α-PPP and its derivatives are selective partial releasers at the human norepinephrine transporter: A pharmacological characterization of interactions between pyrrolidinopropiophenones and uptake1 and uptake2 monoamine transporters

Article abstract of DOI:10.1016/j.neuropharm.2021.108570

While classical cathinones, such as methcathinone, have been shown to be monoamine releasing agents at human monoamine transporters, the subgroup of α-pyrrolidinophenones has thus far solely been characterized as monoamine transporter reuptake inhibitors. Herein, we report data from previously undescribed α-pyrrolidinopropiophenone (α-PPP) derivatives and compare them with the pharmacologically well-researched α-PVP (α-pyrrolidinovalerophenone). Radiotracer-based in vitro uptake inhibition assays in HEK293 cells show that the investigated α-PPP derivatives inhibit the human high-affinity transporters of dopamine (hDAT) and norepinephrine (hNET) in the low micromolar range, with α-PVP being ten times more potent. Similar to α-PVP, no relevant pharmacological activity was found at the human serotonin transporter (hSERT). Unexpectedly, radiotracer-based in vitro release assays reveal α-PPP, MDPPP and 3Br-PPP, but not α-PVP, to be partial releasing agents at hNET (EC₅₀ values in the low micromolar range). Furthermore, uptake inhibition assays at low-affinity monoamine transporters, i.e., the human organic cation transporters (hOCT) 1–3 and human plasma membrane monoamine transporter (hPMAT), bring to light that all compounds inhibit hOCT1 and 2 (IC₅₀ values in the low micromolar range) while less potently interacting with hPMAT and hOCT3. In conclusion, this study describes (i) three new hybrid compounds that efficaciously block hDAT while being partial releasers at hNET, and (ii) highlights the interactions of α-PPP-derivatives with low-affinity monoamine transporters, giving impetus to further studies investigating the interaction of drugs of abuse with OCT1-3 and PMAT.

Full text of DOI:10.1016/j.neuropharm.2021.108570

Neuropharmacology 190 (2021) 108570
Contents lists available at ScienceDirect
Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm
α
-PPP and its derivatives are selective partial releasers at the human
norepinephrine transporter
A pharmacological characterization of interactions between pyrrolidinopropiophenones
and uptake1 and uptake2 monoamine transporters
Julian Maiera, Laurin Rautera, Deborah Rudina, Marco Nielloa, Marion Holya,
Diethart Schmidb, Joseph Wilsonc, Bruce E. Bloughc, Brenda M. Gannond
,
,
e
Kevin S. Murnaned e, Harald H. Sittea
,
,f,*
a
¨
Medical University of Vienna, Center for Physiology and Pharmacology, Institute of Pharmacology, Wahringerstraße 13A, 1090, Vienna, Austria
b
¨
Medical University of Vienna, Center for Physiology and Pharmacology, Institute of Physiology, Wahringerstraße 13A, 1090, Vienna, Austria
c Research Triangle Institute, Center for Drug Discovery, Research Triangle Park, NC, USA
d Mercer University College of Pharmacy, Mercer University Health Sciences Center, Department of Pharmaceutical Sciences, Atlanta, GA, USA
e Louisiana State University Health Sciences Center, Shreveport, Department of Pharmacology Toxicology & Neuroscience and Louisiana Addiction Research Center,
Shreveport, LA, USA
f
¨
AddRess Centre for Addiction Research and Science, Medical University of Vienna, Wahringerstraße 13A, 1090, Vienna, Austria
A R T I C L E I N F O
A B S T R A C T
Keywords:
While classical cathinones, such as methcathinone, have been shown to be monoamine releasing agents at human
α
-PVP
monoamine transporters, the subgroup of
α-pyrrolidinophenones has thus far solely been characterized as
New psychoactive substances
Human monoamine transporters
Partial release
monoamine transporter reuptake inhibitors. Herein, we report data from previously undescribed
nopropiophenone ( -PPP) derivatives and compare them with the pharmacologically well-researched
-pyrrolidinovalerophenone). Radiotracer-based in vitro uptake inhibition assays in HEK293 cells show that the
investigated -PPP derivatives inhibit the human high-affinity transporters of dopamine (hDAT) and norepi-
nephrine (hNET) in the low micromolar range, with -PVP being ten times more potent. Similar to -PVP, no
relevant pharmacological activity was found at the human serotonin transporter (hSERT). Unexpectedly,
radiotracer-based in vitro release assays reveal -PPP, MDPPP and 3Br-PPP, but not -PVP, to be partial releasing
α
-pyrrolidi-
α
α
-PVP
(
α
Organic cation transporter
PMAT
α
α
α
α
α
agents at hNET (EC50 values in the low micromolar range). Furthermore, uptake inhibition assays at low-affinity
monoamine transporters, i.e., the human organic cation transporters (hOCT) 1–3 and human plasma membrane
monoamine transporter (hPMAT), bring to light that all compounds inhibit hOCT1 and 2 (IC50 values in the low
micromolar range) while less potently interacting with hPMAT and hOCT3. In conclusion, this study describes (i)
three new hybrid compounds that efficaciously block hDAT while being partial releasers at hNET, and (ii)
highlights the interactions of
α-PPP-derivatives with low-affinity monoamine transporters, giving impetus to
further studies investigating the interaction of drugs of abuse with OCT1-3 and PMAT.
1. Introduction
Substances Conventions (Baumann and Volkow, 2016; Maier et al., 2018a;
UNODC, 2021), are oftentimes deliberately consumed as cheap, legal alter-
natives to established drugs of abuse or, unwittingly, as adulterants of promi-
nent street drugs (Maier et al., 2018b; WDR, 2020). Synthetic cathinones are
Newpsychoactivesubstances(NPS), drugsofabusethatarenotcontrolled
by the 1961 and 1971 United Nations Narcotic Drugs and Psychotropic
Abbreviations: MAT, monoamine transporter; OCT, organic cation transporter; hDAT, human dopamine transporter; hNET, human norepinephrine transporter;
hSERT, human serotonin transporter; hOCT1-3, human organic cation transporter 1–3; hPMAT, human plasma membrane monoamine transporter; -PPP, -pyr-
α
α
rolidinopropiophenone; α-PVP, α-pyrrolidinovalerophenone.
¨
* Corresponding author. Medical University of Vienna, Center for Physiology and Pharmacology, Institute of Pharmacology, Wahringerstrasse 13a, A-1090 Vienna,
Austria.
E-mail address: harald.sitte@meduniwien.ac.at (H.H. Sitte).
https://doi.org/10.1016/j.neuropharm.2021.108570
Received 18 January 2021; Received in revised form 31 March 2021; Accepted 12 April 2021
Available online 20 April 2021
0028-3908/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

J. Maier et al.
Neuropharmacology 190 (2021) 108570
NPS, targeting monoaminergic neurotransmission, that have been illicitly
producedandsoldundertheumbrellaterms“bathsalts”and“plantfood”since
the2000s(Kelly, 2011;Simmonsetal., 2018). Whilenotmanytoxicologically
confirmed fatalities associated with the sole consumption of synthetic cath-
inones have been reported, they were the third most frequently seized NPS
substance group after synthetic cannabinoids and ketamine derivatives in
recent years (Beck et al., 2016; WDR, 2020; Wright and Harris, 2016; Zaami
etal.,2018).Cathinoneandmethcathinonehavebeenplacedunderschedule1
of the controlled substances act in the UN Convention on Psychotropic Sub-
stances, which includes stereoisomers but not derivatives of scheduled sub-
stances. Thus, clandestine chemists can bypass legal obstacles by synthesizing
unscheduled derivatives. One major challenge in the field of NPS is that it is
exceedingly dynamic – e.g., even though ten additional, new synthetic cath-
inones were placed into schedule 1 by the U.S. drug enforcement administra-
tionin2014,manymorederivativeshaveappearedonillicitmarketseversince
(DEA, 2014; Majchrzak et al., 2018).
Cathinone ((2S)-2-amino-1-phenylpropan-1-one) is, in addition to the
less potent cathine ((1S,2S)-2-amino-1-phenylpropan-1-ol), the main
activealkaloidoftheshrubCathaedulis(khat)(Ahmedandel-Qirbi, 1993).
The first synthetic cathinone derivative, methcathinone (2-(methyl-
amino)-1-phenylpropan-1-one), was described by Hyde et al., in 1928 and
marketed as an antidepressant and stimulant (Hyde et al., 1928; Simmons
et al., 2018). Structurally, cathinones are β-phenethylamines with an
α
-ketone chain next to the aromatic ring (differentiating them from e.g.
amphetamine, see Fig. 1) (Kelly, 2011). A further characterization of de-
rivatives into four groups is common (Majchrzak et al., 2018): (i) cath-
inones with simple N-alkyl-groups and/or aromatic ring substituents (such
as mephedrone), (ii) cathinones with a 3,4-methylenedioxy-substituted
aromatic ring (e.g. methylone), (iii) compounds with an N-pyrrolidinyl
group and aromatic substitution other than 3,4-methylenedioxy and (iv)
cathinones containing both N- pyrrolidinyl and 3,4-methylenedioxyl sub-
stituents. Compounds from groups three and four can be further classified
Fig. 1. The chemical structures of the herein investigated compounds and the
closely related amphetamine and cathinone. The chemical structure of (A)
amphetamine (1-phenylpropan-2-amine), (B) cathinone ((2S)-2-amino-1-phe-
nylpropan-1-one) – the
α
-ketone chain, differentiating it from amphetamine, is
highlighted in red, (C)
α
-pyrrolidinovalerophenone ( -PVP; 1-phenyl-2-pyrroli-
α
din-1-ylpentan-1-one), (D)
α-pyrrolidinopropiophenone (α-PPP; 1-phenyl-2-
as
without 3,3-methylenedioxyl (MD) substituents – such as
rolidinovalerophenone; 1-phenyl-2-pyrrolidin-1-ylpentan-1-one),
-Pyrrolidinohexiophenone; 1-phenyl-2-pyrrolidin-1-ylhexan-1-one),
-PBP -Pyrrolidinobutiophenone; 1-phenyl-2-pyrrolidin-1-ylbuta-
α
-pyrrolidinophenones, that come in various structures – with and
-PVP ( -pyr-
-PHP
pyrrolidin-1-ylpropan-1-one), (E) 3′,4′-Methylenedioxy-alpha-pyrrolidinopro-
piophenone (MDPPP; 1-(1,3-benzodioxol-5-yl)-2-pyrrolidin-1-ylpropan-1-one),
α
α
α
(F) 3′-bromo-
α-pyrrolidinopropiophenone (3-Br PPP; 1-(3-bromophenyl)-2-
(
α
pyrrolidin-1-ylpropan-1-one), (G) 4′-bromo-
α
-pyrrolidinopropiophenone (4-Br
-pyr-
α
(α
PPP; 1-(4-bromophenyl)-2-pyrrolidin-1-ylpropan-1-one), (H) 4′-methyl-
α
n-1-one) or
α-PPP (α-Pyrrolidinopropiophenone; 1-phenyl-2-pyrrolidi-
rolidinopropiophenone (4-Me PPP; 1-(4-methylphenyl)-2-pyrrolidin-1-ylpro-
pan-1-one).
n-1-ylpropan-1-one) derivatives.
Generally, compounds interacting with human monoamine trans-
porters (MATs) can be classified as either substrate-type releasing agents
that cause the efflux of monoamines via reverse transport or as non-
transported inhibitors that bind to MATs in the outward-facing confor-
mation, blocking monoamine reuptake (Sitte and Freissmuth, 2015).
Additionally, (i) allosteric ligands, binding to an alternative site of the
MAT, (ii) atypical inhibitors, ligands that do not produce
inhibitor-typical effects and (iii) partial releasers, causing less efflux
than full releasing agents, have been described (Bhat et al., 2019;
Hasenhuetl et al., 2019; Niello et al., 2020; Rothman et al., 2012).
N-alkyl-group and/or aromatic ring containing cathinones and cath-
inones with a 3,4-methylenedioxy-substituted aromatic ring are MAT
substrates and releasing agents at (at least one) MAT, while inhibiting
the other(s) with varying potencies. On the other hand, cathinone de-
rivatives containing N-pyrrolidinyl group and aromatic or 3,4-methyle-
nedioxyl substituents have been characterized as reuptake inhibitors of
the human dopamine (hDAT; SLC6A3), norepinephrine (hNET; SLC6A2)
and (oftentimes less pronounced) serotonin (hSERT; SLC6A4) MATs
(Eshleman et al., 2017; Simmler et al., 2013). In contrast to the high
affinity, low capacity MATs, the organic cation transporters 1–3
(hOCT1-3, SLC22A1-3; OCTs) and plasma membrane monoamine
transporter (hPMAT, SLC29A4) have a low affinity but a high capacity
for monoamine transport. Due to the important role they – together with
the MATs – play in maintaining a monoaminergic equilibrium in the
central nervous system, recent research has increasingly focused on their
interaction with various drugs (Gasser, 2019; Mayer et al., 2018, 2019).
Meltzer et al. (2006), inspired by the chemical structure of bupropion
and in search of selective dopamine (and norepinephrine) transporter
inhibitors, were among the first to systematically examine
structure-activity relationships of α-pyrrolidinophenones (1-(4-Methyl-
phenyl)-2-pyrrolidin-1-yl-pentan-1-one) (Meltzer et al., 2006). More
recently, because of the increasing number of cathinone derivatives
appearing on illicit markets, thorough analyses of the effects of various
substituents on the potency to interact with MATs have been conducted
in vitro (Bonano et al., 2015; Eshleman et al., 2013; Kolanos et al., 2015;
Rickli et al., 2015; Sakloth et al., 2015; Simmler et al., 2013). Herein, we
provide a pharmacodynamic analysis of the effects of five previously
unreported or un- or undercharacterized α-PPP derivatives (see Fig. 1,
α
-PVP was included as a control substance) at hDAT, hNET and hSERT,
as well as hOCT1-3 and hPMAT, painting a complete picture of
compound-monoamine transporter interaction profiles. Consequently,
this study provides structure-activity relationship analyses for
α-PPP
derivatives, identifying partial releasing agents at hNET and classifying
their interaction with the hOCTs and hPMAT. This might potentially
lend inspiration to further investigations of this interesting chemical
scaffold with yet unknown translational potential.
2. Material and methods
2.1. Reagents and chemicals
The compounds (Fig. 1) were synthesized as described in Fig. 2.
2

J. Maier et al.
Neuropharmacology 190 (2021) 108570
Bromination of the desired commercially available substituted propio-
phenones (1a-1e) with bromine in methylene chloride afforded the
Cells were then preincubated with increasing concentrations of the
compounds, dissolved in Milli-Q H2O or DMSO (dimethyl sulfoxide),
α
-bromo intermediates (2a-2e) in good yields. Conversion to the corre-
diluted in 50
μL of KHB (for a final concentration of 1%) for 10 min.
sponding phenyl-substituted pyrrolidinyl-1-propanones (D-H) by reac-
tion with pyrrolidine in anhydrous ethanol, and immediate
hydrochloride salt afforded the hydrochloride salts in 52%–58% yield
(over 3 steps). The target compounds were immediately converted to
their hydrochloride salts to avoid decomposition. Additional informa-
tion is available in the supplemental material (Appendix A). Racemic
Uptake solution (50
μL), containing KHB, the test compounds and
tritiated substrate (DAT: 0.1
μ
M [3H]DA; NET: 0.05
μ
M [3H]MPP+;
SERT: 0.1
μ
M [3H]5-HT; OCT1-3: 0.03
μ
M [3H]MPP+; PMAT: 0.05
μM
[3H]MPP+), was then added to the wells. Uptake was terminated by
removal of tritiated substrate and by washing the cells with 100 μL ice-
cold KHB after 60 s (DAT and SERT), 180 s (NET) or 600 s (OCT1-3,
α
-PVP was graciously provided by the NIDA Drug Supply Program. [3H]
PMAT). Thereafter, 200 μL of Ultima GoldTM XR scintillation cocktail
1-methyl-4-phenylpyridinium ([3H]MPP+; 80–85
μ
Ci x mmolꢀ 1) was
(PerkinElmer, MA, USA) was added, the plates were shaken and tritiated
substrate uptake was determined in a Wallac 1450 MicroBeta TriLux
Liquid Scintillation Counter & Lumi (GMI, Ramsey, MN, USA). Non-
specific tritiated substrate uptake was measured in the presence of
purchased from American Radiolabeled Chemicals (St. Louis, MO, USA),
[3H]Dopamine (55
μ
Ci x mmolꢀ 1) and [3H]5-HT (28.3
μ
Ci x mmolꢀ 1
)
were purchased from PerkinElmer (Boston, MA, USA) and utilized for
the radiotracer uptake inhibition and release assays. Other chemicals
and cell culture supplies were obtained from Sigma-Aldrich (St. Louis,
MO, USA) and cell culture dishes were ordered from Sarstedt (Nuem-
brecht, Germany).
100
500
μ
M mazindole (DAT), 1 mM MPP+ (NET), 50
μM paroxetine (SERT),
μ
M corticosterone (OCT1-3) and 100 μM decynium-22 (PMAT) and
subtracted from the data. Uptake in the presence of test drugs was
plotted in relation to uptake in the presence of vehicle.
2.2. Cell culture
2.4. Superfusion release assays
For the MATs, stable monoclonal human embryonic kidney
(HEK293) cell lines, overexpressing the proteins of interest, were
established as previously described (Ilic et al., 2020; Mayer et al., 2017).
Stable polyclonal HEK293 cell lines, expressing OCT 1–3 and PMAT,
were established as follows: HEK293 cells were transfected with the
desired plasmid employing the jetPRIME transfection method, accord-
ing to the manufacturer’s instructions (VWR International GmbH,
Vienna, Austria). High selection pressure was maintained for approxi-
Superfusion experiments allowfor the timely resolution of MAT-mediated
substrate efflux, eliminating the possibility of substrate reuptake by clearing
released tritiated substrate via continuous flow (Steinkellner et al., 2016).
Cells, grown on glass-coverslips, were preloaded with 0.05
μ
M [3H]MPP+
(NET and DAT) or 0.4
μ
M [3H]5-HT (SERT) for 20 min at 37 ◦C. Afterwards,
the coverslips were placed in cylindrical chambers and superfused with KHB
(0.7 mL x minꢀ 1), kept at a constant temperature of 25 ◦C, for 40 min to
establish a stable basal release. Three 2-min-fractions of basal release were
then collected in 10 mL counting vials. Subsequently, cells were superfused
mately a week by adding 200
μ
L geneticin (G418, 50 mg x mLꢀ 1). Cells
were then sorted with FACS and 500,000 cells were chosen according to
expression levels.
with monensin (10 μM) or vehicle for four fractions. The ionophore monensin
can be used to distinguish non-transported inhibitors from substrate-type
releasers because it dissipates the sodium gradient that is essential for SLC6
neurotransmitter-sodium-symporter function. When coupled with an inhibi-
tor, monensindoesnotaugmentitseffects, contrarytoitsusageinconjunction
with a releasing agent. Afterwards, exposition of the cells to the test com-
Before uptake inhibition and release assays, cells were maintained in
high glucose- (4.5 g x Lꢀ 1) and L-glutamine-containing (584 mg x Lꢀ 1
)
Dulbecco’s Modified Eagle Medium with 10% heat-inactivated Fetal
Bovine Serum (FBS), penicillin (100 U x 100 mLꢀ 1) and streptomycin
(100 mg x 100 mLꢀ 1) added. For maintaining the selection process,
geneticin (50 mg x mLꢀ 1) was utilized. Cells were grown in 10-cm cell
culture dishes in an incubator at 37 ◦C and 5% CO2. The day prior to
uptake inhibition and batch release experiments, cells were seeded onto
PDL-coated (poly-D-lysine) 96-well plates at a density of approximately
0.2 × 106 cells x mLꢀ 1. For superfusion release assays, cells were, at the
same density, seeded onto PDL-coated glass coverslips, as previously
described (Maier et al., 2018b).
pounds (30
that the compounds, at best, would be moderate to weak releasers) or control
compounds(knownreleasing agentsatNET, DAT:10 MD-amphetamineand
SERT: 3 M para-chloroamphetamine, pCA) was started and five fractions
μM, a concentration this high was used because it was estimated
μ
μ
were collected. Eventually, cells were lysed with 1% SDS (sodium dodecyl
sulfate). Tritiated substrate of each fraction was quantified with a
beta-scintillation counter (PerkinElmer, Waltham, MA, USA). The released
amount of tritiated substrate for each fraction was expressed as percentage of
tritium at the beginning of the fraction.
2.3. Uptake inhibition assays
2.5. Batch release assays
At the beginning of the experiment, cells were washed with Krebs-
HEPES-buffer (KHB - 25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2
mM CaCl2, and 1.2 mM MgSO4 and 5 mM D-glucose, pH adjusted to 7.3).
One major disadvantage of superfusion release assays is that a rela-
tively high amount of test compound is necessary to establish a clear
Fig. 2. Route of synthesis for the PPP derivatives. For details, see Methods and Appendix A.
3

J. Maier et al.
Neuropharmacology 190 (2021) 108570
dose-response relationship (Steinkellner et al., 2016). Thus, we made
use of the batch release method to determine the compounds’ efficacy to
cause reverse transport at the high-affinity monoamine transporters
(Steinkellner et al., 2016). HEK293 cells stably expressing hNET, seeded
the Mann-Whitney-U test. P values < 0.05 were considered statistically
significant.
3. Results
in 96-well plates, were pre-loaded with 0.05
μ
M [3H]MPP+, dissolved in
KHB, for 20 min at 37 ◦C. Afterwards, the cells were washed twice with
KHB and equilibrated for 10 min. Next, the cells were incubated with
3.1. The compounds inhibit transporter-mediated uptake in hDAT- and
hNET-expressing HEK293 cells
several concentrations of
α-PPP, MDPPP or 3-Br PPP, or 10 μM
D-amphetamine for 10 min. Subsequently, the supernatant was trans-
ferred to another 96-well plate and 200 L Ultima GoldTM XR scintil-
lation cocktail (PerkinElmer, MA, USA) was added. The cells in the first
96-well plate were lysed with 200 L liquid scintillation cocktail and the
To assess whether the compounds interact with the SLC6 MATs
(hDAT, hNET, hSERT), uptake inhibition assays were conducted (see
Fig. 3). All compounds proved to be fully efficacious inhibitors of uptake
by hDAT and hNET, in the low micromolar range (see Table 1). The
μ
μ
amount of tritium in the cells and the supernatant was assessed with a
Wallac 1450 MicroBeta TriLux Liquid Scintillation Counter & Lumi
(GMI, Ramsey, MN, USA). The release of tritiated neurotransmitter was
expressed as percentage of the total radioactivity of cells and superna-
tant together, normalized to the basal efflux of untreated cells.
reference compound α-PVP is a 10–20 times more potent inhibitor than
PPP-based compounds. Most substances are (close to) equipotent at
inhibiting hDAT and hNET, with only MDPPP having a DAT/NET ratio
of 4.7 and 3-Br and 4-Me PPP around 2 (2.11 and 2.17, respectively). All
compounds are only weak inhibitors of the human serotonin transporter,
with 4-Br PPP being the most potent. Consequently, all investigated
substances display high DAT/SERT ratios (see Table 1).
Non-specific release was determined in the presence of 30
nisoxetine.
μM
Table 1
2.6. Data and statistical analysis
Half-maximal inhibitory concentrations (IC50) at the hDAT, hNET and hSERT
and DAT/SERT ratio for the tested compounds.
IC50 and EC50 values were calculated and plotted with GraphPad
Prism 8.4.3 (GraphPad Software Inc., San Diego, CA, U.S.A.). Half-
maximal inhibitory concentrations (IC50) were determined by non-
linear regression, solving equation Y=Bottom + (Top-Bottom)/(1 +
10^(X-LogEC50)). Transporter ratios were determined by calculating
((1/numerator IC50)/(1/denominator IC50)). Half-maximal effective
concentrations (EC50) were calculated by non-linear regression, solving
equation Y=Bottom + (Top-Bottom)/(1 + 10^((LogEC50-X))). All data
stem from at least three separate experiments (n = 3), in triplicate (i.e.,
for example, nine wells per data point for the uptake inhibition assays),
and are portrayed as mean ± SD. Data from superfusion assays, in the
presence of monensin or vehicle over time, was statistically analyzed
DAT IC50 in
[95%-CI]
μM
NET IC50 in
[95%-CI]
μ
M
SERT IC50 in
[95%-CI]
μ
M
DAT/
SERT ratio
α
-PVP
-PPP
0.017
0.016
207 [156–283]
12176.47
3498.28
764.87
344.74
43.66
[0.0128–0.022]
0.58 [0.34–0.91]
[0.014–0.018]
0.58
α
2029
[0.46–0.72]
1.74
[1616–2607]
283 [213–374]
MDPPP
0.37 [0.32–0.43]
0.38 [0.24–0.62]
1.52 [1.18–1.97]
0.48 [0.38–0.61]
[1.46–2.08]
0.80
3-Br
PPP
4-Br
PPP
4-Me
PPP
131
[0.70–0.91]
1.65
[99.20–174]
66.36
[1.40–1.94]
1.04
[46.28–94.57]
266 [189–379]
554.17
ˇ
[0.95–1.14]
´
with a mixed-effects model, employing Sidak’s correction for multiple
comparisons. Data from the batch release assays were compared with
Fig. 3. Effects of α-PVP, α-PPP (green line), MDPPP (red line), 3-Br PPP (blue line), 4-Br PPP and 4-Me PPP on transporter-mediated uptake of indicated tritiated
substrate in HEK293 cells stably expressing (A) hDAT, (B) hNET and (C) hSERT. All experiments were conducted three separate times, in triplicate.
4

J. Maier et al.
Neuropharmacology 190 (2021) 108570
3.2.
α
-PPP, MDPPP and 3-Br PPP are monoamine releasing agents at the
as caused by releasing agents, reliant on sodium (as is the case in the
SLC6 family of neurotransmitter/sodium symporters) can therefore be
selectively augmented (Scholze et al., 2000). None of the compounds
caused monoamine release at the human dopamine and serotonin
transporters (for all the data and control experiments with known
human norepinephrine transporter
Uptake inhibition assays provide important information regarding
the interaction between a drug and a transporter but are inconclusive in
the sense that both, substrate-type releasing agents and non-transported
inhibitors, cause a concentration-dependent reduction of tritiated sub-
strate to be located (and measured) intracellularly (Scholze et al., 2000).
Dynamic superfusion release assays employ the Na+/H+ ionophore
monensin to distinguish between the two. Monensin causes increased
sodium influx, thus dissipating the sodium gradient. Outward transport,
releasing agents, as well as α-PVP, see Appendices B and C). Release at
hNET was significantly increased by monensin for
3-Br PPP (see Fig. 4).
α-PPP, MDPPP and
Fig. 4. Effects of (A, B, C) α-PPP, (D, E, F) MDPPP and (G, H, I) 3-Br PPP on transporter-mediated release of preloaded radiolabeled substrate from HEK293 cells
stably expressing (A, D, G) hDAT, (B, E, H) hSERT and (C, F, I) hNET. As indicated, after 6 min of basal release collection, monensin or vehicle was added and after an
ˇ
´
additional 6 min the compound of interest + monensin or vehicle was superfused onto the cells. A statistical analysis with a mixed-effects model, employing Sidak’s
correction for multiple comparisons confirmed significant differences between monensin + compound and vehicle + compound at the indicated time points, thus
validating both compounds’ releasing capabilities. * denotes p < 0.05, **p < 0.01 and ***p < 0.001 (exact values: α-PPP: p = 0.0097, 0.0002, 0.0239, respectively,
DF = 189; MDPPP: p = 0.007789, 0.009121, 0.006736, DF = 156; 3-Br PPP: p = 0.0282, 0.0066, 0.0084, DF = 180). All the other compounds did not cause release at
the investigated transporters, see Appendix B. For control experiments with known full releasing agents, see Appendix C. All experiments were conducted three
separate times, in triplicate.
5

J. Maier et al.
Neuropharmacology 190 (2021) 108570
3.3. The potency of
release at hNET
α
-PPP, MDPPP- and 3-Br PPP-caused monoamine
thearomaticringcaninfluencecompounds’ abilitytointeractwiththehuman
5-HTtransporter(Ricklietal.,2015;Sahaetal.,2015,2019).Therankorderof
the investigated compounds concerning their potency to inhibit hSERT is 4-Br
Subsequently, we conducted dose-response release assays to assess the
potency of the three compounds that cause monoamine release at hNET (see
Fig. 5). As we have previously reported (Seidel et al., 2005), concentrations
past saturation can cause monoamine release to subside again and show a
downward slope for high concentrations, akin to blockers (i.e. a Gaussian
PPP > 3-Br PPP > 4-Me PPP > MDPPP > α-PPP, confirming that a bromide,
more than a methyl or methylenedioxyl aromatic, substituent increases the
serotonergicproperties(Eshlemanetal., 2017;Ricklietal., 2015).Still, except
for 4-Br PPP, all investigated substances were not exceedingly potent hSERT
inhibitors, thus resulting in high hDAT/hSERT ratios, which has been previ-
bell-shaped curve).
(EC50:0.98 M,95%CI:0.68–1.42
0.19–0.55
α
-PPP (EC50: 0.70
μM, 95% CI: 0.52–0.95
μ
M), MDPPP
ously reported for α-PVP and other PPP derivatives (Eshleman et al., 2013).
μ
μ
M)and3-BrPPP(EC50:0.34
μ
M,95%CI:
High hDAT/hSERT ratios, i.e. the ability of compounds to potently inhibit
hDAT while interacting less with hSERT, have been associated with rein-
forcing effects and increased abuse liability of drugs (Baumann et al., 2011;
Weeetal.,2005).Thisishighlyindicativeofasignificantpotentialforabuseof
the herein investigated compounds, although it might be less pronounced for
the bromide substituted derivatives. In addition to in vitro studies, a myriad of
in vivo investigations have corroborated the abuse liability of cathinones
(Gannon et al., 2018; Ray et al., 2019; Riley et al., 2020).
μ
M) are potent, selective releasing agents at the human norepi-
nephrine transporter. Due to the significant difference between the peak of
efflux caused by the test compounds and the comparator substance
D-amphetamine, a full releasing agent at hNET, α-PPP, MDPPP and 3-Br PPP
canbeclassifiedaspartialreleasersathNET,withamaximalefficacyof68.73,
59.33 and 63.90% respectively, compared to D-amphetamine. For experi-
ments with 4-Me and 4-Br PPP, which did not show releasing properties, see
Appendix D.
Recent research suggests that, in addition to the SLC6 MATs, the
understudied OCTs and PMAT play an important role in monoaminergic
neurotransmission and equilibration (Mayer et al., 2018). While hOCT2
and 3, as well as hPMAT, are abundantly expressed in the central ner-
vous system, hOCT1 is mostly expressed in liver and kidney, suggesting
an influence in excretion or reuptake of cationic compounds (Koepsell,
2020; Wang, 2016). No previous structure-activity relationship analyses
for cathinones have explored hOCT1-3 and hPMAT. While the con-
ducted analysis of six compounds only allows for the formulation of
hypotheses to further investigate in future studies, several findings seem
worthy of further scrutiny. It appears to be the case that compounds with
3.4. The interaction of the compounds with hOCT 1–3 and hPMAT
Uptake inhibition assays were conducted to assess whether the
compounds additionally interact with the human low-affinity mono-
amine transporters. All compounds inhibit uptake by the human organic
cation transporters 1 and 2 with similar potencies in the low micromolar
range (see Fig. 6, Tables 2 and 3). The substances do not interact with
the human organic cation transporter 3 at pharmacologically relevant
concentrations. Half-maximal inhibitory concentration values at hPMAT
are in the micromolar range across compounds. For control experiments
with proven potent inhibitors of the OCTs and PMAT, see Appendix E.
a bulky
α
-chain are less potent inhibitors of the human organic cation
transporter 3 but more potent hPMAT inhibitors (
α
-PVP). Aromatic
substituents seem to affect the ability to inhibit hOCT3 and hPMAT
uptake differently, while seemingly not altering their potency at hOCT1
and 2. Most strikingly, the phenethylamine structure in general seems to
allow compounds to potently inhibit hOCT1 and 2 but not hOCT3. Liu
and colleagues employed a machine learning approach and concluded
that hOCT1 and 2 preference by ligands could not be clearly distin-
guished, which can be explained by their high degree of amino acid
sequence homology (70%) (Liu et al., 2016; Sala-Rabanal et al., 2013).
hOCT3 shares less sequence identity (50%) with the other OCTs and it
has been previously shown that many unselective inhibitors and sub-
strates of the organic cation transporters are the least efficacious at
hOCT3 (Sala-Rabanal et al., 2013). Similarly, we have found that hOCT1
and 2 but not hOCT3 share similar affinities for many ligands. Salient
features of known hOCT ligands are their cationic/neutral charge, high
degrees of hydrophobicity and greater sp3 character (Liu et al., 2016;
Sala-Rabanal et al., 2013). While the herein investigated compounds
show relatively similar IC50 values at hOCT1 and 2, their relative po-
tency, reflected in the DAT/OCTs and NET/OCTs ratios, differs sub-
stantially (see Table 3). Due to its high potency to inhibit hDAT and
hNET and comparatively decreased ability to inhibit the OCTs, the
4. Discussion
We herein characterized five PPP derivatives, detailing their in-
teractions with human monoamine transporters. Their pharmacological
properties were compared with the thoroughly studied and classified
α
-PVP. All compounds were found to be potent (i.e. IC50 in the low
micromolar range) inhibitors of hDAT and hNET while displaying much
lower potency at hSERT. As has been previously shown, the reference
compound α-PVP is a 10–20 times more potent inhibitor than PPP-based
compounds (Glennon and Dukat, 2017; Kolanos et al., 2015). As ex-
pected, none of the compounds caused monoamine efflux at hDAT and
hSERT, however, to our surprise, some of the compounds indeed caused
hNET-mediated efflux (see Figs. 4 and 5). This study is the first to show
that α-pyrrolidinophenones, in this case the parent compound α-PPP,
3-Br PPP and MDPPP, that have previously been described as pure up-
take inhibitors, can act as selective and potent releasing agents at the
human norepinephrine transporter. It might even be the case that,
additionally, 4-Br PPP and 4-Me PPP could be very weak partial hNET
releasers, causing too little efflux to be detected with the employed
methods. The second novel finding concerns the previously unexplored
interaction of cathinones with the low-affinity monoamine transporters
hOCT1-3 and hPMAT. Remarkably, all compounds potently inhibited
uptake at the human organic cation transporters 1 and 2, as well as at
hPMAT, but not at hOCT3.
ability of
ditions where small amounts of
necessarily the case for -PPP and its derivatives, which have transporter
ratios closer to unity. Thus, the OCTs might be important CNS targets,
particularly for -PPP derivatives.
It was, to our knowledge, previously unreported that
α-PVP to inhibit the OCTs might be less relevant under con-
α
-PVP are consumed. This is not
α
α
α
-pyrrolidino-
Concerning the structure-activity relationship of the investigated com-
pounds, several findings are noteworthy. As previously described, it was
phenone derivatives can act as partial releasing agents at hNET. Previ-
ous studies classified the pyrrolidine ring-containing cathinones as pure
uptake inhibitors (Brandt et al., 2020; Eshleman et al., 2017). Eshleman
et al. (2017) used a similar superfusion assay but we, in addition, (i)
made use of the ionophore monensin to prove the substrate status of
compounds, (ii) confirmed results with another method (batch release
assays in our case) and (iii) normalized the data to basal efflux. Eshle-
man et al. (2017), on the other hand, normalized to efflux caused by a
highly potent known releasing agent, therefore potentially not seeing
the effects of a “weaker”, partial releaser. Our results suggest that the
confirmed that an increase in chain length of the α-side chain results in
increased potency to inhibit hDAT and hNET uptake (α-PVP vs. α-PPP)
(Eshleman et al., 2017; Kolanos et al., 2015). A methylenedioxyl substituent
seems to slightly bolster the compounds’ ability to inhibit monoamine uptake
by hDAT, with MDPPP displaying an increased potency compared to α-PPP,
while slightly decreasing its potency to inhibit hNET (Eshleman et al., 2017;
Gannonetal.,2018).Still,itisnoteworthythatalltheinvestigatedcompounds
areequipotentorclosetoequipotent(MDPPP, 3-Brand4-MePPP)concerning
theirabilitytoinhibithDATandhNET(Eshlemanetal., 2017). Substituentsof
6

J. Maier et al.
Neuropharmacology 190 (2021) 108570
Fig. 5. Dose-response release assays with (A) -PPP, (B) MDPPP and (C) 3-Br
α
PPP, portrayed as violin plots (with individual values in dots) of the different
concentrations of the compounds used, as percentage of total efflux. KHB,
nisoxetine and D-amphetamine were used as control substances. The Mann-
Whitney-U test was employed to compare between the highest amount of efflux
caused by the investigated compounds and peak efflux caused by D-amphet-
amine, a known full releaser. **** denotes p < 0.0001 (exact values: p =
0.000041, sum of ranks: 45 vs. 126). For experiments with 4-Me PPP and 4-Br
PPP, which did not show release at hNET, see Appendix D. All experiments were
conducted three separate times, in triplicate.
pyrrolidine ring does not seem to be the limiting factor for MAT trans-
port. Nevertheless, it remains enigmatic which substituents are the
decisive factors to distinguish non-transported inhibitors from trans-
ported releasing agents (Sandtner et al., 2016). Norepinephrine
releasing agents or uptake inhibitors have been employed as anorectic
drugs for the last decades, although, due to pronounced side-effects, the
focus of non-invasive therapeutic interventions has mostly shifted to
compounds with different pharmacodynamic properties (Garcia Ram-
irez et al., 2020; Milano et al., 2020). The anorectic diethylpropion is a
prodrug, which acts via its active metabolite ethcathinone, that has
previously been described as a potent hNET releasing agent (with
additional weaker releasing effects at hSERT) as well as an hDAT uptake
inhibitor (Simmler et al., 2014; Yu et al., 2000). The herein classified
α
-pyrrolidinophenones, α-PPP, 3-Br PPP and MDPPP, show a similar
pharmacodynamic profile, and can be regarded as hybrid compounds
that are partial releasing agents at hNET but potent inhibitors of hDAT
and weaker inhibitors of hSERT. Partial releasers produce less efflux
than full releasers (Bhat et al., 2019) (see Fig. 5, compounds compared
to D-amphetamine). It is hypothesized that partial releasers either bind
to a distinct site compared to a full releaser, thus constituting allosteric
modulators, or reside for a longer period of time at the same binding site,
slowing down efflux (Hasenhuetl et al., 2019; Niello et al., 2020;
Rothman et al., 2012). Compared to full releasers, partial releasers at
hDAT might have lower abuse potential (Rothman et al., 2012). It re-
mains unclear whether and potentially how partial releasing agents at
hNET could be employed pharmacologically and clinically. In general,
major potential challenges for the utilization of phenethylamine de-
rivatives as anorectic drugs are their psychostimulant effects and un-
clear abuse liability (Garcia-Mijares et al., 2009). Still, the cathinone
derivative bupropion certainly constitutes a pharmacological and
medical success story (Costa et al., 2019). Recent data suggests that, in a
self-administration paradigm, higher doses of α-PPP elicit robust rein-
forcing effects (Nagy et al., 2020). In vivo data for MDPPP suggests an
abuse liability higher than cocaine, thus rendering it unattractive as a
therapeutic option (Gannon et al., 2018). 3-Br PPP has been shown to
have more pronounced serotonergic effects and might therefore
constitute a more appropriate chemical scaffold for derivatives to be
investigated in potential future in vitro and in vivo studies. While it often
is the case in the field of NPS that (former) medical drugs serve as
inspiration to clandestine chemists, to reverse this pattern would
certainly be an interesting twist (Maier et al., 2018b).
5. Conclusion
The five investigated
hibitors of uptake at hDAT, hNET, hOCT1, hOCT2 and hPMAT, with
negligible potency at hSERT and hOCT3. Three compounds, -PPP,
MDPPP and 3-Br PPP have been shown to be potent partial releasing
agents at hNET, a feature previously unknown for -pyrrolidinophe-
α-pyrrolidinophenone derivatives all are in-
α
α
nones, thus rendering them alluring hybrid compounds. Because of the
fact that only five compounds were available for this investigation, in-
formation about potential structure-activity relationships (especially at
the hOCTs and hPMAT) can only be seen as hypotheses-generating. The
strength of this study is that it provides a complete classification of a
group of compounds concerning their interaction with low- and high-
(caption on next column)
7

J. Maier et al.
Neuropharmacology 190 (2021) 108570
Fig. 6. Effects of
α-PVP,
α
-PPP (green line), MDPPP (red line), 3-Br PPP (blue line), 4-Br PPP and 4-Me PPP on transporter-mediated uptake of tritiated MPP+ in
HEK293 cells stably expressing (A) hOCT1, (B) hOCT2, (C) hOCT3 and (D) hPMAT. For control experiments with known potent inhibitors, see Appendix E. All
experiments were conducted three separate times, in triplicate.
Table 2
Table 3
Half-maximal inhibitory concentrations (IC50) for the human organic cation
transporters 1–3 (hOCT1-3) and the plasma membrane monoamine transporter
(hPMAT).
hDAT/hOCT1 and 2, as well as hNET/hOCT1 and 2 ratios. Higher numbers
indicate more potent inhibition of the numerator than the denominator.
DAT/OCT1
ratio
DAT/OCT2
ratio
NET/OCT1
ratio
NET/OCT2
ratio
OCT1 IC50 in
M [95%-CI]
OCT2 IC50 in
M [95%-CI]
OCT3 IC50 in
M [95%-CI]
PMAT IC50 in
M [95%-CI]
μ
μ
μ
μ
α
-PVP
-PPP
77.06
2.24
2.59
1.66
0.38
1.90
330.59
12.26
15.05
17.13
4.88
81.88
2.24
0.55
0.79
0.35
0.88
351.25
12.26
3.20
α
α
-PVP
-PPP
1.31
5.62
1631
13.42
MDPPP
3-Br PPP
4-Br PPP
4-Me
[1.06–1.64]
1.30
[3.89–7.92]
7.11
[1209–2286]
1050
[9.76–18.70]
75.48
8.14
α
4.49
[1.10–1.55]
0.96
[5.47–9.23]
5.57
[854–1311]
326 [261–410]
[61.43–93.10]
21.86
16.92
7.81
MDPPP
PPP
[0.77–1.13]
0.63
[4.48–6.92]
6.51
[17.21–28.18]
60.62
3-Br
PPP
4-Br
PPP
4-Me
PPP
332 [265–419]
149 [120–183]
433 [378–496]
[0.49–0.81]
0.57
[5.54–7.64]
7.41
[45.47–81.51]
17.53
editing, Visualization. Marion Holy: Investigation, Writing – review &
editing. Diethart Schmid: Investigation, Writing – review & editing.
Joseph Wilson: Investigation, Resources, Writing – review & editing,
Visualization. Bruce E. Blough: Conceptualization, Resources, Writing
– review & editing, Visualization. Brenda M. Gannon: Conceptualiza-
tion, Resources, Writing – review & editing. Kevin S. Murnane:
Conceptualization, Resources, Writing – review & editing. Harald H.
Sitte: Conceptualization, Methodology, Resources, Writing – review &
editing, Supervision, Project administration, Funding acquisition.
[0.46–0.71]
0.91
[5.54–9.83]
8.12
[12.39–28.53]
36.89
[0.66–1.27]
[6.19–10.60]
[27.17–51.00]
affinity monoamine transporters. This might give impetus to future
studies exploring the interaction of NPS with hOCT1-3 and hPMAT.
CRediT authorship contribution statement
Julian Maier: Conceptualization, Methodology, Formal analysis,
Investigation, Data curation, Writing – original draft, Writing – review &
editing, Visualization, Project administration. Laurin Rauter: Formal
analysis, Investigation, Data curation, Writing – review & editing,
Visualization. Deborah Rudin: Investigation, Writing – review & edit-
ing, Visualization. Marco Niello: Investigation, Writing – review &
Declaration of competing interest
None.
8

J. Maier et al.
Neuropharmacology 190 (2021) 108570
(alpha-PVP) influences potency at dopamine transporters. ACS Chem. Neurosci. 6,
1726–1731.
Acknowledgements
Liu, H.C., Goldenberg, A., Chen, Y., Lun, C., Wu, W., Bush, K.T., Balac, N., Rodriguez, P.,
Abagyan, R., Nigam, S.K., 2016. Molecular properties of drugs interacting with
SLC22 transporters OAT1, OAT3, OCT1, and OCT2: a machine-learning approach.
J. Pharmacol. Exp. Therapeut. 359, 215–229.
This research was supported by the Vienna Science and Technology
Fund (WWTF) [CS 15–033] (HHS), Austrian Science Fund (FWF)
¨
[W1232] (HHS) and [DOC33-B27] (HHS and JM), Theodor Korner
Maier, J., Mayer, F.P., Brandt, S.D., Sitte, H.H., 2018a. DARK classics in chemical
neuroscience: aminorex analogues. ACS Chem. Neurosci. 9, 2484–2502.
Maier, J., Mayer, F.P., Luethi, D., Holy, M., Jantsch, K., Reither, H., Hirtler, L.,
Hoener, M.C., Liechti, M.E., Pifl, C., Brandt, S.D., Sitte, H.H., 2018b. The
psychostimulant (+/-)-cis-4,4’-dimethylaminorex (4,4’-DMAR) interacts with
human plasmalemmal and vesicular monoamine transporters. Neuropharmacology
138, 282–291.
Fonds 2020 (JM), the Swiss National Science Foundation (SNSF P2BSP3
191740) (DR) and the National Institute on Drug Abuse [DA040907]
(KSM and BEB) and [DA046215] (KSM). The authors thank Joanne
Wang for the hPMAT cDNA.
Majchrzak, M., Celinski, R., Kus, P., Kowalska, T., Sajewicz, M., 2018. The newest
cathinone derivatives as designer drugs: an analytical and toxicological review.
Forensic Toxicol. 36, 33–50.
Appendix A. Supplementary data
Mayer, F.P., Luf, A., Nagy, C., Holy, M., Schmid, R., Freissmuth, M., Sitte, H.H., 2017.
Application of a combined approach to identify new psychoactive street drugs and
decipher their mechanisms at monoamine transporters. Curr Top Behav Neurosci 32,
333–350.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.neuropharm.2021.108570.
Mayer, F.P., Schmid, D., Holy, M., Daws, L.C., Sitte, H.H., 2019. Polytox" synthetic
cathinone abuse: a potential role for organic cation transporter 3 in combined
cathinone-induced efflux. Neurochem. Int. 123, 7–12.
References
Ahmed, M.B., el-Qirbi, A.B., 1993. Biochemical effects of Catha edulis, cathine and
cathinone on adrenocortical functions. J. Ethnopharmacol. 39, 213–216.
Baumann, M.H., Clark, R.D., Woolverton, W.L., Wee, S., Blough, B.E., Rothman, R.B.,
2011. In vivo effects of amphetamine analogs reveal evidence for serotonergic
inhibition of mesolimbic dopamine transmission in the rat. J. Pharmacol. Exp.
Therapeut. 337, 218–225.
Mayer, F.P., Schmid, D., Owens, W.A., Gould, G.G., Apuschkin, M., Kudlacek, O.,
Salzer, I., Boehm, S., Chiba, P., Williams, P.H., Wu, H.H., Gether, U., Koek, W.,
Daws, L.C., Sitte, H.H., 2018. An unsuspected role for organic cation transporter 3 in
the actions of amphetamine. Neuropsychopharmacology 43, 2408–2417.
Meltzer, P.C., Butler, D., Deschamps, J.R., Madras, B.K., 2006. 1-(4-Methylphenyl)-2-
pyrrolidin-1-yl-pentan-1-one (Pyrovalerone) analogues: a promising class of
monoamine uptake inhibitors. J. Med. Chem. 49, 1420–1432.
Baumann, M.H., Volkow, N.D., 2016. Abuse of new psychoactive substances: threats and
solutions. Neuropsychopharmacology 41, 663–665.
Milano, W., De Biasio, V., Di Munzio, W., Foggia, G., Capasso, A., 2020. Obesity: the new
global epidemic. Pharmacological treatment, opportunities and limits for
personalized therapy. Endocr Metab Immune Disord Drug Targets.
Nagy, E.K., Overby, P.F., Olive, M.F., 2020. Reinforcing effects of the synthetic cathinone
alpha-pyrrolidinopropiophenone (alpha-PPP) in a repeated extended access binge
paradigm. Front. Psychiatr. 11, 862.
´
¨
Beck, O., Franzen, L., Backberg, M., Signell, P., Helander, A., 2016. Toxicity evaluation of
α
-pyrrolidinovalerophenone (α-PVP): results from intoxication cases within the
STRIDA project. Clin. Toxicol. 54, 568–575.
Bhat, S., Newman, A.H., Freissmuth, M., 2019. How to rescue misfolded SERT, DAT and
NET: targeting conformational intermediates with atypical inhibitors and partial
releasers. Biochem. Soc. Trans. 47, 861–874.
Niello, M., Gradisch, R., Loland, C.J., Stockner, T., Sitte, H.H., 2020. Allosteric
modulation of neurotransmitter transporters as a therapeutic strategy. Trends
Pharmacol. Sci. 41, 446–463.
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., 2015. Quantitative structure-activity
relationship analysis of the pharmacology of para-substituted methcathinone
analogues. Br. J. Pharmacol. 172, 2433–2444.
Ray, A., Chitre, N.M., Daphney, C.M., Blough, B.E., Canal, C.E., Murnane, K.S., 2019.
Effects of the second-generation "bath salt" cathinone alpha-
Brandt, S.D., Walters, H.M., Partilla, J.S., Blough, B.E., Kavanagh, P.V., Baumann, M.H.,
2020. The psychoactive aminoalkylbenzofuran derivatives, 5-APB and 6-APB, mimic
the effects of 3,4-methylenedioxyamphetamine (MDA) on monoamine transmission
in male rats. Psychopharmacology (Berl).
pyrrolidinopropiophenone (alpha-PPP) on behavior and monoamine neurochemistry
in male mice. Psychopharmacology (Berl) 236, 1107–1117.
Rickli, A., Hoener, M.C., Liechti, M.E., 2015. Monoamine transporter and receptor
interaction profiles of novel psychoactive substances: para-halogenated
amphetamines and pyrovalerone cathinones. Eur. Neuropsychopharmacol 25,
365–376.
Costa, R., Oliveira, N.G., Dinis-Oliveira, R.J., 2019. Pharmacokinetic and
pharmacodynamic of bupropion: integrative overview of relevant clinical and
forensic aspects. Drug Metab. Rev. 51, 293–313.
Riley, A.L., Nelson, K.H., To, P., Lopez-Arnau, R., Xu, P., Wang, D., Wang, Y., Shen, H.W.,
Kuhn, D.M., Angoa-Perez, M., Anneken, J.H., Muskiewicz, D., Hall, F.S., 2020. Abuse
potential and toxicity of the synthetic cathinones (i.e., "Bath salts"). Neurosci.
Biobehav. Rev. 110, 150–173.
DEA, 2014. Schedules of Controlled Substances: Temporary Placement of 10 Synthetic
Cathinones into Schedule I accessed: https://www.federalregister.gov/documents/
2014/01/28/2014-01172/schedules-of-controlled-substances-temporary-placeme
nt-of-10-synthetic-cathinones-into-schedule-i. (Accessed 18 January 2021).
Eshleman, A.J., Wolfrum, K.M., Hatfield, M.G., Johnson, R.A., Murphy, K.V.,
Janowsky, A., 2013. Substituted methcathinones differ in transporter and receptor
interactions. Biochem. Pharmacol. 85, 1803–1815.
Rothman, R.B., Partilla, J.S., Baumann, M.H., Lightfoot-Siordia, C., Blough, B.E., 2012.
Studies of the biogenic amine transporters. 14. Identification of low-efficacy "partial"
substrates for the biogenic amine transporters. J. Pharmacol. Exp. Therapeut. 341,
251–262.
Eshleman, A.J., Wolfrum, K.M., Reed, J.F., Kim, S.O., Swanson, T., Johnson, R.A.,
Janowsky, A., 2017. Structure-activity relationships of substituted cathinones, with
transporter binding, uptake, and release. J. Pharmacol. Exp. Therapeut. 360, 33–47.
Gannon, B.M., Baumann, M.H., Walther, D., Jimenez-Morigosa, C., Sulima, A., Rice, K.C.,
Collins, G.T., 2018. The abuse-related effects of pyrrolidine-containing cathinones
are related to their potency and selectivity to inhibit the dopamine transporter.
Neuropsychopharmacology 43, 2399–2407.
Saha, K., Li, Y., Holy, M., Lehner, K.R., Bukhari, M.O., Partilla, J.S., Sandtner, W.,
Sitte, H.H., Baumann, M.H., 2019. The synthetic cathinones, butylone and
pentylone, are stimulants that act as dopamine transporter blockers but 5-HT
transporter substrates. Psychopharmacology (Berl) 236, 953–962.
Saha, K., Partilla, J.S., Lehner, K.R., Seddik, A., Stockner, T., Holy, M., Sandtner, W.,
Ecker, G.F., Sitte, H.H., Baumann, M.H., 2015. ’Second-generation’ mephedrone
analogs, 4-MEC and 4-MePPP, differentially affect monoamine transporter function.
Neuropsychopharmacology 40, 1321–1331.
Garcia-Mijares, M., Bernardes, A.M., Silva, M.T., 2009. Diethylpropion produces
psychostimulant and reward effects. Pharmacol. Biochem. Behav. 91, 621–628.
Garcia Ramirez, A.V., Filho, D.R., Zotarelli Filho, I.J., 2020. Meta-analysis and approach
of the real impact of anorexigenic drugs in the obesity in humans: the last five years
of the randomized studies. Curr. Diabetes Rev. 16, 750–758.
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., 2015. Steric parameters, molecular
modeling and hydropathic interaction analysis of the pharmacology of para-
substituted methcathinone analogues. Br. J. Pharmacol. 172, 2210–2218.
Sala-Rabanal, M., Li, D.C., Dake, G.R., Kurata, H.T., Inyushin, M., Skatchkov, S.N.,
Nichols, C.G., 2013. Polyamine transport by the polyspecific organic cation
transporters OCT1, OCT2, and OCT3. Mol. Pharm. 10, 1450–1458.
Sandtner, W., Stockner, T., Hasenhuetl, P.S., Partilla, J.S., Seddik, A., Zhang, Y.W.,
Cao, J., Holy, M., Steinkellner, T., Rudnick, G., Baumann, M.H., Ecker, G.F.,
Newman, A.H., Sitte, H.H., 2016. Binding mode selection determines the action of
ecstasy homologs at monoamine transporters. Mol. Pharmacol. 89, 165–175.
Scholze, P., Zwach, J., Kattinger, A., Pifl, C., Singer, E.A., Sitte, H.H., 2000. Transporter-
mediated release: a superfusion study on human embryonic kidney cells stably
expressing the human serotonin transporter. J. Pharmacol. Exp. Therapeut. 293,
870–878.
Gasser, P.J., 2019. Roles for the uptake2 transporter OCT3 in regulation of dopaminergic
neurotransmission and behavior. Neurochem. Int. 123, 46–49.
Glennon, R.A., Dukat, M., 2017. Structure-activity relationships of synthetic cathinones.
Curr Top Behav Neurosci 32, 19–47.
Hasenhuetl, P.S., Bhat, S., Freissmuth, M., Sandtner, W., 2019. Functional selectivity and
partial efficacy at the monoamine transporters: a unified model of allosteric
modulation and amphetamine-induced substrate release. Mol. Pharmacol. 95,
303–312.
Hyde, J.F., Browning, E., Adams, R., 1928. SYNTHETIC HOMOLOGS OF d,l-EPHEDRINE.
J. Am. Chem. Soc. 50, 2287–2292.
Ilic, M., Holy, M., Jaentsch, K., Liechti, M.E., Lubec, G., Baumann, M.H., Sitte, H.H.,
Luethi, D., 2020. Cell-based radiotracer binding and uptake inhibition assays: a
comparison of in vitro methods to assess the potency of drugs that target monoamine
transporters. Front. Pharmacol. 11.
Seidel, S., Singer, E.A., Just, H., Farhan, H., Scholze, P., Kudlacek, O., Holy, M.,
Koppatz, K., Krivanek, P., Freissmuth, M., Sitte, H.H., 2005. Amphetamines take two
to tango: an oligomer-based counter-transport model of neurotransmitter transport
explores the amphetamine action. Mol. Pharmacol. 67, 140–151.
Simmler, L.D., Buser, T.A., Donzelli, M., Schramm, Y., Dieu, L.H., Huwyler, J.,
Chaboz, S., Hoener, M.C., Liechti, M.E., 2013. Pharmacological characterization of
designer cathinones in vitro. Br. J. Pharmacol. 168, 458–470.
Kelly, J.P., 2011. Cathinone derivatives: a review of their chemistry, pharmacology and
toxicology. Drug Test. Anal. 3, 439–453.
Koepsell, H., 2020. Organic cation transporters in health and disease. Pharmacol. Rev.
72, 253–319.
Kolanos, R., Sakloth, F., Jain, A.D., Partilla, J.S., Baumann, M.H., Glennon, R.A., 2015.
Structural modification of the designer stimulant alpha-pyrrolidinovalerophenone
9

J. Maier et al.
Neuropharmacology 190 (2021) 108570
Simmler, L.D., Rickli, A., Hoener, M.C., Liechti, M.E., 2014. Monoamine transporter and
receptor interaction profiles of a new series of designer cathinones.
Neuropharmacology 79, 152–160.
Wang, J., 2016. The plasma membrane monoamine transporter (PMAT): structure,
function, and role in organic cation disposition. Clin. Pharmacol. Ther. 100,
489–499.
Simmons, S.J., Leyrer-Jackson, J.M., Oliver, C.F., Hicks, C., Muschamp, J.W., Rawls, S.
M., Olive, M.F., 2018. DARK classics in chemical neuroscience: cathinone-derived
psychostimulants. ACS Chem. Neurosci. 9, 2379–2394.
WDR, 2020. accessed. https://wdr.unodc.org/wdr2020/en/cross-cutting.html.
(Accessed 18 January 2021).
Wee, S., Anderson, K.G., Baumann, M.H., Rothman, R.B., Blough, B.E., Woolverton, W.L.,
2005. Relationship between the serotonergic activity and reinforcing effects of a
series of amphetamine analogs. J. Pharmacol. Exp. Therapeut. 313, 848–854.
Wright, T.H., Harris, C., 2016. Twenty-one cases involving alpha-
Sitte, H.H., Freissmuth, M., 2015. Amphetamines, new psychoactive drugs and the
monoamine transporter cycle. Trends Pharmacol. Sci. 36, 41–50.
Steinkellner, T., Mayer, F.P., Hofmaier, T., Holy, M., Montgomery, T., Eisenrauch, B.,
Freissmuth, M., Sitte, H.H., 2016. Tracer flux measurements to study outward
transport by monoamine neurotransmitter transporters. Neurotransmitter
Transporters 23–40.
pyrrolidinovalerophenone (α-PVP). J. Anal. Toxicol. 40, 396–402.
Yu, H., Rothman, R.B., Dersch, C.M., Partilla, J.S., Rice, K.C., 2000. Uptake and release
effects of diethylpropion and its metabolites with biogenic amine transporters.
Bioorg. Med. Chem. 8, 2689–2692.
UNODC, 2021. What Are NPS? accessed: https://www.unodc.org/LSS/Page/NPS.
(Accessed 18 January 2021).
`
Zaami, S., Giorgetti, R., Pichini, S., Pantano, F., Marinelli, E., Busardo, F.P., 2018.
Synthetic cathinones related fatalities: an update. Eur. Rev. Med. Pharmacol. Sci. 22,
268–274.
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