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

4-Phosphoryloxy- N , N -dimethyltryptamine (psilocybin) is a naturally occurring tertiary amine found in many mushroom species. Psilocybin is a prodrug for 4-hydroxy- N , N -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- N -methyltryptamine (baeocystin) and the quaternary ammonium 4-phosphoryloxy- N , N , N -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 in vitro and in vivo 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 in vitro 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- N , N -dimethyltryptamine (psilacetin) induced head twitch responses (ED 50 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.