Scientists Synthesize and Test the Magic Mushroom Compounds Baeocystin, Norbaeocystin, Norpsilocin, and Aeruginascin

This work is a giant step forward for psychedelic research.

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In a February 2020 study published in the Journal of Natural Products, Sherwood et al. describe their work synthesizing and studying four lesser-known magic mushroom (aka psilocybin mushroom) compounds; aeruginascin, norpsilocin, baeocystin, and norbaeocystin.1

Until now, no one was examining these lesser-known compounds. Of the four, norpsilocin was just discovered in 2017 by Lenz et al.,2 but hasn’t been studied further. The other compounds have received no research attention, despite their discovery decades ago. The authors of the paper discuss several reasons why this work could not be done in the past:

  • “…their lack of availability in pure form.”
  • The need for “…producing useful amounts…”
  • The need for full analytical characterization.
  • …difficulty in purification…
  • “Previous methods used to study [these compounds] have typically required either indirect instrumental observation on crude mushroom extracts or multistep isolation processes providing small quantities of purified material.”

In this work, the authors overcome all the above-stated hurdles, opening the door for further research with these compounds in the scientific community.

Synthesis Routes for Four Magic Mushroom Compounds

Below is a high-level overview of the synthetic methods used to produce norbaeocystin, baeocystin, and norpsilocin, as detailed in the paper.

  • Synthesis of acyl chloride from 4-acetyoxyindole and oxalyl chloride.
  • Acyl chloride is reacted with N-benzylmethylamine or dibenzylamine to yield the desired ketoamides.
  • Reduction of the ketoamides using lithium aluminum hydride in tetrahydrofuran (THF) and 2-methyltetrahydrofuran.
  • Removal of the ß-hydroxy intermediate (a reactive impurity that is prone to forming dimers) via filtration through a silica pad.
  • Phosphorylation using sodium hydride, THF, and ortho-xylenyl phosphoryl chloride (o-XPCl).

From that point, norbaeocystin, baeocystin, and norpsilocin were obtained by catalytic hydrogenolysis using either palladium on carbon or a palladium hydroxide catalyst. This was followed by filtration, solvent removal, and precipitation by adjusting the pH to afford solid (and in some cases crystalline) products in good yield. The authors did not discuss the crystallographic features of the psilocybin that was obtained or whether it met the definitions of COMPASS Pathways’ novel crystalline form of psilocybin.

The authors note a key improvement in their method compared to previous work, which eliminates the need to purify the compounds by using chromatography. Their method isolates the compounds by precipitating them from a pH-adjusted aqueous solution via the addition of acetone.

In a separate synthesis, the researchers made aeruginascin by quaternization of psilocybin “using excess methyl iodide in methanol treated with aqueous ammonium hydroxide.” The aeruginascin was collected by filtration and washing with methanol.

Biological Testing of the Magic Mushroom Compounds

The Sherwood et al. study details the results of two biological tests. First, recall that psilocybin is metabolized to psilocin in the body by removal of psilocybin’s phosphate group.3 From this, the researchers hypothesized that baeocystin and norbaeocystin act as prodrugs of the 4-hydroxy compounds (norpsilocin and 4-hydroxytryptamine), which serve as bioactive compounds.

The Head Twitch Response

In the study, the researchers compared baeocystin to psilocybin using the head twitch response (HTR) in mice. The results showed that psilocybin induced HTR in a dose-dependent manner. However, baeocystin was indistinguishable from the control mice that were given saline. The authors concluded that,

…baeocystin alone would likely not induce 5-HT2A receptor-mediated psychoactive effects in vivo.

Notably, mushroom expert Paul Stamets recently described a similar lack of psychoactive activity upon ingesting pure baeocystin. In an interview with Joe Rogan, Stamets says he took 10 mg of baeocystin but did not feel high. He said,

I was ready for liftoff. I was hoping for liftoff, I know what liftoff feels like, and I didn’t get it.

The authors attribute the lack of activity by norpsilocin in the HTR test to the action of monoamine oxidase enzymes (MAO). MAOs use oxygen atoms to remove amine groups from molecules. They noted that secondary amines tend to be broken down readily by MAOs compared to tertiary amines.Therefore, they hypothesize that norpsilocin (secondary amine) gets degraded faster in the body compared to psilocin (tertiary amine).

The authors summed up their observations by saying,

…baeocystin and norpsilocin may be rendered inactive by metabolism before reaching targets in the central nervous system.

Activation of the 5-HT2A Receptor

Scientists know that the dephosphorylation of psilocybin makes psilocin able to penetrate the blood-brain barrier (BBB).3 Getting into the brain allows psilocin to interact with 5-HT2A receptors, causing the psychedelic effect.5

The researchers hypothesized that the reason baeocystin did not induce the HTR was that it couldn’t cross the BBB. So, they tested norpsilocin to see if it activated the serotonin 5-HT2A receptor. For this, they used a test that measures the Gq-mediated calcium flux at 5-HT2A.

The calcium flux test data indicated that norpsilocin was almost a full agonist and more potent at the human and mouse 5-HT2A receptor compared to psilocin. The Emax for norpsilocin at the human 5-HT2A was 93% of serotonin compared to psilocin whose Emax was 73%. For the mouse receptor, Emax for norpsilocin was 99% of serotonin compared to psilocin which had an Emax of 72%.

Additionally, the EC50 values (i.e., the potency) for norpsilocin and psilocin at the human 5-HT2A were 8.4 nM and 4.3 nM, respectively. In this test, the lower the EC50, the greater the potency. Although norpsilocin’s EC50 was greater than that of psilocin, the difference (~4.0 nM) is considered negligible. Thus, the authors described the compounds as approximately equipotent. For the mouse 5-HT2A, the EC50 = 19.0 nM for norpsilocin and 9.9 nM, for psilocin.

In summation, the authors stated,

Norpsilocin was thus as potent if not more efficacious compared to psilocin in Gq-mediated calcium flux at the 5-HT2A receptor.

Summary

Although the HTR animal model suggested that baeocystin (or it’s metabolite, norpsilocin) was not a centrally acting psychedelic, the cellular assays demonstrated that norpsilocin acts as a potent agonist at human 5-HT2A receptors. The latter is traditionally correlated with a psychedelic effect.

The scientists resolved the HTR and 5-HT2A data by explaining that norpsilocin (although potently active at 5-HT2A) may not cross the BBB. In the case of psilocybin, its dephosphorylation makes the resulting psilocin able to penetrate the BBB. It is unclear how the minor structural difference between psilocybin and psilocin would lead to substantial changes in BBB permeability.

Overall, the authors hypothesize that baeocystin and norpsilocin are deactivated by MAOs before reaching 5-HT2A receptors in the central nervous system.

Why is this Study So Important to Psychedelic Research?

This collaborative work by scientists from the Usona Institute, the University of California San Diego, and the Medical College of Wisconsin gives a significant boost to research into the chemistry and pharmacology of psychedelics.

This study has advanced psychedelic science by providing general synthesis routes for four of the minor tryptamine compounds present in naturally occurring magic mushrooms. For the first time, this gives scientists meaningful access to these compounds for further study. The authors also conducted the first biological screenings of baeocystin and norpsilocin.

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Jáchym Fibír
7 months ago

“Remember that psilocin is a secondary amine, and norpsilocin is a primary amine.” Nope. Psilocin is a tertiary amine and norpsilocin is secondary.

Andrew Chadeayne
3 months ago

Thanks for this great article. This is fantastic work by Sherwood et al.! Very exciting to see some progress making and studying the “minor” components in magic mushrooms. It does seem at little odd that the authors synthesized aeruginasin, pointed out that it could be important to the wood lover paralysis phenomenon, but then didn’t do any biological studies to investigate the properties of aeruginascin. I also found it interesting that they didn’t study aeruginascin’s active metabolite, 4-hydroxy-N,N,N-trimethyltryptammonium. The table from the abstract of their paper (pasted below) sort of highlights the unmet need for studying this compound by using… Read more »

Alex Sherwood
3 months ago

Hi Andrew! It didn’t make it into the paper, but I presented this at the ISRP conference in New Orleans last October: We did initially run a pilot experiment comparing aeruginascin to bufotenidine in mice. With aeruginascin, there was some evidence of hind paw weakness/hindlimb abduction at 10 mg/kg and especially 30 mg/kg. There was, however, no evidence of muscle relaxation or ataxia. In comparison, bufotenidine produced fatal respiratory depression in mice at 10 mg/kg IP. In general, the data didn’t seem to support the hypothesis that bufotenidine and aeruginascin (or its putative metabolite) operate by the same mechanism in vivo. A… Read more »

Andrew Chadeayne
3 months ago
Reply to  Alex Sherwood

Thank you for the great follow up comments. This is very interesting. We have seen similar differences between bufotenidine (5-OH-TMT) and 4-OH-TMT (aeruginascin’s putative active metabolite) in cellular assays measuring binding affinity. While 5-OH-TMT has a high affinity for 5-HT3 (and not 5-HT2a), 4-OH-TMT has a significant binding affinity at 5-HT2a but not 5-HT3. So, there’s a big difference between the 4-OH and 5-OH isomers! We expect that our paper on these results will publish in the next 1-2 weeks. In the meantime, I would be happy to share our data. We could also send you some (analytically pure) 4-OH-TMT… Read more »

    References
  1. Sherwood AM, Halberstadt AL, Klein AK, et al. Synthesis and Biological Evaluation of Tryptamines Found in Hallucinogenic Mushrooms: Norbaeocystin, Baeocystin, Norpsilocin, and Aeruginascin. J Nat Prod. February 2020. doi:10.1021/acs.jnatprod.9b01061
  2. Lenz C, Wick J, Hoffmeister D. Identification of ω-N-Methyl-4-hydroxytryptamine (norpsilocin) as a Psilocybe natural product. Journal of Natural Products. 2017;80(10):2835-2838. doi:10.1021/acs.jnatprod.7b00407
  3. Horita A, Weber L. Dephosphorylation of psilocybin to psilocin by alkaline phosphatase. Proceedings of the society for experimental biology and medicine. 1961;106(1):32-34. doi:10.3181/00379727-106-26228
  4. Ferguson GG, Keller WJ. Monoamine oxidase inhibiting activity of a series of (±)-4-methoxy-β-hydroxyphenethylamines. Journal of Pharmaceutical Sciences. 1975;64(8):1431-1432. doi:10.1002/jps.2600640844
  5. Madsen MK, Fisher PM, Burmester D, et al. Psychedelic effects of psilocybin correlate with serotonin 2A receptor occupancy and plasma psilocin levels. Neuropsychopharmacology. January 2019:1. doi:10.1038/s41386-019-0324-9