Effects of N-Alkyl-4-Methylamphetamine Optical Isomers on Plasma Membrane Monoamine Transporters and Abuse-Related Behavior

Abstract

4-Methylamphetamine (4-MA) is an emerging drug of abuse that acts as a substrate at plasma membrane transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT), thereby causing nonexocytotic release of monoamine transmitters via reverse transport. Prior studies by us showed that increasing the N-alkyl chain length of N-substituted 4-MA analogues converts 4-MA from a transportable substrate (i.e., releaser) at DAT and NET to a nontransported blocker at these sites. Here, we studied the effects of the individual optical isomers of N-methyl-, N-ethyl-, and N- n-propyl 4-MA on monoamine transporters and abuse-related behavior in rats because action/function might be related to stereochemistry. Uptake inhibition and release assays were conducted in rat brain synaptosomes whereas electrophysiological assessments of drug-transporter interactions were examined using cell-based biosensors. Intracranial-self-stimulation in rats was employed to assess abuse potential in vivo. The experimental evidence demonstrates that S(+) N-methyl 4-MA is a potent and efficacious releaser at DAT, NET, and SERT with the highest abuse potential among the test drugs, whereas R(-) N-methyl 4-MA is a less potent releaser with reduced abuse potential. The S(+)ethyl analogue has decreased efficacy as a releaser at DAT but retains full release activity at NET and SERT with a reduction in abuse-related effects; the R(-)ethyl analogue has a similar profile but is less potent. S(+) N-Propyl 4-MA is a nontransported blocker at DAT and NET but an efficacious releaser at SERT, whereas the R enantiomer is almost inactive. In conclusion, the S enantiomers of the N-alkyl 4-MA analogues are most potent. Lengthening the N-alkyl chain converts compounds from potent nonselective releasers showing abuse-related effects to more selective SERT releasers with no apparent abuse potential.

Keywords: ICSS; Mephedrone; amphetamine; bath salts; dopamine transporter (DAT); drug abuse; norepinephrine transporter (NET); serotonin transporter (SERT); synthetic cathinones.

Figure 1

Two-dimensional visualization of potencies of compounds obtained in synaptosomes and Ca²⁺ assays. The monoamine transporter tested is indicated as N, S, and D for NET, SERT and DAT, respectively. Left panel, compound producing full release in the synaptosome assay and producing signals in the Ca²⁺ assay were included in the analysis. The correlation analysis was performed between the EC₅₀ to produce release in the synaptosome assay and the EC₅₀ to produce signals in the Ca assay. Right panel, compounds that produced partial or no release in the synaptosome assay and that do not produce signals in the Ca²⁺ assay were included in the analysis. The correlation analysis was done between the IC₅₀ to block neurotransmitter’s uptake in the synaptosome assay and the IC₅₀ to block the signal induced by neurotransmitters in the Ca²⁺ assay.

Figure 2

Effects of N-alkyl-4-MA isomers on ICSS frequency-rate curves. Abscissae: frequency of electrical brain stimulation in log Hz. Ordinates: reinforcement rate expressed as percentage of maximum control reinforcement rate (% MCR). Drug doses are indicated in units of mg kg⁻¹. Filled symbols represent frequencies at which ICSS rates after drug administration were statistically different from rates after saline administration as determined by two-way ANOVA followed by Holm-Sidak post hoc test, p < 0.05. All data are mean ± SEM for 6 rats.

Figure 3

Summary of ICSS data for drug effects collapsed across all frequencies. Abscissae: drug dose in mg kg⁻¹. Ordinates: percentage of baseline number of stimulations per component delivered across all brain stimulation frequencies. Points indicate ICSS rate increasing/decreasing effects, relative to Sal (saline). All data show mean ± SEM for 6 rats.

Scheme 1

Reagents and conditions: (a) S(−)propylene oxide or R(+)propylene oxide, CuI, THF, N₂, −35 °C, 2 h; (b) MsCl, Et₃N, CH₂Cl₂, 0 °C to rt, 12 h; (c) (i) NH₂R, DMF, 50 °C, 24 h, (ii) HCl/Et₂O.