Psychedelic-like Activity of Norpsilocin Analogues

Abstract

Primary metabolites of mushroom tryptamines, psilocybin and baeocystin (i.e., psilocin and norpsilocin), exhibit potent agonist activity at the serotonin 2A receptor (5-HT_2A) in vitro but differ in their 5-HT_2A-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-HT_2A, 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.

Introduction

The pharmacology of naturally occurring psychedelics, notably tryptamines from Psilocybe mushrooms and other natural sources, has been a focal point of scientific inquiry.^1,2 Psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine or 4-PO-DMT) and its active metabolite, psilocin (4-hydroxy-N,N-dimethyltryptamine or 4-HO-DMT), are well-studied for their pronounced serotonin 2A receptor (5-HT_2A) mediated psychoactive effects and potential therapeutic properties.³ Compared to psilocin, norpsilocin (4-hydroxy-N-methyltryptamine or 4-HO-NMT) is a tryptamine metabolite also found in Psilocybe mushrooms that presents a structural variation in its amine group (Figure 1). Despite exhibiting comparable in vitro agonist activity at the 5-HT_2A receptor, the in vivo psychoactive potential of the two compounds is markedly different. While the central nervous system (CNS) mediated psychedelic effects of psilocin are well-established, in part attributable to its tertiary dimethylamine structure, norpsilocin—with a corresponding secondary methylamine—does not produce comparable psychedelic-like effects.^4,5 This disparity prompts questions regarding the molecular characteristics that impart CNS bioavailability and the subsequent receptor interactions requisite for psychedelic-like effects of tryptamines.⁶ Specifically, of interest is the role of the amine substituent in governing pharmacokinetic and pharmacodynamic properties in norpsilocin analogues.⁷

Figure 1.

Tryptamine natural products with disparate bioavailability or psychoactivity, highlighting the role of hydroxyl and amine substitutions.

In addition to the properties imparted by the amine substituents, a notable structural feature on active metabolites of Psilocybe tryptamines is the 4-position hydroxyl group, which potentially contributes to their physical properties and pharmacokinetics.⁸ Experimental evidence indicates that this 4-hydroxyl group might participate in an intramolecular hydrogen bond with the distal amine, masking its charge, reducing its basicity, and increasing the lipophilicity of psilocin.^9,10 Such a hydrogen bond may also lead to preferential structural conformations, potentially impacting the receptor binding dynamics and resistance to metabolic degradation.

The significance of both the presence and position of the hydroxyl group becomes further evident when comparing psilocin to N,N-dimethyltryptamine (DMT) and 5-hydroxy-N,N-dimethyltryptamine (bufotenine or 5-HO-DMT) (Figure 1). DMT, devoid of a hydroxyl group, produces powerful but ephemeral psychoactive effects when administered through inhalation or injection, yet exhibits no discernible oral bioavailability alone due to its rapid metabolism by monoamine oxidases (MAOs) in the gut and liver.^6,11,12 In traditional practices, DMT is often consumed in conjunction with beta-carboline MAO inhibitors to produce the ayahuasca brew, enabling its psychoactive potential upon oral administration.^13,14 Bufotenine, a 5-hydroxy positional isomer of psilocin, is unable to participate in an intramolecular hydrogen bond and presents less pronounced, sometimes negligible, psychoactive effects in humans despite its structural closeness to psilocin.¹⁵ Evidence suggests that limited in vivo activity of bufotenine may be a result of reduced CNS bioavailability, but not receptor engagement, possibly analogous to similar oberservations with norpsilocin.^16,17 Taken together, psilocin’s 4-hydroxyl group and its interaction with the distal dimethylamine group likely imparts both oral bioavailability through enhanced metabolic stability and improved CNS penetration through masking of the charge on the amine.

Given the importance of the 4-hydroxyl group and its interaction with the amine in influencing the pharmacokinetics of psilocin, we examined the structure–activity relationships (SARs) of a series of norpsilocin analogues (Figure 2). To this end, norpsilocin derivatives were synthesized, focusing on variations in the amine functionality to introduce more lipophilic substituents in place of the methyl group on norpsilocin. Based on these modifications, we hypothesized that the compounds would exhibit increased central activity, either by reduced metabolic liability or enhanced blood-brain barrier penetration—provided 5-HT_2A receptor engagement was maintained. The specific substitutions chosen were driven by their relative ease of synthesis and by general principles of medicinal chemistry, concentrating on variations in size, rigidity, lipophilicity, and electrostatic properties. SAR studies were undertaken to elucidate the impact of these modifications on CNS 5-HT_2A-mediated psychedelic-like effects in the mouse head twitch response (HTR) assay, as well as on various receptor affinities and activities in vitro. The findings aimed to provide additional insight about the relationships between certain structural features and bioactivity of psychedelic tryptamines.

Figure 2.

Synthesis of norpsilocin-related analogues with nomenclature and corresponding partition coefficient (cLogP) values calculated using Chemdraw Professional 22.2 (2023, PerkinElmer Informatics).

Results and Discussion

The 4-HO-NxT series was synthesized following the established Speeter-Anthony method as outlined in Figure 2 from 4-acetoxyindole (1) via divergent acyl chloride intermediate 2. Substitution with various primary amines provided ketoamides 3X which were reduced with lithium aluminum hydride to give the target compounds; each was prepared as the corresponding fumarate or succinate salts. The reaction sequence was consistent with previously reported methods.^18,4,19,20 Production of 4-HO-NALT, however, was an exception and proved to be a challenge in the lithium aluminum hydride reduction step, as a portion of the allyl product was reduced, resulting in an alkane impurity (4-HO-NPT) that cocrystallized with the product and had to be removed utilizing flash column chromatography. Otherwise, formation of the succinate or fumarate salts followed by recrystallization or trituration enabled efficient purification for most of the other 4-HO-NxT compounds, typically avoiding chromatographic purifications over the entire reaction sequence. The resulting overall yields ranged between 13%–55% with a purity range of 96.2%–99.8% by ultraperformance liquid chromatography (UPLC) peak area for final products.

While stoichiometric equivalents of the diacids were used in the salt formation steps, inspection of the integration values in the proton NMR spectra (Supporting Information pg 13–44) provided evidence that 4-HO-NET, 4-HO-NPT, 4-HO-NALT, and 4-HO-NtBT preferred crystallizing as the corresponding (2:1) hemisuccinate or hemifumarate salts, whereas 4-HO-NBnT, 4-HO-NcHT, 4-HO-NnBT, and 4-HO-NiPT crystallized as the corresponding 1:1 salts. Similarly, norpsilocin was shown by single-crystal X-ray diffraction to crystallize as a 2:1 hemifumarate salt.²¹ In contrast, the tertiary dimethylamine, 5-MeO-DMT, was previously found to form the 1:1 succinate salt preferentially.²² Taken together, the amine substituents in tryptamines may play a role in the preference for salt formation stoichiometry and may influence the crystallization behavior.

The respective calculated partition coefficients (cLogP) values for individual compounds are depicted in Figure 2 and range from approximately 1.4–2.9, compared to 0.9 for norpsilocin. Previous studies suggest that the optimal partition coefficient for blood-brain barrier penetration is within the range of 1.5–2.7.²³ With a cLogP of 0.9, norpsilocin may not be lipophilic enough to permeate the blood-brain barrier and, therefore, cannot reach the CNS. As depicted in Figure 2, with extension of the secondary amine substituent, an expected increase in cLogP is observed.

Head Twitch Response Studies

In order to examine the potential for these novel compounds to elicit psychoactive effects in the mammalian CNS, we first employed the mouse HTR assay, serving as an indication for psychedelic-like effects and CNS bioavailability. The HTR assay provides a reliable assessment of the ability of a compound to cross the blood-brain barrier and exert 5-HT_2A receptor-mediated behavioral responses consistent with psychedelic-like activity in humans.^24,25 The HTR was determined in groups of male and female mice for 30 min following subcutaneous administration of norpsilocin analogues. Aside from the bulky N-butyl and N-cyclohexyl substitutions, all compounds produced dose-related increases in HTRs vs vehicle controls (Figure 3, Figure S1, Table S1), with dose–response curves exhibiting the typical inverted U shape, characteristic of other psychedelic tryptamines.^5,26,27 4-HO-NALT, 4-HO-NBnT, and 4-HO-NET displayed similar potencies, producing half maximal responses (ED₅₀) at doses ranging from 1.1 to 1.4 mg/kg, while 4-HO-NPT and 4-HO-NiPT were less potent (ED₅₀ = 2.9–3.3 mg/kg, Figure 3A, Table 1). Interestingly, the maximum effect (E_max) varied among the compounds (Table 1). 4-HO-NALT had the highest maximum effect on HTR (77 HTR events), which was ∼1.5–3-fold higher than the maximum counts observed for other active compounds. Norpsilocin previously failed to induce psychedelic-like HTR activity using the same assay setup used presently, whereas psilocin was active (ED₅₀ = 0.11 mg/kg, E_max = 23 HTR events).⁵ Despite favorable cLogP values, 4-HO-NnBT, 4-HO-NtBT, and 4-HO-NcHT were also inactive for inducing the HTR, which was instead likely a result of the relatively weak 5-HT_2A receptor activity described below. The time-course for effects of the compounds on HTR also differed, with 4-HO-NPT, 4-HO-NALT, and 4-HO-NBnT showing peak effects within 5–15 min, while 4-HO-NET and 4-HO-NiPT had a slightly delayed onset to peak of HTR frequency at some doses up to 15–25 min post drug administration (Figure 3B–D, Figure S1A–B).

Figure 3.

Dose–response and time-course data for the HTR induced by norpsilocin analogues. (A) Dose–response curves for effects on the HTR (n = 5–6 mice/dose) and representative time-course plots for HTR activity of (B) 4-HO-NET, (C) 4-HO-NPT, and (D) 4-HO-NALT.

Table 1. Potencies (ED₅₀) and Maximum Efficacies or Effects (E_max) of Norpsilocin and Analogues for HTR in Mice^a.

	4-HO-DMTb	4-HO-NMTb	4-HO-NET	4-HO-NPT	4-HO-NALT	4-HO-NiPT	4-HO-NnBT	4-HO-NtBT	4-HO-NcHT	4-HO-NBnT
ED50(mg/kg s.c.)	0.11	>30	1.4	2.9	1.1	3.3	>30	>30	>30	1.2
C.I. (95%)	0.09–0.19		0.9–2.4	2.7–3.1	0.9–1.3	2.6–4.4				1.0–1.5
Emax(HTR events/30 min)	23		39.8	52.1	76.6	27.5				26.1

^aData are expressed as means, with 95% confidence intervals below.

^bData for psilocin (4-HO-DMT) and norpsilocin (4-HO-NMT) are from a previously published report which used identical testing conditions.⁵

The mouse HTR experiments indicated that extending the N-methyl group of norpsilocin to N-ethyl was adequate to promote CNS-mediated psychedelic-like activity, in support of our initial bioavailability hypothesis. There was an approximately 10-fold (7-fold on a molar basis) decrease in potency compared to psilocin, which has a reported HTR ED₅₀ potency of 0.11–0.17 mg/kg,^5,27 compared to 1.1–1.4 mg/kg for the most potent compounds in the present series. The individual maximum HTR events at any dose (i.e., E_max) was variable for the series; notably, the E_max for 4-HO-NALT (77 HTR events) greatly surpassed that of psilocin and the other active norpsilocin analogues in this study, all ranging from 20 to 40 maximal events. Similarly, 4-HO-NPT and 4-HO-NiPT were comparable in potencies in the HTR assay, though 4-HO-NPT was observed to produce roughly double the maximal HTR events (52 vs 27) compared to 4-HO-NiPT. However, it is unclear if 4-HO-NiPT reached full maximum for stimulation of HTR in this case. Taken together, differences in mouse HTR potency and efficacy might be attributed to factors such as the drugs’ distinct pharmacokinetics and metabolism, multiple receptor effects, and/or induced cellular signaling cascades, making it a considerable challenge to pinpoint the exact reasons for the observed behavioral variations. Nevertheless, the potency of psychedelic compounds in the mouse HTR assay is a remarkably accurate predictor of approximate doses that induce psychedelic activity in humans.²⁴

Bulky aliphatic groups like butyl, tert-butyl, and cyclohexyl negated HTR activity. In contrast to norpsilocin, we hypothesized that the lack of observed behavioral effects was likely not attributable to poor bioavailability but, rather, the receptor pharmacodynamics. For the HTR-active compounds, indirect information about drug kinetics may be inferred from examination of behavioral responses over time (Figure 3B–D), as some differences in the time-course for HTR between compounds were observed. HTR for 4-HO-NALT, for instance, peaked early and descended promptly, while 4-HO-NET peaked somewhat later. This discrepancy in HTR timing between 4-HO-NALT and 4-HO-NET may hint at differential absorption and distribution kinetics, with the maximum HTR occurrence being delayed by 10 min for 4-HO-NET compared to 4-HO-NALT at equivalent doses. Regardless of potential pharmacokinetic differences, the HTR data overall support that secondary amine norpsilocin analogues can be modified to impart CNS activity.

In addition to measures of HTR, temperature change (°C) from pre to post session and locomotor activity (distance traveled in cm) across the session were also monitored in mouse assays. All of the compounds produced dose-related hypothermia at the two highest tested doses (10 and 30 mg/kg, Figure S2, Table S1), which is similar to what was previously reported for norpsilocin.⁵ Conversely, only half of the compounds significantly reduced locomotor activity over the test session (Figure S3, Figure S4, Table S1). Similar to our previous study of the 5-HT_1A-mediated effects of psilocin analogues, the hypolocomotor and hypothermic effects of norpsilocin analogues occurred at ∼10-fold higher doses than those that produced significant increases in basal HTR, and these effects corresponded with onset of the descending limb of the HTR dose–response curve. In contrast to other HTR active compounds, 4-HO-NiPT did not display a descending limb up to 30 mg/kg. However, given that 4-HO-NiPT induced significant hypothermia at the highest dose tested (E_max = −1.7 °C), it is likely that higher doses would reveal the descending limb of the inverted U HTR curve for this compound as observed for other HTR active compounds. Overall, these compounds had similar profiles compared to other mushroom tryptamines, including norpsilocin, for hypothermic and hypolocomotor effects. However, the specific involvement of 5-HT_1A or other receptors in these high dose effects of norpsilocin analogues remains to be determined.

Radioligand Competition Binding Assays

While a concentrated emphasis on 5-HT_2A receptor-mediated behavior (i.e., HTR) provided valuable insights, the intricacies of psychedelic actions may involve more than just singular receptor interactions. At the 5-HT_2A receptor, the significance of a drug’s impact on differential second messenger signaling cascades,²⁸ receptor residence time,²⁹ receptor polymorphisms,³⁰ receptor heterodimerizations,³¹ or even whereabout on a neuron that the ligand–receptor engagement occurs,³² is still not fully understood in the context of psychedelic mode of action or therapeutic potential. Moreover, it is evident that the broader pharmacological profiles of these compounds encompass interactions with multiple targets (e.g., 5-HT_1A), further emphasizing the complexity of their mechanisms of action.^26,33−35 Concurrent to the HTR analysis, the National Institute of Mental Health Psychoactive Drug Screening Program (PDSP) was utilized to conduct extensive binding affinity and functional activity screening with norpsilocin analogues. The in vitro screening revealed numerous additional, sometimes high affinity and potent, interactions across a number of psychoactive receptors beyond 5-HT_2A. These non-5-HT_2A activities will be acknowledged with the caveat that interpretation of such multifaceted and simultaneous CNS receptor engagements poses inherent challenges for interpretation in whole animal biological systems.

To assess the diversity of target interactions displayed by norpsilocin analogues, competition binding assays were conducted by the PDSP. A primary “hit” at any target of interest was defined by >50% inhibition of radioligand binding at the screening concentration of 10 μM. Table 2 portrays all targets where ligands displayed >50% inhibition of binding and were subsequently subjected to full concentration–response experiments (inhibition constants listed in Figure 4 and Figure S5). The examined norpsilocin analogues displayed discernible binding affinities across the majority of the 5-HT receptor subtypes tested; however, no compounds interacted with 5-HT_1A or 5-HT₃ receptors at 10 μM concentrations in initial primary screens.

Table 2. Comprehensive Receptor Target Profile of Norpsilocin Analogues^a.

Receptor	Subtype
Serotonergic	5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT5A, 5-HT6, 5-HT7A
Adrenergic	α1A, α1B, α1D, α2A, α2B, α2C, β1, β2
Dopaminergic	D1, D2, D3, D4, D5
GABAergic	GABAA, PBR, BZP (rat brain site)
Glutamatergic	NMDA, NR2B, Kainate (rat brain)
Histaminergic	H1, H2, H3, H4
Monoaminergic transporter	DAT, NET, SERT
Muscarinic	M1, M2, M3 M4, M5
Nicotinic acetylcholine	α2β2, α2β4, α 3β 2, α3β4, α4β2, α2β2 (rat brain), α7
Opioidergic	δ, μ, κ
Sigmaergic	σ1, σ2

^aHits (bolded text) were defined by one or more compounds exhibiting >50% average inhibition at a 10 μM test concentration (a single experiment with quadruplicate determinations) and advanced to full competition binding assays to determine inhibition constants.

Figure 4.

pK_i (−log K_i) heatmap plot for inhibition constants (K_i, nM) of norpsilocin analogues in competition binding assays for human 5-HT receptors and select sites. Light gray boxes represent inhibition constants >1 μM from full dose–response experiments, and dark gray boxes indicate <50% inhibition in the primary radioligand binding screen. Each inhibition constant was derived from n = 2–3 experiments with triplicate determinations, and values for 95% confidence intervals are shown below each K_i value. Radioligands used, reference control compound used, and control K_i values for each receptor appear in Table S2 while data for other hits across the series is shown in Figure S5.³⁶

The lack of 5-HT_1A affinity of norpsilocin shown here contrasts with our previous report showing discernible affinity of norpsilocin for 5-HT_1A (K_i = 29–86 nM),⁵ which may highlight an anomaly in the primary radioligand binding screens for this receptor site. On the other hand, in the same study norpsilocin had weak 5-HT_1A agonist activity in a Tango assay and low potency for 5-HT_1A-mediated effects in mice, similar to what was observed in the present studies of norpsilocin analogues (Figures S3–S5). Therefore, the findings with norpsilocin suggest that functional activity of norpsilocin analogues are similarly weak for 5-HT_1A-mediated effects.

Outside of the discrepancy with binding to 5-HT_1A, the affinity profile of norpsilocin was similar to our previously reported results.⁵ Norpsilocin analogues had comparable inhibition constants across 5-HT₂ receptor subtypes with few exceptions (Figure 4). Notably, 4-HO-NtBT did not have affinity for 5-HT_2A, consistent with our observed lack of HTR in mouse studies. Additionally, 4-HO-NtBT and 4-HO-NiPT both had μM affinities for binding at 5-HT_2C while all other compounds had inhibition constants in the nM range. Interestingly, norpsilocin, 4-HO-NET, and 4-HO-NALT were the only analogues that competed for binding at 5-HT_1B. Furthermore, inhibition constants across other 5-HT receptors for 4-HO-NET, 4-HO-NPT, and 4-HO-NALT were mostly in the nM range, while all other compounds were either inactive up to 10 μM or generally in the μM range for affinity. Additionally, affinities observed for 5-HT_2A and 5-HT_2C were reduced compared to 5-HT_2B, likely due to the use of agonist-labeled ³H-LSD for 5-HT_2B and antagonist-labeled ³H-ketanserin and ³H-mesulergine for 5-HT_2A and 5-HT_2C, respectively.³⁷ Overall, the compounds primarily targeted 5-HT receptors with nonselective, variable affinities.

Outside of 5-HT receptor subtypes, norpsilocin derivatives did not demonstrate appreciable affinity for adrenergic, dopaminergic, GABAergic, glutamatergic, muscarinic, nicotinic acetylcholine, or opioidergic receptors and displayed negligible affinity for monoamine transporters. As depicted in the Figure 4 and Figure S5 heatmaps, binding affinities for these other non-5-HT receptors sporadically reached the micromolar range for several compounds; however, there were a few exceptions. For example, 4-HO-NALT exhibited an affinity of 467 nM for the H₁ receptor. Intriguingly, both 4-HO-NcHT and 4-HO-NBnT demonstrated substantial (40–250 nM) affinity for σ₁ and σ₂ receptors.

Lastly, inspection of the binding affinity profiles for 4-HO-NBnT and 4-HO-NcHT provides another interesting insight. The two compounds stand out as candidates for interrogating previously proposed interplay between 5-HT and σ receptors to induce neuroplasticity.^38,39 While both drug molecules reveal comparable affinities at σ receptors, 4-HO-NBnT distinctively exhibits high affinity and activity at 5-HT receptors, while 4-HO-NcHT does not. This suggests a congruent binding modality for both compounds at σ receptors, while differing substantially in their interactions at 5-HT receptors. Likewise, similar comparisons with 4-HO-NBnT and 4-HO-NALT may be warranted as both display high affinity and activity at serotonin receptors, while data for the allyl compound suggest it is essentially devoid of affinity for σ receptors. Future studies examining the σ receptor pharmacology of this series of compounds are thus warranted.

Functional Activity at 5-HT₂ Receptors

In addition to radioligand binding studies to determine inhibition constants for norpsilocin analogues at pharmacologically relevant targets, this series of compounds was also evaluated by the PDSP in 5-HT₂ receptor functional assays in vitro. Specifically, we evaluated the structure–activity relationships for Gα_q-mediated calcium flux at human 5-HT₂ receptors, which revealed specific trends. For the 5-HT_2A receptor subtype, the potency rank order by half-maximal effective concentration (EC₅₀) was 4-HO-NBnT > 4-HO-NALT > norpsilocin >4-HO-NET > 4-HO-NPT > 4-HO-NiPT > 4-HO-NnBT > 4-HO-NcHT > 4-HO-NtBT (Figure 5, Figure S6A,B). Most compounds were full agonists and reached an E_max value between 80 and 90% relative to 5-HT, but the N-benzyl derivative was slightly less efficacious at 70%.

Figure 5.

Heatmap plots of the potencies (A, pEC₅₀, EC₅₀) and maximal effects (B, E_max) of norpsilocin analogues in Gα_q-mediated calcium flux assays at human 5-HT₂ receptors. pEC₅₀ = −log EC₅₀ values. EC₅₀ values with 95% confidence intervals noted below in parentheses for 2–3 experiments with triplicate determinations are listed in each box. “x” denotes no activity, and “*” with italicized text denotes potentially ambiguous EC₅₀ and/or E_max values. Concentration–response curves are shown in Figure S6.

For the 5-HT_2B receptor, the EC₅₀ rank order was similar to 4-HO-NBnT > norpsilocin >4-HO-NALT > 4-HO-NET > 4-HO-NPT > 4-HO-NnBT > 4-HO-NiPT > 4-HO-NtBT > 4-HO-NcHT (Figure 5, Figure S6C,D). Most of the compounds exhibited partial agonist activity at the 5-HT_2B receptor with E_max values between 68 and 70%; however, 4-HO-NALT and 4-HO-NBnT were outliers: 4-HO-NALT exhibited 88% maximal response compared to 5-HT, and in contrast, 4-HO-NBnT produced an E_max of only 11%, indicating very weak partial agonist or no activity in this assay.

Regarding the 5-HT_2C receptor, norpsilocin displayed the highest potency, followed by 4-HO-NBnT then 4-HO-NALT (Figure 5, Figure S6E,F). 4-HO-NnBT and 4-HO-NiPT were weak partial agonists at 5-HT_2C. Finally, the sterically bulky substituted compounds 4-HO-NtBT and 4-HO-NcHT were inactive at 5-HT_2C in this assay. Similar to observations at 5-HT_2A, individual potency and efficacy values varied with structure at 5-HT_2C, with norpsilocin and 4-HO-NBnT producing approximately 90% maximal stimulation relative to 5-HT, compared to 4-HO-NALT and 4-HO-NPT, which were partial agonists at 54% and 25%, respectively. Previous studies have indicated that 5-HT_2C receptor activation may counteract the 5-HT_2A receptor-induced HTR behavior in rodents, and therefore the partial agonist efficacy of 4-HO-NALT and 4-HO-NPT at 5-HT_2C may be a contributing factor in the high E_max values observed here in the HTR assay.^40,41 Importantly, the data for norpsilocin for functional activity across 5-HT₂ receptors was similar to previously reported values.^4,5

Overall, 4-HO-NBnT, 4-HO-NALT, 4-HO-NET, and norpsilocin consistently showed high potency for stimulation of Gα_q-mediated calcium flux at human 5-HT₂ receptor subtypes, while the bulkiest aliphatic substituted compounds 4-HO-NtBT and 4-HO-NcHT had lower or negligible activity. It is noteworthy that the 5-HT_2A receptor functional activity trends were similar to the potency trends observed in the mouse HTR assays. Norpsilocin was the lone exception, as norpsilocin was one of the most potent compounds at 5-HT_2A receptors in vitro yet inactive for HTR in vivo, further supporting our initial hypothesis of poor CNS permeability for this compound. The data also suggest that inactivity of the NnBT, NtBT, and NcHT analogues for mouse HTR may be related to their reduced pharmacodynamic effects at 5-HT_2A (EC₅₀ = 32–154 nM and E_max = 17–58%) rather than CNS permeability, as seems to be the case for norpsilocin. Future pharmacokinetic studies comparing the plasma to brain ratio of norpsilocin vs HTR active and inactive analogues would be informative to further support the hypothesis regarding CNS bioavailability and 5-HT_2A pharmacodynamics proposed herein. The observed potency of compounds for 5-HT_2A vs 5-HT_2B was consistently 2- to 4-fold greater, whereas potency for 5-HT_2A vs 5-HT_2C was consistently (10–20)-fold greater. Such consistent potency ratios suggest that active compounds have comparable binding modes at 5-HT₂ receptor subtypes, possibly related to the phylogenetic similarities across 5-HT_2A/B/C receptor orthosteric binding sites.^29,42

Despite the parallel changes in potency at 5-HT₂ receptor subtypes across the tryptamine series, the reduced efficacy of 4-HO-NBnT, particularly at the 5-HT_2B receptor, highlights a potentially unique receptor interaction with the benzyl-substituted compound. The incorporation of benzyl substitutions in drug design has been recognized for nonspecifically enhancing potency by increasing lipophilicity alone;⁴³ however, in the case of psychedelics, N-benzyl substitutions appear to also impart specific 5-HT_2A receptor interactions. This is especially evident in the emergence of the N-benzylmethoxy (NBOMe) phenethylamine series, particularly the 25x-NBOMe derivatives, which have considerably higher potency and unexpected properties compared to their non-N-benzyl-substituted counterparts.⁴⁴⁻⁴⁶ For instance, 25I-NBOMe, one of the most studied in the series, has high potency at the 5-HT_2A receptor, along with serious toxicological side effects atypical of classical psychedelics.⁴⁷ Subsequent studies might evaluate the impact of analogous substitutions on the phenyl ring of 4-HO-NBnT to ascertain additional structure–activity relationships, adding to recent reports evaluating related substituted N-benzyltryptamines.^48,49

Functional Activity at 5-HT_1,4,5,6,7 Receptors

In addition to the 5-HT₂ receptor calcium mobilization assays, the functional agonist activity of the norpsilocin analogues was also screened across an array of other non 5-HT₂ receptor subtypes using the GPCR Tango assay measuring G protein-independent β-arrestin recruitment (Figure 6, Figure S7).¹ Relative to serotonin, comparable activity was identified with one or more norpsilocin analogues at the 5-HT_1B, 5-HT_1D, 5-HT_1E, 5-HT_5A, and 5-HT_7A receptors.

Figure 6.

Functional agonist activity screening heatmap plots for norpsilocin analogues at 5-HT₁, 5-HT₄, 5-HT₅, 5-HT₆, and 5-HT₇ receptors using the GPCR Tango assay measuring β-arrestin recruitment. “x” denotes no activity up to 50 μM, and “*” with italicized text denotes activity trends with ambiguous EC₅₀ and/or E_max values. If a stable top was not reached by 2 consecutive points, the maximum effect observed is reported as E_max. Assays represent 1–2 experiments run in triplicate. The associated concentration–response curves are shown in Figure S7.

In contrast, somewhat weaker activity was detected for certain ligands at the 5-HT_1A, 5HT_1F, and 5-HT₆ receptors, with an absence of any detectible activity at the 5-HT₄ receptors for any ligands, except for norpsilocin at the highest concentration. Similarly, several compounds displayed effects only at the highest concentrations tested and did not produce full curves, so EC₅₀ and E_max values were too ambiguous to calculate.

Only norpsilocin and 4-HO-NALT exhibited measurable, albeit weak, activity at the 5-HT_1A receptor in contrast to 2 μM for the serotonin reference. This receptor subtype is known to play roles in mood, anxiety, and cognition, making this observation of interest for potential CNS-mediated effects.⁵⁰ Many classical psychedelics, including psilocin, exhibit a distinct binding affinity for both 5-HT_1A and 5-HT_2A receptors.⁵¹ Concurrent activation of 5-HT_1A receptors, however, can attenuate some of the psychedelic effects in rodents and humans.^26,52−54 In the context of the norpsilocin derivatives, there appears to be a pronounced selectivity favoring the 5-HT_2A receptor over 5-HT_1A receptors. Such selectivity could result in differential pharmacological effects, depending on the balance of activation across other serotonin receptor subtypes. However, it is important to exercise caution in interpreting these preliminary high-throughput screening findings, and additional studies focusing on 5-HT_1A receptor interactions are warranted to fully understand the implications for CNS-mediated effects. In mouse studies, all norpsilocin analogues induced hypothermia at high doses similar to other tryptamines, including psilocin and related analogues.⁵ On the other hand, only 4-HO-NET, 4-HO-NPT, 4-HO-NnBT, and 4-HO-NBnT reduced the total locomotor activity over the 30 min testing session, suggesting that these compounds may not recruit 5-HT_1A-mediated hypolocomotor effects as potently as other psychedelic tryptamines.

Excluding 4-HO-NtBT and 4-HO-NcHT, which were inactive, comparable agonist activities were detected across all other derivatives at the 5-HT_1B receptor, ranging from 1 to 4 μM, compared to 0.4 μM with the serotonin reference. The 5-HT_1B receptor has been shown to possibly influence behaviors such as aggression and response to antidepressant treatments.⁵⁵ For the 5-HT_1D receptor, norpsilocin, 4-HO-NET, and 4-HO-NALT displayed β-arrestin recruitment activity at potencies similar to those produced by serotonin, with EC₅₀ values ranging from 1 to 3 μM compared to 1 μM for the endogenous ligand. This receptor subtype has been implicated in the vasoconstriction phase of migraine headaches.⁵⁶

At the 5-HT_1E receptor, most compounds tested, except for 4-HO-NtBT and 4-HO-NcHT, exhibited potencies between 1 and 5 μM, relative to the serotonin reference potency of 0.42 μM. The physiological role of the 5-HT_1E receptor remains less defined compared to other subtypes, and few selective ligands are currently known; yet, DNA sequencing has shown that this receptor has high evolutionary conservation (albeit it is not found in rodents) suggesting an important physiological role.⁵⁷ Given the relatively high 5-HT_1E potency and efficacy by several structures, these scaffolds may be of interest in the future design of new 5-HT_1E ligands, should a utility be determined.^58,59

The 5-HT_7A receptor trends contrasted with activity at other receptors, where the n-butyl compound 4-HO-NnBT exhibited the highest potency at 1 μM, closely followed by norpsilocin, 4-HO-NiPT, and 4-HO-NBnT, each ranging from 1 to 2 μM, compared to 0.42 μM for 5-HT. The 5-HT_7A receptor has been associated with circadian rhythm modulation, thermoregulation, and potentially mood disorders.^60,61 The pronounced potency of 4-HO-NnBT at this receptor stands out, given its weaker performance at most other targets examined. In summary, the current evaluation illustrates that subtle structural alterations in 4-HO-NxT derivatives can engender diverse functional profiles across various 5-HT receptor subtypes. Overall, the compounds had several potentially relevant activities across 5-HT receptor subtypes in screening assays assessing G protein-independent signaling, and further evaluation of the G protein-mediated signaling properties of these compounds is warranted.

Conclusions

In the present study, a series of norpsilocin analogues were synthesized and evaluated with the goal of enhancing CNS bioavailability compared to norpsilocin itself. Our initial hypothesis, that increasing lipophilicity through extensions of the methylamine group would restore CNS effects, was supported by results from mouse HTR experiments. The N-ethyl, N-allyl, and N-benzyl derivatives—4-HO-NET, 4-HO-NALT, and 4-HO-NBnT—displayed psychedelic-like effects in the HTR assay with 10-fold weaker potencies relative to psilocin and varying maximal efficacies. The in vitro pharmacological profiling highlighted diverse SARs across the panel of compounds. While the trends at 5-HT₂ receptors aligned with the in vivo results, interactions at numerous other serotonin receptor subtypes may also be relevant to the pharmacology of the compounds. Overall, subtle modifications to the N-substituent of the tryptamine core profoundly impacted the pharmacological target profiles of these psychedelic-inspired compounds. The restored central activity in correlation with enhanced lipophilicity provides insights into structural features governing bioavailability of monoalkyl tryptamines. While predicting therapeutic relevance requires extensive further study, these scaffolds offer intriguing starting points for design of optimized serotonin receptor ligands and tools. The results exemplify how natural product derivatives can serve both as biological probes and possible leads for medication discovery efforts.

Methods

General Experimental Methods

Reactions were performed using commercially obtained solvents. Unless otherwise stated, all commercially obtained reagents were used as received. Reactions were monitored by UPLC-UV-high-resolution mass spectrometry (HRMS) (described below) and thin-layer chromatography (TLC) using EMD/Merck silica gel 60 F254 precoated plates (0.25 mm). Flash column chromatography (FCC) was performed using prepackaged RediSep Rf silica columns on a CombiFlash Rf system (Teledyne ISCO Inc.). ¹H and ¹³C NMR spectra were recorded on a Bruker Avance 400 (at 400 and 101 MHz, respectively) and are reported relative to internal CHCl₃ or D₂O signals. Analytical UPLC was performed with a Waters Acquity I-Class UPLC utilizing Waters HSS T3 column (2.5 μm, 2.1 mm × 30 mm) run in gradient mode with H₂O (0.1% formic acid) and acetonitrile (0.1% formic acid) mobile phases at 0.6 mL/min. Samples were diluted in acetonitrile or water to approximately 1 mg/mL and 0.1 μL injected. Chromatographic peaks were detected and integrated by diode array detector at 269 nm. High-resolution mass spectra were obtained on a Waters G2-XS QTof instrument in electrospray ionization (ESI) positive mode.

General Procedure A to Ketoamides 3X

To a flame-dried round-bottom flask (RBF) under argon, acid chloride 2(4,19,20) (1 equiv) was dissolved in anhydrous tetrahydrofuran (THF) (11 vol). The solution was cooled with an ice bath before adding a 2 M solution of primary amine (1.2 equiv) in THF. After stirring for 15 min, anhydrous triethylamine (1.23 equiv) was introduced dropwise. The reaction mixture was warmed to room temperature and stirred overnight under argon. The mixture was then transferred to a separatory funnel and combined with a 1:1 mixture of EtOAc and water. The organic layer was decanted, and the aqueous phase was extracted with EtOAc twice. The combined organic extracts were washed successively with 1 N HCl, water, and brine and then dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure. Purification of the resultant solid was achieved through flash column chromatography, recrystallization, or trituration as needed.

General Procedure B to 4-HO-NxTs

In a round-bottom flask under argon, a 2 M solution of ketoamide 3X (1 equiv) was prepared in 2-methyltetrahydrofuran (2-Me-THF). With the flask equipped with a reflux condenser, a 2.4 M THF solution of lithium aluminum hydride (4 equiv) was added dropwise, ensuring a gentle reflux was maintained. External heat was applied, and the mixture was refluxed for 4 h to overnight, with reaction completion monitored by UPLC-HRMS. After adding additional THF (20 volumes) to the mixture, it was cooled with an ice bath. Sodium sulfate decahydrate was added until the exothermic reaction ceased, followed by a 30 min stir under argon. Addition of a 20% methanol in dichloromethane (DCM) solution (60 volumes) facilitated filtration through a silica pad. The pad was further washed with the methanol/DCM solution (60 volumes). After rotary evaporation of the filtrate, the residue was dissolved in refluxing acetone, and either fumaric or succinic acid (1 equiv) was added in a single portion, leading to a white precipitate. After cooling, the precipitate was collected by filtration. If required, additional purification was performed using FCC of the free base or trituration of the salt in refluxing EtOH.

3-(2-(Ethylamino)-2-oxoacetyl)-1H-indol-4-yl Acetate (3E)

Prepared using General Procedure A with acid chloride 2 (1.33 g, 5.00 mmol) and ethylamine. The product was purified via flash chromatography with 20–30% EtOAc in hexanes, yielding a beige powder (1.0 g, 73%). UPLC purity: 98.3%. Melting point: 127–129 °C. Rf = 0.36 in 60% EtOAc/heptanes. HRMS (ESI) m/z [M + Na]⁺ calcd for C₁₄H₁₄N₂NaO₄⁺ 297.0846, found 297.0886. ¹H NMR (400 MHz, CDCl₃): δ 9.74 (1H, s), 8.72 (1H, d, J = 3.4 Hz), 7.45 (1H, ddd, J = 5.8, 2.2, 1.5 Hz), 7.15 (1H, dd, J = 8.0, 8.0 Hz), 7.02 (1H, d, J = 8.1 Hz), 6.89 (1H, d, J = 7.7 Hz), 3.39 (2H, qd, J = 7.3, 5.9 Hz), 2.52 (3H, s), 1.24 (3H, t, J = 7.3 Hz). ¹³C NMR (101 MHz, CDCl₃): δ 179.8, 171.8, 162.8, 144.5, 140.4, 138.6, 124.6, 119.4, 116.6, 112.7, 110.8, 34.5, 21.9, 14.7.

3-(2-Oxo-2-(propylamino)acetyl)-1H-indol-4-yl Acetate (3P)

Prepared using General Procedure A with acid chloride 2 (1.96 g, 7.37 mmol) and propylamine. The crude product was recrystallized from 90 mL of a 10% isopropyl alcohol (IPA) solution in diisopropyl ether, yielding a white solid (1.15 g, 53%). UPLC purity: 99.5%. Melting point: 125–127 °C. Rf = 0.37 in 60% EtOAc/heptanes. HRMS (ESI) m/z [M + Na]⁺ calcd for C₁₅H₁₆N₂NaO₄⁺ 311.1002, found 311.1032. ¹H NMR (400 MHz, CDCl₃): δ 9.36 (1H, s), 8.79 (1H, d, J = 3.3 Hz), 7.44 (1H, ddd, J = 6.5, 2.3, 1.5 Hz), 7.16 (1H, dd, J = 8.0, 80. Hz), 7.06 (1H, d, J = 8.1 Hz), 6.88 (1H, d, J = 7.7 Hz), 3.29 (2H, dt, J = 7.7, 6.4 Hz), 2.49 (3H, s), 1.61 (2H, qt, J = 7.8, 7.0 Hz), 0.96 (3H, t, J = 7.4 Hz). ¹³C NMR (101 MHz, CDCl₃): δ 179.9, 171.6, 162.8, 144.6, 140.2, 138.5, 124.8, 119.6, 116.8, 112.9, 110.7, 41.4, 22.9, 21.9, 11.7.

3-(2-(Allylamino)-2-oxoacetyl)-1H-indol-4-yl Acetate (3AL)

Prepared using General Procedure A with acid chloride 2 (2.02 g, 7.60 mmol) and allylamine. The crude product was purified by slurrying in a 10% IPA solution in diisopropyl ether at 55 °C for 1 h, then stirring at room temperature overnight under argon. This yielded a beige solid (1.51 g, 69%). UPLC purity: 98.0%. Melting point: 151–152 °C. Rf = 0.36 in 60% EtOAc/heptanes. HRMS (ESI) m/z [M + Na]⁺ calcd for C₁₅H₁₄N₂NaO₄⁺ 309.0846, found 309.0890. ¹H NMR (400 MHz, CDCl₃): δ 9.79 (1H, s), 8.64 (1H, d, J = 3.3 Hz), 7.56 (1H, ddd, J = 6.5, 2.3, 1.5 Hz), 7.11 (1H, dd, J = 8.0, 8.0 Hz), 6.97 (1H, d, J = 8.1 Hz), 6.87 (1H, d, J = 7.7 Hz), 5.86 (1H, ddt, J = 16.0, 11.0, 5.0 Hz), 5.21 (2H, m), 3.95 (2H, dd, J = 6.2, 5.4 Hz), 2.50 (3H, s). ¹³C NMR (101 MHz, CDCl₃): δ 179.5, 171.8, 162.8, 144.4, 140.4, 138.5, 133.4, 124.7, 119.4, 117.2, 116.6, 112.6, 110.9, 42.0, 21.9.

3-(2-(Isopropylamino)-2-oxoacetyl)-1H-indol-4-yl Acetate (3iP)

Prepared using General Procedure A with acid chloride 2 (2.03 g, 7.64 mmol) and isopropylamine. The crude product was recrystallized from 50 mL of a 10% IPA solution in diisopropyl ether. An additional crop of crystals was harvested from the remaining mother liquor, and a subsequent recrystallization using 40 mL of a 10% IPA solution in isopropyl ether yielded a white powder (1.19 g, 54%). UPLC purity: 99.4%. Melting point: 155–157 °C. Rf = 0.54 in 60% EtOAc/heptanes. HRMS (ESI) m/z [M + Na]⁺ calcd for C₁₅H₁₆N₂NaO₄⁺ 311.1002, found 311.1032. ¹H NMR (400 MHz, CDCl₃): δ 9.64 (1H, s), 8.79 (1H, d, J = 3.3 Hz), 7.30 (1H, dd, J = 8.6, 2.0 Hz), 7.19 (1H, m), 7.07 (1H, d, J = 8.1 Hz), 6.92 (1H, d, J = 7.7 Hz), 4.13 (1H, septd, J = 7.0, 7.0 Hz), 2.55 (3H, s), 1.29 (6H, d, J = 6.6 Hz). ¹³C NMR (101 MHz, CDCl₃): δ 180.0, 171.7, 162.0, 144.6, 140.3, 138.5, 124.7, 119.5, 116.7, 112.8, 110.8, 41.8, 22.7, 21.9.

3-(2-(Butylamino)-2-oxoacetyl)-1H-indol-4-yl Acetate (3nB)

Prepared using General Procedure A with acid chloride 2 (1.96 g, 7.38 mmol) and butylamine. The crude product was purified via an overnight slurry in 50 mL of a 10% IPA solution in diisopropyl ether, resulting in a light yellow powder (1.39 g, 62%). UPLC purity: 99.0%. Melting point: 129–130 °C. Rf = 0.57 in 60% EtOAc/heptanes. HRMS (ESI) m/z [M + Na]⁺ calcd for C₁₆H₁₈N₂NaO₄⁺ 325.1159, found 325.1146. ¹H NMR (400 MHz, CDCl₃): δ 9.66 (1H, s), 8.74 (1H, d, J = 3.3 Hz), 7.46 (1H, dd, J = 6.2, 1.3 Hz), 7.15 (1H, m), 7.03 (1H, d, J = 8.1 Hz), 6.90 (1H, d, J = 7.7 Hz), 3.35 (2H, td, J = 7.6, 6.9), 2.52 (3H, s), 1.59 (2H, tt, J = 8.2, 7.4), 1.40 (2H, tq, J = 8.5 6.9 Hz), 0.96 (3H, t, J = 7.3 Hz). ¹³C NMR (101 MHz, CDCl₃): δ 179.9, 171.7, 162.8, 144.5, 140.0, 138.6, 124.7, 119.5, 116.7, 112.8, 110.8, 39.4, 31.6, 21.9, 20.4, 13.9.

3-(2-(tert-Butylamino)-2-oxoacetyl)-1H-indol-4-yl Acetate (3tB)

Prepared using General Procedure A with acid chloride 2 (2.06 g, 7.77 mmol) and tert-butylamine. The crude product was purified by slurrying in a 10% IPA solution in diisopropyl ether at 55 °C for 1 h, followed by cooling to room temperature with overnight stirring under argon. This yielded a pale yellow solid (1.44 g, 61%). UPLC purity: 95.7%. Melting point: 208–210 °C. Rf = 0.64 in 60% EtOAc/heptanes. HRMS (ESI) m/z [M + Na]⁺ calcd for C₁₆H₁₈N₂NaO₄⁺ 325.1159, found 325.1197. ¹H NMR (400 MHz, CDCl₃): δ 9.60 (1H, s), 8.72 (1H, d, J = 3.3 Hz), 7.28 (1H, s), 7.13 (1H, m), 7.02 (1H, d, J = 8.1 Hz), 6.87 (1H, d, J = 7.7 Hz), 2.50 (3H, s), 1.43 (9H, s). ¹³C NMR (101 MHz, CDCl₃): δ 180.8, 171.8, 162.2, 144.6, 140.2, 138.5, 124.7, 119.6, 116.7, 112.6, 110.8, 51.5, 28.7, 22.0.

3-(2-(Cyclohexylamino)-2-oxoacetyl)-1H-indol-4-yl Acetate (3cH)

Prepared using General Procedure A with acid chloride 2 (1.83 g, 6.90 mmol) and cyclohexylamine. The crude product was purified by slurrying in a 10% IPA solution in diisopropyl ether at 55 °C for 1 h, then stirring at room temperature overnight under argon. This yielded a white powder (1.75 g, 77%). UPLC purity: 99.5%. Melting point: 215–218 °C. Rf = 0.62 in 60% EtOAc/heptanes. HRMS (ESI) m/z [M + Na]⁺ calcd for C₁₈H₂₀N₂NaO₄⁺ 351.1315, found 351.1361. ¹H NMR (400 MHz, CDCl₃): δ 9.32 (1H, s), 8.83 (1H, d, J = 3.3 Hz), 7.30 (1H, dd, J = 8.4, 1.7 Hz), 7.18 (1H, m), 7.09 (1H, d, J = 8.1 Hz), 6.89 (1H, d, J = 7.7 Hz), 3.77 (1H, m), 2.51 (3H, s), 1.95 (2H, m), 1.71 (4H, m), 1.31 (4H, m). ¹³C NMR (101 MHz, CDCl₃): δ 180.1, 171.6, 161.8, 144.7, 140.2, 138.4, 124.8, 119.6, 116.8, 113.1, 110.6, 48.7, 33.0, 25.7, 25.1, 21.9.

3-(2-(Benzylamino)-2-oxoacetyl)-1H-indol-4-yl Acetate (3Bn)

Prepared using General Procedure A with acid chloride 2 (2.02 g, 7.60 mmol) and benzylamine. The crude product was purified by slurrying in a 10% IPA solution in diisopropyl ether at 55 °C for 1 h, then stirring at room temperature overnight under argon. This yielded an off-white powder (1.36 g, 53%). UPLC purity: 97.0%. Melting point: 158–159 °C. Rf = 0.54 in 60% EtOAc/heptanes. HRMS (ESI) m/z [M + Na]⁺ calcd for C₁₈H₂₄N₂NaO₄⁺ 359.1002, found 359.1022. ¹H NMR (400 MHz, CDCl₃): δ 9.58 (1H, s), 8.70 (1H, d, J = 3.3 Hz), 7.77 (1H, dd, J = 6.2, 1.6 Hz), 7.32 (5H, m), 7.12 (1H, m), 6.99 (1H, d, J = 8.1 Hz), 6.88 (1H, d, J = 7.7 Hz), 4.52 (2H, d, J = 6.1 Hz), 2.49 (3H, s). ¹³C NMR (101 MHz, CDCl₃): δ 179.5, 171.8, 162.8, 144.5, 140.4, 138.5, 137.6, 129.0, 128.0, 124.7, 119.5, 116.7, 110.8, 43.7, 21.9.

3-(2-(Ethylamino)ethyl)-1H-indol-4-ol Hemisuccinate (4-HO-NET)

Prepared using General Procedure B from 3E (0.850 g, 3.10 mmol), yielding a white solid (0.46 g, 67%). Purity: 99.0%. Melting point: 222–223 °C (as the hemisuccinate salt), HRMS (ESI) m/z [M + H]⁺ calcd for C₁₂H₁₇N₂O⁺ 205.1335, found 205.1335.

¹H NMR (400 MHz, D₂O) δ 7.17 (1H, m), 7.09 (2H, m), 6.56 (1H, dd, J = 5.8, 2.8 Hz), 3.49 (2H, m), 3.24 (2H, m), 3.07 (2H, q, J = 7.3 Hz), 2.40 (2H, s), 1.24 (3H, t, J = 7.3 Hz). ¹³C NMR (101 MHz, D₂O) δ 182.3, 145.0, 138.8, 123.3, 123.1, 123.0, 115.9, 108.9, 104.5, 103.7, 48.5, 42.7, 34.1, 23.2, 10.3.

3-(2-(Propylamino)ethyl)-1H-indol-4-ol Hemifumarate (4-HO-NPT)

Prepared using General Procedure B from 3P (1.0 g, 3.5 mmol), yielding a white solid (0.57 g, 60%). UPLC purity: 96.29%. Melting point: 213–214 °C (as the hemifumarate salt). HRMS (ESI) m/z [M + H]⁺ calcd for C₁₃H₁₉N₂O⁺ 219.1492, found 219.1498. ¹H NMR (400 MHz, D₂O): δ 7.13 (1H, s), 7.06 (2H, m), 6.56–6.50 (2H, m), 3.34 (2H, t, J = 6.8 Hz), 3.21 (2H, t, J = 6.9 Hz), 2.94 (2H, t, 7.7 Hz), 1.62 (2H, sx, J = 7.6 Hz), 0.90 (3H, t, J = 7.5 Hz). ¹³C NMR (101 MHz, D₂O): δ 174.5, 149.9, 138.8, 135.3, 123.3, 123.1, 115.9, 108.9, 104.5, 103.7, 48.9, 48.8, 23.1, 18.5, 10.0.

3-(2-(Allylamino)ethyl)-1H-indol-4-ol Hemifumarate (4-HO-NALT)

Prepared using General Procedure B from 3AL (1.0 g, 3.5 mmol). As a result of an intractable impurity caused by reduction of the olefin, the freebase material was purified utilizing FCC with a gradient of 50%–100% 3:1:0.2 EtOAc/EtOH/NH₄OH in hexanes, then converted to the fumarate salt to provide a gray solid (0.18 g, 19%). UPLC purity: 99.68%. Melting point: 200–202 °C (as the hemifumarate salt). HRMS (ESI) m/z [M + H]⁺ calcd for C₁₃H₁₇N₂O⁺ 217.1335, found 217.1335. ¹H NMR (400 MHz, D₂O): δ 7.15 (1H, s), 7.07 (2H, m), 6.55 (1H, dd, J = 5.6, 2.8 Hz), 6.51 (1H, s), 5.83 (1H, td, J = 6.7, 5.6 Hz), 5.45 (1H, s), 5.42 (1H, d, J = 5.5 Hz), 3.62 (2H, d, J = 6.7 Hz), 3.39 (2H, t, J = 6.8 Hz), 3.24 (2H, t, J = 6.9 Hz). ¹³C NMR (101 MHz, D₂O): δ 174.5, 149.9, 138.8, 135.3, 127.4, 123.4, 123.3, 123.1, 115.9, 108.8, 104.5, 103.7, 49.3, 48.3, 23.1.

3-(2-(Isopropylamino)ethyl)-1H-indol-4-ol Fumarate (4-HO-NiPT)

Prepared using General Procedure B from 3iP (1.0 g, 3.5 mmol), yielding a white solid (0.84 g, 73%). UPLC purity: 97.57%. Melting point: 255–257 °C (as the fumarate salt). HRMS (ESI) m/z [M + H]⁺ calcd for C₁₃H₁₉N₂O⁺ 219.1492, found 219.1496. ¹H NMR (400 MHz, D₂O): δ 7.16 (1H, s), 7.08 (2H, m), 6.65 (2H, m), 6.55 (1H, m), 3.40 (3H, m), 3.22 (2H, t, J = 6.8 Hz), 1.27 (6H, d, J = 6.6 Hz). ¹³C NMR (101 MHz, D₂O): δ 171.9, 149.9, 138.8, 134.7, 123.3, 123.1, 115.9, 108.9, 104.5, 103.7, 50.4, 46.0, 23.4, 18.0.

3-(2-(Butylamino)ethyl)-1H-indol-4-ol Fumarate (4-HO-NnBT)

Prepared using General Procedure B from 3nB (1.0 g, 3.3 mmol), yielding a white solid (0.95 g, 88%). UPLC purity: 98.64%. Melting point: 247–251 °C (as the fumarate salt). HRMS (ESI) m/z [M + H]⁺ calcd for C₁₄H₂₁N₂O⁺ 233.1648, found 233.1649. ¹H NMR (400 MHz, D₂O): δ 7.18 (1H, s), 7.09 (2H, m), 6.65 (2H, m), 6.56 (1H, m), 3.41 (2H, t, J = 6.8 Hz), 3.26 (2H, t, J = 6.8 Hz), 3.03 (2H, t, J = 6.9 Hz), 1.61 (2H, qu, J = 7.0 Hz), 1.33 (2H, sx, J = 7.0 Hz), 0.89 (3H, t, J = 7.3 Hz). ¹³C NMR (101 MHz, D₂O): δ 172.2, 149.9, 138.8, 134.8, 123.4, 123.1, 108.9, 104.5, 103.7, 48.8, 47.2, 27.4, 23.1, 19.1, 12.7.

3-(2-(tert-Butylamino)ethyl)-1H-indol-4-ol Hemifumarate (4-HO-NtBT)

Prepared using General Procedure B from 3tB (1.0 g, 3.3 mmol). The crude fumarate salt was further purified by trituration in refluxing EtOH to yield a light gray solid (0.46 g, 48%). UPLC purity: 97.58%. Melting point: 247–249 °C (as the hemifumarate salt). HRMS (ESI) m/z [M + H]⁺ calcd for C₁₄H₂₁N₂O⁺ 233.1648, found 233.1647. ¹H NMR (400 MHz, D₂O): δ 7.16 (1H, s), 7.08 (2H, m), 6.55 (1H, m), 6.52 (1H, s), 3.37 (2H, t, J = 6.8 Hz), 3.20 (2H, t, J = 6.9 Hz), 1.32 (9H, s). ¹³C NMR (101 MHz, D₂O): δ 174.4, 149.8, 138.8, 135.2, 123.2, 123.2, 115.9, 109.1, 104.6, 103.7, 56.9, 43.0, 24.8, 23.9.

3-(2-(Cyclohexylamino)ethyl)-1H-indol-4-ol Fumarate (4-HO-NcHT)

Prepared using General Procedure B from 3cH (1.0 g, 3.1 mmol), yielding an off-white solid (0.73 g, 64%). UPLC purity: 96.61%. Melting point: 253–255 °C (as the fumarate salt). HRMS (ESI) m/z [M + H]⁺ calcd for C₁₆H₂₃N₂O⁺ 259.1805, found 259.1805.

¹H NMR (400 MHz, D₂O): δ 7.15 (1H, s), 7.07 (2H, m), 6.68 (2H, m), 6.55 (1H, m), 3.40 (2H, t, J = 6.8 Hz), 3.22 (2H, t, J = 6.8 Hz), 3.1 (1H, m), 2.02 (2H, m), 1.78 (2H, m), 1.27 (5H, m). ¹³C NMR (101 MHz, D₂O): δ 171.5, 149.9, 138.8, 134.6, 123.3, 123.1, 115.9, 109.0, 104.5, 103.7, 56.9, 45.7, 30.2, 28.8, 24.5, 23.9, 23.3.

3-(2-(Benzylamino)ethyl)-1H-indol-4-ol Fumarate (4-HO-NBnT)

Prepared using General Procedure B from 3Bn (1.0 g, 3.0 mmol), yielding a white solid (0.75 g, 66%). UPLC purity: 99.46%. Melting point: 146–149 °C (as the fumarate salt). HRMS (ESI) m/z [M + H]⁺ calcd for C₁₇H₁₉N₂O⁺ 267.1492, found 267.1491. ¹H NMR (400 MHz, D₂O): δ 7.42 (3H, m), 7.34 (2H, m), 7.14 (1H, s), 7.06 (2H, m), 6.50 (2H, m), 4.18 (2H, s), 3.40 (2H, t, J = 6.8 Hz), 3.23 (3H, m). ¹³C NMR (101 MHz, D₂O): δ 174.6, 149.9, 138.8, 135.3, 130.6, 129.5, 129.1, 123.4, 115.8, 108.7, 104.5, 103.7, 50.7, 48.7, 48.1, 25.9, 23.1.

Drugs

All drugs were dissolved in 0.9% saline and administered subcutaneously (s.c.) as the weight of the salt form, at an injection volume of 0.01 mL/g body weight.

Mouse Studies

Studies were conducted as previously described.^5,26,62 Male and female C57BL/6J mice (8 weeks old, The Jackson Laboratory #000664, 24 total with 12 male and 12 female mice/drug) were group-housed for facility acclimation (3–5 per cage for 1–2 weeks) and housed in a 12:12 light-dark cycle (0700 local time = lights on) with ad libitum access to food and water. Mice were housed at the NIDA IRP facilities in Baltimore, MD, USA, and all experiments described herein were approved by the NIDA IRP Animal Care and Use Committee.

After acclimation to the vivarium facility, a temperature transponder (14 × 2 mm, model IPTT-300, Bio Medic Data Systems or BMDS, Inc., Seaford, DE, USA) was implanted in each mouse under brief isoflurane immobilization. Implanted transponders facilitate remote body temperature recordings before and after each experiment with a hand-held reader (BMDS, DAS-8027-IUS). After the implant procedure, mice were allowed at least 1 week recovery prior to the start of experiments and associated drug treatments.

To avoid tolerance to behavioral effects of the test drugs, mice were tested only once every 1–2 weeks.^63,64 Testing was conducted between 1000–1700 local time. Each test session started with a short 5 min period of acclimation to the testing arenas, followed by baseline temperature recording. Mice were next injected s.c. with appropriate doses of test drug or vehicle and returned to the test chambers for a 30 min session. During the test session, locomotor activity was recorded continuously using open field chambers equipped with photobeam arrays (Coulbourn Instruments, Holliston, MA, USA), modified with cylindrical inserts and custom floor panels that facilitate video recordings (GoPro Hero 7 Black camera - 960p resolution at 120 frames per sec) of each mouse to later quantify HTR activity via a commercially available software platform (Clever Sys Inc. Reston, VA, USA).⁶²

Mean total number of HTRs over the 30 min session, change in body temperature from pre to post session (Δ °C), and total distance traveled (cm) for each drug dose for the full session were evaluated via one-way analysis of variance (ANOVA) with Dunnett’s posthoc test (p < 0.05). For posthoc analyses, all doses were compared to respective vehicle controls (0 mg/kg). Dose–response relationships for HTR were fit using bell-shaped curve fits for compounds with biphasic responses and with three parameter fits for other active compounds. The ascending phase of the HTR curve for each drug was used to determine ED₅₀ potency values from sigmoidal nonlinear regression fits.

PDSP Pharmacological Target Screening Assays

Assays were performed as described in the PDSP assay protocol book which can be found online.³⁶ Raw data from PDSP experiments were replotted in GraphPad Prism (Version 9) to determine affinity, potency, and maximum effect values for each compound across assays. Affinity values were determined using one site K_i while potency/maximum values were determined using three-parameter nonlinear regression fits.

Glossary

Abbreviations

Serotonin

5-HT

Serotonin 1A receptor

5-HT_1A

Serotonin 1B receptor

5-HT_1B

Serotonin 1D receptor

5-HT_1D

Serotonin 1E receptor

5-HT_1E

Serotonin 2A receptor

5-HT_2A

Serotonin 2B receptor

5-HT_2B

Serotonin 2C receptor

5-HT_2C

Serotonin 3 receptor

5-HT₃

Serotonin 5A receptor

5-HT_5A

Serotonin 6 receptor

5-HT₆

Serotonin 7A receptor

5-HT_7A

Head twitch response

HTR

Central nervous system

CNS

Norpsilocin or 4-Hydroxy-N-methyltryptamine

4-HO-NMT

Psilocin or 4-Hydroxy-N,N-dimethyltryptamine

4-HO–DMT

Psilocybin or 4-Phosphoryloxy-N,N-dimethyltryptamine

4-PO–DMT

Bufotenine or 5-Hydroxy-N,N-dimethyltryptamine

5-HO–DMT

N,N-dimethyltryptamine

DMT

4-Hydroxy-N-ethyltryptamine

4-HO-NET

4-Hydroxy-N-propyltryptamine

4-HO-NPT

4-Hydroxy-N-isopropyltryptamine

4-HO-NiPT

4-Hydroxy-N-n-butyltryptamine

4-HO-NnBT

4-Hydroxy-N-tert-butyltryptamine

4-HO-NtBT

4-Hydroxy-N-allyltryptamine

4-HO-NALT

4-Hydroxy-N-cyclohexyltryptamine

4-HO-NcHT

4-Hydroxy-N-benzyltryptamine

4-HO-NBnT

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.3c00610.

• Dose–response effects of norpsilocin analogues to produce HTR, hypothermia, and hypolocomotion in mice; CNS receptor radioligands and control compound K_i values; time-course for HTR induced by norpsilocin analogues; dose–response for temperature change across the session induced by norpsilocin analogues; dose–response for total locomotor activity across the session induced by norpsilocin analogues; time-course for locomotor activity effects of norpsilocin analogues; concentration–response curves of norpsilocin analogues for Gα_q-mediated calcium flux at human 5-HT₂ receptors; functional assessment of norpsilocin analogues using the Tango β arrestin recruitment assay across non-5-HT₂ 5-HT receptors; proton and carbon-13 NMR spectra for all final compounds and intermediates (PDF)

Author Contributions

Study design: All authors. Chemical synthesis and analysis: E.K.B., S.E.W., A.M.S. Mouse experiments: G.C.G. Manuscript was initially drafted by A.M.S. and critically reviewed by M.H.B., G.C.G., E.K.B., and S.E.W. The final version was approved by all authors.

This work was supported by NIDA Intramural Research Program Grant No. DA-000522-16 and Cooperative Research and Development Agreement between NIDA & Usona (M.H.B.). We gratefully acknowledge the NIMH PDSP (Contract No. HHSN-271-2018-00023-C) for providing receptor binding data and functional potency as well as efficacy data in support of this study. The NIMH PDSP is Directed by Bryan L. Roth M.D., Ph.D. at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD, USA.

The authors declare no competing financial interest.