Psychedelic-like Activity of Norpsilocin Analogues.

Primary metabolites of mushroom tryptamines, psilocybin and baeocystin (i.e., psilocin and norpsilocin), exhibit potent agonist activity at the serotonin 2A receptor (5-HT2A) in vitro but differ in their 5-HT2A-mediated effects in vivo. In particular, psilocin produces centrally mediated psychedelic effects in vivo, whereas norpsilocin, differing only by the loss of an N-methyl group, is devoid of psychedelic-like effects. These observations suggest that the secondary methylamine group in norpsilocin impacts its central nervous system (CNS) bioavailability but not its receptor pharmacodynamics. To test this hypothesis, eight norpsilocin derivatives were synthesized with varied secondary alkyl-, allyl-, and benzylamine groups, primarily aiming to increase their lipophilicity and brain permeability. Structure-activity relationships for the norpsilocin analogues were evaluated using the mouse head-twitch response (HTR) as a proxy for CNS-mediated psychedelic-like effects. HTR studies revealed that extending the N-methyl group of norpsilocin by a single methyl group, to give the corresponding secondary N-ethyl analogue (4-HO-NET), was sufficient to produce psilocin-like activity (median effective dose or ED50 = 1.4 mg/kg). Notably, N-allyl, N-propyl, N-isopropyl, and N-benzyl derivatives also induced psilocin-like HTR activity (ED50 = 1.1-3.2 mg/kg), with variable maximum effects (26-77 total HTR events). By contrast, adding bulkier tert-butyl or cyclohexyl groups in the same position did not elicit psilocin-like HTRs. Pharmacological assessments of the tryptamine series in vitro demonstrated interactions with multiple serotonin receptor subtypes, including 5-HT2A, and other CNS signaling proteins (e.g., sigma receptors). Overall, our data highlight key structural requirements for CNS-mediated psychedelic-like effects of norpsilocin analogues.

Identification of 5-HT2A receptor signaling pathways associated with psychedelic potential.

Serotonergic psychedelics possess considerable therapeutic potential. Although 5-HT2A receptor activation mediates psychedelic effects, prototypical psychedelics activate both 5-HT2A-Gq/11 and ß-arrestin2 transducers, making their respective roles unclear. To elucidate this, we develop a series of 5-HT2A-selective ligands with varying Gq efficacies, including ß-arrestin-biased ligands. We show that 5-HT2A-Gq but not 5-HT2A-ß-arrestin2 recruitment efficacy predicts psychedelic potential, assessed using head-twitch response (HTR) magnitude in male mice. We further show that disrupting Gq-PLC signaling attenuates the HTR and a threshold level of Gq activation is required to induce psychedelic-like effects, consistent with the fact that certain 5-HT2A partial agonists (e.g., lisuride) are non-psychedelic. Understanding the role of 5-HT2A Gq-efficacy in psychedelic-like psychopharmacology permits rational development of non-psychedelic 5-HT2A agonists. We also demonstrate that ß-arrestin-biased 5-HT2A receptor agonists block psychedelic effects and induce receptor downregulation and tachyphylaxis. Overall, 5-HT2A receptor Gq-signaling can be fine-tuned to generate ligands distinct from classical psychedelics.

Identification of 5-HT2A Receptor Signaling Pathways Responsible for Psychedelic Potential.

Serotonergic psychedelics possess considerable therapeutic potential. Although 5-HT2A receptor activation mediates psychedelic effects, prototypical psychedelics activate both 5-HT2A-Gq/11 and ß-arrestin2 signaling, making their respective roles unclear. To elucidate this, we developed a series of 5-HT2A-selective ligands with varying Gq efficacies, including ß-arrestin-biased ligands. We show that 5-HT2A-Gq but not 5-HT2A-ß-arrestin2 efficacy predicts psychedelic potential, assessed using head-twitch response (HTR) magnitude in male mice. We further show that disrupting Gq-PLC signaling attenuates the HTR and a threshold level of Gq activation is required to induce psychedelic-like effects, consistent with the fact that certain 5-HT2A partial agonists (e.g., lisuride) are non-psychedelic. Understanding the role of 5-HT2A-Gq efficacy in psychedelic-like psychopharmacology permits rational development of non-psychedelic 5-HT2A agonists. We also demonstrate that ß-arrestin-biased 5-HT2A receptor agonists induce receptor downregulation and tachyphylaxis, and have an anti-psychotic-like behavioral profile. Overall, 5-HT2A receptor signaling can be fine-tuned to generate ligands with properties distinct from classical psychedelics.

Swim in the Chiral Pool: MDMA and MDA Enantiomers from Alanine-Derived Precursors.

A divergent two-step process has provided access to optically pure enantiomers of MDMA and MDA, clinically relevant phenylisopropylamine entactogens. Target compounds were synthesized from commercially available alanine-derived aziridines. Critical process parameters were identified, and the reactions were optimized to avoid chromatographic purifications toward gram-scale isolations, providing (R)-(-)-MDMA, (S)-(+)-MDMA, (R)-(-)-MDA, and (S)-(+)-MDA each in greater than 98% purity by UPLC, >99% enantiomeric excess, and net yields between 50 and 60% for the complete process.

5-MeO-DMT modifies innate behaviors and promotes structural neural plasticity in mice.

Serotonergic psychedelics are gaining increasing interest as potential therapeutics for a range of mental illnesses. Compounds with short-lived subjective effects may be clinically useful because dosing time would be reduced, which may improve patient access. One short-acting psychedelic is 5-MeO-DMT, which has been associated with improvement in depression and anxiety symptoms in early phase clinical studies. However, relatively little is known about the behavioral and neural mechanisms of 5-MeO-DMT, particularly the durability of its long-term effects. Here we characterized the effects of 5-MeO-DMT on innate behaviors and dendritic architecture in mice. We showed that 5-MeO-DMT induces a dose-dependent increase in head-twitch response that is shorter in duration than that induced by psilocybin at all doses tested. 5-MeO-DMT also substantially suppresses social ultrasonic vocalizations produced during mating behavior. 5-MeO-DMT produces long-lasting increases in dendritic spine density in the mouse medial frontal cortex that are driven by an elevated rate of spine formation. However, unlike psilocybin, 5-MeO-DMT did not affect the size of dendritic spines. These data provide insights into the behavioral and neural consequences underlying the action of 5-MeO-DMT and highlight similarities and differences with those of psilocybin.

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-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 ß-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.

The crystal structure of baeocystin.

The title compound, baeocystin or 4-phosphor-yloxy-N-methyl-tryptamine {systematic name: 3-[2-(methylazaniumyl)ethyl]-1H-indol-4-yl hydrogen phosphate}, C11H15N2O4P, has a single zwitterionic mol-ecule in the asymmetric unit. The mol-ecule has an intra-molecular N-H⋯O hydrogen bond between the ammonium cation and the hydro-phosphate anion. In the crystal, the mol-ecules are linked by N-H⋯O and O-H⋯O hydrogen bonds into a three-dimensional network.

Psilocybin: Characterization of the Metastable Zone Width (MSZW), Control of Anhydrous Polymorphs, and Particle Size Distribution (PSD).

Psilocybin, a serotonergic agonist, was granted a "breakthrough therapy" status by the Food and Drug Administration for clinical trials involving major depressive disorder and treatment-resistant depression. The direct phosphorylation of psilocin to psilocybin that uses a fast crystallization associated with a kinetically controlled process resulted in a smaller particle size distribution. Herein, the measurement of the metastable zone width (MSZW) and nucleation induction enabled a thermodynamically controlled crystallization process, which leads to the formation of a crystal structure with stronger interactions, controlled particle size distribution (PSD), and improved impurity profile. Employing a high-resolution inline microscopy viewer allowed the real-time monitoring of the crystallization process and the measurement of the particle size. We also present a comprehensive study of the formation of polymorph B (trihydrate), polymorph A (anhydrate), and polymorph H (anhydrate) using water recrystallization, which indicates that the formation of polymorph B (trihydrate) is independent of the crystallization method. However, polymorphs A and H are dependent on the mode of drying: drying at room temperature under vacuum gives rise to mainly polymorph A, and when heated even at relatively low temperatures, a mixture of polymorphs A and H beings to form.

Psilocybin: crystal structure solutions enable phase analysis of prior art and recently patented examples.

Psilocybin {systematic name: 3-[2-(dimethylamino)ethyl]-1H-indol-4-yl dihydrogen phosphate} is a zwitterionic tryptamine natural product found in numerous species of fungi known for their psychoactive properties. Following its structural elucidation and chemical synthesis in 1959, purified synthetic psilocybin has been evaluated in clinical trials and has shown promise in the treatment of various mental health disorders. In a recent process-scale crystallization investigation, three crystalline forms of psilocybin were repeatedly observed: Hydrate A, Polymorph A, and Polymorph B. The crystal structure for Hydrate A was solved previously by single-crystal X-ray diffraction. This article presents new crystal structure solutions for the two anhydrates, Polymorphs A and B, based on Rietveld refinement using laboratory and synchrotron X-ray diffraction data, and density functional theory (DFT) calculations. Utilizing the three solved structures, an investigation was conducted via Rietveld method (RM) based quantitative phase analysis (QPA) to estimate the contribution of the three different forms in powder X-ray diffraction (PXRD) patterns provided by different sources of bulk psilocybin produced between 1963 and 2021. Over the last 57 years, each of these samples quantitatively reflect one or more of the hydrate and anhydrate polymorphs. In addition to quantitatively evaluating the composition of each sample, this article evaluates correlations between the crystal forms present, corresponding process methods, sample age, and storage conditions. Furthermore, revision is recommended on characterizations in recently granted patents that include descriptions of crystalline psilocybin inappropriately reported as a single-phase `isostructural variant.' Rietveld refinement demonstrated that the claimed material was composed of approximately 81% Polymorph A and 19% Polymorph B, both of which have been identified in historical samples. In this article, we show conclusively that all published data can be explained in terms of three well-defined forms of psilocybin and that no additional forms are needed to explain the diffraction patterns.

Discriminative-Stimulus Effects of Synthetic Cathinones in Squirrel Monkeys.

BACKGROUND: Synthetic cathinones display overlapping behavioral effects with psychostimulants (e.g., methamphetamine [MA]) and/or entactogens (e.g., 3,4-methylenedioxymethaphetamine [MDMA])-presumably reflecting their dopaminergic and/or serotonergic activity. The discriminative stimulus effects of MDMA thought to be mediated by such activity have been well characterized in rodents but have not been fully examined in nonhuman primates. METHODS: The present studies were conducted to systematically evaluate the discriminative stimulus effects of 5 abused synthetic cathinones (methylenedioxypyrovalerone [MDPV], α-pyrrolidinovalerophenone [α-PVP], methcathinone [MCAT], mephedrone, and methylone) in adult male squirrel monkeys trained to distinguish intramuscular injections of MA (0.1 mg/kg; n = 4) or MDMA (0.6 mg/kg; n = 4) from vehicle. RESULTS: Each training drug produced dose-dependent effects and, at the highest dose, full substitution. MDMA produced predominantly vehicle-like responding in the MA-trained group, whereas the highest dose of MA (0.56 mg/kg) produced partial substitution (approximately 90% appropriate lever responding in one-half of the subjects) in the MDMA-trained group. MDPV, α-PVP, and MCAT produced full substitution in MA-trained subjects, but, at the same or higher doses, only substituted for MDMA in one-half of the subjects, consistent with primarily dopaminergically mediated interoceptive effects. In contrast, mephedrone and methylone fully substituted in MDMA-trained subjects but failed to fully substitute for the training drug in MA-trained subjects, suggesting a primary role for serotonergic actions in their interoceptive effects. CONCLUSIONS: These findings suggest that differences in the interoceptive effects of synthetic cathinones in nonhuman primates reflect differing compositions of monoaminergic actions that also may mediate their subjective effects in humans.

Chemoenzymatic Synthesis of 5-Methylpsilocybin: A Tryptamine with Potential Psychedelic Activity.

A novel analogue of psilocybin was produced by hybrid chemoenzymatic synthesis in sufficient quantity to enable bioassay. Utilizing purified 4-hydroxytryptamine kinase from Psilocybe cubensis, chemically synthesized 5-methylpsilocin (2) was enzymatically phosphorylated to provide 5-methylpsilocybin (1). The zwitterionic product was isolated from the enzymatic step with high purity utilizing a solvent-antisolvent precipitation approach. Subsequently, 1 was tested for psychedelic-like activity using the mouse head-twitch response assay, which indicated activity that was more potent than the psychedelic dimethyltryptamine, but less potent than that of psilocybin.

Synthesis and Characterization of 5-MeO-DMT Succinate for Clinical Use.

To support clinical use, a multigram-scale process has been developed to provide 5-MeO-DMT, a psychedelic natural product found in the parotid gland secretions of the toad, Incilius alvarius. Several synthetic routes were initially explored, and the selected process featured an optimized Fischer indole reaction to 5-MeO-DMT freebase in high-yield, from which the 1:1 succinate salt was produced to provide 136 g of crystalline active pharmaceutical ingredient (API) with 99.86% peak area by high-performance liquid chromatography (HPLC) and a net yield of 49%. The report provides in-process monitoring, validated analytical methods, impurity formation and removal, and solid-state characterization of the API essential for subsequent clinical development.

Synthesis and Biological Evaluation of Tryptamines Found in Hallucinogenic Mushrooms: Norbaeocystin, Baeocystin, Norpsilocin, and Aeruginascin.

A general synthetic method was developed to access known tryptamine natural products present in psilocybin-producing mushrooms. In vitro and in vivo experiments were then conducted to inform speculations on the psychoactive properties, or lack thereof, of the natural products. In animal models, psychedelic activity by baeocystin alone was not evident using the mouse head twitch response assay, despite its putative dephosphorylated metabolite, norpsilocin, possessing potent agonist activity at the 5-HT2A receptor.

Assessment of rimonabant-like adverse effects of purported CB1R neutral antagonist / CB2R agonist aminoalkylindole derivatives in mice.

BACKGROUND: Cannabinoids may be useful in the treatment of CNS disorders including drug abuse and addiction, where both CB1R antagonists / inverse agonists and CB2R agonists have shown preclinical efficacy. TV-5-249 and TV-6-41, two novel aminoalkylindoles with dual action as neutral CB1R antagonists and CB2R agonists, previously attenuated abuse-related effects of ethanol in mice. PURPOSE: To further characterize these drugs, TV-5-249 and TV-6-41 were compared with the CB1R antagonist / inverse agonist rimonabant in assays relevant to adverse effects and cannabinoid withdrawal. PROCEDURES AND FINDINGS: The cannabinoid tetrad confirmed that TV-5-249 and TV-6-41 were devoid of CB1R agonist effects at behaviorally-relevant doses, and neither of the novel drugs induced rimonabant-like scratching. Generalized aversive effects were assessed, and rimonabant and TV-5-249 induced taste aversion, but TV-6-41 did not. Schedule-controlled responding and observation of somatic signs were used to assess withdrawal-like effects precipitated by rimonabant or TV-6-41 in mice previously treated with the high-efficacy CB1R agonist JWH-018 or vehicle. Rimonabant and TV-6-41 dose-dependently suppressed response rates in all subjects, but TV-6-41 did so more potently in JWH-018-treated mice than in vehicle-treated mice, while rimonabant equally suppressed responding in both groups. Importantly, rimonabant elicited dramatic withdrawal signs, but TV-6-41 did not. CONCLUSIONS: These findings suggest differences in both direct adverse effects and withdrawal-related effects elicited by rimonabant, TV-5-249, and TV-6-41, which could relate to neutral CB1R antagonism, CB2R agonism, or a combination of both. Both mechanisms should be explored and exploited in future drug design efforts to develop pharmacotherapies for drug dependence.

Scalable Regioselective and Stereoselective Synthesis of Functionalized (E)-4-Iodobut-3-en-1-ols: Gram-Scale Total Synthesis of Fungal Decanolides and Derivatives.

A reliable protocol to synthesize both racemic and chiral (E)-4-iodobut-3-en-1-ols from aldehydes or epoxides, respectively, containing various aromatic and aliphatic substitutions has been established. The utility of these compounds was then demonstrated by providing access to known fungal decanolides as well as novel aromatic macrocycles. The protocol provided a gram-scale route to (-)-aspinolide A and (-)-5-epi-aspinolide A utilizing a catalytic Nozaki-Hiyama-Kishi reaction to close the macrolide in the final step in 65-84% yields.

Modular Approach to pseudo-Neoclerodanes as Designer κ-Opioid Ligands.

Informed by previous semisynthetic work on salvinorin A, a modular total synthesis has been developed capable of producing novel compounds targeting the κ-opioid receptor. The strategy has permitted the deliberate simplification and introduction of functionality about the target molecule to provide access to molecular features on salvinorin A otherwise unattainable by semisynthesis. Using this approach, a potent pseudo-neoclerodane κ-opioid receptor ligand (2) has been realized.

Semisynthesis and Kappa-Opioid Receptor Activity of Derivatives of Columbin, a Furanolactone Diterpene.

Columbin (1) is a furanolactone diterpene isolated from the roots of Jateorhiza and Tinospora species. These species generally grow in Asia and Africa and have been used in folk medicine for their apparent analgesic and antipyretic activities. Columbin (1) is of particular interest due to its structural similarity to the known kappa-opioid receptor (KOR) agonist salvinorin A. Given that the KOR is of interest in the study of many serious diseases, such as anxiety, depression, and drug addiction, obtaining natural or semisynthetic molecules with KOR activity recently has gained much interest. For this reason, in the present study, derivatives of 1 were designed and synthesized using known structure-activity relationships of salvinorin A at KORs. The structures of the columbin analogues prepared were elucidated by NMR spectroscopy and mass spectroscopy, and their KOR activity was investigated in vitro by inhibition of forskolin-induced cAMP accumulation. Slight improvements in KOR activity were observed in columbin derivatives over their parent compound. However, despite the structural similarities to salvinorin A, neither columbin (1) nor its derivatives were potent KOR ligands. This work represents not only the first evaluation of columbin (1) at the KOR but also one of the first works to explore synthetic strategies that are tolerated on the columbin core.

Addressing Structural Flexibility at the A-Ring on Salvinorin A: Discovery of a Potent Kappa-Opioid Agonist with Enhanced Metabolic Stability.

Previous structure-activity studies on the neoclerodane diterpenoid salvinorin A have demonstrated the importance of the acetoxy functionality on the A-ring in its activity as a κ-opioid receptor agonist. Few studies have focused on understanding the role of conformation in these interactions. Herein we describe the synthesis and evaluation of both flexible and conformationally restricted compounds derived from salvinorin A. One such compound, spirobutyrolactone 14, was synthesized in a single step from salvinorin B and had similar potency and selectivity to salvinorin A (EC50 = 0.6 ± 0.2 nM at κ; >10000 nM at µ and δ). Microsomal stability studies demonstrated that 14 was more metabolically resistant than salvinorin A. Evaluation of analgesic and anti-inflammatory properties revealed similar in vivo effects between 14 and salvinorin A. To our knowledge, this study represents the first example of bioisosteric replacement of an acetate group by a spirobutyrolactone to produce a metabolically resistant derivative.