This in vitro and in vivo study investigates novel analogues of the designer drug 5-(2-methylaminopropyl) benzofuran (5-MAPB) as potential MDMA-like monoamine releasers. The research reveals that the S isomers of 5- and 6-(2-methylaminobutyl)benzofuran (5-MABB and 6-MABB) exhibit efficacy as releasing agents for serotonin, norepinephrine, and dopamine transporters. In contrast, the R isomers show reduced potency in inducing behavioural effects, suggesting the aminoalkyl benzofuran scaffold is a potential template for developing compounds with MDMA-like properties.
3,4-Methylenedioxymethamphetamine (MDMA) has shown efficacy as a medication adjunct for treating post-traumatic stress disorder (PTSD). However, MDMA is also used in non-medical contexts that pose risk for cardiovascular and neurological complications. It is well established that MDMA exerts its effects by stimulating transporter-mediated release of the monoamines, 5-hydroxytryptamine (5-HT), norepinephrine, and dopamine. Current research efforts are aimed at developing MDMA-like monoamine releasers with better efficacy and safety profiles. To this end, we investigated neurochemical and behavioral effects of novel analogs of the designer drug, 5-(2-methylaminopropyl)benzofuran (5-MAPB). We used in vitro transporter assays in rat brain synaptosomes to examine transmitter uptake inhibition and releasing properties for enantiomers of 5-(2-methylaminobutyl)benzofuran (5-MABB) and 6-(2-methylaminobutyl)benzofuran (6-MABB) as compared to MDMA. We then tested these same compounds in male Sprague-Dawley rats trained to discriminate MDMA (1.5 mg/kg) from saline. In vitro results revealed that S isomers of 5- and 6-MABB are efficacious releasing agents at transporters for 5-HT (SERT), norepinephrine (NET), and dopamine (DAT). By contrast, R isomers are efficacious releasers at SERT, partial releasers at NET, but lack releasing activity at DAT. In vivo results showed that all compounds produce dose-dependent increases in MDMA-lever responding and full substitution at the highest dose tested. The diminished NET and DAT releasing activities for R isomers of 5- and 6-MABB are associated with reduced potency for inducing behavioral effects. Collectively, these findings indicate that the aminoalkyl benzofuran scaffold may be a viable template for developing compounds with MDMA-like properties.
Papers cited by this study that are also in Blossom
Carhart-Harris, R. L., Wall, M. B., Erritzoe, D. et al. · International Journal of Neuropsychopharmacology (2013)
Jerome, L., Feduccia, A. A., Wang, J. B. et al. · Psychopharmacology (2020)
Liechti, M. E., Baumann, C., Gamma, A. et al. · Neuropsychopharmacology (2000)
Papers in Blossom that reference this study
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.
Despite the clinical utility of MDMA, the drug is associated with certain cardiovascular risks and metabolic side effects. Developing a therapeutic alternative with MDMA-like monoamine releasing activity is of interest. Our in vitro and in vivo findings indicate that the aminoalkyl benzofuran scaffold may be useful for developing compounds with MDMA-like properties.
3,4-Methylenedioxymethamphetamine (MDMA), also known as "Ecstasy", is a phenethylamine derivative with structural similarity to the psychostimulant, methamphetamine, and the psychedelic, mescaline. Recently, MDMAassisted psychotherapy has received considerable attention, and phase III clinical trials with MDMA are ongoing for the treatment of post-traumatic stress disorder (PTSD) (). Individuals with PTSD experience intrusive memories of traumatic events which evoke emotional reactions, flashbacks, hypervigilance, and negative thinking. The increased empathy and pro-social effects of MDMA serve to reduce anxiety and facilitate conversation, which help patients to confront traumatic experiences. In phase II clinical trials, where PTSD symptoms were assessed using the CAPS-IV scale, results demonstrated that symptom severity was significantly decreased for up to 12 months after MDMA assisted-psychotherapy. Though clinical outcomes with MDMA are promising, there remains cause for concern. The non-medical (i.e., recreational) use of MDMA has a well-documented history of cardiovascular and neurological complications. Prolonged non-medical use of MDMA can induce altered 5-hydroxytryptamine (5-HT) receptor densities and 5-HT depletions in the brain (see. Neuroimaging studies reveal decreases in 5-HT-related proteins in human MDMA users that may reflect loss of nerve terminals. While the risk for 5-HT deficits is greatest after high-dose MDMA exposure, even low-dose administration in controlled settings can produce elevations in blood pressure, heart rate, and stress hormones in humans. Additionally, the metabolism of MDMA causes irreversible inhibition of cytochrome 2D6, which leads to non-linear accumulation of the drug (de la. Thus, there is room for improvement in developing new MDMA-like medications. MDMA exerts its pharmacological effects by interacting with monoamine transporter proteins expressed on nerve cells. In particular, MDMA acts as a transportable substrate at transporters for 5-HT (SERT), norepinephrine (NET), and dopamine (DAT), thereby stimulating transporter-mediated release of 5-HT, norepinephrine, and dopamine. Studies in rodents reveal that pro-social effects of MDMA are related to SERT-mediated release of 5-HT, whereas cardiovascular and reinforcing effects likely involve NET and DAT. Clinical investigations show that blockade of SERT with 5-HT selective reuptake inhibitors can significantly blunt subjective effects of MDMA in humans, supporting involvement of SERT-mediated 5-HT release in the therapeutic attributes of the drug. Aminoalkyl benzofuran derivatives, such as 5-(2-aminopropyl)benzofuran (5-APB) and its N-methylated counterpart 5-(2-methylaminopropyl)benzofuran (5-MAPB) (see Figure), are new psychoactive substances (NPS) that act as transporter substrates analogous to MDMA. Members of our team previously showed that 5-APB and 5-MAPB are releasers at SERT, NET, and DAT, with 3-fold greater potency than MDMA. Consistent with their transporter releasing properties, 5-APB and 5-MAPB elevate extracellular 5-HT and dopamine in rodent brain, and engender MDMA-like discriminative stimulus effects in rats. 5-APB and 5-MAPB also exhibit agonist activity at 5-HT 1A , 5-HT 2A , and 5-HT 2C receptors, which may contribute to their pharmacology. With regard to medication development, one potential attribute of benzofurans is that they are metabolized differently than MDMA and may not induce inhibition of cytochrome 2D6. Moreover, benzofuran compounds have more sustained acute effects when compared to MDMA (see, which might be advantageous in some clinical circumstances. In the present study, we examined the pharmacology of novel derivatives of 5-MAPB and its positional isomer 6-(2-methylaminopropyl)benzofuran (6-MAPB). We used in vitro transporter assays in rat brain synaptosomes to evaluate uptake inhibition and releasing activity for enantiomers of 5-(2-methylaminobutyl)benzofuran (5-MABB) and 6-(2methylaminobutyl)benzofuran (6-MABB) (Figure). In separate experiments, in vivo drug discrimination methods were used to characterize interoceptive stimulus effects of the drugs in rats trained to discriminate MDMA from saline. Based on previous findings with cathinonebased compounds, we hypothesized that the increased alpha-carbon chain lengths for 5-and 6-MABB would produce 5-HT releasers with reduced activity at NET and DAT.
For the in vitro transporter assays, 24 adult male Sprague-Dawley rats (Envigo, Frederick, MD, USA) were used. Rats weighing 250-300 g were group housed at the NIDA Intramural Research Program (IRP) animal facility in a temperature (22.2 ± 1.1 o C) and humidity (45 ± 10%) controlled room, on a 12 h light-dark cycle (lights on at 0700) with free access to food and water. For the in vivo drug discrimination experiments, eight adult male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA, USA) were used. Rats were individually housed at the Western Michigan University animal facility in a temperature (20 ± 2 °C) and humidity (50 ± 5%) controlled room, on a 12 h light-dark cycle (lights on at 0700). For the drug discrimination studies, animals were provided free access to water in their home cages and fed restricted diets of commercial rodent chow (Purina®, Richmond, IN, USA) to maintain body weights at approximately 90% of free-feeding weights (380-460 g). All procedures were reviewed and approved by the Institutional Animal Care and Use Committees of the NIDA IRP (Baltimore, MD, USA) and Western Michigan University (Kalamazoo, MI, USA) and were Future studies will examine the effects of these compounds in female rats. In Vitro Transporter Assays. Synaptosome preparation. Rats were killed by CO 2 narcosis, and synaptosomes were prepared from brains using standard procedures. In brief, synaptosomes were prepared from caudate tissue for DAT assays, or from whole brain minus caudate and cerebellum for NET and SERT assays. Brain tissue was homogenized in ice-cold 10% sucrose followed by centrifugation at 1000 g for 10 min. The resulting supernatant containing crude synaptosomes was kept on ice until use in uptake or release procedures. Uptake inhibition and release assays were carried out as previously described.
For uptake inhibition assays, 5 nM [ 3 H]dopamine, 10 nM [ 3 H]norepinephrine, or 5 nM [ 3 H]5-HT was used as the radiolabeled transmitter for DAT, NET, or SERT, respectively. To optimize uptake for a single transporter, unlabeled blockers were included that prevented uptake of [ 3 H]transmitter by competing transporters. Specifically, 50 nM 1-(2-(diphenylmethoxy)ethyl)-4-(3-phenylpropyl)piperazine (GBR12935) was added to the NET and SERT assays to block DAT, whereas 100 nM nomifensine was added to the SERT assays to block NET. Uptake assays were initiated by adding 100 μL of tissue to 900 μL Krebs-phosphate buffer (126 mM NaCl, 2.4 mM KCl, 0.83 mM CaCl 2 , 0.8 mM MgCl 2 , 0.5 mM KH 2 PO 4 , 0.5 mM Na 2 SO 4 , 11.1 mM glucose, 0.05 mM pargyline, 1 mg/mL bovine serum albumin, and 1 mg/mL ascorbic acid, pH 7.4) containing test Release assays. For release assays, 9 nM [ 3 H]MPP+ was used as the radiolabeled substrate for DAT and NET, whereas 5 nM [ 3 H]5-HT was used for SERT. All buffers used in the release assay contained 1 μM reserpine to block vesicular uptake of substrates. The selectivity of release assays was optimized for a single transporter by including unlabeled blockers to prevent the uptake of [ 3 H]MPP+ or [ 3 H]5-HT by competing transporters. Specifically, 100 nM desipramine was added to DAT assays to block NET, while 100 nM citalopram was added to DAT and NET assays to block SERT. Synaptosomes were preloaded with radiolabeled substrate in Krebs-phosphate buffer for 1 h. 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 by liquid scintillation counting. Effects of test drugs on release are expressed as percent maximum release, with maximal release (i.e., 100% Emax) defined as the release produced by tyramine at doses that evoke the efflux of all 'releasable' tritium by synaptosomes (10 µM tyramine for DAT and NET assay conditions, and 100 µM tyramine for SERT assay conditions). Data analysis. Effects of test drugs on release and uptake inhibition were analyzed by nonlinear regression using GraphPad Prism 7 (GraphPad Scientific, San Diego, CA, USA). Dose-response values for the uptake inhibition and release were fit to the equation, Y(x) = Ymin+(Ymax -Ymin) / (1+ 10exp[(logP 50logx)] × n), where x is the concentration of the compound tested, Y(x) is the response measured, Ymax is the maximal response, P 50 is either IC 50 (the concentration that yields half-maximal uptake inhibition response) or EC 50 (the concentration that yields half-maximal release response), and n is the Hill slope parameter. Compounds displaying <30% of Emax releasing efficacy were considered inactive in the release assay, since uptake inhibitors can evoke this degree of partial release.
Apparatus. Training and testing were conducted in eight sound-attenuated operant conditioning chambers (ENV-001, Med Associates Inc., St. Albans, Vermont, USA) equipped with three retractable levers, a food pellet delivery mechanism, a house light, and fan. All experimental events and data collection were programmed with Med-PC software (versions IV and V, Med Associates Inc.). Food reinforcers were 45 mg Dustless Precision Pellets® (Product# F0021, Bio-Serv Inc., Flemington, New Jersey, USA). Preliminary Training. Rats were initially acclimated to operant conditioning chambers for a single 60 min session in which no levers were present, and food pellets were dispensed on a 60-second fixed time interval. Subsequently, rats were trained to lever press the center lever on a fixed ratio (FR) 1 schedule that was gradually incremented to FR 20. Substitution tests were then conducted with R and S isomers of 5-MABB and 6-MABB, administered at 0.32, 0.64, 1.28, and 2.56 mg/kg, I.P., 30 min prior to the session. The order of doses for each compound was counterbalanced among rats. Data Analysis. The number of sessions to criteria for each subject was calculated as the number of training sessions completed to meet the aforementioned criteria for stimulus control. Percent drug lever was calculated by dividing the number of responses emitted on the MDMAassociated lever by the total number of responses on both levers and multiplying by 100. Response rates were expressed as the number of responses emitted per sec throughout a test session. Full substitution was defined as 80% or greater responses on the MDMA-associated lever; partial substitution was defined as 20-79% responses on the MDMA lever; no substitution was defined as less than 20% responses on the MDMA lever. Dose response curves were plotted for each compound and nonlinear regressions were performed to determine ED 50 s. For each test compound assessed, response rate was analyzed with a repeated measures analysis of variance. Statistical significance was determined at alpha of p <0.05. All statistical and graphical analyses were performed using the GraphPad Prism version 7 software (GraphPad Software, Inc., La Jolla, CA, USA).
We first characterized the effects of MDMA and the enantiomers of 5-and 6-MABB in uptake inhibition assays carried out in rat brain synaptosomes. Figuredepicts the concentration-response curves for the test compounds to inhibit uptake of [ 3 H]neurotransmitters at DAT, NET and SERT, whereas Tablesummarizes potency estimates for each compound at each transporter (i.e., IC 50 values). In previous publications (e.g.,, we have highlighted the relationship between potency at DAT versus SERT using the DAT/SERT ratio. For uptake inhibition assays, this metric is defined as (DAT IC 50 ) -1 / (SERT IC 50 ) -1 . As shown previously, MDMA acted as a fully efficacious uptake inhibitor at DAT, NET, and SERT, with somewhat greater potency (i.e., left-shifted concentration-response curve) for SERT. R-and S-5-MABB had uptake inhibition profiles analogous to MDMA, with more potent effects at SERT relative to DAT and NET. The 5-MABB enantiomers displayed potencies that were generally similar to MDMA at all transporters. The enantiomers of 6-MABB also acted as fully efficacious uptake inhibitors at DAT, NET, and SERT, but these compounds were more potent at inhibiting uptake at DAT. In particular, R-6-MABB had a DAT/SERT ratio of 15.4, indicative of substantial selectivity for DAT over SERT. Although uptake inhibition assays are useful for identifying compounds that interact with monoamine transporters, such assays cannot differentiate between non-transportable uptake inhibitors versus transportable substrates which evoke neurotransmitter releaseFreissmuth 2015;. Thus, we next examined the effects of MDMA and benzofuran compounds in release assays designed to detect drug-induced efflux of [ 3 H]substrates from preloaded synaptosomes. Figuredepicts the effects of MDMA and the enantiomers of 5and 6-MABB in release assays optimized for DAT, NET, and SERT, whereas Tablesummarizes potency estimates for each compound at each transporter (i.e., EC 50 values). The efficacy of the compounds is also noted in Table, expressed as a percentage of maximal release, Emax. For the release assays, the DAT/SERT ratio is defined as (DAT EC 50 ) -1 / (SERT EC 50 ) -1 . The S enantiomers of 5-and 6-MABB acted as efficacious releasing agents at DAT, NET, and SERT. S-5-MABB displayed selectivity towards SERT, while S-6-MABB was nonselective across the three transporters. It is noteworthy that the release efficacies for S-6-MABB at DAT and NET ranged from 72-81% of maximal, which is somewhat less than the full efficacy effects of S-5-MABB and MDMA at these transporters. Importantly, the R enantiomers of 5-and 6-MABB displayed hybrid transporter activity, characterized by full efficacy releasing effects at SERT (102-104% Emax), partial releasing activities at NET (58-66% Emax), and minimal release at DAT (<30% Emax). We previously showed that increasing the alpha-carbon chain length of cathinone compounds, from methyl to ethyl, produces hybrid transporter compounds that exhibit fully efficacious releasing effects at SERT coupled with lack of releasing activity at DAT.
In drug discrimination experiments using MDMA as the training drug, all eight rats met the initial criteria for stimulus control with an average of 34 (± 15) sessions.
In recent years, MDMA has gained attention as an effective adjunct for treating PTSD, and therapeutic effects of the drug are thought to involve transporter-mediated release of monoamine neurotransmitters, especially 5-HT. Despite the promising clinical outcomes with MDMA, there are legitimate health concerns with the medical use of the drug, including cardiovascular stimulation and metabolic inhibition of cytochrome P450 enzymes. Accordingly, current medication development efforts are aimed at finding new chemical scaffolds with MDMA-like monoamine releasing activity (see. As an example, a number of research groups have suggested that ring-substituted cathinone derivatives might be effective MDMA-like treatments for PTSD and other indications, such as depression and substance use disorders. Here, we examined the neurochemical and behavioral effects of benzofuran derivatives structurally related to the monoamine releasers, 5-and 6-MAPB. We found that S isomers of 5-and 6-MABB are efficacious releasing agents at SERT, NET, and DAT, similar to the mechanism of MDMA. By contrast, the R isomers of 5-and 6-MABB are efficacious releasers only at SERT, with reduced substrate activity at NET and DAT. Results from drug discrimination experiments in rats demonstrated that enantiomers of 5-and 6-MABB fully substitute for the MDMA stimulus cue, and the diminished NET and DAT releasing activities for R isomers coincide with reduced potency in vivo. Taken together, the findings point to the further exploration of aminoalkyl benzofuran analogs as MDMA-like monoamine releasers with therapeutic potential. Our uptake inhibition assays in rat brain synaptosomes reveal for the first time that both enantiomers of 5-and 6-MABB are fully efficacious inhibitors at DAT, NET, and SERT. The R and S enantiomers of 5-MABB are more potent at inhibiting SERT-mediated uptake relative to DAT-mediated uptake, with DAT/SERT ratios less than 1. By contrast, the R and S isomers of 6-MABB are more potent as uptake inhibitors at DAT relative to SERT, and this selectivity profile is especially true for S-6-MABB, which exhibits a DAT/SERT ratio of 15.4. Our uptake inhibition results with MABB compounds are consistent with prior findings examining the effects of various benzofuran NPS in cells transfected with human DAT, NET, or. In particular,demonstrated that racemic 6-APB has higher DAT/SERT selectivity when compared to racemic 5-APB, andshowed similar results for 6-MAPB versus 5-MAPB. Thus, the more potent effects on DAT-mediated uptake inhibition seem to be conserved for 6-APB, 6-MAPB, and 6-MABB. While uptake inhibition assays are useful for identifying transporter ligands, such assays are unable to discriminate non-transportable uptake inhibitors from transportable substrate-type releasers. Here, we used release assays in rat brain synaptosomes to characterize transporter-mediated releasing activities for the enantiomers of 5-and 6-MABB. We found that the S isomers of 5-and 6-MABB are fully efficacious releasers at DAT, NET, and SERT, whereas the R isomers are fully efficacious releasers only at SERT. The R isomers of 5-and 6-MABB act as partial releasers at NET and are devoid of releasing activity at DAT (i.e., <30% efficacy). The release findings with R isomers of MABB are consistent with data showing that increased alpha carbon chain length of ring-substituted cathinones (e.g., butylone) can decrease releasing efficacy at NET, and eliminate release activity at DAT (see. Moreover, the findings suggest that decreased releasing activity of certain racemic compounds (i.e., partial releasers) may be driven chiefly by their R isomers, as suggested by the findings of others. For instance,recently demonstrated that S enantiomers of ring-substituted cathinones are fully efficacious releasers at human monoamine transporters, whereas their R counterparts are not. With regard to the development of novel MDMA-like medications, more studies are needed to examine the enantiomer-specific releasing effects of phenalkylamines, cathinones, and benzofuran ligands. Our study is the first to examine the discriminative stimulus effects of 5-and 6-MABB. We found that both enantiomers of 5-and 6-MABB engender MDMA-like discriminative stimulus effects, and S enantiomers are slightly more potent than R enantiomers in this regard. Although no published drug discrimination studies have examined effects of racemic 5-or 6-MABB,investigated the effects of racemic 5-APB in groups of rats trained to discriminate MDMA, methamphetamine, cocaine, or the psychedelic compound 2,5-dimethoxy-4-methylphenyl isopropylamine (DOM), from saline. They found that 5-APB fully substituted for MDMA and partially substituted for methamphetamine, cocaine, and DOM. Based on their results,concluded that 5-APB discrimination is mediated by both dopamine and serotonin neuronal systems. To further assess the duration of action and active dose range of benzofuran compounds,also measured the effects of these substances on locomotor activity in mice. Their findings indicated that 5-APB has a slightly different locomotor activity profile from MDMA, with 5-APB inducing more rapid and sustained increases in motor activity when compared to MDMA. In the present study, we observed greater behavioral potency for S enantiomers of 5-and 6-MABB when compared to their R enantiomers. It is tempting to speculate that reduced NET and DAT releasing activities for R enantiomers might contribute to their reduced potency in drug discrimination experiments. However, the R enantiomers of both 5-and 6-MABB are also somewhat less potent at inducing SERT-mediated 5-HT release when compared to the S enantiomers. Our drug discrimination findings showing more potent effects of S enantiomers of MABB compounds align with the known behavioral profile of MDMA enantiomers, where S-MDMA is more potent than R-MDMA. In a study where rats were trained to discriminate MDMA enantiomers from saline, the serotonergic psychedelics, DOM, lysergic acid diethylamide (LSD), and mescaline, partially substituted for S-MDMA, whereas the psychostimulants, amphetamine, and cocaine failed to substitute for either enantiomer. Given the pharmacological specificity of drug discrimination, the prior findings with MDMA indicate serotonergic neural systems are more salient to the discriminative stimulus effects of S-MDMA than to those of R-MDMA, whereas dopaminergic actions seem less relevant to the discrimination of either isomer. More studies are warranted to determine the precise neurobiological underpinning of the discriminative stimulus effects of MABB enantiomers and other MDMA-like entactogen compounds. In summary, the results obtained from the present study affirm that enantiomers of 5-and 6-MABB are monoamine releasing agents that produce MDMA-appropriate lever responding and fully substitute for MDMA at sufficient doses. Further research is needed to discern which transporters and receptors are most important for the discriminative stimulus effects of these benzofuran derivatives and their enantiomers, but our current findings reveal that SERTmediated release of 5-HT is a prominent feature shared by all compounds tested. The assessment of selective 5-HT or dopamine receptor antagonists would help differentiate the relative importance of 5-HT versus dopamine receptors in modulating the MDMA-like stimulus effects of benzofuran derivatives. Based on the structural and pharmacological similarities between MDMA and various benzofurans, it would also be of interest to assess the stimulant effects of the compounds. Objective assessments of locomotor activity and drug self-administration would be valuable to determine if the R and S enantiomers of 5-and 6-MABB differ with respect to central nervous system stimulant effects. Finally, it must be mentioned that we only studied the effects of drugs in male rats, and future investigations are warranted to investigate the pharmacology of these compounds on female rats. The present study contributes to the growing literature on the in vivo pharmacological actions of benzofuran molecules. Further evaluation of these substances is warranted and may aid in development of medications that retain MDMAlike properties with fewer adverse effects.
Compounds were provided by Tactogen and the NIDA Drug Supply Program.
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Vargas, M. V., Hatzipantelis, C. J., Dunlap, L. E. et al. · ACS Chemical Neuroscience (2026)