Hybrid dopamine uptake blocker-serotonin releaser ligands: A new twist on transporter-focused therapeutics

Article abstract of DOI:10.1021/ml500113s

As part of our program to study neurotransmitter releasers, we report herein a class of hybrid dopamine reuptake inhibitors that display serotonin releasing activity. Hybrid compounds are interesting since they increase the design potential of transporter related compounds and hence represent a novel and unexplored strategy for therapeutic drug discovery. A series of N-alkylpropiophenones was synthesized and assessed for uptake inhibition and release activity using rat brain synaptosomes. Substitution on the aromatic ring yielded compounds that maintained hybrid activity, with the two disubstituted analogues (PAL-787 and PAL-820) having the most potent hybrid activity.

Full text of DOI:10.1021/ml500113s

Letter
pubs.acs.org/acsmedchemlett
Hybrid Dopamine Uptake Blocker−Serotonin Releaser Ligands: A
New Twist on Transporter-Focused Therapeutics
Bruce E. Blough,*,† Antonio Landavazo,† John S. Partilla,‡ Michael H. Baumann,‡ Ann M. Decker,†
Kevin M. Page,† and Richard B. Rothman‡,§
†Center for Drug Discovery, Discovery-Science-Technology, RTI International, 3040 Cornwallis Road, Research Triangle Park, North
Carolina 27709, United States
‡Medicinal Chemistry Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health,
Baltimore, Maryland 21224, United States
S
* Supporting Information
ABSTRACT: As part of our program to study neuro-
transmitter releasers, we report herein a class of hybrid
dopamine reuptake inhibitors that display serotonin releasing
activity. Hybrid compounds are interesting since they increase
the design potential of transporter related compounds and
hence represent a novel and unexplored strategy for
therapeutic drug discovery. A series of N-alkylpropiophenones
was synthesized and assessed for uptake inhibition and release
activity using rat brain synaptosomes. Substitution on the aromatic ring yielded compounds that maintained hybrid activity, with
the two disubstituted analogues (PAL-787 and PAL-820) having the most potent hybrid activity.
KEYWORDS: Dopamine uptake inhibitors, serotonin releasers, serotonin receptor, cocaine, methamphetamine
lasma membrane biogenic amine transporters (BATs)
terminate aminergic signaling by transporting previously
pounds are called neurotransmitter releasers, such as the DA/
NE releaser methcathinone 2a.2 Reuptake inhibition and
release are the primary modes of action for most BAT ligands.
Both types of BAT compounds can elevate synaptic
concentrations of neurotransmitters, and this enhancement of
neurotransmission can have profound in vivo effects. For
instance, the stimulatory in vivo effects of DAT ligands are well-
known as demonstrated by the ability of DA reuptake blockers
and releasers to induce locomotor activity via elevating synaptic
DA.2−4 Despite their shared acute physiological effects,
mechanistic differences between reuptake inhibitors and
releasers lead to dissimilar kinetics, downstream effects, and
feedback control.5 In particular, a reuptake inhibitor depends
on ongoing exocytotic release of neurotransmitter to achieve its
effects, while a releaser causes rapid efflux of a cytoplasmic pool
of neurotransmitter that is independent from exocytosis. These
differences in mechanism can lead to opposing changes in
biological regulation. As a specific example, the administration
of the DA uptake inhibitor cocaine upregulates striatal DATs,6
as does bupropion,7 while administration of the DA releaser
methamphetamine downregulates striatal DATs.8 Therefore,
while uptake inhibitors and releasers can both elevate synaptic
concentrations of neurotransmitter, the mechanisms and
pharmacological consequences of their use are different.5
P
released neurotransmitters, dopamine, norepinephrine, and
serotonin (DA, NE, and 5-HT transported via DAT, NET,
and SERT, respectively), from the synaptic cleft back into the
neuronal cytoplasm. One of the most successful strategies for
drug discovery over the past 30 years has been the development
of BAT ligands, which block the reuptake of amine neuro-
transmitters, thereby elevating synaptic concentrations of these
neurotransmitters. At the molecular level, BAT ligands block
reuptake by either inhibiting endogenous transmitter binding or
preventing the transporter protein from undergoing obligatory
conformational changes. From a therapeutic perspective, these
compounds are known as reuptake inhibitors, an example of
which is the DA/NE reuptake inhibitor bupropion 1 (Figure
1).1 Alternatively, other BAT ligands can act as transporter
substrates that induce neurotransmitter release. These com-
Received: September 5, 2013
Accepted: April 15, 2014
Published: April 15, 2014
Figure 1. Propiophenone compounds.
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
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Transporter ligand discovery and development efforts have
traditionally focused on the development of a drug within a
given mechanistic class of BAT ligand, either a compound that
inhibits reuptake or one that induces neurotransmitter release.
Most transporter ligands recruit one mechanism or the other.
This pattern suggests that BAT ligands possess general
structural features that cause them to act as either releasers
or uptake inhibitors, but not both. However, given the relatively
recent confirmation that a compound must be a substrate in
order to induce transporter-mediated release,9 the possibility
exists that a BAT ligand might be a substrate of one transporter
but not of another and instead act as a reuptake inhibitor. In
turn, this implies that it might be possible to find compounds
with mixed (i.e., hybrid) transporter activity, whereby certain
ligands induce release at one transporter but block reuptake at
another.
Hybrid compounds are intriguing for several reasons. First,
the kinetics of ligand−transporter interaction may be different
for releasers versus reuptake blockers. A releaser mimics an
endogenous neurotransmitter and acts quickly at the target
transporter, possibly because it interacts at a well-defined
domain suited for neurotransmitter translocation. However, an
uptake inhibitor can have much slower on- and off-rates of
interaction with transporter proteins. Second, the profile of
synaptic neurotransmitter change should differ according to
molecular mechanism since an uptake inhibitor relies on
ongoing exocytotic release to achieve its effects, while a releaser
overrides the system and triggers neurotransmitter efflux
regardless of the state of the system. Third, the effects of
chronic use may also be different since neuroadaptive changes
are related to acute drug mechanism, and in some cases may
have the opposite effects, an example of which is the long-term
effect of BAT ligands on DATs described above. Thus, the in
vivo properties of a dual DA-5-HT uptake inhibitor may be
different than a dual DA-5-HT releaser, which in turn may be
different than a DA uptake inhibitor-5-HT releaser or a DA
releaser-5-HT uptake inhibitor. These hybrid possibilities
increase the drug design potential for transport related
compounds and represent a novel strategy for therapeutic
discovery.
Indeed, a compound with hybrid transporter activity has
already been identified in the Rothman laboratory: 2-(N-
ethylamino)propiophenone (2b), the bioactive metabolite of
the clinically available appetite suppressant diethylpropion
(2c).10 This analogue of both 1 and 2a was found to be a DA
reuptake inhibitor with a Ki value of 1014 nM and a 5-HT
releaser with an EC50 value of 2118 nM. To our knowledge this
was the first compound identified to have hybrid activity, albeit
weak and structural analogues did not have the same hybrid
activity. As part of our program to study neurotransmitter
releasers, other hybrid compounds have been identified from
our screening data. For example, we recently reported that the
(−)-isomer of the abused street drug 3,4-methylenedioxy-N-
ethylamphetamine also has hybrid transporter activity.11
Here we describe the first example of a family of related
compounds with hybrid uptake inhibitor-releaser activity:
substituted 2-(N-cyclopropylamino)propiophenones (3a−h).
These BAT ligands are DA reuptake inhibitors with 5-HT
releasing activity. The lead structure, 2-(N-cyclopropylamino)-
3′-chloropropiophenone (3a) was originally synthesized as part
of a set of bupropion related compounds and was found to be a
DA reuptake inhibitor with an IC50 of 265 nM and a 5-HT
uptake inhibitor with an IC50 of 3180 nM.1 This compound
elicited unique in vivo behavioral activity related to drug abuse,
specifically a slower onset and longer duration of action in
generalization to cocaine. Such a profile is well suited for a DA
agonist pharmacotherapy for treating addiction to cocaine,
methamphetamine, nicotine, and other drugs of abuse.
Compound 3a was originally thought to be a DA/5-HT uptake
inhibitor, but a more detailed analysis of the transporter activity
uncovered the 5-HT releasing activity. This suggests 3a was
originally mischaracterized as a SERT blocker but is in reality a
SERT substrate that induces 5-HT release. All of the other
compounds except for the cyclobutyl compound (data not
shown) in the original bupropion series were found to be
uptake blockers as originally described. A series of additional
analogues was then synthesized and tested for transporter
activity to validate the activity across the structural class, all of
which were found to have the same unique transporter profile.
The synthesis of 3a−3h is shown in Scheme 1. Commercially
available propiophenones 4a−h were brominated using
a
Scheme 1. Synthesis of Hybrid Analogues
a
(a) Br2, MeOH; (b) cyclopropylamine.
bromine in methanol or dichloromethane followed by
amination with cyclopropylamine to form compounds 3a−h
with good yields in both steps. The N-propyl and N-ethyl
bupropion analogues (6 and 7, respectively) were synthesized
as previously described.1 Compounds 8, 9, 10, and 12 were
synthesized as described in the Supporting Information.
Compound 11 was synthesized as described in the literature.12
The optical isomers of 3a were synthesized using a modified
procedure used to synthesize the optical isomers of bupropion,
but using cyclopropylamine instead of tert-butylamine.13,14
The transporter activity of all compounds was assessed using
the previously described protocol for identifying releasers and
uptake inhibitors, using synaptosomes generated from rat brain
homogenate.15,16 Compounds were binned as releasing
substrates or uptake inhibitors by assessment in both
synaptosomal release and uptake inhibition assays. Once the
functional activity of a compound was determined, dose−
response curves were completed for the binned activity.15
The activity of these compounds is shown in Table 1.
Contrary to the original report by Carroll et al.,1 in our SERT
rat synaptosome assays, the lead compound 3a was found to be
a 5-HT releaser with an EC50 of 1328 nM. Potencies and
mechanism of activity at the other two transporters were
roughly the same compared to the original report. The optical
isomers of 3a were examined in order to assess enantiospecific
effects, but the potencies were found to be the same. It is
uncertain if this observation is a real effect of the two
enantiomers, or due to epimerization to the racemic mixture
under the assay conditions. Bupropion has been reported to
epimerize rapidly, but more detailed analysis suggests it does so
slowly under physiological conditions.17 Stirring the enan-
tiomers in water or alcohol failed to induce much epimerization
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Table 1. Comparison of Dopamine, Serotonin, and Norepinephrine Transporter Uptake Inhibition and Release Data for a
a
Series of N-Alkylpropiophenones Using Rat Synaptosomes
b
b
DAT
SERT
inhibition IC50
(nM)
inhibition IC50
PAL # compd
Z
R1
R2
X
Y
release EC50 (nM)
(nM)
release EC50 (nM)
1328 148
433
3a
O
CH(CH2CH2)
H
Cl
H
533 61
c
c
265 94
3180 170
1122
1123
363
361
586
588
591
743
744
787
820
304
434
426
429
550
(−)-3a
(+)-3a
6
O
CH(CH2CH2)
CH(CH2CH2)
nC3H7
H
Cl
Cl
Cl
Cl
Br
H
294 24
562 152
733 163
O
H
H
369 40
O
H
H
793 66
>10k
>10k
7
O
C2H5
H
H
5920 621
386 29
3b
3c
O
CH(CH2CH2)
CH(CH2CH2)
CH(CH2CH2)
CH(CH2CH2)
CH(CH2CH2)
CH(CH2CH2)
CH(CH2CH2)
H
H
H
621 141
1067 203
1014 118
1632 161
667 92
O
H
CH3
OCH3
H
H
1104 69
2372 175
2011 178
1103 884
425 22
3d
3e
O
H
H
O
H
Cl
CH3
Cl
CH3
H
3f
O
H
H
3g
3h
8
O
H
Cl
356 96
O
H
Cl
592 31
181 31
H,H
O
H
Cl
11.8 0.7
46.8 4.0
120
6
9
CH3
H
Cl
H
410 38
10
11
12
H,H
O
CH3
CH3
CH3
C2H5
H
H
225
9
4698 479
CH3
H
H
411 98
>10K
>10K
H,H
H
H
H
2596 408
a
b
c
ND = not determined; PAL = phenyl amine library. Data are represented as means SD and are the result of N = 3 performed in triplicate. Data
from Carroll et al.1
(by optical rotation) but could occur under the assay
conditions. Other compounds from the original set of
bupropion analogues from Carroll et al. were tested, two of
which are included in Table 1. The N-propyl analogue (6) was
found to be a selective DA uptake inhibitor with an IC50 of 793
nM. The N-ethyl analogue (7) was found to have much weaker
activity than 3a and did not show hybrid activity as did the
originally reported hybrid compound (unsubstituted analogue
2b).
Substitution on the aromatic ring yielded compounds with a
variety of potencies, but all were found to maintain hybrid DA
reuptake/5-HT releaser activity. The site of ring substitution
did not significantly alter activity, with 3a and 3e, as well as 3c
and 3f, having approximately the same activity despite being
substituted in the 3- and 4-positions, respectively. The
disubstituted compounds 3g and 3h were the most potent in
the series. The 3,4-dichloro analogue 3g and 3-chloro-4-methyl
analogue 3h had roughly the same potencies for DA reuptake
inhibition as for 5-HT release. These compounds had the most
potent 5-HT release EC50 values of 356 and 181 nM,
respectively, an order of magnitude more potent than the
lead compound 3a. All of the compounds were also active at
NET, either as weak uptake inhibitors or as partial releasers
(data not shown).
5-HT releasing activity. Alkyl extension alpha to the nitrogen to
form phenylpentanamine (12) eliminated both DA and 5-HT
release. Interestingly, the release potencies weaken for
compounds 8−12 as the overall structures get larger. 3-
Chloroamphetamine (8) has a 5-HT release EC50 value of 120
nM and the EC50 values weaken to 410 nM and then 4698 nM
for 9 and 10, respectively. Increasing the size further makes the
compound inactive. Similarly, 3-chloroamphetamine (8) has a
DA release EC50 value of 11.8 nM, and the EC50 values weaken
progressively to an EC50 value of 411 nM for 11. Increasing the
size of the ligand molecule past a critical point serves to flip the
activity to a DAT uptake inhibitor. Since release activity
depends on transportability, these trends suggest that the larger
the structure, the less likely the compound can be transported
by either DAT or SERT and thus be a releaser. The N-
cyclopropyl and N-cyclobutyl analogues retain their ability to
be transported through SERT but not DAT thus leading to
hybrid activity.
The agonist activity of the compounds at the 5-HT2A, 5-
HT2B, 5-HT2C, and 5-HT1A G-protein-coupled receptors
(GPCRs) was determined in calcium mobilization assays and
in a 5-HT2A β-arrestin assay. These GPCRs were chosen due to
the long-standing association of these receptors with the
pharmacology of abused drugs, in particular 5-HT releasers.
Agonists of these receptors would be considered off-target
liabilities. The 10 μM agonist screens revealed that all of the
hybrid compounds were inactive at the 5-HT1A, 5-HT2A, 5-
HT2B, and 5-HT2C receptors. Taken together, the screening
data indicate that these hybrid compounds may not elicit the
adverse effects often associated with drug abuse. Thus, the
hybrid compounds synthesized in this report could represent a
Hybrid activity was preserved with aromatic substitution, but
other changes resulted in the compounds either becoming DA/
5-HT releasers or DAT/SERT uptake inhibitors. Smaller
structures such as 3-chloroamphetamine (8), 3′-chloro-2-
methylaminopropiophenone (9), and N-methyl-1-phenyl-2-
butanamine (10) were found to be DA and 5-HT releasers.
The addition of an oxygen to 10 to form 2-methylaminobutyr-
ophenone (11) maintained DA release activity but eliminated
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ACS Medicinal Chemistry Letters
Letter
new structural class that could be examined as treatments for
drug addiction.
ABBREVIATIONS
■
BAT, biogenic amine transporter; DA, dopamine; 5-HT,
serotonin; DAT, dopamine transporter; SERT, serotonin
transporter; 5-HT2A, serotonin 2A receptor; 5-HT2B, serotonin
2B receptor; 5-HT2C, serotonin 2C receptor; 5-HT1A, serotonin
1A receptor
While the potency of these compounds may not appear
substantial (i.e., midnanomolar to micromolar range), this
potency range agrees with that of clinically used compounds
exhibiting similar structures. For example, the clinically used
antidepressant and smoking cessation compound bupropion
has about the same level of potency.1 The optimal selectivity for
DAT reuptake inhibition versus SERT release is unknown due
to the differences in kinetics, mechanisms of neurotransmitter
elevation, and effects after chronic use. The mixed mechanistic
profile of these compounds makes such an assessment more
complicated, compared to developing a DA/5-HT releaser or a
DA/5-HT reuptake inhibitor, but the differences in mechanistic
profile make the study of these compounds intriguing,
especially regarding chronic use.
REFERENCES
■
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ASSOCIATED CONTENT
■
S
* Supporting Information
Selected experimental procedures. This material is available free
of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*(B.E.B.) Tel: 919-541-1244. Fax: 919-541-6499. E-mail: beb@
rti.org.
Present Address
§(R.B.R.) Department of Psychiatry, MedStar St. Mary’s
Hospital, 25500 Point Lookout Road, Leonardtown, Maryland
20650, United States.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
Funding was provided by the National Institute on Drug Abuse
(DA12970). Aspects of this work were supported by the
Intramural Research Program, National Institute on Drug
Abuse, NIH.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank Dr. Ivy Carroll for the original samples
of 3a, 6, and 7.
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