Discovery and Structure–Activity Relationships of 2,5-Dimethoxyphenylpiperidines as Selective Serotonin 5-HT2A Receptor Agonists

Classical psychedelics such as psilocybin, lysergic acid diethylamide (LSD), and N,N-dimethyltryptamine (DMT) are showing promising results in clinical trials for a range of psychiatric indications, including depression, anxiety, and substance abuse disorder. These compounds are characterized by broad pharmacological activity profiles, and while the acute mind-altering effects can be ascribed to their shared agonist activity at the serotonin 2A receptor (5-HT2AR), their apparent persistent therapeutic effects are yet to be decidedly linked to activity at this receptor. We report herein the discovery of 2,5-dimethoxyphenylpiperidines as a novel class of selective 5-HT2AR agonists and detail the structure–activity investigations leading to the identification of LPH-5 [analogue (S)-11] as a selective 5-HT2AR agonist with desirable drug-like properties.


■ INTRODUCTION
Serotonin is involved in a multitude of functions in the central nervous system (CNS) as well as in the periphery, where a total of 14 different receptor subtypes mediate the effects of the neurotransmitter. 1,2Agonists of 5-HT 2A R such as psilocybin, LSD, and DMT are often referred to as psychedelics due to their perturbation of perception and state of mind. 3,4−13 In addition to their 5-HT 2A R agonist activity, classical psychedelics target numerous other serotonin receptor subtypes and in some cases other monoaminergic receptors as well. 14,15While their shared 5-HT 2A R agonism is believed to be responsible for their acute psychedelic effects, the persistent therapeutic benefits following a single administration of these drug are yet to be causally linked to activation of 5-HT 2A R. 3,4 Thus, it remains an open question whether the broad activity profiles of the classical psychedelics are required for therapeutic efficacy.This conundrum has in recent years spawned significant research efforts aimed at developing selective 5-HT 2A R agonists to probe their possible therapeutic applications. 16esearch into new ligands selectively targeting the 5-HT 2A R has taken many avenues.−29 Overall, decorating the 2,5-dimethoxyphenethylamine scaffold (often referred to as "2C-X's") with a lipophilic substituent in the 4′position usually confers increased agonist potency at 5-HT 2 receptors, including the 5-HT 2A R, as seen, for example, in 2-(4bromo-2,5-dimethoxyphenyl)ethan-1-amine (2C-B) (1) (Figure 1).The N-benzyl phenethylamine (NBOMe) class of compounds derived from N-benzylation of the 2C-X's include some of the most potent 5-HT 2A R agonists reported to date.Some of these also exhibit pronounced selectivity for 5-HT 2A R over the two other 5-HT 2 R subtypes, 5-HT 2B R and 5-HT 2C R. 30,31 However, concerns about their safety profiles may hinder the clinical development of compounds from the NBOMe class. 32epresentatives of the NBOMe class have been used as tool compounds to investigate the hypothesis that biased agonists mediating selective activation of specific 5-HT 2A R-coupled downstream pathways may hold therapeutic advantages compared to nonbiased receptor agonists.These investigations have led too the development of biased agonists with promising in vitro profiles, but their clinical potential is yet to be investigated. 31,33nvestigations into receptor−ligand interactions of phenethylamines have guided the design of new ligands and provided information about the importance of the ethylamine chain conformation in relation to their 5-HT 2A R activity. 15,29The functional properties of conformationally restricted 2C-X analogues like (R)-(3-bromo-2,5-dimethoxybicyclo[4.2.0]octa-1,3,5-trien-7-yl)methanamine (TCB-2) (2) and (1R,2S)-2-(4-bromo-2,5-dimethoxyphenyl)cyclopropan-1-amine (DMPCA) (3) (Figure 1) show that agonist potency at 5-HT 2A R is very dependent on the spatial orientation of the ethylamine chain, 29,34−36 with bioactivity typically residing primarily in a single enantiomer of such conformationally restrained compounds, as is also the case for 4-substituted 2,5dimethoxy amphetamines. 23,35,37Several potent 5-HT 2A R agonists have emerged from these efforts, whereas selectivity for the 5-HT 2A R toward the other 5-HT 2 Rs remains an unresolved challenge.
In the present work, we were inspired by previous work on restricted phenethylamine structures to investigate the effects of introducing related conformational restraint on the 2C-X scaffold via a bridge between the benzylic position and the nitrogen atom (illustrated in Figure 1). 38RESULTS AND DISCUSSION Initially, we targeted the 4-, 5-, and 6-membered congeners of 2C-B (1) (Figure 2) seeking to investigate the effects of restraining the ethylamino side chain at various bond angles, thus also probing the importance of the spatial orientation of the secondary amine.The racemate of phenylpiperidine 6 was reported by Nichols and coworkers in 2013 as an intermediate in the synthesis of a series of structurally constrained NBOMes, but the authors did not report any pharmacological data for this compound. 39iven the close structural similarity between the orthosteric sites in 5-HT 2A R and 5-HT 2C R, 40 we chose to characterize the functional properties of the analogues at these two receptor subtypes in a fluorescence-based Ca 2+ imaging assay in our development of the structure−activity relationships in the pursuit of selective 5-HT 2A R agonists. 41,42e found 4 to be a potent albeit unselective full agonist at 5-HT 2A R and 5-HT 2C R (EC 50 = 1.6 and 5.8 nM, respectively) (Figure 3 and Table 1).This profile was very similar to that of 2C-B (1) with EC 50 values of 1.6 and 4.1 nM at 5-HT 2A R and 5-HT 2C R, respectively.The two enantiomers of 5, 5 eu , and 5 dis , were both potent and unselective high-efficacious partial agonists at 5-HT 2A R (EC 50 = 5.3 and 7.7 nM) and also potent agonists at 5-HT 2C R (EC 50 = 26 and 18 nM), but notably displayed different efficacies at this receptor (R max = 16% and 73%) (Figure 3).The two enantiomers of the 6membered analogue 6 displayed decreased agonist potencies at 5-HT 2A R compared to 4 and 5, but interestingly, the trend of differential efficacies at 5-HT 2A R and 5-HT 2C R observed for the enantiomers of 5 was even more pronounced for 6.Eutomer 6 eu displayed partial agonism (R max = 37%) and an EC 50 of 69 nM on 5-HT 2A R, while being devoid of measurable agonist activity at the 5-HT 2C R (Figure 3 and 4, Table 1).When tested as an antagonist at 5-HT 2C R, 6 eu mediated concentration-dependent inhibition of the 5-HT EC 90 -induced response through the receptor, with an IC 50 value of 640 nM.In comparison, distomer 6 dis displayed moderately potent partial agonism at both 5-HT 2A R (EC 50 = 370 nM) and 5-HT 2C R (EC 50 = 1,900 nM).
Encouraged by the profile of 6 eu , we set out to investigate whether the structure−activity relationships of 2,5-dimethoxyphenethylamines (2C-X family) would translate to the corresponding phenylpiperidine series.In the first series of analogues we probed the effects of various substituents in the 4-position, including other halogens, trifluoromethyl (TFM), nitrile, alkyl (methyl, ethyl, and n-butyl), and thioalkyl (methyl-, ethyl-, and isopropylthio) substituents, as several of the corresponding 2C-Xs have been reported to be significantly more potent than their 4-bromo-substituted analogues. 15The structures and functional properties of these analogues at 5-HT 2A R and 5-HT 2C R in the Ca 2+ /Fluo-4 assay are given in Table 1.
Several interesting observations can be made from the data in Table 1.Overall, there was a marked difference in the agonist potencies displayed by these analogs at 5-HT 2A R, clearly indicating that the 4-substituent is an important determinant of the activity of the 2,5-dimethoxyphenylpiperidines.Furthermore, the functional 5-HT 2A R-over-5-HT 2C R selectivity observed for 6 eu was retained in the eutomers across the entire series.The R max values displayed by all eutomers of 8−17 at 5-HT 2A R were between 25 and 92%, whereas their efficacies at 5-HT 2C R were much lower (R max ∼ 0−20%).
Removal of the 4-substituent was detrimental for receptor activity, as 7 displayed very weak 5-HT 2A R agonist activity (tested as a racemic mixture).Exchange of the 4-bromosubstituent in 6 eu for chloro-or iodo-substituents provided analogues 8 eu and 9 eu displaying comparable agonist potencies at 5-HT 2A R. In contrast, the 4-cyano group in 10 eu was unfavorable for both 5-HT 2A R and 5-HT 2C R activities, whereas the TFM-substituted derivative 11 eu possessed ∼20-fold higher agonist potency at 5-HT 2A R than 6 eu .Moreover, 11 eu did not elicit measurable agonist activity at 5-HT 2C R and displayed an IC 50 value of 320 nM at this receptor when tested as an antagonist (Table 1 and Figure 5).The three alkyl-substituted  phenylpiperidines 12 eu , 13 eu , and 14 eu displayed comparable agonist activities at 5-HT 2A R with EC 50 values of 100, 41, and 270 nM, respectively.While 13 eu thus maintained potency comparable to 6 eu , the compound also exhibited measurable agonist activity at the 5-HT 2C R. The thiomethyl and thioethyl derivatives 15 eu and 16 eu were also slightly more potent 5-HT 2A R agonists than 6 eu , whereas the thioisopropyl derivative 17 eu was less potent.Like the ethyl analogue, 13 eu , 15 eu , and 16 eu did not match the functional subtype selectivity exhibited by 6 eu for 5-HT 2A R toward 5-HT 2C R.
In general, the eutomers of the halogen-and TFMsubstituted analogues 6, 8, 9, and 11 displayed negligible agonist activity at 5-HT 2C R, whereas the eutomers of the alkyl and thioalkyl derivatives 12−17 all evoked detectable agonist responses at this receptor.In contrast to the differential functionalities exhibited by the eutomers at 5-HT 2A R and 5-HT 2C R, the profiles of the distomers of 8−17 were similar at the two receptors, where they were weak-to-moderately potent partial agonists with 3−13 fold higher agonist potencies at 5-HT 2A R (EC 50 range: 110−2,200 nM) than at 5-HT 2C R (EC 50 range: 640−10,000 nM).
From this initial screening of different 4-substituents, the TFM-substituted 11 eu seemed to be the most promising lead for a selective 5-HT 2A R agonist from the phenylpiperidine series, so we decided to investigate the effects of further modifications to this compound.Before doing so, we established the absolute configuration of the distomer to be (R)-11, via X-ray crystallography (see the Methods section and Supporting Information for details), thereby showing the eutomer of 11 to be (S)-11.In all cases, the first eluting enantiomer on chiral high-performance liquid chromatography (HPLC) was also the most potent 5-HT 2A R agonist and the least efficacious 5-HT 2C R agonist of the relative enantiomers.Thus, we tentatively assign the (S)-configuration to the eutomers of all the phenylpiperidines reported in this study and correspondingly the (R)-configuration to all of the distomers.
Next, we investigated the effects of alkylation of the secondary amine as well as the impact of modifications to the 2-MeO and 5-MeO substituents on the phenyl ring in 11.
To facilitate direct comparisons between the phenylethylamine (2C-X) and phenylpiperidine scaffolds, 2C-TFM (18), one of the most potent 5-HT 2A R agonists from the 2C-X family published to date, 21 was included as a reference.The structures of 18 and phenylpiperidines 19-26 are given in Figure 4, and their functional properties at 5-HT 2A R and 5-HT 2C R in the Ca 2+ /Fluo-4 assay are given in Table 2.
Glennon and colleagues have reported that sequential Nmethylation of 2C−B (1) gives analogues with 10-fold reduced affinities at 5-HT 2A R. 43 Analogously, the N-methyl and N-ethyl derivatives 19 and 20 were both substantially less potent than 11.Further extension of the methyl/ethyl group in this scaffold has been investigated by Nichols et al. 39 The racemic NBOMe analogue of 6 exhibited a K i value of 2 μM at the 5-HT 2A R in a [ 3 H]-ketanserin competition binding assay, and thus Nsubstitution of the phenylpiperidine scaffold appears to be unfavored for 5-HT 2A R activity.
Deletion of both methoxy groups on the phenyl ring was detrimental to activity, as compound 21 displayed negligible agonist activity at both 5-HT 2A R and 5-HT 2C R, when tested as a racemic mixture (Table 2).Deletion of the 5-MeO in (S)-11 led to a 20-fold drop in agonist potency at 5-HT 2A R (22), whereas deletion of the 2-MeO group (23) led to a more than 500-fold drop in potency.These effects are even more pronounced than what we previously have observed for desmethoxy analogues of 2C-B (1) and 1-(4-bromo-2,5dimethoxyphenyl)propan-2-amine (DOB). 44Extension of either MeO group to an EtO group was somewhat tolerated with respect to agonist potency at 5-HT 2A R. Thus, the 2-EtO analogue 24 eu and the 5-EtO analogue 25 eu displayed 10-and  1 and S2.
3-fold higher EC 50 values, respectively, than (S)-11 at 5-HT 2A R, whereas the 2,5-di-EtO-analogue 26 eu displayed a 30fold higher EC 50 value than (S)-11 at the receptor (Table 2).24 eu and 25 eu displayed 70-and 30-fold higher agonist potencies at 5-HT 2A R than at 5-HT 2C R, respectively, but both EtO analogues induced robust activation of the latter receptor.The negligible agonist efficacy displayed by (S)−11 at 5-HT 2C R suggests that both the 2-and 5-MeO groups in (S)-11 are important for its low intrinsic activity at this receptor.
In summary, in this last series, we observed the same overall trend again; that the 5-HT 2A R agonist activity primarily resides in one enantiomer (Table 2).However, none of the structural modifications presented in Figure 4 proved beneficial in terms of agonist potency at 5-HT 2A R or selectivity toward 5-HT 2C R, when compared to (S)-11.
2C-TFM ( 18) is a very potent partial agonist of both 5-HT 2A R and 5-HT 2C R, with a 10-fold selectivity for 5-HT 2A R (Figure 4 and Table 2).The phenethylamine side chain in this 2C-X analogue is inherently flexible, allowing it to adopt numerous different conformations.Restricting the conformationally flexibility of the phenethylamine side chain by incorporating it into a piperidine ring in (R)-11 leads to a 100-fold drop in agonist potency at both 5-HT 2A R and 5-HT 2C R. With the other enantiomer, (S)-11, we only see a 4fold drop in agonist potency at 5-HT 2A R accompanied by the absence of measurable agonist efficacy at 5-HT 2C R. We speculate that (S)-11 is unable to adopt a conformation capable of eliciting substantial 5-HT 2C R activation, thus converting the compound into a competitive antagonist or a very low-efficacious agonist at this receptor, so low that its agonist activity is not detectable in the Ca 2+ /Fluo-4 assay.This Table 1.Functional Properties of 1, 4-17 at 5-HT 2A R and 5-HT 2C R a a Functional properties exhibited by 4−17 at stable 5-HT 2A R-and 5-HT 2C R-HEK293 cell lines in the Ca 2+ /Fluo-4 assay.eu: eutomer, dis: distomer.EC 50 values are given in nM, and R max values are given as % of the 5-HT R max .For the compounds also tested in antagonist mode at 5-HT 2C R (using 5-HT EC 90 as an agonist), IC 50 values are given in nM.All data are based on at least 3 independent experiments.n.a.: no agonist activity: the compound displayed no significant agonist activity or negligible levels of agonist activity at 5-HT 2C R at concentrations up to 50 μM.w.a.: weak agonist activity: the compound only elicited significant agonist responses at micromolar concentrations, so a complete concentration−response curve could Table 1.continued not be obtained.n.d.: not determinable: the R max value for the compound could not be determined since a complete concentration− response curve was not obtained in the tested concentration range (up to 50 μM).See Table S2 for full details on the data reported in Table 1.profile is also seen with compounds 6, 7 and 8 for which the eutomers at the 5-HT 2A R are also de facto antagonists at the 5-HT 2C R in this assay.
Based on the above investigations, compound (S)-11 was selected for further characterization and renamed LPH-5.
Agonism at the 5-HT 2B R has been linked to cardiac valvular fibrosis, 45 and in 2023, the FDA issued a regulatory guidance protocol for the clinical development of new 5-HT 2A R agonists, wherein this pharmacological relationship is specifically mentioned as a safety concern. 46In a fluorescence-based Ca 2+ imaging assay, LPH-5 was found to be a moderately potent partial 5-HT 2B R agonist (EC 50 : 190 nM; R max : 65%), thus exhibiting an ∼60 fold selectivity for 5-HT 2A R over 5-HT 2B R (Figure 6).As for the de facto antagonist activity displayed by (S)-11 (and by the eutomers of several other analogues in this series) at 5-HT 2C R, the transient nature of the agonist-induced response and the resulting lack of equilibrium conditions in the Ca 2+ /Fluo-4 assay mean that the obtained IC 50 values in the assay are not applicable for calculations of K i or K b values for the compounds.While absolute EC 50 and IC 50 values should not be compared directly, the 100-fold difference in the average EC 50 (3.2nM) and IC 50 (320 nM) values displayed by (S)-11 at 5-HT 2A R and 5-HT 2C R, respectively, is nevertheless noteworthy.Thus, we propose that (S)-11, in addition to its functional selectivity arising from its distinct intrinsic agonist activities at 5-HT 2A R and 5-HT 2C R, also displays substantial potency-based subtype selectivity at the two receptors.In this context, it should be noted that psilocin (the active metabolite of psilocybin currently being investigated in numerous clinical trials) has been reported to be an equipotent and equiefficacious agonist at 5-HT 2A R, 5-HT 2B R, and 5-HT 2C R. 47 Subsequently, the binding affinities of LPH-5 at all three 5-HT 2 Rs were determined in a [ 125 I]-1-(4-iodo-2,5-dimethoxyphenyl)propan-2-amine ([ 125 I]DOI) competition binding assay.The selectivity trend observed in the functional data was mirrored in these experiments, as LPH-5 displayed a K i value of 1.3 nM for 5-HT 2A R and a K i value of 13 nM at both 5-HT 2B R and 5-HT 2C R (Figure S2 and Table S3).Encouraged by the selectivity profile exhibited by LPH-5 at the 5-HT 2 Rs, a broad screen with the compound was performed (see Table S4 for the full list of the targets).The data from this screen indicates the LPH-5 possesses binding affinities at the respective targets in the high nanomolar (>100 nM) or micromolar ranges, and we conclude that it will be possible to obtain selective activation of 5-HT 2A R with LPH-5 at a suitable dose/exposure of the compound.
We then went on to characterize LPH-5 with respect to lipophilicity (Log P, see Table S5) and membrane permeability (MDR1-MDCKII, see Table S6) (Figure 6).LPH-5 displayed high bidirectional membrane permeability, with an efflux ratio of 0.94 and a Log P value of 3.45.Ligand efficiency (LE) and ligand lipophilicity efficiency (LLE) are two simple metrics often used to evaluate the properties of ligands at an early stage of the discovery phase. 48Using the pEC 50 value of 8.49 obtained for LPH-5 at 5-HT 2A R gives a LE of 0.6 and a LLE of 5, which in both cases are favorable values for a CNS-targeted compound. 49

■ CONCLUSION
In conclusion, we have investigated the structure−activity relationships of 2,5-dimethoxy-phenylpiperidines as a new class of 5-HT 2A R agonists and found that LPH-5 is a potent and selective 5-HT 2A R agonist with desirable drug-like properties.
■ METHODS Experimental Section.General Experimental Details.All reactions were performed under an atmosphere of argon unless  18), (R)-11, and (S)-11 at 5-HT 2A R and 5-HT 2C R, and concentration−inhibition relationship for (S)-11 tested in antagonist mode at 5-HT 2C R using 5-HT EC 90 as agonist (bottom, right).Data are averaged data given as mean ± SEM values based on at least three independent determinations performed in duplicate, see Table S2.
otherwise indicated.Reagents and starting materials were obtained from commercial sources and used as received.Solvents were of chromatography grade or dried either by an SG Water solvent purification system (DCM, DMF, THF) or with 3 Å molecular sieves (DMSO, toluene, MeCN, Et 2 O, EtOH, DME, and MeOH).Reactions sensitive to water were run in flame-or oven-dried (150 °C) glassware under N 2 or argon.Purification by column chromatography and dry column vacuum chromatography (DCVC) was performed following standard procedures using Merck Kieselgel 60 (40−63 μm or 15−40 μm mesh, respectively.Microwave heated reactions were performed using a Biotage Initiator apparatus in a sealed vial using an external surface sensor for temperature monitoring. Thin-Layer Chromatography (TLC).For TLC analysis, precoated silica gel 60 F254 plates purchased from Merck were used.EtOAc, n-heptane, acetone, toluene, DCM, Et2O, MeOH, Et 3 N, and mixtures thereof were used as eluents.Visualization of the compounds was achieved with UV light (254 nm), iodine on silica or potassium permanganate, anisaldehyde, ninhydrin, or ferric chloride stains.The denoted retention factors (R f ) were rounded to the nearest 0.05.
High-Performance Liquid Chromatography (HPLC) Methods.As a part of the essential characterization of new chemical entities, all reported final compounds were assayed for purity by HPLC.All compounds reported are >95% pure by HPLC analysis (see the Supporting Information for representative spectra).HPLC retention times (t R ) for all final compounds are reported in minutes (min) and were determined by different methods, given in parentheses.
Method A (Analytical HPLC).HPLC was recorded on a Thermo Scientific Dionex 3000 UltiMate instrument connected to a Thermo Scientific Dionex 3000 diode array detector using a Gemini-NX 3 μm C18 110A (250 × 4. Method C (Preparative HPLC).Preparative HPLC was performed on a Thermo Scientific Dionex 3000 ultimate instrument connected to a Thermo Scientific Dionex 3000 photodiode array detector using a Gemini-NX 5u RP C18 column (250 × 21.2 mm) with UV detection at 254 and 280 nm.MP A: 0.1% TFA, 100% H 2 O (v/v).MP B: 0.1% TFA, Table 2. Functional Potencies of 11 and 18−28 at 5-HT 2A R and 5-HT 2C R a a Functional properties exhibited by 11 and 18−26 at stable 5-HT 2A Rand 5-HT 2C R-HEK293 cell lines in the Ca 2+ /Fluo-4 assay.eu: eutomer, dis: distomer.EC 50 values are given in nM, and R max as % of the 5-HT R max .All data are based on at least 3 independent experiments.n.a.: no agonist activity.The compound displayed no significant agonist activity or negligible levels of agonist activity at 5-HT 2C R at concentrations up to 50 μM.w.a.: weak agonist activity: the compound only elicited significant agonist responses at micromolar concentrations, so a complete concentration−response curve could not be obtained.n.d.: not determinable.The R max value for the compound could not be determined since a complete concentration− response curve was not obtained in the tested concentration range (up to 50 μM).As 19 and 20 are direct derivatives of compounds (S)-11 and (R)-11, absolute configuration has been assigned to the individual enantiomers of these compounds.See Table S2 for full details on the data reported in Table 2. Method D (Chiral HPLC).Enantiomeric excess (ee) of the desired enantiomers was determined using a Thermo Scientific Dionex 3000 UltiMate instrument connected to a Thermo Scientific Dionex 3000 diode array detector using an analytical Phenomenex Lux 5 Amylose-2 (250 × 4.6 mm) chiral column with UV detection at 205, 210, 254, and 280 nm.MP A: 0.1% diethylamine in heptane (v/v).MP B: 0.1% diethylamine in EtOH (v/v).Flow rate: 10.0 mL/min using an isocratic gradient: 10% MP B.
High-Resolution Mass Spectrometry (HRMS).Analysis was performed by matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF).Analysis was performed in positive ion mode with MALDI ionization on a Thermo QExactive Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with an AP-SMALDI 10 ion source (TransmitMIT, Giessen, Germany) and operated with a mass resolving power of 140,000 at m/z 200.2,5-Dihydroxybenzoic acid was used as matrix and lock mass for internal mass calibration, providing a mass accuracy of 3 ppm or better.Samples were prepared using 2,5-dihydroxybenzoic acid as the matrix.
Melting Point (MP).Melting point was measured for recrystallized compounds on a Stanford Research System OptiMelt capillary melting point apparatus with visual inspection, and the values are reported in a range rounded to the nearest 0.5 °C.

General Procedure A. The Coupling of Sulfonyl
Hydrazines with Boronic Acid to Give Phenyl Azetidines and Phenyl Pyrrolidines.Using the procedure reported by Ley and coworkers, a flame-dried microwave vial, backfilled with argon gas, was charged with Boc-protected sulfonylhydrazone (1 equiv), boronic acid (2 equiv), and dry Cs 2 CO 3 (1.1 equiv). 50The contents of the vial were sealed and subjected to high vacuum for 2 h before re-establishing the argon atmosphere.The contents of the vial were suspended in anhydrous 1,4-dioxane (0.12 M).The suspension was thoroughly degassed before capping the vial.The reaction was heated to 110 °C under vigorous stirring.After 18 h, the reaction was allowed to cool to ambient temperature and filtered over a plug of Celite.The filtrate was concentrated in vacuo and immediately subjected to purification by flash column chromatography (1:2 EtOAc/heptane) giving the desired compound with minor impurities as a clear oil.Crude carboxylate was dissolved in 4 M HCl in dioxane (1 mL) and stirred at ambient temperature for 24 h.The pure amine hydrochloride was precipitated out by the addition of Et 2 O (25 mL) and isolated by decantation giving the title compound.
General Procedure B-1.The Separation of Enantiomeric Mixtures of Free Amines.Analytical amounts of the racemate were dissolved in a mixture of MeOH, EtOH, and diethylamine (10:17:0.1)and separated by enantiomeric separation method 1, 2, or 3 unless otherwise specified.The hydrochloride salts were prepared by dissolving the products in a minimum amount of Et 2 O and treating the solution with 4 M HCl in dioxane.The precipitate was isolated by decantation and redissolved in the minimum amount of MeOH.Et 2 O was added dropwise until nucleation was observed, and the solution was allowed to crystallize at −4 °C overnight giving the pure title compound as white or off-white solids.Enantiomeric excess (ee) of the desired enantiomer was determined using Chiral HPLC Method 1 (ee > 95%).
General Procedure B-2.The Separation of Enantiomeric Mixtures of Boc-Protected Amines.5−19 mg of the racemate was dissolved in a suitable mixture of the corresponding mobile phases and separated by chiral HPLC using a suitable combination of colomns, mobile phases, and flow rates as specified in conditions 1, 2, 3, 4, or 5 below.Enantiomeric excess (ee) of the desired enantiomer was determined using the same system (ee > 95%).In all cases, the eutomer eluted first.General Procedure C. The Suzuki Coupling of Pyridyl Boronic Acids and 3-Bromopyridine.A flame-dried roundbottom flask equipped with a stir bar and a cooler, backfilled with N 2 gas, was charged with the appropriate boronic acid (1 equiv), 3-bromopyridine (1.1 equiv), triphenylphosphine (0.15 equiv), and DME (10 M). 2 M aqueous Na 2 CO 3 (2.7 equiv) was added followed by Pd/C (0.15 mmol).The reaction was stirred at 80 °C for 17 h under a N 2 atmosphere.The reaction was allowed to cool to ambient temperature and then filtered through a pad of Celite.The filtrate was diluted with H 2 O (50 mL) and EtOAc (50 mL).The phases were separated, and the aqueous phase further extracted with EtOAc (3 × 50 mL).The combined organic phases were washed with H 2 O and brine before being dried over MgSO 4 , filtered, and evaporated in vacuo.The crude product was purified by flash column chromatography to give the title compound.

Journal of Medicinal Chemistry
General Procedure D. Hydrogenantion of Phenylpyridines Using ThalesNano H-Cube.Phenylpyridine was dissolved in glacial AcOH (0.01M).ThalesNano H-Cube was loaded with a fresh catalyst cartridge (Pd(OH) 2 /C).The apparatus was set to run at 100 °C and 80 Barr.The reaction was followed by TLC.Upon complete consumption of starting material, AcOH was removed in vacuo.
General Procedure E. The Suzuki Coupling of Pyridyl Boronic Acids with Aryl Bromides.A flame-dried microwave vial equipped with a stir bar and backfilled with argon gas was charged with the appropriate boronic acid (1 equiv) and [1,1′-bis(di-tert-butylphosphino)ferrocene]dichloropalladium-(II) (5 mol %) within a glovebox and tightly sealed.Aqueous degassed K 3 PO 4 solution (0.9 M, 1.5 equiv) was added, followed by addition of the corresponding aryl bromide (1 equiv) in degassed dioxane (0.3 M).The resulting mixture was heated by microwave irradiation at 100 °C for 1 h, then allowed to cool to ambient temperature, filtered through a silica pad, further eluted with EtOAc (50−100 mL), and then evaporated in vacuo.The residue was purified by flash column chromatography using mobile phase mixtures of petroleum ether/EtOAc.
General Procedure F1.Hydrogenation of Phenylpyridines Using Parr Apparatus.Phenylpyridine (1 equiv) was dissolved in glacial AcOH (2.0 M) in a hydrogenation flask.PtO 2 (0.1 equiv) was added, and the reaction vessel was shaken under 4 bar H 2 pressure on a Parr apparatus for 24 h.The reaction mixture was washed through a pad of Celite with EtOAc (25 mL), and the filtrate was basified using 10% aq.NaOH solution (150 mL).The phases were separated, and the aqueous layer was further extracted with EtOAc (2 × 50 mL).The combined organic phases were dried over MgSO 4 , filtered, and evaporated in vacuo.
General Procedure F2.Hydrogenation of Phenylpyridines Using Pressure Reactor.To a stirred solution of the substrate (1 equiv) in glacial AcOH (0.25 M) was added PtO 2 (15 mol %).The reaction mixture was hydrogenated at ambient temperature under 10 bar H 2 pressure in a Buchi tinyclave steel pressure reactor.After 16 h, the reaction mixture was filtered through a syringe filter and evaporated in vacuo.The residue was partitioned between DCM and aq.sat.NaHCO 3 .The organic phase was separated, and the aqueous phase was further extracted with DCM (2×).Combined organic extracts were dried over Na 2 SO 4 , filtered, and evaporated in vacuo.
General Procedure G. Introduction of Boc-Protecting Group.A round-bottom flask was charged with the amine (1 equiv), Boc 2 O (1.5 equiv), and DCM (0.1 M) before Et 3 N (2 equiv) was added.The resulting mixture was stirred for 1 h at ambient temperature and then evaporated to dryness in vacuo.The residue was purified by flash column chromatography using mobile phase mixtures of Petroleum ether/EtOAc.
General Procedure H. Cleavage of Boc-Protecting Group.A round-bottom flask was charged with the Bocportected amines (1 equiv), and ethereal HCl (40 equiv) was added.The solution was stirred for 3−7 days at ambient temperature to achieve full conversion eventually giving the desired product as a white precipitate.The slurry was centrifuged, and the ethereal layer discarded.The resulting solids were washed with Et 2 O and evaporated to dryness in vacuo.
General Procedure I. Bromination of Phenols.A flamedried round-bottom flask, equipped with a stir bar and a rubber septum, backfilled with argon gas, was charged with the corresponding phenol (1 equiv) in DCM and AcOH (2:1, 0.1 M), and elemental bromine was added (1.05 equiv) dropwise at 0 °C.The reaction mixture was slowly warmed to ambient temperature overnight and then evaporated directly in vacuo.The residue was purified by flash column chromatography using mobile phase mixtures of petroleum ether/EtOAc. 51eneral Procedure J. Methylation of Phenols.A flamedried microwave vial equipped with a stir bar and backfilled with argon gas was charged with the corresponding phenol (1 equiv) in acetone (0.25 M).K 2 CO 3 (8 equiv) was added followed by methyl iodide (6 equiv).The vial was sealed, and the reaction mixture was stirred for 5 h at 60 °C, then evaporated directly in vacuo, and partitioned between DCM and H 2 O. Phases were separated, and the aqueous phase was further extracted with DCM.Combined organic extracts were dried over Na 2 SO 4 , filtered, and evaporated in vacuo.The residue was purified by flash column chromatography using mobile phase mixtures of petroleum ether/EtOAc.General Procedure K. Synthesis of Alkylsulfanes from 1,4-Dibromo-2,5-Dimethoxybenzenes.A flame-dried round-bottom flask equipped with a stirr bar and a rubber septum, backfilled with argon gas, was charged with 1,4dibromo-2,5-dimethoxybenzene (1 equiv) in dry THF (0.4 M).The resulting solution was cooled to −78 °C.and 2.5 M n-BuLi in hexanes (1.1 equiv) was added dropwise.The reaction mixture was stirred at this temperature for 1 h before the dropwise addition of the appropriate disulfide (1.1 equiv).The mixture was allowed to warm to ambient temperature, stirred for 1 h, and then quenched with 1 M HCl.The mixture was concentrated under reduced pressure to half of the initial volume.Et 2 O was added, and phases were separated.The organic phase was washed with H 2 O and NaHCO 3 and then concentrated in vacuo.The residue was purified by flash column chromatography using mobile phase mixtures of petroleum ether/EtOAc.General Procedure L. Reductive Amination.A flamedried round-bottom flask equipped with a stirr bar and a rubber septum was charged with the amine (1 equiv) and MeOH (61.5 mM).The corresponding aldehyde (5 equiv) was added, followed by one drop of AcOH.The resulting mixture was cooled to 0 °C, and then NaCNBH 3 (3 equiv) was added.The reaction mixture was allowed to warm up to room temperature overnight.DCM was added, and the mixture was washed with 1 M NaOH and brine.The organic phase was dried over anhydrous Na 2 SO 4 , filtered, and evaporated.The residue was taken up in Et 2 O and treated with ethereal 2 M HCl (5 equiv).The resulting suspension was centrifugated.The supernatant was discarded, and the solid was washed with ether and evaporated to dryness in vacuo.
General Procedure M. Ethylation of Phenols.A flamedried microwave vial equipped with a stir bar and backfilled with argon gas was charged with the corresponding phenol (1 equiv) in acetone (0.25 M).K 2 CO 3 (8 equiv) was added followed by bromoethane (5 equiv).The vial was sealed, and the reaction mixture was stirred for 5 h at 60 °C, then evaporated directly in vacuo, and partitioned between DCM and H 2 O. Phases were separated, and the aqueous phase was further extracted with DCM.Combined organic extracts were dried over Na 2 SO 4 , filtered, and evaporated in vacuo.The residue was purified by flash column chromatography using mobile phase mixtures of petroleum ether/EtOAc.Enantiomeric Separation Method 1. Chiral HPLC.Analytical amounts of racemate were dissolved in a mixture of MeOH, EtOH, and diethylamine (10:17:0.1)and separated, unless otherwise specified, on a Thermo Scientific Dionex 3000 UltiMate instrument connected to a Thermo Scientific Dionex 3000 diode array detector by a Phenomenex Lux 5 Amylose-2 (250 × 10 mm) chiral column with UV detection at 205, 210, 254, and 280 nm.MP A: 0.1% diethylamine in heptane (v/v).MP B: 0.1% diethylamine in EtOH (v/v).Flow rate: 10.0 mL/min using an isocratic gradient of 30−10% MP B. Loadings were between 1 and 3 mL per injection (3−5 mg/ mL).Enantiomeric excess was determined on an identical instrument using a Phenomenex Lux 5 Amylose-2 (250 × 4.6 mm) chiral column (HPLC Method D).
Enantiomeric Separation Method 3. Resolution by Chiral Salt Formation and Crystallization.Racemic amine (1 equiv) was dissolved in MeOH (0.5 M) at room temperature and added over 5 min to a boiling solution of L(+)tartaric acid (1 equiv) in MeOH (70 mM).Upon complete addition, the reaction was left to cool to room temperature for 48 h yielding white crystalline solids which were isolated by filtration.The filtrate was left at 4 °C overnight giving a second crop of solids, isolated by filtration.Crops were combined, redissolved in boiling MeOH (40 mL), and allowed to cool to room temperature giving white solids, which were again subjected to recrystallization from boiling MeOH (20 mL) eventually giving clear prismatic crystals (5% total yield, 96% enantiomeric excess).

3-(4-Chloro-2,5-dimethoxyphenyl)piperidine (8).
To a flame-dried round-bottom flask, backfilled with argon gas, was charged with 7 (500 mg, 1.93 mmol), N-chlorosuccinimide (310 mg, 2.32 mmol) and MeCN.The solution was cooled (0 °C), TiCl 4 (0.2 mL, 1.93 mmol) was slowly added, and the reaction was stirred for 10 min.The cooling source was removed, and the reaction was stirred on for an additional 5 min before being quenched with MeOH (8 mL).The reaction was allowed to warm to ambient temperature and then basified (≈pH 9) with aq.NaOH solution (10% v/v) under precipitation of white solid.The solution was clarified by filtration through a fritted glass funnel and washed through with EtOAc (50 mL).The filtrate was washed with sat.aq.Na 2 CO 3 (50 mL) and brine (50 mL), dried over MgSO 4 , filtered, and concentrated in vacuo to give the crude-free base with minimal impurities as a yellow solid (529 mg, 94% crude yield).Analytical amounts of the racemic mixture were separated and isolated as the two individual enantiomers as their hydrochloride salts with minor impurities using general procedure B-1, method 1, using an isocratic gradient of 30% MP B. Enantiomer  ■ COMPOUND 9

3-(4-Iodo-2,5-dimethoxyphenyl)piperidine (9).
A flame-dried round-bottom flask, equipped with a stir bar, backfilled with argon gas, was charged with 7 (500 mg, 1.9 mmol), TEA (0.53 mL, 3.8 mmol), and DCM.The reaction mixture was cooled to 0 °C in an ice bath, and trifluoroacetic anhydride (483.06 mg, 2.3 mmol) was carefully added under vigorous stirring.The reaction was stirred for 5 min at 0 °C before being allowed to warm to ambient temperature and stirred for approximately 40 min.The reaction was monitored by TLC.Upon completion, the reaction was quenched with H 2 O (20 mL) and phases were separated.The aqueous layer was further extracted with EtOAc (2 × 50 mL).The combined organic layers were washed with H 2 O (50 mL) and brine (50 mL), then dried over MgSO 4 , filtered, and concentrated in vacuo to give the crude trifluoroacetamide in quantative yield.TLC R f = 0.5 (33% EtOAc in heptane v/v).The crude product was dissolved in MeOH (20 mL) and purged with a flow of argon gas.The reaction was cooled to 0 °C in an ice bath and shielded from light with aluminum foil.AgNO 3 (355 mg, 2.09 mmol) was added in one portion followed by I 2 (578 mg, 2.28 mmol) in several small portions.The reaction was stirred at 0 °C for 1.75 h and then washed through a plug of Celite into a mixture of ice and sat.aq.NaHSO 3 .The mixture was allowed to warm to ambient temperature, and organics were evaporated in vacuo.The remaining aqueous mixture was extracted with EtOAc (3 × 50 mL).The combined organic phases were washed with H 2 O (50 mL) and brine (50 mL), dried over MgSO 4 , then filtered, and concentrated in vacuo, giving the crude iodide as a yellow oil.Major impurities were removed by flash column chromatography (33% EtOAc in heptane v/v).The protected iodide was suspended in MeOH (15 mL), and 25% aq.NaOH solution (2 mL) was added.The reaction was gently warmed until a clear solution was obtained and then left to stir for approximately 2 h until TLC analysis showed complete deprotection of the amine.The reaction was concentrated in vacuo and partitioned between a mixture of EtOAc, DCM, and H 2 O (100 mL) (1:1:2, v/v).The aqueous phase was further extracted with DCM (2 × 50 mL).The combined organic phases were washed with H 2 O (50 mL) and brine (50 mL), dried over MgSO 4 , then filtered, and concentrated in vacuo to give the pure iodide (471 mg, 71%) as clear oil.Analytical amounts of the racemic mixture were separated and isolated as the two individual enantiomers as their hydrochloride salts using general procedure B-1, method 1, using an isocratic gradient of 30% MP B. Enantiomer 1: Rt 6.95, Enantiomer 2: Rt 10.163.MP 252−255 °C; TLC R f = 0.15 (5% TEA and 10% MeOH in EtOAc v/v/v); 1 H NMR (400 MHz, CDCl 3 ) δ 7.38 (s, 1H), 6.87 (s, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 3.48−3.38(m, 3H), 3.12 (t, J = 13.0Hz, 1H), 3.06 (td, J = 11.2, 9.9, 2.2 Hz, 1H), 2.14−2.06(m, 1H), 2.02− 1.87 (m, 3H); 13

tert-Butyl 3-(2,5-dimethoxyphenyl)piperidine-1-carboxylate.
A flame-dried round-bottom flask, equipped with a stir bar, backfilled with argon gas, was charged with 7 (1 g, 3.87 mmol) and di-tert-butyl dicarbonate (931 mg, 4.26 mmol).The contents of the vessel were suspended in a mixture of TEA in DCM (1:10 v/v) (12 mL).The reaction was stirred at room temperature for 18 h.The reaction was monitored by TLC.Upon complete conversion to carboxylate, the reaction was concentrated in vacuo.Major impurities were removed by flash column chromatography (20% EtOAc in heptane v/v) to give the protected amine as a clear oil in quantitative yield.The

STEREOCHEMISTRY
The distomer of 11 was crystallized as the D-tartaric acid salt yielding prismatic crystals allowing elucidation of its absolute configuration in reference to the known stereochemistry of the tartrate (Figure 7).The crystallized compound was identified as the (R) enantiomer of 11.Thus, the eutomer of 11 was assigned to be the (S)-enantiomer.

Journal of Medicinal Chemistry
The relation between relative retention times on chiral-HPLC and potency on 5-HT 2A R and 5-HT 2C R remains constant over the series, with the eutomer at the 5-HT 2A R eluting first, followed by the distomer.Furthermore, the first eluting enantiomer also exhibited the lowest agonist activity at the 5-HT 2C R. Based on this, we tentatively assign the absolute stereochemistry of the phenylpiperidine series to follow that of compound 11, e.g., that the eutomers have the (S)configuration and the distomers have the (R)-configuration.
■ CRYSTALLOGRAPHY X-Ray Crystallographic Analysis of the Distomer of Compound 11 in Complex with (2S,3S)-Tartaric Acid.Single crystals suitable for X-ray diffraction studies were grown from a solution in methanol.A single crystal was mounted and immersed in a stream of nitrogen gas [T = 123(1) K].Data were collected, using graphite-monochromated MoKα radiation (λ = 0.71073 Å) on a Bruker D8 Venture diffractometer.Data collection and cell refinement were performed using the Bruker Apex2 Suite software. 53Data reduction using SAINT and multiscan correction for absorption using SADABS-2016-2 was performed within the Apex2 Suite. 54,55The crystal data, data collection, and the refinement data are given in Table S1 of this manuscript along with the structure solution and refinement of the deutomer to be (R)-11 as salt with (2S,3S)tartaric acid.Fractional atomic coordinates, a list of anisotropic displacement parameters, and a complete list of geometrical data have been deposited in the Cambridge Crystallographic Data Centre (CCDC 2321050).
■ PHARMACOLOGY Ca 2+ Imaging Assays.The functional characterization of the compounds at human 5-HT 2A R and 5-HT 2C R in a Ca 2+ / Fluo-4 assay was performed essentially as previously described. 41,42Stable 5-HT 2A R-and 5-HT 2C R-HEK293 cells were split into poly-D-lysine-coated black-walled 96-well plates with clear bottom (6 × 10 4 cells/well). 41The following day, the culture medium was aspirated, and the cells were incubated in 50 μL of assay buffer Hank's Buffered Saline Solution (HBSS) containing 20 mM HEPES, 1 mM CaCl 2 , 1 mM MgCl 2, and 2.5 mM probenecid, pH 7.4] supplemented with 6 mM Fluo−4/AM at 37 °C for 1 h.Then, the buffer was aspirated, the cells were washed once with 100 μL of assay buffer, and then 100 μL of assay buffer was added to the cells.The 96-well plate was assayed in a FLEXStation 3 (Molecular Devices, Crawley, UK) measuring emission [in fluorescence units (FU)] at 525 nm caused by excitation at 485 nm before and up to 90 s after addition of 33.3 μL of compound solution in assay buffer.For compound testing in antagonist mode at 5-HT 2C R, the compound was added with the assay buffer onto the cells and incubated for 5 min before assaying of the plate in the FLEXStation 3 using 5-HT (EC 90 ) as an agonist.The compound was characterized in duplicate at least three times at both cell lines.
The functional characterization of (S)-11 at human 5-HT 2B R in a Calcium No Wash PLUS assay was performed by Eurofins.Briefly, 5-HT 2B R-HEK293 cells were seeded in a total volume of 20 μL into black-walled, clear-bottoom, poly-Dlysine-coated 384-well microplates.On the day of the assay, the culture medium was aspirated and the cells were loaded with 20 μL of dye solution (1× dye loading buffer consisting of 1× dye, 1× additive A, and 2.5 mM probenecid in HBSS/20 mM HEPES) and incubated for 30−60 min at 37 °C.After this incubation, 10 μL of HBSS/20 mM HEPES was added to the wells, and the cells were incubated for 30 min at room temperature.The 384-well plate was assayed in a FLIPR-Tetra (Molecular Devices) measuring fluorescence over 2 min before and after addition of 10 μL of agonist solution (in HBSS/20 mM HEPES).The compound was characterized in duplicate three times at the cell line.
Radioligand Binding Assays.The binding affinities of (S)-11 at human 5-HT 2A R, 5-HT 2B R, and 5-HT 2C R were determined in [ 125 I]DOI competition binding assay by Eurofins.Membrane homogenates from HEK293 cells transfected with the three respective receptors were incubated for 60 min at 22 °C with 0.1 nM [ 125 I]DOI in the absence or presence of the test compound in assay buffer (50 mM Tris-HCl, 5 mM MgCl 2 , 10 μM pargyline, 0.1% ascorbic acid, pH 7.4).Nonspecific binding was determined in the presence of 1 μM DOI.Following incubation, the samples were filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% polyethylenimine and rinsed several times with ice cold wash buffer (50 mM Tris-HCl, pH 7.4) using a 96-sample cell harvester (Unifilter, Packard).The filters were dried and then counted for radioactivity in a scintillation counter (Topcounter, Packard) using a scintillation cocktail (Microscint-0, Packard).The compound was characterized in duplicate at least three times at each receptor.
The binding affinities of (S)-11 at various other targets were estimated in radioligand binding competition assays by Eurofins.The assays were conventional competition binding assays performed with specific radioligands for the respective targets.Specific details for the assays are given in Table S4.
Full experimental details and additional figures and characterization; extended experimental details, copies of NMR spectra, HPLC chromatograms, and additional assay data (PDF) Molecular formula strings (CSV) Nitrogen atoms are in blue, fluorine atoms green, and oxygen atoms red. 52

Figure 3 .
Figure 3. Functional properties exhibited by 1, 4, 5, and 6 at stable 5-HT 2A R-and 5-HT 2C R-HEK293 cell lines in a Ca 2+ /Fluo-4 assay.Data are given as mean ± standard deviation (S.D.) values and are from representative experiments performed in duplicate out of at least 3 independent experiments, see Tables1 and S2.

Figure 6 .
Figure 6.Overview of the functional properties, membrane permeability, Log P, LE, and LLE of LPH-5.