Investigation of the Structure–Activity Relationships of Psilocybin Analogues

This study investigated 17 psilocybin analogs and found similar activation patterns of the 5-HT2 receptors. The mouse studies also found similar behavioral (head-twitch) responses. Some analogs did show different activation patterns than expected.

Abstract

“The 5-HT2A receptor is thought to be the primary target for psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine) and other serotonergic hallucinogens (psychedelic drugs). Although a large amount of experimental work has been conducted to characterize the pharmacology of psilocybin and its dephosphorylated metabolite psilocin (4-hydroxy-N,N-dimethyltryptamine), there has been little systematic investigation of the structure-activity relationships (SAR) of 4-substituted tryptamine derivatives. In addition, structural analogs of psilocybin containing a 4-acetoxy group, such as 4-acetoxy-N,N-dimethyltryptamine (4-AcO-DMT), have appeared as new designer drugs, but almost nothing is known about their pharmacological effects. To address the gap of information, SAR studies were conducted with 17 tryptamines containing a variety of symmetrical and asymmetrical N,N-dialkyl substituents and either a 4-hydroxy or 4-acetoxy group. Calcium mobilization assays were conducted to assess functional activity at human and mouse 5-HT2 subtypes. Head-twitch response (HTR) studies were conducted in C57BL/6J mice to assess 5-HT2A activation in vivo. All of the compounds acted as full or partial agonists at 5-HT2 subtypes, displaying similar potencies at 5-HT2A and 5-HT2B receptors, but some tryptamines with bulkier N-alkyl groups had lower potency at 5-HT2C receptors and higher 5-HT2B receptor efficacy. In addition, O-acetylation reduced the in vitro 5-HT2A potency of 4-hydroxy-N,N-dialkyltryptamines by about 10-20-fold but did not alter agonist efficacy. All of the compounds induce head twitches in mice, consistent with an LSD-like behavioral profile. In contrast to the functional data, acetylation of the 4-hydroxy group had little effect on HTR potency, suggesting that O-acetylated tryptamines may be deacetylated in vivo, acting as pro-drugs. In summary, the tryptamine derivatives have psilocybin-like pharmacological properties, supporting their classification as psychedelic drugs.”

Authors: Adam K. Klein, Muhammad Chatha, Lauren J. Laskowski, Emilie I. Anderson, Simon D. Brandt, Stephen J. Chapman, John D. McCorvy & Adam L. Halberstadt

Notes

“One of the main findings of these studies is that psilocin and its homologues activate calcium mobilization via 5-HT2A with high efficacy and nanomolar potency, whereas the acetate esters have about 10-fold lower potency.”

Acetate esthers are a special class of acetate (a salt formed by the combination of acetic acid with a base) that “have the general formula CH3CO2R, where R is an organyl group“.

Summary

Psilocybin, a hallucinogenic mushroom, is rapidly dephosphorylated to psilocin, a drug with similar effects to lysergic acid diethylamide (LSD) and mescaline. Psilocin has a higher potency at the receptor level than psilocybin.

Hofmann and colleagues isolated psilocybin and psilocin in 1957, and several 4-substituted tryptamines were synthesized. These compounds were found to be potent psychedelic drugs, and 4-AcO-DMT was proposed as an alternative prodrug for psilocybin in scientific studies.

A number of 4-substituted tryptamines have been detected in Europe, including 4-AcO-DIPT, 4-AcO-DMT, 4-AcO-MET, and 4-HO-DPT. These chemicals produce psilocybin-like psychedelic effects.

There is a lack of information regarding the pharmacological and behavioral properties of 4-acetoxy-N,N-dialkyltryptamines, and little systematic investigation of the effect of N-alkyl substitution on their activity. This study focused on activity at the 5-HT2A receptor, which is thought to be the primary target for psilocybin and other psychedelic drugs in humans and rodents.

Mice were housed in a climate-controlled room on a reverse-light cycle and provided with ad libitum access to food and water. All experiments were approved by the UCSD Institutional Animal Care and Use Committee.

Drugs were administered intraperitoneally at a volume of 5 mL/kg, and in vitro at 10 mM concentration before serial dilution. Psilocin was dissolved in water containing 5 mM tartaric acid (pH 5.0), and all other compounds were dissolved in isotonic saline.

A head-mounted neodymium magnet was attached to the cranium of mice, and their head twitches were recorded using a magnetometer coil. The head twitches were identified based on sinusoidal wavelets and at least three sequential head movements with a frequency of 40 Hz.

Head twitches were analyzed using one-way analyses of variance (ANOVA) and Tukey’s studentized range method. Median effective doses and 95% confidence intervals were calculated using nonlinear regression.

5-HT2 receptor functional experiments were performed with Flp-In T-REx 293 cells expressing either human 5-HT2A (h5-HT2A), mouse 5-HT2A (m5-HT2A), human 5-HT2B (h5-HT2B), or human 5-HT2C INI (h5-HT2C). Calcium flux was measured using a FLIPRTETRA cellular screening system. The relative activity of 5-HT was determined by the ratio of Emax over EC50 parameter estimates.

4-Hydroxy-N,N-dialkyltryptamines Induce the Head-

Previous studies have shown that psilocin induces the HTR in C57BL/6J mice with ED50 = 0.17 mg/kg, which is equivalent to 0.81 mol/kg. The HTR follows an inverted-U-shaped doseresponse function and is related to the steric properties of the alkyl groups on the amine nitrogen. We examined the relationship between the potency of HTR and the steric properties of amine substituents. The results showed that the two were negatively correlated.

O-Acetylation did not reliably alter the potency of psilocin or its homologues, but did increase in some cases and reduce in others.

Figure 2 depicts the time course of effects on the HTR for a subset of the compounds. The maximal response typically occurred during the first 10 min after drug administration.

All of the 4-substituted tryptamines stimulated calcium mobilization via activation of human and mouse 5-HT2A receptors, and were highly efficacious 5-HT2A agonists, with the exception of the O-acetylated tryptamines 4-AcO-DMT and 4-AcO-DIPT.

Results at the 5-HT2B receptor were similar to those at the 5-HT2A receptor, although efficacy at 5-HT2B was more variable, ranging from 22.1 to 97.4%.

The tryptamines had lower potency at 5-HT2C receptors compared to 5-HT2A receptors, and N-alkyl chain length had striking effects on 5-HT2C activity.

N,N-dialkyltryptamines containing either a hydroxy or acetoxy group at the 4-position have similar pharmacological activity to LSD, but have different potencies in mice and humans.

Psilocybin acts as a prodrug for psilocin, which has a potency 100-fold higher than psilocybin at h5-HT2A receptors. 4-AcO-DMT and its homologues may also act as prodrugs, but controlled biotransformation studies are necessary to definitively show that.

4-Hydroxy- and 4-acetoxy-N,N-dialkyltryptamines reportedly produce very similar effects in humans, but 4-AcO-DMT and 4-AcO-DET produce larger peak responses in the HTR assay than their O-desacetyl counterparts. This may be because the 4-acetoxy group facilitates brain uptake.

These are the first studies conducted with the N-allyl-N-methyl-substituted tryptamine 4-HO-MALT. It had about the same potency and efficacy as 4-HO-MPT at 5-HT2 subtypes, and had a potency similar to that of 4-HO-MPT in the HTR assay.

Tryptamine hallucinogens interact with the 5-HT2B receptor, and some medications linked to cardiac-valve disorders also activate 5-HT2B. Repeated, daily use of low doses of hallucinogens may pose a risk of valvular heart disease, especially considering the greater 5-HT2B agonist efficacy of 4-HO-DPT and 4-HO-DIPT.

The size of the N-alkyl group in psilocin has little effect on the potency of psilocin at 5-HT2A or 5-HT2B, whereas the potency at 5-HT2C declines when there is a relatively bulky substituent on the terminal amine. The N,N-dialkyl substitution pattern does not appear to be an important determinant of 5-HT2A agonist potency, but the size of the alkyl chains does seem to affect potency in the HTR assay.

In summary, 4-substituted N,N-dialkyltryptamines activate 5-HT2A receptors in vitro and in vivo, and are likely serving as prodrugs for the corresponding 4-hydroxytryptamines. Additionally, these compounds also act as 5-HT2B agonists, which is a potentially worrisome property.

Notes

These studies were funded by NIDA, the Medical College of Wisconsin, the UCSD T32 Fellowship in Biological Psychiatry & Neuroscience, and the Veteran’s Administration VISN 22 Mental Illness Research, Education, and Clinical Center.