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4755
above, concurrent elevation of extracellular 5-HT reduces
amphetamine-induced stimulant and rewarding effects without
reducing -amphetamine increases in extracellular DA, and with-
out altering the ability of -amphetamine to decrease cocaine
self-administration.3 Seen from this perspective, concurrent eleva-
tion of extracellular 5-HT can reduce the abuse liability of
D
-
was measured in an in vitro calcium mobilization assay using cells
over-expressing the 5-HT2A receptor. All of the compounds were
either purchased or synthesized in our laboratory. Tryptamines
4a, 4b, 4d, 4g, 4h, 4j, 4l, and 4m were purchased commercially.
Tryptamines 4c, 4e, 4f, 4i, 4k, 4n, 4o, 4p and 4q were synthesized
by reacting the requisite substituted indole with oxalyl chloride
followed by reaction with either ammonia or ethylamine in diox-
ane and subsequent reduction with lithium aluminum hydride
D
D
D
-
amphetamine-like agents without altering the therapeutic effect,
which is mediated by the increased release of DA.
This type of profile was observed with PAL-287 (compound 1, 1-
naphthyl-2-aminopropane), a universal BAT releaser evaluated as
our original proof of principle.22 PAL-287 was found to induce
transporter mediated release of DA, 5-HT, and NE with EC50 values
of 12.6 nM, 3.4 nM, and 11.1 nM, respectively. In vivo microdialysis
in rats demonstrated that PAL-287 (1-3 mg/kg, iv) increased extra-
cellular DA and 5-HT with the effects on 5-HT being somewhat
greater. PAL-287 induced substantially less locomotor stimulation
than (+)-amphetamine, a drug that increases only extracellular DA.
PAL-287 did not produce serotonergic ‘neurotoxicity’ and had little
or no reinforcing properties in rhesus monkeys trained to self-
administer cocaine. Yet, PAL-287 produced a dose-dependent
decrease in responding for cocaine when infused at a dose of
1.0 mg/kg/h. Although the addition of 5-HT releasing activity to a
DA releaser decreases behavioral selectivity (less separation
between cocaine vs food responding behavior),17,18 the clinical
implications of these findings are not clear, in view of the fact that
patients treated with various serotonergic medications do not
appear to suffer from non-specific disruptions of behaviors related
to the reward pathways. Our results with PAL-287 support the
hypothesis that a non-amphetamine substrate at DATs and SERTs
will release DA and 5-HT from neurons in vivo, be minimally rein-
forcing, and also suppress ongoing cocaine self-administration.
The inclusion of NE release in a pharmacotherapy for the treat-
ment of substance abuse is less clear. Dual DA/NE releasing agents,
such as amphetamine and phentermine, continue to be safely used
in the clinic.3 NE reuptake inhibitors, either administered alone or
as dual NE/5-HT reuptake inhibitors, have been safely used as anti-
depressants.23 Despite the favorable safety record associated with
using NE reuptake inhibitors and releasing agents in the clinic,
some studies suggest an undesirable cardiotoxic component may
exist with NE elevation. Ephedrine and analogs, which have been
shown to be primarily NE releasing agents,24 have been linked to
adverse cardiovascular events including sudden cardiac death.25
Furthermore, NE has been shown to increase oxidative stress on
the cardiac cells of rats by auto-oxidation,26 and myocardial ische-
mia has been shown to be enhanced by NE elevation mediated by
superoxide anion radicals.27 Because of these issues, one goal of
our releaser medications development program has been to iden-
tify compounds that induce DA and 5-HT release without having
NE releasing properties in order to avoid cardiotoxicity complica-
tions. We hypothesized that such a molecule would maximize
therapeutic effects while minimizing potential toxicities. Herein,
we report the identification of the first class of dual DA/5-HT
releasers with at least 10-fold less releasing activity at the NE
transporter (NET) over the DAT and SERT.
(Scheme 1).31 The racemic
a-alkyl tryptamines 7a, 7b, 7c, 7d, 7e,
and 7f were synthesized by nitro olefin formation followed by lith-
ium ammonium hydride reduction (Scheme 2).32 The optically
active tryptamines (ꢀ)-7d and (+)-7d were synthesized by reacting
the N-protected 3-bromoindole with n-butyllithium, then adding
(+)- or (ꢀ)-propylene oxide to form optically active alcohols 8
(Scheme 3, shown for (+)-7d).33 The secondary alcohol was then
converted to the amine by forming the tosylates 9 followed by
azide displacement, azide reduction, and finally deprotection to
form the optically active tryptamines (ꢀ)-7d and (+)-7d.
All but one compound, 4-methoxytryptamine (4f) were 5-HT-
releasing substrates (Table 1). Twenty-two of the twenty-four 5-
HT releasers were very potent, with EC50 values under 140 nM.
The most potent 5-HT releaser was 7-chlorotryptamine (4p) with
an EC50 value of 8 nM. This is comparable to PAL-287, with an
EC50 value of 4 nM. It was also one of the most selective 5-HT
releasers, as were all the 7-substituted analogs. Tryptamines are
not normally known to possess strong stimulant activity, yet ten
of the compounds had EC50 values for DA release of less than
165 nM. All five fluoro derivatives tested, 4-fluorotryptamine
(4e), 5-fluorotryptamine (4j), 6-fluorotryptamine (4l), 5-fluoro-
a-
methyltryptamine (4c), and 5-fluoro- -ethyltryptamine (7f) were
a
potent DA releasers with EC50 values of 106 nM, 82.3 nM,
104 nM, 31.8 nM and 150 nM, respectively. The other five com-
pounds were tryptamine (4a), 6-methoxytryptamine (4m),
methyltryptamine (7a), 5-chloro- -methyltryptamine (7b) and
(S)- -ethyltryptamine ((+)-7d).
a-
a
a
Surprisingly, most of the compounds were not active as NE
releasers. To date we have screened over 1000 small arylalkylam-
ines for activity at all three biogenic amine transporters and nor-
mally NE release parallels DA release, and is usually slightly
more potent. However in the case of the tryptamines, the NE
releasing activity of the DA releasers was weaker in almost every
case, and often by at least an order of magnitude (>10-fold). This
activity profile implies that these compounds do not interact with
the NET as well as they interact with the DAT. The NE release
potencies for (S)-
(4e), 6-fluorotryptamine (4l), 5-fluoro-
and 5-chloro- -methyltryptamine (7b) were 10-, 11-, 30-, and
a-ethyltryptamine ((+)-7d), 4-fluorotryptamine
a-ethyltryptamine (7f),
a
64-, and 36-fold weaker than their DA release potencies. In addi-
tion, several compounds including 5-chlorotryptamine (4h), 5-
bromotryptamine (4i), and 6-methyltryptamine (4n) were inactive
as NE releasers at 10 lM but maintained reasonable potencies as
DA releasers (EC50 values < 500 nM). In addition, these compounds
were found to have fairly robust 5-HT releasing potencies with
EC50 values from 12.9 nM to 75 nM making them the first dual
selective DA/5-HT releasers discovered in our project.
From a structure activity relationship perspective, N-methyla-
tion did not improve the selectivity or potency compared to trypt-
amine (4a vs 4b). N-ethylation caused the scaffold to lose its DA
releasing activity (4a vs 4c). Alkylation of the 1-position (nitrogen
on the indole ring, 4d) also caused the scaffold to lose its DA releas-
ing activity. N-alkylation at either the side chain nitrogen or the
indole ring does not appear to be a viable approach for optimizing
Twenty-five tryptamines were studied in BAT release and
uptake inhibition assays as well as in a 5-HT 2A (5-HT2A) receptor
assay since tryptamines are known to interact with the 5-HT2A
receptor and 5HT2A agonists are thought to be hallucinogenic.28
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 homoge-
nate.29,30 Compounds were binned as releasing substrates or
uptake inhibitors by assessment in both synaptosomal release
assays and uptake inhibition assays. Once the functional activity
of a compound was determined, dose response curves were com-
pleted for the binned activity. 5-HT2A receptor agonist activity
dual DA/5-HT releasing activity.
a-Alkylation of tryptamine to
form -methyltryptamine (7a), similar to the difference between
a
phenethylamine and amphetamine, caused the DA releasing
potency to improve 2-fold and the NE-releasing potency to