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The N -benzylphenethylamines (NBOMes) are a class of ligands from which compounds with impressive selectivity for the serotonin 2A receptor (5-HT 2A R) over the closely related serotonin 2C receptor (5-HT 2C R) have emerged. These include 4-(2-((2-hydroxybenzyl)amino)ethyl)-2,5-dimethoxybenzonitrile (25CN-NBOH, 1 ) and 2-(2,5-dimethoxy-4-bromobenzyl)-6-(2-methoxyphenyl)piperidine (DMPMBB, 2 ). The present work entails the synthesis and characterization of ligands wherein the structures of these two molecules have been fused. The desired compounds were accessed by a six-step synthetic procedure followed by the chiral resolution of the resulting racemic mixtures, giving one active (( S , S )- 3 ) and three essentially inactive stereoisomers. In silico experiments support that one of the four possible stereoisomers would be active. Further in silico investigations showed that 1 , 2 , and ( S , S )- 3 share a common binding mode, further supporting the shared stereochemistry between the active enantiomer (( S , S )- 3 ) and 2 ., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

The 2,5-dimethoxyphenethylamine (2,5-PEA) scaffold is recognized as a motif conferring potent agonist activity at the serotonin 2A receptor (5-HT 2A R). The 2,5-dimethoxy motif is present in several classical phenethylamine psychedelics such as 2,4,5- trimethoxyamphetamine (TMA-2), 2,5-dimethoxy-4-methylamphetamine (DOM), 2,5-dimethoxy-4-iodoamphetamine (DOI), 2,5- dimethoxy-4-bromoamphetamine (DOB), 2,5-dimethoxy-4-bromophenethylamine (2C-B), and 2,5-dimethoxy-4-iodophenethylamine (2C-I), and it has previously been suggested that this structural motif is essential for 5-HT 2A R activation. In the present study, we present data that challenges this assumption. The 2- and 5-desmethoxy derivatives of 2C-B and DOB were synthesized, and their pharmacological profiles were evaluated in vitro at 5-HT 2A R and 5-HT 2C R in binding and functional assays and in vivo by assessing their induction of the head-twitch response in mice. Elimination of either the 2- or 5-methoxy group leads to a modest drop in binding affinity and functional potency at 5-HT 2A R and 5-HT 2C R, which was more pronounced upon removal of the 2-methoxy group. However, this trend was not mirrored in vivo , as removal of either methoxy group resulted in significant reduction in the ability of the compounds to induce the head-twitch response in mice. Thus, the 2,5-dimethoxyphenethylamine motif appears to be important for in vivo potency of phenethylamine 5-HT 2A R agonists, but this does not correlate to the relative affinity and potency of the ligands at the recombinant 5-HT 2A R.

Herein, we report the development of bitopic ligands aimed at targeting the orthosteric binding site (OBS) and a metastable binding site (MBS) within the same receptor unit. Previous molecular dynamics studies on ligand binding to the β 2 -adrenergic receptor (β 2 AR) suggested that ligands pause at transient, less-conserved MBSs. We envisioned that MBSs can be regarded as allosteric binding sites and targeted by homobivalent bitopic ligands linking two identical pharmacophores. Such ligands were designed based on docking of the antagonist ( S )-alprenolol into the OBS and an MBS and synthesized. Pharmacological characterization revealed ligands with similar potency and affinity, slightly increased β 2 /β 1 AR-selectivity, and/or substantially slower β 2 AR off-rates compared to ( S )-alprenolol. Truncated bitopic ligands suggested the major contribution of the metastable pharmacophore to be a hydrophobic interaction with the β 2 AR, while the linkers alone decreased the potency of the orthosteric fragment. Altogether, the study underlines the potential of targeting MBSs for improving the pharmacological profiles of ligands.