<i>N</i> -Benzyl-tryptamine Derivatives as Serotonin 5-HT _2A Receptor (5-HT _2A R) Agonists: Focus on Different In Vitro 5-HT _2A R Activity Profiles

<i>N</i>-Benzyl-derived phenethylamines are highly active (psychedelic) 5-HT_2AR (serotonin 2A receptor) agonists used as biochemical tools and on the drug market. The impact of adding an <i>N</i>-benzyl substituent to the tryptamine core structure has scarcely been studied. A recent systematic exploration of <i>N</i>-benzyl-substituted (5-MeO-)tryptamines revealed a surprisingly low activity in a Ca²⁺ mobilization assay for certain analogues. Considering the increased number of reports on biased agonism at the 5-HT_2AR, with particular examples in the group of phenethylamines containing an <i>N</i>-benzyl substituent, a series of 16 of these (5-MeO-)tryptamine derivatives (14 of which containing an <i>N</i>-benzyl group) was evaluated in two additional assays. Specifically, two highly similar yet complementary NanoBiT assays, monitoring the recruitment of β-arrestin 2 (βarr2) or miniGα_q to the 5-HT_2AR, were applied. The functional data allowed assessment of the impact of different substituents on the compounds' 5-HT_2AR activity: (i) the introduction of an <i>N</i>-benzyl group on the (5-MeO-)tryptamine core; (ii) the substitution of the <i>N</i>-benzyl at different positions; and (iii) the presence of a 5-MeO group on the tryptamine core structure. Interestingly, the <i>N</i>-benzyl-substituted (5-MeO-)tryptamines were more active in βarr2 than in the miniGα_q recruitment assay, and biased agonism was assessed by both quantitative and qualitative methods. Aside from the estimation of structure-activity relationships and biased agonism, the current data set allowed for a comparison of the functional data between different assays, hence providing a better understanding of the translatability of results from one assay to the other.