Novel Benzofuran Derivatives Induce Monoamine Release and Substitute for the Discriminative Stimulus Effects of 3,4-Methylenedioxymethamphetamine

MDMA (3,4-methylenedioxymethamphetamine) combines entactogenic and stimulant properties and has shown promise as an adjunct to psychotherapy for post-traumatic stress disorder. Its primary pharmacology involves acting as a transportable substrate at the serotonin (SERT), norepinephrine (NET), and dopamine (DAT) transporters to evoke monoamine release. Despite clinical benefits, non-medical use and some therapeutic exposures carry cardiovascular and neurological risks and MDMA metabolism irreversibly inhibits CYP2D6, motivating efforts to develop alternative compounds that preserve therapeutic monoamine-releasing actions while improving safety. This study set out to characterise novel aminoalkyl benzofuran derivatives — enantiomers of 5-(2-methylaminobutyl)benzofuran (5-MABB) and 6-(2-methylaminobutyl)benzofuran (6-MABB) — using in vitro transporter assays in rat brain synaptosomes and in vivo drug discrimination in male rats trained to distinguish MDMA from saline. Johnson and colleagues hypothesised that lengthening the alpha-carbon chain (relative to some related cathinones) would yield compounds biased towards serotonin release with reduced NET and DAT activity, potentially yielding MDMA-like effects with different peripheral or dopamine-mediated liabilities.

The study combined in vitro synaptosome assays and in vivo drug discrimination. For in vitro work, 24 adult male Sprague-Dawley rats provided brain tissue for synaptosome preparations. Caudate tissue was used for DAT assays and whole brain minus caudate and cerebellum for NET and SERT assays. Synaptosomes were prepared by homogenisation in sucrose and centrifugation, then used in uptake inhibition and release protocols. Uptake inhibition assays employed radiolabelled transmitters: 5 nM [3H]dopamine for DAT, 10 nM [3H]norepinephrine for NET, and 5 nM [3H]5-HT for SERT. Selective blockers were included to isolate each transporter (for example, GBR12935 to block DAT activity in NET and SERT assays). Release assays used 9 nM [3H]MPP+ for DAT/NET and 5 nM [3H]5-HT for SERT; buffers contained 1 µM reserpine to block vesicular uptake and competing transporter blockers to optimise selectivity. Release was quantified after preloading synaptosomes and exposing them to test drugs; tyramine was used to define 100% maximal releasable tritium (Emax). Compounds producing <30% of Emax were considered inactive as releasers, recognising that uptake inhibitors can spur partial efflux. Concentration–response data were fitted by nonlinear regression to derive IC50 values for uptake inhibition and EC50 values for release; IC50 is the concentration producing half-maximal inhibition of uptake and EC50 is the concentration producing half-maximal release. Analyses used GraphPad Prism 7. For in vivo testing, eight adult male Sprague-Dawley rats were trained in operant conditioning chambers to discriminate MDMA (1.5 mg/kg, training dose) from saline. Rats were food restricted to ~90% of free-feeding weight and trained on fixed-ratio schedules until stimulus control criteria were met. Test sessions assessed substitution of R and S isomers of 5- and 6-MABB administered intraperitoneally at 0.32, 0.64, 1.28, and 2.56 mg/kg given 30 min before sessions; dose order was counterbalanced. Primary behavioural endpoints were percent responding on the MDMA-associated lever (full substitution defined as ≥80%, partial 20–79%, none <20%) and response rate. ED50s were estimated from dose–response curves and response rates were analysed by repeated-measures ANOVA with significance at p < 0.05.

Uptake inhibition assays showed that MDMA functioned as a fully efficacious inhibitor of DAT, NET, and SERT, with relatively greater potency at SERT. Both enantiomers of 5-MABB had uptake inhibition profiles similar to MDMA, generally more potent at SERT than at DAT or NET. Enantiomers of 6-MABB were also fully efficacious uptake inhibitors across transporters but were comparatively more potent at DAT; the R-6-MABB isomer had a DAT/SERT ratio of 15.4, indicating notable DAT selectivity in the uptake inhibition metric. Release assays distinguished substrate-type releasers from mere uptake inhibitors. S enantiomers of both 5- and 6-MABB acted as efficacious releasers at SERT, NET, and DAT. S-5-MABB exhibited selectivity towards SERT, whereas S-6-MABB behaved in a nonselective manner across transporters; the release efficacies of S-6-MABB at DAT and NET were reported in the range of 72–81% of maximal (Emax), somewhat below the full-efficacy levels of S-5-MABB and MDMA. In contrast, the R enantiomers displayed hybrid profiles: they produced full-efficacy release at SERT (102–104% Emax), partial release at NET (58–66% Emax), and minimal release at DAT (<30% Emax). These in vitro patterns align with the hypothesised effect of increasing alpha-carbon chain length producing compounds with reduced DAT-mediated release. In drug discrimination, all eight rats achieved stimulus control in a mean of 34 ± 15 training sessions. When tested, each enantiomer produced dose-dependent increases in responding on the MDMA-associated lever and reached full substitution at the highest dose tested. S enantiomers were slightly more behaviourally potent than R enantiomers. The diminished NET and DAT releasing activity of the R isomers corresponded with their reduced potency in the discrimination paradigm. The extracted text indicates ED50 values were estimated from dose–response curves but specific numerical ED50s and detailed response-rate data were not present in the provided excerpt.

Johnson and colleagues interpret the data as showing that aminoalkyl benzofuran derivatives can act as monoamine releasers with MDMA-like interoceptive effects in rats. S enantiomers of 5- and 6-MABB resemble MDMA mechanistically, producing efficacious release at SERT, NET, and DAT, whereas R enantiomers are essentially SERT-preferring releasers with reduced or absent DAT activity and only partial NET release. Behaviourally, both enantiomers fully substituted for MDMA in discrimination testing, but S isomers were modestly more potent, and the weaker NET/DAT releasing activity of R isomers coincided with lower potency in vivo. The authors situate these findings alongside prior work on benzofuran NPS and on alpha-carbon chain effects in cathinones, noting conserved patterns whereby increased chain length can diminish NET/DAT releasing efficacy. They suggest that stereoselective pharmacology may underlie partial-releaser properties observed for some racemic compounds, and that enantiomer-specific evaluation is important for drug development. Limitations acknowledged by the investigators include the exclusive use of male rats and the absence of data on locomotor stimulant effects, reinforcing potential next steps such as testing receptor antagonists to parse 5-HT versus dopamine contributions, locomotor and self-administration assays to assess stimulant or reinforcing liabilities, and studies in female subjects. Overall, the authors conclude that the aminoalkyl benzofuran scaffold warrants further exploration as a template for MDMA-like monoamine releasers, while emphasising the need for additional work to determine which transporters and receptors most critically mediate the discriminative and other behavioural effects reported.