Receptor interaction profiles of novel N-2-methoxybenzyl (NBOMe) derivatives of 2,5-dimethoxy-substituted phenethylamines (2C drugs)

Rickli and colleagues situate their work within the pharmacology of classic serotonergic hallucinogens, which include tryptamines (for example psilocin, DMT), ergolines (LSD), and phenethylamines (mescaline). Earlier research indicates that hallucinogenic effects are primarily mediated via the 5-HT2A receptor but can be modulated by other serotonin receptors, monoamine transporters and trace amine-associated receptors. The authors note that many synthetic tryptamine derivatives have emerged as recreational novel psychoactive substances and that small structural changes can substantially alter pharmacology and toxicity, so in vitro receptor interaction profiling is important for anticipating psychoactive effects and clinical risks. The present study set out to characterise the in vitro receptor and transporter interaction profiles of a series of recreationally used tryptamines (DiPT, 4-OH-DiPT, 4-OH-MET, 5-MeO-AMT, 5-MeO-MiPT) alongside classic hallucinogens (psilocin, DMT, LSD, and mescaline). The investigators measured binding affinities at human monoamine receptors, functional activation at 5-HT2A and 5-HT2B receptors, inhibition of human monoamine uptake transporters (NET, DAT, SERT), and transporter-mediated monoamine release, with MDMA included as a comparator in transporter assays. LSD and mescaline were included to allow direct comparison within the same assays and cell batches.

The compounds tested (psilocin, LSD, DMT, mescaline, MDMA, DiPT, 4-OH-DiPT, 4-OH-MET, 5-MeO-AMT, 5-MeO-MiPT) were obtained from commercial suppliers, used as racemates and had purity ≥98%. Radioligand binding assays were performed using membrane preparations of HEK293 cells overexpressing human transporters or receptors (human genes except TAAR1 where rodent genes were used). Binding was measured by displacement of radiolabeled selective ligands at concentrations equal to Kd. A range of radioligands and competitor ligands were used for NET, SERT, DAT, 5-HT1A, 5-HT2A, 5-HT2C, adrenergic α1/α2, D1–D3, H1 and TAAR1. IC50 values were obtained from three to five independent 10-point concentration–response curves using nonlinear regression and converted to Ki (affinity) values via the Cheng–Prusoff equation. Functional activity at the 5-HT2A receptor was assessed in mouse embryonic fibroblasts (NIH-3T3) expressing human 5-HT2A using a FLIPR calcium mobilisation assay. Cells were loaded with a fluorescent calcium dye and challenged with test substances; EC50 values were derived from concentration–response curves and efficacy was expressed relative to 5-HT set at 100%. A parallel FLIPR-based calcium assay in HEK293 cells expressing human 5-HT2B measured 5-HT2B activation using Fluo-4 dye and similar analysis. Inhibition of human NET, DAT and SERT was measured in HEK293 cells stably expressing the respective transporters. Cells were preincubated with test compounds, then exposed to radiolabeled monoamines ([3H]NE, [3H]DA, [3H]5-HT) and uptake quantified after separation via centrifugation through silicone oil. Nonspecific uptake was determined using specific inhibitors (nisoxetine for NET, mazindol for DAT, fluoxetine or citalopram for SERT). IC50 values were calculated from variable-slope sigmoidal dose–response fits. Transporter-mediated monoamine release was probed by preloading transporter-expressing HEK293 cells with radiolabeled monoamines and then exposing them to a single high concentration (100 μM) of test compounds. Release was stopped after 15 min for [3H]5-HT and [3H]DA and after 45 min for [3H]NE; nonspecific pseudo-efflux controls used transporter inhibitors. ANOVA followed by Dunnett's test compared compound-induced release to controls; substances producing significantly greater outflow were classified as monoamine releasers. Cytotoxicity at the high test concentration (100 μM) was assessed after 4 h using the ToxiLight adenylate kinase release assay to ensure cell integrity during pharmacological assays. Where relevant, previously published data for LSD and mescaline obtained in the same laboratory and cell batches were included for comparison.

All tested tryptamines bound to and activated 5-HT2A receptors at mostly submicromolar concentrations, but binding affinity to 5-HT2A was lower for all tryptamines (including psilocin and DMT) than for LSD; mescaline was the least potent. For the entire panel there was no significant correlation between 5-HT2A Ki and 5-HT2A EC50 (Spearman R s = 0.4, p>0.1, n=10), and the same lack of correlation held when analysing only tryptamines (R s = 0.4, p>0.1, n=7). However, among the tryptamines, 5-HT2A binding affinity correlated with estimated psychoactive human doses (R s = 0.9, p<0.05, n=7), whereas 5-HT2A activation potency did not. Activation efficacies varied markedly: LSD and psilocin were partial agonists with 28% and 16% activation efficacy respectively, while several tryptamines exhibited high efficacies (reported up to 480% for DiPT and 5-MeO-MiPT). 5-MeO-AMT produced the highest 5-HT2A activation potency among the tryptamines tested. Selectivity of 5-HT2A over 5-HT2C binding was low for all compounds (binding ratios <10). Only 5-MeO-AMT and 4-OH-DiPT activated 5-HT2B at submicromolar concentrations. Binding to non-serotonergic monoamine receptors and transporters was generally weak for the tryptamines. DiPT and 4-OH-MET showed submicromolar affinity to SERT. LSD uniquely bound adrenergic α1 receptors with submicromolar affinity and exhibited submicromolar affinity at α2 and dopaminergic D1–D3 receptors; LSD also bound rat TAAR1. DMT showed moderate α1-adrenergic binding (Ki = 1.3 mM as reported in the extract). Several compounds (psilocin, DMT, 4-OH-MET, 5-MeO-AMT) had low-micromolar affinity to rat TAAR1. In transporter inhibition assays, most substances except mescaline and LSD inhibited uptake at least at one transporter. Psilocin, DMT, DiPT and 4-OH-DiPT inhibited SERT with IC50 values in the low micromolar range, similar to MDMA. These compounds, along with 4-OH-MET, also inhibited NET but with lower potency than MDMA. DAT inhibition was absent or very weak for the tryptamines, contrasting with MDMA which showed stronger DAT inhibition. In the transporter-mediated release experiments, DMT induced 5-HT release and 5-MeO-AMT induced both 5-HT and DA release. None of the other tested substances acted as substrate releasers under the conditions used. Cytotoxicity testing revealed no cell damage after 4 h incubation at 100 μM in the adenylate-kinase release assay. The authors also report structure–activity observations: α-methylation and 5-methoxylation tended to increase 5-HT2A potency (e.g. 5-MeO-AMT), 4-hydroxylation increased 5-HT2A binding (psilocin vs DMT; 4-OH-DiPT vs DiPT), and N-substitutions (symmetrical vs asymmetrical N-alkylation) altered 5-HT2 receptor affinity and activity. The extract notes that previously published data for LSD and mescaline, produced in the same laboratory, were included for direct comparison.

The investigators interpret their findings to indicate that all tested tryptamines are partial or full 5-HT2A receptor agonists and that many also interact with the serotonin transporter, which may contribute to MDMA-like properties in addition to classical hallucinogenic effects. They emphasise clear differences between tryptamines and LSD: LSD showed higher 5-HT2A binding affinity, greater potency at 5-HT1A receptors, and notable submicromolar interactions with adrenergic and dopaminergic receptors, whereas tryptamines generally lacked sizeable adrenergic or dopaminergic receptor interactions. Functionally, the tryptamines tended to be full 5-HT2A agonists with high activation efficacies, whereas LSD, psilocin and DMT were partial agonists with lower efficacies in the assay used. The authors relate 5-HT2A binding affinity to reported human psychoactive doses for the tryptamines (significant correlation), suggesting that lower 5-HT2A affinity corresponds with higher required doses compared with LSD. They caution that 5-HT2A activation potency did not correlate with human doses and that binding affinity and functional activation in vitro do not always align, noting that different in vitro assays probe different second-messenger pathways and may not fully capture mechanisms mediating subjective effects. Limitations acknowledged by the study team include the usual constraints of in vitro pharmacology: factors such as absorption, protein binding, brain penetration and metabolism are not captured and will influence in vivo potency and toxicity. The authors note, for example, that LSD may have superior brain penetration compared with tryptamines. They also indicate that the relevance of TAAR1 binding for subjective and reinforcing properties requires further study. In their concluding interpretation the authors state that the in vitro profiles predict that the tested tryptamines will exert both hallucinogenic and MDMA-like effects in humans, and that differences from LSD (notably the lack of strong adrenergic and dopaminergic interactions) may underlie differing clinical and subjective profiles. The investigators recommend further work to address pharmacokinetic and in vivo factors that determine clinical effects and toxicity.