Design, Synthesis, and Characterization of 4-Undecylpiperidine-2-carboxamides as Positive Allosteric Modulators of the Serotonin (5-HT) 5-HT_2C Receptor

Abstract

An impaired signaling capacity of the serotonin (5-HT) 5-HT_2C receptor (5-HT_2CR) has been implicated in the neurobehavioral processes that promote relapse vulnerability in cocaine use disorder (CUD). Restoration of the diminished 5-HT_2CR signaling through positive allosteric modulation presents a novel therapeutic approach. Several new molecules with the 4-alkylpiperidine-2-carboxamide scaffold were designed, synthesized, and pharmacologically evaluated, leading to the discovery of selective 5-HT_2CR positive allosteric modulators (PAMs). Compound 16 (CYD-1–79) potentiated 5-HT-evoked intracellular calcium release in cells stably expressing the human 5-HT_2CR but not the 5-HT_2AR cells. A topographically distinct allosteric site was identified based on the newly solved 5-HT_2CR structure. Compound 16 modulated 5-HT_2CR-mediated spontaneous ambulation, partially substituted for the training dose of the 5-HT_2CR agonist WAY163909, synergized with a low dose of WAY163909 to substitute fully for the stimulus effects of WAY163909, and attenuated relapse vulnerability as assessed in a rodent self-administration model, indicating its therapeutic promise for CUD.

Graphical Abstract

INTRODUCTION

The United States is reeling from a public health crisis driven by the misuse of psychoactive drugs, the pervasive trajectory to substance use disorders (SUDs), and the limited access to efficacious treatment strategies. A 61% increase in adults initiating cocaine use coupled with the highest cocaine overdoses reported since 2006 reveal alarming trends¹ and signal the potential for a resurgence in the incidence of cocaine use disorder (CUD), an acquired brain disorder marked by habitual cocaine-seeking despite adverse consequences. Core challenges in recovering from CUD include the risk for relapse promoted in part by the attentional orientation toward motivationally relevant, drug-associated stimuli (cue reactivity) that support drug-seeking during abstinence.² New, effective, and accessible modalities for the treatment of CUD are needed to combat relapse risk to alleviate the chronic psychological and physical burdens of this disorder.

Dysfunctional serotonin (5-hydroxytryptamine; 5-HT) neurotransmission contributes to the neurobiological states that precipitate relapse events in CUD and is an important target in the quest to develop effective medications for CUD.^2,3 Serotonin is involved in essential brain functions as diverse as appetite, cognition, reward, and motor control.⁴ The actions of 5-HT are transduced by 14 receptor subtypes, which are classified into seven receptor families based upon the conjunction of genetic and molecular structure, intracellular transduction mechanisms, and pharmacological criteria. The 5-HT₂ receptor (5-HT₂R) subtype family of 5-HT receptors is composed of three G protein-coupled receptors (GPCRs), 5-HT_2AR, 5-HT_2BR, and 5-HT_2CR, that share a high level of amino acid sequence homology within transmembrane regions.⁵ In particular, hypofunctional 5-HT_2CR neurotrans-mission is implicated as a core mediator of relapse vulnerability, and selective 5-HT_2CR agonists are efficacious to blunt cocaine cue reactivity as well as the rewarding effects of cocaine in preclinical models^2,3,6–12 but lack abuse liability on their own.^13–15 However, achieving receptor subtype selectivity versus the highly homologous 5-HT_2AR and 5-HT_2BR is an essential requisite for the successful development of 5-HT_2CR ligands because activation of the related 5-HT_2A/2BR systems can result in significant adverse CNS (5-HT_2AR) and cardiovascular (5-HT_2BR) effects.¹⁶ Despite advances in the past decade,^17,18 attaining high 5-HT_2CR selectivity remains a challenge yet to be solved. For instance, dexfenfluramine is a 5-HT_2CR agonist that was previously approved for antiobesity therapy. However, this drug was later withdrawn from the market because of side effects caused by its potent agonist activity at the 5-HT_2BR.¹⁶ Lorcaserin, a conformationally restricted analogue of dexfenfluramine, is a 5-HT_2CR agonist that has recently been approved by the FDA for treatment of obesity. However, lorcaserin is classified as a Schedule IV drug due to potential side effects, and like most other 5-HT_2CR agonists, its selectivity for 5-HT_2CR (K_i = 15 nM) is only modest with a 7.5-fold selectivity over the 5-HT_2AR (112 nM) and 11.6-fold selectivity versus the 5-HT_2BR (174 nM).¹⁹

Most therapeutic compounds that target GPCRs interact at the orthosteric site, which accommodates the endogenous ligand, to control downstream intracellular signaling. However, with an increasing emphasis on cellular functional screens, a growing number of allosteric modulators of GPCRs have been discovered and are under investigation as potential medications. These small molecules do not bind to the orthosteric ligand site but instead act at a topographically distinct allosteric site on the receptor and either potentiate or inhibit the binding or signaling of an orthosteric ligand. There are theoretical reasons that allosteric ligands may be preferred therapeutics, including the prospects for increased subtype selectivity, avoidance of receptor desensitization, better control of physiological systems, and upper ceiling effects, as well as separate control of affinity and efficacy of orthosteric ligands.²⁰ In recent years, multiple allosteric modulators of GPCRs have been developed and predicted to have robust effects in a variety of CNS disorders.^21–23 The recent preclinical indications of efficacy, coupled with the launch of cinacalcet²⁴ and maraviroc²⁵ as the first marketed GPCR allosteric modulators, provide strong validation of the clinical utility of both positive and negative allosteric modulators, respectively. The validated proof of concept is fueling the discovery of highly selective ligands for other GPCRs that have been previously intractable. Therefore, allosteric modulators of 5-HT_2CR present a novel pharmacological strategy to fine-tune binding or signaling in response to endogenous 5-HT or synthetic ligands in a site-and event-specific manner. Such a strategy may address issues concerning receptor subtype selectivity, toxicity, desensitization, and long-term changes in receptor up or down regulation that orthosteric ligands may promote.²⁶

An analogue of the antibiotic lincomycin, PNU-69176E (1, Figure 1), was identified via a chemical library screen as a 5-HT_2CR positive allosteric modulator (PAM).²⁷ Nevertheless, little effort has been made on structural modifications of chemical lead 1 to optimize its PAM activity, and thus the relevant structure–activity relationship (SAR) studies remain sparse. Structurally, 1 consists of two moieties directly attached to the piperidine-2-carboxamide core scaffold, an undecyl lipophilic tail (LT) and a polar head (PH) moiety including a complex α-D-galactopyranoside, neither of which are optimal for displaying drug-like properties such as cLogP, tPSA, and bioavailability [see Supporting Information (SI), Table S1]. We have established the only reported method to readily access 1 and its diastereomer and demonstrated that 1 exhibits a profile of 5-HT_2CR PAM activity with no intrinsic agonist activity at the 5-HT_2CR or 5-HT_2AR.²⁸ While the lead 5-HT_2CR PAM recently described was shown to suppress locomotor activity, food intake, and body weight gain,²⁹ the impact of 5-HT_2CR PAMs remains to be explored with in vivo models to uncover interaction with exogenous 5-HT_2CR stimulation and to determine efficacy to suppress relapse vulnerability in CUD. These combined observations clearly justify our structural optimization campaign to further explore the SAR of structural modifications of 1 and develop new 5-HT_2CR putative PAMs with an enhanced pharmacological profile for the treatment of CUD. Herein, we describe our efforts for the rational design, chemical synthesis, and pharmacological evaluation of novel, simplified 5-HT_2CR PAMs based on 1 as the chemical lead. The identification of small molecule 5-HT_2CR PAMs affords novel chemical probes for elucidating the neurobiology of 5-HT_2CR signaling and new opportunities for proof of concept studies to develop pharmacotherapies for CUD arising from deficient 5-HT_2CR signaling capacity.

Figure 1.

Initial design strategy for novel 5-HT_2CR positive allosteric modulators based on 1.

RESULTS AND DISCUSSION

Chemistry.

Very little is known about allosteric modulation of the 5-HT_2CR, and the binding site of 1 remains unclear. In our drug design strategy, we speculate that 1 consists of three possible moieties important for binding, including the previously described PH and LT, and the N–H of the core piperidine, as depicted in Figure 1. This hypothesis was formulated initially on the basis of the available pharmacophore-directed homology modeling of the human 5-HT_2CR^30,31 and the limited SAR studies on 1 that showed that shorter alkyl chains (methyl to n-hexyl groups) or the undecyl derivatives with the headgroup of N-phenyl, N,N-diethyl, N-[2-(dimethylamino)ethyl]-N-methyl, or N-(4-acetoamido-1-naphthylsulfonyl) in place of the specific polar sugar moiety failed to exert positive allosteric modulation on 5-HT_2CR.²⁷ This suggests that the PH and LT are likely important pharmacophore groups required for allosteric modulation. Considering the poor drug-like properties of 1, our initial effort was directed to simplify and optimize the PH. The N–H of the piperidine was kept intact so that the target compounds could form HCl salts for improved aqueous solubility.

We first designed a series of new analogues to optimize the PH by keeping the n-undecyl LT intact while replacing the sugar moiety of 1 with a variety of simple amino alcohols (Scheme 1). These simplified PHs structurally resemble fragments of the polar α-D-galactopyranoside moiety of 1, which presumably undergo selective binding via hydrogen bonding or dipole–dipole interactions near the membrane surface. These new analogues, with simplified and readily accessible PHs, have enhanced drug-like properties (Table S1) and development potential. According to our previously established synthetic route,²⁸ a four-step protocol affords 3, which undergoes Sonogashira coupling with 1-undecyne, resulting in the alkynylated picolinic derivative 4. Metal-catalyzed hydrogenation readily provided the (cis-2,4)-piperidyl carboxylate 5. Boc-protection and hydrolysis of 5 provided 6 as the key intermediate in high yield on the multigram scale (68% yield from 3). Direct deprotection of 6 afforded the parent amino acid 7. With 6 in hand, amidation coupling with a variety of amino alcohols using HBTU/DIPEA followed by Boc-deprotection with TFA generated a series of novel derivatives 8–22 (Table 1) with diverse, simplified PHs in 63–88% yields (2 steps, Scheme 1). It was noted that the diastereomers 12–15 could be separated through preparative TLC (PTLC) when the phenyl amino alcohols were employed as the PHs. The other compounds were inseparable enantiomeric or diastereomeric mixtures of isomers by TLC.

Scheme 1.

Synthesis of PNU-69176E Analogs with Simplified Polar Heads (PH)^a

^aReagents and conditions: (a) see ref 28; (b) 1-undecyne, CuI, Pd(PPh₃)₂Cl₂, Et₃N, rt, 12 h, 94%; (c) H₂ (60 psi), PtO₂, HCl/MeOH/H₂O, rt, 16 h, 95%; (d) (i) (Boc)₂O, Et₃N, MeOH, rt, 16 h, 90%; (ii) LiOH, THF/H₂O (v/v = 2:1), rt, 48 h, 85%; (e) (i) amino alcohols, HBTU, DIPEA, DMF, rt, 16 h; (ii) TFA, CH₂Cl₂, rt, 63–88% (two steps).

Table 1.

Effects of 4-Alkylpiperdine-2-carboxamides (1 nM) on 5-HT-Induced Ca_i²⁺ Release in h5-HT_2CR-CHO Cells

^aMaximum 5-HT-induced Ca_i²⁺ release (E_max) in the presence of 1 nM of test compound; the screen utilized concentrations of 5-HT (vehicle, 10^–11 to 10^–6 M) to establish the E_max of 5-HT in the presence of the test compound as described in Figure S2.

^*indicates p < 0.05. The E_max for the test compound plus 5-HT was normalized to the E_max for 5-HT alone. Subsequent post hoc comparisons between means for E_max were made using an unpaired t test with Welch’s correction (GraphPad Prism). All statistical analyses were conducted with an experiment-wise error rate of α = 0.05.

To further explore the SAR of the LT attached to the core piperidinyl scaffold, replacement of the long alkyl chain with other saturated moieties (cyclohexyl, cyclohexylethyl or 4-methylcyclohexylethyl) were investigated. The 2-amino-1-phenyl-1,3-propanediol PHs consistently resulted in separable isomers when coupled with the piperidine scaffold bearing an undecyl tail; therefore, these polar heads were retained with the cyclohexyl tails with the hope that the isomers would again be separable by TLC. As shown in Scheme 2, Suzuki coupling of 3 with phenylboronic acid or Sonogashira coupling of 3 with ethynylbenzene or 1-ethynyl-4-methylbenzene afforded requisite substituted picolinic derivatives 23–25. Metal-catalyzed hydrogenation of compounds on a Parr hydrogenator at 60 psi in the presence of 0.4 equiv of PtO₂ and 1 equiv of 37% aq HCl readily provided compounds 26–28. Boc-protection and hydrolysis of 26–28 provided 29–31 as the key intermediates in high yield on the multigram scale. Amidation coupling with (1S,2S)-or (1R,2R)-(–)-2-amino-1-phenyl-1,3-propanediol followed by removal of Boc groups with TFA furnished a series of novel derivatives 32–37 with compact LTs in 83–86% yields (two steps). As previously observed, each amidation coupling reaction also produced a pair of diastereoisomers, which could be separated by PTLC after the Boc-deprotection. It was our expectation that the compact LTs would result in final compounds that could form crystals suitable for X-ray diffraction. To our delight, two of the six compounds were crystallized and characterized. As shown in Figure 2, the absolute stereochemistry of compounds 32 and 35 was unambiguously determined by their X-ray crystallography analyses (Table S2, Figure S1), from which the stereochemistry of the other diastereoisomers could also be deduced as described in our previous work.²⁸

Scheme 2.

Synthesis of PNU-69176E Analogs with Compact Lipophilic Tails (LT)^a

^aReagents and conditions: (a) Suzuki coupling, (i) phenylboronic acid, Pd(PPh₃)₄, Na₂CO₃, EtOH/H₂O/PhMe = 2:1:1, reflux; (ii) H₂SO₄, methanol, reflux, 98% (two steps); or Sonogashira coupling, ethynylbenzene or 1-ethynyl-4-methylbenzene, CuI, Pd(PPh₃)₂Cl₂, Et₃N, rt, 12 h, 88% to 97%; (b) (i) H₂ (60 psi), PtO₂, HCl/MeOH/H₂O, rt, 16 h, 97–98%; (ii) (Boc)₂O, Et₃N, MeOH, rt, 16 h, 96–98%; (c) LiOH, THF/H₂O (v/v = 2:1), rt, 48 h, quantitative yield; (d) (i) amino alcohols, HBTU, DIPEA, DMF, rt, 16 h; (ii) TFA, CH₂Cl₂, rt, 83–86% (two steps).

Figure 2.

Determination of absolute configuration of chiral carbons 2 and 4 of the piperdinyl ring. X-ray crystallography structures of analogues (2S,4R)-32 and (2R,4S)-35.

In Vitro Ca_i²⁺ Release Assay in h5-HT_2CR-CHO Cells.

The best-characterized intracellular pathway of 5-HT_2CR signaling is the activation of phospholipase Cβ (PLCβ) via G_αq/11 proteins followed by the production of inositol-1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG), leading to increased Ca_i²⁺ release from intracellular stores.³² Therefore, in vitro functional characterization of these synthetic compounds can be determined by utilizing a fluorescence-based, live cell Ca_i²⁺ release assay to measure activation of the 5-HT_2CR signaling pathway. The Ca_i²⁺ levels can be regarded as a primary readout, as has been utilized in a multitude of 5-HT_2CR drug discovery projects.³³ Our assay was conducted in Chinese hamster ovary (CHO) cells stably transfected with the human 5-HT_2CR (unedited INI isoform; h5-HT_2CR-CHO cells).^28,34,35 The maximum capacity of 5-HT to promote Ca_i²⁺ release (E_max) was established and set to 100% response. All compounds are reported relative to the E_max of 5-HT. Previous titration studies with compound 1 revealed an upward shift of 5-HT-evoked Ca_i²⁺ release at the concentration effective to evoke 20% response (EC₂₀), while 1 nM of 1 produced a leftward shift of the 5-HT-evoked Ca_i²⁺ response curve.²⁸ Therefore, all compounds were initially screened at 1 nM for their ability to enhance 5-HT-induced Ca_i²⁺ release evoked by increasing concentrations of 5-HT in h5-HT_2CR-CHO cells (Figure S2). The cells were pretreated for 15 min with each test compound to establish an absence of intrinsic agonist activity in each assay. The analysis of novel compounds on maximum 5-HT-induced Ca_i²⁺ release in h5-HT_2CR-CHO cells is summarized in Table 1.

None of the 24 new analogs screened exhibited intrinsic efficacy at 1 nM to induce Ca_i²⁺ release in h5-HT_2CR-CHO cells in the absence of 5-HT (Figure S2). Compound 7, the deprotected amino acid of the key intermediate 6 (Scheme 1), exhibited no significant PAM activity (Table 1). Topographical analysis of 1 revealed 2-or 3-carbon long chains to the nearest heteroatom (O and Cl) in the sugar-like PH. Therefore, the selection of PHs emphasized the incorporation of −OH(s) two carbons away from the amide linker. The coupling of ethanolamine with the key intermediate afforded 8. Similar to 7, no significant PAM activity with 8 was observed (Table 1). Incorporation of two −OH groups (a 1,3-propandiol fragment, 9) provided a negligible increase in 5-HT-induced Ca_i²⁺ release, though statistically significant. Various analogues that retained the 1,3-propanediol fragment with the addition of a methyl or phenyl group (10–15) did not provide for the desired PAM activity (Table 1). However, compound 14, which was a separable isomer with the (2S,4R) configuration at the piperidine ring and the (1R,2R) configuration for the PH moiety, displayed significant activity consistent as a negative allosteric modulator (NAM, Figure 3A, Table 1). Next, the use of an additional carbon spacer between the amide linker and the most distal −OH was explored through the incorporation of a 1,2-propanediol fragment (16). The glycol moiety proved fruitful. Compound 16 displayed significant PAM activity and increased the 5-HT-evoked Ca_i²⁺ release ∼23% above the 5-HT E_max value (Figure 3B). The enantiomerically pure versions of the PH of 16 were incorporated into the scaffold and were tested and showed a stereochemical preference for PAM activity. Compound 16a, the R-variant at the PH, lacked appreciable PAM activity (Figure 3C), while the S-configuration (16b), like 16, displayed significant PAM activity (Table 1). Incorporation of a terminal −Cl abolished statistically significant activity (17). Additional attempts to incorporate various hydrogen bond donor/acceptor moieties as PHs did not produce compounds with PAM activity [N-ethyl morpholino (18), methyl esters of threonine (19), serine (20), tyrosine (21), and tryptophan (22)]. The lactate dehydrogenase assay was employed to predict the cytotoxicity of compounds at 0.1 nM and 1 μM; none of the tested compounds exhibited values in this assay indicative of cellular toxicity (Table S3).

Figure 3.

Effects of select compounds on Ca_i²⁺ release in live h5-HT_2CR-CHO cells in the absence (black circles) and in the presence of the test compound (red triangles) against the concentration–response curve for 5-HT, vehicle (blue circle), or vehicle in the presence of test compound (green triangle). (A) Compound 14 (1 nM), representative example of a potential NAM; (B) compound 16 (1 nM), representative example of a potential PAM; (C) compound 16a (1 nM), representative example of an inactive compound. The maximum 5-HT-induced Ca_i²⁺ release in the absence of the test compounds was set as 100%. The E_max of 5-HT in the presence of the test compounds are listed in Table 1. Concentration–response curves for remaining compounds are in Figure S2.

Compound 16 promoted an upward shift of 5-HT-evoked Ca_i²⁺ release at multiple concentrations (Figure 4A) without a leftward shift, an indication that 16 promotes improved 5-HT efficacy but did not impact 5-HT potency at the 5-HT_2CR (Figure 4A). To determine the selectivity of 16 for allosteric modulation of the 5-HT_2CR over the 5-HT_2AR, multiple concentrations of 16 were employed to assess 5-HT-evoked Ca_i²⁺ release in h5-HT_2AR-CHO cells (Figure 4B).^28,34 Compound 16 produced no change in the 5-HT E_max or EC₅₀ at the 5-HT_2AR (Figure 4B). These data provide the conceptual framework for the continued development of selective PAMs of the 5-HT_2CR given the ability of 16 to affect 5-HT efficacy at the 5-HT_2CR (Figure 4A), but not the 5-HT_2AR (Figure 4B).

Figure 4.

Effects of 16 on Ca_i²⁺ release in live h5-HT_2CR-CHO cells (A) and h5-HT_2AR-CHO cells (B) against the concentration–response curve for 5-HT. (A) Compound 16 (1 pM, 1 nM, 1 μM) demonstrates an upward shift in h5-HT_2CR-CHO cells. (B) Compound 16 (1 pM, 1 nM, 1 μM) does not alter 5-HT-induced signaling in h5-HT_2AR-CHO cells. The maximum 5-HT-induced Ca_i²⁺ release in the absence of the test compounds was set as 100%.

The compounds synthesized according to Scheme 2 retained the 1-phenyl-1,3-propandiol PH in an effort to produce separable isomers to further explore the impact of changes made to the LT. Preserving the PH used in the potential NAM 14, which produced a decrease in 5-HT-mediated Ca_i²⁺ release, while changing the LT from the undecyl tail to an ethylcyclohexyl moiety, afforded a NAM that promoted a slight but significant decrease in 5-HT-induced Ca_i²⁺ release (32, 96.0% ± 1.9%). All other attempts to utilize various LTs failed to produce any compounds with allosteric effects (34–37). Exploration of the remaining chemical space, in particular with regard to the LT, will be reported in due course.

Molecular Docking to 5-HT_2CR.

To explore a potential binding mode and provide additional lines of evidence that the described compounds are allosteric modulators, molecular docking using the Schrödinger Drug Discovery Suite was employed to identify the allosteric binding site relative to the orthosteric binding site (see SI for details on docking protocol). Until recently, the only solved structures of serotonin receptors were the 5-HT_1BR and 5-HT_2BR.^36–38 However, the very recently disclosed 5-HT_2CR structure has afforded the opportunity to conduct the first allosteric-5-HT_2CR docking study.³⁹ With compound 16b displaying positive allosteric modulation, molecular docking using the active state of the 5-HT_2CR was used to search the extracellular portion of the receptor for a suitable allosteric site.

Figure 5A depicts a global view of the 5-HT_2CR and the docked endogenous ligand 5-HT (pink space-fill) in the orthosteric site engaging residues consistent with previous reports based on site-directed mutagenesis studies.⁴¹ The red cluster overlay of 16b (red wire-representation of top five poses using Schrödinger Glide scoring function) highlighted a possible binding pocket for the proposed PAM as being topologically distinct from the orthosteric site.

Figure 5.

Binding poses of (A) global view of 5-HT_2CR (PDB 6BQG) with 5-HT (pink space-fill representation) at the orthosteric site and 16b (red stick representation overlay of top five poses) at the proposed allosteric site; (B) representative pose of 16b (red stick representation) engaged with residues of proposed allosteric site (superscript designation represents Ballesteros–Weinstein notation);⁴⁰ (C) representative pose of 16a (red stick representation).

The allosteric site (Figure 5A) is near extracellular loop 2 (EL2) accommodating the PH of the proposed PAM, an observation also made when describing similar binding modes of other class A GPCRs.⁴² Figure 5B depicts the 1,2-diol PH moiety with the S-configuration engaging in a bidentate, H-bonding interaction with the backbone carbonyl L209 of EL2, a residue conserved in the 5-HT₂R subfamily. Additionally, the ionizable N atom of the piperidine ring engages in an H-bond with the −OH side chain of S334 of transmembrane helix VI (TMH VI), which is not present in either the 5-HT_2AR or 5-HT_2BR, thus suggesting a predicted molecular basis for the 5-HT_2CR subtype functional selectivity over the 5-HT_2AR as described in our cell-based studies. The predicted bridging effect of 16b between EL2 and TMH VI may result in a drawing in of the extracellular portion of TMH VI and, thereby, promote the outward shift of the intracellular portion of TMH VI, consistent with reports that this helical movement is associated with the active state of the receptor.^39,43,44 Compound 16a, containing the 1,2-diol PH with the R-configuration, does not form the same EL2-to-TMH VI bridge (Figure 5C), providing a potential explanation as to why 16a is inactive in the described cell-based assay.

In Vitro Assessment of Off-target Effects of Compound 16.

Preliminary data from a broad-panel assay generously provided by the National Institute of Mental Health (NIMH) Psychoactive Drug Screening Program (PDSP)⁴⁵ showed that the most promising PAM, compound 16 assessed at 10 μM displayed no significant ability to displace binding to a variety of related receptors (Table 2). Specifically, at 10 μM, 16 did not displace binding of radioligands to 5-HT receptors (5-HT_1AR, 5-HT_1BR, 5-HT_1DR, 5-HT_1ER, 5-HT_2AR, 5-HT_2BR, 5-HT_2CR, 5-HT₃R, 5-HT_5AR, 5-HT₆R, 5-HT₇R), dopamine receptors (D₁, D₂, D₄, D₅), or monoamine transporters [5-HT transporter (SERT) and norepinephrine transporter (NET)]. Compound 16 (10 μM) shows weak to no displacement of binding to other evaluated receptors and monoamine transporters (Table S4). Compound 16 (10 μM) did display significant inhibition (defined as greater than 50%) of radioligand binding at the dopamine transporter (DAT; 66.3%), dopamine D₃ receptor (71.7%), or α_2A and α_2B receptors (85.1% and 80.7%, respectively). However, the concentration (10 μM) tested in the radioligand displacement assay is significantly higher than the concentrations of 16 (1 pM, 1 nM, 1 μM) screened for activity in the functional assay. Therefore, in total, compound 16 appears to be promising with regard to displaying minimal off-target effects, a commonly postulated advantage of allosteric modulators of GPCRs.²⁰ Moreover, 16 showed little to no interaction with the orthosteric sites of the tested 5-HT receptor subtypes.

Table 2.

Displacement of Radioligand Binding by Compound 16 (10 μM) in a Broad Panel of Receptors and Transporters^a

receptor or transporter	radioligand	% inhib (10 μm) b	Ki (μM)c
5-HTIA	[3H]-8-OH-DPAT	25.7	e
5-HT1B	[3H]-GR125743	–18.1	e
5-HT1D	[3H]-GR125743	5.5	e
5-HT1E	[3H]-5-HT	0.7	e
5-HT2A	[3H]-ketanserin	37.1d	e
5-HT2B	[3H]-LSD	15.1d	e
5-HT2C	[3H]-mesulergine	9.8d	e
5-HT3	[3H]-LY278584	27.0	e
5-HT5A	[3H]-LSD	17.2	e
5-HT6	[3H]-LSD	18.1	e
5-HT7	[3H]-LSD	26.8	e
D1	[3H]-SCH23390	293	e
D2	[3H]-N-methylspiperone	28.8	e
D3	[3H]-N-methylspiperone	71.4	4.7
D4	[3H]-N-methylspiperone	–14.2	e
D5	[3H]-SCH23390	323	e
DAT	[3H]-WIN35428	66.3	1.8
SERT	[3H]-citalopram	24.8	e

^aNational Institute of Mental Health (NIMH) Psychoactive Drug Screening Program (PDSP).⁴⁵ Receptor binding profiles were generously provided by NIMH PDSP, Contract no. HHSN-271–2013-00017-C. The 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.

^bData represent mean % inhibition (n = 4) for compound 16 assessed for displacement of binding at targets. Inhibition of binding >50% at 10 μM of compound 16 resulted in determination of K_i.

^cData represent K_i (μM) values obtained from nonlinear regression of radioligand competition isotherms. K_i values are calculated from best fit IC₅₀ values using the Cheng–Prusoff equation.

^dData for 5-HT₂ receptor subtypes represent the average of the mean % inhibition (n = 8) for compound 16.

^eNot tested.

In Vivo Pharmacokinetic (PK) Profile of Compound 16.

The PK profile of 16 was assessed in a pilot study to investigate whether this class of molecules exhibits favorable drug-like properties. The PK parameters of 16 were evaluated in rats after single iv (5 mg/kg) or po (10 mg/kg) administration. As summarized in Table 3, Compound 16 displays a decent half-life (T_1/2) after administration of 5 mg/kg iv (t_1/2 = 6.59 ± 0.26 h) or 10 mg/kg po (5.82 ± 0.37 h). The time of maximum concentration (T_max) after 10 mg/kg po was 3.3 ± 0.58 h. The plasma clearance (CL) of 16 was 5.37 ± 0.61 L h^–1 kg^–1 after 5 mg/kg iv, while the volume of distribution plasma distribution (V_ss) was 35.07 ± 4.66 L/kg. Compound 16 displayed a maximum serum concentration (C_max) of 68.1 ± 6.8 ng/mL after 10 mg/kg po, while 16 exhibited moderate plasma exposure (AUC_0–inf) after administration of 5 mg/kg iv (939 ± 108 ng·h·mL^–1) or 10 mg/kg po (737 ± 56 ng·h·mL^–1). Decent oral bioavailability (F) was observed (10 mg/kg po; 39.1%). We also calculated the central nervous system (CNS) multiparameter optimization (MPO) value for compound 16 to be 4.5; collective scores range from 0 to 6 with higher MPO scores predicted to be more desirable for CNS medication candidates (Table S5).⁴⁶ Based upon these data, compound 16 was selected for subsequent evaluation of in vivo efficacy.

Table 3.

In Vivo Pharmacokinetic Profile of Compound 16^a

dose (mg/kg)	T1/2 (h)	Tmax (h)	CL (L h–1 kg–1)	Vss (L/kg)	Cmax (ng/mL)	AUC0–inf (ng·h·mL–1)	F (%)
5, iv	6.59 ± 0.26	b	5.37 ± 0.61	35.07 ± 4.66		939 ± 108
10, po	5.82 ± 0.37	3.3 ± 0.58	b	b	68.1 ± 6.8	737 ± 56	39.1

^aValues are the average of three runs. Vehicle, DMSO/Tween 80/saline (v/v 1:3:6). T_1/2, half-life; T_max, time of maximum concentration; CL, plasma clearance; V_ss, volume of distribution; C_max, maximum concentration; AUC_0–inf, area under the plasma concentration–time curve; F, oral bioavailability.

^bNot determined.

Effects of Compound 16 on Spontaneous Locomotor Activity.

Our candidate 5-HT_2CR PAM could be expected to enhance the effects of endogenous (5-HT) or exogenous 5-HT_2CR agonists in vivo. Selective and nonselective agonists of the 5-HT_2CR induce dose-dependent suppression of spontaneous locomotor activity in rodents, which is reversed by selective 5-HT_2CR antagonists.^9,47,48 In the present experiment, we tested the hypothesis that 16 would evoke a modest suppression of motor activity in adult male Sprague–Dawley rats similar to the observations made for a recently evaluated candidate 5-HT_2CR PAM.²⁹ We assessed the effects of compound 16 (0.5, 1, or 5 mg/kg ip) on spontaneous locomotor activity over a 90 min session. As the effects of compound 16 were confined to the initial 30 min of the session, Figure 6 presents ambulations totaled for that 30 min. A main effect of compound 16 was observed for mean total ambulations (±SEM) [F_(3,25) = 3.44, p = 0.03]. A priori comparisons revealed that 5 mg/kg of compound 16 significantly reduced mean total ambulations (±SEM) vs saline (p < 0.05; Figure 6A). A main effect of compound 16 treatment was also observed for mean total vertical activity (±SEM) in 30 min [F_(3,25) = 9.14, p = 0.0003]. A priori comparisons revealed that 5 mg/kg of compound 16 significantly reduced mean total vertical activity (±SEM) vs saline (p < 0.05; data not shown). Ambulation and vertical activity are often positively correlated; however, these measures are actually independent of one another, can be pharmacologically dissociated, and appear to be mediated by different neural circuitry.^49–52

Figure 6.

Effects of compound 16 on spontaneous locomotor activity. (A) Groups of rats (n = 7–8/group) naive to the activity monitors were injected with a single dose of saline or compound 16 (0.5, 1, or 5 mg/kg; ip) immediately prior to the start of locomotor assessment. (B) Groups of rats (n = 9–10/group) naive to the activity monitors were injected with a single dose of the 5-HT_2CR antagonist SB242084 (1 mg/kg; ip; 15 min pretreatment) and the 5-HT_2CR agonist WAY163909 (1 mg/kg; ip) in combination with compound 16 (5 mg/kg; ip) immediately prior to the start of locomotor assessment. The mean total ambulations (±SEM) in 30 min are plotted. *p < 0.05.

Figure 6B illustrates the allosteric effects of compound 16 (5 mg/kg) in combination with the selective 5-HT_2CR orthosteric agonist WAY163909 (1 mg/kg) on spontaneous locomotor activity in the presence or absence of the 5-HT_2CR antagonist SB242084 (1 mg/kg). Pretreatment with SB242084 inhibits WAY163909-evoked suppression of ambulations, but not vertical activity (data not shown). A main effect of treatment was observed for ambulations totaled across the 30 min session [F_(3,34) = 8.25, p = 0.0003]. A priori comparisons revealed that the combination of compound 16 plus WAY163909 reduced total ambulations versus WAY163909 alone (p < 0.05; Figure 6B); this effect was prevented by SB242084 (p < 0.05; Figure 6B). A main effect of treatment was observed for vertical activity totaled across the 30 min session [F_(3,34) = 19.19, p < 0.0001]; a priori comparisons revealed that the combination of compound 16 plus WAY163909 reduced vertical activity versus WAY163909 alone (p < 0.05; data not shown); this effect was only marginally prevented by SB242084 pretreatment (p < 0.05; data not shown).

These data demonstrate that the 5-HT_2CR is engaged in the effects of compound 16 to suppress motor activity in the presence of exogenous 5-HT_2CR stimulation with WAY163909. Furthermore, compound 16 may augment endogenous 5-HT_2CR signaling in vivo to suppress spontaneous ambulations, as might be expected of a PAM.

Effects of Compound 16 in a WAY163909 vs Saline Drug Discrimination Assay.

We employed the drug discrimination assay to explore the impact of compound 16 in the context of endogenous or exogenous 5-HT_2CR stimulation. The drug discrimination assay is widely recognized as one of the major methods in neuropharmacology and drug discovery.^53–55 This assay has face validity for modeling the subjective effects of drugs that penetrate the blood–brain barrier as well as to assess the influence of the novel compounds on the interoceptive effects of receptor-selective agonists or other ligands, including allosteric modulators.^56–59 Drug discrimination procedures have been extensively used to characterize the neuropharmacological profile of novel small molecules as well as 5-HT₂R subtype agonists, including 5-HT_2CR agonists (e.g., m-chlorophenylpiperazine, Ro 60–0175).^60–63 Here, we trained rats to discriminate 0.75 mg/kg of the selective 5-HT_2CR agonist WAY163909 from saline in a two-lever drug discrimination protocol. The mean number of sessions required to meet the criterion for acquisition of the WAY163909 (0.75 mg/kg, ip; 15 min pretreatment) versus saline discrimination was 44 training sessions (range 39–57). During dose–response tests, WAY163909 (0.125–1.0 mg/kg, ip) elicited a dose-dependent increase in WAY163909-appropriate responding whereas saline resulted in <10% WAY163909-appropriate responding (Figure 7A, closed circles). Greater than 95% WAY163909-appropriate responding was observed at the training dose (0.75 mg/kg, ip). Response rates were stable across all test doses of WAY163909 (Figure 7A, open triangles). The time course of WAY163909-appropriate responding at 0.75 mg/kg (Figure S3) indicated that the stimulus effects of the training dose of WAY163909 (0.75 mg/kg, ip) are maximal at a 15 min pretreatment time and decline until 120 min at which time rats cannot discriminate this dose of WAY163909 (Figure S3). The dose predicted to elicit 50% of WAY163909-lever responding (ED₅₀) in rats is 0.53 mg/kg.

Figure 7.

Dose–response relationship of WAY163909, compound 16, and the combination in a WAY163909 vs saline drug discrimination assay. The dose–response curves (n = 7–9/dose) for (A) WAY163909 (ip) and (B) compound 16 (ip) are presented. Closed circles (solid lines) denote the mean percentage of WAY163909-appropriate responding (±SEM; left axis), and open triangles (dotted lines) denote the mean responses/min (±SEM; right axis). *p < 0.05 vs 0.75 mg/kg WAY163909. (C) The results of substitution tests in which WAY163909 alone or in combination with compound 16 are presented. The dotted line represents the % WAY163909-lever response observed for the training dose of WAY163909 (0.75 mg/ kg). *p < 0.05 vs 0.5 mg/kg of compound 16; p < 0.05 vs 0.5 mg/kg of WAY163909.

Compound 16 alone did not stimulate Ca_i²⁺ release in live h5-HT_2CR-CHO cells, indicating an inability to act as a 5-HT_2CR agonist in vitro; therefore, we hypothesized that compound 16 would not substitute fully for the full 5-HT_2CR agonist WAY163909 in rats trained to discriminate WAY163909 from saline. Compound 16 (0.125–0.5 mg/kg; ip) evoked a maximum of 45% ± 20% (SEM) WAY163909-appropriate responding at a dose of 1 mg/kg (Figure 7B, closed circles). All doses of 16 elicited WAY163909-lever responding that was significantly different than the training dose (0.75 mg/ kg, p < 0.05); this lack of substitution of 16 suggests a dissociation between the stimulus effects of the full 5-HT_2CR agonist WAY163909 and compound 16. Response rates (Figure 7B, open triangles) were stable across the lowest doses of compound 16 (0.125–0.5 mg/kg). At the highest dose of compound 16 tested (1 mg/kg), seven out of the nine rats tested completed the lever press response criterion; the response rate for these rats was significantly decreased relative to the training dose of WAY163909 (p < 0.05). While this observation may reflect motor suppression evoked by 16, the maximal dose of 16 (1 mg/kg) employed in drug discrimination studies was not associated with suppressed ambulations (Figure 6A). In general, responding in an operant task is relatively more sensitive than is locomotor activity, and it is important to note that modifications in response rates are dissociable from response choice.^60,64

The observation that compound 16 partially substituted for WAY163909 may not be unexpected given that the effects of endogenous 5-HT could be enhanced by a 5-HT_2CR PAM. Coupled with data that compound 16 does not have intrinsic activity in the h5-HT_2CR-CHO assay, these data support the contention that compound 16 does not exhibit appreciable efficacy as a 5-HT_2CR agonist and are consistent with other findings regarding the discriminative stimulus effects of allosteric modulators.⁶⁵

We also postulated that a 5-HT_2CR PAM would enhance the effects of the 5-HT_2CR agonist WAY163909 to evoke its discriminative stimulus effects. To further probe this premise, rats were injected with low doses of WAY163909 (0.5 mg/kg) in combination with compound 16 (0.5 mg/kg; Figure 7C); each evokes ∼30% WAY163909-appropriate responding with rates of ∼30 responses/min. Interestingly, the coadministration of 0.5 mg/kg of WAY163909 plus 0.5 mg/kg of compound 16 synergized to evoke a full substitution for WAY163909 (∼95% WAY163909-lever responding). The response rate seen with the combination (15 ± 2.0 responses/min) did not differ from 0.5 mg/kg of WAY163909 (31.0 ± 4.4 responses/min), but was significantly lower than 0.5 mg/kg of compound 16 (27.4 ± 5.8 responses/min) administered alone (p < 0.05). Taken together, these data suggest that compound 16 acts in vivo to augment 5-HT_2CR agonist-mediated signaling in vivo.

Effects of Compound 16 on Cocaine Cue Reactivity.

We employed a rat model of cocaine self-administration to establish the efficacy of compound 16 to suppress relapse vulnerability assessed by responding for the discrete cue complex paired with cocaine intake (cue reactivity).^8,9,12,66,67 Selective 5-HT_2CR agonists effectively suppress cocaine cue reactivity and cocaine intake in the self-administration paradigm,^{9,12,47,68,69} with no evidence of abuse liability on their own.^13–15 Relapse during abstinence from cocaine use is a major hurdle for the successful treatment of CUD; an effective pharmacotherapy that improves inhibitory control in the context of exposure to cocaine-associated cues would represent a first-in-class drug useful for those suffering from CUD.^2,3

The self-administration assay has the greatest face validity for modeling human drug-taking and cue-reactivity and for therapeutic drug discovery. Cocaine self-administration training consisted of 14 daily 180 min sessions during which rats were trained to lever press for cocaine infusions (0.75 mg/kg per 0.1 mL infusion, iv).^6,8,9,11 Schedule completions on the active lever resulted in delivery of a cocaine infusion paired simultaneously with illumination of the house and stimulus lights and activation of the infusion pump (discrete cue complex paired with cocaine delivery); inactive lever presses produced no scheduled consequences. Following stable acquisition, rats were reintroduced to the self-administration chambers 24 h later and assayed in a cue reactivity test session comprised of two sequential components. The first component (10 min) evaluated whether rats would exhibit differential levels of lever presses when placed in the context in the absence of the discrete cue complex. The second component (60 min) was signaled by a single, nonresponse contingent delivery of the discrete cue complex presented immediately at the termination of the first component.⁶

We assessed the effects of compound 16 (1 mg/kg; ip) on cocaine cue reactivity across both components of the test session. A main effect of treatment [(F_(1,14) = 7.28, p = 0.0173], time [(F_(13,182) = 82.07, p < 0.0001], and a treatment × time interaction [(F_(13,182) = 4.58, p < 0.0001] for previously active lever presses was observed. Previously active lever presses reinforced by the discrete cue complex were significantly lower following pretreatment with 16 vs saline (Figure 8; p < 0.05); compound 16 did not significantly alter inactive lever presses vs saline (data not shown). These in vivo data provide the first reported in vivo evidence that a putative 5-HT_2CR PAM suppresses a key facet of relapse vulnerability in CUD.

Figure 8.

Effects of compound 16 on cocaine cue reactivity in rats (n = 7/treatment group). Mean (±SEM) previously active lever are presented for the cue reactivity test session following administration for saline or compound 16. The first component of the cue reactivity session (initial 10 min) evaluated lever presses in the absence of the discrete cue complex. The second component was signaled by the single delivery of the discrete cue complex (denoted by arrow). Presses on the previously active lever in the 60 min second component were reinforced by the discrete cue complex; inactive lever presses were recorded but produced no scheduled consequences. Rats were injected with saline (1 mL/kg, ip) or compound 16 (1 mg/kg; ip) 15 min prior to a cocaine cue reactivity test session. Compound 16 suppressed cue reactivity vs saline (*p < 0.05).

CONCLUSIONS

A series of structurally simplified analogues have been rationally designed and synthesized based on the 5-HT_2CR PAM 1. The allosteric modulatory SAR of the 5-HT_2CR was explored using a fluorescence-based, 5-HT-evoked Ca_i²⁺ release assay. The stereochemistry of the synthesized compounds was secured by X-ray crystallographic analysis of compounds 32 and 35. Simplification of the PH of 1 is tolerable or even favorable for potentiation of 5-HT_2CR-evoked Ca_i²⁺ release. None of the compounds exhibited 5-HT_2CR agonist efficacy when tested alone, and 16 (CYD-1–79) potentiated 5-HT-evoked Ca_i²⁺ release in h5-HT_2CR-CHO cells with receptor subtype selectivity versus the 5-HT_2AR. Radioligand binding results suggest that these compounds do not bind to the orthosteric site of the 5-HT_2CR; a topographically distinct site was identified by our molecular docking analyses. Compound 16 exhibited a favorable overall pharmacokinetic profile such as decent oral bioavailability and half-life, and a respectable CNS MPO value (4.5 out of 6). Compound 16 evoked a modulation of 5-HT_2CR-mediated spontaneous ambulations, partially substituted for the training dose of WAY163909, synergized with a low dose of WAY163909 to substitute fully for the stimulus effects of this selective 5-HT_2CR agonist, and attenuated cocaine cue reactivity in rats. Our current knowledge of the selectivity and specificity of compound 16 is driving our future studies to develop drug-like 5-HT_2CR PAMs with constrained off-target effects.

Our success in the chemical design, synthesis, and pharmacological evaluation of these new molecules opens new doors to a greater understanding of 5-HT_2CR allosteric modulation and the development of novel pharmacotherapeutics for CUD characterized by deficient 5-HT_2CR signaling capacity.^2,3 To the best of our knowledge, this report includes the only in vivo investigation of a 5-HT_2CR PAM in a proof-of-concept study toward the treatment of CUD and is the only study that proposes allosteric binding sites for candidate PAMs on the 5-HT_2CR. As impaired signaling capacity of the 5-HT_2CR may contribute to the neurobehavioral processes that underlie chronic health issues such as depression, impulsivity disorders, obesity, and schizophrenia, the full potential for novel molecules with actions as selective 5-HT_2CR PAMs remains to be elucidated. Continued medicinal chemistry efforts toward structural optimization, in particular at the LT, and in vitro and in vivo cellular evaluations are underway.

EXPERIMENTAL SECTION

Chemistry.

General.

All commercially available starting materials and solvents were reagent grade and used without further purification. Reactions were performed under a nitrogen atmosphere in dry glassware with magnetic stirring. Preparative column chromatography was performed using silica gel 60, particle size 0.063–0.200 mm (70–230 mesh, flash). Analytical TLC was carried out employing silica gel 60 F254 plates (Merck, Darmstadt). Visualization of the developed chromatograms was performed with detection by UV (254 nm). NMR spectra were recorded on a Bruker-600 (¹H, 600 MHz; ¹³C, 150 MHz) spectrometer or Bruker-300 (¹H, 300 MHz; ¹³C, 75 MHz). ¹H and ¹³C NMR spectra were recorded with TMS as an internal reference. Chemical shifts were expressed in ppm, and J values were given in Hz. High-resolution mass spectra (HRMS) were obtained from Thermo Fisher LTQ Orbitrap Elite mass spectrometer. Parameters include the following: Nano ESI spray voltage was 1.8 kV; capillary temperature was 275 °C, and the resolution was 60 000; ionization was achieved by positive mode. Melting points were measured on a Thermo Scientific electrothermal digital melting point apparatus and were uncorrected. Purity of final compounds was determined by analytical HPLC, which was carried out on a Shimadzu HPLC system (model CBM-20A LC-20AD SPD-20A UV/vis). HPLC analysis conditions were as follows: Waters μBondapak C18 (300 mm × 3.9 mm); flow rate 0.5 mL/min; UV detection at 270 and 254 nm; linear gradient from 30% acetonitrile in water (0.1% TFA) to 100% acetonitrile (0.1% TFA) in 20 min followed by 30 min of the last-named solvent. All newly synthesized compounds have been fully characterized with ¹H NMR, ¹³C NMR, HRMS, and HPLC analyses to ensure a purity >95% prior to the biological evaluations.

(cis-2,4)-4-Undecylpiperidine-2-carboxylic acid (7).

Compound 6 (54 mg, 0.14 mmol) was dissolved in CH₂Cl₂ (1 mL). TFA (0.25 mL) was added to the solution at rt. The reaction mixture was stirred for 2 h at rt, and the reaction was judged complete by TLC. The reaction mixture was then concentrated under reduced pressure to afford an oily , which was partitioned between CH₂Cl₂ (15 mL) and a saturated solution of NaHCO₃ (5 mL). The organic layer was treated with anhydrous Na₂SO₄ (5 mL), filtered, and concentrated under reduced pressure. The crude mixture was loaded on a PTLC plate (hexanes/ EtOAc; 5:1) which afforded 7 (22.5 mg, 40%) as a white solid; mp 185–187 °C. ¹H NMR (300 MHz, CDCl₃) δ 9.55 (s, 1H), 8.77 (s, 1H), 3.44 (t, J = 12.4 Hz, 2H), 3.01–2.76 (m, 1H), 2.34 (d, J = 13.2 Hz, 1H), 1.81 (d, J = 11.5 Hz, 1H), 1.64–1.42 (m, 2H), 1.30 (s, 21H), 0.93 (t, J = 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 174.12, 59.64, 43.72, 36.17, 35.05, 33.50, 31.91, 29.68, 29.64, 29.62, 29.34, 28.70, 26.48, 22.68, 14.09. HRMS (ESI) calcd for C₁₇H₃₃NO₂ [M + H]⁺ 284.2584; found 284.2580.

cis-N-(2-Hydroxyethyl)-4-undecylpiperidine-2-carboxamide (8).

To a solution of 6 (70 mg, 0.18 mmol) and 2-aminoethanol (11 mg, 0.18 mmol) in 4 mL of DMF was added HBTU (89 mg, 0.23 mmol) and DIPEA (58 mg, 0.45 mmol). The resulting mixture was stirred at room temperature for 16 h. The DMF was removed under vacuum to give a brown oily residue, which was partitioned between CH₂Cl₂ (50 mL) and 10% citric aqueous solution (10 mL). The organic layer was separated and washed with saturated aqueous NaHCO₃ (10 mL). After drying over anhydrous Na₂SO₄, the solvent was removed under vacuum to give an oily residue. This residue was purified with a silica gel column (5% MeOH in CH₂Cl₂) and afforded the Boc-protected amide (58 mg, 75%). The amide (58 mg, 0.13 mmol) was dissolved in CH₂Cl₂ (1 mL), followed by the addition of TFA (250 μL). The resulting mixture was stirred at room temperature. After 2 h, TLC showed that the starting material had disappeared. The solvent was removed under vacuum to give an oily residue. The residue was partitioned between CH₂Cl₂ (30 mL) and saturated NaHCO₃ aqueous solution (10 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated to give an oily residue. This residue was purified with a silica gel column (eluting with 10% MeOH in CH₂Cl₂), affording 8 (34 mg, 73%) as a colorless amorphous gel. ¹H NMR (600 MHz, CDCl₃) δ 7.54 (d, 1H, J = 4.8 Hz), 4.11 (br s, 2H), 3.71 (s, 2H), 3.46 (d, 2H, J = 10.2 Hz), 3.32 (m, 1H), 3.24 (d, 1H, J = 12.0 Hz), 2.78 (t, 1H, J = 11.4 Hz), 2.09 (d, 1H, J = 12.6 Hz), 1.73 (d, 1H, J = 13.2 Hz), 1.48 (br s, 1H), 1.25 (m, 22H), 0.88 (t, 3H, J = 7.2 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 173.5, 61.2, 60.0, 45.1, 42.2, 36.8, 36.0, 35.3, 31.9, 31.4, 29.8, 29.7, 29.6, 29.4 (4C), 26.4, 22.7, 14.1. HRMS Calcd for C₁₉ H₃₈ N₂ O₂: [M + H]⁺ 327.3006; found 327.3010.

cis-N-(1,3-Dihydroxypropan-2-yl)-4-undecylpiperidine-2-carboxamide (9).

Compound 9 (50 mg) was prepared in 76% yield (2 steps) by a procedure similar to that used to prepare compound 8. The title compound was obtained as a colorless amorphous gel. ¹H NMR (600 MHz, CD OD) δ ¹H NMR (600 MHz, CDCl + CD OD) δ 3.89 (t, 1H, J = 4.2 Hz), 3.66 (m, 4H), 3.15 (m, 1H), 2.62 (m, 1H), 1.99 (d, 1H, J = 12.0 Hz), 1.71 (d, 1H, J = 12.6 Hz), 1.43 (m, 1H), 1.26 (m, 20H), 1.02 (m, 2H), 0.88 (t, 3H, J = 6.6 Hz). ¹³C NMR (150 MHz, CDCl₃ + CD₃OD) δ 174.5, 60.9, 60.1, 52.1, 45.1, 36.7, 36.4, 35.6, 32.1, 31.5 (2C), 29.4, 29.3 (3C), 29.0 (2C), 26.0, 22.3, 13.5. HRMS Calcd for C₂₀H₄₀N₂O₃: [M + H]⁺ 357.3112; found 357.3119.

cis-N-((2S,3S)-1,3-Dihydroxybutan-2-yl)-4-undecylpiperidine-2-carboxamide (10).

Compound 10 (48 mg) was prepared in 71% yield (2 steps) by a procedure similar to that used to prepare compound 8. The title compound was obtained as a colorless amorphous gel. ¹H NMR (600 MHz, CDCl₃) δ 7.59 (m, 1H), 4.78 (br s, 3H), 4.06 (m, 1H), 3.74 (m, 3H), 3.5 (m, 1H), 3.22 (m, 1H), 2.73 (q, 1H, J = 13.8 Hz), 2.06 (m, 1H), 1.74 (t, 1H, J = 13.8 Hz), 1.47 (m, 1H), 1.25 (m, 19H), 1.15 (m, 5H), 0.87 (t, 3H, J = 7.2 Hz). ¹³C NMR (150 MHz, CDCl₃): δ 173.7, 172.9, 67.6, 67.4, 63.4, 60.4, 59.7, 55.5, 45.3, 44.8, 36.8, 36.2, 36.0, 35.6, 35.2, 31.9, 31.3, 30.9, 29.8, 29.6, 29.4, 26.5, 26.4, 22.7, 20.4, 14.1. HRMS Calcd for C₂₁H₄₂N₂O₃: [M + H]⁺ 371.3274; found 371.3270.

cis-N-((2R,3R)-1,3-Dihydroxybutan-2-yl)-4-undecylpiperidine-2-carboxamide (11).

Compound 11 (52 mg) was prepared in 77% yield (2 steps) by a procedure similar to that used to prepare compound 8. The title compound was obtained as a colorless amorphous gel. ¹H NMR (600 MHz, CDCl₃) δ 7.27 (m, 1H), 4.10 (m, 1H), 3.77 (m, 6H), 3.31 (dd, 1H, J = 2.4 Hz, 12.0 Hz), 3.25 (dd, 1H, J = 2.4 Hz, 11.4 Hz), 3.15 (m, 1H), 2.65 (t, 1H, J = 12.6 Hz), 2.05 (m, 1H), 1.68 (m, 1H), 1.43 (m, 1H), 1.25 (m, 19H), 1.17 (m, 3H), 1.08 (m, 2H), 0.88 (t, 3H, J = 7.2 Hz). ¹³C NMR (150 MHz, CDCl₃): δ 174.8, 174.2, 67.8, 67.7, 63.7, 61.0, 60.4, 54.9, 45.7, 45.3, 37.0, 36.9, 36.0, 35.7, 32.2, 31.9, 29.9, 29.7, 29.4, 26.5, 26.4, 22.7, 20.5, 20.4, 14.1. HRMS Calcd for C₂₁H₄₂N₂O₃: [M + H]⁺ 371.3274; found 371.3258.

(2R,4S)-N-((1S,2S)-1,3-Dihydroxy-1-phenylpropan-2-yl)-4-undecylpiperidine-2-carboxamide ((2R,4S)-12) and (2S,4R)-N-((1S,2S)-1,3-Dihydroxy-1-phenylpropan-2-yl)-4-undecylpiperidine-2-carboxamide ((2S,4R)-13).

Compounds (2R,4S)-12 (14 mg, 35%) and (2S,4R)-13 (15 mg, 35%) were prepared (2 steps) by a procedure similar to that used to prepare compound 8. These two isomers could be separated by preparative TLC as colorless amorphous gels. (2R,4S)-12. ¹H NMR (600 MHz, CDCl₃ + CD₃OD) δ 7.37 (m, 2H), 7.32 (t, 2H, J = 7.2 Hz), 7.24 (t, 1H, J = 6.6 Hz), 4.98 (m, 1H), 4.03 (d, 1H, J = 4.2 Hz), 3.69 (m, 1H), 3.62 (m, 1H), 3.10 (d, 2H, J = 11.4 Hz), 2.60 (t, 1H, J = 12.0 Hz), 1.78 (d, 1H, J = 12.6 Hz), 1.67 (d, 1H, J = 12.6 Hz), 1.33 (m, 1H), 1.26 (m, 20H), 1.18 (m, 1H), 1.01 (m, 1H), 0.88 (t, 3H, J = 6.6 Hz), 0.75 (t, 1H, J = 12.6 Hz). ¹³C NMR (150 MHz, CDCl₃ + CD₃OD) δ 174.7, 141.2, 128.0 (2C), 127.3, 125.8 (2C), 72.3, 62.1, 60.2, 56.2, 45.2, 36.7, 36.1, 35.5, 31.9, 31.7, 29.6, 29.5 (2C), 29.4 (2C), 29.1, 26.1, 22.5, 13.8. HRMS Calcd for C₂₆H₄₄N₂O₃: [M + H]⁺ 433.3425; found 433.3415. (2S,4R)-13. ¹H NMR (600 MHz, CDCl₃) δ 7.41 (m, 1H), 7.38 (m, 1H), 7.31 (t, 2H, J = 7.2 Hz), 7.25 (m, 1H), 5.06 (s, 1H), 4.08 (m, 1H), 3.80 (m, 4H), 3.22 (d, 1H, J = 10.8), 3.03 (d, 1H, J = 12.0 Hz), 2.54 (t, 1H, J = 12.0 Hz), 1.77 (d, 1H, J = 12.0 Hz), 1.61 (d, 1H, J = 12.6 Hz), 1.27 (m, 20H), 1.13 (m, 2H), 0.95 (m, 1H), 0.88 (t, 3H, J = 6.6 Hz), 0.81 (m, 1H). ¹³C NMR (150 MHz, CDCl₃) δ 173.6, 141.4, 128.2 (2C), 127.4, 125.8 (2C), 73.3, 63.2, 60.0, 56.4, 44.9, 36.7, 36.2, 35.2, 31.8, 31.6, 29.7, 29.6 (3C), 29.3 (2C), 26.2, 22.6, 14.0. HRMS Calcd for C₂₆H₄₄N₂O₃: [M + H]⁺ 433.3425; found 433.3427.

(2S,4R)-N-((1R,2R)-1,3-Dihydroxy-1-phenylpropan-2-yl)-4-undecylpiperidine-2-carboxamide ((2S,4R)-14) and (2R,4S)-N-((1R,2R)-1,3-Dihydroxy-1-phenylpropan-2-yl)-4-undecylpiperidine-2-carboxamide ((2R,4S)-15).

Compounds (2S,4R)-14 (35 mg, 43%) and (2R,4S)-15 (37 mg, 45%) were prepared (2 steps) by a procedure similar to that used to prepare compound 8. These two isomers could be separated by preparative TLC as colorless amorphous gels. (2S,4R)-14. ¹H NMR (600 MHz, CDCl₃ + CD₃OD) δ 7.67 (br s, 1H), 7.40 (d, 2H, J = 7.8 Hz), 7.31 (t, 2H, J = 7.8 Hz), 1.24 (t, 1H, J = 7.8 Hz), 4.94 (d, 1H, J = 4.8 Hz), 4.10 (m, 1H), 3.67 (dd, 1H, J = 6.0 Hz, 11.4 Hz), 3.52 (dd, 1H, J = 6.0 Hz, 11.4 Hz), 3.17 (d, 1H, J = 12.6 Hz), 2.71 (m, 1H), 1.93 (d, 1H, J = 13.2 Hz), 1.75 (d, 1H, J = 13.8 Hz), 1.46 (br s, 1H), 1.28 (s, 20H), 1.14 (m, 1H), 0.97 (q, 1H, J = 12.6 Hz), 0.89 (t, 3H, J = 7.2 Hz). ¹³C NMR (150 MHz, CDCl₃): δ 172.9, 141.8, 128.0 (2C), 127.3, 126.0 (2C), 71.7, 61.4, 59.5, 56.7, 44.5, 36.5, 35.5, 35.1, 31.7, 30.6, 29.6 (4C), 29.4, 29.1, 26.1, 22.4, 13.5. HRMS Calcd for C₂₆H₄₄N₂O₃: [M + H]⁺ 433.3425; found 433.3427. (2R,4S)-15. ¹H NMR (600 MHz, CDCl₃) δ 7.40 (d, 1H, J = 8.4 Hz), 7.35 (d, 2H, J = 7.2 Hz), 7.28 (m, 2H), 7.21 (t, 1H, J = 7.8 Hz), 5.02 (d, 1H, J = 3.0 Hz), 4.52 (br s, 2H), 4.08 (m, 1H), 3.77 (m, 1H), 3.71 (m, 1H), 3.13 (dd, 1H, J = 1.8 Hz, 12.0 Hz), 2.94 (d, 1H, J = 11.4 Hz), 2.43 (m, 1H), 1.71 (d, 1H, J = 12.6 Hz), 1.56 (d, 1H, J = 12.0 Hz), 1.26 (m, 20H), 0.99 (m, 2H), 0.88 (t, 3H, J = 7.2 Hz), 0.73 (q, 1H, J = 12.0 Hz). ¹³C NMR (150 MHz, CDCl₃): δ 173.8, 141.7, 128.2 (2C), 127.4, 125.9 (2C), 72.9, 63.0, 60.0, 56.3, 44.9, 36.8, 36.4, 35.4, 31.9, 31.8, 29.8, 29.7 (4C), 29.4, 26.3, 22.7, 14.1. HRMS Calcd for C₂₆H₄₄N₂O₃: [M + H]⁺ 433.3425; found 433.3427.

cis-4-Undecylpiperidine-2-carboxylic Acid (2,3-Dihydroxypropyl)amide (16).

Compound 16 (45 mg) was prepared in 68% yield (2 steps) by a procedure similar to that used to prepare compound 8. The title compound was obtained as a colorless amorphous gel. ¹H NMR (300 MHz, CDCl₃) δ 7.49 (m, 1H), 3.82 (m, 4H), 3.50 (m, 3H), 3.36 (m, 1H), 3.29 (d, 1H, J = 10.5 Hz), 3.15 (m, 1H), 2.66 (t, 1H, J = 11.1 Hz), 2.03 (m, 1H), 1.69 (d, 1H, J = 12.9 Hz), 1.42 (m, 1H), 1.25 (br s, 20 H), 1.02 (m, 2H), 0.88 (t, 3H, J = 6.6 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 175.2, 70.9 (2C), 63.8, 63.7, 60.4, 45.6, 42.0, 37.0, 36.7, 35.8, 32.2, 31.9, 29.8, 29.7, 29.4, 26.5, 22.7, 14.1. HRMS Calcd for C₂₀H₄₀N₂O₃: [M + H]⁺ 357.3112; found 357.3116.

cis-N-((R)-2,3-Dihydroxypropyl)-4-undecylpiperidine-2-carboxamide (17).

Compound 17 (54 mg) was prepared in 80% yield (2 steps) by a procedure similar to that used to prepare compound 8. The title compound was obtained as a colorless amorphous gel. ¹H NMR (300 MHz, CDCl₃) δ 7.54 (m, 1H), 3.99 (br s, 3H), 3.77 (d, 1H, J = 2.1 Hz), 3.56 (m, 1H), 3.50 (m, 1H), 3.35 (s, 1H), 3.33 (m, 1H), 3.29 (d, 1H, J = 5.4 Hz), 3.15 (d, 1H, J = 5.4 Hz), 2.66 (t, 1H, J = 6.0 Hz), 2.02 (d, 1H, J = 5.7 Hz), 1.69 (d, 1H, J = 6.3 Hz), 1.43 (br s, 1H), 1.25 (br s, 20 H), 1.04 (m, 2H), 0.88 (t, 3H, J = 3.6 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 175.0, 70.8, 63.8 (2C), 60.4, 45.5, 42.0, 37.0, 36.7, 35.7, 32.1, 31.9, 29.9, 29.7, 29.4, 26.5, 22.7, 14.1. HRMS Calcd for C₂₀H₄₀N₂O₃: [M + H]⁺ 357.3112; found 357.3113.

cis-N-((S)-2,3-Dihydroxypropyl)-4-undecylpiperidine-2-carboxamide (18).

Compound 18 (44 mg) was prepared in 67% yield (2 steps) by a procedure similar to that used to prepare compound 8. The title compound was obtained as a colorless amorphous gel. ¹H NMR (300 MHz, CDCl₃) δ 8.34 (m, 1H), 5.77 (br s, 3H), 3.82 (s, 2H), 3.52 (m, 4H), 3.20 (dd, 1H, J = 3.0 Hz), 2.89 (m, 1H), 2.12 (d, 1H, J = 4.5 Hz), 1.79 (d, 1H, J = 5.1 Hz), 1.57 (br s, 1H), 1.36 (m, 1H), 1.25 (br s, 22H), 0.88 (t, 3H, J = 3.6 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 171.6, 70.8, 70.6, 63.7, 59.1, 44.6, 42.3, 36.5, 34.7, 32.0, 29.9, 29.7, 29.4, 26.5, 22.7, 14.1. HRMS Calcd for C₂₀H₄₀N₂O₃: [M + H]⁺ 357.3112; found 357.3115.

cis-N-((S)-3-Chloro-2-hydroxypropyl)-4-undecylpiperidine-2-carboxamide (19).

Compound 19 (43 mg) was prepared in 63% yield (2 steps) by a procedure similar to that used to prepare compound 8. The title compound was obtained as a colorless amorphous gel. ¹H NMR (300 MHz, CDCl₃) δ 7.38 (br s, 1H), 3.89 (br s, 3H), 3.53 (m, 3H), 3.36 (m, 2H), 3.16 (m, 1H), 2.69 (t, 1H, J = 11.4 Hz), 2.07 (m, 1H), 1.70 (m, 1H), 1.43 (m, 1H), 1.26 (br s, 20H), 1.06 (m, 2H), 0.88 (t, 3H, J = 6.6 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 175.2, 70.7, 70.5, 60.4, 46.4 (2C), 45.6, 45.5, 43.0, 42.9, 36.9, 36.6, 35.7, 35.6, 32.0, 31.9, 29.8, 29.7, 29.6, 29.4, 26.4, 22.7, 14.1. HRMS Calcd for C₂₀H₃₉ClN₂O₂: [M + H]+ 375.2773; found 375.2776.

cis-N-(3-Morpholinopropyl)-4-undecylpiperidine-2-carboxamide (20).

Compound 20 (55 mg) was prepared in 74% yield (2 steps) by a procedure similar to that used to prepare compound 8. The title compound was obtained as a colorless amorphous gel. ¹H NMR (600 MHz, CD₃OD) δ 4.05 (d, 2H, J = 12.6 Hz), 3.79 (m, 3H), 3.48 (d, 2H, J = 10.8 Hz), 3.37 (m, 2H), 3.22 (m, 5H), 2.98 (m, 1H), 2.21 (d, 1H, J = 13.2 Hz), 1.94 (m, 3H), 1.66 (br s, 1H), 1.25 (m, 22H), 0.84 (t, 3H, J = 7.2 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 169.2, 63.8, 58.0, 54.8, 52.1, 51.9, 43.6, 36.2, 36.1, 35.8, 33.9, 33.4, 31.7, 29.5 (2C), 29.4 (2C), 29.2, 28.0, 26.2, 23.7, 22.4, 13.2. HRMS Calcd for C₂₄H₄₇N₃O₂: [M + H]⁺ 396.3585; found 396.3588.

Methyl((cis-2,4)-4-undecylpiperidine-2-carbonyl)-l-allothreoninate (21).

Compound 21 (14 mg) was prepared as a whitish wax in 87.5% yield (2 steps) by a procedure similar to that used to prepare compound 8. ¹H NMR (300 MHz, CDCl₃) δ 7.43 (t, J = 9.8 Hz, 1H), 4.58 (td, J = 9.1, 2.6 Hz, 1H), 4.44–4.30 (m, 1H), 3.78 (d, J = 1.1 Hz, 3H), 3.30 (dd, J = 11.6, 2.7 Hz, 1H), 3.22–3.11 (m, 1H), 2.68 (tt, J = 11.6, 2.6 Hz, 1H), 2.45 (s, 3H), 2.11 (d, J = 13.4 Hz, 1H), 1.70 (d, J = 12.9 Hz, 1H), 1.52–1.37 (m, 1H), 1.27 (s, 22H), 1.11–0.98 (m, 2H), 0.89 (t, J = 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 174.76, 171.48, 67.99, 67.80, 60.91, 60.71, 57.07, 56.89, 52.54, 45.86, 45.70, 36.98, 36.64, 35.95, 32.52, 32.34, 31.92, 29.81, 29.68, 29.64, 29.35, 26.45, 22.69, 20.05, 14.12. HRMS (ESI) calcd for C₂₂ H₄₂ N₂ O₄ [M + H]⁺ 399.3217; found 399.3208.

Methyl((cis-2,4)-4-undecylpiperidine-2-carbonyl)-l-serinate (22).

Compound 22 (16 mg) was prepared as a whitish wax in 89.0% yield (2 steps) by a procedure similar to that used to prepare compound 8. ¹H NMR (300 MHz, CDCl₃) δ 7.51 (dd, J = 20.8, 7.7 Hz, 1H), 4.70–4.58 (m, 1H), 4.07–3.83 (m, 2H), 3.79 (s, 3H), 3.25 (d, J = 11.5 Hz, 1H), 3.15 (d, J = 12.1 Hz, 1H), 2.83 (s, 2H), 2.66 (t, J = 12.2 Hz, 1H), 2.07 (t, J = 10.4 Hz, 1H), 1.69 (d, J = 13.0 Hz, 1H), 1.41 (s, 1H), 1.27 (s, 20H), 1.14–0.94 (m, 2H), 0.89 (t, J = 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 174.61, 174.21, 170.98, 170.92, 62.86, 60.64, 54.48, 54.45, 52.67, 45.88, 45.65, 37.03, 36.99, 36.77, 36.54, 35.99, 35.85, 32.57, 32.51, 31.92, 29.81, 29.68, 29.64, 29.35, 26.45, 26.40, 22.69, 14.12. HRMS (ESI) calcd for C₂₁H₄₅N₂O₃ [M + H]⁺ 385.3061; found 385.3058.

Methyl((cis-2,4)-4-undecylpiperidine-2-carbonyl)-L-tyrosinate (23).

Compound 23 (32 mg) was prepared as a white solid in 86.0% yield (2 steps) by a procedure similar to that used to prepare compound 8; mp 129.0–129.6 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.07 (dd, J = 36.9, 8.4 Hz, 1H), 6.95 (dd, J = 8.4, 3.6 Hz, 2H), 6.67 (dd, J = 13.6, 8.5 Hz, 2H), 4.91–4.75 (m, 1H), 3.74 (d, J = 1.8 Hz, 3H), 3.24–3.03 (m, 3H), 2.95 (td, J = 14.2, 7.0 Hz, 1H), 2.61 (ddd, J = 12.1, 9.8, 5.6 Hz, 1H), 1.99 (t, J = 14.3 Hz, 1H), 1.74–1.55 (m, 1H), 1.27 (s, 22H), 1.10–0.93 (m, 3H), 0.89 (t, J = 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 174.38, 173.99, 172.24, 172.20, 155.80, 130.34, 130.17, 126.93, 126.70, 115.66, 115.61, 60.52, 60.39, 53.01, 52.94, 52.40, 45.79, 45.46, 37.22, 36.95, 36.91, 36.65, 35.90, 35.75, 32.37, 32.29, 31.92, 29.81, 29.68, 29.66, 29.36, 26.43, 26.37, 22.69, 14.13. HRMS (ESI) calcd for C₂₇H₄₄N₂O₄ [M + H]⁺ 461.3374; found 461.3362.

Methyl((cis-2,4)-4-undecylpiperidine-2-carbonyl)-l-tryptophanate (24).

Compound 21 (43 mg) was prepared as a white solid in 83% yield (2 steps) by a procedure similar to that used to prepare compound 8; mp 112.6–113.8 °C. 1H NMR (300 MHz, CDCl₃) δ 8.56 (d, J = 13.5 Hz, 1H), 7.56 (dd, J = 7.6, 3.3 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H), 7.15 (ddd, J = 14.8, 10.9, 6.9 Hz, 3H), 6.97 (s, 1H), 4.96 (td, J = 10.1, 5.7 Hz, 1H), 3.71 (d, J = 5.6 Hz, 3H), 3.43–3.23 (m, 2H), 3.12 (dt, J = 11.5, 3.1 Hz, 1H), 3.05–2.92 (m, 1H), 2.62–2.46 (m, 1H), 2.08–1.93 (m, 1H), 1.59 (d, J = 11.0 Hz, 2H), 1.29 (s, 20H), 1.21–1.11 (m, 2H), 1.06–0.82 (m, 4H). ¹³C NMR (75 MHz, CDCl₃) δ 174.22, 174.10, 172.49, 172.42, 136.15, 136.12, 127.73, 127.70, 122.81, 122.72, 122.11, 119.47, 119.39, 118.69, 118.47, 111.42, 111.32, 110.12, 110.03, 60.66, 60.61, 52.61, 52.52, 52.34, 45.77, 37.05, 36.97, 36.74, 36.71, 36.01, 35.94, 32.65, 32.46, 31.94, 29.85, 29.82, 29.66, 29.37, 27.71, 27.57, 26.44, 22.71, 14.14. HRMS (ESI) calcd for C₂₉H₄₅N₃O₃ [M + H]⁺ 484.3534; found 484.3517.

(2R,4S)-4-(2-Cyclohexylethyl)-N-((1R,2R)-1,3-dihydroxy-1-phenylpropan-2-yl)piperidine-2-carboxamide ((2R,4S)-35) and (2S,4R)-4-(2-Cyclohexylethyl)-N-((1R,2R)-1,3-dihydroxy-1-phenylpropan-2-yl)-piperidine-2-carboxamide ((2S,4R)-34).

Compounds (2R,4S)-35 (53 mg, 44%) and (2S,4R)-34 (50 mg, 42%) were prepared from 33 in 2 steps by a procedure similar to that used to prepare compound 8. These two isomers could be separated by preparative TLC as colorless amorphous gels. Compound 35. ¹H NMR (600 MHz, CDCl₃) δ 7.77 (d, 1H, J = 6.0 Hz), 7.32 (d, 2H, J = 7.2 Hz), 7.26 (m, 2H), 7.19 (t, 1H, J = 7.2 Hz), 5.50 (br s, 2H), 4.97 (d, 1H), 4.12 (d, 1H, J = 3.0 Hz), 3.74 (m, 2H), 3.39 (d, 1H, J = 11.4 Hz), 3.03 (d, 1H, J = 9.0 Hz), 2.50 (m, 1H), 1.69 (m, 6H), 1.58 (d, 1H, J = 9.6 Hz), 1.19 (m, 10H), 0.97 (m, 1H), 0.86 (m, 2H), 0.74 (q, 1H, J = 12.0 Hz). ¹³C NMR (150 MHz, CDCl₃): δ 172.3, 141.5, 128.1 (2C), 127.3, 125.8 (2C), 72.6, 62.7, 59.2, 56.5, 44.3, 37.8, 35.2, 34.9, 34.0, 33.7, 33.4 (2C), 30.3, 26.7, 26.4 (2C). HRMS Calcd for C₂₃H₃₆N₂O₃: [M + H]⁺ 389.2799; found 389.2802. Compound 34. ¹H NMR (600 MHz, CDCl₃ + CD₃OD) δ 7.69 (br s, 1H), 7.39 (d, 2H, J = 7.8 Hz), 7.31 (t, 2H, J = 7.8 Hz), 7.23 (t, 1H, J = 7.8 Hz), 4.97 (d, 1H, J = 4.2 Hz), 4.09 (m, 1H), 3.70 (m, 1H), 3.5 (m, 1H), 3.15 (dd, 1H, J = 2.4 Hz, 12.0 Hz), 3.11 (d, 1H, J = 12.0 Hz), 2.63 (m, 1H), 1.80 (m, 1H), 1.69 (m, 7H), 1.20 (m, 10H), 0.89 (m, 1H), 0.82 (q, 1H, J = 12.6 Hz). ¹³C NMR (150 MHz, CDCl₃): δ 174.5, 141.9, 127.9 (2C), 127.2, 125.9 (2C), 71.5, 61.5, 60.1, 56.2, 45.0, 37.7, 36.3, 35.8, 33.9, 33.2 (2C), 31.7, 29.3, 26.5, 26.2 (2C). HRMS Calcd for C₂₃H₃₆N₂O₃: [M + H]⁺ 389.2799; found 389.2802.

(2S,4S)-N-((1S,2S)-1,3-Dihydroxy-1-phenylpropan-2-yl)-4-(2-(4-methylcyclohexyl)ethyl)piperidine-2-carboxamide ((2R,4S)-36) and (2S,4R)-N-((1R,2S)-1,3-Dihydroxy-1-phenylpropan-2-yl)-4-(2-(4-methylcyclohexyl)ethyl)piperidine-2-carboxamide ((2S,4R)-37).

Compounds (2R,4S)-36 (40 mg, 41%) and (2S,4R)-37 (42 mg, 43%) were prepared from 33 in 2 steps by a procedure similar to that used to prepare compound 8. These two isomers could be separated by preparative TLC as colorless amorphous gels. Compound 36. ¹H NMR (300 MHz, CDCl₃) δ 7.32 (m, 5H), 4.99 (d, 1H, J = 7.2 Hz), 4.08 (m, 1H), 3.70 (m, 1H), 3.62 (m, 1H), 3.34 (m, 1H), 3.09 (m, 2H), 2.61 (m, 1H), 1.46 (m, 4H), 1.27 (m, 13H), 0.89 (m, 3H), 0.76 (q, 1H, J = 12.6 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 175.6, 142.2, 128.5, 127.8, 126.4, 72.4, 62.4, 60.9, 56.9, 45.8, 38.0, 37.0, 36.5, 35.7, 35.1, 34.7, 33.8, 33.7, 33.3, 32.7, 31.1 (2C), 30.7, 29.2, 29.1, 22.8, 20.4. HRMS Calcd for C₂₄H₃₈N₂O₃: [M + H]⁺ 403.2955; found 403.2955. Compound 37. ¹H NMR (600 MHz, CDCl₃) δ 7.31 (m, 5H), 7.22 (t, 1H, J = 7.2 Hz), 5.03 (d, 1H, J = 3.6 Hz), 4.08 (m, 1H), 3.93 (br s, 3H), 3.78 (m, 1H), 3.72 (m, 1H), 3.13 (dd, 1H, J = 2.4 Hz, 12.0 Hz), 2.99 (d, 1H, J = 10.8 Hz), 2.48 (m, 1H), 1.74 (m, 1H), 1.67 (m, 1H), 1.60 (m, 2H), 1.44 (m, 3H), 1.20 (m, 10H), 0.91 (d, 3H, J = 6.6 Hz), 0.87 (m, 2H), 0.78 (q, 1H, J = 12.6 Hz). ¹³C NMR (150 MHz, CDCl₃): δ 174.0, 141.6, 128.2, 127.4, 125.9, 73.1, 63.1, 60.2, 56.4, 50.5, 45.1, 37.5, 36.4, 35.7, 35.3, 34.6, 34.2, 34.0, 33.3, 32.9, 32.0, 30.8, 30.1, 28.7, 22.6, 20.2. HRMS Calcd for C₂₄H₃₈N₂O₃: [M + H]⁺ 403.2955; found 403.2953.

(2S,4R)-4-Cyclohexyl-N-((1R,2R)-1,3-dihydroxy-1-phenylpropan-2-yl)piperidine-2-carboxamide (38) and (2R,4S)-4-Cyclohexyl-N-((1R,2R)-1,3-dihydroxy-1-phenylpropan-2-yl)piperidine-2-carboxamide (39).

Compounds 38 (70 mg, 40%) and 39 (74 mg, 43%) were prepared from 33 (2 steps) by a procedure similar to that used to prepare compound 8. These two isomers could be separated by preparative TLC as colorless amorphous gel. Compound 38. ¹H NMR (600 MHz, CDCl₃) δ 7.48 (d, 1H, J = 8.4 Hz), 7.38 (d, 2H, J = 7.2 Hz), 7.28 (m, 2H), 7.22 (t, 1H, J = 7.8 Hz), 4.98 (d, 1H, J = 4.8 Hz), 4.30 (br s, 3H), 4.08 (m, 1H), 3.70 (m, 1H), 3.62 (m, 1H), 3.11 (dd, 1H, J = 2.4 Hz, 12.0 Hz), 2.97 (m, 1H), 2.46 (m, 1H), 1.72 (m, 2H), 1.59 (m, 4H), 1.13 (m, 5H), 0.99 (m, 2H), 0.84 (m, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 174.5, 141.7, 128.2 (2C), 127.4, 126.2 (2C), 72.9, 62.7, 61.0, 56.6, 45.5, 42.7, 41.0, 33.3, 29.8, 28.6 (2C), 26.6, 26.5 (2C). HRMS Calcd for C₂₁H₃₂N₂O₃: [M + H]⁺ 361.2486; found 361.2488. Compound 39. ¹H NMR (600 MHz, CDCl₃+CD₃OD) δ 7.48 (s, 1H), 7.37 (d, 2H, J = 7.2 Hz), 7.30 (m, 2H), 7.23 (t, 1H, J = 7.2 Hz), 4.99 (d, 1H, J = 3.6 Hz), 4.10 (m, 1H), 3.72 (m, 1H), 3.62 (m, 1H), 3.13 (m, 2H), 2.58 (dt, 1H, J = 3.0 Hz, 12.6 Hz), 1.75 (m, 3H), 1.66 (m, 4H), 1.15 (m, 6H), 0.93 (m, 2H), 0.87 (q, 1H, J = 12.0 Hz). ¹³C NMR (150 MHz, CDCl₃): δ 173.9, 141.7, 128.0 (2C), 127.2, 125.8 (2C), 71.9, 62.0, 60.0, 56.3, 45.1, 42.7, 41.0, 33.5, 29.8, 29.7, 28.7, 26.5, 26.4 (2C). HRMS Calcd for C₂₁H₃₂N₂O₃: [M + H]⁺ 361.2486; found 361.2487.

In Vitro Pharmacology.

Intracellular Calcium (Ca_i²⁺) Release Assay in Live h5-HT₂R-CHO Cells.

Chinese hamster ovary (CHO) cells stably transfected with human unedited (INI) h5-HT_2CR (h5-HT_2CR-CHO cells) or the human 5-HT_2AR (h5-HT_2AR-CHO cells) were a generous gift of Drs. Kelly A. Berg and William P. Clarke (University of Texas Health Science Center, San Antonio). Cells were grown at 37 °C, 5% CO₂, and 85% relative humidity environment in GlutaMax-MEM medium (Invitrogen, Carlsbad CA) containing 5% fetal bovine serum (Atlanta Biologicals, Atlanta GA) and 100 μg/mL hygromycin (Mediatech, Manassas VA) and were passaged when they reached 80% confluence. The Ca_i²⁺ release assay was performed according to our recent publications.^9,28,70 Briefly, cells (150 μL; passages 6–16) were plated in serum-replete medium at a density of 14 000–16 000 (FlexStation 3; Molecular Devices) or 30 000 cells/ well (FLIPR^TETRA; Molecular Devices) in black-wall 96-well culture plates with optically clear flat bottoms. To ensure even plating of cells, the source reservoir was frequently agitated or triturated, and plates were maintained on a rotary shaker at low speed for 20 min after plating and returned to the incubator overnight. Approximately 24 h following plating, the medium was replaced with serum-free (SF) GlutaMax-MEM medium supplemented with 20 nM to 100 μM putrescine (Sigma-Aldrich, St. Louis, MO), 20 nM to 100 μM progesterone (Sigma-Aldrich), and 1:100 ITS (1000 mg/L human recombinant insulin, 550 mg/L human recombinant transferrin, 0.67 mg/L selenious acid; Corning Inc., Corning, NY) (SF+ medium). Following a 3 h incubation, SF+ medium was replaced with 40 μL of Hank’s balanced saline solution (HBSS; without CaCl₂ or MgCl₂, pH 7.4) plus 40 μL of Calcium 4 dye solution (FLIPR No-wash kit, Molecular Devices, Sunnyvale, CA, part no. R8142) supplemented with 2.5 mM of water-soluble probenicid (Sigma) to inhibit extracellular transport of the dye. Plates were incubated for 60 min at 37 °C followed by 15 min at room temperature in the dark. Drug dilutions were prepared at 5× final concentration in 1× HBSS; delivery of compound (20 μL/well) was followed 15 min later by 5-HT (10 pM to 10 μM; 25 μL/well). A baseline was established for each well before addition of the test compound and again before addition of 5-HT. The fluorescence read after compound addition was used to establish potential intrinsic agonist activity of the compounds. The fluorescence read following the addition of 5-HT was used to assess allosteric modulation of 5-HT-evoked Ca_i²⁺ release. Fluorescence was measured using a FlexStation 3 (Molecular Devices) or FLIPR^TETRA (130 gain, 60% intensity, 0.3 s exposure). For the FlexStation 3, a 17 s baseline was established before addition of compounds following which fluorescence was recorded every 1.7 s for 240 s. Maximum peak height was determined by the SoftMax software (Pro 5.4.5) for each well. For the FLIPR^TETRA, a 10 s baseline was established before addition of compounds following which fluorescence was recorded every 1 s for 120 s following compound or for 360 s following 5-HT. Maximum peak height was determined by ScreenWorks 4.0 software for each well. After the final readings, cells were fixed in 2% paraformaldehyde (Sigma) overnight. The maximum 5-HT-induced Ca_i²⁺ release (E_max) in the presence of test compound was determined using 4-parameter nonlinear regression analysis (GraphPad Prism 7.04) and calculated from at least three biological replicates, each conducted in technical triplicates. The E_max for the test compound plus 5-HT was normalized to the E_max for 5-HT alone. Subsequent post hoc comparisons between means for E_max were made using an unpaired t test with Welch’s correction (GraphPad Prism). All statistical analyses were conducted with an experiment-wise error rate of α = 0.05.

In Vivo Pharmacokinetics and Behavior.

Pharmacokinetic Analysis.

Male Sprague–Dawley rats were randomly assigned into control and treatment groups (n = 3/group). Rats in the control group were injected with vehicle [DMSO/Tween 80/saline (v/v 1:3:6)], and rats in the treatment groups were administered compound 16 at 5 mg/ kg iv or 10 mg/kg po. Blood samples (0.3 mL) were collected from the retro-orbital sinus vein before dosing and at 5 min and 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.0, 9.0, and 24 h postdosing for iv administration and 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.0, 9.0, and 24 h postdosing for po administration. Blood samples were placed in heparinized tubes and immediately centrifuged at 12 000g for 5 min at 4 °C. All samples were stored at −20 °C until analysis.

The pharmacokinetic parameters of compound 16 were calculated according to a noncompartmental model using WinNonlin (Pharsight Corporation, ver 5.3, Mountain View, CA, USA). The peak concentration (C_max) and time of peak concentration (T_max) were directly obtained by visual inspection of the plasma concentration– time profile. The elimination rate constant (λ) was obtained by the least-squares fitted terminal log–linear portion of the slope of the plasma concentration–time profile. The elimination half-life (T_1/2) was evaluated according to 0.693/λ. The area under the plasma concentration–time curve from 0 to time t (AUC_0–t) was evaluated using the linear trapezoidal rule and further extrapolated to infinity (AUC_0–inf) according to the following equation: AUC_0–inf = AUC_0–t + C_last/λ. The pharmacokinetic parameters were presented as mean ± SD.

Effects of Compound 16 on Spontaneous Locomotor Activity. Animals.

A total of 67 male Sprague–Dawley rats (Harlan, Inc., Indianapolis, IN) weighing 225–325 g at the start of the experiments were used. Rats were housed two per cage and allowed to acclimate for 5–7 days in a colony room at a constant temperature (21–23 °C) and humidity (45–50%) on a 12 h light–dark cycle (lights on 0700–1900 h). Food and water were available ad libitum. Rats were handled for 1 week prior to the start of behavioral testing. All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (2011) and with the approval of the UTMB Institutional Animal Care and Use Committee. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques, when available.

Drugs.

WAY163909 [(7b-R,10a-R)-1,2,3,4,8,9,10,10a-octahydro-7bH-cyclopenta[b][1,4]diazepino[6,7,1hi]indole] was a gift from Pfizer, Inc. (New York, NY) and dissolved in 0.9% NaCl (vehicle employed for comparison to WAY163909). SB242084 [6-chloro-5-methyl-1-[[2-(2-methylpyrid-3-yloxy)pyrid-5-yl]carbamoyl]indoline dihydrochloride; Sigma Chemical Co., St. Louis, MO, USA] was dissolved in saline containing 10 mmol/L citric acid (Sigma Chemical Co.) and 8% 2-hydroxypropyl-β-cyclodextrin (Trappsol Hydroxpropyl Beta Cyclodextrin, pharmaceutical grade, Cyclodextrin Technologies Development Inc., High Springs, FL, USA) with the final pH of the solution adjusted to 5.6. SB242084, WAY163909, and compound 16 were injected intraperitoneally (ip) at a volume of 1 mL/kg.

Locomotor Activity Assessments.

Locomotor activity was monitored and quantified under low light conditions using a modified open field activity system (San Diego Instruments, San Diego, CA) according to previous publications with minor modifications.⁴⁷ Clear plexiglass chambers (40 × 40 × 40 cm³) were surrounded by a 4 × 4 photobeam matrix positioned 4 cm from the chamber floor. Total ambulations were quantified as the sum of consecutive photobeam breaks that occurred within the inner central 16 × 16 cm² perimeter and in the surrounding outer peripheral 16 × 16 cm² perimeter of the activity monitor. Vertical activity counts (rearing) were quantified as the sum of breaks in the upper row of 16 photobeams positioned 16 cm from the monitor floor.

1. A between-subjects design was employed to study the efficacy of compound 16 to impact spontaneous motor activity. Rats (n = 29) were administered vehicle (saline, 1 mL/kg, ip) or compound 16 (0.5, 1, or 5 mg/kg, ip) immediately prior to placement in activity monitors on the test day; ambulations and vertical activity counts were monitored in 5 min bins for 90 min.

2. A between-subjects design was used to investigate the selectivity of compound 16 to allosterically modulate WAY163909-mediated suppression of spontaneous motor activity. Rats (n = 38) were pretreated with SB242084 (1 mg/kg, ip) or vehicle (8% 2-hydroxypropyl-β-cyclodextrin) 15 min prior to ip administration of WAY163909 (1 mg/kg) plus compound 16 (5 mg/kg) and immediately placed into activity monitors on the test day; ambulations and vertical activity counts were monitored in 5 min bins for 90 min.

Statistical Analyses.

Locomotor activity data are presented as mean total ambulations (±SEM) summed in 30 min. The main effect of treatment on total ambulations or vertical activity was analyzed with a one-way analysis of variance using the GLM procedure (SAS version 9.4 for Windows). Subsequent a priori comparisons to vehicle were made with Dunnett’s procedure (experiment i) or a Bonferroni multiple comparisons test (experiment ii). All statistical analyses were conducted with an experiment-wise error rate of α = 0.05.

Effects of Compound 16 in a WAY163909 versus Saline Drug Discrimination Assay. Animals.

Male Sprague–Dawley rats (n = 9; Harlan, Inc.) weighing 300–325 g at the beginning of the experiment were housed two per cage in a temperature-(21–23 °C) and humidity-controlled (45–50%) environment; lighting was maintained under a 12 h light–dark cycle (0700–1900 h). Rats were maintained at 80–90% of their free-feeding weights by restricting access to water. Rats received water during daily training sessions (5–6 mL/rat/session), in the afternoon several hours after training (20 min), and over the weekend (36 h). Experiments were conducted during the light phase of the light–dark cycle (between 0900 and 1200 h) and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with the approval of the Institutional Animal Care and Use Committee at University of Texas Medical Branch.

Drug Discrimination Procedures.

Standard two-lever, water-reinforced drug discrimination procedures were used (Med Associates, St. Albans, USA). Each chamber was equipped with a water-filled dispenser mounted equidistantly between two retractable response levers on the wall and housed in a light-and sound-proof cubicle. Illumination came from a 28-V house light; ventilation and masking noise were provided by a ventilation fan. A computer with Med-PC IV software was used to run programs and record all experimental events.

Rats were trained to discriminate an injection of WAY163909 (0.75 mg/kg; 1.0 mL/kg, ip) from saline (1.0 mL/kg, ip) administered 15 min before start of training sessions (see Supporting Information for WAY163909 time course). Daily sessions lasted 15 min and were conducted Monday through Friday. During the phase of errorless training, only the stimulus-appropriate (drug or saline) lever was present. Training began under a fixed ratio 1 (FR1) schedule of water reinforcement, and the FR requirement was incremented until all animals were responding reliably under an FR20 schedule for each experimental condition. For half of the rats, left lever responses were reinforced after WAY163909 administration, whereas right lever responses were reinforced after saline administration; conditions were reversed for the remaining animals. During this phase of training, WAY163909 and saline were administered irregularly with the restriction that neither condition prevailed for more than three consecutive sessions. After responding stabilized, both levers were presented simultaneously during 15 min training sessions. The rats were required to respond on the stimulus-appropriate (correct) lever to obtain water reinforcement. There were no programmed consequences for responding on the incorrect lever. This phase of training continued until the performance of all rats attained criterion (defined as mean accuracies of at least 80% stimulus-appropriate responding for ten consecutive sessions).

Test sessions were initiated and conducted once or twice per week following attainment of criterion. Training sessions were run during the intervening days to maintain discrimination accuracy. Rats were required to maintain accuracies of at least 80% correct for saline and WAY163909 maintenance sessions, which immediately preceded a test. During test sessions, animals were placed in the chambers and, upon completion of 20 responses on either lever, a single reinforcer was delivered and the house lights were turned off. The rat was removed from the chamber, returned to the colony, and allowed free access to water for 15 min beginning 15–30 min after the end of each test. The test sessions were terminated after 15 min if the rats did not complete 20 responses on either lever; only data from rats that accomplished the FR20 during test sessions within 15 min were employed in data analysis.

Time course, substitution and combination tests were conducted. To determine the time course of WAY163909, rats were pretreated with WAY163909 (0.75 mg/kg, ip) 15, 30, 60, or 120 min prior to placement into the chambers. In substitution tests, rats were administered WAY163909 (0.125, 0.25, 0.5, 0.75, or 1.0 mg/kg, ip), compound 16 (0.125, 0.25, 0.5, 1.0 mg/kg, ip), or saline 15 min prior to test. In combination tests, rats were tested for lever selection following simultaneous ip administration of compound 16 (0.5 mg/ kg) and WAY163909 (0.5 mg/kg) and placed in the operant chamber 15 min later. The doses of WAY163909 and compound 16 employed were based upon the results of the substitution tests as producing <50% drug-appropriate responding. Full substitution was defined as ≥80% drug-appropriate responding and not statistically different from the training drug, and partial substitution as ≥40% and <80% drug-appropriate responding.

Statistical Analyses.

Accuracy was defined as the percentage of correct responses to total responses before the delivery of the first reinforcer. During test sessions, performance was expressed as the percentage of drug-lever responses to total responses upon completion of an FR20 on either lever. The response rate (responses per minute) was calculated as the total number of responses emitted before completion of the first FR20 divided by the number of minutes taken to complete the first ratio. For compound 16 dose–response tests, Student’s t-test for repeated measures was used to compare the percentage of drug-lever responding and response rate during test sessions with the corresponding values for the previous saline session (substitution tests). A two-way ANOVA for repeated measures was employed to analyze the effects of pretreatment with compound 16 (factor 1) and WAY163909 dose (factor 2; SAS for Windows, version 9.4). A priori comparisons were calculated to compare the effects of each dose of WAY163909 in the presence versus absence of compound 16 using a Bonferonni-corrected test. Log-probit analyses were used to estimate the dose of WAY163909 predicted to elicit 50% WAY163909-appropriate responding (ED₅₀).⁴⁸ Only data from animals that completed the FR20 during the test sessions were used. All statistical analyses were conducted with an experiment-wise error rate of α = 0.05.

Effects of Compound 16 on Cocaine Cue Reactivity. Animals.

Male, outbred Sprague–Dawley rats (n = 17; Harlan, Houston, TX) weighing 250–275 g upon arrival were housed two per cage under a 12-h light–dark cycle with controlled temperature (21–23 °C) and humidity (40–50%). Animals were acclimated for 7 days to the colony room prior to the start of handling and experimental procedures. All experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (2011) and with the University of Texas Medical Branch Institutional Animal Care and Use Committee approval.

Drugs.

(–)-Cocaine (National Institute on Drug Abuse, Research Triangle Park, NC) was dissolved in 0.9% NaCl.

Cocaine Self-Administration and Cue Reactivity Analyses.

Cocaine self-administration studies employed standard operant conditioning chambers (Med-Associates, Inc., St. Albans, VT, USA) housed in ventilated, sound-attenuating cubicles with fans (Med-Associates, Inc.). Each chamber was outfitted with two retractable response levers, a stimulus light above each response lever, a houselight opposite the levers, and a magazine-type pellet dispenser. The cocaine infusions were delivered via syringes attached to infusion pumps (Med Associates, Inc.) located outside the cubicles. The infusion pumps were connected to liquid swivels (Instech, Plymouth Meeting, PA, USA) that were fastened to the catheters via polyethylene 20 tubing encased inside a metal spring leash (Plastics One, Roanoke, VA).

Rats were anesthetized (8.6 mg/kg of xylazine, 1.5 mg/kg of acepromazine, 43 mg/kg of ketamine in bacteriostatic saline) and implanted with intravenous catheters with back mounts and allowed to recover for 5–7 days.^6,8,9,47 Catheter patency was maintained by daily flushes with a solution of 0.1 mL of bacteriostatic saline containing heparin sodium (10 U/mL; American Pharmaceutical Partners, East Schaumburg, IL), streptokinase (0.67 mg/mL; Sigma Chemical), and ticarcillin disodium (66.67 mg/mL; Research Products International, Mt. Prospect, IL) immediately following daily cocaine self-administration sessions.

Cocaine self-administration training consisted of 14 daily 180 min sessions during which rats were trained to lever press for cocaine infusions (0.75 mg/kg per 0.1 mL infusion).^6,8,9,47 Schedule completions on the active lever resulted in delivery of a cocaine infusion over a 6 s period paired simultaneously with illumination of the house and stimulus lights and activation of the infusion pump (discrete cue complex paired with cocaine delivery); inactive lever presses produced no scheduled consequences. Following reinforcer delivery, the stimulus light as well as the infusion pump were inactivated; the house light remained on for an additional 20 s to indicate a timeout period during which lever presses had no scheduled consequences. Rats were trained on a FR1 schedule of reinforcement and progressed to an FR5 schedule after achieving seven infusions per hour with less than 10% variability for three consecutive days. Upon achieving stability on the FR5 schedule (less than 10% variability for a minimum of three consecutive days), rats were pseudorandomly assigned to either vehicle or compound 16 and returned to their home cages for the appropriate time period. Rats that exhibited diminished catheter patency were excluded from analysis (n = 3).

Rats were reintroduced to the self-administration chambers 24 h after the last self-administration session and assayed in a test session comprised of two sequential components. In the first component, responses on either lever were recorded, but no discrete cues (e.g., stimulus light, pump) were present nor delivered during the 10 min period. The second component was signaled by the single delivery of the discrete cue complex presented at the termination of the first 10 min component. To assess cocaine cue reactivity, presses on the previously active lever in the 60 min second component were reinforced by the discrete cue complex on an FR1; inactive lever presses were recorded but produced no scheduled consequences.⁶ Rats received an injection of vehicle (0.9% NaCl, 1 mL/kg, ip) or compound 16 (1.0 mg/kg, ip) 15 min prior to the cue reactivity session.

Statistical Analyses.

The data from the self-administration and cue reactivity phases were analyzed separately. For the cue reactivity tests, a two-way ANOVA for repeated measures was employed to analyze the effects of treatment with compound 16 (factor 1) and time (factor 2; SAS for Windows, version 9.4) on previously active or inactive lever presses. A priori comparisons were calculated to compare the effects of compound 16 at each time point using Fishers LSD test. All statistical analyses were conducted with an experiment-wise error rate of a α = 0.05.

ABBREVIATIONS USED

5-HT

serotonin

5-HT_2AR

5-HT_2A receptor

5-HT_2BR

5-HT_2B receptor

5-HT_2CR

5-HT_2C receptor

CNS

central nervous system

GPCR

G protein-coupled receptor

FDA

U.S. Food and Drug Administration

PAM

positive allosteric modulator

NAM

negative allosteric modulator

SAR

structure–activity relationship

tPSA

total polar surface area

HBTU

N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate

DIPEA

N,N-diisopropylethylamine

TFA

trifluoroacetic acid

PTLC

preparative thin layer chromatographic

PLCβ

phospholipase Cβ

IP3

inositol-1,4,5-trisphosphate

DAG

diacylglycerol

CHO

Chinese hamster ovary

CCR5

C–C chemokine receptor type 5

PLD

phospholipase D

PK

pharmacokinetics

PD

pharmacodynamics

ADMET

absorption, distribution, metabolism, excretion, and toxicity

EL

extracellular loop

TMH

transmembrane helix

MPO

multi-parameter optimization

TMS

tetramethylsilane

DMF

dimethylformamide