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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-HT2A). 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-HT2A and the serotonin 1A receptor (5-HT1A). The same compounds displayed affinity for 5-HT2A and 5-HT1A in mouse brain tissue in vitro and exhibited agonist efficacy in assays examining 5-HT2A-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 (ED50 0.11–0.29 mg/kg) indicative of psychedelic-like activity. Head twitches were blocked by 5-HT2A antagonist pretreatment, supporting 5-HT2A involvement. Both secondary and tertiary amines decreased body temperature and locomotor activity at higher doses, the effects of which were blocked by 5-HT1A 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.

Novel synthetic opioids continue to emerge on recreational drug markets worldwide. In response to legislative bans on fentanyl analogues, non-fentanyl structural templates, such as 2-benzylbenzimidazoles ('nitazenes'), are being exploited to create new -opioid receptor (MOR) agonists. Here, we pharmacologically characterize an emerging cyclic analogue of etonitazene, called N-pyrrolidino etonitazene (etonitazepyne), using in vitro and in vivo methods. A series of analytically confirmed fatalities is described to complement preclinical findings. Radioligand binding assays in rat brain tissue revealed that N-pyrrolidino etonitazene has high affinity for MOR (Ki=4.09 nM) over -opioid (Ki=959 nM) and -opioid (Ki=980 nM) receptors. In a MOR--arrestin2 activation assay, N-pyrrolidino etonitazene displayed high potency (EC50=0.348 nM), similar to etonitazene (EC50=0.360 nM), and largely exceeding the potencies of fentanyl (EC50=14.9 nM) and morphine (EC50=290 nM). When administered s.c. to male Sprague Dawley rats, N-pyrrolidino etonitazene induced opioid-like antinociceptive, cataleptic, and thermic effects. Its potency in the hot plate test (ED50=0.0017 mg/kg) was 10fold and 2,000-fold greater than fentanyl (ED50=0.0209 mg/kg) and morphine (ED50=3.940 mg/kg), respectively. Twenty-one overdose fatalities associated with N-pyrrolidino etonitazene were found to contain low blood concentrations of the drug (median=2.2 ng/mL), commonly in the context of polysubstance use. N-Pyrrolidino etonitazene was reported as a cause of death in at least two cases, demonstrating toxicity in humans. We demonstrate that N-pyrrolidino etonitazene is an extremely potent MOR agonist that is likely to present high risk to users. Continued vigilance is required to identify and characterize emergent 2benzylbenzimidazoles, and other non-fentanyl opioids, as they appear in the marketplace.

Synthetic cathinones are a class of new psychoactive substances that induce psychostimulant effects and pose risk for hospitalizations, overdose, and death. At the present time, derivatives of the synthetic cathinone, methylone, are being confiscated in nonmedical (i.e., recreational) drug markets worldwide. In particular, eutylone is a newly emerging methylone analog that possesses ethyl groups at the α-carbon and amine positions. Little information is available about the pharmacological effects of eutylone, but based on its structure, we surmised that the compound interacts with monoamine transporters in the brain. To test this hypothesis, we compared the effects of eutylone and its structural isomers, dibutylone and pentylone, using in vitro transporter assays in rat brain synaptosomes and in vivo locomotor activity assessments in mice. All drugs displayed dose-related inhibition of [ 3 H]neurotransmitter uptake at dopamine transporters (DAT) and norepinephrine transporters (NET), but effects at DAT were 10-fold more potent (IC 50 = 120 nM). Eutylone and pentylone inhibited uptake at serotonin transporters (SERT), while dibutylone did not. Additionally, eutylone and pentylone displayed weak partial releasing actions at SERT which achieved 50% of maximal response. All drugs stimulated dose-related locomotion in mice, and eutylone was most potent and efficacious in this regard (ED 50 = 2 mg/kg, sc). Our results demonstrate that eutylone is a hybrid transporter compound with uptake inhibition properties at DAT and NET but substrate activity at SERT. The effects of eutylone are similar to those produced by pentylone, which suggests that eutylone will exhibit abuse liability and pose risks for psychostimulant side-effects in human users.

Analogues of 4-phosphoryloxy-N,N-dimethyl­tryptamine (psilocybin) are being sold on recreational drug markets and developed as potential medications for psychedelic-assisted therapies. Many of these tryptamine-based psilocybin analogues produce psychedelic-like effects in rodents and humans primarily by agonist activity at serotonin 2A receptors (5-HT2A). However, the comprehensive pharmacological target profiles for these compounds compared to psilocybin and its active metabolite 4-hydroxy-N,N-dimethyl­tryptamine (psilocin) are unknown. The present study determined the receptor binding profiles of various tryptamine-based psychedelics structurally related to psilocybin across a broad range of potential targets. Specifically, we examined tryptamine psychedelics with different 4-position (hydroxy, acetoxy, propionoxy) and N,N-dialkyl (dimethyl, methyl-ethyl, diethyl, methyl-propyl, ethyl-propyl, diisopropyl, methyl-allyl, diallyl) substitutions. Further, the psilocybin analogue 4-propionoxy-N,N-dimethyl­tryptamine (4-PrO-DMT) was administered to mice in experiments measuring head twitch response (HTR), locomotor activity, and body temperature. Overall, the present pharmacological profile screening data show that the tryptamine psychedelics target multiple serotonin receptors, including serotonin 1A receptors (5-HT1A). 4-Acetoxy and 4-propionoxy analogues of 4-hydroxy compounds displayed somewhat weaker binding affinities but similar target profiles across 5-HT receptors and other identified targets. Additionally, differential binding screen profiles were observed with N,N-dialkyl position variations across several non-5-HT receptor targets (i.e., alpha receptors, dopamine receptors, histamine receptors, and serotonin transporters), which could impact in vivo pharmacological effects of the compounds. In mouse experiments, 4-PrO-DMT displayed dose-related psilocybin-like effects to produce 5-HT2A-mediated HTR (0.3–3 mg/kg s.c.) as well as 5-HT1A-mediated hypothermia and hypolocomotion (3–30 mg/kg s.c.). Lastly, our data support a growing body of evidence that the 5-HT2A-mediated HTR induced by tryptamine psychedelics is attenuated by 5-HT1A receptor agonist activity at high doses in mice.

Psychedelics are a class of drugs that produce unique subjective effects via agonist actions at the 5-hydroxytryptamine 2A receptor (5-HT 2A ). The 5-HT 2A -mediated head twitch response (HTR) in rodents is used as a reliable proxy for psychedelic drug activity in humans, but existing methods for measuring HTRs require surgery or time-consuming visual scoring. In the present work, we validated a simple noninvasive method for quantitating HTRs using computer-based analysis of experimental video recordings. Male C57BL/6J mice received injections of the 5-HT 2 receptor agonist (±)2,5-dimethoxy-4-iodoamphetamine (DOI; 0.03−3 mg/kg, s.c.) and were placed into cylindrical arenas. High frame rate videos were recorded via cameras mounted above the arenas. Antagonist experiments, which entailed pretreatment with the 5-HT 2A antagonist M100907 (0.01 or 0.1 mg/kg s.c.) prior to DOI (1 mg/kg s.c.), were also recorded. The experimental videos were analyzed for HTRs using a newly developed feature of a commercial software package and compared to visual scoring carried out by trained observers. As expected, DOI produced dose-related increases in HTRs, which were blocked by M100907. Computer scoring was positively correlated with visual scoring, and no statistical difference between the two methods was found. The software captured nearly all visually observed HTRs, false positives induced by other behaviors (e.g., grooming) were rare and easily identified, and results were improved by optimizing lighting conditions. Our findings demonstrate the utility of combining high frame rate video recordings with commercial software analyses to measure HTRs, validating an additional reliable method to study psychedelic-like drug activity in mice.

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