Structure-activity relationships of serotonergic 5-MeO-DMT derivatives: insights into psychoactive and thermoregulatory properties

Puigseslloses and colleagues situate this work within two converging developments: renewed research interest in the therapeutic potential of psychedelics and the rapid emergence of novel psychoactive substances (NPS) derived from tryptamines. The introduction reviews structural features of tryptamines (indole core, ethyl side chain, terminal amino group), notes that 5-methoxy substitution (5-MeO-) is common among recreationally used analogues, and highlights gaps in knowledge — specifically a lack of systematic dose/concentration–response and structure–activity relationship (SAR) studies that probe how N,N-terminal substitutions affect interactions with serotonin receptors and the serotonin transporter (SERT). The authors also underscore contradictory or incomplete evidence regarding the relative roles of 5-HT2A and 5-HT1A receptors in hallucinogenic and thermoregulatory responses. This study therefore aims to characterise a series of amino-substituted 5-MeO-tryptamines by combining computational docking, in vitro pharmacology (binding, functional assays, uptake/release, electrophysiology) and in vivo mouse behavioural tests. The stated objectives are to (i) map activity at 5-HT1A and 5-HT2A receptors and SERT, (ii) test psychedelic-like behaviours, thermoregulatory and locomotor effects in mice and explore correlations with in vitro measures, (iii) perform an SAR analysis focused on N,N-substitution, and (iv) determine the contributions of 5-HT1A and/or 5-HT2A to the observed in vivo effects.

The investigators used a multimodal experimental strategy. In vitro work comprised radioligand binding to human 5-HT1A, 5-HT2A and SERT membrane preparations, 5-HT2A-mediated calcium mobilization in CHO-K1 cells, [3H]5-HT uptake inhibition assays, transporter-mediated release assays (batch and superfusion), and whole-cell patch-clamp electrophysiology in HEK293 cells expressing human SERT. Binding experiments used established radioligands and were performed in duplicate across at least three independent experiments; functional and transport assays were replicated across multiple independent experiments as detailed in the Methods. Molecular docking used 5-HT-bound crystal structures (PDB IDs 7E2Y and 7WC4) and MOE2020 with a template-guided protocol; ligand efficiency and GBVI/WSA ΔG scoring were reported. Behavioural testing was performed in male Swiss CD-1 mice (6–8 weeks, 30–35 g). Animals were housed under standard conditions and group allocation was block-randomized; experimenters were blinded to treatments. Main behavioural endpoints were the head twitch response (HTR; video-recorded and scored by two blinded observers), core body temperature (rectal probe measured 60 min after injection), and horizontal locomotor activity (HLA; 30 min tracking). Dose–response studies used intraperitoneal administration of tryptamines at 0.3–30 mg/kg with group sizes of N = 8–10. Antagonist experiments probed receptor specificity using subcutaneous ketanserin (1 mg/kg, 5-HT2A antagonist) or WAY100635 (1 mg/kg, 5-HT1A antagonist) administered prior to tryptamine. Data analysis used non-linear regression to derive IC50, EC50, Emax and pED50 values; Ki values were calculated via the Cheng–Prusoff equation. Statistical testing included one-way ANOVA with appropriate post-hoc tests, mixed-effects models with Šidák correction for release assays, and nonparametric tests where normality was not met. Correlations between in vitro and in vivo metrics were assessed by Pearson correlation. Outliers were removed using ROUT (Q = 0.1%), alpha was set at 0.05, and sample-size planning was performed with GPower to target 80% power.

Binding and functional receptor data indicated that all tested 5-MeO-tryptamines displaced radioligands at 5-HT1A and 5-HT2A in the nanomolar range and acted as full agonists at 5-HT2A in calcium-mobilization assays. Notably, 5-MeO-pyr-T showed the highest affinity and selectivity for 5-HT1A, whereas allyl-amino derivatives (5-MeO-MALT, 5-MeO-DALT) displayed higher 5-HT2A binding. SERT affinities were in the low micromolar range and Ki values inversely correlated with molecular volume (R2 = 0.6112, P < 0.01), indicating steric effects on transporter binding. Uptake inhibition assays showed micromolar potency across compounds, with 5-MeO-pyr-T the most potent inhibitor. Release assays with monensin revealed that 5-MeO-DMT, 5-MeO-MET, 5-MeO-DET and 5-MeO-pyr-T evoked transporter-mediated 5-HT release, although none matched the maximal efflux produced by the positive control p-chloroamphetamine (pCA). Superfusion concentration–response assays for 5-MeO-pyr-T gave an EC50 = 5.70 ± 1.59 µM. Whole-cell patch-clamp recordings in hSERT-expressing HEK293 cells showed inward currents for the four most active releasers, consistent with substrate translocation. For reference, 5-HT itself had an EC50 ≈ 0.05 µM. Reported parameters for the test drugs included: 5-MeO-DET EC50 = 3.5 µM, Emax = 0.26; 5-MeO-DMT EC50 = 2.6 µM, Emax ≈ 0.41; 5-MeO-MET EC50 = 13 µM, Emax ≈ 0.46; 5-MeO-pyr-T EC50 = 0.09 µM, Emax ≈ 0.49. Molecular docking supported a preferential interaction with 5-HT1A over 5-HT2A. Docking models showed conserved engagement of the indole scaffold with Thr121 (5-HT1A) and hydrogen-bonding/electrostatic interactions between the protonated amino group and Asp116, interactions that were reduced or absent in 5-HT2A models. The 5-HT1A pocket was described as larger and better able to accommodate varying N-substituents, whereas 5-HT2A conformations tended to interact in more solvent-exposed regions. In vivo, every 5-MeO-tryptamine elicited HTR in mice with varying potency (ED50) and Emax; dose–response profiles were often inverted-U shaped or reached a plateau. In vitro 5-HT2A calcium-flux potency correlated with HTR potency (P < 0.05, R2 = 0.5644) and Emax (P < 0.05, R2 = 0.4194). Smaller molecular volume also correlated with greater HTR (P < 0.05, R2 = 0.4082). Ketanserin pretreatment blocked HTR, consistent with 5-HT2A dependence, whereas pretreatment with the 5-HT1A antagonist WAY100635 increased HTR counts relative to tryptamine alone, indicating that 5-HT1A activation attenuates HTR. Core body temperature decreased dose-dependently 60 min after injection for all compounds. Potency to induce hypothermia correlated with 5-HT1A binding affinity (P < 0.05, R2 = 0.5067), and maximal hypothermic effect inversely correlated with maximal HTR (P < 0.001, R2 = 0.7620). WAY100635 attenuated the hypothermic responses, supporting 5-HT1A mediation. Horizontal locomotor activity was reduced in a dose-dependent manner (hypolocomotion), and hypolocomotion potency correlated with hypothermic potency (P < 0.01, R2 = 0.5904).

Puigseslloses and colleagues interpret their data as providing an SAR framework for 5-methoxy-substituted tryptamines focusing on N,N-terminal substitutions, with implications for both NPS surveillance and potential therapeutic development. They emphasise that most compounds examined have higher affinity for 5-HT1A than for 5-HT2A, and that docking and binding data converge on key interactions in 5-HT1A (indole–Thr121 and amino–Asp116) that are less stabilising in 5-HT2A. The authors propose that isopropyl-terminal substitutions reduce 5-HT1A affinity, whereas N,N-allyl groups may increase 5-HT2A affinity; bulkier N substituents were associated with greater SERT affinity. Regarding transporter pharmacology, the study found that smaller 5-MeO-tryptamines are more likely to act as SERT substrates and partial releasers, with 5-MeO-pyr-T identified as the most potent partial releaser in this panel. The investigators note that partial transporter-mediated release could be therapeutically interesting, but they acknowledge that 5-HT1A autoreceptor feedback could counteract synaptic 5-HT increases and that further work is needed to determine in vivo therapeutic relevance. On behavioural pharmacology, all tested compounds were full agonists at 5-HT2A in vitro yet varied in their capacity to elicit HTR. The authors highlight two key and related observations: (i) in vitro 5-HT2A potency correlates with HTR potency and efficacy, and (ii) 5-HT1A activation attenuates HTR while mediating hypothermia and hypolocomotion. They therefore emphasise an opposite modulatory role for 5-HT1A and 5-HT2A in psychedelic-like and thermoregulatory responses. The presence of compounds that are 5-HT2A agonists but produce little HTR is discussed as noteworthy; the authors suggest possibilities such as signalling bias (Gq versus beta-arrestin pathways) or intracellular receptor activation tied to lipophilicity and neuroplasticity, and they call for follow-up studies to clarify mechanisms. The authors acknowledge limitations and uncertainties that warrant further research: mechanistic work to explain why some 5-HT2A agonists show low HTR despite nanomolar receptor activity, investigation of downstream signalling bias (for example beta-arrestin versus Gq), and studies assessing neuroplasticity-relevant outcomes. They also note that in vivo therapeutic implications of partial SERT release require additional corroboration. Finally, the discussion frames the findings as supplying actionable pharmacological data for regulatory agencies dealing with NPS and as a foundation for selecting 5-MeO-tryptamines for future therapeutic investigation.