Receptor Interaction Profiles of 4-Alkoxy-3,5-Dimethoxy-Phenethylamines (Mescaline Derivatives) and Related Amphetamines

This neurochemistry paper (2022) explored the effects of several mescaline derivatives, scalines and 3C-scalines, on monoamine receptors in vitro. Scalines and 3C-scalines interacted with the 5-HT_2A and 5-HT_2C receptors, with preference to the 5-HT_2A receptor, and bound with higher affinities (up to 63-fold and 34-fold increase, respectively) when compared to mescaline.

Abstract

“3,4,5-Trimethoxyphenethylamine (mescaline) is a psychedelic alkaloid found in peyote cactus. Related 4-alkoxy-3,5-dimethoxy-substituted phenethylamines (scalines) and amphetamines (3C-scalines) are reported to induce similarly potent psychedelic effects and are therefore potential novel therapeutics for psychedelic-assisted therapy. Herein, several pharmacologically uninvestigated scalines and 3C-scalines were examined at key monoamine targets in vitro. Binding affinity at human serotonergic 5-HT_1A, 5-HT_2A, and 5-HT_2C, adrenergic α_1A and α_2A, and dopaminergic D₂ receptors, rat and mouse trace amine-associated receptor 1 (TAAR1), and human monoamine transporters were assessed using target specific transfected cells. Furthermore, activation of human 5-HT_2A and 5-HT_2B receptors, and TAAR1 was examined. Generally, scalines and 3C-scalines bound with weak to moderately high affinity to the 5-HT_2A receptor (K_i = 150–12,000 nM). 3C-scalines showed a marginal preference for the 5-HT_2A vs the 5-HT_2C and 5-HT_1A receptors whereas no preference was observed for the scalines. Extending the 4-alkoxy substituent increased 5-HT_2A and 5-HT_2C receptors binding affinities, and enhanced activation potency and efficacy at the 5-HT_2A but not at the 5-HT_2B receptor. Introduction of fluorinated 4-alkoxy substituents generally increased 5-HT_2A and 5-HT_2C receptors binding affinities and increased the activation potency and efficacy at the 5-HT_2A and 5-HT_2B receptors. Overall, no potent affinity was observed at non-serotonergic targets. As observed for other psychedelics, scalines and 3C-scalines interacted with the 5-HT_2A and 5-HT_2C receptors and bound with higher affinities (up to 63-fold and 34-fold increase, respectively) when compared to mescaline.”

Authors: Karolina E. Kolaczynska, Dino Luethi, Daniel Trachsel, Marius C. Hoener & Matthias E. Liechti

Summary

1 INTRODUCTION

Serotonin modulates vital central nervous system processes like appetite, sexual activity, memory, attention, or sleep through interactions with various 5-HT receptors. The 5-HT2 receptor subtype is a key pharmacological target for therapeutic drugs including antidepressants, anxiolytics, and antipsychotics. Ligands for each receptor isoform have been synthesized, but lack subtype selectivity. The 5-HT2A and 5-HT2C receptors are both involved in the induction of psychedelic effects associated with classical psychedelics like LSD and psilocybin, as well as novel derivatives thereof. They both mediate their effects via Gq-protein-mediated activation of phospholipase C (PLC), which leads to calcium release and further downstream effects. Psychedelic phenethylamines can be classified into three distinct groups based on their aryl substitution pattern, and most active compounds contain two methoxy groups allocated at the 2- or 3-position and at the 5- or 6-position.

Mescaline, a natural ingredient of the psychoactive cactus peyote, was discovered in 1897 and its methyl congener, 3,4,5-trimethoxyamphetamine, was synthesized in 1947. The thorough investigation of the structure-activity relationship (SAR) of 3,4,5-trisubstituted phenethylamines has been slow largely due to early reports of their relatively weak human potencies. However, the focus has shifted even more to 2,4,5-trisubstituted derivatives when Alexander Shulgin discovered that some of these substances are active at doses well below 10 mg.

The 2,4,6-trisubstituted derivatives are even less investigated than the 2,4,5-trisubstituted derivatives, but they seem to show a more shared SAR with the 2,4,5-series than the 3,4,5-trisubstituted series. Furthermore, conformational restriction of the MeO groups in both series leads to increased potencies. Another significant structural modifier is the presence of an -methyl (-Me) group. This group has a small effect on binding affinity of 2,4,5-trisubstituted derivatives at 5-HT2A/2C receptors. In vivo, introduction of an -Me group into the 2,4,5-series has noteworthy effects on drug discrimination experiments and head-twitch response. These effects can be explained by an increase in hydrophobic properties and metabolic stability, as well as a stronger intrinsic activity at the receptor.

The R-configuration of amphetamines behaved as the more potent enantiomers, with a higher affinity to the 5-HT2A receptor and higher functional potency and functional efficacy (intrinsic activity) than the S-configuration.

Animal and human observations with 2,4,5-trisubstituted derivatives are in strong contrast to what has been observed for scalines and 3C-scalines. The effects of 4-position substituents on 5-HT2A/2C receptor interaction properties are not entirely understood and require further investigation. Significant changes have been achieved with mescaline derivatives bearing larger carbon chain lengths at the 4-alkoxy position, including escaline (15), isoproscaline (IP; 22), proscaline (24), allylescaline (AL; 30), and methallylescaline (MAL; 32).

The nomenclature for naming derivatives with a structural modification of the 4-substituent of mescaline involves a common name in respect to their 4-substituent, such as escaline (15), fluoroescaline (16), proscaline (24), allylescaline (30) etc.

In light of the renewed interest in psychedelic substances, we investigated the binding and activation properties of different mescaline derivatives at human monoaminergic receptors and at trace amine-associated receptor 1.

2.1 Drugs

The 3,5-dimethoxy-4-substituted phenethylamines and 3,5-dimethoxy-4-substituted amphetamines were synthesized as racemates and provided as hydrochloride salts for pharmacological testing by ReseaChem. The purities of all substances were >98%.

2.2 Radioligand Receptor and Transporter Binding Assays

Radioligand binding affinity for monoamine receptors and transporters was assessed using cell line derived membrane preparations overexpressing respective monoamine receptors or transporters. The specific binding of radioligand to the target site was defined by measuring the difference between total binding and nonspecific binding.

The following radioligands and their respective competitors were used: 8-OH-DPAT, Ketanserin, Mesulergine, RO5166017, Prazosin, Rauwolscine, Phentolamine, hD2 receptor, N-methyl-[3H] nisoxetine, citalopram, WIN35,428 and indatraline.

2.3 Activity at the Serotonin 5-HT2A Receptor

NIH-3T3 cells expressing human 5-HT2A receptor were seeded in poly-D-lysine-coated 96-well plates and incubated with dye solution for 1 h at 37°C. The rise in fluorescence was measured and EC50 values were calculated from the concentration-response curves.

2.4 Activity at the Serotonin 5-HT2B Receptor

To assess the functional activity of the serotonin 5-HT2B receptor, HEK 293 cells were seeded at a density of 50,000 cells per well in 96-well poly-D-lysine-coated plates overnight at 37°C, and then exposed to 25 l of test drugs diluted in assay buffer online.

2.5 Activity at the Human TAAR1

To assess the functional activity of the human TAAR1, HEK 293 cells expressing recombinant human TAAR1 were grown in 250 ml falcon culture flasks containing 30 ml of high glucose DMEM at 37°Cand5%CO2/ 95% air.

2.6 Monoamine Uptake Transporter Inhibition

HEK 293 cells stably transfected with the human serotonin, norepinephrine or dopamine transporters were used to examine the activity of the scalines and 3C-scalines at monoamine transporters at pharmacologically relevant concentrations. The cells were incubated with the test drug, vehicle control, or transporter-specific inhibitors for 10 min, and then transferred to 500 l microcentrifuge tubes containing 50 l of 3M KOH and 200 l silicon oil. Monoamine uptake was quantified by liquid scintillation counting.

2.7 Statistical Analysis

Calculations and analyses were performed using Prism 7.0a (GraphPad, San Diego, CA, United States). Receptor affinity binding (Ki) was determined using the Cheng-Prusoff equation and maximal activation activity (efficacy) was compared to control using 1-way ANOVA analysis of variance followed by a Dunett’s multiple-comparison test.

4.1 5-HT Receptor Binding

Smaller lipophilic 4-substituents at the 4-position of 2,5-dimethoxy substituted phenethylamines and amphetamines lead to agonistic properties, while larger lipophilic substituents at the 4-position lead to antagonistic effects at the 5-HT2A/2C receptors.

In vitro studies of 3,4,5-trisubstituted phenethylamines and amphetamines have shown that they have the lowest 5-HT2A receptor affinities among psychedelic phenethylamines, but SAR investigations of 4-alkoxysubstituted 3,5-dimethoxyphenethylamines have revealed a similar trend.

4.1.1 5-HT1A Receptor Binding

Only a few phenethylamine derivatives slightly augmented the binding affinity at the 5-HT1A receptor when compared to mescaline, indicating that the receptor does not tolerate this steric expansion.

4.1.2 5-HT2A Receptor Binding

All tested phenethylamine derivatives displayed an increased affinity at the 5-HT2A receptor compared to mescaline, except for DFIP (23). However, these 5-HT2A receptor interactions were less potent when compared to other psychedelic phenethylamines. It is well established that phenethylamine psychedelics induce their psychoactive effects mainly by agonistic action at the 5-HT2A receptor. However, different downstream signaling cascades, biased agonism, and other pharmacological targets may contribute to the subjective effects.

The most promising modifications of amphetamines were 4-trifluoromethoxy (TFM, 13), 4-methallyloxy (MAL, 32), and 4-benzyloxy (BZ, 33), which resulted in 17- to 63-fold higher affinity at the 5-HT2A receptor.

All examined structural modifications on the amphetamine derivatives increased affinity at the 5-HT2A receptor when compared to TMA (6). The most potent amphetamine derivatives, 3C-P (4-propyloxy substituent) and 3C-AL (4-allyloxy substituent), showed at least a 10-fold increase in 5-HT2A receptor affinity compared with 6.

4.1.4 5-HT Receptor Subtype Selectivity

Most 3,4,5-substituted phenethylamine and amphetamine derivatives had moderate to high preference for the 5-HT2A over the 5-HT1A receptor, similar to psychedelic 2C derivatives investigated earlier.

The tested derivatives showed similar affinities at the 5-HT2A and 5-HT2C receptors, with some compounds being slightly more selective for one or the other receptor subtype. However, other factors such as interactions with other monoamine receptors, lipophilicity, receptor activation, functional selectivity, and metabolism could also affect potency.

In general, the extension of the carbon chain or fluorination of the 4-alkyloxy moiety in the 3,4,5-substituted series moderately increased the binding affinity at the 5-HT1A receptor for some phenethylamine derivatives, while the number of fluorine atoms at the 4-alkyloxy moiety proportionally increased the binding affinity at the 5-HT2A and 5-HT2C receptor. A previous investigation revealed that the introduction of an -Me group causes slight increases in binding affinity at the 5-HT2A but not 5-HT2C receptors.

4.2 Activation Potency and Efficacy at the 5-HT2A and 5-HT2B Receptors

The 3-C-scalines TFM (13), MAL (32), and BZ (33), had high 5-HT2A receptor affinities and high activation potency, suggesting that they may be potentially psychedelic in humans. However, a discrepancy between binding and activation was observed for various substances, suggesting that other signaling events may better predict the potency.

5 CONCLUSION

We pharmacologically examined a series of 4-alkoxy-substituted 3,5-dimethoxyphenethylamines and 4-alkoxy-substituted 3,5-dimethoxy-amphetamines in vitro for their serotonergic and adrenergic receptor interactions. Our results suggest therapeutic potential for several novel mescaline derivatives as well.