The biogenic amine transporter activity of vinylogous amphetamine analogs

Article abstract of DOI:10.1039/c6md00245e

A series of vinylogous amphetamine analogs was synthesized and examined for their activity at biogenic amine transporters and serotonin-2 receptor (5-HT₂) subtypes. (1S,3E)-1-Methyl-4-phenyl-but-3-enylamine (S-6) is a potent dual dopamine/serotonin (DA/5-HT) releaser with no activity at 5-HT₂ receptors. This unique profile of actions suggests that analog S-6 is a viable lead compound for identifying new structural classes of DA/5-HT releasers with therapeutic benefit and reduced abuse liability.

Full text of DOI:10.1039/c6md00245e

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RESEARCH ARTICLE
The biogenic amine transporter activity of
vinylogous amphetamine analogs†‡
Cite this: DOI: 10.1039/c6md00245e
Ann M. Decker,a John S. Partilla,b Michael H. Baumann,b Richard B. Rothman§b and
a
*
Bruce E. Blough
Received 30th April 2016,
Accepted 6th June 2016
A series of vinylogous amphetamine analogs was synthesized and examined for their activity at biogenic
amine transporters and serotonin-2 receptor (5-HT2) subtypes. (1S,3E)-1-Methyl-4-phenyl-but-3-
enylamine (S-6) is a potent dual dopamine/serotonin (DA/5-HT) releaser with no activity at 5-HT2 recep-
tors. This unique profile of actions suggests that analog S-6 is a viable lead compound for identifying new
structural classes of DA/5-HT releasers with therapeutic benefit and reduced abuse liability.
DOI: 10.1039/c6md00245e
www.rsc.org/medchemcomm
apy whereby patients are administered less potent and less
addictive stimulant-like medications.6 BAT releasers repre-
Introduction
Plasma membrane biogenic amine transporters (BATs) regu-
late neuronal signaling in the central nervous system by
transporting previously released monoamine neurotransmit-
ters – dopamine, norepinephrine, and serotonin (DA, NE,
and 5-HT transported via DAT, NET, and SERT, respectively)
– from the synapse back to the neuronal cytoplasm.1 Li-
gands that interact with BATs are divided into two general
classes: reuptake inhibitors and substrate-type releasers (for
a review2). Both types of ligands elevate extracellular neuro-
transmitter concentrations but act via different mechanisms.
Reuptake inhibitors bind to transporters and block
sent one class of compounds being evaluated as potential ag-
onist medications (for a review7).
Several studies have demonstrated the ability of SIJ+)-
amphetamine (1, Fig. 1), which has a high selectivity for re-
leasing DA relative to 5-HT, to act as an agonist therapy for
stimulant dependence. Chronic treatment with SIJ+)-
amphetamine in rhesus monkeys results in a selective dose-
dependent decrease in cocaine self-administration compared
to food-maintained responding using progressive-ratio,8
choice,9 and second-order10 schedules. In a double-blind,
placebo-controlled clinical trial, treatment with SIJ+)-
amphetamine results in a decrease in cocaine use,11 which is
consistent with other clinical trials testing agonist treatments
(for a review12). However, a significant limitation of using
SIJ+)-amphetamine as a medication is its abuse potential due
to activation of mesolimbic dopamine neurons.13
Previous evidence suggests that deficits in both DA and
5-HT are associated with withdrawal symptoms and that ele-
vations in extracellular 5-HT can counteract the stimulant
and reinforcing effects of DA (dual deficit model of stimulant
addiction).14 One possible advantage of using dual DA/5-HT
releasers as agonist medications is their combined ability to
provide the necessary stimulant-like properties required for
therapeutic efficacy (i.e., DA release) while reducing abuse lia-
bility (5-HT release). As such, multiple lines of evidence show
that 5-HT elevations can reduce drug seeking behavior. In
transporter-mediated
reuptake
of
neurotransmitters.3
Substrate-type releasers bind to the substrate site on the
transporters, are transported inside the neuron, and pro-
mote neurotransmitter efflux by carrier-mediated exchange.4
Disruption of BAT function plays an important role in the
pathophysiology of many neurological diseases such as de-
pression, anxiety, Parkinson's disease, schizophrenia, and
psychostimulant addiction.5
Psychostimulants, like cocaine and methamphetamine,
are addictive drugs that target BATs in the central and pe-
ripheral nervous systems to cause a variety of harmful physio-
logical effects in humans. One potential strategy to treat
psychostimulant addiction is called agonist substitution ther-
a Center for Drug Discovery, RTI International, 3040 E. Cornwallis Road, Research
Triangle Park, NC 27709, USA. E-mail: beb@rti.org; Fax: +919 541 6499;
Tel: +919 541 1244
b Medicinal Chemistry Section, Intramural Research Program, National Institute
on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA
† The authors declare no competing interests.
‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/
c6md00245e
§ Present address. Department of Psychiatry, MedStar St. Mary's Hospital, 25500
Point Lookout Road, Leonardtown, MD 20650, USA.
Fig. 1 SIJ+)-amphetamine and PAL-287.
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vivo studies conducted in rats reveal that 5-HT release de-
creases the stimulant effects of amphetamine-type drugs15
and that fenfluramine (5-HT releaser) dose-dependently at-
tenuates cue-reinstated cocaine-seeking behavior.16 The re-
duction in drug-seeking behavior observed in rats translates
to humans in that fenfluramine significantly reduces cocaine
craving in abstinent cocaine-dependent patients.17 Further,
in preliminary clinical trials, co-administration of the anorec-
tics phentermine (DA releaser) and fenfluramine (Fen–Phen)
shows promise in treating cocaine and alcohol dependence
thus supporting the idea of using dual DA/5-HT releasers as
therapeutics.18
Based on this evidence, our laboratory decided to focus
our efforts on dual DA and 5-HT releasers. As part of this
program, we identified 1-naphthyl-2-aminopropane (PAL-
287¶ 2) as our lead compound (Fig. 1).19 PAL-287 releases
radiolabeled neurotransmitters from DAT, SERT, and NET
with EC50 values of 12.6 nM, 3.4 nM, and 11.1 nM, respec-
tively (Table 1). In vivo microdialysis experiments in rats
corroborate the in vitro data by showing that PAL-287 (1–3
mg kg−1 i.v.) increases extracellular DA and 5-HT in the
frontal cortex, with effects on 5-HT being larger (464% in-
crease compared to 133% increase).20 Furthermore, in rats,
PAL-287 causes significantly less motor stimulation com-
pared to SIJ+)-amphetamine, which has 71-fold greater po-
tency to release DA compared to 5-HT; importantly high
doses of PAL-287 do not cause depletion of cortical 5-HT.20
In rhesus monkeys trained to self-administer cocaine, PAL-
287 produces a dose-dependent decrease in cocaine self-
administration and significantly decreases cocaine- versus
food-maintained responding at 1.0 mg kg−1 h−1.21 Overall,
the data collected with the non-amphetamine analog, PAL-
287, support the hypothesis that dual DA/5-HT releasers
possess the therapeutic effects of amphetamine-type
releasers while being minimally reinforcing.
To begin to investigate the structure–activity requirements
of the naphthyl moiety of PAL-287, a series of vinylogous
PAL-287 analogs were synthesized and evaluated for trans-
porter activity. BAT activity was measured as done previously
using synaptosomes prepared from rat brain homogenates
according to the protocol developed by Rothman and co-
workers.22 Compounds were first screened in uptake inhibi-
tion and release assays to determine the exact mode of drug
action. Compounds active in both assays are releasers while
compounds active only in the uptake inhibition assay are up-
take inhibitors. Active compounds were then fully character-
ized by running 8-point concentration response curves in the
assay corresponding to their mechanism of action. Substrate
reversal experiments were conducted to validate substrate ac-
tivity. The analogs were also tested for agonist activity at the
serotonin-2 receptor subtypes (5-HT2A, 5-HT2B, 5-HT2C) using
in vitro calcium mobilization assays in transfected HEK293
cells as previously described.23 These receptors are associated
with the pharmacology of abused drugs, as 5-HT2A agonists
are thought to be hallucinogenic24,25 while 5-HT2B agonists
are associated with valvular heart disease and pulmonary hy-
pertension;26,27 activity at these receptors would be consid-
ered off-target liabilities. On the other hand, agonists at
5-HT2C may be beneficial as potential pharmacotherapies for
drug abuse and appetite suppression.28,29
Methods
Biological assays
DAT, NET, and SERT assays. All animal studies were
conducted in facilities fully accredited by the Association for
the Assessment and Accreditation of Laboratory Animal Care
(AAALAC) and experiments were performed in accordance
with the Institutional Care and Use Committee (IACUC) of
the National Institute on Drug Abuse Intramural Research
Program (NIDA IRP). Rats were euthanized by CO2 narcosis,
and brains were processed to yield synaptosomes as previ-
ously described.22 Synaptosomes were prepared from rat stri-
atum for the DAT assays, whereas synaptosomes were pre-
pared from whole brain minus striatum and cerebellum for
the NET and SERT assays.
For uptake inhibition assays, 5 nM [3H]DA, 10 nM [3H]nor-
epinephrine and 5 nM [3H]5-HT were used to assess transport
activity at DAT, NET, and SERT, respectively. The selectivity
of uptake assays was optimized for a single transporter by in-
cluding unlabeled blockers to prevent uptake of [3H]transmit-
ter by competing transporters. Uptake inhibition assays were
initiated by adding 100 μl of tissue suspension to 900 μL
Krebs-phosphate buffer (126 mM NaCl, 2.4 mM KCl, 0.83
mM CaCl2, 0.8 mM MgCl2, 0.5 mM KH2PO4, 0.5 mM Na2SO4,
11.1 mM glucose, 0.05 mM pargyline, 1 mg mL−1 bovine se-
rum albumin, and 1 mg mL−1 ascorbic acid, pH 7.4)
containing test drug and [3H]transmitter. Uptake inhibition
assays were terminated by rapid vacuum filtration through
Whatman GF/B filters, and retained radioactivity was quanti-
fied by liquid scintillation counting. Concentration–response
curves were generated to yield IC50 values.
For release assays, 9 nM [3H]1-methyl-4-phenylpyridinium
([3H]MPP+) was used as the radiolabeled substrate for DAT
and NET, while 5 nM [3H]5-HT was used as a substrate for
SERT. All buffers used in the release assays contained 1 μM
reserpine to block vesicular uptake of substrates. The selectiv-
ity of release assays was optimized for a single transporter by
including unlabeled blockers to prevent the uptake of [3H]
MPP+ or [3H]5-HT by competing transporters. Synaptosomes
were preloaded with radiolabeled substrate in Krebs-
phosphate buffer for 1 h (steady state). Release assays were
initiated by adding 850 μL of preloaded synaptosomes to 150
μL of test drug. Release was terminated by vacuum filtration
and retained radioactivity was quantified as described for up-
take inhibition. Concentration–response curves were gener-
ated to yield EC50 values.
Substrate reversal experiments were conducted to validate
substrate activity. The releasing ability of test compounds
was tested at an EC80 concentration in the absence and
¶ PAL = phenyl amine library.
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Table 1 Structure–activity of a series of vinylogous amphetamine analogs for releasing radiolabeled substrates from DAT, SERT and NET
EC50 (nM SD)a
b
5-HT2Ab EC50
(nM SEM)
,
5-HT2B EC50
(nM SEM)
,
5-HT2Cb EC50
(nM SEM)
,
PAL
#
Group
DAT
SERT
NET
7.1
( ) AMP
287
869
870
871
872
875
873
904
905
906
881
893
1
2
4
24.8 3.5
12.6 0.4
997 220
443 100
660 190
272 44
1770 94
3.4 0.2
384 110
756 79
288 + 84
1
IA
IA
IA
11.1 0.9
2980 490
784 190
496 160
239 59
138 260
279 24
621 190
170 32
211 42
308 100
301 76
466c nM
>10k
40c nM
IA
23c nM (20%)
I
I
I
IA
S-4
R-4
6
S-6
R-6
8
S-8
R-8
10
11
4950 1400
>10k
IA
IA
IA
IA
II
II
II
III
III
III
IV
IV
54
40
8
9
5620 1700
IA
IA
IA
IA
IA
206 26
540 87
109 29
646 70
663 86
1156 160
>10k
>10k
IA
IA
900 100
304 110
1416 180
666 160
1114 150
1860 76
1600 320
>10k
IA
IA
IA
>10k
>10k
IA
IA
IA
IA
>10k
IA
IA
IA
a
b
EC50 release values were determined as described in Methods. Each value is mean SD (n = 3). Calcium mobilization EC50 values were
determined as described in Methods. Each value is mean SEM (at least n = 3). c Data from ref. 19. IA = Inactive at 10 μM.
presence of an uptake inhibitor (250 nM GBR1209 for DAT,
166 nM desipramine for NET, 100 nM fluoxetine for SERT). If
the test agent was a releaser, the uptake inhibitor reduced
the effect of the test agent. If the test agent was an uptake in-
hibitor, the addition of a second uptake inhibitor led to ei-
ther no change or an increased effect in the release assay.
Calcium mobilization assays. Three individual HEK293
cell lines stably expressing the human 5-HT2A, 5-HT2B, and
5-HT2C receptors were used. The day before the assay, cells
were plated into 96-well black-walled assay plates (2A –
40 000 cells per well; 2B and 2C – 35 000 cells per well) in
DMEM-HG supplemented with 10% fetal bovine serum, 100
units of penicillin and streptomycin, and 15 mM HEPES.
The cells were incubated overnight at 37 °C, 5% CO2. Prior
(GraphPad Prism 6.0, GraphPad Software, Inc., San Diego,
CA).
Compound synthesis. The synthesis of all novel com-
pounds is provided in the ESI.‡
Results & discussion
Four groups of PAL-287 vinylogous analogs were synthesized
in which the naphthyl moiety was replaced with phenyl
alkynyl, phenyl (E)-alkenyl, or phenyl (Z)-alkenyl moieties
(Fig. 2). Group I consisted of the racemic alkynyl isostere 4,
as well as the two chiral isosteres, S-4 and R-4. Group II
consisted of the racemic and chiral (E)-alkenyl isosteres (6,
S-6, R-6), while group III consisted of the corresponding (Z)-
alkenyl isosteres (8, S-8, R-8). The analogs of group IV (10,
11) have one less carbon between the phenyl ring and the
amine group. All analogs were synthesized in three or four
steps from commercially available materials. Scheme 1 shows
the synthesis of the (S)-stereoisomers from groups I–III. To
synthesize group I alkyne S-4, commercially available alcohol
R-3 was converted to the tosylate, which underwent displace-
ment with inversion of configuration to the azide, which was
reduced under Staudinger conditions to provide S-4. The
same commercially available starting alcohol R-3 was selec-
tively reduced with lithium aluminum hydride (LAH) or
Lindlar's catalyst to afford the corresponding (E)- or (Z)-ole-
fins, R-5 and R-7, respectively. These olefins were then
converted to amines S-6 and S-8, respectively, using the same
three step tosylation/azide formation/Staudinger reduction
steps. The (R)-stereoisomers and the racemates for groups I–
III were synthesized using the same pathway starting with
the corresponding commercially available (S)-alcohol and ra-
cemic alcohol, respectively. Scheme 2 shows the synthesis of
group IV analogs (10, 11) from commercially available alcohol
9 using the same pathway as the other analogs, except that
mesylation was performed instead of tosylation due to stabil-
ity issues.
to the assay, Calcium
5 dye (Molecular Devices) was
reconstituted according to the manufacturer instructions.
The reconstituted dye was diluted 1 : 40 in pre-warmed (37
°C) assay buffer (1× HBSS, 20 mM HEPES, 2.5 mM proben-
ecid, pH 7.4 at 37 °C). Growth medium was removed and
the cells were gently washed with 100 μL of pre-warmed
(37 °C) assay buffer. The cells were incubated for 45 mi-
nutes at 37 °C, 5% CO2 in 200 μL of the diluted Calcium 5
dye. Serial dilutions of the test compounds were prepared
in 1% DMSO/assay buffer, aliquoted into 96-well polypropyl-
ene plates, and warmed to 37 °C. After the dye-loading in-
cubation period, the cells were pre-treated with 25 μL of
9% DMSO/assay buffer and incubated for 15 min at 37 °C.
After the pre-treatment incubation period, the plate was
read with a FlexStation II (Molecular Devices). Calcium-
mediated changes in fluorescence were monitored every
1.52 seconds over a 60 second time period, with the
FlexStation II adding 25 μL of test compound dilutions at
the 19 second time point (excitation at 485 nm, detection
at 525 nm). Peak kinetic reduction (SoftMax, Molecular De-
vices) relative fluorescent units (RFU) were plotted against
compound concentration. Data were fit to the appropriate
three-parameter logistic curve to generate EC50 values
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Fig. 2 Vinylogous amphetamine analogs synthesized for this study.
Scheme 1 Synthesis of group I alkynes, group II (E)-alkenes and group III (Z)-alkenes.
Table 1 shows the transporter data for the analogs. All
compounds were active as DAT and NET releasers with vary-
ing potencies and all but two compounds, 10 and 11, were ac-
tive as SERT releasers. At the DAT, group I alkynes had simi-
lar potencies with S-4 being the most potent with an EC50
value of 443 nM. At the SERT, the alkynes also had similar
potencies with R-4 being the most potent with an EC50 value
of 288 nM. At the NET, alkyne R-4 was the most potent
(EC50 = 496 nM) and 4 was the least potent (EC50 = 2980 nM).
The group II (E)-alkenes were potent at all three transporters,
swith EC50 values less than 540 nM. At the DAT, group II (E)-
alkenes had similar potencies with R-6 being the least potent
(EC50 = 540 nM) and S-6 being the most potent with an EC50
value of 206 nM. At the SERT and NET, S-6 was the most ac-
tive compound in this group with EC50 values of 40 nM and
138 nM, respectively. At the DAT, group III (Z)-alkenes were
less than 1500 nM, with S-8 being the most potent at 304
nM. At the SERT, analogs 8 and S-8 had similar potencies
with 8 being slightly more potent (EC50 = 646 nM). At the
NET, S-8 was the most potent analog with an EC50 value of
170 nM. Group IV analogs were inactive at SERT and rela-
tively weak releasers at DAT with 10 being the most potent
(EC50 = 666 nM). However, both analogs had similar poten-
cies at the NET (EC50 ∼ 300 nM).
Scheme 2 Synthesis of group IV analogs.
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From a structure–activity perspective, all compounds were
substrates for the transporters, indicating that the trans-
porters can translocate larger structures than previously be-
lieved.30 All compounds, except group IV, were dual DA/5-
HT releasers, but with varying degrees of transporter selec-
tivity. The group I alkynes did not exhibit much selectivity
for releasing 5-HT compared to DA as the racemic analog 4
and R-4 were only 2.6-fold and 2.3-fold, respectively, more
potent at the DAT. S-4 was essentially equipotent at the
DAT and SERT. The group II alkenes were all more selective
at SERT relative to DAT with 5-fold greater potency at SERT.
This group was interesting because the activities at the
transporters were all very similar, indicating no differences
between chiral isomers, R-6 and S-6, and the racemate 6.
The group III (Z)-olefins were similar to the group I alkynes
as the analogs did not show much SERT/DAT selectivity. The
racemic analog 8 and R-8 had similar potencies for releasing
5-HT relative to DA (1.4- and 1.2-fold, respectively) while S-8
was slightly more potent for releasing DA relative to 5-HT
(2.2-fold). Removing a carbon between the alkene and the
amine (group IV) resulted in analogs that were selective at
DAT and NET. These compounds were inactive at SERT indi-
cating they could not bind to the site of translocation; this
activity profile suggests the compounds may be weak
stimulants.
As part of our program to develop neurotransmitter
releasers as therapeutic agents, we have analyzed over 1000
small molecule phenethylamines for BAT activity and found
that NE release almost always parallels DA release with
slightly higher potency. Recently, we identified a group of
tryptamines that are >10-fold selective for releasing DA rela-
tive to NE.23 While most of the vinylogous analogs follow
the DA/NE release trend, a few compounds show selectivity
for DAT or NET. Group I racemic alkyne 4 was 3-fold more
potent at releasing DA compared to NE with EC50 values of
997 nM and 2980 nM, respectively. Analog S-4 followed the
typical trend at DAT and NET with EC50 values of 660 nM
and 496 nM, respectively, while activity at DAT and NET for
analog R-4 was reversed with EC50 values of 443 nM and
784 nM, respectively. All group II (E)-alkenes and two group
III (Z)-alkenes followed the typical trend; however, group III
(Z)-alkene R-8 was 6.7-fold selective for releasing NE relative
to DA with EC50 values of 211 nM and 1416 nM, respec-
tively. Both group IV analogs, 10 and 11, were more potent
at NET compared to DAT, but with different selectivities
(2.2-fold and 3.7-fold, respectively).
acteristics. S-6 has 5-fold 5-HT/DA release potency, similar to
PAL-287, which has 3.7-fold selectivity. S-6 has 3.5-fold higher
potency for 5-HT release compared to NE release, which is
similar to PAL-287's 3.3-fold selectivity, and both compounds
have almost equal DA/NE release potencies.
The vinylogous analogs were also evaluated for agonist
activity at 5-HT2A, 5-HT2B, and 5-HT2C receptors using
in vitro calcium mobilization assays (Table 1). Overall, the
analogs had varying degrees of weak activity at all three re-
ceptors, making them much more like S(+)-amphetamine
(inactive in all three assays) than PAL-287 (Table 1). Previ-
ous functional studies reveal that PAL-287 is a full agonist
at 5-HT2A and 5-HT2B receptors (EC50 = 466 nM and 40 nM,
respectively) and a partial agonist (EC50 = 2.3 nM, EMAX
=
19
20%) at 5-HT2C
.
At 5-HT2A, the vinylogous analogs were all
less potent than PAL-287 as most of them were inactive (S-6,
10, 11) or had EC50 values > 10 μM (4, R-4, R-6, R-8).
The remaining analogs had potencies in the micromolar
range. Analog S-8, which had an EC50 value of 1600 nM and
EMAX of 102%, was the most potent and efficacious analog.
The racemic analog 8 had a similar potency (EC50 = 1860
nM) and efficacy (EMAX = 90%). The only other compounds
that were active were alkyne S-4 and racemic alkene 6,
which had a 2.7-fold and 3-fold reduction in potency, re-
spectively, compared to 8. These compounds were also not
as efficacious and had EMAX values in the lower 80% range.
At 5-HT2B, all the analogs were inactive. This was interesting
because PAL-287 was active at 5-HT2B as an agonist with an
EC50 value of 40 nM. At 5-HT2C, only group III (Z)-alkenes
8 and S-8 were weak agonists with activity less than 50% of
the control 5-HT EMAX at 10 μM. The most active transporter
compound (S-6) was inactive at all three receptors, indicat-
ing that this compound may not produce the typical effects
associated with agonist activity at the 5-HT2 receptors; how-
ever, the full in vivo effects of S-6 would need to be evalu-
ated in appropriate assays.
As noted, elevations in extracellular 5-HT can counteract
the stimulant and reinforcing effects of DA release14 in a dual
DA/5-HT releaser leading to non-stimulant, non-addictive
therapeutics, but the potential psychedelic effects observed
with many 5-HT releasers remains an issue. Neurotransmitter
releasers such as PAL-287 and S-6 possess transporter profiles
using rat brain synaptosomes that are similar to psychedelic
compounds such as 3,4-methylenedioxymethamphetamine
(MDMA)31 and the “bath salt” mephedrone.32 Similar relative
profiles are also observed in engineered cell-based trans-
porter assays containing over-expressed transporter pro-
teins.33 While the biogenic amine transporters play a role in
the psychoactivity of these psychedelics, a complete rationale
for these effects remains unclear. The 5-HT selective releaser
fenfluramine was not euphoric at therapeutic doses (20 mg)
but hallucinogenic episodes were observed at high doses (240
mg),34 implying that hallucinogenic effects are not primarily
transporter related. While similar studies have not yet been
conducted on PAL-287, anecdotal internet reports suggest
that PAL-287 does not possess strong psychoactive effects.
Group II (E)-alkenes were the most active compounds
compared to the other three groups. The most potent analog
at the DAT, SERT, and NET was (E)-alkene S-6 with EC50
values of 206 nM, 40 nM, and 138 nM, respectively. This ana-
log retains the same configuration as SIJ+)-amphetamine, has
the same number of carbons between the phenyl and amine
groups as PAL-287, and has a similar steric conformation as
PAL-287, compared to the more sterically hindered (Z)-ole-
fins. While PAL-287 is 10-fold more potent than S-6 at all
three transporters, the compounds share some activity char-
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Regardless, further therapeutic development of dual DA/5-HT
releasers would need to include behavioral assessment in-
cluding drug discrimination to avoid such side-effects, espe-
cially drug candidates that are 5-HT selective.
4 A. E. Fleckenstein, T. J. Volz, E. L. Riddle, J. W. Gibb and
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Another important development issue is serotonin deple-
tion and neurotoxicity. Many serotonin releasers deplete
stores of 5-HT potentially leading to neurotoxicity with
chronic use. The 5-HT releasers fenfluramine, MDMA, and
p-chloroamphetamine (PCA) all cause long-term 5-HT deple-
tion;35 PCA is even used as a behavioral tool to help un-
mask the effects of 5-HT on behavior.36 However, not all
5-HT releasers cause depletion and neurotoxicity. Meta-
chlorophenylpiperazine (mCPP), while equipotent with fen-
fluramine, was found to lack the 5-HT depleting effects of
fenfluramine37 as was our laboratory's main lead com-
pound, PAL-287.38 As part of the devlopment process, dual
DA/5-HT releaser drug candidates will need to be assessed
for 5-HT depletion in order to determine any potential neu-
rotoxicity associated with chronic use.
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16 J. J. Burmeister, E. M. Lungren and J. L. Neisewander,
Psychopharmacology, 2003, 168, 146.
17 L. Buydens-Branchey, M. Branchey, J. Hudson, M.
Rothman, P. Fergeson and C. McKernin, Am J Addict.,
1998, 7, 142.
18 R. B. Rothman, T. Gendron and P. Hitzig, J. Subst. Abuse
Treat., 1994, 11, 273.
Conclusions
In conclusion, eleven vinylogous PAL-287 analogs with less
conformational restriction were studied, resulting in the dis-
covery of a new series of amphetamine analogs that have
transporter activity. All of the analogs were substrates for the
BATs, lending support to the notion that transporters are
able to translocate larger structures than previously believed.
Of the eleven analogs, three compounds (6, S-6, R-6) were po-
tent releasers of DA, 5-HT, and NE, with S-6 being the most
potent with EC50 values of 206 nM, 40 nM, and 138 nM, re-
spectively. This analog has the same configuration as SIJ+)-
amphetamine, and has a similar steric conformation as PAL-
287 due to the same number of carbons between the phenyl
and amine groups. Although S-6 is 10-fold less potent than
PAL-287, the two compounds share similar transporter selec-
tivities. Analog S-6 was also inactive in in vitro 5-HT2 calcium
mobilization assays, indicating no potential in vivo effects.
The unique activity profile for S-6 suggests that this com-
pound represents a new lead for identifying neurotransmitter
releasers with therapeutic potential.
19 R. B. Rothman, B. E. Blough and M. H. Baumann, Trends
Pharmacol. Sci., 2006, 27, 612.
20 R. B. Rothman, B. E. Blough, W. L. Woolverton, K. G.
Anderson, S. S. Negus, N. K. Mello, B. L. Roth and M. H.
Baumann, J. Pharmacol. Exp. Ther., 2005, 313, 1361.
21 S. S. Negus, N. K. Mello, B. E. Blough, M. H. Baumann and
R. B. Rothman, J. Pharmacol. Exp. Ther., 2007, 320, 627.
22 R. B. Rothman, M. Katsnelson, N. Vu, J. S. Partilla, C. M.
Dersch, B. E. Blough and M. H. Baumann, Eur. J.
Pharmacol., 2002, 447, 51.
23 B. E. Blough, A. Landavazo, J. S. Partilla, A. M. Decker, K. M.
Page, M. H. Baumann and R. B. Rothman, Bioorg. Med.
Chem. Lett., 2014, 24, 4754.
Acknowledgements
24 D. E. Nichols, Pharmacol. Ther., 2004, 101, 131.
25 J. González-Maeso, N. V. Weisstaub, M. Zhou, P. Chan, L.
Ivic, R. Ang, A. Lira, M. Bradley-Moore, Y. Ge, Q. Zhou, S. C.
Sealfon and J. A. Gingrich, Neuron, 2007, 53, 439.
26 R. B. Rothman and M. H. Baumann, Expert Opin. Drug Saf.,
2009, 8, 317.
This research was supported by the National Institute on
Drug Abuse Project DA12970 (BEB) and the Intramural Re-
search Program, National Institute on Drug Abuse, NIH.
27 J. M. Launay, P. Hervé, K. Peoc'h, C. Tournois, J. Callebert,
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