Investigation of the Structure-Activity Relationships of Psilocybin Analogues

Klein and colleagues situate this work within renewed scientific and public interest in psilocybin and related 4-substituted tryptamines. The introduction notes that psilocybin is rapidly dephosphorylated to psilocin and that psilocin is widely accepted as the active species at serotonergic targets, especially the 5-HT2A receptor, which is implicated in the psychedelic effects of LSD, mescaline and related compounds. Although many 4-hydroxy and 4-acetoxy tryptamines have been described historically and more recently have appeared as recreational designer drugs, the authors identify a gap in systematic structure-activity relationship (SAR) data for N,N-dialkyl substitutions and for O-acetylated analogues such as 4-AcO-DMT. To address this gap, the investigators examined a panel of 16 4-substituted N,N-dialkyltryptamines containing either a 4-hydroxy or 4-acetoxy group. Their primary aims were to characterise functional agonism at human and mouse 5-HT2 receptor subtypes in vitro using calcium mobilization assays, and to assess 5-HT2A-mediated behavioural activity in vivo using the mouse head-twitch response (HTR). The combined in vitro and in vivo approach was intended both to map how N-alkyl substitution affects receptor activity and to evaluate whether the O-acetylated compounds behave as prodrugs of the corresponding 4-hydroxy tryptamines.

Adult male C57BL/6J mice (6–8 weeks old) were used for behavioural studies; animals were housed under controlled conditions and experiments were approved by the institutional animal care committee. The HTR was measured with a surgically implanted head-mounted neodymium magnet and an external magnetometer coil. After a one-week recovery from implantation, mice were dosed intraperitoneally (IP, 5 mL/kg) and head-twitch activity was recorded for 30 minutes in a coil-enclosed glass cylinder. Head twitches were identified by waveform criteria (sinusoidal wavelets, ≥3 sequential head movements at ≥40 Hz, amplitude above noise, duration <0.15 s and stable baseline) and counts were analysed by one-way ANOVA with post hoc Tukey tests. Median effective doses (ED50) and 95% confidence intervals were obtained by nonlinear regression. Significance was set at α = 0.05. In vitro functional assays measured Gq-mediated calcium flux in Flp-In T-REx 293 cells expressing inducible human 5-HT2A, mouse 5-HT2A, human 5-HT2B or human 5-HT2C (INI) receptors. Cells were loaded with Fluo-4 Direct dye, stimulated with serial dilutions of test compounds and fluorescence changes recorded on a FLIPR TETRA. Peak responses were normalised to the maximal response produced by 5-HT (set to 100%) and baseline (0%). Dose-response curves were fit with sigmoidal functions and relative activity (RA) was reported as log(Emax/EC50) when appropriate. Sixteen test compounds were evaluated, including both 4-hydroxy and 4-acetoxy N,N-dialkyltryptamines; several compounds and psilocin were sourced from prior laboratory stocks and commercial suppliers, with identities and >95% purity confirmed by mass spectrometry and NMR. For in vitro work compounds were dissolved in DMSO at 10 mM before dilution; for behavioural experiments psilocin was dissolved in mildly acidified water and other compounds in isotonic saline. Statistical and curve-fitting analyses used GraphPad Prism software.

All 4-hydroxy-N,N-dialkyltryptamines tested produced head twitches in C57BL/6J mice, with dose–response functions that were typically inverted-U shaped. Psilocin produced an ED50 of 0.17 mg/kg (0.81 μmol/kg). Across the panel, HTR potencies varied: for symmetrical N,N-dialkyl series the rank order was psilocin (ED50 = 0.81 μmol/kg) > 4-HO-DET (1.56 μmol/kg) > 4-HO-DPT (2.47 μmol/kg) > 4-HO-DIPT (3.46 μmol/kg), and for selected asymmetric analogues 4-HO-MET (0.65 μmol/kg) > 4-HO-MPT (1.92 μmol/kg) > 4-HO-MIPT (2.97 μmol/kg). Steric bulk on the amine correlated with reduced behavioural potency: for eight N,N-dialkyl-4-hydroxytryptamines the sum of Charton’s upsilon steric parameters for the two amine substituents was negatively correlated with −log ED50 in the HTR (R = −0.8283, p = 0.011). The 4-acetoxy analogues were also active in the HTR. O-acetylation did not produce a consistent shift in behavioural potency across the series; in general there was little difference between 4-hydroxy compounds and their acetate esters in the HTR, although individual cases showed increased or decreased potency. In vitro calcium-mobilisation assays showed that all 4-substituted tryptamines stimulated human and mouse 5-HT2A receptors. The 4-hydroxy compounds were highly potent at h5-HT2A (EC50 ≈ 1–10 nM) and generally highly efficacious (Emax 90–100% relative to 5-HT). The O-acetylated tryptamines were weaker at 5-HT2A, with potency reduced approximately an order of magnitude to 10–40-fold versus the 4-hydroxy counterparts; two acetates had notably reduced efficacy (4-AcO-DMT Emax = 79.2%, 4-AcO-DIPT Emax = 74.6%). At h5-HT2B, potency patterns were similar to 5-HT2A but efficacy varied widely (Emax range ≈ 22.1–97.4%). Substitution patterns influenced 5-HT2B efficacy: N-ethyl–N-propyl, N,N-dipropyl and N,N-diisopropyl substitutions were associated with high 5-HT2B Emax (~90–100%), whereas shorter alkyl chains tended to yield lower efficacy. The 5-HT2C receptor showed lower potency overall and greater sensitivity to N-alkyl bulk: N,N-diisopropyl substitution produced high efficacy at 5-HT2C (4-HO-DIPT Emax = 92.1%) but markedly reduced potency (4-HO-DIPT EC50 = 1408 nM). Consequently, some compounds displayed strong selectivity for 5-HT2A over 5-HT2C (for example, 4-HO-DPT ≈ 129-fold; 4-HO-DIPT ≈ 206-fold).

Klein and colleagues interpret their results as demonstrating that 4-hydroxy N,N-dialkyltryptamines are potent, efficacious 5-HT2A agonists in vitro and reliably induce the 5-HT2A-mediated head-twitch response in mice, supporting their classification as psychedelic drugs. The acetylated analogues showed substantially lower potency in cell-based 5-HT2A assays (roughly 10–40-fold), yet produced HTR potencies comparable to the corresponding 4-hydroxy compounds. The authors view this in vitro/in vivo discrepancy as consistent with the hypothesis that 4-acetoxy tryptamines act as prodrugs that are deacetylated to the active 4-hydroxy species in peripheral tissues or blood prior to central action, but they note that definitive biotransformation studies are required to confirm this mechanism. Several mechanistic explanations for the behavioural findings are discussed: esterification could increase lipophilicity and thereby enhance brain uptake or absorption from the injection site, analogous to how heroin increases brain levels of 6-monoacetylmorphine versus direct 6-MAM dosing. The paper also reports the first experimental characterisation of 4-HO-MALT (N-methyl-N-allyl substitution), which showed similar receptor and behavioural profiles to 4-HO-MPT, implying that a single allyl substituent on the terminal amine does not markedly impair 5-HT2A activity. Safety considerations receive attention because many compounds in the panel are potent h5-HT2B agonists. The investigators highlight that 5-HT2B activation has been implicated in drug-induced valvular heart disease and pulmonary hypertension with some chronically administered drugs, and they caution that repeated or daily use of potent 5-HT2B agonists (for example via microdosing) could present cardiac risk. In particular, several longer-chain N-alkyl analogues (e.g., 4-HO-DPT and 4-HO-DIPT) showed greater 5-HT2B efficacy than psilocin. Finally, the authors conclude that N,N-dialkyl substitution has limited influence on 5-HT2A and 5-HT2B potency but can reduce 5-HT2C potency when bulky substituents are present, and that increasing steric bulk tends to lower in vivo HTR potency. They propose that these SAR findings will help predict the psychoactive potential of new tryptamine derivatives based on substitution patterns on the terminal amine and the indole ring. The paper emphasises the need for further work on metabolism and long-term safety to fully characterise the pharmacology of O-acetylated tryptamines.