
==== Front
Mol Psychiatry
Mol Psychiatry
Molecular Psychiatry
1359-4184
1476-5578
Nature Publishing Group UK London

38486047
2506
10.1038/s41380-024-02506-8
Article
Structure-activity relationships of serotonergic 5-MeO-DMT derivatives: insights into psychoactive and thermoregulatory properties
http://orcid.org/0000-0003-2542-972X
Puigseslloses Pol 12
http://orcid.org/0000-0002-9791-249X
Nadal-Gratacós Núria 12
Ketsela Gabriel 2
http://orcid.org/0000-0002-5487-6505
Weiss Nicola 1
Berzosa Xavier 2
Estrada-Tejedor Roger 2
Islam Mohammad Nazmul 3
Holy Marion 3
http://orcid.org/0000-0002-0518-5791
Niello Marco 34
Pubill David 1
Camarasa Jordi 1
http://orcid.org/0000-0002-5078-366X
Escubedo Elena 1
http://orcid.org/0000-0002-1339-7444
Sitte Harald H. 356
http://orcid.org/0000-0001-8904-7398
López-Arnau Raúl raullopezarnau@ub.edu

1
1 https://ror.org/021018s57 grid.5841.8 0000 0004 1937 0247 Department of Pharmacology, Toxicology and Therapeutic Chemistry, Pharmacology Section and Institute of Biomedicine (IBUB), Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain
2 https://ror.org/04p9k2z50 grid.6162.3 0000 0001 2174 6723 Pharmaceutical Chemistry Group (GQF), IQS School of Engineering, Universitat Ramon Llull, 08017 Barcelona, Spain
3 https://ror.org/05n3x4p02 grid.22937.3d 0000 0000 9259 8492 Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Wäehringerstrasse 13A, 1090 Vienna, Austria
4 https://ror.org/042t93s57 grid.25786.3e 0000 0004 1764 2907 Genetics of Cognition Lab, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy
5 https://ror.org/00xddhq60 grid.116345.4 0000 0004 0644 1915 Hourani Center for Applied Scientific Research, Al-Ahliyya Amman University, Amman, Jordan
6 https://ror.org/05n3x4p02 grid.22937.3d 0000 0000 9259 8492 Center for Addiction Research and Science, Medical University Vienna, Waehringer Strasse 13A, 1090 Vienna, Austria
14 3 2024
14 3 2024
2024
29 8 23462358
4 10 2023
23 2 2024
28 2 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Recent studies have sparked renewed interest in the therapeutic potential of psychedelics for treating depression and other mental health conditions. Simultaneously, the novel psychoactive substances (NPS) phenomenon, with a huge number of NPS emerging constantly, has changed remarkably the illicit drug market, being their scientific evaluation an urgent need. Thus, this study aims to elucidate the impact of amino-terminal modifications to the 5-MeO-DMT molecule on its interactions with serotonin receptors and transporters, as well as its psychoactive and thermoregulatory properties. Our findings demonstrated, using radioligand binding methodologies, that all examined 5-MeO-tryptamines exhibited selectivity for 5-HT1AR over 5-HT2AR. In fact, computational docking analyses predicted a better interaction in the 5-HT1AR binding pocket compared to 5-HT2AR. Our investigation also proved the interaction of these compounds with SERT, revealing that the molecular size of the amino group significantly influenced their affinity. Subsequent experiments involving serotonin uptake, electrophysiology, and superfusion release assays confirmed 5-MeO-pyr-T as the most potent partial 5-HT releaser tested. All tested tryptamines elicited, to some degree, the head twitch response (HTR) in mice, indicative of a potential hallucinogenic effect and mainly mediated by 5-HT2AR activation. However, 5-HT1AR was also shown to be implicated in the hallucinogenic effect, and its activation attenuated the HTR. In fact, tryptamines that produced a higher hypothermic response, mediated by 5-HT1AR, tended to exhibit a lower hallucinogenic effect, highlighting the opposite role of both 5-HT receptors. Moreover, although some 5-MeO-tryptamines elicited very low HTR, they still act as potent 5-HT2AR agonists. In summary, this research offers a comprehensive understanding of the psychopharmacological profile of various amino-substituted 5-MeO-tryptamines, keeping structural aspects in focus and accumulating valuable data in the frame of NPS. Moreover, the unique characteristics of some 5-MeO-tryptamines render them intriguing molecules as mixed-action drugs and provide insight within the search of non-hallucinogenic but 5-HT2AR ligands as therapeutical agents.

Subject terms

Neuroscience
Psychology
Molecular biology
issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

The use of tryptamines has seen an increased growth in recent years, parallel to the growth in “modern shamanism,” a new trend in drug experimentation consisting of the exploration of the inner-self [1]. In this sense, the so-called New Psychoactive Substances (NPS) phenomenon has gained great popularity globally [2]. These substances tend to be analogs of existing controlled drugs or newly synthesized chemicals that mimic the psychoactive effects of substances that are already under control [3–5]. Since their structure slightly differs from their banned analogs, they can be sold without legal implications in certain countries labeled as “research chemicals” [6, 7]. Still, as the illicit market continuously evolves and new tryptamine derivatives appear, there is a lack of comprehensive regulation for these compounds and their legal status often remains unclear.

As for the recreational use of tryptamines, low doses are required to produce psychotropic phenomena, altering sensory perception and mood, and they have been associated with various cases of intoxications and even fatalities [8]. Tryptamines derivatives consist of an indole scaffold, an amino group and an ethyl side chain, resembling the structure of serotonin [9]. Researchers point out that substitution in position 5 of the indole ring is considered to increase potency compared to other substituted and non-substituted tryptamines [10–12], although the clinical effects reported are similar between them [8].

Further, the Psychonaut Project in 2002 revealed that 5-methoxy-substituted tryptamines, such as 5-MeO-DMT or 5-MeO-DALT, were being widely experimented by users [13]. Moreover, other 5-methoxy-substituted tryptamines, such as 5-MeO-DIPT (also called “foxy methoxy”) and 5-MeO-MIPT (“moxy”) have been described in several case studies, reporting fatalities and intoxications [14–16]. Other 5-methoxy analogs such as 5-MeO-MET [17], 5-MeO-NIPT, 5-MeO-EIPT, 5-MeO-MALT [18], and 5-MeO-pyr-T [19] have also appeared on the drug market over the past 10 years.

On the other hand, a renewed interest on the pharmacology of hallucinogens has re-emerged, as new evidence points towards their potential use as therapeutics for treating several mental disorders, such as depression, anxiety, substance abuse and obsessive-compulsive behaviors [20–24]. Despite the growing interest in the therapeutic properties of tryptamines, there are few dose/concentration-response studies in vitro and in vivo that enable a detailed analysis of the mechanisms of action of novel compounds [25], especially 5-methoxy-substituted tryptamines [11].

Serotonin 5-HT2A and 5-HT1A receptors (5-HT2AR and 5-HT1AR) were shown to be the main target of psychedelics, including tryptamine derivatives, being such interaction the main responsible for their hallucinogenic effects [26, 27]. Moreover, other studies have also demonstrated reasonable 5-HT uptake inhibition, or even 5-HT releasing properties for some 5-MeO-substituted and non-substituted tryptamines [28–30]. In fact, mixed action molecules that combine 5-HT uptake inhibition with actions at 5-HT receptors might overcome the limitations of selective serotonin reuptake inhibitors (SSRIs) when treating mood disorders [31–34].

Most of the structure-activity relationship (SAR) studies on tryptamines have focused on substitutions on the indole ring [10, 35–37]. There has, however, been little systematic investigation on the substitution patterns on the terminal amino group. Moreover, previous research has demonstrated that some 5-MeO-tryptamines are able to interact with 5-HT2AR, but with higher affinity and/or potency for 5-HT1AR [29, 35, 38–40], while other authors reported opposite results [28]. Furthermore, literature about the role of 5-HT1AR in the hallucinogenic response induced by psychedelics (i.e., LSD, DOI, tryptamine derivatives) is scarce and oftentimes contradictory [25, 27, 39, 41], as well as their thermoregulatory effects [25, 41–44].

Therefore, the aim of this study was to (i) characterize the pharmacological profile of different amino-substituted 5-MeO-tryptamines at different 5-HT receptors and the serotonin transporter (SERT), using a combination of computational and in vitro assays; (ii) evaluate in vivo their psychedelic effects, thermoregulatory response and locomotor behavior in mice as well as any correlation between both in vivo and in vitro results; (iii) perform a SAR study; (iv) and finally, evaluate the contribution of 5-HT1AR and/or 5-HT2AR to psychedelic effects and thermoregulatory responses in mice.

Materials and methods

Subjects

Male Swiss CD-1 mice (Janvier, Le Genest, France) weighing 30–35 g (6–8 weeks old) were used for the behavioral experiments. The animals were housed in polycarbonate cages with wood-derived bedding and temperature-controlled conditions (22 ± 1 °C) under a 12 h light/dark cycle and had free access to food and drinking water. All the studies were carried out following the ARRIVE guidelines [45]. Experimental groups were randomized (block randomization) and researchers were blinded to the group allocation, the outcome assessment and the data analysis. Animal care and experimental protocols were approved by the Animal Ethics Committee of the University of Barcelona under the supervision of the Autonomous Government of Catalonia, in accordance with the guidelines of the European Community Council (2010/63/EU). The sample size was determined using GPower software. The minimal significance was set at 0.05 and statistical power at 0.8.

Drugs and materials

5-MeO-tryptamines were synthesized as hydrochloride salts (Supplementary Fig. 1), identified and characterized as described in the Supplementary Material. WAY100635 maleate and ketanserin tartrate were obtained from Tocris (Bio-Techne R&D Systems, S.L.U., Madrid, Spain). Solutions for injection were freshly prepared daily in isotonic saline solution (0.9% NaCl, pH 7.4). The radioligands [3H]5-HT (33.2 Ci/mmol), [3H]ketanserin (22.8 Ci/mmol), [3H]8-OH-DPAT (200 Ci/mmol), [3H]imipramine (40 Ci/mmol) and the membrane preparations expressing human 5-HT1A and 5-HT2A receptors (h5-HT1AR and h5-HT2AR) were purchased from Perkin Elmer, Inc. (Waltham, MA, USA). p-Chloroamphetamine (pCA) and paroxetine were purchased from Sigma Aldrich. All other reagents were of analytical grade and purchased from several commercial sources. See the Supplementary Material for buffers and solutions composition.

Uptake inhibition, release, and electrophysiology assays with HEK293 cells

Uptake inhibition assays

The [3H]5-HT uptake inhibition assay was performed as described [46]. See the Supplementary Material for cell culture procedures. The medium was removed from each well and replaced with 200 μL/well of Krebs-HEPES-Buffer (KHB). The cells were pre-incubated for 5 min with different concentrations of drug in KHB at 50 μL/well. Subsequently, the solution was removed and cells were incubated with the drug dilutions and [3H]5-HT in KHB for 1 min. The incubation solution was aspirated and the cells were washed with ice-cold KHB and lysed with 1% sodium dodecyl sulfate (SDS). The lysate was collected into vials and scintillation liquid was added. The radioactivity was quantified using a beta-scintillation counter (Perkin Elmer, Waltham, MA, USA). Non-specific uptake was determined in the presence of 30 μM paroxetine. Data were expressed as percentage of control uptake (absence of tryptamine). Assays were carried out per triplicate for at least three independent experiments.

Batch release assays

Batch release is a functional radiometric assay to assess transporter-mediated efflux. Cells were preloaded with 0.1 µM [3H]5-HT in KHB for 20 min at 37 °C. Subsequently, cells were washed three times with KHB and equilibrated for 10 min in KHB or KHB+monensin (10 µM). Next, cells were incubated with a concentration around and a concentration above their determined IC50 values in KHB or in KHB+monensin and the resulting supernatant was transferred to a new well every 2 min. 10 µM pCA and 0.05 µM paroxetine were used as positive and negative controls, respectively. Liquid scintillation cocktail was added both to the wells containing cells (200 µL) and to the transferred supernatant (100 µL). Total radioactivity determined from/in the remaining lysate of the cells plus the transferred supernatant was set as 100%, and the radioactivity determined in each fraction was expressed as a percentage thereof. Assays were performed per duplicate for five independent experiments.

Superfusion release assays

Concentration-response release assays were performed using a superfusion assay [47]. Cells were preloaded with 0.1 µM [3H]5-HT in KHB for 20 min at 37 °C. Afterwards, cells were manually and gently washed once with KHB, and superfused with KHB at a rate of 0.5 mL/min for 10 min at room temperature (RT), to establish a stable basal release. Three 2 min-fractions of basal release were collected in 10 mL counting vials before exposing cells to various concentrations of 5-MeO-pyr-T (five fractions). Subsequently, the cells were superfused with 1% SDS for three final fractions to determine total radioactivity present at cells at the end of the experiment. 2 mL of scintillation cocktail was added to each vial, radioactivity was measured and expressed as percentage of released radioactivity in relation to the total radioactivity present at the beginning of that fraction. Experiments were performed per triplicate for five independent experiments.

Transporter-mediated currents

Whole-cell patch clamping was used to measure the transporter-mediated currents and performed as previously described [31]. In brief, cells were clamped at −60 mV and substrate-induced transporter currents were recorded at RT using an Axopatch 700B amplifier and pClamp 11.2 software (MDS Analytical Technologies, Sunnyvale, CA, USA). Cells were continuously superfused by a DAD-12 superfusion system and an 8-tube perfusion manifold (ALA Scientific Instruments in Farmingdale, NY, USA), which allowed to apply a physiological external solution. The pipette solution mimics the internal ionic composition of a cell. The recorded currents were filtered at 1 kHz and digitized at 10 kHz using a Digidata 1550 (MDS Analytical Technologies) before being analyzed using Clampfit 10.2 software from Molecular Devices located in San Jose, CA, USA. The elicited currents by the test drugs were normalized to the current amplitude elicited by a saturating concentration of 5-HT (10 µM) applied to the same cell. Passive holding currents were subtracted, and the traces were filtered using a 50-Hz digital Gaussian low-pass filter for analysis purposes. Five independent experiments were performed.

Radioligand binding and calcium mobilization assays

Binding assays were performed as described previously [48, 49]. Briefly, membrane preparations expressing h5-HT1AR, h5-HT2AR or hSERT (see Supplementary Material) were incubated with radiolabeled selective ligands 0.4 nM [3H]8-OH-DPAT, 1 nM [3H]ketanserin or 3 nM [3H]imipramine, respectively. The drugs were diluted in the corresponding binding buffer and tested at increasing concentrations in duplicate. Binding reactions were performed in tubes containing the drug dilutions; 25 μl of the corresponding radioligand; and the membranes (10 μg and 5 μg/500 μL for 5-HT1AR and 5-HT2AR, respectively, and 15 μg/100 μL for hSERT), all diluted in binding buffer. Non-specific binding was determined in the presence of 5-HT (10 μM) for 5-HT receptors; and paroxetine (3 μM) for hSERT and subtracted from total binding. Incubation was performed for 1 h at 27 oC for 5-HT1A/2AR and 22 oC for hSERT, according to the manufacturer’s protocol (Perkin Elmer, Inc., Waltham, MA, USA) and previous studies - saturation and kinetic experiments [48–50], respectively. The binding reactions were stopped by rapid filtration through GF/B glass microfiber filters pre-soaked with 0.5% polyethyleneimine and washing with ice-cold wash buffer. The filters were placed into vials and scintillation cocktail was added. The trapped radioactivity was quantified. Specific binding was defined as the difference between total binding (binding buffer alone) and non-specific binding. Experiments were conducted per duplicate for at least three independent experiments.

5-HT2AR functional assays were performed with CHO/K1 cells expressing human 5-HT2AR using the Invitrogen™ Fluo-4 NW Calcium Assay Kit (Thermo Fisher, Waltham, MA, USA), as described in the manufacturer’s protocol. Briefly, cells were loaded into black 96-well plates with probenecid and the dye provided by the manufacturer. Compounds solutions were added to the wells and fluorescence was quantified through a fluorescence intensity plate reader (VICTOR Nivo Multimode Plate Reader, Perkin Elmer). Emax was defined as percentage of the response induced by 5-HT (10−4 M). All determinations were conducted per triplicate for at least three independent experiments.

Molecular docking

Structural models for human 5-HT1AR and 5-HT2AR, complexed with 5-HT, were retrieved from the Protein Data Bank, PDBID: 7E2Y [51] and PDBID: 7WC4 [52], respectively. All models were prepared using the QuickPrep protocol implemented in MOE2020 software (Molecular Operating Environment, Montreal, Canada), including the ligand. Molecular docking was conducted using MOE2020 software. The placement method was guided to the interaction site described experimentally by following the template docking protocol, defining the 5-HT indole moiety as scaffold. The GBVI/WSA ΔG score function was applied to quantify the free energy of binding of the docking conformations, setting the total number of conformations to 100. Ligand Efficiency (LE) was calculated as the quotient between S and the number of heavy atoms, to assess the binding affinity independently of the molecular size. The docking protocol was validated by reproducing the interaction mechanism described for 5-HT in the PDB complexes.

Behavioral studies

Head twitch response (dose-response and antagonist experiments)

HTR studies were adapted from [26]. Mice (N = 8–10 per group) were injected intraperitoneally (i.p.) with saline or the appropriate tryptamine dose (0.3, 1, 3, 10, or 30 mg/kg) and placed into the observation arena (25 × 25 × 40 cm). A camera mounted above the observation cage recorded the mice for the next 10 min after the injection. Another batch of animals (N = 8–10) received subcutaneous injections of either saline or the respective antagonist (WAY100635 1 mg/kg for 5-HT1AR or ketanserin 1 mg/kg for 5-HT2AR). After a 10-min interval, the animals were i.p. injected with the corresponding tryptamine (10 mg/kg) and placed in the observation arena. Animal behavior was recorded for the next 10 min after the last injection, as mentioned before. Video recordings were examined by two blind observers blinded to the treatments, who assessed the number of head twitches, defined as a rapid rotational jerk of the head which is not contiguous with any grooming behavior [39].

Core body temperature measurements

Rectal temperature of mice (N = 8–10 per group) was measured 60 min after i.p. injection of the corresponding compound (see previous section “Head twitch response”). This time point was chosen according to the maximal effect observed in pilot experiments. A thermocouple probe (YS451, Panlab, Barcelona, Spain) connected to a digital thermometer (TMP812RS, Panlab, Barcelona, Spain) was inserted 2 cm into the rectum and steady temperature readout was obtained after 10 s of insertion.

Horizontal locomotor activity

The HLA test was carried out according to a previous study [53]. The same animals (N = 8–10 per group) used for the assessment of HTR were recorded for 30 min in an observation arena, under low-light conditions with white noise. Video recordings were processed using a tracking software (Smart 3.0 Panlab, Barcelona, Spain) to measure the total traveled distance.

Data analysis

Data were expressed as means±standard deviation (SD). Competition, release and functional assays, and substrate-induced current curves were plotted and fitted by non-linear regression to obtain the IC50, EC50, and Emax values. For behavioral studies, data were fitted by non-linear regression and pED50 and Emax values (with the corresponding 95% CI) were estimated by means of a software (GraphPad Prism 10) that further extrapolates the data. The Cheng-Prusoff equation was used to calculate Ki (affinity): Ki = EC50/(1 + [radioligand-concentration/Kd]) [54]. HTR curves were fitted using a Gaussian equation as described [55]. Data from batch release assays were statistically analyzed with a mixed-effects model, employing Šidák’s correction for multiple comparisons. Total release at each concentration and the behavioral experiments with the corresponding antagonists were statistically analyzed using one-way ANOVA, followed by Tukey’s post-hoc test. Molecular volume (van der Waals) was calculated using a grid approximation (spacing 0.75 Å). logP was calculated using Molinspiration Cheminformatics software. For in vivo studies, one-way ANOVA with Dunnet’s post-hoc test was used to compare all conditions to saline in dose-response experiments when data were normally distributed. Otherwise, a non-parametric test (Kruskall-Wallis with Dunn’s post-hoc test) was used. Alpha was set at 0.05. Outliers were excluded following ROUT’s method (Q = 0.1%). All statistical analyses and Pearson correlations were conducted using GraphPad Prism 10 software (GraphPad software, San Diego, CA, USA).

Results

5-MeO-DMT derivatives show nanomolar affinity for 5-HT1A receptors and are full 5-HT2AR agonists

The binding affinities for 5-HT1AR, 5-HT2AR, SERT, and EC50 and Emax values for 5-HT2AR-mediated calcium-flux are summarized in Table 1. All the tested compounds displaced [3H]8-OH-DPAT and [3H]ketanserin binding at nanomolar concentrations (see also Supplementary Fig. 2). Isopropyl-amino compounds (5-MeO-NIPT, 5-MeO-MIPT, 5-MeO-EIPT, 5-MeO-DIPT) showed lower affinity at 5-HT1AR compared to the other compounds tested. In contrast, 5-MeO-pyr-T showed the highest affinity and selectivity for 5-HT1AR. Regarding the affinity for 5-HT2AR, allyl-amino derivatives (5-MeO-MALT and 5-MeO-DALT) showed higher binding affinities. All the tested compounds showed full agonism for 5-HT2AR in calcium mobilization assays, (Fig. 1a). All tryptamines tested displayed low micromolar affinity at SERT, and the obtained Ki values inversely correlated with the volume of the compounds (R2 = 0.6112, P < 0.01; Supplementary Fig. 3).Table 1 Summary of the results obtained for the tested 5-MeO-tryptamines, with their structure and physicochemical properties.

Compounds	5-MeO-DMT	5-MeO-MET	5-MeO-DET	5-MeO-pyr-T	5-MeO-MALT	5-MeO-DALT	5-MeO-NIPT	5-MeO-MIPT	5-MeO-EIPT	5-MeO-DIPT	
Chemical structure											
Molecular volume (Å3)	237.63	252.00	273.25	262.37	266.38	300.13	255.25	274.50	290.00	308.38	
Octanol-water p. coefficient (logP)	2.33	2.71	3.08	2.73	2.97	3.62	2.76	3.00	3.38	3.68	
5-HT1AR	
Ki ± SD (nM)	2.57 ± 0.09	3.11 ± 0.51	4.93 ± 0.62	0.577 ± 0.195	5.96 ± 0.47	3.26 ± 0.39	15.7 ± 2.3	24.8 ± 7.6	18.4 ± 8.4	15.8 ± 1.3	
5-HT2AR	
Ki ± SD (nM)	105 ± 22	94.1 ± 16.7	128 ± 4	373 ± 59	52.9 ± 9	71.7 ± 14.8	123 ± 38	147 ± 32	151 ± 26	399 ± 49	
SERT	
Ki ± SD (nM)	14,510 ± 2925	7710 ± 3378	10,410 ± 970	3006 ± 354	4015 ± 417	1189 ± 96	8590 ± 1706	2869 ± 928	1776 ± 83	1618 ± 475	
5-HT uptake inhibition	
IC50 ± SD (nM)	50,068 ± 31,457	30,102 ± 12,585	60,012 ± 28,013	2765 ± 1431	44,053 ± 11,421	22,313 ± 4688	32,110 ± 11,497	29,768 ± 3918	22,172 ± 5870	24,215 ± 1977	
5-HT2AR calcium mobilization	
EC50 ± SD (nM);

Emax ± SD (%5-HT)

	5.28 ± 1.87

100 ± 5

	4.46 ± 0.06

102 ± 5

	17.1 ± 5.1

102 ± 7

	13.5 ± 2.2

92 ± 4

	4.95 ± 0.47

99 ± 9

	11.3 ± 3.3

99 ± 2

	13.2 ± 3.6

91 ± 1

	5.88 ± 2.39

96 ± 1

	23.6 ± 1.6

103 ± 4

	6.21 ± 1.25

99 ± 6

	
HTR	
pED50 (ED50 mg/kg)

(95% CI pED50)

Emax (HTR events)

(95% CI)

	0.685 (4.84)

(0.499–0.871)

38.1

(31.7–44.5)

	0.435 (2.72)

(0.188–0.682)

28.4

(23.5–33.2)

	0.365 (2.32)

(0.018–0.711)

9.73

(7.61–11.85)

	0.863 (7.29)

(0.626–1.080)

10.0

(6.9–13.1)

	−0.099 (0.796)

((−0.283)–0.085)

27.6

(23.2–32.0)

	0.619 (4.16)

(0.419–0.818)

10.4

(8.5–12.4)

	0.686 (4.85)

(0.154–1.217)

7.58

(5.46–9.71)

	−0.102 (0.791)

((−0.344)–0.141)

14.19

(11.7–16.68)

	1.03 (10.6)

(0.87–1.18)

9.27

(7.10–11.44)

	−0.322 (0.477)

−(0.626–0.017)

10.5

(8.5–12.5)

	
Change in body temperature	
pED50 (ED50 mg/kg)

(95% CI pED50)

Emax (ΔT ºC)

(95% CI)

	0.677 (4.75)

(0.307–1.046)

−1.63

−(2.15–1.11)

	0.678 (4.76)

(0.268–1.088)

−1.72

−(2.26–1.17)

	0.856 (7.18)

(0.697–1.016)

−4.18

−(4.77–3.60)

	0.461 (2.89)

(0.257–0.664)

−4.31

−(4.88–3.74)

	1.01 (10.2)

(0.80–1.22)

−2.75

−(3.45–2.06)

	1.13 (13.6)

(1.04–1.23)

−2.63

−(3.02–2.23)

	0.867 (7.35)

(0.720–1.013)

−4.24

−(4.86–3.61)

	1.17 (14.8)

(0.951–1.388)

−3.62

−(4.57–2.67)

	1.18 (15.0)

(0.991–1.358)

−4.32

−(5.33–3.30)

	1.17 (14.8)

(0.93–1.40)

−3.98

−(5.2–2.75)

	
HLA (hypolocomotion) pED50 (ED50 mg/kg) (95% CI)	−0.011 (0.976)

((−0.327)–0.305)

	0.229 (1.70)

(0.094–0.364)

	0.679 (4.78)

(0.216–1.142)

	0.205 (1.60)

(0.011–0.399)

	0.865 (7.32)

(0.529–1.201)

	0.794 (6.22)

(0.533–1.055)

	0.292 (1.96)

(0.161–0.422)

	0.817 (6.57)

(0.568–1.067)

	0.683 (4.82)

(0.466–0.900)

	0.655 (4.52)

(0.314–0.997)

	
Emax (% reduction)

(95% CI)

	41.72

(32.18–51.26)

	66.79

(58.20–75.37)

	63.56

(37.58–89.53)

	71.25

(60.71–81.79)

	85.40

(55.18–115.6)

	73.11

(57.45–88.78)

	77.64

(69.78–85.50)

	82.78

(62.16–103.4)

	68.02

(54.57–81.46)

	55.32

(38.20–72.44)

	
Titles and means in bold.

In vitro results for 5-HT1AR, 5-HT2AR, SERT binding affinities, 5-HT2AR-mediated calcium mobilization (Emax and EC50) and 5-HT uptake inhibition (IC50). In vivo HTR studies, change in body temperature and HLA. In vitro results are presented as means ± SD for N ≥ 3. In vivo results (N = 8–10) are presented as Emax and pED50 with 95% CI. ED50 values are also shown (between parentheses) for clarity purposes.

Fig. 1 In vitro assays.

a 5-HT2AR-mediated calcium mobilization assay of the tested 5-MeO-tryptamines and reference compounds 5-HT (full agonist) and dopamine (DA; partial agonist). Data are expressed as means ± SD for N ≥ 3 experiments. b 5-HT uptake inhibition at SERT. Data are expressed as percentage of control uptake (absence of tryptamine), as means ± SD for N ≥ 3 experiments. c–f Effects of 5-MeO-DMT, 5-MeO-MET, 5-MeO-DET and 5-MeO-pyr-T on transport-mediated batch release of preloaded [3H]5-HT from HEK293 cells stably expressing SERT. *p < 0.05, **p < 0.01, ***p < 0.001 vs release in absence of monensin (mixed-effects model, employing Šidák’s correction; N = 5). g–j Whole-cell patch clamp experiments used to identify tryptamine-induced SERT-mediated inwardly directed currents in HEK293 cells (N = 5). k–n Representative single-cell traces showing currents elicited by 10 µM of 5-MeO-DMT, 5-MeO-MET, 5-MeO-DET, and 5-MeO-pyr-T. Data are presented as means ± SD for N = 5 independent experiments. o Concentration-response relationship of 5-MeO-pyr-T measured in superfusion release assays at different concentrations, as percentage of total efflux (N = 5). KHB and pCA were used as control substances. *p < 0.05, **p < 0.01, ***p < 0.001 versus KHB, ###p < 0.001 vs pCA (Tukey’s test).

5-MeO-DMT, 5-MeO-MET, 5-MeO-DET and 5-MeO-pyr-T act as partial releasers at SERT

Our results demonstrated that all the compounds tested are able to inhibit [3H]5-HT uptake in the micromolar range, with 5-MeO-pyr-T displaying the highest potency (Table 1 and Fig. 1b).

Compounds were tested in a release assay using the ionophore monensin (Fig. 1c–f and Supplementary Fig. 4). Monensin causes a rise in intracellular Na+ and an alkalization of the interior of the cell, thereby augmenting transporter-mediated efflux [56]. 5-MeO-DMT, 5-MeO-MET, 5-MeO-DET and 5-MeO-pyr-T all showed to be capable of evoking 5-HT release, despite incapable of reaching the same level of efflux triggered by pCA 3 µM (Fig. 1c–f). Followed by the latter, 5-MeO-MIPT and 5-MeO-EIPT evoked a lower release at SERT (<10% total cpm) (Supplementary Fig. 4d, e).

By conducting whole-cell patch clamp experiment on HEK293 cells expressing hSERT, we evaluated the capacity of the four compounds evoking the highest 5-HT release, 5-MeO-DMT, 5-MeO-DET, 5-MeO-MET and 5-MeO-pyr-T, to be transported by hSERT (Fig. 1g–j). All four compounds elicited inwardly-directed currents suggesting their translocation into cytosol. We compared their induced currents to a saturating concentration of 5-HT (10 µM) (Fig. 1k–n). Consistent with previous studies, 5-HT showed an EC50 in the low µM range (EC50 = 0.05 µM) [57, 58]. 5-MeO-DET produced a blunted current (EC50 = 3.5 µM; Emax = 0.26). 5-MeO-DMT, 5-MeO-MET, and 5-MeO-pyr-T all produced Emax in the 40–50% range (5-MeO-DMT: Emax = 0.41; 5-MeO-MET: Emax = 0.46; 5-MeO-pyr-T: Emax = 0.49). While 5-MeO-MET showed a moderate activity (EC50 = 13 µM), 5-MeO-DMT and 5-MeO-pyr-T showed activity in the low micromolar range (EC50 = 2.6 µM; EC50 = 0.09 µM, respectively) and partial efficacy compared to 5-HT.

Since 5-MeO-pyr-T was the most potent compound inhibiting 5-HT uptake and elicited 5-HT release at lower concentrations, the potency of 5-MeO-pyr-T to evoke 5-HT release was studied (Fig. 1o), obtaining the following results: EC50 = 5.70 ± 1.59 µM (see Supplementary Table 1 for statistical results).

Molecular docking reveals a better interaction with 5-HT1AR over 5-HT2AR

Tryptamines classically interact with 5-HT1AR and 5-HT2AR. Here, we found a pronounced heterogeneity in the interaction of different derivatives with 5-HT1AR and 5-HT2AR. Therefore, we relied on in silico experiments to establish structure-activity relationships.

Consistently with our in vitro experiments, molecular docking of tryptamine analogs showed a better interaction of the studied compounds with 5-HT1AR than with 5-HT2AR (Fig. 2a). Although being flexible structures, the analysis of 5-HT1AR and 5-HT2AR binding pockets revealed that entry cavities are significantly different (Fig. 2b).Fig. 2 Interaction mechanism of 5-MeO-tryptamines at 5-HT receptors.

a Ligand efficiency of the compounds when bound to 5-HT1AR or 5-HT2AR. b Binding pockets of 5-HT1AR (blue) and 5-HT2AR (purple). c Interaction between the indole scaffold and Thr121 of 5-MeO-DMT. d, e Predicted binding mechanism of 5-MeO-MET and 5-MeO-DIPT at 5-HT1AR. Green zones correspond to hydrophobic regions and purple zones correspond to hydrophilic regions within the pocket. f, g Spatial orientation of 5-MeO-MET and 5-MeO-DIPT in the 5-HT2AR pocket.

5-HT1AR shows a bigger cavity, allowing the establishment of a better interaction with the molecules under study in the receptor’s back pocket. For all compounds tested, the indole scaffold reaches the inner part of the receptor and strongly interacts with Thr121 via hydrogen bond (Fig. 2c). The interaction mechanism at 5-HT1AR is stabilized by the formation of one or two hydrogen bonds between the amino group and Asp116 (Fig. 2d). In addition, electrostatic interactions between the positively charged amino-protonated molecules and the negatively charged residue also contribute to the binding affinity, as described for tryptamine binding to 5-HT receptors [59, 60]. Additionally, the arrangement of the alkyl chains from the amino group also plays a role in stabilizing the molecule, e.g., in 5-MeO-MET, the ethyl group is located in a highly hydrophobic region that may contribute to stabilizing the interaction mechanism. Contrarily, although 5-MeO-DIPT shows the interaction with Asp116, the hydrocarbon chains are located in a hydrophilic region (Fig. 2e). This fact may contribute to reducing the binding energy.

Interestingly, the aforementioned interactions between the amino group and the corresponding residue (Asp116 in 5-HT1AR) are greatly reduced when ligands are docked at 5-HT2AR (Fig. 2f–g), preventing a strong interaction with Asp155 [59]. Moreover, the hydrogen bond between the indole moiety and the Thr residue (Thr121 in 5-HT1AR; Thr160 in 5-HT2AR) is non-existent at 5-HT2AR. Attending the crystal structure available in the Protein Data Bank, the electrostatic features of residues in the binding cavity of 5-HT2AR differ from the pattern defined in 5-HT1AR, predictably affecting the recognition process of ligands. In fact, at 5-HT2AR, conformations tend to interact in the solvent-exposed region.

Potency at 5-HT2AR-mediated calcium-flux correlates to HTR potency

All the 5-MeO-tryptamines tested induced HTR in mice (Fig. 3) with varying potencies (ED50) and Emax values (Table 1). The HTR dose-response profiles followed an inverted U-shape or reached a plateau (Fig. 3a–j and Supplementary Fig. 5). Statistical data are shown in Supplementary Table 2.Fig. 3 Head Twitch Response.

a–j Number of head-twitch events during a 10-min period for all the tested tryptamines. Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs control group (Kruskall-Wallis with Dunn’s test). N = 8–10 mice per group. k Correlation between HT2AR-mediated calcium mobilization potency (in vitro) and HTR potency (in vivo), with 95% CI. l Representative example of the number of head twitches after 5-MeO-pyr-T injection (i.p., 10 mg/kg) with or without 5-HT1AR or 5-HT2AR antagonist pretreatment, WAY100635 (s.c., 1 mg/kg; WAY) or ketanserin (s.c., 1 mg/kg; KS), respectively. Data are presented as means ± SD. ***p < 0.001 vs control group, ##p < 0.01, ###p < 0.001 vs group receiving only 5-MeO-pyr-T (ANOVA with Tuckey’s test). N = 8–10 mice per group.

Interestingly, we found a correlation between in vitro 5-HT2AR-mediated calcium mobilization potency and HTR potency (P < 0.05, R2 = 0.5644; Fig. 3k) and Emax (P < 0.05, R2 = 0.4194; Supplementary Fig. 6). In addition, the smaller the size of the tryptamines (e.g., 5-MeO-DMT, 5-MeO-MET) the higher was the HTR observed (P < 0.05, R2 = 0.4082; Supplementary Fig. 7).

5-HT1AR modulates both psychedelic and hypothermic effects

To evaluate for target specificity, we also evaluated the HTR response elicited by tryptamine derivatives following a pretreatment with the selective 5-HT2AR antagonist ketanserin. Consistent with a dependence of HTR to 5-HT2AR agonism [39], administering ketanserin prior to tryptamine injection prevented HTR response. However, when administering the selective 5-HT1AR antagonist WAY100635 followed by tryptamine injection, the number of head twitches increased significantly compared to tryptamine derivative alone. Figure 3l shows the results obtained for 5-MeO-pyr-T as a representative example. Results and statistical data for all 5-MeO-tryptamines are presented in Supplementary Fig. 8 and Supplementary Table 3, respectively.

Regarding the thermoregulatory effects induced by 5-MeO-tryptamines, there is a significant decrease in core body temperature measured 60 min after injection (Table 1 and Fig. 4a–j). Dose-response curves and statistical data are provided in the Supplementary Material (Supplementary Table 2 and Supplementary Fig. 9). A correlation exists between in vivo potency at inducing hypothermia and in vitro binding affinity at 5-HT1AR (P < 0.05, R2 = 0.5067; Fig. 4k). Moreover, we found an inverse correlation between maximal effects of HTR and hypothermic response (P < 0.001, R2 = 0.7620; Fig. 4l).Fig. 4 Hypothermic response.

a–j Change in core body temperature 60 min post injection. Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs control saline group (ANOVA with Dunnet’s test or Kruskall-Wallis with Dunn’s test). N = 8–10 mice per group. k Correlation between affinity for 5-HT1AR (in vitro) and potency in the hypothermic response (in vivo). l Correlation between maximal effects in the hypothermic response and HTR. Discontinuous lines represent 95% CI. m, n Representatives examples (5-MeO-pyr-T and 5-MeO-EIPT, i.p., 10 mg/kg) of the core body temperature measured after 60 min of tryptamine injection with or without WAY100635 pretreatment (s.c., 1 mg/kg; WAY). Data are shown as means ± SD. **p < 0.01, ***p < 0.001 vs control saline group. ##p < 0.01, ###p < 0.001 vs group receiving only tryptamine (ANOVA with Tuckey’s test). N = 8–10 mice per group.

When administering WAY100635 prior to tryptamine injection, the decrease in body temperature was lower compared to the group receiving the tested compound alone. Figure 4m, n shows the results obtained for 5-MeO-pyr-T and 5-MeO-EIPT as representative examples. Results derived from the core body temperature study for all the 5-MeO-tryptamines are presented in Supplementary Fig. 10. Statistical data are shown in Supplementary Table 3.

5-MeO-DMT derivatives induce hypolocomotion

5-MeO-tryptamines induced a dose-dependent decrease of HLA in mice (Fig. 5). Table 1 summarizes the hypolocomotion potencies for all tryptamines tested. Dose-response curves and statistical data are shown in Supplementary Fig. 11 and Supplementary Table 4, respectively. Hypothermic response and hypolocomotion potencies are correlated (P < 0.01, R2 = 0.5904; Supplementary Fig. 12).Fig. 5 Horizontal locomotor activity.

a–j Total distance traveled in a 30-min period. Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs control saline group (ANOVA with Dunnet’s test or Kruskall-Wallis with Dunn’s test). N = 8–10 mice per group.

Discussion

As the NPS market continuously evolves and new cases related to new synthetic tryptamines emerge, it is crucial for health and legal authorities to continue monitoring and address the effects associated with their recreational use [3, 4]. Thus, our study intends to provide useful information to the implicated organisms by characterizing novel 5-MeO-tryptamines through a SAR study based on the N,N-substitutions of the synthetic tryptamines, which may also offer valuable data to predict the pharmacological effects of structurally similar tryptamines that might appear in the future. In addition, a better understanding of the pharmacological profile of this class of tryptamines will offer valuable knowledge for the potential use of these compounds as promising therapeutical agents [21, 52, 61].

One of the aims of the present study was to explore how specific N,N-alkyl and N,N-allyl substitutions on 5-MeO-tryptamines affect the key 5-HT receptors responsible for regulating psychedelic effects, along with their interaction with SERT. Although all tested 5-MeO-tryptamines showed nanomolar affinity for 5-HT1AR, we found that 5-MeO-tryptamines with isopropyl-terminal amino groups showed lower affinities compared to the rest of the molecules, in line with previous studies [62]. The binding mode of all molecules was examined by means of docking calculations, reproducing a strong interaction between the indole scaffold and the Thr121 in the inner part of 5-HT1AR, and also the critical placement of the amino group interacting with Asp116. Hydrophobic interactions may also contribute to stabilization of the molecule in the binding pocket. The loss of these interactions with isopropyl derivatives may explain the lower affinity of these tryptamines.

In contrast to 5-HT1AR, the binding mechanisms predicted for all compounds at 5-HT2AR did not show a key interaction. The change in the pocket’s volume would allow free movement of the ligand, hampering the recognition and the fit of the ligand to the binding cavity. Moreover, the electrostatic features of residues in the binding cavity of 5-HT2AR differs from the pattern defined in 5-HT1AR, predictably affecting the recognition process of ligands. These in silico predictions are in accordance with our in vitro findings, which reveal a higher affinity for 5-HT1AR over 5-HT2AR for 5-MeO-tryptamines, as previously described in the literature for several tryptamine derivatives [63–65]. Although our findings show that the length of the alkyl chains does not have an impact on the affinity for 5-HT2AR, as previously described [36, 62], our binding results suggest the presence of N, N-allyl groups increase affinity for this receptor.

Regarding SERT interaction, steric effects seem to have an impact on the affinity for such transporter, as suggested previously for different molecules [66, 67]. In fact, we found a significant correlation between the size of the molecule and the experimental Ki values: molecules with bulkier substituents on the N position possess higher affinities for SERT. Similarly, a study on quaternary ammonium salts of 4-substituted tryptamines [68] reported increase in affinity for SERT binding and potency for 5-HT uptake inhibition when increasing the bulk of the ammonium unit.

Our results demonstrated that some tryptamines were able to promote 5-HT transported-mediated release, with 5-MeO-DMT, 5-MeO-MET, 5-MeO-DET and 5-MeO-pyr-T showing significant releasing properties among the tested compounds. The slight to none 5-HT releasing activity observed for 5-MeO-DIPT, 5-MeO-MIPT, and 5-MeO-DALT is in accordance with those previously reported [29]. In fact, it is known that large molecules targeting particular monoamine transporters face steric interactions and tend to result in pure blockers, while smaller compounds tend to be better releasers as they can be transported through the membrane more easily [28, 69, 70]. Some authors [28] also suggested that addition of 5-methoxy groups to the chemical scaffold of tryptamines resulted in less active 5-HT releasers, which could explain the weak releasing properties observed for the tested compounds. Since monoamine transporters use the sodium gradient across cell membranes to concentrate their substrates in the cytosol [60], electrophysiology experiments are a useful tool to identify substrates [71]. Therefore, whole-cell patch clamp experiments were performed to study the capacity of 5-MeO-DMT, 5-MeO-MET, 5-MeO-DET and 5-MeO-pyr-T to elicit SERT-mediated inward current. Our results showed that the tested compounds are partial SERT substrates, with 5-MeO-pyr-T being the most potent. Concentration-response release assays were subsequently performed for 5-MeO-pyr-T to assess the 5-HT releasing potency of this compound. The significant difference between the peak of efflux caused by 5-MeO-pyr-T and the positive control pCA allows the classification of this tryptamine as a 5-HT partial releaser, a mechanism that is gaining a lot of interest for their potential therapeutic use [31, 72–74]. Although it does not seem that 5-HT releasing is the prime mechanism of the tested compounds, it may still play a supportive role. Moreover, the likely interaction with 5-HT1A autoreceptors (negative inhibitory feedback) may play an opposite role in the increased 5-HT levels at the synaptic cleft, but not in the transporter-mediated efflux itself. Therefore, further studies are needed in order to corroborate if such 5-HT releasing mechanism has implications in the likely in vivo therapeutical effects of novel psychedelics/tryptamines.

All the tested tryptamines induced HTR in mice, as a measure of its potential hallucinogenic effects in humans [75]. This psychedelic response is known to be mainly triggered after activation of 5-HT2AR [26, 27], as ketanserin (5-HT2AR antagonist) pretreatment completely blocks the head twitch behavior. In general, small amino substituents in 5-MeO-tryptamines tend to produce more head twitches, existing a correlation between molecular volume and HTR efficacy. Since some tryptamines showed low activity in the HTR, we tested the in vitro functionality at 5-HT2AR through calcium mobilization assays. Our results showed that all the tested 5-MeO-tryptamines act as full agonists at 5-HT2AR. Yet, potencies vary from one compound to another, correlating to both potency and efficacy in eliciting HTR: potent compounds inducing in vitro calcium mobilization tend to exhibit higher potency and higher efficacy in the HTR. On the other hand, 5-HT2AR agonists that produce little or no hallucinogenic effects are gaining a lot of interest due to their potential as antidepressants [76]. In this sense, 5-MeO-DIPT, 5-MeO-NIPT, 5-MeO-EIPT, and especially, 5-MeO-pyr-T induce very low HTR but still have 5-HT2AR-mediated calcium mobilization potency at nanomolar range. Further studies are needed to elucidate the mechanisms that explain these particularities (i.e., beta-arrestin versus Gq pathways after 5-HT2AR activation [77, 78]). Moreover, recent studies demonstrate that lipophilic tryptamines (e.g., 5-MeO-DMT) exhibit greater abilities to promote neuroplasticity, a key factor in the treatment of mood disorders, due to intracellular 5-HT2AR activation [61]. Therefore, some of the compounds tested in the present study, which are more lipophilic than 5-MeO-DMT and produce low HTR, could be promising candidates for future studies focused on their neuroplasticity properties, with the aim of searching novel psychedelics for treating mood disorders.

Another important site of action for tryptamine derivatives is 5-HT1AR [65, 79, 80]. It has been proposed that 5-HT1AR ligands can modulate 5-HT2AR-mediated effects [42, 81]. For example, some studies describe that pretreatment with the 5-HT1AR agonist 8-OH-DPAT attenuates HTR [27, 82]. Yet, the role of 5-HT1AR in the hallucinogenic effects is still object of debate and controversy. Fantegrossi and coworkers [39, 83] used the 5-HT1AR selective antagonist WAY100635 prior to injection of 5-MeO-DIPT and N,N-DPT, and described a partial attenuation in the HTR for both tryptamine compounds. A more recent study [25] reported no enhancing or inhibition in the HTR when pretreating with WAY100635 followed by psilocyn administration. Contrarily, Glatfelter and coworkers [41] recently reported the 5-HT2AR-mediated HTR is attenuated by 5-HT1AR agonist activity. The discrepancy between the different findings concerning the functional interaction between 5-HT1AR and 5-HT2AR could be attributed to the use of different doses of WAY100635, mouse strain and/or the tryptamine derivative itself. In line with the latter observations, our results indicate that pretreating animals with the 5-HT1AR antagonist followed by administration of 10 mg/kg of tryptamine (the dose in which the HTR effects are significant and close to the Emax) induce a significant increase in the HTR, which points out that 5-HT1AR activation attenuates the HTR behavior. Confirming the role of 5-HT1AR in the psychedelic experience, Pokorny and coworkers [81] found out that 5-HT1AR agonists such as buspirone reduce psilocybin-induced symptoms in humans, including visual hallucinations, derealization and depersonalization, via activation of 5-HT1AR and/or an interaction between 5-HT1A and 5-HT2A receptors, suggesting particular 5-HT1AR agonists could be useful for the treatment of schizophrenia and visual hallucinations in Parkinson’s disease.

Several studies have reported a hyperthermic effect on rodents and humans induced by hallucinogenic drugs, suggesting the 5-HT2AR involvement in the raise of body temperature [43, 84, 85]. Nevertheless, 5-MeO-tryptamines have also a high affinity for 5-HT1AR, a receptor also known to be involved in central body temperature regulation [86–88]. In fact, 5-HT1AR agonists have been reported to cause a dose-dependent hypothermia in rodents [89]. In an attempt to further characterize the pharmacological profile of 5-MeO-tryptamines, we monitored core body temperature after drug administration. Our results showed a dose-dependent hypothermic effect on core body temperature, in agreement with previous studies [42]. The use of WAY100635 (5-HT1AR antagonist) for pretreating mice attenuated the significant hypothermic effects induced by a dose of 10 mg/kg of the corresponding tryptamine, thus confirming the implication of 5-HT1AR on temperature regulation, as reported previously [25, 41, 42]. In fact, we found that high affinity for 5-HT1AR correlates with greater potencies at inducing hypothermic effects in vivo.

As mentioned before, activation of 5-HT1AR can cause a decrease in the 5-HT2AR-mediated effects. In line with this, our in vivo observations showed an inverse correlation between the HTR and hypothermic maximal effects. This finding further corroborates the opposite role of both 5-HT receptors: 5-MeO-tryptamines that produce more 5-HT1AR-related effects (hypothermia) induce less psychedelic-like effects through 5-HT2AR.

All the tested 5-MeO-tryptamines decreased HLA in mice, as previously reported for other tryptamine derivatives [53, 80, 90]. Potency in inducing hypolocomotion was found to be correlated with hypothermic potency, as HLA is known to be also mediated through 5-HT1AR activation [91].

In summary, the present study examined the pharmacology and behavioral effects of 5-methoxy-substituted tryptamines with a range of modifications on the amino position. 5-MeO-tryptamines analogs possess nanomolar affinity for 5-HT1AR and 5-HT2AR, which oppositely modulate the hallucinogenic response. Moreover, all tryptamines tested induced a remarkable hypothermic response in mice, an effect mediated by 5-HT1AR. Although some 5-MeO-tryptamines exhibited low HTR activity, all the tested compounds showed full agonism at 5-HT2AR. 5-MeO-tryptamine derivatives with bulkier substituents on the N position showed higher affinities for SERT and some tryptamines, especially 5-MeO-pyr-T, act as partial 5-HT releasers. Finally, and as mentioned before, the understanding of the pharmacological profile of this class of tryptamines will provide useful information for future studies in the field of drug therapy.

Supplementary information

SUPPLEMENTARY INFORMATION

Supplementary information

The online version contains supplementary material available at 10.1038/s41380-024-02506-8.

Acknowledgements

This study was supported by Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación/10.13039/501100011033 (PID2019-109390RB-I00), Plan Nacional sobre Drogas (2020I051), and European Union (EU) Home Affairs Funds, NextGenPS project (number: 101045825). JC, DP, RLA, and EE belong to 2021SGR00090 from Generalitat de Catalunya. PP received a doctoral scholarship grant from Generalitat de Catalunya (AGAUR), 2022 FISDU 00004. This study was further supported by a grant from the Austrian Science Fund/FWF (grant P35589 to HHS and MN). We would like to thank Dr. Gemma Navarro Brugal for her expert technical advice in performing the intracellular calcium measurements.

Author contributions

PP and GK performed, acquired data, and analyzed in vivo experiments. PP, GK, NNG, NW, MNI, and MH performed, acquired data and analyzed in vitro experiments. PP, NNG, GK, and XB synthetized and performed analytical characterization of compounds. PP, RET, and EE performed and analyzed in silico studies. MN and HH designed and supervised release and electrophisiology assays. DP designed and supervised functional in vitro assays. JC and EE contributed to data analysis and curation. PP and RLA wrote the manuscript. DP, EE, HH, and MN helped to write and critically revised the manuscript. RLA conceived and designed the project.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

1. Catalani V Corkery JM Guirguis A Napoletano F Arillotta D Zangani C Psychonauts’ psychedelics: a systematic, multilingual, web-crawling exercise Eur Neuropsychopharmacol. 2021 49 69 92 10.1016/j.euroneuro.2021.03.006 33857740
Catalani V, Corkery JM, Guirguis A, Napoletano F, Arillotta D, Zangani C, et al. Psychonauts’ psychedelics: a systematic, multilingual, web-crawling exercise. Eur Neuropsychopharmacol. 2021;49:69–92.33857740
2. EMCDDA. European monitoring centre for drugs and drug addiction (EMCDDA). European drug report: trends and developments. Publications Office of the European Union, 2022.
3. Reuter P Pardo B Can new psychoactive substances be regulated effectively? An assessment of the British Psychoactive Substances Bill Addiction. 2017 112 25 31 10.1111/add.13439 27220685
Reuter P, Pardo B. Can new psychoactive substances be regulated effectively? An assessment of the British Psychoactive Substances Bill. Addiction. 2017;112:25–31.27220685
4. Shafi A Berry AJ Sumnall H Wood DM Tracy DK New psychoactive substances: a review and updates Ther Adv Psychopharmacol. 2020 10 2045125320967197 10.1177/2045125320967197 33414905
Shafi A, Berry AJ, Sumnall H, Wood DM, Tracy DK. New psychoactive substances: a review and updates. Ther Adv Psychopharmacol. 2020;10:2045125320967197.33414905
5. Soussan C Kjellgren A The users of novel psychoactive substances: online survey about their characteristics, attitudes and motivations Int J Drug Policy. 2016 32 77 84 10.1016/j.drugpo.2016.03.007 27184218
Soussan C, Kjellgren A. The users of novel psychoactive substances: online survey about their characteristics, attitudes and motivations. Int J Drug Policy. 2016;32:77–84.27184218
6. Gibbons S ‘Legal Highs’ – novel and emerging psychoactive drugs: a chemical overview for the toxicologist Clin Toxicol. 2012 50 15 24 10.3109/15563650.2011.645952
Gibbons S. ‘Legal Highs’ – novel and emerging psychoactive drugs: a chemical overview for the toxicologist. Clin Toxicol. 2012;50:15–24.
7. Musselman ME Hampton JP ‘not for human consumption’: a review of emerging designer drugs Pharmacotherapy. 2014 34 745 57 10.1002/phar.1424 24710806
Musselman ME, Hampton JP. ‘not for human consumption’: a review of emerging designer drugs. Pharmacotherapy. 2014;34:745–57.24710806
8. Tittarelli R Mannocchi G Pantano F Romolo FS Recreational use, analysis and toxicity of tryptamines Curr Neuropharmacol. 2015 13 26 46 10.2174/1570159X13666141210222409 26074742
Tittarelli R, Mannocchi G, Pantano F, Romolo FS. Recreational use, analysis and toxicity of tryptamines. Curr Neuropharmacol. 2015;13:26–46.26074742
9. Nichols DE Structure-activity relationships of serotonin 5-HT2A agonists Wiley Interdiscip Rev Membr Transp Signal. 2012 1 559 79 10.1002/wmts.42
Nichols DE. Structure-activity relationships of serotonin 5-HT2A agonists. Wiley Interdiscip Rev Membr Transp Signal. 2012;1:559–79.
10. Klein LM Cozzi NV Daley PF Brandt SD Halberstadt AL Receptor binding profiles and behavioral pharmacology of ring-substituted N,N-diallyltryptamine analogs Neuropharmacology. 2018 142 231 9 10.1016/j.neuropharm.2018.02.028 29499272
Klein LM, Cozzi NV, Daley PF, Brandt SD, Halberstadt AL. Receptor binding profiles and behavioral pharmacology of ring-substituted N,N-diallyltryptamine analogs. Neuropharmacology. 2018;142:231–9.29499272
11. Kozell LB Eshleman AJ Swanson TL Bloom SH Wolfrum KM Schmachtenberg JL Pharmacologic activity of substituted tryptamines at 5-HT 2A R, 5-HT 2C R, 5-HT 1A R, and SERT J Pharmacol Exp Ther. 2023 385 62 75 10.1124/jpet.122.001454 36669875
Kozell LB, Eshleman AJ, Swanson TL, Bloom SH, Wolfrum KM, Schmachtenberg JL, et al. Pharmacologic activity of substituted tryptamines at 5-HT 2A R, 5-HT 2C R, 5-HT 1A R, and SERT. J Pharmacol Exp Ther. 2023;385:62–75.36669875
12. Rogawaski M Aghajanian G Serotonin autoreceptors on dorsal raphe neurons: structure-activity relationships of tryptamine analogs J Neurosci. 1981 1 1148 54 10.1523/JNEUROSCI.01-10-01148.1981 6793698
Rogawaski M, Aghajanian G. Serotonin autoreceptors on dorsal raphe neurons: structure-activity relationships of tryptamine analogs. J Neurosci. 1981;1:1148–54.6793698
13. Scherbaum N Schifano F Bonnet U New psychoactive substances (NPS)—a challenge for the addiction treatment services Pharmacopsychiatry 2017 50 116 22 10.1055/s-0043-102059 28444659
Scherbaum N, Schifano F, Bonnet U. New psychoactive substances (NPS)—a challenge for the addiction treatment services. Pharmacopsychiatry 2017;50:116–22.28444659
14. Shimizu E Watanabe H Kojima T Hagiwara H Fujisaki M Miyatake R Combined intoxication with methylone and 5-MeO-MIPT Prog Neuropsychopharmacol Biol Psychiatry. 2007 31 288 91 10.1016/j.pnpbp.2006.06.012 16876302
Shimizu E, Watanabe H, Kojima T, Hagiwara H, Fujisaki M, Miyatake R, et al. Combined intoxication with methylone and 5-MeO-MIPT. Prog Neuropsychopharmacol Biol Psychiatry. 2007;31:288–91.16876302
15. Tanaka E Kamata T Katagi M Tsuchihashi H Honda K A fatal poisoning with 5-methoxy-N,N-diisopropyltryptamine, Foxy Forensic Sci Int. 2006 163 152 4 10.1016/j.forsciint.2005.11.026 16406422
Tanaka E, Kamata T, Katagi M, Tsuchihashi H, Honda K. A fatal poisoning with 5-methoxy-N,N-diisopropyltryptamine, Foxy. Forensic Sci Int. 2006;163:152–4.16406422
16. Wilson JM McGeorge F Smolinske S Meatherall R A foxy intoxication Forensic Sci Int. 2005 148 31 6 10.1016/j.forsciint.2004.04.017 15607587
Wilson JM, McGeorge F, Smolinske S, Meatherall R. A foxy intoxication. Forensic Sci Int. 2005;148:31–6.15607587
17. European Monitoring Centre for Drugs and Drug Addiction, Europol, New drugs in Europe, 2012 : EMCDDA–Europol 2012 annual report on the implementation of Council Decision 2005/387/JHA. Publications Office; 2013. Available from: doi/10.2810/99367
18. European Monitoring Centre for Drugs and Drug Addiction, Europol, EMCDDA–Europol 2014 annual report on the implementation of Council Decision 2005/387/JHA : in accordance with Article 10 of Council Decision 2005/387/JHA on the information exchange, risk assessment and control of new psychoactive substances: implementation reports. Publications Office; 2015. Available from: doi/10.2810/112317
19. European Monitoring Centre for Drugs and Drug Addiction, Europol, EMCDDA–Europol 2017 annual report on the implementation of Council Decision 2005/387/JHA : in accordance with Article 10 of Council Decision 2005/387/JHA on the information exchange, risk assessment and control of new psychoactive substances. Publications Office; 2018. Available from: doi/10.2810/759319
20. Bogenschutz MP Ross S Therapeutic applications of classic hallucinogens Curr Top Behav Neurosci. 2016 36 361 91 10.1007/7854_2016_464
Bogenschutz MP, Ross S. Therapeutic applications of classic hallucinogens. Curr Top Behav Neurosci. 2016;36:361–91.
21. Cameron LP Patel SD Vargas MV Barragan EV Saeger HN Warren HT 5-HT2ARs mediate therapeutic behavioral effects of psychedelic tryptamines ACS Chem Neurosci. 2023 14 351 8 10.1021/acschemneuro.2c00718 36630260
Cameron LP, Patel SD, Vargas MV, Barragan EV, Saeger HN, Warren HT, et al. 5-HT2ARs mediate therapeutic behavioral effects of psychedelic tryptamines. ACS Chem Neurosci. 2023;14:351–8.36630260
22. Heifets BD Olson DE Therapeutic mechanisms of psychedelics and entactogens Neuropsychopharmacology. 2024 49 104 18 10.1038/s41386-023-01666-5 37488282
Heifets BD, Olson DE. Therapeutic mechanisms of psychedelics and entactogens. Neuropsychopharmacology. 2024;49:104–18.37488282
23. McClure-Begley TD Roth BL The promises and perils of psychedelic pharmacology for psychiatry Nat Rev Drug Discov. 2022 21 463 73 10.1038/s41573-022-00421-7 35301459
McClure-Begley TD, Roth BL. The promises and perils of psychedelic pharmacology for psychiatry. Nat Rev Drug Discov. 2022;21:463–73.35301459
24. Reckweg JT Uthaug MV Szabo A Davis AK Lancelotta R Mason NL The clinical pharmacology and potential therapeutic applications of 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) J Neurochem. 2022 162 128 46 10.1111/jnc.15587 35149998
Reckweg JT, Uthaug MV, Szabo A, Davis AK, Lancelotta R, Mason NL, et al. The clinical pharmacology and potential therapeutic applications of 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT). J Neurochem. 2022;162:128–46.35149998
25. Erkizia-Santamaría I Alles-Pascual R Horrillo I Meana JJ Ortega JE Serotonin 5-HT2A, 5-HT2c and 5-HT1A receptor involvement in the acute effects of psilocybin in mice. In vitro pharmacological profile and modulation of thermoregulation and head-twich response Biomed Pharmacother. 2022 154 113612 10.1016/j.biopha.2022.113612 36049313
Erkizia-Santamaría I, Alles-Pascual R, Horrillo I, Meana JJ, Ortega JE. Serotonin 5-HT2A, 5-HT2c and 5-HT1A receptor involvement in the acute effects of psilocybin in mice. In vitro pharmacological profile and modulation of thermoregulation and head-twich response. Biomed Pharmacother. 2022;154:113612.36049313
26. Carbonaro TM Eshleman AJ Forster MJ Cheng K Rice KC Gatch MB The role of 5-HT2A, 5-HT2C and mGlu2 receptors in the behavioral effects of tryptamine hallucinogens N,N-dimethyltryptamine and N,N-diisopropyltryptamine in rats and mice Psychopharmacology. 2015 232 275 84 10.1007/s00213-014-3658-3 24985890
Carbonaro TM, Eshleman AJ, Forster MJ, Cheng K, Rice KC, Gatch MB. The role of 5-HT2A, 5-HT2C and mGlu2 receptors in the behavioral effects of tryptamine hallucinogens N,N-dimethyltryptamine and N,N-diisopropyltryptamine in rats and mice. Psychopharmacology. 2015;232:275–84.24985890
27. Shahar O Botvinnik A Esh-Zuntz N Brownstien M Wolf R Lotan A Role of 5-HT2A, 5-HT2C, 5-HT1A and TAAR1 receptors in the head twitch response induced by 5-hydroxytryptophan and psilocybin: translational implications Int J Mol Sci. 2022 23 14148 10.3390/ijms232214148 36430623
Shahar O, Botvinnik A, Esh-Zuntz N, Brownstien M, Wolf R, Lotan A, et al. Role of 5-HT2A, 5-HT2C, 5-HT1A and TAAR1 receptors in the head twitch response induced by 5-hydroxytryptophan and psilocybin: translational implications. Int J Mol Sci. 2022;23:14148.36430623
28. Blough BE Landavazo A Decker AM Partilla JS Baumann MH Rothman RB Interaction of psychoactive tryptamines with biogenic amine transporters and serotonin receptor subtypes Psychopharmacology. 2014 231 4135 44 10.1007/s00213-014-3557-7 24800892
Blough BE, Landavazo A, Decker AM, Partilla JS, Baumann MH, Rothman RB. Interaction of psychoactive tryptamines with biogenic amine transporters and serotonin receptor subtypes. Psychopharmacology. 2014;231:4135–44.24800892
29. Nagai F Nonaka R Satoh Hisashi Kamimura K The effects of non-medically used psychoactive drugs on monoamine neurotransmission in rat brain Eur J Pharmacol. 2007 559 132 7 10.1016/j.ejphar.2006.11.075 17223101
Nagai F, Nonaka R, Satoh Hisashi Kamimura K. The effects of non-medically used psychoactive drugs on monoamine neurotransmission in rat brain. Eur J Pharmacol. 2007;559:132–7.17223101
30. Sogawa C Sogawa N Tagawa J Fujino A Ohyama K Asanuma M 5-Methoxy-N,N-diisopropyltryptamine (Foxy), a selective and high affinity inhibitor of serotonin transporter Toxicol Lett. 2007 170 75 82 10.1016/j.toxlet.2007.02.007 17382495
Sogawa C, Sogawa N, Tagawa J, Fujino A, Ohyama K, Asanuma M, et al. 5-Methoxy-N,N-diisopropyltryptamine (Foxy), a selective and high affinity inhibitor of serotonin transporter. Toxicol Lett. 2007;170:75–82.17382495
31. Mayer FP Niello M Cintulova D Sideromenos S Maier J Li Y Serotonin-releasing agents with reduced off-target effects Mol Psychiatry. 2023 28 722 32 10.1038/s41380-022-01843-w 36352123
Mayer FP, Niello M, Cintulova D, Sideromenos S, Maier J, Li Y, et al. Serotonin-releasing agents with reduced off-target effects. Mol Psychiatry. 2023;28:722–32.36352123
32. Blier P Piñeyro G el Mansari M Bergeron R de Montigny C Role of somatodendritic 5-HT autoreceptors in modulating 5-HT neurotransmission Ann N. Y Acad Sci 1998 861 204 16 10.1111/j.1749-6632.1998.tb10192.x 9928258
Blier P, Piñeyro G, el Mansari M, Bergeron R, de Montigny C. Role of somatodendritic 5-HT autoreceptors in modulating 5-HT neurotransmission. Ann N. Y Acad Sci. 1998;861:204–16.9928258
33. Nackenoff AG Simmler LD Baganz NL Pehrson AL Sánchez C Blakely RD Serotonin transporter-independent actions of the antidepressant vortioxetine as revealed using the sert met172 mouse ACS Chem Neurosci 2017 8 1092 1100 10.1021/acschemneuro.7b00038 28272863
Nackenoff AG, Simmler LD, Baganz NL, Pehrson AL, Sánchez C, Blakely RD. Serotonin transporter-independent actions of the antidepressant vortioxetine as revealed using the sert met172 mouse. ACS Chem Neurosci. 2017;8:1092–1100.28272863
34. Rothman RB Baumann MH Therapeutic and adverse actions of serotonin transporter substrates Pharm Ther 2002 95 73 88 10.1016/S0163-7258(02)00234-6
Rothman RB, Baumann MH. Therapeutic and adverse actions of serotonin transporter substrates. Pharm Ther. 2002;95:73–88.
35. Cozzi NV Daley PF Receptor binding profiles and quantitative structure-affinity relationships of some 5-substituted-N,N-diallyltryptamines Bioorg Med Chem Lett 2016 26 959 64 10.1016/j.bmcl.2015.12.053 26739781
Cozzi NV, Daley PF. Receptor binding profiles and quantitative structure-affinity relationships of some 5-substituted-N,N-diallyltryptamines. Bioorg Med Chem Lett. 2016;26:959–64.26739781
36. Klein AK Chatha M Laskowski LJ Anderson EI Brandt SD Chapman SJ Investigation of the structure-activity relationships of psilocybin analogues ACS Pharm Transl Sci 2021 4 533 42 10.1021/acsptsci.0c00176
Klein AK, Chatha M, Laskowski LJ, Anderson EI, Brandt SD, Chapman SJ, et al. Investigation of the structure-activity relationships of psilocybin analogues. ACS Pharm Transl Sci. 2021;4:533–42.
37. Rickli A Moning OD Hoener MC Liechti ME Receptor interaction profiles of novel psychoactive tryptamines compared with classic hallucinogens Eur Neuropsychopharmacol 2016 26 1327 37 10.1016/j.euroneuro.2016.05.001 27216487
Rickli A, Moning OD, Hoener MC, Liechti ME. Receptor interaction profiles of novel psychoactive tryptamines compared with classic hallucinogens. Eur Neuropsychopharmacol. 2016;26:1327–37.27216487
38. Arunotayanun W Dalley JW Huang XP Setola V Treble R Iversen L An analysis of the synthetic tryptamines AMT and 5-MeO-DALT: emerging ‘Novel Psychoactive Drugs Bioorg Med Chem Lett 2013 23 3411 5 10.1016/j.bmcl.2013.03.066 23602445
Arunotayanun W, Dalley JW, Huang XP, Setola V, Treble R, Iversen L, et al. An analysis of the synthetic tryptamines AMT and 5-MeO-DALT: emerging ‘Novel Psychoactive Drugs. Bioorg Med Chem Lett. 2013;23:3411–5.23602445
39. Fantegrossi WE Harrington AW Kiessel CL Eckler JR Rabin RA Winter JC Hallucinogen-like actions of 5-methoxy-N,N-diisopropyltryptamine in mice and rats Pharm Biochem Behav 2006 83 122 9 10.1016/j.pbb.2005.12.015
Fantegrossi WE, Harrington AW, Kiessel CL, Eckler JR, Rabin RA, Winter JC, et al. Hallucinogen-like actions of 5-methoxy-N,N-diisopropyltryptamine in mice and rats. Pharm Biochem Behav. 2006;83:122–9.
40. Gatch MB Forster MJ Janowsky A Eshleman AJ Abuse liability profile of three substituted tryptamines J Pharmacol Exp Ther 2011 338 280 9 10.1124/jpet.111.179705 21474568
Gatch MB, Forster MJ, Janowsky A, Eshleman AJ. Abuse liability profile of three substituted tryptamines. J Pharmacol Exp Ther. 2011;338:280–9.21474568
41. Glatfelter GC Naeem M Pham DNK Golen JA Chadeayne AR Manke DR Receptor binding profiles for tryptamine psychedelics and effects of 4-propionoxy- N,N -dimethyltryptamine in mice ACS Pharm Transl Sci 2023 6 567 77 10.1021/acsptsci.2c00222
Glatfelter GC, Naeem M, Pham DNK, Golen JA, Chadeayne AR, Manke DR, et al. Receptor binding profiles for tryptamine psychedelics and effects of 4-propionoxy- N,N -dimethyltryptamine in mice. ACS Pharm Transl Sci. 2023;6:567–77.
42. Glatfelter GC Pottie E Partilla JS Sherwood AM Kaylo K Pham DNK Structure–activity relationships for psilocybin, baeocystin, aeruginascin, and related analogues to produce pharmacological effects in mice ACS Pharm Transl Sci 2022 5 1181 96 10.1021/acsptsci.2c00177
Glatfelter GC, Pottie E, Partilla JS, Sherwood AM, Kaylo K, Pham DNK, et al. Structure–activity relationships for psilocybin, baeocystin, aeruginascin, and related analogues to produce pharmacological effects in mice. ACS Pharm Transl Sci. 2022;5:1181–96.
43. Jiang XL Shen HW Yu AM Potentiation of 5-methoxy-N,N-dimethyltryptamine-induced hyperthermia by harmaline and the involvement of activation of 5-HT1A and 5-HT2A receptors Neuropharmacology 2015 89 342 51 10.1016/j.neuropharm.2014.10.013 25446678
Jiang XL, Shen HW, Yu AM. Potentiation of 5-methoxy-N,N-dimethyltryptamine-induced hyperthermia by harmaline and the involvement of activation of 5-HT1A and 5-HT2A receptors. Neuropharmacology. 2015;89:342–51.25446678
44. Williams MT Herring NR Schaefer TL Skelton MR Campbell NG Lipton JW Alterations in body temperature, corticosterone, and behavior following the administration of 5-methoxy-diisopropyltryptamine (‘Foxy’) to adult rats: a new drug of abuse Neuropsychopharmacology 2007 32 1404 20 10.1038/sj.npp.1301232 17047665
Williams MT, Herring NR, Schaefer TL, Skelton MR, Campbell NG, Lipton JW, et al. Alterations in body temperature, corticosterone, and behavior following the administration of 5-methoxy-diisopropyltryptamine (‘Foxy’) to adult rats: a new drug of abuse. Neuropsychopharmacology. 2007;32:1404–20.17047665
45. Percie du Sert N Hurst V Ahluwalia A Alam S Avey MT Baker M The ARRIVE guidelines 2.0: updated guidelines for reporting animal research J Cereb Blood Flow Metab 2020 40 1769 77 10.1177/0271678X20943823 32663096
Percie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, et al. The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. J Cereb Blood Flow Metab. 2020;40:1769–77.32663096
46. Nadal-Gratacós N Alberto-Silva AS Rodríguez-Soler M Urquizu E Espinosa-Velasco M Jäntsch K Structure–activity relationship of novel second-generation synthetic cathinones: mechanism of action, locomotion, reward, and immediate-early genes Front Pharm 2021 12 749429 10.3389/fphar.2021.749429
Nadal-Gratacós N, Alberto-Silva AS, Rodríguez-Soler M, Urquizu E, Espinosa-Velasco M, Jäntsch K, et al. Structure–activity relationship of novel second-generation synthetic cathinones: mechanism of action, locomotion, reward, and immediate-early genes. Front Pharm. 2021;12:749429.
47. Brugnoli FR Holy M Niello M Maier J Hanreich M Menzel M Development and validation of an automated microfluidic perfusion platform for parallelized screening of compounds in vitro Basic Clin Pharm Toxicol 2023 133 535 47 10.1111/bcpt.13940
Brugnoli FR, Holy M, Niello M, Maier J, Hanreich M, Menzel M, et al. Development and validation of an automated microfluidic perfusion platform for parallelized screening of compounds in vitro. Basic Clin Pharm Toxicol. 2023;133:535–47.
48. Duart-Castells L Nadal-Gratacós N Muralter M Puster B Berzosa X Estrada-Tejedor R Role of amino terminal substitutions in the pharmacological, rewarding and psychostimulant profiles of novel synthetic cathinones Neuropharmacology 2021 186 108475 10.1016/j.neuropharm.2021.108475 33529677
Duart-Castells L, Nadal-Gratacós N, Muralter M, Puster B, Berzosa X, Estrada-Tejedor R, et al. Role of amino terminal substitutions in the pharmacological, rewarding and psychostimulant profiles of novel synthetic cathinones. Neuropharmacology. 2021;186:108475.33529677
49. Nadal-Gratacós N Lleixà E Gibert-Serramià M Estrada-Tejedor R Berzosa X Batllori X Neuropsychopharmacology of emerging drugs of abuse: meta-and para-halogen-ring-substituted α-PVP (“flakka”) derivatives Int J Mol Sci 2022 23 2226 10.3390/ijms23042226 35216339
Nadal-Gratacós N, Lleixà E, Gibert-Serramià M, Estrada-Tejedor R, Berzosa X, Batllori X, et al. Neuropsychopharmacology of emerging drugs of abuse: meta-and para-halogen-ring-substituted α-PVP (“flakka”) derivatives. Int J Mol Sci. 2022;23:2226.35216339
50. Sucic S Dallinger S Zdrazil B Weissensteiner R Jørgensen TN Holy M The N terminus of monoamine transporters is a lever required for the action of amphetamines J Biol Chem 2010 285 10924 38 10.1074/jbc.M109.083154 20118234
Sucic S, Dallinger S, Zdrazil B, Weissensteiner R, Jørgensen TN, Holy M, et al. The N terminus of monoamine transporters is a lever required for the action of amphetamines. J Biol Chem. 2010;285:10924–38.20118234
51. Xu P Huang S Zhang H Mao C Zhou XE Cheng X Structural insights into the lipid and ligand regulation of serotonin receptors Nature 2021 592 469 73 10.1038/s41586-021-03376-8 33762731
Xu P, Huang S, Zhang H, Mao C, Zhou XE, Cheng X, et al. Structural insights into the lipid and ligand regulation of serotonin receptors. Nature. 2021;592:469–73.33762731
52. Cao D Yu J Wang H Luo Z Liu X He L Structure-based discovery of nonhallucinogenic psychedelic analogs Science 2022 375 403 11 10.1126/science.abl8615 35084960
Cao D, Yu J, Wang H, Luo Z, Liu X, He L, et al. Structure-based discovery of nonhallucinogenic psychedelic analogs. Science. 2022;375:403–11.35084960
53. Tylš F Páleníček T Kadeřábek L Lipski M Kubešová A Horáček J Sex differences and serotonergic mechanisms in the behavioural effects of psilocin Behav Pharmacol 2016 27 309 20 10.1097/FBP.0000000000000198 26461483
Tylš F, Páleníček T, Kadeřábek L, Lipski M, Kubešová A, Horáček J. Sex differences and serotonergic mechanisms in the behavioural effects of psilocin. Behav Pharmacol. 2016;27:309–20.26461483
54. Yung-Chi C Prusoff WH Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction Biochem Pharm 1973 22 3099 108 10.1016/0006-2952(73)90196-2 4202581
Yung-Chi C, Prusoff WH. Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharm. 1973;22:3099–108.4202581
55. Halberstadt AL Chatha M Chapman SJ Brandt SD Comparison of the behavioral effects of mescaline analogs using the head twitch response in mice J Psychopharmacol 2019 33 406 14 10.1177/0269881119826610 30789291
Halberstadt AL, Chatha M, Chapman SJ, Brandt SD. Comparison of the behavioral effects of mescaline analogs using the head twitch response in mice. J Psychopharmacol. 2019;33:406–14.30789291
56. Scholze P Zwach J Kattinger A Pifl C Singer EA Sitte HH Transporter-mediated release: a superfusion study on human embryonic kidney cells stably expressing the human serotonin transporter J Pharm Exp Ther 2000 293 870 8
Scholze P, Zwach J, Kattinger A, Pifl C, Singer EA, Sitte HH. Transporter-mediated release: a superfusion study on human embryonic kidney cells stably expressing the human serotonin transporter. J Pharm Exp Ther. 2000;293:870–8.
57. Bhat S Niello M Schicker K Pifl C Sitte HH Freissmuth M Handling of intracellular K+ determines voltage dependence of plasmalemmal monoamine transporter function Elife 2021 10 e67996 10.7554/eLife.67996 34061030
Bhat S, Niello M, Schicker K, Pifl C, Sitte HH, Freissmuth M, et al. Handling of intracellular K+ determines voltage dependence of plasmalemmal monoamine transporter function. Elife. 2021;10:e67996.34061030
58. Bhat S Hasenhuetl PS Kasture A El-Kasaby A Baumann MH Blough BE Conformational state interactions provide clues to the pharmacochaperone potential of serotonin transporter partial substrates J Biol Chem 2017 292 16773 86 10.1074/jbc.M117.794081 28842491
Bhat S, Hasenhuetl PS, Kasture A, El-Kasaby A, Baumann MH, Blough BE, et al. Conformational state interactions provide clues to the pharmacochaperone potential of serotonin transporter partial substrates. J Biol Chem. 2017;292:16773–86.28842491
59. Kristiansen K Kroeze WK Willins DL Gelber EI Savage JE Glennon RA A highly conserved aspartic acid (Asp-155) anchors the terminal amine moiety of tryptamines and is involved in membrane targeting of the 5-HT2A serotonin receptor but does not participate in activation via a ‘salt-bridge disruption’ mechanism J Pharm Exp Ther 1996 293 735 46
Kristiansen K, Kroeze WK, Willins DL, Gelber EI, Savage JE, Glennon RA, et al. A highly conserved aspartic acid (Asp-155) anchors the terminal amine moiety of tryptamines and is involved in membrane targeting of the 5-HT2A serotonin receptor but does not participate in activation via a ‘salt-bridge disruption’ mechanism. J Pharm Exp Ther 1996;293:735–46.
60. Kristensen AS Andersen J Jørgensen TN Sørensen L Eriksen J Loland CJ SLC6 neurotransmitter transporters: structure, function, and regulation Pharm Rev 2011 63 585 640 10.1124/pr.108.000869 21752877
Kristensen AS, Andersen J, Jørgensen TN, Sørensen L, Eriksen J, Loland CJ, et al. SLC6 neurotransmitter transporters: structure, function, and regulation. Pharm Rev. 2011;63:585–640.21752877
61. Vargas MV Dunlap LE Dong C Carter SJ Tombari RJ Jami SA Psychedelics promote neuroplasticity through the activation of intracellular 5-HT2A receptors Science 2023 379 700 6 10.1126/science.adf0435 36795823
Vargas MV, Dunlap LE, Dong C, Carter SJ, Tombari RJ, Jami SA, et al. Psychedelics promote neuroplasticity through the activation of intracellular 5-HT2A receptors. Science. 2023;379:700–6.36795823
62. Mckenna DJ Repke DB Lo L Peroutka SJ Differential interactions of indolealkylamines with 5-hydroxytryptamine receptor subtypes Neuropharmacology 1990 29 193 8 10.1016/0028-3908(90)90001-8 2139186
Mckenna DJ, Repke DB, Lo L, Peroutka SJ. Differential interactions of indolealkylamines with 5-hydroxytryptamine receptor subtypes. Neuropharmacology. 1990;29:193–8.2139186
63. Fantegrossi WE Murnane KS Reissig CJ The behavioral pharmacology of hallucinogens Biochem Pharm 2008 75 17 33 10.1016/j.bcp.2007.07.018 17977517
Fantegrossi WE, Murnane KS, Reissig CJ. The behavioral pharmacology of hallucinogens. Biochem Pharm. 2008;75:17–33.17977517
64. Halberstadt AL Nichols DE Geyer MA Behavioral effects of α,α,β,β-tetradeutero-5-MeO-DMT in rats: comparison with 5-MeO-DMT administered in combination with a monoamine oxidase inhibitor Psychopharmacol (Berl) 2012 221 709 18 10.1007/s00213-011-2616-6
Halberstadt AL, Nichols DE, Geyer MA. Behavioral effects of α,α,β,β-tetradeutero-5-MeO-DMT in rats: comparison with 5-MeO-DMT administered in combination with a monoamine oxidase inhibitor. Psychopharmacol (Berl). 2012;221:709–18.
65. López-Arnau R Camarasa J Carbó M Nadal-Gratacós N Puigseslloses P Espinosa-Velasco M 3,4-Methylenedioxy methamphetamine, synthetic cathinones and psychedelics: from recreational to novel psychotherapeutic drugs Front Psychiatry 2022 13 990405 10.3389/fpsyt.2022.990405 36262632
López-Arnau R, Camarasa J, Carbó M, Nadal-Gratacós N, Puigseslloses P, Espinosa-Velasco M, et al. 3,4-Methylenedioxy methamphetamine, synthetic cathinones and psychedelics: from recreational to novel psychotherapeutic drugs. Front Psychiatry. 2022;13:990405.36262632
66. Niello M Cintulova D Hellsberg E Jäntsch K Holy M Ayatollahi LH para-Trifluoromethyl-methcathinone is an allosteric modulator of the serotonin transporter Neuropharmacology 2019 161 107615 10.1016/j.neuropharm.2019.04.021 31028773
Niello M, Cintulova D, Hellsberg E, Jäntsch K, Holy M, Ayatollahi LH, et al. para-Trifluoromethyl-methcathinone is an allosteric modulator of the serotonin transporter. Neuropharmacology. 2019;161:107615.31028773
67. Saha K Partilla JS Lehner KR Seddik A Stockner T Holy M Second-generation’ mephedrone analogs, 4-MEC and 4-MePPP, differentially affect monoamine transporter function Neuropsychopharmacology 2015 40 1321 31 10.1038/npp.2014.325 25502630
Saha K, Partilla JS, Lehner KR, Seddik A, Stockner T, Holy M, et al. Second-generation’ mephedrone analogs, 4-MEC and 4-MePPP, differentially affect monoamine transporter function. Neuropsychopharmacology. 2015;40:1321–31.25502630
68. Glatfelter GC Pham DNK Walther D Golen JA Chadeayne AR Baumann MH Synthesis, structural characterization, and pharmacological activity of novel quaternary salts of 4-substituted tryptamines ACS Omega 2022 7 24888 94 10.1021/acsomega.2c03476 35874244
Glatfelter GC, Pham DNK, Walther D, Golen JA, Chadeayne AR, Baumann MH, et al. Synthesis, structural characterization, and pharmacological activity of novel quaternary salts of 4-substituted tryptamines. ACS Omega. 2022;7:24888–94.35874244
69. Sandtner W Stockner T Hasenhuetl PS Partilla JS Seddik A Zhang Y-W Binding mode selection determines the action of ecstasy homologs at monoamine transporters Mol Pharm 2016 89 165 75 10.1124/mol.115.101394
Sandtner W, Stockner T, Hasenhuetl PS, Partilla JS, Seddik A, Zhang Y-W, et al. Binding mode selection determines the action of ecstasy homologs at monoamine transporters. Mol Pharm. 2016;89:165–75.
70. Reith MEA Blough BE Hong WC Jones KT Schmitt KC Baumann MH Behavioral, biological, and chemical perspectives on atypical agents targeting the dopamine transporter Drug Alcohol Depend 2015 147 1 19 10.1016/j.drugalcdep.2014.12.005 25548026
Reith MEA, Blough BE, Hong WC, Jones KT, Schmitt KC, Baumann MH, et al. Behavioral, biological, and chemical perspectives on atypical agents targeting the dopamine transporter. Drug Alcohol Depend. 2015;147:1–19.25548026
71. Hilber B Scholze P Dorostkar MM Sandtner W Holy M Boehm S Serotonin-transporter mediated efflux: a pharmacological analysis of amphetamines and non-amphetamines Neuropharmacology 2005 49 811 9 10.1016/j.neuropharm.2005.08.008 16185723
Hilber B, Scholze P, Dorostkar MM, Sandtner W, Holy M, Boehm S, et al. Serotonin-transporter mediated efflux: a pharmacological analysis of amphetamines and non-amphetamines. Neuropharmacology. 2005;49:811–9.16185723
72. Bhat S Newman AH Freissmuth M How to rescue misfolded SERT, DAT and NET: targeting conformational intermediates with atypical inhibitors and partial releasers Biochem Soc Trans 2019 47 861 74 10.1042/BST20180512 31064865
Bhat S, Newman AH, Freissmuth M. How to rescue misfolded SERT, DAT and NET: targeting conformational intermediates with atypical inhibitors and partial releasers. Biochem Soc Trans. 2019;47:861–74.31064865
73. Maier J Rauter L Rudin D Niello M Holy M Schmid D α-PPP and its derivatives are selective partial releasers at the human norepinephrine transporter Neuropharmacology 2021 190 108570 10.1016/j.neuropharm.2021.108570 33864800
Maier J, Rauter L, Rudin D, Niello M, Holy M, Schmid D, et al. α-PPP and its derivatives are selective partial releasers at the human norepinephrine transporter. Neuropharmacology. 2021;190:108570.33864800
74. Niello M Gradisch R Loland CJ Stockner T Sitte HH Allosteric modulation of neurotransmitter transporters as a therapeutic strategy Trends Pharm Sci 2020 41 446 63 10.1016/j.tips.2020.04.006 32471654
Niello M, Gradisch R, Loland CJ, Stockner T, Sitte HH. Allosteric modulation of neurotransmitter transporters as a therapeutic strategy. Trends Pharm Sci. 2020;41:446–63.32471654
75. Halberstadt AL Chatha M Klein AK Wallach J Brandt SD Correlation between the potency of hallucinogens in the mouse head-twitch response assay and their behavioral and subjective effects in other species Neuropharmacology 2020 167 107933 10.1016/j.neuropharm.2019.107933 31917152
Halberstadt AL, Chatha M, Klein AK, Wallach J, Brandt SD. Correlation between the potency of hallucinogens in the mouse head-twitch response assay and their behavioral and subjective effects in other species. Neuropharmacology. 2020;167:107933.31917152
76. Dong C Ly C Dunlap LE Vargas MV Sun J Hwang IW Psychedelic-inspired drug discovery using an engineered biosensor Cell 2021 184 2779 92 10.1016/j.cell.2021.03.043 33915107
Dong C, Ly C, Dunlap LE, Vargas MV, Sun J, Hwang IW, et al. Psychedelic-inspired drug discovery using an engineered biosensor. Cell. 2021;184:2779–92.33915107
77. Pottie E Stove CP In vitro assays for the functional characterization of (psychedelic) substances at the serotonin receptor 5-HT2AR J Neurochem 2022 162 39 59 10.1111/jnc.15570 34978711
Pottie E, Stove CP. In vitro assays for the functional characterization of (psychedelic) substances at the serotonin receptor 5-HT2AR. J Neurochem 2022;162:39–59.34978711
78. Poulie CBM Pottie E Simon IA Harpsøe K D’Andrea L Komarov IV Discovery of β-Arrestin-Biased 25CN-NBOH-Derived 5-HT2AReceptor Agonists J Med Chem 2022 65 12031 43 10.1021/acs.jmedchem.2c00702 36099411
Poulie CBM, Pottie E, Simon IA, Harpsøe K, D’Andrea L, Komarov IV, et al. Discovery of β-Arrestin-Biased 25CN-NBOH-Derived 5-HT2AReceptor Agonists. J Med Chem. 2022;65:12031–43.36099411
79. Araújo AM Carvalho F Bastos M de L Guedes de Pinho P Carvalho M The hallucinogenic world of tryptamines: an updated review Arch Toxicol 2015 89 1151 73 10.1007/s00204-015-1513-x 25877327
Araújo AM, Carvalho F, Bastos M, de L, Guedes de Pinho P, Carvalho M. The hallucinogenic world of tryptamines: an updated review. Arch Toxicol. 2015;89:1151–73.25877327
80. Halberstadt AL Koedood L Powell SB Geyer MA Differential contributions of serotonin receptors to the behavioral effects of indoleamine hallucinogens in mice J Psychopharmacol 2011 25 1548 61 10.1177/0269881110388326 21148021
Halberstadt AL, Koedood L, Powell SB, Geyer MA. Differential contributions of serotonin receptors to the behavioral effects of indoleamine hallucinogens in mice. J Psychopharmacol. 2011;25:1548–61.21148021
81. Pokorny T Preller KH Kraehenmann R Vollenweider FX Modulatory effect of the 5-HT1A agonist buspirone and the mixed non-hallucinogenic 5-HT1A/2A agonist ergotamine on psilocybin-induced psychedelic experience Eur Neuropsychopharmacol 2016 26 756 66 10.1016/j.euroneuro.2016.01.005 26875114
Pokorny T, Preller KH, Kraehenmann R, Vollenweider FX. Modulatory effect of the 5-HT1A agonist buspirone and the mixed non-hallucinogenic 5-HT1A/2A agonist ergotamine on psilocybin-induced psychedelic experience. Eur Neuropsychopharmacol. 2016;26:756–66.26875114
82. Berendsen HHG Broekkamp CLE Behavioural evidence for functional interactions between 5-HT-receptor subtypes in rats and mice Br J Pharm 1990 101 667 73 10.1111/j.1476-5381.1990.tb14138.x
Berendsen HHG, Broekkamp CLE. Behavioural evidence for functional interactions between 5-HT-receptor subtypes in rats and mice. Br J Pharm. 1990;101:667–73.
83. Fantegrossi WE Reissig CJ Katz EB Yarosh HL Rice KC Winter JC Hallucinogen-like effects of N,N-dipropyltryptamine (DPT): Possible mediation by serotonin 5-HT1A and 5-HT2A receptors in rodents Pharm Biochem Behav 2008 88 358 65 10.1016/j.pbb.2007.09.007
Fantegrossi WE, Reissig CJ, Katz EB, Yarosh HL, Rice KC, Winter JC. Hallucinogen-like effects of N,N-dipropyltryptamine (DPT): Possible mediation by serotonin 5-HT1A and 5-HT2A receptors in rodents. Pharm Biochem Behav. 2008;88:358–65.
84. Leonard JB Anderson B Klein-Schwartz W Does getting high hurt? Characterization of cases of LSD and psilocybin-containing mushroom exposures to national poison centers between 2000 and 2016 J Psychopharmacol 2018 32 1286 94 10.1177/0269881118793086 30182795
Leonard JB, Anderson B, Klein-Schwartz W. Does getting high hurt? Characterization of cases of LSD and psilocybin-containing mushroom exposures to national poison centers between 2000 and 2016. J Psychopharmacol. 2018;32:1286–94.30182795
85. Nisijima K Yoshino T Yui K Katoh S Potent serotonin (5-HT)2A receptor antagonists completely prevent the development of hyperthermia in an animal model of the 5-HT syndrome Brain Res 2001 890 23 31 10.1016/S0006-8993(00)03020-1 11164765
Nisijima K, Yoshino T, Yui K, Katoh S. Potent serotonin (5-HT)2A receptor antagonists completely prevent the development of hyperthermia in an animal model of the 5-HT syndrome. Brain Res. 2001;890:23–31.11164765
86. Bill DJ Knight M Forster EA Fletche A Direct evidence for an important species difference in the mechanism of 8‐OH‐DPAT‐induced hypothermia Br J Pharm 1991 103 1857 64 10.1111/j.1476-5381.1991.tb12342.x
Bill DJ, Knight M, Forster EA, Fletche A. Direct evidence for an important species difference in the mechanism of 8‐OH‐DPAT‐induced hypothermia. Br J Pharm. 1991;103:1857–64.
87. Cryan JF Kelliher P Kelly JP Leonard BE Comparative effects of serotonergic agonists with varying efficacy at the 5-HT(1A) receptor on core body temperature: Modification by the selective 5-HT(1A) receptor antagonist WAY 100635 J Psychopharmacol 1999 13 278 83 10.1177/026988119901300310 10512084
Cryan JF, Kelliher P, Kelly JP, Leonard BE. Comparative effects of serotonergic agonists with varying efficacy at the 5-HT(1A) receptor on core body temperature: Modification by the selective 5-HT(1A) receptor antagonist WAY 100635. J Psychopharmacol. 1999;13:278–83.10512084
88. Newman-Tancredi A Depoortère R Carilla-Durand E Tarayre JP Kleven M Koek W NLX-112, a highly selective 5-HT1A receptor agonist: Effects on body temperature and plasma corticosterone levels in rats Pharm Biochem Behav 2018 165 56 62 10.1016/j.pbb.2017.11.002
Newman-Tancredi A, Depoortère R, Carilla-Durand E, Tarayre JP, Kleven M, Koek W, et al. NLX-112, a highly selective 5-HT1A receptor agonist: Effects on body temperature and plasma corticosterone levels in rats. Pharm Biochem Behav. 2018;165:56–62.
89. Hedlund PB Kelly L Mazur C Lovenberg T Sutcliffe JG Bonaventure P 8-OH-DPAT acts on both 5-HT1A and 5-HT7 receptors to induce hypothermia in rodents Eur J Pharm 2004 487 125 32 10.1016/j.ejphar.2004.01.031
Hedlund PB, Kelly L, Mazur C, Lovenberg T, Sutcliffe JG, Bonaventure P. 8-OH-DPAT acts on both 5-HT1A and 5-HT7 receptors to induce hypothermia in rodents. Eur J Pharm. 2004;487:125–32.
90. Abiero A Ryu IS Botanas CJ Custodio RJP Sayson LV Kim M Four novel synthetic tryptamine analogs induce head-twitch responses and increase 5-HTR2a in the prefrontal cortex in mice Biomol Ther 2020 28 83 91 10.4062/biomolther.2019.049
Abiero A, Ryu IS, Botanas CJ, Custodio RJP, Sayson LV, Kim M, et al. Four novel synthetic tryptamine analogs induce head-twitch responses and increase 5-HTR2a in the prefrontal cortex in mice. Biomol Ther. 2020;28:83–91.
91. Krebs-Thomson K Ruiz EM Masten V Buell M Geyer MA The roles of 5-HT1A and 5-HT2 receptors in the effects of 5-MeO-DMT on locomotor activity and prepulse inhibition in rats Psychopharmacology 2006 189 319 29 10.1007/s00213-006-0566-1 17013638
Krebs-Thomson K, Ruiz EM, Masten V, Buell M, Geyer MA. The roles of 5-HT1A and 5-HT2 receptors in the effects of 5-MeO-DMT on locomotor activity and prepulse inhibition in rats. Psychopharmacology. 2006;189:319–29.17013638
