The dopamine, serotonin and norepinephrine releasing activities of a series of methcathinone analogs in male rat brain synaptosomes

Article abstract of DOI:10.1007/s00213-018-5063-9

Rationale: Novel synthetic “bath salt” cathinones continue to appear on the street as abused and addictive drugs. The range of subjective experiences produced by different cathinones suggests that some compounds have primarily dopaminergic activity (possi

Full text of DOI:10.1007/s00213-018-5063-9

Psychopharmacology
https://doi.org/10.1007/s00213-018-5063-9
ORIGINAL INVESTIGATION
The dopamine, serotonin and norepinephrine releasing activities
of a series of methcathinone analogs in male rat brain synaptosomes
Bruce E. Blough1 & Ann M. Decker1 & Antonio Landavazo1 & Ojas A. Namjoshi1 & John S. Partilla2
Michael H. Baumann2 & Richard B. Rothman2
&
Received: 30 May 2018 /Accepted: 2 October 2018
#
Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
Rationale Novel synthetic Bbath salt^ cathinones continue to appear on the street as abused and addictive drugs. The range of
subjective experiences produced by different cathinones suggests that some compounds have primarily dopaminergic activity
(possible stimulants) while others have primarily serotonergic activity (possible empathogenics). An understanding of the
structure activity relationships (SARs) of these compounds will help in assessing the likely behavioral effects of future novel
structures, and to define potential therapeutic strategies to reverse any reinforcing effects.
Objectives A series of methcathinone analogs was systematically studied for their activity at the dopamine and serotonin
transporters. Compound structures varied at the aromatic group, either by substituent or by replacement of the phenyl ring with
a naphthalene or indole ring.
Methods A novel, high-yielding synthesis of methcathinone hydrochlorides was developed which avoids isolation of the
unstable free bases. Neurotransmitter transporter release activity was determined in rat brain synaptosomes as previously report-
ed. Compounds were also screened for activity at the norepinephrine transporter.
Results Twenty-eight methcathinone analogs were analyzed and fully characterized in dopamine and serotonin transporter
release assays. Compounds substituted at the 2-position (ortho) were primarily dopaminergic. Compounds substituted at the 3-
position (meta) were found to be much less dopaminergic, with some substituents favoring serotonergic activity. Compounds
substituted at the 4-position (para) were found to be far more serotonergic, as were disubstituted compounds and other large
aromatic groups. One exception was the fluoro-substituted analogs which seem to favor the dopamine transporter.
Conclusions The dopaminergic to serotonergic ratio can be manipulated by choice of substituent and location on the aromatic
ring. It is therefore likely possible to tweak the subjective and reinforcing effects of these compounds by adjusting their structure.
Certain substituents like a fluoro group tend to favor the dopamine transporter, while others like a trifluoromethyl group favor the
serotonin transporter.
.
.
.
Keywords Biogenic amine transporters Rat brain synaptosomes BBath salts^ Methcathinones
Introduction
This article belongs to a Special Issue on Bath Salts
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00213-018-5063-9) contains supplementary
material, which is available to authorized users.
Synthetic Bbath salt^ cathinones continue to be a serious abuse
problem due to the powerful stimulant, hallucinogenic, and
addictive effects they produce in humans. The CY 2017
DEA BEmerging Threats^ report indicates that there were
369 confirmed reports of Bbath salts,^ the most prevalent be-
ing N-ethylpentylone. Structurally smaller Bbath salts^ were
also observed including N-ethyl-4-chloromethcathinone,
ethylone, and dibutylone. Methcathinone analogs, including
the original Bbath salt^ methylone, along with newer
methcathinones such as methylmethcathinone, and
chloromethcathinone were also found. The evidence shows
* Bruce E. Blough
beb@rti.org
1
Center for Drug Discovery, RTI International, 3040 E. Cornwallis
Road, Research Triangle Park, NC 27709, USA
2
Designer Drug Research Unit, Intramural Research Program,
National Institute on Drug Abuse, National Institutes of Health,
Baltimore, MD 21224, USA

Psychopharmacology
that clandestine laboratories continue to make and distribute
novel cathinone analogs making the study of their properties
imperative.
transported (Rudnick and Clark 1993), while substrate-type
releasers bind to the substrate site on the transporters, are
transported inside the neuron, and promote neurotransmitter ef-
flux by transporter-mediated exchange (Fleckenstein et al. 2007).
While both types of compounds elevate synaptic neurotransmit-
ters, a releaser also depolarizes the membrane as ions move back
through the transporter, while reuptake inhibitors do not (Sulzer
et al. 2005). The SERT and NET also have voltage-dependent
transient currents with ion channel-like properties that may be
relevant to their function, and these currents can be blocked by
reuptake inhibitors such as fluoxetine and cocaine (Li et al.
2002). These differences in mechanism are substantial, likely
induce divergent effects between mechanistic classes, and prob-
ably underlie the differences in therapeutic potential of releasers
and reuptake inhibitors.
Given the potential abuse liability of Bbath salts^ along
with the ever-changing illicit drug market, it is becoming in-
creasingly important to understand the pharmacology of these
drugs at BATs. An understanding of the mechanism of action
of these emerging bath salts, and their specific BAT profiles,
might also aid in the discovery of treatment medications. Our
research laboratories have studied neurotransmitter releasers
as a potential therapeutic for cocaine addiction and abuse since
1999 (Rothman et al. 2008). These efforts included the dis-
covery of novel N-cyclopropyl and N-cyclobutylcathinones as
hybrid dopamine reuptake inhibitor/serotonin releasers
(Blough et al. 2014a), and the pursuit of the DA releasing
stimulant phenmetrazine as a potential pharmacotherapy
(Banks et al. 2013; Rothman et al. 2002). Many compound
classes, including methcathinones, have been considered as
potential therapeutics and were studied as part of our research
(Reith et al. 2015). Herein, we report the systematic study of a
series of methcathinones, and their interactions at the DAT,
SERT, and NET.
As a class of abused drugs, cathinones are a recent phe-
nomenon exploding on the street in the early 2000s, but bio-
active compounds have actually been studied for many years.
Methcathinone (1) was first synthesized in 1928 as an ephed-
rine homolog (Hyde et al. 1928). It was later patented by Parke
Davis in 1957 for its analeptic or Brestorative^ properties
(L’Italien et al. 1957). In 1975, the UN found that the N-
demethylated analog cathinone (2) was the active ingredient
in Catha edulis, a plant used widely in Africa and the Middle
East as a stimulant (Patel 2000). In the early 1980s,
methcathinone was examined as a stimulant and was found
to substitute for amphetamine in drug-discrimination assays in
rats (Glennon et al. 1987). Methcathinone was also found to
dose-dependently maintain self-administration in baboons
(Kaminski and Griffiths 1994). Several methcathinone ana-
logs were reported in the literature prior to 2004, including
bromomethcathinones (Foley and Cozzi 2003) and methylone
(Peyton and Shulgin 1996), all of which were primarily stud-
ied as potential antidepressants. These efforts may have been a
follow-up on the success of the cathinone antidepressant and
smoking cessation drug bupropion (Wellbutrin®, Zyban®)
(Carroll et al. 2014). Interestingly, as an extension of the
bupropion success, cathinone analogs of bupropion have been
studied as potential therapeutics for cocaine and nicotine
abuse (Carroll et al. 2009; Carroll et al. 2010).
Since the explosion of the cathinones as a popular class of
recreational drug around 2010, laboratories around the world
began to characterize these compounds pharmacologically,
resulting in numerous reports identifying mechanism of action
and SAR (Baumann et al. 2013; Bonano et al. 2015; Del Bello
et al. 2015; Eshleman et al. 2017; Glennon and Dukat 2017;
Gregg et al. 2015; Hutsell et al. 2016; Luethi et al. 2018; Rickli
et al. 2015; Saha et al. 2015; Schindler et al. 2016; Shalabi et al.
2017; Shekar et al. 2017; Simmler et al. 2013, 2014). The pri-
mary mechanism of action of the cathinones as a class appears to
be their interactions at the biogenic amine transporters (BATs),
which help regulate monoaminergic signaling in the central ner-
vous system (CNS). The primary function of BATs is to transport
previously released monoamine neurotransmitters—dopamine,
norepinephrine, and serotonin (DA, NE, and 5-HT transported
via DAT, NET, and SERT, respectively)—from the synapse back
to the neuronal cytoplasm (Masson et al. 1999). BATs can also
participate in release of the same neurotransmitters in a non-
calcium-dependent manner in the opposite direction (Sulzer
et al. 2005). Ligands that interact with BATs are classified as
either reuptake inhibitors or substrate-type releasers; both types
of ligands act to elevate extracellular neurotransmitter concentra-
tions but they act via different mechanisms (Rothman et al.
2008). Reuptake inhibitors bind to transporters and block
transporter-mediated reuptake of neurotransmitters without being
Methods
Compounds
Compounds 1 and 2 were acquired from the NIDA Drug
Supply. Phenacylamine was purchased from Sigma-Aldrich
and converted to its hydrochloride salt. All other compounds
were synthesized following the route described in Scheme 1.
The synthesis and analytical information of all novel com-
pounds is provided in the electronic supplementary informa-
tion (ESI). All compounds were > 95% pure when tested.
DAT, SERT, and NET rat brain synaptosome assays
The DAT, SERT, and NET reuptake inhibition and release
assays were conducted according to the methods of
Rothman and Baumann (Partilla et al. 2016). Male CD

Psychopharmacology
Scheme 1 General synthesis of
methcathinones
O
O
O
O
H
Br2
BnNHCH3
ACE-Cl
HCl
Br
N
N
3
4
5
1
Sprague-Dawley rats (300–400 g) were maintained in facili-
ties fully accredited by the AAALAC and experiments were
performed in accordance with the Institutional Animal Care
and Use Committee at Mispro Biotech. A total of 25 rats were
used for the experiments. Rats were euthanized by CO2 nar-
cosis and brains were processed to yield synaptosomes as
previously described. Synaptosomes were prepared from rat
striatum for the DAT assays, whereas synaptosomes were pre-
pared from whole brain minus striatum and cerebellum for the
SERT and NET assays. Initial screening assays were conduct-
ed at a final test compound concentration of 10 μM.
For neurotransmitter reuptake inhibition assays, 5 nM
[3H]DA, 5 nM [3H]5-HT, and 10 nM [3H]NE were used to
assess activity at the DAT, SERT, and NET, respectively. The
selectivity of the SERT assay was optimized for SERT by
including unlabeled blockers to prevent uptake of [3H]5-HT
by competing transporters (100 nM nomifensine to block
NET, 50 nM GBR12935 to block DAT). The selectivity of
the NETassay was optimized for NET by including unlabeled
blockers to prevent uptake of [3H]NE by competing trans-
porters (50 nM GBR12935 to block DAT). No blockers are
needed for the DAT assay. The assay was initiated by adding
100 μL of the synaptosomal prep to 900 μL of Krebs-
phosphate buffer (12.6 mM NaCl, 0.24 mM KCl, 0.05 mM
KH2PO4, 0.11 mM CaCl2, 0.083 mM MgCl2, 0.05 mM
Na2SO4, 11.1 mM glucose, 0.05 mM pargyline, 13.8 mM
Na2HPO4, 1 mg/mL ascorbic acid, and 1 mg/mL BSA, pH
7.4) containing test compound and [3H]neurotransmitter.
Non-specific binding (NSB) was determined in the presence
of 1 μM indatraline and total binding (TB) was determined in
the presence of vehicle. After 15 min at RT (DAT), 30 min at
RT (SERT), or 10 min at 37 °C (NET), the assay was termi-
nated by rapid vacuum filtration/washing through GF/B filters
(Brandel harvester). The retained radioactivity was quantified
by a PerkinElmer TopCount.
SERT). No blockers are needed for the DAT release assay.
Synaptosomes were preloaded (steady state) with
radiolabeled substrate in Krebs-phosphate buffer
(12.6 mM NaCl, 0.24 mM KCl, 0.05 mM KH2PO4,
0.11 mM CaCl2, 0.083 mM MgCl2, 0.05 mM Na2SO4,
11.1 mM glucose, 0.05 mM pargyline, 13.8 mM
Na2HPO4, 1 mg/mL ascorbic acid, pH 7.4) for 30 min
(DAT, NET) or 1 h (SERT). Release assays were initiated
by adding 850 μL of preloaded synaptosomes to 150 μL
of test compound prepared in Krebs-phosphate buffer
containing 1 mg/mL BSA. NSB was determined in the
presence of tyramine (10 μM for DAT/NET and 100 μM
for SERT) and TB was determined in the presence of
vehicle. Assays were terminated by rapid vacuum
filtration/washing through GF/B filters on a Brandel
(Gaithersburg, MD, USA) harvester and retained radioac-
tivity was quantified by a PerkinElmer TopCount.
Substrate activity for releasers was confirmed by detecting
a significant reversal of the releasing effect of the test com-
pound in the presence of reuptake inhibitors (at least N = 2).
The release assays were run in the same manner as described
except that the EC80 concentration of each test compound was
tested in the presence and absence of a known reuptake inhib-
itor (100× IC50). The reuptake inhibitor will bind to the trans-
porter and block the releaser from being transported; thus, the
releaser’s ability to release neurotransmitter will be dimin-
ished, as shown by a reduction in % release in the assay.
GBR12935 (82 nM) is the known blocker for the DAT sub-
strate reversal assay, while citalopram (450 nM) is the known
blocker for the SERT substrate reversal assay.
Data analysis
Maximal binding (MB) was calculated with the equation
MB = TB − NSB. Specific bound (SB) was calculated with
the equation SB = test compound signal − NSB. Percent of
MB was calculated with the equation % MB = (1 − SR/
MR) × 100. To determine compound potencies in the release
assays, % MR was plotted against the log of compound con-
centration. Data were fit to a three-parameter logistic curve to
generate EC50 values (GraphPad Prism, GraphPad Software,
Inc., San Diego, CA). For the substrate reversal assays, % MB
was calculated for the test compound with and without the
known reuptake inhibitor. Student’s t test was performed to
determine if the release values were significant.
For neurotransmitter release assays, 5 nM [3H]DA,
2 nM [3H]5-HT, and 5 nM [3H]MPP+ were used to assess
activity at the DAT, SERT, and NET, respectively. All
buffers used in the release assays contained 1 μM reser-
pine to block vesicular uptake of substrates. The same
blockers are used for the SERT assay to optimize SERT
selectivity as listed in the reuptake inhibition methods.
The selectivity of the NET assay was optimized for NET
by including unlabeled blockers to prevent uptake of
[3H]MPP+ by competing transporters (100 nM
GBR12935 to block DAT, 100 nM citalopram to block

Psychopharmacology
Table 1 DAT-, SERT-, and NET-mediated release activity of 2-phenyl substituted methcathinones
NE Release
% @ 10 Mb
DA Release
EC50, nMa
5-HT Release
EC50, nMa
DAT
Selectivityc
Compound
R
methcathinone
(R = H)
1
2
49.9 3.1
83.1 6.8
208 13
48.7 8.8
>10,000
4,270 270
6,100 750
>10,000
100%
95%
96%
85%
69%
33%
NDd
86
73
cathinone
6
phenylacylamine
>48
>205
n/a
7
F
>10,000
8
CF3
>10,000
9
OCF3
CH3
>10,000
>10,000
n/a
10
97.9 11
347 130
3.5
a Values are mean S.D. of at least N = 3 performed in duplicate as described in the methods
b Values are the mean % release of N = 2 10 μM screens performed in duplicate
c DAT selectivity is calculated as SERT EC50 / DAT EC50
d ND not determined
Results
methcathinones by amination with the secondary amine N-
benzylmethylamine, followed by debenzylation with ACE-
Cl, allowed us to form the hydrochloride salts directly and
completely avoid isolation of the unstable free base.
Methcathinone synthesis
The methcathinone analogs were synthesized as shown in
Scheme 1. The synthesis is described for the parent scaffold
methcathinone (1) but was the same for all of the
methcathinones (see Supplemental Data). Acetophenone was
quantitatively brominated alpha to the ketone to form
bromoketone 4, followed by amination with
methylbenzylamine to form ketoamine 5. The benzyl group
was then removed using 1-chloroethyl chloroformate (ACE-
Cl) to directly form methcathinone hydrochloride analogs in
high yields and purity. Overall yields of the methcathinone
hydrochloride salts from their respective substituted
phenylpropiophenones ranged between 50 and 97%, depend-
ing on the phenyl substitution.
While amination of bromoketones like 4 with large primary
alkylamines, such as t-butylamine, is high yielding and scal-
able, in our experience, amination with smaller primary
alkylamines like methylamine results in highly colored solu-
tions and much lower yields. These observations suggest that
free base dimerization occurs followed by various oxidative
processes. Large α-alkyl groups prevent dimerization and free
base decomposition. Synthesis of the benzyl protected
Dopamine, serotonin, and norepinephrine
transporter activity
The synthesized methcathinones were evaluated for DAT,
SERT, and NET activity according to the protocol developed
by Rothman and co-workers (Partilla et al. 2016) using rat
brain synaptosomes prepared from rat brain homogenates.
Compounds were initially screened in DA, 5-HT, and NE
reuptake inhibition and release assays to assess mechanism
of action. According to the protocol, compounds active in
both the reuptake inhibition and releaser assays are binned
mechanistically as releasers while compounds only active in
the reuptake inhibition assay are binned as reuptake inhibitors.
We have never observed compounds only active in the release
assay. All of the compounds in this study were found to be
releasers or were inactive. Active releasers at DA and 5-HT
were then fully characterized for release potency at each trans-
porter by running 8-point concentration response curves; sub-
strate reversal experiments were conducted to validate sub-
strate activity. NE release was not fully characterized;

Psychopharmacology
therefore, the screening data is presented as % release at
10 μM. Since the ratio of DAT and SERT release has been
shown throughout the literature to be important for abuse lia-
bility and psychoactive behavioral effects, we calculated this
ratio with the equation DAT selectivity = SERT EC50 / DAT
EC50.
analog 8 appears active as a NE releaser without having
DAT or SERT activity suggesting it may be a selective NE
releaser.
Table 2 shows DAT, SERT, and NET release activity for 3-
substituted methcathinones. Compared to the parent
methcathinone 1, adding a bromide to the 3-position (11) led
Table 1 shows DAT, SERT and NET release activity for
phenacylamine (6), cathinone (2), methcathinone (1), and sev-
eral 2-substituted methcathinones. Methcathinone (1) was 86-
fold more selective for releasing DA relative to 5-HT with
EC50 values of 49.9 nM and 4270 nM, respectively. The
unsubstituted cathinones phenacylamine (6) and cathinone
(2) were both potent DA releasers with EC50 values of
208 nM and 83.1 nM, respectively, which are 4.2-fold and
1.7-fold less potent compared to 1. Adding substituents at
the 2-position led to a variety of results. Compared to
methcathinone 1, DAT activity remained unchanged for the
2-F analog (7, EC50 = 48.7 nM) while SERT activity was
completely lost. The 2-CH3 analog 10 was 2-fold less potent
as a DA releaser with an EC50 value of 97.9 nM, but was 12-
fold more potent as a 5-HT releaser (EC50 = 347 nM).
Interestingly, addition of a CF3 group (8) or a OCF3 group
(9) to the 2-position resulted in a complete loss of transporter
substrate activity at both DAT and SERT. All of the com-
pounds were active as potent NET releasers, except for the
2- OCF3 analog 9 which was weak. Surprisingly, the 2-CF3
to an almost 2-fold more potent compound at DAT (EC50 =
28.0 nM) and a 31-fold more potent compound at SERT
(EC50 = 137 nM). The 3-Cl analog 12 was approximately
the same potency as 1 at DAT with an EC50 = 46.8 nM, but
was about an order of magnitude more potent as a 5-HT re-
leaser with an EC50 = 410 nM. The 3-F analog 13 was
equipotent at releasing DA compared to 1 but was 2.9-fold
more potent at releasing 5-HT (EC50 = 1460 nM). The
trifluoromethyl analog 14 was 26-fold less potent at releasing
DA but was 14-fold more potent at releasing 5-HT (EC50
=
297 nM) compared to 1. The 3-OCF3 analog 15 was also less
potent at DAT but only by 15-fold and kept the increase in
SERT potency with an EC50 value of 188 nM. Compared to 1,
the 3-CH3 analog 16 was equipotent at the DAT with an EC50
value of 70.6 nM but was 14.6-fold more potent at releasing 5-
HTwith an EC50 value of 292 nM. Having a methoxy group at
the 3-position (17) resulted in a 2.6-fold increase in potency
(EC50 = 129 nM) at DAT and a 14-fold decrease in potency
(EC50 = 306 nM) at SERT. The 3-nitro analog 18 was 25-fold
less potent and 3-fold more potent at the DAT and the SERT,
Table 2 DAT-, SERT-, and NET-mediated release activity of 3-phenyl substituted methcathinones
NE Release
% @ 10 Mb
DA Release
EC50, nMa
5-HT Release
EC50, nMa
DAT
Selectivityc
Compound
R
Br
11
12
13
14
15
16
17
18
28.0 4.1
46.8 4.0
64.8 8.6
1,290 370
729 150
70.6 22
137 75
410 38
112%
94%
100%
78%
79%
94%
68%
86%
4.9
8.8
22
Cl
F
1,460 640
297 81
CF3
OCF3
CH3
OCH3
NO2
0.2
0.3
4.1
2.4
1.1
188 35
292 39
129
8
306 42
1,230 290
1,400 590
a Values are mean S.D. of at least N = 3 performed in duplicate as described in the methods
b Values are the mean % release of N = 2 10 μM screens performed in duplicate
c DAT selectivity is calculated as SERT EC50 / DAT EC50

Psychopharmacology
respectively, compared to 1. All of the compounds were active
as NET releasers. While difficult to accurately predict based
on the screening data, the 3-CF3 analog 14 and possibly the 3-
OCF3 analog 15 appear to be SERT/NET selective releasers.
The 3-nitro analog 18 appears to be a selective NE releaser.
Table 3 shows DAT, SERT, and NET release activity data
for a series of 4-substituted methcathinones. Compared to the
parent methcathinone (1), the 4-Br analog 19 was equipotent
at DAT and 100-fold more potent at SERT with an EC50 value
of 42.5 nM. The 4-Cl analog 20 had similar release potencies
at DATand SERTcompared to the 4-Br analog 19. Relative to
1, the 4-F analog 21 was 2.4-fold less potent at DAT and 2.9-
fold more potent at SERT with EC50 values of 119 nM and
1450 nM, respectively. The trifluoromethyl analog 22 and
trifluoromethoxy analog 23 resulted in weak DA releasers
(EC50 > 4 μM) but 15-fold and 36-fold more potent 5-HT
releasers, respectively, indicating that large groups are not
well tolerated at the 4-position for DAT translocation.
Placing a methoxy group at the 4-position forming 25 resulted
in an 18-fold less potent DA releaser (EC50 = 881 nM) and a
22-fold more potent 5-HT releaser (EC50 = 195 nM). The 4-
methyl substituted compound known as mephedrone (24) was
not examined as part of this study but was included in the
Bonano et al. study (Bonano et al. 2015). Clearly, the methyl
substituent results in equipotent DA release and 36-fold more
potent 5-HT release compared to 1; however, because com-
pound 24 was not tested as part of the current study, making
such a comparison has limitations. All of the compounds were
potent NE releasers.
Table 4 shows the DAT, SERT, and NET release activity
data for 1-naphthylmethcathinone, 2-naphthylmethcathinone,
3-indoylmethcathinone, and several disubstituted
methcathinone analogs. The analogs with other aromatic
groups were evaluated for transporter activity and all were
found to be almost equipotent at the DATand > 100-fold more
potent at the SERT compared to 1. The 1-naphthyl analog 26
was equipotent at the DAT compared to 1 with an EC50 of
55.2 nM, but was 198-fold more potent at the SERT with an
EC50 of 21.6 nM. Similarly, the 2-naphthyl analog 27 was
equipotent at the DAT with an EC50 of 33.8 but was 158-
fold more potent at the SERT with an EC50 of 27.1 nM. The
3-indole analog 28 was almost 2-fold less potent at the DAT
with an EC50 of 92.8 nM but was 103-fold more potent at the
SERT with an EC50 of 41.3 nM. Several disubstituted analogs
were synthesized and tested in the rat brain synaptosome as-
says. Compared to methcathinone 1, the 3,4-dichloro analog
29 was 3.6-fold less potent at releasing DA and was 57-fold
more potent at releasing 5-HT. The 3,4-difluoro analog 30 was
Table 3 DAT-, SERT-, and NET-mediated release activity of 4-phenyl substituted methcathinones
NE Release
% @ 10 Mb
DA Release
EC50, nMa
5-HT Release
EC50, nMa
DAT
Selectivityc
Compound
R
Br
19
20
21
22
23
24
25
67.8 12
74.7 15
42.5 26
71.1 12
1,450 360
270 97
78%
102%
107%
NDf
0.6
0.9
Cl
Fd
119 18
12
d
CF3
4,230 1,500
7,510 2,100
49.1 8.3e
881 220
0.06
0.02
2.4
OCF3
CH3
118 71
NDf
118 26e
94%
101%
d
OCH3
195 86
0.2
a Values are mean S.D. of at least N = 3 performed in duplicate as described in the methods
b Values are the mean % release of N = 2 10 μM screens performed in duplicate.
c DAT selectivity is calculated as SERT EC50 / DAT EC50
d In the course of our studies, these compounds were included as part of a separate methcathinone study (Bonano et al. 2015). Values reported in this table
were generated by our laboratory
e Values are from (Bonano et al. 2015)
f ND not determined

Psychopharmacology
Table 4 DAT-, SERT-, and NET-mediated release activity of other methcathinones
NE Release
% @ 10 Mb
DA Release
EC50, nMa
5-HT Release
EC50, nMa
DAT
Selectivityc
Compound
X
-
Y
26
27
28
29
30
31
55.2 12
33.8 3.6
92.8 21
178 33
227 44
124 7.4
21.6 4.4
92%
94%
55%
103%
85%
94%
0.4
0.8
0.4
0.4
4.2
0.3
-
27.1 14
41.3 10
74.4 31
960 450
41.7 14
-
Cl
F
Cl
Cl
F
CH3
a Values are mean S.D. of at least N = 3 performed in duplicate as described in the methods
b Values are the mean % release of N = 2 10 μM screens performed in duplicate
c DAT selectivity is calculated as SERT EC50 / DAT EC50
4.5-fold less potent at DAT and was 4-fold more potent at
releasing 5-HT. The 3-chloro, 4-methyl analog 31 was 2.5-
fold less potent at DAT and was 102-fold more potent at
SERT. All of the compounds except for the 3-indoyl analog
28 appear to be potent NE releasers.
at the 3-position at the SERT, other than the finding that all of
the substitutions were more potent as 5-HT releasers than 1.
The DAT activity rank order for the 4-substituted analogs
(Table 3) was H = CH3 > Br ~ Cl > F > OCH3 ≫ CF3 > OCF3,
indicating that at the 4-position, small electron-withdrawing
groups are preferred for DAT translocation. The SERT rank
order was Br > Cl > OCH3 > CH3 = OCF3 ~ CF3 > F ≫ H, in-
dicating that SERT translocation also prefers small electron
withdrawing groups but can tolerate larger electron donating
groups as well. As with the 3-substituted analogs, all were
much more potent as 5-HT releasers than methcathinone.
As observed with other releaser scaffolds (Blough 2008;
Blough et al. 2014a; b) the DAT/SERT ratio was greatly in-
fluenced by the choice of phenyl substituent and location of
substitution. Methcathinone, cathinone, and phenylacylamine
were all DATselective, as were both 2-substituted compounds
with transporter activity, the 2-fluoro compound 7 and the 2-
methyl compound 10. The 3-substituted compounds were
much less DAT selective with all but the 3-F analog 13 being
less than 10-fold DAT selective. Two of the compounds were
selective 5-HT releasers, the 3-CF3 analog 14 and the 3-OCF3
compound 15. Interestingly, these two substituents rendered
the scaffold inactive at both transporters when substituted at
the 2-position. All but two of the 4-substituted compounds (21
and 24) were selective 5-HT releasers. The same two substit-
uents that were inactive when at the 2-position were the most
serotonergic, the 4-CF3 analog 22 and the 4-OCF3 analog 23.
These trends suggest that the serotonin transporter can
Discussion
All of the BAT active methcathinones were neurotransmitter
releasers. These observations fit with the mechanistic theory
that neurotransmitter releasers are transporter substrates, since
all of these compounds are small molecular weight, sterically
small phenyl amines (Reith et al. 2015; Sulzer et al. 2005).
The location and type of phenyl substituent had a large effect
on the potency of a compound as a DATor SERT releaser. For
the 2-substituted compounds (Table 1), the DAT rank order
was H = F > CH3 ≫ CF3 ~ OCF3 indicating that sterically
smaller groups are preferred by the DAT compared to the
bulkier groups. The SERT rank order was CH3 ≫ H, F, CF3,
OCF3, indicating that SERT translocation requires small elec-
tron donating groups at the 2-position. In the case of 3-
substituted analogs (Table 2), the DAT rank order was Br >
Cl = H > F ~ CH3 > OCH3 > OCF3 > NO2 ~ CF3, indicating
that small electron withdrawing groups are preferred most
by the DAT. The SERT rank order was Br ~ OCF3 > Cl ~
CH3 ~ OCH3 ~ CF3 > NO2 ~ F ≫ H. This rank order provides
no clear preference for steric or ionic character in a substituent

Psychopharmacology
accommodate and translocate 3,4-substituted phenyl analogs
better than the dopamine transporter but that the serotonin
transporter cannot accommodate a 2-substituent very well
due to steric and/or electronic effects.
SERT can have empathogenic or entactogenic properties, such
as 3,4-methylenedioxymethamphetamine (MDMA). DAT selec-
tivity can therefore be used as a guide for both abuse liability as
well as potential psychoactive effects.
Within a specific substituent, the trend shows that there is
relatively more activity as a 5-HT releaser going from 2- to 3-
to 4-substitution. For example, the DAT selectivity decreased
for the fluoro compounds from 205 to 22 to 12. The 3-bromo
and 4-bromo compounds have a DAT selectivity of 4.9 and
0.6 respectively, as do the same chloro analogs, which have
ratios of 8.8 and 0.9. The CF3 and OCF3 analogs were inactive
as 2-substituted compounds, but had DAT selectivities of ~
0.2 at the 3-position and ~ 0.04 at the 4-position. These chang-
es in DAT selectivity generally reflect weaker release activity
at the DAT. The CF3 analog data were found to be roughly the
same as that reported by Cozzi et al. and show the same sub-
stitution pattern trend (Cozzi et al. 2013). The 2-substituted
compounds were not very active at any of the transporters,
with EC50 values of 8 μM. The relative selectivity for the 3-
CF3 and 4-CF3 analogs was 0.6 and ≪ 0.04. One substituent
that did not appear to vary much in selectivity was the CH3
group, which had a DAT selectivity of 3.5, 4.1, and 2.4 at the
2-, 3-, and 4-positions respectively. The EC50 data did not
differ substantially between the three regioisomers and the
compounds were all slightly dopaminergic.
Table 4 lists several disubstituted compounds as well as
compounds in which the phenyl ring is replaced by an indole
or naphthalene ring. Interestingly, all of the compounds with
the exception of the 3,4-difluoro analog had roughly the same
DAT selectivity, ranging from 0.3 to 0.8. The 3,4-difluoro
compound 29 was the only DAT selective compound, which
correlates with observations that fluoro compounds are gener-
ally selective DA releasers. These results indicate that
disubstitution at the 3- and 4-positions results in a small loss
of DA release potency but a large increase in 5-HT release
potency. This observation substantiates the notion that the
SERT has additional room at the site of translocation than
the DAT.
DAT selectivity of neurotransmitter releasers is important be-
cause it appears to affect their abuse potential. Wee et al. showed
that the reinforcing properties of releasers in rhesus monkeys
varied with DAT selectivity, the most selective DAT compounds
being more reinforcing compared to analogs that were non-
selective (Wee et al. 2005). A similar trend was observed when
studying the same set of releasers in locomotor stimulation in
rats, with the more DAT selective compounds demonstrating
more forward locomotor activity than less selective compounds
(Baumann et al. 2011). These observed trends continue to be
validated as novel releaser bath salts have emerged (Glennon
and Dukat 2017; Simmler et al. 2013, 2014). DAT selectivity
also appears to affect the psychoactive effects of a releaser.
Highly DAT selective releasers such as methamphetamine are
potent locomotor stimulants. Releasers more selective for
Based on the in vitro data, one might expect that 3-
fluoromethcathinone (13) would be a locomotor stimulant
similar to methamphetamine because it is a highly potent,
DAT selective compound (Simmler et al. 2013). Indeed, 3-
fluoromethcathinone was found to induce locomotor stimula-
tion and produced similar discriminative effects as metham-
phetamine in rats (Gatch et al. 2015). Methcathinones such as
the 4-CF3 analog 22 and 4-OCF3 analog 23 with low DAT
selectivity might be less abused empathogens similar to
MDMA. Such compounds have yet to be explored to our
knowledge, but may generalize behaviorally to MDMA. The
behavioral outcomes of analogs with similar DAT and SERT
activity are difficult to predict. The 2-naphthyl analog of
methcathinone 27 is roughly equipotent at DAT and SERT,
and one might predict weak reinforcing and rewarding prop-
erties. Compound 27 was modestly self-administered in rats
but induced conditioned place preference in mice similar to
methamphetamine which suggests more complicated behav-
ioral effects (Botanas et al. 2017).
While most of the focus of these compounds has been on
their DAT and SERT activity, the noradrenergic properties of
methcathinones might also play a significant role in their phar-
macology (Hysek et al. 2011; Rothman et al. 2001). NE
releasers have been shown to affect cocaine self-
administration (Banks et al. 2014; Kohut et al. 2017) and
drugs elevating NE do not appear to be reinforcing (Wee
and Woolverton 2004). In our experience with rat brain syn-
aptosomes, NET release potencies generally vary in a similar
fashion as DAT release potencies and compounds tend to be
slightly more potent as NE releasers (unpublished). We gen-
erally observed the same trends with the methcathinones
based on percent release, but with several notable exceptions.
The 2-CF3 analog 8 and 3-nitro analog 18 both appear to be
selective NE releasers. This was unexpected but they may
represent probe compounds for the study of the effects of
NE release in various aspects of drug-seeking behavior such
as reinforcement and reward. Finally, the NE release potency
of the 3-indoyl analog 28 appears to be much weaker than its
DAT release potency compared to other bioisosteric analogs
such as the 2-naphthyl analog 27. This trend was also ob-
served in a study of tryptamines (Blough et al. 2014b) and
remains the only releaser scaffold which consistently releases
DA more potently than NE.
Conclusion
The work presented herein is an SAR study of the DAT, SERT,
and NET properties of a set of 28 methcathinone analogs. All

Psychopharmacology
evaluation of bupropion analogues as potential pharmacotherapies
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of the active compounds were found to be neurotransmitter
releasers, as opposed to reuptake inhibitors. The choice of
phenyl substituent had a dramatic effect on the DAT, SERT,
and NET releasing activity of the analogs, as did the site of
phenyl substitution. These differences demonstrate an ability
to manipulate the DAT, SERT, and NET releasing potencies as
well as the DAT selectivity of each analog, which plays a
critical role in abuse liability and potential psychoactive ef-
fects. A rapid, high-yielding, and scalable synthesis of
methcathinone hydrochloride analogs was also presented.
Del Bello F, Sakloth F, Partilla JS, Baumann MH, Glennon RA (2015)
Ethylenedioxy homologs of N-methyl-(3,4-methylenedioxyphenyl)-2-
aminopropane (MDMA) and its corresponding cathinone analog
methylenedioxymethcathinone: interactions with transporters for sero-
tonin, dopamine, and norepinephrine. Bioorg Med Chem 23:5574–
5579
Funding information This research was supported by the National
Institute on Drug Abuse project DA12970 (BEB) and the Intramural
Research Program, National Institute on Drug Abuse, NIH project DA-
00522 (MHB).
Eshleman AJ, Wolfrum KM, Reed JF, Kim SO, Swanson T, Johnson RA,
Janowsky A (2017) Structure-activity relationships of substituted
cathinones, with transporter binding, uptake, and release. J
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Fleckenstein AE, Volz TJ, Riddle EL, Gibb JW, Hanson GR (2007) New
insights into the mechanism of action of amphetamines. Annu Rev
Pharmacol 47:681–698
Compliance with ethical standards
Conflict of interest The authors declare that there is no conflict of
interest.
Foley KF, Cozzi N (2003) Novel aminopropiophenones as potential an-
tidepressants. Drug Dev Res 60:252–260
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