antibody.
MMMA. These results were complemented by a flow cytometric
measurement of cleaved (activated) caspase-3, a critical effector
of most of the apoptotic pathways (Table 3). For R-(-)-HHMA
and (()-HHMA, the activity of caspase-3 increased to a
maximal value after exposure to 0.1 mM, a dose below the TC50
value (0.3 mM). For aminoalcohol 9 and DA, maximal induction
of caspase-3 activity required a higher dose (1 mM), whereas
for (()-MMMA, a significant increase in caspase-3 activity was
observed at 10 mM, in the absence of any detectable cytotox-
icity. These results suggest that induction of necrosis is dominant
over that of apoptosis in ROS-sensitive SH-SY5Y cells. In these
cells, induction of proapoptotic events such as caspase-3
activation would not be followed by the detection of apoptosis.
Important, although HHMA racemate and its R-enantiomer
exhibited close toxicological profiles, no conclusion could be
drawn about the enantioselective toxicity. For a complete
comparison and evaluation of the enantioselective toxicity of
both enantiomers, the synthesis of optically pure S-(+)-
enantiomer is required. This synthesis is envisioned in our
laboratory and will be reported in due course.
The catechol toxicophore is neither present in MDMA, the
precursor of HHMA, nor in MMMA for which the two phenolic
groups are protected. For MMMA, no toxicity was detected in
the bacterial plate assay, whereas, at high doses, toxic effects
could be detected in the flow cytometric assay measuring PI
uptake. We can then speculate that the toxicity observed with
MMMA is not related to increased oxidative stress. Conse-
Conclusions
Despite several years of intensive efforts, the mechanisms
by which MDMA destroys brain serotonin (5-HT) axon
terminals remain to be fully elucidated. In particular, the

218 Chem. Res. Toxicol., Vol. 23, No. 1, 2010
Felim et al.
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catechol so that in studies dealing with MDMA toxicity, there
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Acknowledgment. We thank Dr. M.-B. Fleury, Emeritus
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sions. We also thank Carmen Navarro for her technical
assistance. This research was made possible thanks to grants
MEC (Spain) BIO2007-65662, EC VI FP projects A-Cute-Tox,
and LSHB-CT-2004-512051 (to J.E.O’C.), and to the joint
financial support of the Mission Interministe´rielle de Lutte contre
la Drogue et la Toxicomanie (MILDT) and the Institut National
de la Sante´ et de la Recherche Me´dicale (INSERM) (Appel a`
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Supporting Information Available: Experimental proce-
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pounds 6 and 8; 1H and 13C NMR spectra of (()-HHMA, (()-
MMMA, and R-(-)-HHMA and its synthetic intermediates,
together with aminoalcohol derivatives 7 and 9; analytical HPLC
chromatograms of (()-HHMA, (()-MMMA, R-(-)-HHMA,
and of aminoalcohol 9. This material is available free of charge
via the Internet at http://pubs.acs.org.
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