Structure–Activity Relationships for Psilocybin, Baeocystin, Aeruginascin, and Related Analogues to Produce Pharmacological Effects in Mice

4-Phosphoryloxy-<i>N</i>,<i>N</i>-dimethyltryptamine (psilocybin) is a naturally occurring tertiary amine found in many mushroom species. Psilocybin is a prodrug for 4-hydroxy-<i>N</i>,<i>N</i>-dimethyltryptamine (psilocin), which induces psychedelic effects via agonist activity at the serotonin (5-HT) 2A receptor (5-HT_2A). Several other 4-position ring-substituted tryptamines are present in psilocybin-containing mushrooms, including the secondary amine 4-phosphoryloxy-<i>N</i>-methyltryptamine (baeocystin) and the quaternary ammonium 4-phosphoryloxy-<i>N</i>,<i>N</i>,<i>N</i>-trimethyltryptamine (aeruginascin), but these compounds are not well studied. Here, we investigated the structure-activity relationships for psilocybin, baeocystin, and aeruginascin, as compared to their 4-acetoxy and 4-hydroxy analogues, using <i>in vitro</i> and <i>in vivo</i> methods. Broad receptor screening using radioligand binding assays in transfected cells revealed that secondary and tertiary tryptamines with either 4-acetoxy or 4-hydroxy substitutions display nanomolar affinity for most human 5-HT receptor subtypes tested, including the 5-HT_2A and the serotonin 1A receptor (5-HT_1A). The same compounds displayed affinity for 5-HT_2A and 5-HT_1A in mouse brain tissue <i>in vitro</i> and exhibited agonist efficacy in assays examining 5-HT_2A-mediated calcium mobilization and β-arrestin 2 recruitment. In mouse experiments, only the tertiary amines psilocin, psilocybin, and 4-acetoxy-<i>N</i>,<i>N</i>-dimethyltryptamine (psilacetin) induced head twitch responses (ED₅₀ 0.11-0.29 mg/kg) indicative of psychedelic-like activity. Head twitches were blocked by 5-HT_2A antagonist pretreatment, supporting 5-HT_2A involvement. Both secondary and tertiary amines decreased body temperature and locomotor activity at higher doses, the effects of which were blocked by 5-HT_1A antagonist pretreatment. Across all assays, the pharmacological effects of 4-acetoxy and 4-hydroxy compounds were similar, and these compounds were more potent than their 4-phosphoryloxy counterparts. Importantly, psilacetin appears to be a prodrug for psilocin that displays substantial serotonin receptor activities of its own.