Synthesis, Structural Characterization, and Pharmacological Activity of Novel Quaternary Salts of 4-Substituted Tryptamines.

Aeruginascin (4-phosphoryloxy-N,N,N-trimethyltryptammonium) is an analogue of psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine) that has been identified in several species of psilocybin-containing mushrooms. Our team previously reported the synthesis, structural characterization, and biological activity of the putative metabolite of aeruginascin (4-hydroxy-N,N,N-trimethyltryptammonium; 4-HO-TMT) and its potential prodrug (4-acetoxy-N,N,N-trimethyltryptammonium; 4-AcO-TMT). Here, we report the synthesis, structural characterization, and pharmacological activity of several quaternary tryptammonium analogues of 4-HO-TMT and 4-AcO-TMT, namely, 4-hydroxy-N,N-dimethyl-N-ethyltryptammonium (4-HO-DMET), 4-hydroxy-N,N-dimethyl-N-n-propyltryptammonium (4-HO-DMPT), and 4-hydroxy-N,N-dimethyl-N-isopropyltryptammonium (4-HO-DMiPT), as well as their hypothesized prodrugs 4-acetoxy-N,N-dimethyl-N-ethyltryptammonium (4-AcO-DMET), 4-acetoxy-N,N-dimethyl-N-n-propyltryptammonium (4-AcO-DMPT), and 4-acetoxy-N,N-dimethyl-N-isopropyltryptammonium (4-AcO-DMiPT). Compounds were synthesized using established methods, and structures were characterized by single-crystal X-ray diffraction. Test compounds were screened for in vitro pharmacological activity at a variety of receptors and transporters to determine potential targets of action. None of the compounds exhibited measurable affinity for the serotonin 2A receptor (5-HT2A), but several analogues had low micromolar affinity (K i) for the serotonin 1D receptor (5-HT1D) and serotonin 2B receptor (5-HT2B), where they appeared to be weak partial agonists with low micromolar potencies. Importantly, 4-HO-DMET, 4-HO-DMPT, and 4-HO-DMiPT displayed sub-micromolar affinity for the serotonin transporter (SERT; 370-890 nM). The same 4-hydroxy analogues had low to sub-micromolar potencies (IC50) for inhibition of 5-HT uptake at SERT in transfected cells (3.3-12.3 μM) and rat brain tissue (0.31-3.5 μM). Overall, our results show that quaternary tryptammonium analogues do not target 5-HT2A sites, suggesting the compounds lack psychedelic-like subjective effects. However, certain 4-hydroxy quaternary tryptammonium analogues may provide novel templates for exploring structure-activity relationships for selective actions at SERT.

Introduction

Emerging clinical evidence suggests that psychedelics, such as psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine), have therapeutic potential in treating various psychiatric disorders.[1−5] Psychedelics interact with a number of G protein-coupled receptors in preclinical models, but the primary subjective effects of psychedelics in humans seem to be mediated by agonist actions at the serotonin 2A receptor (5-HT2A).[2,6] Whether the acute subjective effects of psychedelics are related to their long-term therapeutic attributes is still unresolved, but the current interest in psychedelics provides unique opportunities for medication development. To this end, the basic structures of psilocybin and related tryptamine compounds are amenable to structure–activity relationship (SAR) studies that could identify candidate medications for treating specific diseases.

Hallucinogenic mushrooms containing psilocybin are known to contain other tryptamines with unexplored biological activity.[7] Norbaeocystin (4-phosphoryloxytryptamine), baeocystin (4-phosphoryloxy-N-methyltryptamine), norpsilocin (4-hydroxy-N-methyltryptamine), and aeruginascin (4-phosphoryloxy-N,N,N-trimethyltryptammonium) are some of the tryptamines structurally related to psilocybin and its active metabolite, psilocin (4-hydroxy-N,N-dimethyltryptamine), found in psilocybin-containing mushrooms.[8−12] The pharmacological activities of baeocystin and norpsilocin were recently studied by Sherwood et al., who showed that neither compound is active in the mouse head twitch assay, a behavioral assay used to predict psychedelic drug activity.[7,13] Biological effects of the quaternary ammonium analogue of psilocybin—aeruginascin (Figure )—have not been explored, despite reports of enhanced psychedelic-like subjective effects after consumption of Inocybe aeruginascens, which contains aeruginascin and psilocybin.[11,12] Aeruginascin also appears to be one of a few tryptamines found in Psilocybe cubensis, a popular species of hallucinogenic mushroom used recreationally and as a religious sacrament.[14]

Figure 1

Structures of aeruginascin as well as putative active metabolite, synthetic prodrug, and novel derivatives thereof included in the study.

In the present study, we describe the synthesis and structural characterization of analogues of the hydrolysis product of aeruginascin, as well as potential prodrugs for these molecules (Figure and Figure ). Members of our team have previously published the structural characterization and serotonergic receptor binding affinities of the putative active metabolite of aeruginascin (4-hydroxy-N,N,N-trimethyltryptammonium; 4-HO-TMT) and its potential prodrug (4-acetoxy-N,N,N-trimethyltryptammonium; 4-AcO-TMT).[15] Here, we report the in vitro pharmacological activity of six novel analogues of 4-HO-TMT and 4-AcO-TMT. We found that none of the compounds display measurable affinity for 5-HT2A, but some of the compounds have low micromolar affinity (i.e., Ki) for the serotonin 1D receptor (5-HT1D) and the serotonin 2B receptor (5-HT2B), as well as sub-micromolar affinity at the serotonin transporter (SERT). These affinities translated to weak partial agonist activities at 5-HT1D and 5-HT2B receptors. Further, compounds were moderate uptake blockers in SERT assays but not dopamine transporter (DAT) assays.

Figure 2

Crystal structures of the aeruginascin active metabolite 4-HO-TMT, synthetic prodrug 4-AcO-TMT, and novel derivatives thereof included in the study.

Results and Discussion

Synthesis of Aeruginascin Analogues

The synthesis of the putative metabolite of aeruginascin (4-HO-TMT) and its potential prodrug (4-AcO-TMT) were reported previously.[15] Six new compounds were prepared by procedures modified from those in the prior report. The 4-acetoxy prodrug derivatives (4-AcO-DMET, 4-AcO-DMPT, 4-AcO-DMiPT) were synthesized by refluxing excess alkyl iodide (iodoethane, 1-iodopropane, 2-iodopropane) in a tetrahydrofuran solution of psilacetin (4-acetoxy-N,N,-dimethyltryptamine; 4-AcO-DMT). The prodrug derivatives were hydrolyzed to their corresponding metabolite analogues (4-HO-DMET, 4-HO-DMPT, 4-HO-DMiPT) by refluxing them in a 5:1 acetic acid/water solution. Detailed syntheses and characterization of all compounds are contained in the Supporting Information.

Structural Characterization of Aeruginascin Analogues

All compounds were structurally characterized by single-crystal X-ray diffraction. All operations were performed on a Bruker D8 Venture CMOS diffractometer, using Mo Kα radiation with a TRIUMPH monochromator. Details of data collection, refinement, and structural parameters are contained in the Supporting Information. Files can be obtained from the Cambridge Crystallographic Database Centre (CCDC 2123853–2123858). The structures of only five quaternary tryptammonium compounds have been reported previously.[15−18]

Pharmacological Activity of Aeruginascin Analogues

All compounds were tested for in vitro activity by the National Institute of Mental Health, Psychoactive Drug Screening Program (NIMH PDSP). The initial primary screening used a fixed concentration of each test compound (10 μM) to detect inhibition of radioligand binding at 46 different pharmacological targets.[19] Follow-up secondary screening, which provided dose–response curves and Ki affinity values, was carried out for those compounds exhibiting >50% inhibition of radioligand binding in the 10 μM primary screens (Table ).

Table 1

Pharmacological Target Profiles and Binding Affinity of Quaternary Salts of 4-Substituted Tryptamines at Identified Human 5-HT Receptors and Monoamine Transporters

compound	targets identified in 10 μM PDSP screena	5-HT1DKi (μM)	5-HT2BKi (μM)	DAT Ki (μM)	SERT Ki (μM)
		[3H]GR125743	[3H]LSD	[3H]WIN35428	[3H]citalopram
4-HO-TMT	DAT, 5-HT2B, 5-HT1D	4.86	1.28	7.92
4-AcO-TMT	DAT, 5-HT2B		1.17	4.35
4-HO-DMPT	DAT, SERT, 5-HT2B, 5-HT1D	2.38	1.09	8.75	0.83
4-HO-DMET	SERT, 5-HT2B		1.35		0.89
4-HO-DMiPT	SERT				0.37
4-AcO-DMPT	none
4-AcO-DMET	DAT			>10
4-AcO-DMiPT	DAT			>10

Targets that exhibited >50% average inhibition. Ergotamine tartrate Ki value at 5-HT1D = 2.97 nM. SB 206553 Ki value at 5-HT2B = 4.86 nM. GBR 12909 Ki value at DAT = 2.89 nM. Amitriptyline Ki value at SERT = 6.36 nM.

Primary screening at monoamine transporters revealed that 4-HO-TMT, 4-AcO-TMT, 4-HO-DMPT, 4-AcO-DMET, and 4-AcO-DMiPT competed for radioligand binding at DAT, while 4-HO-DMPT, 4-HO-DMET, and 4-HO-DMiPT competed for radioligand binding at SERT. Primary screening also revealed that 4-HO-TMT, 4-AcO-TMT, 4-HO-DMPT, and 4-HO-DMET competed for radioligand binding at 5-HT2B, while 4-HO-TMT and 4-HO-DMPT also competed at 5-HT1D. Notably, 4-AcO-DMiPT displayed no inhibition of radioligand binding >50% at any of the 46 sites tested. 4-HO-DMPT inhibited >50% of specific radioligand binding at the greatest number of sites (4) at the 10 μM primary screening concentration.

Interestingly, primary screens showed no inhibition of binding to 5-HT2A or the serotonin 1A receptor (5-HT1A) for any of the compounds, in contrast to our previous study which showed 4-HO-TMT had micromolar affinity for these sites.[15] The discrepancy between 5-HT2A binding results in previous experiments versus the present work could be explained by the use of an agonist radioligand ([125I]DOI) in the prior report versus an antagonist radioligand ([3H]ketanserin) in the current work. It is well-established that the specific radioligand used for labeling receptor proteins can markedly influence the measured affinity of competing test compounds.[20−26] However, it has also been shown that affinity for psychedelics to compete for [3H]ketanserin binding correlates with their psychoactive dose in humans.[27] Because none of the tryptammonium analogues inhibited radioligand binding at 5-HT2A, we hypothesize that these compounds may lack psychedelic effects in vivo.[13]

Radioligands used for secondary screening, Ki values for each test compound, and the cold ligand comparator Ki values are listed in Table . Secondary screening results revealed that the only sub-micromolar Ki values were at SERT. More specifically, 4-HO-DMPT, 4-HO-DMET, and 4-HO-DMiPT all displayed sub-micromolar affinity for SERT labeled with [3H]citalopram (370–890 nM; Table ). 4-HO-TMT and 4-HO-DMPT had affinities of 4.9 and 2.4 μM for 5-HT1D labeled with [3H]GR125743 (Table ). Furthermore, 4-HO-TMT, 4-AcO-TMT, 4-HO-DMPT, and 4-HO-DMET all displayed affinity for [3H]LSD-labeled 5-HT2B (Ki = 1.1–1.4 μM; Table ). Results for 4-HO-TMT and 4-AcO-TMT at 5-HT2B are at odds with previous experiments showing that these compounds had affinities of 120 and >10,000 nM for displacement of [125I]DOI binding at 5-HT2B, respectively.[15] Here, we show that both compounds compete for [3H]LSD binding to 5-HT2B with low micromolar affinity. Discrepancies in the data likely result from differences in radioligand used and assay conditions employed.[25,26] For [3H]WIN35428 binding, 4-HO-TMT, 4-AcO-TMT, and 4-HO-DMPT (4.3–8.7 μM), but not 4-AcO-DMET or 4-AcO-DMiPT (>10 μM), displayed affinity for DAT, demonstrating the relevance of acetoxy versus hydroxy substitution at the 4-position (Table ). Lastly, the radioligand binding assays did not identify any other pharmacological activities for 4-HO-DMiPT except for SERT, indicating that this compound may be selective for that site.

Substitution of an acetoxy for a hydroxy at the 4-position reduced affinity at the four identified binding sites (5-HT1D, 5-HT2B, DAT, SERT). Of the acetoxy esters tested, only 4-AcO-TMT displayed any binding affinity, with Ki values of 1.17 and 4.35 μM at 5-HT2B and DAT, respectively, and showed competition for binding at 5-HT1D unlike its 4-hydroxy counterpart (Table ). These observations are consistent with previous suggestions that 4-phosphoryloxytryptamine prodrugs (e.g., psilocybin) have reduced pharmacological activity compared to their hydrolyzed 4-hydroxy analogues (e.g., psilocin), which are generally considered the active metabolites of 4-phosphoryloxy and 4-acetoxy analogues.[7,28] The three 4-hydroxy compounds with the lowest steric bulk about their quaternary nitrogens, and 4-AcO-TMT, all competed for radioligand binding at 5-HT2B with Ki values around 1 μM. Three of these compounds (4-HO-TMT, 4-HO-DMPT, and 4-AcO-TMT) showed modest binding affinity at DAT. Most notably, increasing the steric bulk about the quaternary nitrogen afforded increased binding at the SERT. The smallest 4-hydroxy compound (4-HO-TMT) showed no competition for radioligand binding at SERT. Increasing the bulk at the quaternary nitrogen by incorporating linear alkyl chains, ethyl (4-HO-DMET) and n-propyl (4-HO-DMPT), engendered increased affinity at SERT, with similar Ki values of 830 and 890 nM, respectively (Table ). Finally, incorporating a branched alkyl chain with a tertiary carbon at the nitrogen, isopropyl (4-HO-DMiPT), further improved SERT binding affinity, showing a Ki of 370 nM (Table ). The steric impact of the quaternary ammonium is an interesting SAR at SERT, with an increase in steric bulk demonstrating improved competition for radioligand binding at this target. Conversely, increasing the steric bulk seems to weaken binding affinity at 5-HT2B and DAT, resulting in 4-HO-DMiPT displaying selective activity at SERT.

The quaternary ammonium compounds were next examined for activity in functional assays measuring agonist potency at 5-HT1D and 5-HT2B, as well as uptake inhibition potency at DAT and SERT. 4-HO-TMT, 4-AcO-TMT, 4-HO-DMPT, 4-HO-DMET, and 4-HO-DMiPT were chosen for the functional assays based on their binding affinities at the four aforementioned sites of action. In fluorescence-based agonist activity assays for 5-HT1D, four of five compounds (excluding 4-HO-DMiPT) had micromolar agonist potencies from 13.9–46.8 μM (Table and Figure A).

Table 2

Potency Estimations for Quaternary Salts of 4-Substituted Tryptamines at Identified Human Monoamine Transporter Targets for Uptake Inhibition Properties and for Agonist Activity at Human 5-HT Receptor Targetsa

compound	DAT uptake	SERT uptake	5-HT1D	5-HT2B
	IC50 (μM)	IC50 (μM)	EC50 (μM)	EC50 (μM)
4-HO-TMT	>100	6.5	46.8	2.7
4-AcO-TMT	>100	12.3	13.9	0.3
4-HO-DMPT	83.7	3.3	32.2	1.3
4-HO-DMET	>100	7.1	14.7	3.0
4-HO-DMiPT	>100	3.9	>100	19.4

GBR 12909 IC50 value at DAT = 77.3 nM. Paroxetine IC50 value at SERT = 34.9 nM. 5-HT EC50 values at 5-HT1D and 5-HT2B = 515.7 and 31.0 nM, respectively.

Figure 3

Functional activity of quaternary tryptammonium analogues at human 5-HT1D (A) and 5-HT2B (B) receptors.

Similar low micromolar potency agonist-like functional activity was also observed for four of five compounds at 5-HT2B, with 4-AcO-TMT exhibiting sub-micromolar potency (Table and Figure B). For the 5-HT receptor functional assays, the reference ligand 5-HT had EC50 potency values of 515.7 and 31.0 nM at 5-HT1D and 5-HT2B, respectively. Relative to 5-HT at these two receptor targets, all five quaternary tryptammonium compounds were weak partial agonists, as none reached maximal efficacy equivalent to 5-HT (Figure ).

We also examined the effects of 4-HO-TMT, 4-AcO-TMT, 4-HO-DMPT, 4-HO-DMET, and 4-HO-DMiPT in fluorescence-based assays assessing uptake inhibition at DAT and SERT. Results revealed that control compounds GBR 12909 (DAT) and paroxetine (SERT) had potencies (i.e., IC50) for inhibition of monoamine uptake of 77.3 and 34.9 nM, respectively. At DAT, the five quaternary tryptammonium compounds displayed weak to no functional transporter uptake activity (84 to >100 μM; Table ). SERT uptake inhibition for all five compounds was greatly improved versus potencies at DAT (3.3–12.3 μM; Table ), but IC50 values were still in the low micromolar range. Given the results for uptake inhibition in transfected cells, we examined uptake inhibition activities in rat brain synaptosomes, a native tissue preparation commonly used to study the effects of psychomotor stimulants drugs.[29,30] In rat brain synaptosomes, all five compounds had >10 μM potencies for uptake inhibition at DAT (Table and Figure A). This is consistent with the weak potencies and affinities seen at these sites in the competition binding and cell-based functional assays. For SERT uptake inhibition activities in rat brain synaptosomes, four out of five compounds displayed sub-micromolar potencies (310–840 nM) with only 4-AcO-TMT displaying potency in the low micromolar range (3.5 μM; Table and Figure B). As predicted from competition binding assays, 4-HO-DMPT, 4-HO-DMET, and 4-HO-DMiPT produced the most potent uptake inhibition in rat brain synaptosomes. All five compounds were SERT selective uptake inhibitors relative to other monoamine transporters, but their potencies varied considerably when compared to the potency of cocaine at SERT (IC50 = 263 nM). In particular, 4-HO-DMiPT displayed indistinguishable potency for SERT uptake inhibition compared to cocaine and was the most potent compound tested in this regard. Despite these intriguing data from cells and synaptosomes, it will be important to determine whether these compounds are capable of entry to the central nervous system (CNS). The highly polar quaternary ammonium component of the molecules might preclude entry into the brain. If these compounds are incapable of penetrating the blood brain barrier, they may represent useful peripherally restricted SERT selective molecules that can be used as pharmacological tools and templates for future research.

Table 3

Potency of Quaternary Salts of 4-Substituted Tryptamines at Synaptosomal Rat Brain Monoamine Transportersa

compound	DAT uptake	SERT uptake
	IC50 (μM)	IC50 (μM)
4-HO-TMT	>10	0.84
4-AcO-TMT	>10	3.5
4-HO-DMPT	>10	0.81
4-HO-DMET	>10	0.59
4-HO-DMiPT	>10	0.31

Cocaine IC50 values at DAT and SERT = 198 nM and 263 nM.

Figure 4

Functional activity for monoamine uptake inhibition of quaternary tryptammonium analogues at DAT (A) and SERT (B) in rat brain synaptosomes.

Conclusion

The synthesis, structural characterization, and pharmacological evaluation of eight novel quaternary tryptammonium analogues of aeruginascin has been performed. Competitive radioligand binding assays at 46 receptors and transporters revealed in vitro binding affinity for some of these compounds at four targets (5-HT1D, 5-HT2B, DAT, SERT) that could mediate pharmacological effects in vivo. The assays also revealed informative SARs, including a decrease in binding affinity at 5-HT1D, 5-HT2B, and DAT with the incorporation of a 4-acetoxy group and with an increase in steric bulk. For this series of compounds, the most substantial binding affinity and noteworthy SAR was observed at SERT. Increasing the steric bulk of the quaternary ammonium unit improves affinity for SERT binding and increases potency for uptake inhibition, and in the case of 4-HO-DMiPT seemingly generates a relatively potent and selective SERT ligand. Future studies should investigate potential in vivo effects of these compounds to determine if they are CNS active.

Methods

PDSP Receptor Screening, Competition Binding Assays, and Functional Assays

Briefly, assays were conducted using cells stably expressing human receptors or transporters of interest listed below:

5-HT1A, 5-HT1B, 5-HT1D, 5-ht1e, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT5A, 5-HT6, 5-HT7A, Alpha1A, Alpha1B, Alpha1D, Alpha2A, Alpha2B, Alpha2C, Beta1, Beta2, Beta3, BZP Rat Brain Site, D1, D2, D3, D4, D5, DAT, DOR, GABAA, H1, H2, H3, H4, KOR, M1, M2, M3, M4, M5, MOR, NET, PBR, SERT, Sigma 1, Sigma 2.

Cellular assays were used to determine relevant targets at a 10 μM screening concentration as well as affinity and functional activity for identified targets using assays described in detail at https://pdsp.unc.edu/pdspweb/content/UNC-CH%20Protocol%20Book.pdf .

Monoamine Transporter Uptake Assays in Rat Brain

Assays were run as described previously with little modifications.[29,30] Briefly, male Sprague–Dawley rats were euthanized by CO2 narcosis, and brains were harvested to prepare synaptosomes for uptake assays. Using the crude synaptosome fraction post-centrifugation, [3H]dopamine or [3H]5-HT was used to assess transport activity at rDAT or rSERT, respectively.