Ethylenedioxy homologs of N-methyl-(3,4-methylenedioxyphenyl)-2-aminopropane (MDMA) and its corresponding cathinone analog methylenedioxymethcathinone: Interactions with transporters for serotonin, dopamine, and norepinephrine

Article abstract of DOI:10.1016/j.bmc.2015.07.035

N-Methyl-(3,4-methylenedioxyphenyl)-2-aminopropane (MDMA; 'Ecstasy'; 1) and its β-keto analog methylone (MDMC; 2) are popular drugs of abuse. Little is known about their ring-expanded ethylenedioxy homologs. Here, we prepared N-methyl-(3,4-ethylenedioxyph

Full text of DOI:10.1016/j.bmc.2015.07.035

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Ethylenedioxy homologs of N-methyl-(3,4-methylenedioxyphenyl)-
2-aminopropane (MDMA) and its corresponding cathinone analog
methylenedioxymethcathinone: Interactions with transporters
for serotonin, dopamine, and norepinephrine
Fabio Del Bello a, , Farhana Sakloth a, John S. Partilla b, Michael H. Baumann b, Richard A. Glennon a,
⇑
a Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Box 980540, Richmond, VA 23298, USA
b Designer Drug Research Unit, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
N-Methyl-(3,4-methylenedioxyphenyl)-2-aminopropane (MDMA; ‘Ecstasy’; 1) and its b-keto analog
methylone (MDMC; 2) are popular drugs of abuse. Little is known about their ring-expanded ethylene-
dioxy homologs. Here, we prepared N-methyl-(3,4-ethylenedioxyphenyl)-2-aminopropane (EDMA; 3),
both of its optical isomers, and b-keto EDMA (i.e., EDMC; 4) to examine their effects at transporters for
serotonin (SERT), dopamine (DAT), and norepinephrine (NET). In general, ring-expansion of the
methylenedioxy group led to a several-fold reduction in potency at all three transporters. With respect
to EDMA (3), S(+)3 was 6-fold, 50-fold, and 8-fold more potent than its R(À) enantiomer at SERT, DAT,
and NET, respectively. Overall, in the absence of a b-carbonyl group, the ethylenedioxy (i.e., 1,4-dioxane)
substituent seems better accommodated at SERT than at DAT and NET.
Received 14 May 2015
Revised 7 July 2015
Accepted 16 July 2015
Available online xxxx
Keywords:
MDMA
MDMC
DAT
Ó 2015 Elsevier Ltd. All rights reserved.
SERT
NET
1. Introduction
The methylenedioxy substituent of MDMA (1) is common to a
number of other ‘clandestine’ and U.S. Schedule I phenylalky-
The phenylalkylamine N-methyl-(3,4-methylenedioxyphenyl)-
2-aminopropane (1; Fig. 1), a compound commonly known as
MDMA, ‘Ecstasy’ or ‘Molly’, is a popular ‘recreational drug’ and a
U.S. Schedule I controlled substance. Its pharmacology and mecha-
nism of action have been extensively investigated.1–4 For example,
MDMA (1) is considered an ‘empathogen’ to distinguish its subjec-
tive effects in humans from those of classical phenylalkylamine
hallucinogens and phenylalkylamine central stimulants5—even
though MDMA, and its individual optical isomers, display some
lamine substances, including certain ‘bath salts’ constituents or
synthetic cathinones.4,11,12 One of the better recognized of the
original bath salts constituents is the b-keto analog of MDMA
(i.e., MDMC, methylone, bk-MDMA; 2) (Fig. 1).13 MDMC (2) pro-
duces MDMA-like neurochemical and behavioral effects in rodents
and acts as a neurotransmitter releaser at the three transporters
mentioned above.14,15 Despite the scheduling of MDMC (2) to
ban its sale and use in the U.S., the drug continues to be confiscated
by law enforcement personnel, often in tablets being sold as
Ecstasy.16
6
degree of stimulant character, for example, MDMA’s actions are
associated with its non-selective neurotransmitter releasing effects
at the membrane transporters for 5-HT, dopamine, and
norepinephrine (i.e., SERT, DAT, and NET, respectively).7,8
Accordingly, pretreatment with medications that block the trans-
mitter-releasing effects of MDMA at SERT, DAT, and NET can signif-
icantly reduce the subjective and cardiovascular actions of MDMA
(1) in human subjects under controlled laboratory conditions.9,10
Expansion of the methylenedioxy ring of MDMA (1) and MDMC
(2) to their larger ethylenedioxy (i.e., 1,4-dioxane) homologs
affords N-methyl-1-(3,4-ethylenedioxyphenyl)-2-aminopropane
(i.e., 3,4-ethylenedioxymethamphetamine, EDMA; 3) and
3,4-ethylenedioxymethcathinone (EDMC; 4) (Fig. 1). Relatively
little is known about the pharmacology of EDMA (3) and its
individual optical isomers have not been reported. Likewise,
EDMC (4) has not been previously investigated.
Given the growing interest in phenylalkylamine analogs as
potential drugs of abuse [reviewed11,12,17], the Drug Enforcement
Administration (DEA) has solicited information on phenylalkylami-
⇑
Corresponding author. Tel.: +1 (804)828 8487.
E-mail address: glennon@vcu.edu (R.A. Glennon).
Current address: School of Pharmacy, Medicinal Chemistry Unit, University of
Camerino, 62032 Camerino, Italy.
nes that are not yet controlled as Schedule
I
substances.18
http://dx.doi.org/10.1016/j.bmc.2015.07.035
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Del Bello, F.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.035

2
F. Del Bello et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
NO2
CH3
CH3
CH3
CH3
CH3
O
HN
HN
HN
O
CH3
a
O
O
O
O
O
O
O
O
5
6
2
1
b
CH3
NH
c
CH3
NH
CH3
CH3
CH3
CH3
HN
O
CH3
CH3
O
O
O
O
O
O
O
O
4
3
S,S(-)7
d
R,R(+)7
Figure 1. Structures of MDMA (1), MDMC (2), and their ethylenedioxy counterparts
EDMA (3) and EDMC (4).
NH2
CH3
NH2
CH3
Specifically listed among these agents are some ethylenedioxy
analogs, including EDMA (3). Hence, we prepared and examined
EDMA (3), its individual optical isomers, and its b-keto (or meth-
cathinone) analog 4, for comparison with MDMA (1) and MDMC
(2), to function as substrates (i.e., as releasers) at SERT, DAT, and
NET.
O
O
O
O
S(+)8
e
R(-)8
2. Chemistry
O
O
CH3
CH3
CH3
CH3
CH3
CH3
HN
O
HN
O
Compounds ( )1 and ( )2 as their hydrochloride (HCl) salts
were on hand from previous studies in our laboratory.
Compound ( )3 was synthesized from 1-(3,4-ethylene-
dioxyphenyl)-2-aminopropane hydrochloride,19 by converting it
into its carbamate analog followed by reduction using LiAlH4.
Compound ( )4 was synthesized by a nucleophilic substitution
CH3
CH3
O
O
O
O
reaction
using
1-(3,4-ethylenedioxyphenyl)-2-bromo-1-
propanone20 and N-methylamine. Both of these reaction sequences
are shown in the Supporting information section.
S(-)9
R(+)
9
f
Compounds S(+)3 and R(À)3 were prepared as shown in
Scheme 1; this is similar to a literature procedure reported for
related phenylalkylamine optical isomers.21 Reduction of
nitroalkene 519 with iron powder followed by heating an aqueous
mixture gave the intermediate ketone 6. The crucial step for this
synthesis was the reductive amination using either S(À)- or
CH3
CH3
HN
HN
CH3
CH3
O
O
R(+)-a-methylbenzylamine and sodium triacetoxyborohydride;
O
O
this reaction selectively afforded S,S(À)7 and R,R(+)7, whose
hydrogenation at 50 psi using 10% Pd/C as catalyst led to the enan-
tiomers S(+)8 and R(À)8, respectively. Acylation with di-tert-butyl
dicarbonate to the N-Boc intermediates S(À)9 and R(+)9, followed
by reduction with LiAlH4 afforded the desired enantiomers S(+)3
and R(À)3, respectively.
S(+)3
R(-)3
Scheme 1. Reagents and conditions: (a) (i) Fe, AcOH, rt; (ii) reflux; (iii) H2O;
-methylbenzylamine, NaBH(OAc)3, DCE, rt; (c) R-(+)- -methylbenzy-
lamine, NaBH(OAc)3, DCE, rt; (d) H2, 10% Pd/C, 50 psi; (e) (BOC)2O, Et3N, DCM, rt;
(b) S-(À)-
a
a
(f) (i) LiAlH4, THF, 0 °C (ii) reflux (iii) HCl/Et2O.
The enantiomeric purity of the isomers of 3, determined by 1H
NMR spectrometry in the presence of the chiral shift reagent
S(+)-2,2,2-trifluoro-1-(9-anthryl)ethanol, was found to be >98%
(detection limit) for both enantiomers. In fact, the 1H NMR
spectrum of the racemate [i.e., ( )3] showed two singlets: one at
d 4.27 and another at d 4.24 for the methylene protons of the
benzodioxane nucleus, whereas only one singlet was observed
for S(+)3 and R(À)3 at d 4.27 and d 4.24, respectively.
in Scheme 2. After protecting the amine function with a Boc group,
affording S(À)11, treatment with 1,2-dibromoethane furnished the
1,4-benzodioxane derivative S(À)9, whose reduction with LiAlH4
gave the desired enantiomer S(+)3.
3. Results and discussion
The S absolute configuration of the enantiomer (+)3, suggested
by analogy to earlier studies,20 was also determined by comparing
the sign of its optical rotation with that of S(+)3 obtained by
The potency of ( )EDMA (3) to release [3H]5-HT (EC50 = 117 nM)
at SERT was approximately six times and three times greater than
its potency to release [3H]MPP+ at DAT and NET (EC50 = 597 and
325 nM, respectively; Table 1). By contrast ( )MDMA (1),
stereoselective
synthesis
starting
from
the
known
S(+)1-(3,4-dihydroxyphenyl)-2-propanamine [S(+)10],22 as shown
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F. Del Bello et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
O
O
CH3
CH3
CH3
CH3
CH3
CH3
HN
O
HN
O
NH2
CH3
HN
CH3
CH3
CH3
CH3
a
c
b
HO
O
HO
O
OH
O
OH
O
S(-)11
S(-)9
S(+)10
S(+)3
Scheme 2. Reagents and conditions: (a) (BOC)2O, MeOÀNa+, rt; (b) BrCH2CH2Br, K2CO3, DMF, rt; (c) (i) LiAlH4, THF, 0 °C; (ii) reflux; (iii) HCl/Et2O.
Table 1
EC50 values for test agents to release [3H]5-HT at SERT, and [3H]MPP+ at DAT and NET
EC50, nM ( SD)a
SERT
DAT
NET
( )EDMA (3)
117 ( 17)
[102 3]
91 ( 20)
[96 5]
573 ( 108)
[97 5]
61 ( 9)
[98 2]
347 ( 38)
[105 3]
242 ( 48)
597 ( 50)
[108 3]
276 ( 28)
[103 2]
14,600 ( 2850)
[106 10]
75 ( 5)
325 ( 61)
[91 2]
239 ( 37)
[94 3]
1952 ( 356)
[85 4]
72 ( 13)
[95 3]
S(+)EDMA [(S)3]
R(À)EDMA [(R)3]
( )MDMA (1)
( )EDMC (4)
[99 1]
496 ( 52)
[105 4]
133 ( 11)
327 ( 54)
[84 4]
152 ( 33)
( )MDMC (2)b
a
EC50 values are mean ( SD) for n = 3–4 experiments performed in triplicate;
efficacy values [in brackets] are % maximal release expressed as mean ( SD) as
described in Methods.
b
EC50 values for MDMC (2) were reported earlier15 and are included for
comparison with EDMC (4).
investigated as a comparator compound, was nearly equipotent as
a releaser at all three neurotransmitter transporters (Table 1). The
present findings with MDMA are consistent with our previous
data15,17 and those reported by Simmler et al.4 and Eshleman
et al.23 who examined the effects of MDMA and related drugs in
human embryonic kidney (HEK) cells transfected with human
SERT, DAT and NET. Thus, the molecular mechanism of action for
MDMA at monoamine transporters is similar in rats and humans.
On the other hand, the potency of ( )MDMA for releasing
monoamines in rat brain synaptosomes shown here (i.e.,
60–70 nM) is greater than its potency in transfected HEK cells
(i.e., 1–20 lM). Such discrepancies in absolute potency could be
related to species differences in drug responsiveness, differences
in release assay methods employed, or the absence of important
neuronal membrane proteins in non-neuronal HEK cells.
Figure 2. Effects of ( )EDMA and its isomers on the release of [3H]5-HT at SERT and
[3H]MMP+ at DAT and NET. Data are % of maximal release expressed as mean SD
for n = 3–4 separate experiments performed in triplicate. ( )MDMA is included as a
references compound.
The data depicted in Table 1 and Figure 2 demonstrate that
( )EDMA (3) exhibited approximately half the potency of
( )MDMA (1) as a releaser at SERT, but was 8-fold and 4.5-fold less
potent as a releaser at DAT and NET, respectively. Thus, ( )EDMA
(3) was slightly more selective for SERT over DAT and NET.
As shown in Figure 2, the S(+) isomer of EDMA [S(+3)] was the
more potent of the two optical isomers, being about 6-fold, 50-fold,
and 8-fold more potent than its R-enantiomer at SERT, DAT, and
NET, respectively. It has been reported previously that S(+)MDMA
is more potent than R(À)MDMA at all three transporters, so the
present data with EDMA support the general concept that the
S-isomers of ring-substituted phenylalkylamines (i.e., ampheta-
mine-related compounds) are more potent than their
R-enantiomers as releasing agents.8 Interestingly, R(À)EDMA was
more SERT-selective when compared to its S(+)-isomer.
Table 1). Unlike what was seen with ( )EDMA (3), ( )EDMC (4) dis-
played no transporter selectivity. Comparing the potencies of
( )EDMC (4) with those of ( )MDMC (2), the former was slightly
less potent at each of the three transporters.
4. Conclusions
For the compounds examined, ring-expansion from
a
methylenedioxy group to an ethylenedioxy group resulted in a
modest (<8-fold) decrease in release potency at SERT, DAT, and
NET in the absence of the benzylic carbonyl group (i.e., comparing
1 with 3), and even less (about 2-fold) in its presence (i.e., compar-
ing 2 with 4). Overall, the ethylenedioxy group appears to be better
( )EDMC (4) was capable of releasing [3H]substrate at SERT,
DAT, and NET (EC50 = 347, 496, and 327 nM, respectively;
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F. Del Bello et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
accommodated at SERT than at DAT or NET. The latter finding is
consistent with a previous finding that SERT accepts larger sub-
stituents in this general position than DAT.24,25 In the absence of
the carbonyl group (i.e., 3), ring-expansion seems better tolerated
at SERT than at DAT and NET, and the S(+) isomer of 3 is more
potent than its R(À) enantiomer at all three transporters.
If the behavioral actions of EDMA (3) are related to its actions at
SERT, DAT, and NET, it might be expected that its effects would be
similar to MDMA (1), but that it would be less potent. Racemic
MDMA is behaviorally active at a total human dose of 80–150 mg
po,26 and limited data indicate that racemic EDMA fails to produce
similar effects in humans at 200 mg, and only a ‘threshold’ effect at
250 mg.26
5.1.2. S(+)1-(3,4-Ethylenedioxyphenyl)-N-methyl-2-aminoprop-
ane hydrochloride [S(+)3]
A solution of S(À)9 (0.05 g, 0.17 mmol) in dry THF (2 mL) was
added in a dropwise manner at 0 °C to a stirred suspension of
LiAlH4 (0.02 g, 0.52 mmol) in dry THF (5 mL). The mixture was
heated at reflux under an N2 atmosphere for 4 h, allowed to cool,
and EtOAc (5 mL) was added, followed by the addition of aqueous
20% NaOH (0.5 mL). The white precipitate was removed by filtra-
tion and the residue was washed with EtOAc (3 Â 5 mL). The com-
bined organic portion was dried (Na2SO4) and the solvent was
evaporated under reduced pressure to give a yellow oil (0.03 g,
84%), which was converted to the hydrochloride salt in anhydrous
Et2O by addition of HCl–saturated anhydrous Et2O. The precipitate
i
Given the data presented here, future studies are warranted to
compare the potencies of EDMA and EDMC to their methylene-
dioxy homologs in animal models of addiction and neurotoxicity.
was collected by filtration and recrystallized from PrOH/Et2O to
give S(+)3 as white crystals (72% yield); mp 192–194 °C;
[a]
25 = +14.5° (c 0.5, H2O); 1H NMR (CDCl3) d 1.34 (d, 3H, CH3),
D
2.71 (s, 3H, CH3) 2.74 (dd, 1H, CH2), 3.25 (dd, 1H, CH2), 3.36 (m,
1H, CH), 4.25 (s, 4H, CH2O), 6.68–6.79 (m, 3H, ArH), 9.62 (br s,
2H, NH+2). Anal. Calcd for (C12H17NO2ÂHCl) C, 59.14; H, 7.44; N,
5.74. Found: C, 58.97; H, 7.41; N, 5.72.
5. Experimental
5.1. Chemistry
5.1.3. R(À)1-(3,4-Ethylenedioxyphenyl)-N-methyl-2-aminopro-
pane hydrochloride [R(À)3]
All commercially available reagents and solvents were
purchased from Sigma–Aldrich Co. (St. Louis, MO) and Platte
Valley Scientific Product List (Gothenburg, NE), and used as deliv-
ered. Melting points were measured in glass capillary tubes
(Thomas–Hoover melting point apparatus) and are uncorrected.
1H NMR spectra were recorded with a Bruker 400 MHz spectrom-
eter. Chemical shifts (d) are reported in parts per million (ppm) rel-
ative to tetramethylsilane as internal standard. Optical rotations
were measured using a Jasco DIP-1000 polarimeter. Reactions
and product mixtures were routinely monitored by thin-layer
chromatography (TLC) on silica gel precoated F254 Merck plates.
Elemental analysis for C, H, and N was performed by Atlantic
Microlabs (Norcross, GA) and determined values were within
0.4% of theory.
The compound was prepared from R(À)9 in the same manner as
S(+)3: 67% yield; [
a
]
25 = À14.2° (c 0.5, H2O). The 1H NMR spectrum
D
was identical to that of S(+)3. Anal. Calcd for (C12H17NO2ÂHCl) C,
59.14; H, 7.44; N, 5.74. Found: C, 58.96; H, 7.46; N, 5.66.
5.1.4. ( )-1-(3,4-Ethylenedioxyphenyl)-2-(methylamino)-1-
propanone hydro- chloride [( )4]
Methylamine in EtOH (0.60 mL, 13.08 mmol) was added to a
stirred
solution
of
1-(3,4-ethylenedioxyphenyl)-2-bromo-
propanone20 (0.88 g, 3.27 mmol) in anhydrous benzene (12 mL)
at room temperature under an N2 atmosphere in a sealed tube.
The reaction mixture was allowed to stir at room temperature
for 36 h, filtered, and the solvent was evaporated under reduced
pressure. The residue was dissolved in Et2O (25 mL) and washed
with H2O (3 Â 5 mL). The organic portions were combined and
acidified with 2 M HCl (10 mL). The aqueous portion was basified
with saturated NaHCO3 (25 mL) and extracted with Et2O
(3 Â 10 mL). The combined organic portions were washed with
brine (3 Â 5 mL), dried (Na2SO4), and evaporated to dryness under
reduced pressure to yield a residue (0.15 g) as a free base that was
converted to the hydrochloride salt and purified by recrystalliza-
tion to afford 0.04 g (6%) of the product as a buff-colored powder;
mp 218–219 °C, absolute EtOH/Et2O; 1H NMR (DMSO-d6) d 1.43 (d,
3H, CH3), 2.57 (s, 3H, CH3), 4.33 (m, 4H, CH2O), 5.07 (m, 1H, CH),
7.05 (d, 1H, ArH), 7.56 (dd, J = 5.7, 1.9 Hz, 2H, ArH), 9.28 (s, 1H,
NH+). Anal. Calcd for (C12H15NO2ÂHCl) C, 55.93; H, 6.26; N, 5.43.
Found: C, 56.01; H, 6.12; N, 5.33.
5.1.1. ( )-1-(3,4-Ethylenedioxyphenyl)-N-methyl-2-aminopro-
pane hydrochloride [( )3]
Triethylamine in anhydrous Et2O (0.4 mL) and ethyl
chloroformate (0.14 mL) in anhydrous Et2O (1 mL) were added to
a
suspension of 1-(3,4-ethylenedioxyphenyl)-2-aminopropane
hydrochloride19 (0.35 g) in anhydrous Et2O (10 mL) and stirred at
room temperature under an N2 atmosphere for 1 h. Reaction mix-
ture was filtered and solvent removed under reduced pressure to
afford the corresponding carbamate as a yellow oil (0.3 g, 79%);
IR (Diamond, cm–1) 3066 (NH), 1754 (N–CO–O–), 1577 (CH2
CH2O). A solution of the above carbamate (0.30 g, 1.13 mmol) in
anhydrous THF (12 mL) was added in a dropwise manner at 0 °C
to a stirred suspension of LiAlH4 (0.13 g, 3.40 mmol) in anhydrous
THF (12 mL). The reaction mixture was heated at reflux under an
N2 atmosphere for 3 h, and then cooled at 0 °C and quenched by
the dropwise addition of Et2O (50 mL), H2O (3 mL) and 15%
NaOH (0.5 mL). The white precipitate was removed by filtration
and the filtrate was dried (Na2SO4). The solvent was evaporated
under reduced pressure to give a yellow oily residue that was dis-
solved in absolute EtOH (5 mL) and converted to the hydrochloride
salt by the addition of saturated solution of HCl/Et2O. The
precipitate was collected by filtration and recrystallized from
iPrOH to afford 0.03 g (11%) of the product as a white solid: mp
150–151 °C; 1H NMR (DMSO-d6) d 1.25 (d, 3H, CH3), 2.54 (s, 3H,
CH3), 2.52–2.58 (m, 1H, CH), 3.08–3.13 (m, 1H CH), 3.26–3.30 (m,
1H, CH), 4.22 (s, 4H, CH2), 6.66 (dd, J = 8.2, 1.9 Hz, 1H, ArH), 6.74
(d, J = 1.9 Hz, 1H, ArH), 6.80 (d, J = 8.2 Hz, 1H, ArH), 7.86 (s, 3H,
NH+3). Anal. Calcd for (C12H17NO2ÂHCl) C, 59.14; H, 7.44; N, 5.74.
Found: C, 58.91; H, 7.42; N, 5.53.
5.1.5. 1-(3,4-Ethylenedioxyphenyl)propan-2-one (6)
A solution of 519 (1.40 g, 6.33 mmol) in glacial AcOH (18 mL)
was added in a dropwise manner at room temperature to a stirred
suspension of Fe powder (4.80 g, 86.40 mmol) in glacial AcOH
(18 mL). The resulting mixture was heated at reflux for 3 h, and
then cooled to room temperature. Excess iron was removed by fil-
tration, and the residue was diluted with H2O (50 mL) and
extracted with DCM (3 Â 30 mL). The combined organic portion
was washed with aqueous 2 N NaOH and dried (Na2SO4). Solvent
was removed under reduced pressure and the residual oil was
chromatographed on a silica gel column (Aldrich silica gel 60)
using hexanes/EtOAc (9:1) as eluent to give 6 as a yellow oil
(78% yield); 1H NMR (CDCl3) d 2.23 (s, 3H, CH3), 3.68 (s, 2H, CH2),
4.27 (s, 4H, CH2O), 6.68-6.72 (m, 2H, ArH), 6.85 (s, 1H, ArH).
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F. Del Bello et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
5.1.6. S,S(À)N-(1-Phenylethyl)-1-(3,4-ethylenedioxyphenyl)-2-
aminopropane hydrochloride [S,S(À)7]
extracts were combined and dried (Na2SO4). Solvent was evapo-
rated under reduced pressure and the residual oil was chro-
matographed on a silica gel column (Aldrich silica gel 60) using
hexanes/EtOAc (8:2) as eluent to give S(À)9 as a colorless oil in
Glacial AcOH (0.22 g, 3.64 mmol) and NaBH(OAc)3 (1.08 g,
5.10 mmol) were added to
a stirred solution of 6 (0.70 g,
3.64 mmol) and S(À) -methylbenzylamine (0.44 g, 3.64 mmol) in
a
45% yield; [
a
]
D
25 = À13.3° (c 0.5, CHCl3); 1H NMR (CDCl3) d 1.09
anhydrous DCE (15 ml). The reaction mixture was allowed to stir
at room temperature under an N2 atmosphere for 24 h and then
basified with aqueous 1 N NaOH to pH 9. The mixture was
extracted with Et2O (3 Â 20 mL), the combined organic portion
was dried (Na2SO4) and solvent was evaporated to give a yellow
oil that was converted to the hydrochloride salt in anhydrous
Et2O by addition of HCl–saturated anhydrous Et2O. The precipitate
(d, 3H, CH3), 1.44 (s, 9H, CH3) 2.56 (dd, 1H, CH2), 2.72 (dd, 1H,
CH2), 3.84 (m, 1H, CH), 4.25 (s, 4H, CH2O), 6.66–6.77 (m, 3H, ArH).
5.1.11. R(+)1-(3,4-Ethylenedioxyphenyl)propan-2-ylcarbamic
acid tert-butyl ester [R(+)9]
This compound was also prepared from R(À)8 in the same
manner employed for the preparation of S(À)9 (Scheme 1);
i
was collected by filtration and recrystallized from PrOH to afford
[a]
25 = +13.0° (c 0.5, CHCl3). The 1H NMR spectrum was identical
D
S,S(À)7 as white crystals in 50% yield; mp 180–182 °C;
to that of S(À)9.
[
a]
D
25 = À34.4° (c 1.0, MeOH); 1H NMR (CDCl3) d 1.39 (d, 3H, CH3),
1.95 (d, 3H, CH3), 2.73 (dd, 1H, CH2), 2.95 (m, 1H, CH), 3.27 (dd,
1H, CH2), 4.20 (s, 4H, CH2O), 4.39 (m, 1H, CH), 6.45 (s, 1H, ArH),
6.47 (d, 1H, ArH) 6.70 (d, 1H, ArH), 7.47 (m, 3H, ArH), 7.68 (d,
2H, ArH), 9.77 (br s, 1H, NH+2), 10.25 (br s, 1H, NH2+).
5.1.12. S(À)1-(3,4-Dihydroxyphenyl)-2-ylcarbamic acid tert-
butyl ester [S(À)11]
Sodium methoxide (0.04 g, 0.74 mmol) in dry MeOH (0.5 mL)
was added to a stirred solution of S(+)1021 (0.15 g, 0.61 mmol)
and di-tert-butyl dicarbonate (0.15 g, 0.69 mmol) in MeOH
(10 mL). The reaction mixture was allowed to stir at room temper-
ature under an N2 atmosphere for 0.5 h. Solvent was evaporated
under reduced pressure, and the residue was diluted with EtOAc
(20 mL). The inorganic salt was removed by filtration and solvent
was removed under reduced pressure. The residual oil was chro-
matographed on a silica gel column (Aldrich silica gel 60) using
hexanes/EtOAc (7:3) as eluent to give S(À)11 as a colorless oil in
5.1.7. R,R(+)N-(1-Phenylethyl)-1-(3,4-ethylenedioxyphenyl)-2-
aminopropane hydrochloride [R,R(+)7]
The compound was prepared in the same manner as S,S(À)7
using R(+)a-methylbenzylamine; [a]
25 = +33.6° (c 1.0, MeOH).
D
The 1H NMR spectrum was identical to that of S,S(À)7.
5.1.8. S(+)1-(3,4-Ethylenedioxyphenyl)-2-aminopropane
hydrochloride [S(+)8]
62% yield; [
a
]
25 = À3.0° (c 1.0, CHCl3); 1H NMR (CDCl3) d 1.08 (d,
D
Pd/C catalyst (10%, 0.35 g) was added to a solution of S,S(À)7
(0.58 g, 1,74 mmol) in MeOH (30 mL). The reaction mixture was
hydrogenated at ca. 50 psi for 40 h and then filtered. The solvent
was evaporated under reduced pressure and the residue was
recrystallized from acetone to give S(+)8 as white crystals in 73%
3H, CH3), 1.44 (s, 9H, CH3) 2.54 (dd, 1H, CH2), 2.65 (dd, 1H, CH2),
3.82 (m, 1H, CH), 6.58 (dd, 1H, ArH), 6.77 (m, 2H, ArH).
5.2. In vitro release assays
yield; mp 166–167 °C;
[a]
25 = +30.1° (c 0.5, H2O); 1H NMR
5.2.1. Subjects
D
(CDCl3) d 1.42 (d, 3H, CH3), 2.79 (dd, 1H, CH2), 3.10 (dd, 1H, CH2),
3.53 (m, 1H, CH), 4.27 (s, 4H, CH2O), 6.74–6.86 (m, 3H, ArH), 8.43
(br s, 3H, NH+3).
Male Sprague-Dawley rats (Charles River, Wilmington, MA,
USA) weighing 250–350 g were housed three per cage with free
access to food and water and maintained on a 12 h light/dark cycle
with lights on from 7:00a.m. to 7:00p.m. Animal facilities were
accredited by the Association for the Assessment and
Accreditation of Laboratory Animal Care, and procedures were car-
ried out in accordance with the Institutional Animal Care and Use
Committee and the National Institutes of Health guidelines on care
and use of animal subjects in research (National Research Council,
2011).
5.1.9. S(+)1-(3,4-Ethylenedioxyphenyl)-2-aminopropane
hydrochloride [R(À)8]
The compound was prepared from R,R(+)7 in the same manner
as S(+)8; [
identical to that of S(+)8.
a
]
25 = À30.6° (c 0.5, H2O). The 1H NMR spectrum was
D
5.1.10. S(À)1-(3,4-Ethylenedioxyphenyl)propan-2-yl carbamic
acid tert-butyl ester [S(À)9]
5.2.2. Procedure
Method 1 (Scheme 1): A solution of S(+)8 (0.15 g, 0.65 mmol) in
aqueous 1 N NaOH (15 mL) was extracted with Et2O (3 Â 10 mL).
The combined organic portion was dried (Na2SO4) and the solvent
was evaporated to give the free base as a yellow oil (0.12 g,
0.62 mmol). Di-tert-butyl dicarbonate (0.14 g, 0.62 mmol) was
added to a solution of the free base and NEt3 (0.10 mL, 0.62 mmol)
in anhydrous DCM (5 mL) at 0 °C under an N2 atmosphere. The
reaction mixture was stirred at room temperature for 1 h and then
quenched with H2O (10 mL). The mixture was extracted with DCM
(3 Â 10 mL) and the combined organic portion was dried (Na2SO4).
Solvent was removed under reduced pressure and the residual oil
was then chromatographed on a silica gel column (Aldrich silica
gel 60) using hexanes/EtOAc (8:2) as eluent to give S(À)9 as a col-
orless oil in 83% yield.
Rats were euthanized by CO2 narcosis, and brains were pro-
cessed to yield synaptosomes as previously described.27 Whole
brain minus caudate and cerebellum was used to prepare synapto-
somes for SERT and NET assays, whereas caudate was used for DAT
assays. One whole brain minus caudate and cerebellum (for SERT
and NET assays) or one pair of caudates (for DAT assays) was
diluted in 10 mL of ice-cold 10% sucrose containing 1 lM reser-
pine. Tissue was homogenized using a Potter-Elvehjem homoge-
nizer, centrifuged at 1000g for 10 min at 4 °C, and supernatants
(i.e., synaptosomal preparations) were retained on ice.
Supernatants were diluted with sucrose solution to yield protein
concentrations of 900
90
lg/mL for SERT and NET assays and
l
g/mL for DAT assays. In the release procedure, 5 nM
[3H]5-HTwas used as a radioligand substrate for SERT whereas
9 nM [3H]-1-methyl-4-phenylpyridinium ([3H]MPP+) was used as
the radiolabeled substrate for NET and DAT. All buffers used in
Method 2 (Scheme 2): 1,2-Dibromoethane (0.04 mL, 0.45 mmol)
was added to a stirred mixture of S(À)11 (0.10 g, 0.38 mmol),
K2CO3 (0.16 g, 1.14 mmol) and dry DMF (5 mL). The reaction
mixture was allowed to stir at room temperature under an N2
atmosphere for 24 h. Water (10 mL) was added and the resulting
mixture was extracted with EtOAc (3 Â 10 mL). The organic
the release assay methods contained 1 lM reserpine to block vesic-
ular uptake of substrates. The selectivity of release assays was opti-
mized for a single transporter by including unlabeled blockers to
prevent the uptake of [3H]5-HT or [3H]MPP+ by competing
Please cite this article in press as: Del Bello, F.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.035

6
F. Del Bello et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
transporters. Synaptosomes were preloaded with radiolabeled sub-
strate in Krebs-phosphate buffer for 1 h (steady state). Release
References and notes
1. Green, A. R.; Cross, A. J.; Goodwin, G. M. Psychopharmacology 1993, 119, 247.
2. Parrott, A. C. Hum. Psychopharmacol. Clin. Exp. 2001, 16, 557.
3. Baylen, C. A.; Rosenberg, H. Addiction 2006, 101, 933.
assays were initiated by adding 850
lL of preloaded synaptosomes
to 150 L of test drug. Eight point dilution curves, with doses rang-
l
ing from 10 to 10,000 nM, were performed in triplicate on three
separate occasions for each test drug. Release was terminated by
vacuum filtration, and retained radioactivity was quantified by liq-
uid scintillation counting. The specific counts per minute (cpm) for
each assay (i.e., total cpm—nonspecific cpm) were 7000, 1700, and
2500 for SERT, NET, and DAT, respectively.
4. Simmler, L. D.; Buser, T. A.; Donzelli, M.; Schramm, Y.; Dieu, L.-H.; Huwyler, J.;
Chaboz, S.; Hoener, M. C.; Liechti, M. E. Br. J. Pharmacol. 2013, 168, 458.
5. Metzner, R. In Through the Gateway of the Heart. Accounts of Experiences with
MDMA and Other Empathogenic Substances; Adamson, S., Ed.; Four Trees
Publications: San Francisco, 1985; pp 1–6.
6. Young, R.; Glennon, R. A. Pharmacol. Biochem. Behav. 2008, 88, 318.
7. Rudnick, G.; Wall, S. C. Proc. Natl. Acad. Sci. U.S.A. 1817, 1992, 89.
8. Setola, V.; Hufeisen, S. J.; Grande-Allen, K. J.; Vesely, I.; Glennon, R. A.; Blough,
B.; Rothman, R. B.; Roth, B. L. Mol. Pharmacol. 2003, 63, 1223.
9. Liechti, M. E.; Vollenweider, F. X. Hum. Psychopharmacol. 2001, 16, 589.
10. Hysek, C. M.; Simmler, L. D.; Ineichen, M.; Grouzmann, E.; Hoener, M. C.;
Brenneisen, R.; Huwyler, J.; Liechti, M. E. Clin. Pharmacol. Ther. 2011, 90, 246.
11. Iversen, L. E. ‘Consideration of the cathinones’. Advisory Council on the Misuse
of Drugs. A report submitted to the Home Secretary of the UK (March 31, 2010).
12. Glennon, R. A. Adv. Pharmacol. 2014, 69, 581.
13. Methylone (bk-MDMA) Critical Review Report, Expert Committee on Drug
Dependence 36th Meeting, World Health Organization, Agenda item 4.14,
2014.
14. Cozzi, N. V.; Sievert, M. K.; Shulgin, A. T.; Jacob, P.; Ruoho, A. E. Eur. J. Pharmacol.
1999, 381, 63.
15. Baumann, M. H.; Ayestas, M. A.; Partilla, J. S.; Sink, J. R.; Shulgin, A. T.; Brandt, S.
D.; Rothman, R. B.; Ruoho, A. E.; Cozzi, N. V. Neuropsychopharmacology 2012, 37,
1192.
5.2.3. Data analysis
Statistical analyses were carried out using GraphPad Prism
(v. 6.0; GraphPad Scientific, San Diego, CA, USA). EC50 values and
corresponding SDs for stimulation of release were calculated based
on non-linear regression analysis. Efficacy for test compounds was
expressed as a percentage of maximal release (i.e.,% Emax), which
was defined as the release produced by 100
lM tyramine for
SERT and 10 M tyramine for DAT and NET. These concentrations
l
of tyramine induce the efflux of all ‘releasable’ tritium from
synaptosomes under the assay conditions described.
16. U.S. Drug Enforcement Administration, Office of Diversion Control, National
Forensic Laboratory Information System Report, June 2014. https://www.nflis.
deadiversion.usdoj.gov.
17. Baumann, M. H.; Partilla, J. S.; Lehner, K. R. Eur. J. Pharmacol. 2013, 698, 1.
18. Rannazzisi, J. T. Federal Register, 71, 203 (October 20 2006), 62017.
19. Vallejos, G.; Fierro, A.; Rezende, M. C.; Sepulveda-Boza, S.; Reyes-Parada, M.
Bioorg. Med. Chem. 2005, 13, 4450.
20. Bockmühl, M.; Erhart, G. US Patent 1964973A, July 3, 1934.
21. Nichols, D. E.; Barfknecht, C. F.; Rusterholz, D. B.; Benington, F.; Morin, R. D. J.
Med. Chem. 1973, 16, 480.
5.2.4. Reagents
[3H]5-HT (specific activity = 30 Ci mmolÀ1) was purchased from
Perkin Elmer (Shelton, CT, USA). [3H]MPP+ (specific
activity = 85 Ci mmolÀ1
)
was
purchased
from
American
Radiolabeled Chemicals (St. Louis, MO, USA). All other chemicals
and reagents were acquired from Sigma–Aldrich (St. Louis, MO,
USA).
22. Milhazes, N.; Cunha-Oliveira, T.; Martins, P.; Garrido, J.; Oliveira, C.; Rego, A. C.;
Borges, F. Chem. Res. Toxicol. 2006, 19, 1294.
23. Eshleman, A. J.; Wofrum, K. M.; Hatfield, M. G.; Johnson, R. A.; Murphy, K. V.;
Janowsky, A. Biochem. Pharmacol. 1803, 2013, 85.
24. Sakloth, F.; Kolanos, R.; Mosier, P. D.; Bonano, J. S.; Banks, M. L.; Partilla, J. S.;
Baumann, M. H.; Negus, S. S.; Glennon, R. A. Br. J. Pharmacol. 2015, 172, 2210.
25. Bonano, J. S.; Banks, M. L.; Kolanos, R.; Sakloth, F.; Barnier, M. L.; Glennon, R. A.;
Cozzi, N. V.; Partilla, J. S.; Baumann, M. H.; Negus, S. S. Br. J. Pharmacol. 2015,
172, 2433.
Acknowledgments
This work was supported in part by PHS Grant DA033930 and
by the Intramural Program of the National Institute on Drug Abuse.
Supplementary data
26. Shulgin, A.; Shulgin, A. Pihkal; Transform Press: Berkeley, CA, 1991.
27. Baumann, M. H.; Dersch, C. M.; Romero, D. V.; Rice, K. C.; Carroll, F. I.; Partilla, J.
S. Synapse 2001, 39, 32.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.07.035.
Please cite this article in press as: Del Bello, F.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.035