"deconstruction" of the abused synthetic cathinone methylenedioxypyrovalerone (MDPV) and an examination of effects at the human dopamine transporter

Article abstract of DOI:10.1021/cn4001236

Synthetic cathinones, β-keto analogues of amphetamine (or, more correctly, of phenylalkylamines), represent a new and growing class of abused substances. Several such analogues have been demonstrated to act as dopamine (DA) releasing agents. Methylenediox

Full text of DOI:10.1021/cn4001236

Research Article
pubs.acs.org/chemneuro
“Deconstruction” of the Abused Synthetic Cathinone
Methylenedioxypyrovalerone (MDPV) and an Examination of Effects
at the Human Dopamine Transporter
Renata Kolanos,† Ernesto Solis, Jr.,‡ Farhana Sakloth,† Louis J. De Felice,*,‡ and Richard A. Glennon*,†
†Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia 23298, United
States
‡Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, Virginia 23298,
United States
S
* Supporting Information
ABSTRACT: Synthetic cathinones, β-keto analogues of amphetamine (or, more correctly, of
phenylalkylamines), represent a new and growing class of abused substances. Several such analogues
have been demonstrated to act as dopamine (DA) releasing agents. Methylenedioxypyrovalerone (MDPV)
was the first synthetic cathinone shown to act as a cocaine-like DA reuptake inhibitor. MDPV and seven
deconstructed analogues were examined to determine which of MDPV’s structural features account(s) for
uptake inhibition. In voltage-clamped (−60 mV) Xenopus oocytes transfected with the human DA
transporter (hDAT), all analogues elicited inhibitor-like behavior shown as hDAT-mediated outward
currents. Using hDAT-expressing mammalian cells we determined the affinities of MDPV and its analogues
to inhibit uptake of [3H]DA by hDAT that varied over a broad range (IC50 values ca. 135 to >25 000 nM). The methylenedioxy
group of MDPV made a minimal contribution to affinity, the carbonyl group and a tertiary amine are more important, and the
extended α-alkyl group seems most important. Either a tertiary amine, or the extended α-alkyl group (but not both), are required
for the potent nature of MDPV as an hDAT inhibitor.
KEYWORDS: bk-Amphetamines, β-keto amphetamines, β-ketophenylalkylamines, hDAT, electrophysiology, “bath salts”, drug abuse
he naturally occurring β-keto analogue of the phenyl-
alkylamine amphetamine, now termed cathinone (1;
Figure 1), was first identified as the major central stimulant
component of the shrub “khat” (Catha edulis) in 1975.1 The
khat plant has been used for many hundreds of years in certain
Middle Eastern countries for its stimulant action.2 Cathinone
analogues (currently termed “synthetic cathinones”) seemingly
erupted on the drug abuse scene just in the past few years.
However, some analogues were known earlier. The first
synthetic cathinone, termed “methcathinone” (2)3 (the N-
methyl counterpart of cathinone or the β-keto analogue of
methamphetamine), was actually a widely abused substance in
the former Soviet Union since the early 1980s under the name
of ephedrone; however, this information was not disseminated
until 1989,4 and was not widely available for many years
thereafter. Another synthetic cathinone is methylone or
MDMC (3), the β-keto analogue of the empathogen MDMA
(or N-methyl-(3,4-methylenedioxyphenyl)-2-aminopropane;
4). This synthetic cathinone analogue was independently
reported by two groups of investigators in the mid-1990s.5,6
In 2010, an alarm was sounded by the British Home Office
that the abuse of synthetic cathinones in Europe was on the
rise.7 One of the popular synthetic cathinones at the time was
referred to as “bath salts” (although known by several other
names) that contained either mephedrone (5), methylenediox-
ypyrovalerone (MDPV; 6), methylone (3), or a combination of
two or more of these and/or other agents.8 MDPV was
originally patented as a central stimulant in 1969,9 but its
identification as a drug of potential abuse did not occur until
T
Figure 1. Chemical structures of cathinone (1), methcathinone (2),
methylone (MDMC; 3), MDMA (4), mephedrone (5), and MDPV
(6).
Received: June 10, 2013
Revised: October 9, 2013
© XXXX American Chemical Society
A
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nearly 40 years later.10 The three most common bath salts
ingredients were emergency scheduled (U.S. Schedule I) in
2011;11 mephedrone and MDPV were permanently scheduled
in 201212, and methylone was permanently scheduled in
2013.13 Recent reports confirm the central stimulant actions of
MDPV.14,15 As of now, 44 synthetic cathinones have been
encountered on the clandestine market.16
Cathinone and methcathinone, like amphetamine and
methamphetamine, have been previously found to act,
primarily, at the human dopamine (DA) membrane transporter
(hDAT) by causing the release of DA.3,17 It was initially
thought that mephedrone and MDPV, being cathinone
analogues, might act in a similar fashion. However, we found
that mephedrone induces hDAT-mediated inward currents
characteristic of a substrate whereas MDPV induces hDAT-
mediated outward currents characteristic of a cocaine-like
inhibitor in cells voltage-clamped to −60 mV.18−20 Consistent
with these findings, others have now found that mephedrone is
(primarily) a DA releasing agent whereas MDPV is an hDAT
reuptake inhibitor.21−24
An early goal of these studies was to determine which
molecular components of MDPV are responsible for its actions
as a cocaine-like agent compared to, for example, methcathi-
none (which acts as a DA-like releasing agent at hDAT). To
address this, we employed two-electrode voltage-clamp
(TEVC) in hDAT-expressing Xenopus laevis oocytes (clamped
to −60 mV) in response to MDPV and the MDPV analogs. In
each recording, an oocyte was first exposed to a short
application of 5 μM DA, followed by 10 μM application for
1 min of either MDPV (Figure 2A) or one of the analogues
MDPV is unique because, although it interacts with the
hDAT, it appears to act in a mechanistically different manner
than other synthetic cathinones already investigated. The
purpose of the present study was to determine what it is
about the structure of MDPV that makes it a mechanistically
different type of synthetic-cathinone entity. We employed a
deconstruction approach of the MDPV molecule to address the
problem. We deconstructed the MDPV molecule, one
structural change at a time (i.e., a chemical cladistics approach)
to examine this; seven deconstructed analogues (7−13) of
MDPV were synthesized and investigated.
Figure 2. MDPV (A) and compound 8 (B) confer long-lasting effects
on hDAT. Currents were recorded in voltage-clamped (−60 mV)
Xenopus laevis oocytes expressing hDAT (using a two-electrode
voltage-clamp technique); results are representative. (A) Exposure to
DA (5 μM) produced an hDAT-mediated inward current. MDPV
application (10 μM, 1 min) induced an outward hDAT-mediated
current that did not return to baseline when the drug was washed out.
Following exposure to MDPV, a 5 μM DA-application induced a
diminished hDAT-mediated inward current. (B) In a different oocyte,
exposure to DA (5 μM) yielded an hDAT-mediated inward current.
Application of compound 8 (10 μM, 1 min) induced an outward
hDAT-mediated current that did not return to baseline when the drug
was washed out. After exposure to compound 8, application of DA (5
μM) produced a diminished hDAT-mediated inward current (though
slightly larger than the current elicited after MDPV application in A).
(Representative tracings for the other six compounds not shown here
are provided in the Supporting Information.)
Some of the target compounds have been previously
reported in the chemical literature; their synthesis was
replicated here, and the targets were isolated as their
hydrochloride salts: 6,26 8,25 9,26 10,27,28 11,29 and 12.27,28 1-
(Benzo[d]-1,3-dioxol-5-yl)pentanone (14) was prepared as
reported by Masazumi et al.30 and brominated with Br2 to
afford 15 (Scheme 1). Compound 15 has been previously
prepared by a different method;31 although its boiling point was
1
not reported, the H NMR spectrum of 15 was identical with
that reported in the literature. Reaction of 15 with pyrrolidine
followed by catalytic reduction afforded 7 (Scheme 1).
(see Figure 2B and Supporting Information). In contrast to the
“synthetic cathinones” methcathinone and mephedrone, which
produce hDAT-mediated inward currents in cells voltage-
clamped to −60 mV, all of the deconstructed analogues,
unexpectedly, behaved in the same manner as MDPV. At 10
μM, all the examined compounds produced qualitatively similar
hDAT-mediated outward currents that were similar in
amplitude (normalized to percent of the amplitude of the
trace induced by the first DA application, Table 1). The
outward current represents the block of an endogenous leak
current and suggests that all the compounds behaved as hDAT
inhibitors. Compound 8 has been previously found to act as a
DA reuptake inhibitor.25
Scheme 1
In these same recordings, however, we sought to determine if
exposure to MDPV and its analogues would affect hDAT
activity. Following washout for 1 min, a subsequent application
of 5 μM DA (after exposure to MDPV or its analogs) elicited
hDAT-mediated inward currents with distinct amplitudes
(Figure 2 and Supporting Information). For example, following
MDPV exposure, DA elicited a diminished hDAT-mediated
inward current (as compared to the first DA application)
(Figure 2A). In contrast, after exposing hDAT to compound 8
B
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“cathinone analogue” to an “amphetamine analogue”, reduced
affinity by nearly 10-fold (7; IC50 = 1,150 nM). In contrast,
compound 8 (IC50 = 205 nM), lacking the methylenedioxy
group of MDPV, possessed an affinity similar to that of MDPV.
The chief structural differences between MDPV and other
cathinone analogues (including, for example, analogues 3, 5,
and cathinone itself) is the presence of an α-n-propyl group and
the pyrrolidine moiety. Shortening the α-n-propyl to an α-
methyl group (i.e., 9; IC50 = 3540 nM) produced a 25-fold
decrease in affinity. Replacement of the pyrrolidine ring of
MDPV with an N,N-dimethylamine (i.e., 10; IC50 = 715 nM)
decreased affinity by 5-fold. A similar decrease in affinity was
observed when 9 was converted to its N,N-dimethylamine
counterpart 11 (IC50 = 22 300 nM). Demethylation of 10 to
secondary amine 12 (IC50 = 1,950 nM) reduced affinity by <3-
fold, whereas primary amine 13 (IC50 = 27 100 nM) displayed
another ∼15-fold decreased affinity.
Table 1. Magnitude of the Effect Produced by MDPV and
Analogues 7−13 (10 μM), and Percent Recovery Following a
Subsequent Application of 5 μM Dopamine
a
b
% response ( SEM)
% recovery ( SEM)
N
MDPV
16.4 (2.4)
15.3 (1.7)
14.8 (2.1)
18.0 (1.2)
14.1 (1.7)
18.7 (2.2)
16.9 (2.0)
19.2 (3.4)
12.1 (1.1)
90.0 (1.6)
25.4 (1.7)
91.9 (1.9)
57.5 (2.4)
102.0 (0.8)
75.7 (4.5)
102.6 (1.5)
7
5
5
6
3
5
5
5
7
8
9
10
11
12
13
a
Percent response (relative to the amplitude of the inward current
b
produced by 5 μM DA. Percent recovery upon application of 5 μM
DA 1 min following the outward current produced by MDPV or 7−
13.
As described, several different structural features of MDPV
are responsible for its potent actions as an hDAT inhibitor (as
measured from the affinity values obtained in the uptake
inhibition assay) and, accordingly, several different structural
features of MDPV confer its long-lasting actions at hDAT (as
seen in the recovery experiment). Following MDPV exposure,
DA elicited an hDAT-mediated inward current 12.1% the size
of the first DA application (Figure 2A, Table 1). The
methylenedioxy group of MDPV, although making a small
contribution, is not a major determinant for the long-lasting
effect on hDAT, since DA recovery was 25.4% (viz. 8). The
carbonyl group of MDPV, although a modest contributor to
uptake inhibition affinity (10-fold lower than MDPV), seems to
play a significant role to produce the long-lasting effect on
hDAT (90% recovery) (viz. 7). Moreover, shortening the α-n-
propyl chain to an α-methyl group (i.e., 9) resulted in a 25-fold
decrease in affinity, which seems related to its pronounced role
to elicit the long-lasting effect on hDAT (DA recovery =
91.9%). Comparing MDPV with 10, and 9 with 11, the
presence of the cyclic ring system seems to contribute to
affinity (∼5−6-fold drop in affinity for both comparisons),
which parallels changes on DA recovery (MDPV (12%) vs 10
(57.5%) and 9 (91.9%) vs 11 (102%)). The secondary and
primary amine counterparts of 10 (i.e., 12 and 13) elicited
increased DA recovery (75.7% for 12 and 102.6% for 13)
agreeing with 3-fold and 14-fold affinity reductions, respec-
tively, as compared to 10. Conversion of the pyrrolidine moiety
of MDPV to its simplest tertiary (i.e., N,N-dimethyl), secondary
(i.e., N-monomethyl), and primary amine counterparts (i.e., 10,
12, and 13, respectively) resulted in relatively small decreases in
affinity, but, taken together, conversion of the pyrrolidine
moiety found in MDPV to its primary amine 13 resulted in a
collective 200-fold decrease in affinity. Similarly, the conversion
from MDPV to analogues with sequential pyrrolidine moiety
modifications (10 to 12 to 13) shifted DA recovery from 12.1%
to 102.6%, suggesting that a tertiary amine is a major
contributor to the potent effect MDPV has at hDAT.
(under the same conditions as Figure 2A), DA produced a
somewhat larger hDAT-mediated inward current as compared
to the current produced by the initial DA application (Figure
2B). Eventually, modifications to the structure of MDPV did
not exhibit a diminished hDAT-mediated inward current and a
full DA recovery was seen (as with compounds 11 and 13 (see
Table 1 and Supporting Information).
By employing hDAT-expressing HEK-293 cells, we deter-
mined the affinities of MDPV and 7−13 to inhibit uptake of
[3H]DA by hDAT (Figure 3), which spanned a 200-fold range.
None of the structural modifications resulted in a compound
with a higher affinity than MDPV (IC50 = 135 nM). Removal of
the carbonyl group of MDPV, essentially converting it from a
It would appear, then, that either (but not necessarily both) a
tertiary amine (as found, for example, in MDPV, 9, and 10) or
an extended side chain (as found, for example, in MDPV and
12) is required for high affinity, but that optimal affinity is
associated with both substituents. The presence of either feature
seems sufficient to convert the hDAT substrate-like nature of
simple methcathinone (2) analogues (inferred by their ability
to induce inward currents at −60 mV) to inhibitor-like agents
(inferred by their ability to induce outward currents).
Figure 3. Affinity (IC50 SEM) of deconstructed analogues 7−13 for
hDAT relative to that of MDPV itself. Each arrow represents a single
molecular modification.
C
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Pd/C (0.05 g), and glacial AcOH (10 mL) was shaken in a Parr
hydrogenator under H2 (ca. 55 psi) at room temperature for 16 h.
During this time, the starting material was consumed and, presumably,
1-(benzo[d]-1,3-dioxol-5-yl)-2-(pyrrolidin-1-yl)pentan-1-ol was
formed as determined by IR analysis. Additional amounts of 10%
Pd/C (0.03 g) and perchloric acid (0.4 mL) were periodically added at
24 h intervals while the reaction mixture was further hydrogenated for
a total 72 h until the intermediate alcohol was completely consumed.
The catalyst was removed by filtration through a Celite pad. The
filtrate was basified with 15% NaOH to pH 9−10 and extracted with
EtOAc (3 × 10 mL). The combined organic portion was washed with
brine (10 mL) and dried (Na2SO4), and solvent was removed under
reduced pressure. The oily residue was converted to its hydrochloride
salt by dissolving it in anhydrous Et2O and adding a saturated solution
of gaseous HCl in anhydrous Et2O. The salt separated as an oil.
Solvent was removed under reduced pressure, and the oily residue
solidified after being dried under high vacuum (0.15 g, 79%). The solid
was recrystallized twice from a mixture of absolute EtOH/anhydrous
Et2O to afford the product (0.06 g, 31%) as beige crystals: mp 135−
137 °C; 1H NMR (DMSO-d6) δ 0.77 (t, J = 7.2 Hz, 3H, CH3), 1.16−
1.34 (m, 2H, CH2), 1.52−1.57 (m, 2H, CH2), 1.89−1.97 (m, 4H,
CH2), 2.75 (dd, J = 13.5, 9.7 Hz, 1H, CH2), 3.07−3.12 (m, 3H, CH2),
3.46−3.49 (m, 3H, CH2), 6.00 (s, 2H, CH2O), 6.75 (t, J = 7.8 Hz, 1H,
ArH), 6.87 (d, J = 7.8 Hz, 1H, ArH), 6.92 (s, 1H, ArH), 10.41 (br s,
1H, NH+). Anal. Calcd for C16H23NO2·HCl·0.25H2O: C, 63.56; H,
8.17; N, 4.63. Found: C, 63.70; H, 7.97; N, 4.63.
All compounds studied here acted similar to MDPV though
their ability to inhibit hDAT uptake is weaker than MDPV.
When comparing uptake assays with current measurements,
note that, unlike current recordings, uptake inhibition is in the
presence of DA and currents measured are under −60 mV
voltage-clamp whereas in the uptake assay cells are unclamped.
One of the major differences among the compounds
investigated herein is their effect on recovery (Table 1). This
needs to be examined in more detail, and was examined here
only with a single fixed (i.e., 5 μM) concentration of DA.
Nevertheless, percent recovery is highly correlated with affinity
(r = 0.928, n = 8, when pIC50 is plotted against % recovery)
(Supporting Information Figure 2). This does not seem to be
related to incomplete washout because the response to MDPV,
the agent showing the least recovery following application of
DA after 1 min of buffer washout, failed to return to baseline
even following >30 min of washing.20 It would seem, then, that
the SAR for recovery is similar to the SAR for affinity in that
those analogues with the highest affinity resulted in the least
recovery.
It can be concluded that (a) the methylenedioxy ring of
MDPV is not a major contributor to its affinity for hDAT, (b)
the carbonyl group of MDPV increases its affinity for hDAT,
and (c) a tertiary amine or, more importantly, the extended side
chain, are sufficient and critical contributors to affinity. From
previous work, we have established that the direction of current
elicited by a compound (inward vs outward) can be used to
determine its action. Inward and outward currents would be
expected to depolarize or hyperpolarize a cell, respectively.
Mechanistically, MDPV acts in a different manner than, for
example, methcathinone or mephedrone at hDAT. The
purpose of this investigation was to specifically identify what
structural features of MDPV account for these differences. The
contribution of MDPV’s structural features to its action to
induce hDAT-mediated outward currents has now been
identified and quantified.
2-Amino-1-(benzo[d]-1,3-dioxol-5-yl)pentan-1-one Hydrochlor-
ide (13). Sodium azide (0.15 g, 2.31 mmol) was added to a solution
of α-bromoketone 15 (0.66 g, 2.31 mmol) in anhydrous MeOH (10
mL), and the reaction mixture was allowed to stir at room temperature
for 12 h. Solvent was removed under reduced pressure. The resulting
solid residue was dissolved in H2O (20 mL) and extracted with EtOAc
(3 × 6 mL). The combined organic portion was dried (Na2SO4), and
the solvent was removed under reduced pressure to afford the α-azido
ketone (0.52 g, 91%) as a dark-yellow oil that was used without further
purification: 1H NMR (CDCl3) δ 0.98 (t, J = 7.4 Hz, 3H, CH3), 1.43−
1.60 (m, 2H, CH2), 1.78−1.91 (m, 2H, CH2), 4.47 (dd, J = 8.4, 5.4
Hz, 1H, CH), 6.07 (s, 2H, CH2O), 6.88 (d, J = 8.2 Hz, 1H, ArH), 7.42
(d, J = 1.6 Hz, 1H, ArH), 7.52 (dd, J = 8.2, 1.6 Hz, 1H, ArH).
Tin(II) chloride dihydrate (0.99 g, 4.38 mmol) was added in one
portion to a solution of the α-azido ketone (0.51 g, 2.19 mmol) in
absolute EtOH (10 mL) at 0 °C (ice-bath), and the reaction mixture
was allowed to stir at room temperature for 2 h. Solvent was removed
under reduced pressure, and the oily residue was partitioned between a
saturated solution of NaHCO3 and EtOAc (100 mL/25 mL). The
organic layer was separated and the aqueous portion was extracted
with EtOAc (2 × 25 mL). The combined organic portion was washed
with brine (25 mL) and dried (Na2SO4), and solvent was removed
under reduced pressure to give a yellow, oily residue. The oil was
converted to its hydrochloride salt and recrystallized from absolute
EtOH to afford 13 (0.26 g, 49%) as white crystals: mp 224−225 °C
Once our studies had been completed, it came to light that
several of the agents we examined here have now appeared on
the clandestine market.16 For example, 8 (already known as α-
PVP), 9 (now known as MDPPP), 11 (bk-MDDMA), and 12
(bk-MBDP) have been confiscated by law enforcement agents.
So, in addition to an investigation of the SAR of MDPV as a
cocaine-like agent, an unintended consequence of the present
investigation is to provide some of the first information on the
mechanism of action of several newly confiscated synthetic
cathinones that have not been previously investigated
mechanistically.
1
(dec); H NMR (DMSO-d6) δ 0.82 (t, J = 7.2 Hz, 3H, CH3), 1.16−
1.29 (m, 1H, CH2), 1.32−1.45 (m, 1H, CH2), 1.66−1.83 (m, 2H,
CH2) 5.02 (dd, J = 7.0, 4.7 Hz, 1H, CH), 6.18 (s, 2H, CH2O), 7.11 (d,
J = 8.2 Hz, 1H, ArH), 7.54 (d, J = 1.6 Hz, 1H, ArH), 7.71 (dd, J = 8.2,
METHODS
■
+
1.6 Hz, 1H, ArH), 8.44 (br s, 3H, NH3 ). Anal. Calcd for C12H15NO3·
Chemistry. All commercially available reagents and solvents were
purchased from Sigma-Aldrich Co. (St. Louis, MO) and used as
delivered. Melting points were measured in glass capillary tubes
HCl: C, 55.93; H, 6.26; N, 5.43. Found: C, 55.97; H, 6.11; N, 5.40.
1-(Benzo[d]-1,3-dioxol-5-yl)-2-bromopentan-1-one (15). Bromine
(0.35 mL, 1.09 g, 6.8 mmol) was added in one portion to a stirred
mixture of 1-(benzo[d]-1,3-dioxol-5-yl)pentan-1-one (14)29 (9.45 g,
45.8 mmol) and freshly sublimed AlCl3 (0.31 g, 2.3 mmol) in
anhydrous Et2O (150 mL) at 0 °C under an N2 atmosphere. After 10
min, the ice-bath was removed. The reaction mixture was allowed to
warm to room temperature, and additional Br2 (2.00 mL, 6.23 g, 39.0
mmol) was added in a dropwise manner over a 5 min period. The
reaction mixture was neutralized by adding a saturated solution of
NaHCO3. The organic layer was separated and dried (Na2SO4), and
solvent was removed under reduced pressure. The crude material was
purified by Kugelrohr distillation to afford the product (11.24 g, 86%)
as a yellow oil: bp 217 °C, 0.6 Torr; 1H NMR (CDCl3) δ 1.00 (t, J =
1
(Thomas-Hoover melting point apparatus) and are uncorrected. H
NMR spectra were recorded with a Bruker 400 MHz spectrometer.
Chemical shifts (δ) are reported in parts per million (ppm) relative to
tetramethylsilane as internal standard. Infrared (IR) spectra were
recorded using a Thermo Scientific Nicolet iS10 FT-IR spectrometer.
The purity of all compounds (>95%), established by elemental
analysis, was performed by Atlantic Microlabs (Norcross, GA) and are
within 0.4% of theory. Reactions and product mixtures were routinely
monitored by thin-layer chromatography (TLC) on silica gel
precoated F254 Merck plates.
1-(1-(Benzo[d]-1,3-dioxol-5-yl)pentan-2-yl)pyrrolidine Hydro-
chloride (7). A mixture of MDPV (6) (0.20 g, 0.64 mmol), 10%
D
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7.4 Hz, 3H, CH3), 1.40−1.62 (m, 2H, CH2), 2.07−2.22 (m, 2H, CH2),
5.09 (dd, J = 7.7, 6.6 Hz, 1H, CH), 6.09 (s, 2H, CH2O), 6.90 (d, J =
8.2 Hz, 1H, ArH), 7.51 (d, J = 1.7 Hz, 1H, ArH), 7.65 (dd, J = 8.2, 1.7
Hz, 1H, ArH).
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Addition of synthetic drugs to Schedule I of the Controlled Substances
Act, pp 138−140 (enacted January 3, 2012; signed July 9, 2012)
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Expression of hDAT in Xenopus laevis Oocytes. Oocytes were
harvested and prepared from adult X. laevis females following standard
procedures.32−34 Stage V−VI oocytes were selected for cRNA
injection within 24 h of isolation. cRNA was transcribed from the
pOTV vector using the mMessage Machine T7 kit (Ambion Inc.,
Austin, TX). Oocytes were injected with 50 ng of hDAT cRNA
(Nanoject AutoOocyteInjector, Drummond Scientific Co., Broomall,
PA) and incubated at 18 °C for 4−8 days in Ringers solution
supplemented with sodium pyruvate (550 μg/mL), streptomycin (100
μg/mL), tetracycline (50 μg/mL), and 5% dialyzed horse serum.
Electrophysiology. Two-electrode voltage-clamp (TEVC) experi-
ments were performed as previously described.35 Recordings were at
room temperature (23−25 °C). Electrodes having a resistance of 1−5
MΩ were filled with 3 M KCl. X. laevis oocytes expressing hDAT were
voltage-clamped to −60 mV with a GeneClamp 500 (Axon
Instruments), and the holding current was recorded using a Clampex
10 (Axon Instruments). Extracellular buffer consisted of (in mM): 120
NaCl, 7.5 HEPES, 5.4 potassium gluconate, and 1.2 calcium gluconate,
pH 7.4. In a typical recording, extracellular buffer was perfused until
stable baseline currents were obtained, followed by experimental drugs
(perfusion duration is indicated by a horizontal line on the trace).
Maintenance of Cells Stably Expressing hDAT (hDAT-HEK).
Cells were prepared in DMEM supplemented with 10% FBS, 2 mM L-
glutamine, penicillin (100 units/mL), streptomycin (100 mg/mL), and
G418.
3
Radiolabeled H-DA Uptake Competition Assay. hDAT-HEK
cells in DMEM suspension were counted, and 200 000 cells were
placed in individual Eppendorf tubes and incubated for 15 min at
room temperature (23−25 °C) with mixtures of 1% [3H]DA. The
compounds were tested (MDPV and analogs) at a broad range of
concentrations. Ice-cold (4 °C) PBS was added to the samples, cells
were centrifuged for 1 min at 2000 rpm, supernatant was removed, and
cells were washed twice with PBS, centrifuged, and supernatant was
removed. Cells were solubilized with Ecoscint H (National
Diagnostics, Atlanta, GA), and the [3H]DA remaining was measured
using a scintillation counter. To determine IC50 values, uptake values
were plotted against concentration and data were fit using the Hill
equation y = Vmax + (Vmin − Vmax) * xn/(kn + xn) using Origin 8
(OriginLab Corporation, Northampton, MA).
ASSOCIATED CONTENT
■
S
* Supporting Information
Representative tracings for currents induced by analogues 7 and
9−13, and the correlation between % DA recovery versus
hDAT affinity. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
(15) Fantegrossi, W. E., Gannon, B. M., Zimmerman, S. M., and Rice,
K. C. (2013) In vivo effects of abused “bath salt” constituent 3,4-
methylenedioxypyrovalerone (MDPV) in mice: Drug discrimination,
thermoregulation, and locomotor activity. Neuropsychopharmacology
38, 563−573.
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Nations Office of Drugs and Crime. A United Nations Publication,
Vienna, Austria.
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amphetamine-like mechanism of action of the alkaloid cathinone.
Biochem. Pharmacol. 35, 3015−3019.
(18) Kolanos, R., Cameron, K. N., Vekariya, R. H., De Felice, L. J.,
and Glennon, R. A. (2011) “Bath salts”: An imitation of methamphet-
amine plus cocaine? Southeast Regional Meeting of American
Chemical Society (SERMACS), Richmond, VA, October 26−29.
(19) Cameron, K., Kolanos, R., Verkariya, R., De Felice, L., and
Glennon, R. A. (2013a) Mephedrone and methylenedioxypyrovaler-
■
Corresponding Authors
*E-mail: glennon@vcu.edu.
*E-mail: ljdefelice@vcu.edu.
Author Contributions
R.K. and F.S. synthesized and characterized the target
compounds, and E.S. performed the assays and analyzed the
data; all provided manuscript input. R.A.G. conceived the
initiation of the project; R.A.G. and J.L.D. oversaw the project,
assisted with data analysis, and wrote the paper.
Funding
This work was supported in part by PHS Grant DA 033930.
Notes
The authors declare no competing financial interest.
E
dx.doi.org/10.1021/cn4001236 | ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Research Article
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dx.doi.org/10.1021/cn4001236 | ACS Chem. Neurosci. XXXX, XXX, XXX−XXX