Fentanyl-Type Antagonist of the μ-Opioid Receptor: Important Role of Axial Chirality in the Active Conformation

Abstract

In recent years, synthetic opioids have emerged as a predominant cause of drug-overdose-related fatalities, causing the “opioid crisis.” To design safer therapeutic agents, we accidentally discovered μ-opioid receptor (MOR) antagonists based on fentanyl with a relatively uncomplicated chemical composition that potentiates structural modifications. Here, we showed the development of novel atropisomeric fentanyl analogues that exhibit more potent antagonistic activity against MOR than naloxone, a morphinan MOR antagonist. Derivatives displaying stable axial chirality were synthesized based on the amide structure of fentanyl. The aS- and aR-enantiomers exerted antagonistic and agonistic effects on the MOR, respectively, and each atropisomer interacted with the MOR by assuming a distinct binding mode through molecular docking. These findings suggest that introducing atropisomerism into fentanyl may serve as a key feature in the molecular design of future MOR antagonists to help mitigate the opioid crisis.

Introduction

Opioid analgesics are conventionally administered in clinical practice. Apart from their medical applications, opioids are frequently misused for their euphoric effects. Substance abuse among young individuals is rapidly escalating and presenting a global cause of concern. According to the Centers for Disease Control and Prevention, over 106,000 people in the USA succumbed to drug overdose in 2021, with 75.4% of the annual overdose-related fatalities attributable to opioids.¹ Currently, detoxification and maintenance therapy are the two most prevalent approaches used to treat opioid-use disorders.² Naloxone, the μ-opioid receptor (MOR) antagonist approved by the Food and Drug Administration, has demonstrated the ability to efficaciously manage opioid misuse and overdose and mitigate relapse. However, naloxone has certain drawbacks, including a brief duration of action³ and weak affinity for δ- and κ-opioid receptors.⁴ To facilitate the provision of alternative therapeutics, it is necessary to develop MOR antagonists with more favorable profiles. To date, numerous antagonists possessing morphinan skeletons have been extensively investigated;^3,5−7 however, little compound superior to naloxone has been obtained. We anticipated that further studies based on the morphinan skeleton, which has already been thoroughly investigated, would not deliver improved results. Therefore, the development of a novel framework that facilitates the assessment of the structure–activity relationships (SARs) concerning antagonistic activity against the MOR will likely prove more fruitful. 4-Anilidopiperidines, μ agonists, have a prominent place due to their high potency, low cardiovascular toxicity, fast onset, and often short duration of action.⁸ Fentanyl, discovered in 1962 by Janssen, is the prototype of the 4-anilidopiperidine class of synthetic opioid analgesics.⁹ It is a full agonist that binds with high affinity to the MOR. It is about 50–100 times more potent than morphine and is characterized by a rapid onset of analgesia and relatively short duration of action.^10,11 However, fentanyl exhibits serious adverse effects, including respiratory depression, muscle rigidity, nausea, sedation, and, with prolonged use, tolerance and addiction.¹⁰ Based on the precedent SAR studies of fentanyl analogues, the potent factors for the analgesic activity are elucidated (Table 1).¹²⁻²⁰

Table 1. Structure and Pharmacological Activity of Fentanyl and Its Derivatives.

^aED₅₀ value according to ref (17).

^bED₅₀ value according to ref (18).

^cED₅₀ value according to ref (19).

As shown in 4-methyl fentanyl and carfentanil, substitution at the 4-position of the piperidine ring should increase the binding affinity by 3.8–30-fold in comparison to fentanyl.^17,18 Additionally, the stereochemistry of the substituents of the piperidine ring plays a crucial role in the potency of these analogues. For example, 3-methylfentanyl, which has two chiral centers, should exist in four stereoisomers. The (+)-cis isomer is 16 times more active than trans isomers and about 100 times more active than its (−)-enantiomer.¹⁹

There is one other thing that is important for the design of potent fentanyl analogues. Recently, we have been interested in axial chirality derived from the Ar−N(CO) axis and its relationship with bioactivities.²¹⁻²⁴ We presumed that fentanyl analogues should exhibit E/Z-isomerism around the N−(C=O) bond (axis 1) and atropisomerism based on the Ar−N(CO) bond (axis 2). Theoretically, they should exist in four stereoisomers as shown in Figure 1. While fentanyl was known to exist predominantly in the E-form, the atropisomeric property has not been investigated.^25,26 We anticipated that the stereoelectronic effect at the 2′ or 6′-positions of the anilino phenyl moiety should restrict the rotation about N−(C=O) and Ar−N(CO) axes to provide atropisomers. We aimed at discussing the relationship between the structure and the MOR interaction in the series of fentanyl analogues substituted in the position 2′ or 6′ of an anilino phenyl ring. This study focused on the stereochemical property of fentanyl analogues to provide atropisomers for the first time. Also, SAR studies and docking studies afford a clue to the future drug design of fentanyl analogues with agonistic/antagonistic activity against MOR. These findings suggest that the introduction of atropisomerism into fentanyl may serve as a key feature in the molecular design of future MOR antagonists to help mitigate the opioid crisis.

Figure 1.

Stereoisomers of fentanyl analogues.

Results and Discussion

Chemistry

Derivatives of fentanyl (1–38) were prepared from 1-(2-phenylethyl)-4-piperidone (39), as shown in Scheme 1. The reductive amination of commercially available compound 39 furnished compounds 40–50, which were successively treated with various acid chlorides to provide fentanyl derivatives 1–38.

Scheme 1. Preparation of Fentanyl Analogues.

Reagents and conditions: (a) AcOH, EtOH, reflux; then, NaBH₃CN, reflux; (b) NaH, DMF, 25 °C.

First, the conformational properties of fentanyl analogues 1–38 were characterized using ¹H NMR spectroscopy (Table 2). Methyl carbamate derivatives 3, and 4, N-alkanoyl derivatives 5–10, 13, 14, 22, 25–30, and 36–38, 4-substituted benzoyl derivatives 2, 11, 24, and 34, and N-heteroaryl derivatives 1, 15, 23, 31–33, and 35 were observed as single isomers at 25 °C irrespective of the substituents of the anilino moiety. However, N-2-substituted-benzoyl derivatives 12 and 16–20, and N-2,6-disubstituted-benzoyl derivative 21 existed as an equilibrium mixture of E/Z-diastereomers in solution (CDCl₃) at the ratio of 89:11–67:33. In each spectrum, the H-4 proton resonance of the piperidine ring in the major diastereomer was located at approximately 4.77–4.27 ppm, 1.22–0.53 ppm downfield from its partner, respectively.²⁷ This downfield shift was ascribed to the anisotropic effect of the carbonyl of the acyl group (Figure 1). Therefore, we presumed that compounds 12 and 16–21 exist in E-amide in preference to Z-amide. Meanwhile, the H-4 proton resonance of the piperidine ring in single isomers 1–11, 13–15, and 22–38 was located at 4.79–4.16 ppm, corresponding to E-amide in 12 and 16–21. Additionally, considering that fentanyl exists in the E-form in solution,^25,26 the conformations of 1–11, 13–15, and 22–38 observed as single isomers were presumed to be E-amide.

Table 2. Physicochemical Properties of Fentanyl Derivatives and Their Activities against the MOR.

^aThe NMR data of fentanyl adopted from ref (26).

To determine the E/Z-amide stereochemistry, an investigation using NOESY spectroscopy was conducted. A correlation between the 6-H of the benzoyl group and 2′-CH₃ of the anilino moiety was observed in the major peak of 17 (see Supporting Information Figures S1 and S2). Therefore, the preference for E-amide was determined. Fortunately, a single crystal for the X-ray crystal structure analysis of compound 34 was obtained, in which 34 possessed the E-form (Figure 2 and see the Supporting Information). Based on these results, the preference for E-amide observed in compounds 1–38 was confirmed. Bulky substituents at the 2-position of the N-benzoyl group increased the ratio of Z-isomers (Table 2). Unfortunately, the separation of E/Z-amide in compounds 12 and 16–21 using nonchiral HPLC failed because the rotational barrier of the N–(C=O) bond (axis 1) was less than that required for the isolation.

Figure 2.

X-ray crystallographic structure of compound 34.

As shown in Figure 1, four stereoisomers [i.e., E/Z-amide rotamers around N–(C=O) bond (axis 1) and (aS)/(aR)-axial isomers based on the Ar−N(CO) bond (axis 2)] were assumed to exist. Although the rotation of axis 1 was too rapid to isolate E/Z-amide, the presence of atropisomers derived from axis 2 was confirmed by isolating the enantiomers of compounds 8, 23, 24, and 30–35via chiral HPLC. The separated atropisomers have opposite [α]_D values from each other. After obtaining the atropisomerically pure isomers, the activation-free energy barrier to rotation (ΔG^⧧) was estimated (Table 2) (see Supporting Information Figures S3–S11). The ΔG^⧧ value of 96 kJ/mol at 37 °C for compound 8, which was stable enough to be isolated, was assumed to be due to the lower bulkiness of the 2′-Me substitute of the anilino moiety. Considering this, it is plausible that the atropisomers of compound 11 with 2′-Me monosubstituted were not isolated via chiral HPLC. Both 2′ and 6′ substitutions in the anilino moiety were necessary to freeze the rotation of the Ar−N(CO) bond (axis 2). The enantiomers of compound 34 at 80 °C with a ΔG^⧧ value of 123 kJ/mol were more stable than those of compound 24 (ΔG^⧧ = 115 kJ/mol at 80 °C). Similarly, the enantiomers of compound 35 with a ΔG^⧧ value of 131 kJ/mol were more stable than those of compound 23 (ΔG^⧧ = 116 kJ/mol at 80 °C) and 31 (ΔG^⧧ = 127 kJ/mol at 80 °C). The increased energy barrier was ascribed to the bulky substituents in the anilino moiety (Cl < Et < i-Pr).

The heteroaryl-substituted series were also investigated. We changed the 2-furoyl of compound 31 to the regioisomer 3-furoyl of compound 32 and the isosteric five-membered heteroaromatic ring 2-thenoyl of compound 33. The enantiomers of 32 and 33 (ΔG^⧧ = 130 and 127 kJ/mol at 100 °C, respectively) were equally as stable as those of compound 31. Meanwhile, the higher stability with a ΔG^⧧ value of approximately >130 kJ/mol was estimated in N-cyclohexanoyl 30 because no racemization was observed at 80 °C for 54 h and decomposition at 100 °C was observed. Considering that the atropisomers of N-butanoyl compound 8 were isolatable with a ΔG^⧧ value of 96 kJ/mol, the N-alkanoyl-substituted compounds are in more sterically restricted form than the N-aroyl-substituted compounds.

SAR Targeting the μ-Opioid Receptor

Fentanyl and its derivatives 1–10, 13, 15, 16, 21–23, 25–33, and 35–38 were evaluated as MOR agonists using opioid-μ-receptor-expressing CHO cells (Table 2, see Supporting Information Figures S12–S15). Fentanyl was used as a control compound. In this case, those with axial chirality were evaluated as racemates. First, the EC₅₀s of the N-alkanoyl- and N-aroyl-substituted series were compared. Among compounds 6–10, the activity was the highest in N-propionyl compound 7 (EC₅₀: 3.40 nM), which is comparable to fentanyl. With the increasing bulkiness of the N-alkanoyl group, the potency decreases considerably. Thus, 8 (EC₅₀: 5.94 nM), 9 (EC₅₀: 14.1 nM), and 10 (EC₅₀: 544 nM) were about 1.7, 4.1, and 160 times less potent than 7, respectively. A similar trend was observed in compounds 26–30. In particular, the marked decrease in the activity of compounds 10 (EC₅₀: 544 nM) and 30 (EC₅₀: 606 nM) indicated that the size of cyclohexane significantly impairs binding to the receptor.

As to N-aroyl-substituted compounds, compound 2 with a 4-nitro substituent at the N-benzoyl group was inactive since the steric and electronic effects of the 4-nitro group at the benzoyl site appear to be a fatal obstacle for the MOR receptor. Considering that compound 16 with a N-2-fluorobenzoyl group (EC₅₀: 121 nM) and compound 21 with a N-2,6-difluorobenzoyl group (EC₅₀: 110 nM) showed low activity, N-benzoyl derivatives seemed unpromising. Therefore, we focused on five-membered heterocycles. While the activity of N-2-pyranoyl-substituted 1 (EC₅₀: 17.6 nM) and N-2-furoyl-substituted 15 (EC₅₀: 9.19 nM) is more potent than that of N-benzoyl derivatives, 2-thenoyl compound 33 showed a marked decrease in potency (EC₅₀: 32,300 nM). Comparing the substitution position of the furanoyl group, a large difference in the potency of 2-furoyl 31 (EC₅₀: 24.5 nM) and 3-furoyl 32 (EC₅₀: 40,000 nM) indicates the importance of the position of oxygen on the furan ring. Comparing the N-propionyl-substituted compounds 7 (EC₅₀: 3.40 nM) and 37 (EC₅₀: 4.2 nM) revealed that the 2′-methyl or 2′-chloro monosubstitute of the anilino moiety produced agonistic activity equal to that of fentanyl. Among the N-propionyl-substituted compounds, in which both ortho-positions of the anilino moiety were substituted, 22 with 2′-chloro,6′-methyl (EC₅₀: 0.2 nM), 36 with 2′-fluoro,6′-methyl (EC₅₀: 0.2 nM), 38 with 2′-fluoro,6′-chloro (EC₅₀: 1.4 nM) showed excellent agonistic activity. In contrast, a decreasing trend in potency was observed in 13 with 2′,6′-dimethyl (EC₅₀: 4.5 nM) and 26 with 2′-ethyl,6′-methyl (EC₅₀: 35 nM). Based on this, a halogen substituent at either ortho-position enhanced the agonistic activity. Comparing the N-2-furoyl-substituted compounds 15 with 2′,6′-dimethyl (EC₅₀: 9.19 nM), 23 with 2′-chloro,6′-methyl (EC₅₀: 5.86 nM), 31 with 2′-ethyl,6′-methyl (EC₅₀: 24.5 nM), and 35 with 2′-isopropyl,6′-methyl (EC₅₀: 49.0 nM), increasing the bulkiness at the ortho-position of the anilino moiety (Me < Et < i-Pr) decreases potency.

Next, the separated enantiomers of 8, 23, 31–33, and 35 were subjected to in vitro assays to examine the difference in potency between the enantiomers (Table 2, see Supporting Information Figures S12–S15). The separated enantiomer (−)-31 exhibited a slight increase in agonistic activity (EC₅₀: 14.0 nM) than its racemate (EC₅₀: 24.5 nM), whereas (+)-31 showed no agonistic activity (Table 2). Therefore, we examined the antagonist activity of (+)-31 against the MOR and observed antagonist activity with an IC₅₀ value of 32.5 nM, 5-fold more potent than naloxone (IC₅₀: 165 nM), which is a control compound as an antagonist for MOR (Table 2, see Supporting Information Figure S16). The distinct way each enantiomer of compound 31 interacted with the target molecule was unusual. The in vitro binding affinities of compound 31 enantiomers to the MOR were examined via a competitive radioligand binding assay to determine whether the agonistic or antagonistic activity could be attributed to the binding of the agonist and antagonist, respectively, to the orthosteric site of the MOR (see Supporting Information Table S1 and Figure S17). Both (+)-31 and (−)-31 exhibited remarkable affinity in the binding experiment at the nanomolar level for the MOR (K_i = 1.86 and 4.96 nM, respectively). They were more potent than [d-Ala2,NMe-Phe4-Gly5-ol]-enkephalin (DAMGO), which is a full agonist at MOR (K_i = 16.5 nM). We confirmed that the agonist/antagonist potency of each atropisomer of 31 was due to the binding affinity for the orthosteric site of the MOR. Therefore, we readdressed the calcium flux assay conducted using MOR-CHO cells (Table 2). The bioactivity of 35 was investigated in which an isopropyl group, instead of an ethyl group, was substituted at the 6′-position of the anilino moiety. (+)-35 did not exhibit agonist activity as strongly as (+)-31. Therefore, we examined the MOR antagonist activity of (+)-35, which exhibited low antagonistic activity (IC₅₀: 2610 nM). We postulated that substitution with the 6′-isopropyl group, which is bulkier than the ethyl group in (+)-31, might block the binding site, decreasing the antagonist’s activity. In contrast to the antagonist effect, (−)-35 exhibited more potent agonist activity (EC₅₀: 6.72 nM) than (−)-31 (EC₅₀: 14.0 nM). Fentanyl derivatives differ in their binding mode to MOR depending on their steric structure, resulting in exactly the opposite activity. Interestingly, compound 23, in which a chloro atom, instead of an ethyl group, was substituted at the 6′-position, showed agonist activity in the racemate form, with both atropisomers exhibiting agonistic activity. The electronic properties of the 6′-chloro substituent might have altered the binding configuration of 23 with the MOR, so the antagonistic activity was lost. In addition, in compound 8, racemic and enantiomeric forms showed comparable agonist activity because atropisomers with 2′-methyl monosubstitution were unstable (ΔG^⧧ = 96 kJ/mol).

Changes in the N-acyl substitution were also investigated. Maintaining the two ortho-position substitutions with ethyl and methyl groups, the 3-furoylated compound 32 and 2-thenoylated compound 33 were examined. While the (−)-atropisomers of these compounds showed weak MOR agonistic activity [(−)-32: EC₅₀ = 1550 nM, (−)-33: EC₅₀ = 12,300 nM], the (+)-enantiomers of 32 and 33 exhibited antagonistic activity, albeit at a low level [(+)-32: IC₅₀ = 1590 nM, (+)-33: IC₅₀ = 3030 nM]. Although the activity of compounds (+)-31, (+)-32, (+)-33, (+)-35 is weak, it is apparent that no agonistic activity was observed (Figures S14 and S15). These results revealed that the N-2-furoyl and alkyl substitutions at the 2′ and 6′ positions of the anilino moiety are important for eliciting antagonistic activity. The methyl and ethyl groups at the ortho-positions on the anilino moiety are the optimal combination to achieve MOR antagonistic activity. Intriguingly, the MOR distinguishes the difference of only one carbon between the methyl and ethyl groups at the ortho-position in each atropisomer and exhibits different responses (agonistic/antagonistic) accordingly.

Murine models were used for the subsequent ethopharmacological analysis of (+)-31 and naloxone. A prior in vitro assay established (+)-31 as a potent MOR antagonist. Morphine injection triggered spontaneous locomotor activity in mice that peaked at 30 min, and the intraperitoneal administration of (+)-31 antagonized the effect of morphine for 120 min as with naloxone (Figure 3A). This antagonistic effect of (+)-31 was comparable to that of naloxone (Figure 3B).

Figure 3.

Ethopharmacological analyses of (+)-31 and naloxone (NLX). (A) Time-dependent alterations after acute administration of morphine (MRP; 10 mg/kg; i.p.) and pretreatment with the opioid receptor antagonist NLX (2 mg/kg; i.p.; pre 30 min) or (+)-31 (2 mg/kg, i.p.; pre 30 min) in MRP-induced hyperlocomotion in mice. Each point represents the mean activity counts with standard error of the mean (SEM) for 10 min (n = 12). (B) Each column represents the mean total locomotor activity counts with the SEM for 120 min (n = 12). Dunnett’s post-test was also applied to each graph.

Determination of Absolute Configuration

Although optically resolved, 23, 31–33, and 35 did not provide single crystals suitable for X-ray diffraction analysis; (+)-31 exhibited characteristic positive Cotton effects at 230 and 265 nm with similar intensities. Compounds (+)-32, (+)-33, and (+)-35 showed similar electronic circular dichroism (ECD) spectral profiles (Figure 4), suggesting that the chirality at the Ar−N(CO) bond (axis 2) correlates with these Cotton effects. Considering the computation costs, we performed the ECD spectral calculations using time-dependent density-functional theory (DFT).²⁸ These spectral calculations were performed on the simplified model compound 35′, in which the phenylethyl group of 35 was replaced with a methyl group. The CAM-B3LYP/def2-TZVP level of theory²⁹ was selected based on our previous data.^30,31 The ECD spectra were constructed considering the Boltzmann distributions calculated using the free energies based on the same calculation conditions as above. The DFT calculations of the aR enantiomer of 35′ (Figure 4F) reproduced the experimental ECD spectra of 35 well (see Supporting Information Tables S2–S5). Therefore, the absolute configurations of (+)-35/(−)-35 were determined to be aS/aR. Accordingly, (+)-31/(−)-31 were also determined to be aS/aR. Similarly, we determined the absolute configurations of the (+)- and (−)-enantiomers of 32 and 33 and concluded that the enantiomers displaying MOR antagonistic activities were in the aS form.

Figure 4.

Comparison of calculated and experimental ECD. The calculated ECD values of the aR and aS isomers are similar to the experimental ECD values of the (−)- and (+)-enantiomers, respectively. (A) Experimental ECD of 23: (+)-23 (red) and (−)-23 (blue). (B) Experimental ECD of 31: (+)-31 (red) and (−)-31 (blue). (C) Experimental ECD of 32: (+)-32 (red) and (−)-32 (blue). (D) Experimental ECD of 33: (+)-33 (red) and (−)-33 (blue). (E) Experimental ECD of 35: (+)-35 (red) and (−)-35 (blue). (F) Calculated TD-DFT-based ECD of 35′: aS-35′ (red) and aR-35′ (blue).

Docking Studies and Molecular Dynamics Simulations

To obtain structural insights into the enantiomers of aR/aS-31 and MOR interactions, we conducted molecular docking studies and molecular dynamics (MD) simulations of the complexes of each of the 31 enantiomers and the MOR (Protein Data Bank [PDB]: 5C1M).³² Recently, Zhuang et al. obtained a cocrystal with fentanyl and MOR (PDB: 8EF5)³³ and reported that fentanyl binds to the same orthosteric site of MOR as morphine does. Therefore, each aR-31 and aS-31 was added to replace fentanyl at the same orthosteric site. The predicted binding positions of the 31 enantiomers and the reported binding conformation of fentanyl were superimposed, and the three binding positions were compared (Figure 5A, see Supporting Information Figures S18 and S19 and Tables S6 and S7). These enantiomers existed as E-conformers similar to fentanyl; however, each enantiomer interacted with the MOR amino acid residues in a distinct manner. The phenylethyl chain of the agonist aR-31 was located between transmembrane (TM)2 and TM3, consistent with the binding position of fentanyl. This confirmed that the interaction of aR-31 with the MOR active site was similar to that of fentanyl.

Figure 5.

Superposition of the predicted aR-31 (blue) and aS-31 (green) binding configurations in the MOR (PDB: 5C1M) and that of fentanyl (red) in the MOR (PDB: 8EF5).

Conversely, aS-31 also bound to the orthosteric site; nonetheless, the phenylethyl group extended in a different direction, between TM2 and TM7, toward TM1. Considering that the distinction between the two ortho-substituents (one carbon difference between the methyl and ethyl groups) causing axial chirality in compound 31 is minute, it is intriguing that the orientation of the phenylethyl group that interacts with MOR differs markedly. To identify the occurrence of such striking differences in the MOR interactions, we investigated the extent to which each enantiomer interacts with the amino acid residues of the MOR via MD pharmacophore analysis. Like fentanyl, both enantiomers exhibited strong ionic interactions with Asp147. In the case of aR, an ortho-methyl group interacted with Tyr148 (100%) and Met151 (95%), and an ortho-ethyl group interacted hydrophobically with Ile296 (100%), Ile322 (99%), and Trp293 (99%). However, in the case of aS, the ortho-methyl group interacted hydrophobically with Ile296 (90%) and Ile322 (94%), and the ortho-ethyl group interacted with Tyr148 (96%), Val236 (92%), and Met151 (86%). On comparing the interactions between the ethyl groups in each enantiomer and the amino acid residues of the MOR, Trp293 (99%) of aR and Val236 (92%) of aS did not interact with the methyl group of the paired enantiomer. This implies that the different interactions with these amino acid residues are key for mediating the agonistic and antagonistic effects.

Conclusions

We successfully created atropisomers of fentanyl analogues by introducing substituents at the 2′ or 6′ position of the anilino moiety and separated the enantiomeric forms. Examination of the affinity at the MOR revealed that one of the atropisomers, the aR-form, of the fentanyl analogues serves as an agonist and the other, the aS-form, as an MOR antagonist. Notably, the atropisomers differ by a single carbon (methyl and ethyl). Docking studies of 31 also revealed that the binding poses of the aR and aS forms occupied the orthosteric sites. Each atropisomer assumes a distinct conformation and interaction pattern owing to its unique chemical structure. This pioneering discovery was achieved by introducing atropisomerism into the fentanyl framework. Although atropisomeric properties are often overlooked, many drugs exhibit latent chirality. Hence, collectively, this study provides a novel perspective and valuable insights for developing pharmaceuticals.

Experimental Section

General Remarks

Experimental materials were procured from commercial suppliers. The nuclear magnetic resonance (NMR) spectra were recorded on a spectrometer (JEOL Ltd., Tokyo, Japan, and Bruker, Billerica, MA, USA) operating at 600 MHz for ¹H NMR and 150 MHz for ¹³C NMR. Chemical shifts are expressed in ppm relative to tetramethylsilane as an internal standard and coupling constants (J) are reported in hertz. The abbreviated representations for the splitting patterns are as follows: singlet (s), doublet (d), triplet (t), quartet (q), quintet (quin), multiplet (m), and broad (br). Infrared (IR) spectra were acquired using a Fourier-transform IR spectrometer equipped with an attenuated total reflectance accessory (ATR; diamond). The high-resolution mass spectra were obtained using the electrospray ionization mode. Melting points were recorded on a melting point apparatus and retained as is. Optical rotations were determined using a digital polarimeter. Analytical thin-layer and column chromatography was performed on precoated, glass-backed silica gel plates (Merck silica gel 60 F₂₅₄: Merck KGaA, Darmstadt, Germany) and silica gel (60 μm), respectively. Extracted solutions were dried over anhydrous Na₂SO₄. Solvents were evaporated under reduced pressure. All compounds are >95% pure by HPLC.

N-Phenyl-1-(2-phenylethyl)-4-piperidinamine (40)³⁴

Compound 40 was prepared according to a modified literature procedure.³⁴ Acetic acid (686 μL, 12 mmol) and aniline (1.82 mL, 20 mmol) were added to a stirred solution of 1-(2-phenylethyl)-4-piperidone (2.03 g, 10 mmol) in ethanol (50 mL). The mixture was stirred under reflux for 2.5 h and cooled to 25 °C, followed by the careful, slow addition of sodium cyanoborohydride (1.26 g, 20 mmol). After stirring under reflux for 5 h, the mixture was treated with 2 N NaOH and concentrated. The concentrate was extracted with ethyl acetate. The extract was washed with 2 N NaOH and brine, dried over Na₂SO₄, and concentrated. The crude product was purified via column chromatography (methanol/dichloromethane = 1:15) to produce 40 as a white solid; yield: 1.81 g (6.45 mmol, 65%). Spectral data were consistent with the results reported in the literature.³⁴

Compounds 41–50 were prepared from the corresponding 1-(2-phenylethyl)-4-piperidone (39) according to a procedure similar to that for preparing 40 from 39.

N-(4-Methoxyphenyl)-1-(2-phenylethyl)-4-piperidinamine (41)

White solid; yield: 275.9 mg (89%). MP: 97 °C. IR (ATR): 3384 cm^–1 (NH). ¹H NMR (600 MHz, CDCl₃): δ 7.30–7.27 (m, 2H), 7.22–7.18 (m, 3H), 6.77 (dt, J = 9.0, 3.0 Hz, 2H), 6.58 (dt, J = 9.0, 3.0 Hz, 2H), 3.74 (s, 3H), 3.23 (tt, J = 10.2, 3.6 Hz, 1H), 2.98–2.96 (m, 2H), 2.83–2.80 (m, 2H), 2.63–2.60 (m, 2H), 2.19 (t, J = 10.2 Hz, 2H), 2.08–2.06 (m, 2H), 1.48 (ddd, J = 24.0, 10.2, 3.6 Hz, 2H). An NH peak was not observed. ¹³C NMR (150 MHz, CDCl₃): δ 152.1, 141.2, 140.3, 128.6, 128.4, 126.0, 115.0, 114.9, 60.6, 55.8, 52.5, 51.0, 50.7, 33.8, 32.6, 30.9. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₀H₂₇N₂O, 311.2118; found, 311.2118.

N-(3-Methoxyphenyl)-1-(2-phenylethyl)-4-piperidinamine (42)

White solid; yield: 210.1 mg (68%). MP: 80–81 °C. IR (ATR): 3265 cm^–1 (NH). ¹H NMR (600 MHz, CDCl₃): δ 7.31–7.28 (m, 2H), 7.22–7.19 (m, 3H), 7.07 (t, J = 8.4 Hz, 1H), 6.26 (ddd, J = 8.4, 2.4, 0.6 Hz, 1H), 6.23 (ddd, J = 8.4, 2.4, 0.6 Hz, 1H), 6.17 (t, J = 2.4 Hz, 1H), 3.78 (s, 3H), 3.56 (br s, 1H), 3.30 (tt, J = 10.2, 3.6 Hz, 1H), 2.97–2.96 (m, 2H), 2.84–2.81 (m, 2H), 2.63–2.61 (m, 2H), 2.20 (td, J = 10.2, 1.8 Hz, 2H), 2.10–2.08 (m, 2H), 1.51 (ddd, J = 24.0, 10.2, 3.6 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 160.9, 148.5, 140.4, 130.0, 128.7, 128.4, 126.0, 106.4, 102.1, 99.3, 60.6, 55.1, 52.4, 50.0, 33.9, 32.6. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₀H₂₇N₂O, 311.2118; found, 311.2118.

N-(2-Methylphenyl)-1-(2-phenylethyl)-4-piperidinamine (43)

White solid; yield: 1.13 g (77%). MP: 61 °C. IR (ATR): 3411 cm^–1 (NH). ¹H NMR (600 MHz, CDCl₃): δ 7.31–7.28 (m, 2H), 7.22–7.19 (m, 3H), 7.10 (td, J = 7.2, 1.2 Hz, 1H), 7.06 (d, J = 7.2 Hz, 1H), 6.65–6.62 (m, 2H), 3.39 (tt, J = 10.8, 3.6 Hz, 1H), 3.39 (br s, 1H), 2.98–2.96 (m, 2H), 2.84–2.82 (m, 2H), 2.64–2.62 (m, 2H), 2.25 (td, J = 10.8, 3.6 Hz, 2H), 2.13 (s, 3H), 2.13–2.12 (m, 2H), 1.55 (ddd, J = 24.0, 10.8, 3.6 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 145.0, 140.4, 130.4, 128.7, 128.4, 127.1, 126.0, 121.9, 116.7, 110.3, 60.6, 52.5, 49.7, 33.9, 32.7, 17.5. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₀H₂₇N₂, 295.2169; found, 295.2170.

N-(2,6-Dimethylphenyl)-1-(2-phenylethyl)-4-piperidinamine (44)

White solid; yield: 944.5 mg (61%). MP 70 °C. IR (ATR): 3367 cm^–1 (NH). ¹H NMR (600 MHz, CDCl₃): δ 7.28 (t, J = 7.8 Hz, 2H), 7.21–7.18 (m, 3H), 6.98 (d, J = 7.8 Hz, 2H), 6.80 (t, J = 7.8 Hz, 1H), 3.03–2.98 (m, 3H), 2.82–2.79 (m, 2H), 2.61–2.58 (m, 2H), 2.27 (s, 6H), 2.05 (td, J = 12.0, 2.4 Hz, 2H), 1.96–1.94 (m, 2H), 1.50 (ddd, J = 24.0, 12.0, 2.4 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 144.7, 140.4, 129.2, 128.8, 128.7, 128.4, 126.0, 121.5, 60.5, 54.3, 53.0, 34.0, 33.9, 19.0. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₁H₂₉N₂, 309.2325; found, 309.2325.

N-(2-Chloro-6-methylphenyl)-1-(2-phenylethyl)-4-piperidinamine (45)

White solid; yield: 311.4 mg (19%). MP 62–63 °C. IR (ATR): 3358 cm^–1 (NH). ¹H NMR (600 MHz, CDCl₃): δ 7.30–7.27 (m, 2H), 7.21–7.17 (m, 3H), 7.18 (dd, J = 7.8, 1.2 Hz, 1H), 7.02 (d, J = 7.8 Hz, 1H), 6.80 (t, J = 7.8 Hz, 1H), 3.59 (br s, 1H), 3.16 (tt, J = 10.8, 3.6 Hz, 1H), 2.98–2.96 (m, 2H), 2.82–2.79 (m, 2H), 2.61–2.58 (m, 2H), 2.31 (s, 3H), 2.09 (t, J = 10.8 Hz, 2H), 1.94–1.92 (m, 2H), 1.56 (ddd, J = 24.0, 10.8, 3.6 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 142.9, 140.4, 131.3, 129.8, 128.7, 128.4, 127.2, 126.8, 126.0, 122.0, 60.6, 54.0, 52.7, 33.9, 33.5, 19.4. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₀H₂₆ClN₂, 329.1779; found, 329.1779.

N-(2-Ethyl-6-methylphenyl)-1-(2-phenylethyl)-4-piperidinamine (46)

Oil; yield: 1.04 g (64%). IR (ATR): 3373 cm^–1 (NH). ¹H NMR (600 MHz, CDCl₃): δ 7.28 (t, J = 7.2 Hz, 2H), 7.20–7.18 (m, 3H), 7.02 (d, J = 7.2 Hz, 1H), 6.99 (d, J = 7.2 Hz, 1H), 6.86 (t, J = 7.2 Hz, 1H), 3.00–2.95 (m, 3H), 2.82–2.79 (m, 2H), 2.64 (q, J = 7.8 Hz, 2H), 2.60–2.57 (m, 2H), 2.28 (s, 3H), 2.04 (td, J = 12.0, 2.4 Hz, 2H), 1.96–1.94 (m, 2H), 1.50 (ddd, J = 24.0, 12.0, 2.4 Hz, 2H), 1.23 (t, J = 7.2 Hz, 3H). An NH peak was not observed. ¹³C NMR (150 MHz, CDCl₃): δ 144.0, 140.4, 135.2, 129.6, 128.71, 128.67, 128.4, 126.6, 126.0, 121.8, 60.5, 54.9, 53.1, 33.9, 24.5, 19.2, 14.4; several signals overlapped. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₂H₃₁N₂, 323.2482; found, 323.2483.

N-(2-Isopropyl-6-methylphenyl)-1-(2-phenylethyl)-4-piperidinamine (47)

Oil; yield: 214.0 mg (64%). IR (ATR): 3373 cm^–1 (NH). ¹H NMR (600 MHz, CDCl₃): δ 7.30–7.27 (m, 2H), 7.20–7.18 (m, 3H), 7.09 (dd, J = 7.8, 1.2 Hz, 1H), 6.98 (dt, J = 7.8 Hz, 1H), 6.92 (t, J = 7.8 Hz, 1H), 3.22 (sep, J = 7.2 Hz, 1H), 3.01–2.99 (m, 2H), 2.91 (tt, J = 10.8, 3.6 Hz, 1H), 2.81–2.79 (m, 2H), 2.60–2.57 (m, 2H), 2.29 (s, 3H), 2.01 (td, J = 11.4, 1.8 Hz, 2H), 1.96–1.94 (m, 2H), 1.51 (ddd, J = 23.4, 12.0, 3.6 Hz, 2H), 1.22 (d, J = 7.2 Hz, 6H). An NH peak was not observed. ¹³C NMR (150 MHz, CDCl₃): δ 143.0, 140.8, 140.4, 130.3, 128.6, 128.4, 128.3, 126.0, 123.8, 122.3, 60.5, 55.8, 53.2, 34.0, 33.8, 27.6, 24.0, 19.3. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₃₃N₂, 337.2638; found, 337.2636.

N-(2-Fluoro-6-methylphenyl)-1-(2-phenylethyl)-4-piperidinamine (48)

Oil; yield: 87.5 mg (28%). IR (ATR): 3369 cm^–1 (NH). ¹H NMR (600 MHz, CDCl₃): δ 7.28 (m, 2H), 7.20 (m, 3H), 6.87 (t, J = 7.8 Hz, 2H), 6.74 (td, J_H–H = 7.8 Hz, J_H–F = 6.0 Hz, 1H), 3.30 (tt, J = 12.0, 4.8 Hz, 1H), 2.96 (m, 2H), 2.81 (m, 2H), 2.60 (m, 2H), 2.24 (s, 3H), 2.13 (t, J = 12.0 Hz, 2H), 2.00 (m, 2H), 1.48 (ddd, J = 24.0, 10.2, 3.6 Hz, 2H). NH peak was not observed. ¹³C NMR (150 MHz, CDCl₃): δ 154.5 (d, ¹J_C–F = 238.4 Hz), 140.4, 133.6 (d, ¹J_C–F = 10.0 Hz), 129.8 (d, ¹J_C–F = 2.9 Hz), 128.7, 128.4, 126.06, 126.04, 126.02, 120.3 (d, ¹J_C–F = 8.6 Hz), 113.6 (d, ¹J_C–F = 21.6 Hz), 60.6, 53.3, 52.6, 33.9, 33.7, 18.1; One C–F coupling is not described. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₀H₂₆FN₂, 313.2075; found, 313.2074.

N-(2-Chlorophenyl)-1-(2-phenylethyl)-4-piperidinamine (49)

Oil; yield: 470.0 mg (30%). IR (ATR): 3415 cm^–1 (NH). ¹H NMR (600 MHz, CDCl₃): δ 7.29 (m, 2H), 7.25 (dd, J = 7.8, 1.8 Hz, 1H), 7.21 (m, 3H), 7.12 (td, J = 7.8, 1.8 Hz, 1H), 6.68 (dd, J = 7.8, 1.8 Hz, 1H), 6.61 (td, J = 7.8, 1.8 Hz, 1H), 4.24 (d, J = 7.8 Hz, 1H), 3.38 (m, 1H), 2.95 (m, 2H), 2.83 (m, 2H), 2.63 (m, 2H), 2.25 (t, J = 10.8 Hz, 2H), 2.10 (m, 2H), 1.59 (ddd, J = 24.0, 10.8, 3.6 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 142.9, 140.4, 129.3, 128.7, 128.4, 127.7, 126.0, 119.2, 116.9, 111.7, 60.6, 52.2, 49.6, 33.9, 32.3. HRMS (ESI): m/z [M + H]⁺ calcd for C₁₉H₂₄ClN₂, 315.1623; found, 315.1623.

N-(2-Chloro-6-fluorophenyl)-1-(2-phenylethyl)-4-piperidinamine (50)

Oil: 46.3 mg (6%). IR (ATR): 3383 cm^–1 (NH). ¹H NMR (600 MHz, CDCl₃): δ 7.29 (m, 2H), 7.20 (m, 3H), 7.08 (dt, J = 8.4, 1.2 Hz, 1H), 6.92 (ddd, J_H–H = 24.0, 1.2 Hz, J_H–F = 8.4 Hz, 1H), 6.69 (td, J_H–H = 8.4 Hz, J_H–F = 5.4 Hz, 1H), 3.80 (s, 1H), 3.59 (m, 1H), 2.93 (m, 2H), 2.81 (m, 2H), 2.61 (m, 2H), 2.19 (t, J = 10.2 Hz, 2H), 2.02 (m, 2H), 1.52 (ddd, J = 24.0, 10.2, 3.6 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 153.4 (d, ¹J_C–F = 241.4 Hz) 140.4, 132.8 (d, ¹J_C–F = 12.9 Hz), 128.7, 128.4, 126.0, 125.0, 124.0 (d, ¹J_C–F = 5.7 Hz), 119.0 (d, ¹J_C–F = 8.7 Hz), 115.0 (d, ¹J_C–F = 21.6 Hz), 60.5, 52.4, 52.2, 33.9, 33.5. HRMS (ESI): m/z [M + H]⁺ calcd for C₁₉H₂₃ClFN₂, 333.1528; found, 333.1528.

N-Phenyl-N-[1-(2-phenylethyl)-4-piperidinyl]-1H-pyrrole-2-carboxamide (1)

Oxalyl chloride (137 μL, 1.6 mmol) was added dropwise to a solution of pyrrole-2-carboxylic acid (133.3 mg, 1.2 mmol) in THF containing DMF (2 drops) at 25 °C. After 14 h, the reaction was concentrated. The resulting oily residue was redissolved in CH₂Cl₂ (2 mL). To a stirred solution of N-phenyl-1-(2-phenylethyl)-4-piperidinamine (40) (122.2 mg, 0.4 mmol) in CH₂Cl₂ (2 mL), sodium hydride (60% in oil) (48 mg, 1.2 mmol) was added at 0 °C under an argon atmosphere. The mixture was stirred at 25 °C for 30 min and treated with the above-mentioned resulting oily residue. After stirring at 25 °C for 3.5 h, the reaction was quenched with water and extracted with CH₂Cl₂. The organic layer was washed with 2 N NaOH and brine, dried over Na₂SO₄, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate/hexane = 1:4) to produce 1 as a white solid; yield: 53.3 mg (36%); MP: 204–205 °C. IR (ATR): 1613 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 9.58 (m, 1H), 7.45–7.42 (m, 3H), 7.26 (t, J = 7.2 Hz, 2H), 7.22–7.16 (m, 5H), 6.79 (dd, J = 6.0, 3.0 Hz, 1H), 5.87 (dd, J = 6.0, 3.0 Hz, 1H), 4.83 (tt, J = 12.0, 2.4 Hz, 1H), 4.60 (s, 1H), 3.06–3.04 (m, 2H), 2.76–2.73 (m, 2H), 2.58–2.55 (m, 2H), 2.21 (td, J = 12.0, 2.4 Hz, 2H), 1.90–1.88 (m, 2H), 1.58 (ddd, J = 24.0, 12.0, 4.2 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 160.9, 140.2, 138.6, 131.3, 129.3, 128.9, 128.6, 128.4, 126.0, 125.5, 120.6, 113.2, 109.7, 60.5, 53.1, 53.0, 33.8, 30.4. HRMS: m/z [M + H]⁺ calcd for C₂₄H₂₈N₃O, 374.2227; found, 374.2228.

4-Nitro-N-phenyl-N-[1-(2-phenylethyl)-4-piperidinyl]benzamide (2)

Sodium hydride (60% in oil) (48 mg, 1.2 mmol) was added to a stirred solution of N-phenyl-1-(2-phenylethyl)-4-piperidinamine (40) (122.2 mg, 0.40 mmol) in CH₂Cl₂ (4 mL) at 0 °C. The mixture was stirred at 25 °C for 20 min and treated with 4-nitrobenzoyl (222.7 mg, 1.2 mmol). After stirring at 25 °C for 8.5 h, the reaction was quenched with 2 N NaOH and extracted with diethyl ether. The organic layer was washed with 2 N NaOH and brine, dried over Na₂SO₄, and concentrated. The concentrate was purified by silica gel column chromatography (dichloromethane/methanol = 20:1) to produce 2 as a yellow solid; 155.7 mg (91%). MP: 152–153 °C. IR (ATR): 1641 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.98 (d, J = 7.8 Hz, 2H), 7.38 (d, J = 7.8 Hz, 2H), 7.27 (t, J = 7.8 Hz, 2H), 7.22–7.20 (m, 3H), 7.18 (t, J = 7.8 Hz, 3H), 7.00 (d, J = 7.8 Hz, 2H), 4.79 (t, J = 12.0 Hz, 1H), 3.10–3.08 (m, 2H), 2.78–2.76 (m, 2H), 2.61–2.58 (m, 2H), 2.26 (t, J = 12.0 Hz, 2H), 1.97 (d, J = 12.0 Hz, 2H), 1.65–1.63 (m, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 168.7, 147.5, 143.1, 138.3, 130.7, 129.1, 128.9, 128.6, 128.4, 128.3, 126.1, 122.9, 60.3, 53.8, 53.0, 33.7, 30.4; several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₆H₂₈N₃O₃, 430.2125; found, 430.2125.

Compounds 3–38 were prepared from the corresponding fentanyl analogue intermediates (40–50) according to a procedure similar to that described for preparing 2 from 40.

Methyl N-(4-Methoxyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]carbamate (3)

White solid; yield: 107.8 mg (73%); MP: 150–151 °C. IR (ATR): 1684 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.27–7.25 (m, 2H), 7.17 (dt, J = 8.4, 1.2 Hz, 1H), 7.16 (dd, J = 8.4, 1.2 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H), 6.86 (dt, J = 8.4, 2.4 Hz, 2H), 4.23 (m, 1H), 3.80 (s, 3H), 3.63 (s, 3H), 3.01–2.99 (m, 2H), 2.74–2.72 (m, 2H), 2.55–2.52 (m, 2H), 2.12 (t, J = 12.0 Hz, 2H), 1.84–1.81 (m, 2H), 1.49 (ddd, J = 25.2, 12.0, 3.0 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 158.8, 156.5, 140.2, 131.0, 128.6, 128.4, 126.0, 114.0, 60.4, 55.4, 54.9, 53.1, 52.8, 33.8, 30.8; several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₂H₂₉N₂O₃, 369.2173; found, 369.2173.

Methyl N-(3-Methoxyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]carbamate (4)

White solid; yield: 50.8 mg (34%). MP: 113–114 °C. IR (ATR): 1703 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.28–7.24 (m, 2H), 7.19 (d, J = 7.2 Hz, 1H), 7.16 (d, J = 7.2 Hz, 2H), 6.86 (dd, J = 8.4, 2.4 Hz, 2H), 6.68 (d, J = 8.4 Hz, 1H), 6.62 (t, J = 2.4 Hz, 1H), 4.23 (t, J = 12.0 Hz, 1H), 3.80 (s, 3H), 3.64 (s, 3H), 3.13–2.93 (m, 2H), 2.82–2.64 (m, 2H), 2.63–2.43 (m, 2H), 2.24–2.03 (m, 2H), 1.86 (d, J = 12.0 Hz, 2H), 1.61–1.54 (m, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 159.8, 156.1, 139.0, 129.3, 128.6, 128.4, 126.1, 122.4, 116.2, 113.0, 60.3, 55.3, 53.1, 52.8, 33.7, 30.7; several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₂H₂₉N₂O₃, 369.2173; found, 369.2170.

N-(3-Methoxyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]hexanamide (5)

Oil; yield: 148.2 mg (91%). IR (ATR): 1650 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.29 (t, J = 7.8 Hz, 1H), 7.25–7.24 (m, 2H), 7.18–7.17 (m, 1H), 7.15 (dd, J = 7.8, 1.2 Hz, 2H), 6.90 (ddd, J = 7.8, 2.4, 1.2 Hz, 1H), 6.66 (d, J = 7.8, 2.4, 1.2 Hz, 1H), 6.60 (t, J = 2.4 Hz, 1H), 4.66 (tt, J = 12.0, 4.2 Hz, 1H), 3.81 (s, 3H), 3.04–3.02 (m, 2H), 2.75–2.73 (m, 2H), 2.57–2.54 (m, 2H), 2.20–2.16 (m, 2H), 1.95 (t, J = 7.2 Hz, 2H), 1.80 (t, J = 7.2 Hz, 2H), 1.58–1.52 (m, 2H), 1.47 (tdd, J = 24.0, 12.0, 2.4 Hz, 1H), 1.36–1.12 (m, 6H), 0.82 (t, J = 7.2 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 172.8, 160.1, 140.1, 139.9, 129.8, 128.6, 128.4, 126.0, 122.7, 116.4, 113.4, 60.3, 55.4, 52.9, 52.0, 34.9, 33.6, 31.5, 30.4, 30.3, 25.2, 22.4, 13.9; several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₆H₃₇N₂O₂, 409.2850; found, 409.2849.

N-(2-Methylphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]acetamide (6)

Oil; yield: 111.5 mg (83%). IR (ATR): 1651 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.28 (dd, J = 7.8, 1.2 Hz, 1H), 7.26 (t, J = 7.8 Hz, 3H), 7.21 (td, J = 7.8, 1.2 Hz, 1H), 7.19–7.15 (m, 3H), 7.05 (dd, J = 7.8, 1.2 Hz, 1H), 4.61 (tt, J = 12.0, 4.2 Hz, 1H), 3.03 (ddd, J = 10.8, 5.4, 3.6 Hz, 1H), 2.95 (ddd, J = 10.8, 5.4, 3.6 Hz, 1H), 2.74–2.72 (m, 2H), 2.55–2.52 (m, 2H), 2.24 (s, 3H), 2.17 (td, J = 12.0, 2.4 Hz, 1H), 2.12 (td, J = 12.0, 2.4 Hz, 1H), 2.04–2.01 (m, 1H), 1.74–1.70 (m, 1H), 1.71 (s, 3H), 1.63 (ddd, J = 24.0, 12.0, 3.6 Hz, 1H), 1.29 (ddd, J = 24.0, 12.0, 3.6 Hz, 1H). ¹³C NMR (150 MHz, CDCl₃): δ 170.5, 140.2, 138.6, 137.0, 131.4, 130.3, 128.6, 128.4, 128.4, 126.9, 126.0, 60.5, 53.5, 53.10, 53.07, 33.8, 31.0, 29.4, 23.0, 18.2. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₂H₂₉N₂O, 337.2274; found, 337.2275.

N-(2-Methyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]propionamide (7)

Oil; yield: 97.7 mg (70%). IR (ATR): 1651 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.29–7.24 (m, 4H), 7.20 (dt, J = 7.8, 1.2 Hz, 1H), 7.16 (t, J = 7.8 Hz, 3H), 7.04 (dd, J = 7.8, 1.2 Hz, 1H), 4.63 (tt, J = 12.0, 3.6 Hz, 1H), 3.03 (ddd, J = 11.4, 5.4, 3.0 Hz, 1H), 2.95 (ddd, J = 11.4, 5.4, 3.0 Hz, 1H), 2.74–2.72 (m, 2H), 2.55–2.52 (m, 2H), 2.22 (s, 3H), 2.17 (td, J = 12.0, 2.4 Hz, 1H), 2.12 (td, J = 12.0, 2.4 Hz, 1H), 2.03–2.00 (m, 1H), 1.85 (ddq, J = 34.2, 16.8, 7.8 Hz, 2H), 1.73–1.69 (m, 1H), 1.62 (ddd, J = 24.0, 12.0, 3.6 Hz, 1H), 1.28 (ddd, J = 24.0, 12.0, 3.6 Hz, 1H), 1.02 (t, J = 7.8 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 173.7, 140.2, 138.1, 137.2, 131.3, 130.5, 128.6, 128.35, 128.30, 126.8, 126.0, 60.5, 53.6, 53.2, 53.1, 33.8, 31.0, 29.4, 28.1, 18.3, 9.4. HRMS: m/z [M + H]⁺ calcd for C₂₃H₃₁N₂O, 351.2431; found, 351.2432.

N-(2-Methylphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]butyramide (8)

Oil; yield: 94.5 mg (65%). IR (ATR): 1650 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.29–7.24 (m, 4H), 7.20 (td, J = 7.8, 1.2 Hz, 1H), 7.16 (t, J = 7.8 Hz, 3H), 7.03 (d, J = 7.8 Hz, 1H), 4.63 (tt, J = 12.0, 3.6 Hz, 1H), 3.02 (ddd, J = 10.8, 5.4, 3.6 Hz, 1H), 2.95 (ddd, J = 10.8, 5.4, 3.6 Hz, 1H), 2.74–2.72 (m, 2H), 2.55–2.52 (m, 2H), 2.22 (s, 3H), 2.17 (td, J = 12.0, 2.4 Hz, 1H), 2.12 (td, J = 12.0, 2.4 Hz, 1H), 2.03–2.00 (m, 1H), 1.88–1.77 (m, 2H), 1.71–1.68 (m, 1H), 1.62 (ddd, J = 24.0, 12.0, 4.2 Hz, 1H), 1.56 (dtd, J = 21.0, 7.8, 1.8 Hz, 2H), 1.27 (ddd, J = 24.0, 12.0, 4.2 Hz, 1H), 0.80 (t, J = 7.8 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 172.9, 140.3, 138.2, 137.2, 131.3, 130.6, 128.7, 128.5, 128.4, 126.8, 126.1, 60.5, 53.6, 53.2, 53.1, 36.5, 33.8, 31.1, 29.3, 18.6, 18.3, 13.8. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₃₃N₂O, 365.2587; found, 365.2587. Chiral HPLC separation: less polar: [α]²⁰_D −19.8 (c 0.080, CHCl₃) as 99% ee; more polar: [α]²⁰_D +19.9 (c 0.070, CHCl₃) as 94% ee. [CHIRALPAK IG, 2-propanol/hexane = 2:8].

N-(2-Methyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]cyclopentanecarboxamide (9)

White solid; yield: 141.4 mg (91%). MP: 112–113 °C. IR (ATR): 1646 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.28–7.24 (m, 4H), 7.21 (dd, J = 7.8, 1.8 Hz, 1H), 7.18 (d, J = 7.8 Hz, 1H), 7.15 (d, J = 7.8 Hz, 2H), 7.04 (d, J = 7.8 Hz, 1H), 4.62 (tt, J = 12.6, 4.2 Hz, 1H), 3.05–2.95 (m, 2H), 2.76–2.73 (m, 2H), 2.56–2.53 (m, 2H), 2.22 (quin, J = 8.4 Hz 1H), 2.22 (s, 3H), 2.18–2.11 (m, 2H), 2.03–2.01 (m, 1H), 1.94 (dq, J = 12.0, 8.4 Hz, 1H), 1.76–1.62 (m, 4H), 1.59–1.48 (m, 3H), 1.43–1.25 (m, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 176.9, 138.2, 137.3, 131.1, 130.7, 128.6, 128.4, 128.2, 126.6, 126.0, 60.4, 53.6, 53.2, 53.1, 42.7, 33.7, 31.8, 30.9, 30.2, 29.3, 26.4, 26.1, 18.5; several signals overlapped. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₆H₃₅N₂O, 391.2744; found, 391.2744.

N-(2-Methyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]cyclohexanecarboxamide (10)

White solid; yield: 89.1 mg (55%); MP: 154–155 °C. IR (ATR): 1647 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.29–7.24 (m, 4H), 7.20 (dd, J = 7.2, 1.8 Hz, 1H), 7.18 (d, J = 7.2 Hz, 1H), 7.15 (d, J = 7.2 Hz, 2H), 7.04 (d, J = 7.2 Hz, 1H), 4.60 (tt, J = 12.0, 3.6 Hz, 1H), 3.04–3.02 (m, 1H), 2.96–2.94 (m, 1H), 2.77–2.72 (m, 2H), 2.55–2.52 (m, 2H), 2.21 (s, 3H), 2.17 (td, J = 12.0, 2.4 Hz, 1H), 2.14 (td, J = 12.0, 2.4 Hz, 1H), 2.02–1.99 (m, 1H), 1.79 (tt, J = 12.0, 3.6 Hz, 1H), 1.70–1.51 (m, 8H), 1.34 (ddd, J = 24.0, 12.0, 3.6 Hz, 1H), 1.25 (ddd, J = 24.0, 12.0, 3.6 Hz, 1H), 1.16 (ddt, J = 24.0, 12.0, 3.6 Hz, 1H), 0.98 (ddt, J = 24.0, 12.0, 3.6 Hz, 1H), 0.82 (ddt, J = 24.0, 12.0, 3.6 Hz, 1H). ¹³C NMR (150 MHz, CDCl₃): δ 176.3, 140.2, 138.0, 137.2, 131.1, 130.3, 128.6, 128.3, 128.2, 126.6, 126.0, 60.4, 53.4, 53.1, 42.5, 33.8, 31.0, 29.9, 29.3, 28.9, 25.7, 25.6, 25.3, 18.4; several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₇H₃₇N₂O, 405.2900; found, 405.2901.

4-Chloro-N-(2-methylphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]benzamide (11)

White solid; yield: 117.8 mg (68%); MP: 110–111 °C. IR (ATR): 1639 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.27 (t, J = 7.2 Hz, 2H), 7.24–7.12 (m, 8H), 7.06 (d, J = 7.2 Hz, 2H), 7.01 (d, J = 7.2 Hz, 1H), 4.72 (t, J = 12.0 Hz, 1H), 3.11–3.09 (m, 1H), 3.00–2.99 (m, 1H), 2.77–2.74 (m, 2H), 2.59–2.56 (m, 2H), 2.26–2.16 (m, 3H), 2.01 (s, 3H), 1.85 (ddd, J = 24.0, 12.0, 3.6 Hz, 1H), 1.73–1.71 (m, 1H), 1.41 (ddd, J = 24.0, 12.0, 3.6 Hz, 1H). ¹³C NMR (150 MHz, CDCl₃): δ 169.3, 140.2, 138.3, 136.7, 135.4, 135.3, 131.4, 130.9, 129.7, 128.6, 128.4, 128.3, 127.6, 126.3, 126.0, 60.4, 55.1, 53.2, 53.1, 33.8, 31.5, 29.2, 18.5. HRMS: m/z [M + H]⁺ calcd for C₂₇H₃₀ClN₂O, 433.2041; found, 433.2042.

2-Chloro-N-(2-methylphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]benzamide (12)

White solid; yield: 20.0 mg (83%); E/Z = 6.1:1; MP: 102–105 °C. IR (ATR): 1638 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ(E-isomer) 7.33–7.30 (m, 1H), 7.27 (t, J = 7.8 Hz, 2H), 7.20–7.16 (m, 4H), 7.10–7.09 (m, 1H), 7.07–7.02 (m, 4H), 6.98 (td, J = 7.8, 1.8 Hz, 1H), 4.77 (tt, J = 12.0, 3.6 Hz, 1H), 3.10–3.08 (m, 1H), 3.01–2.99 (m, 1H), 2.77–2.74 (m, 2H), 2.59–2.56 (m, 2H), 2.29 (s, 3H), 2.28–2.22 (m, 2H), 2.18 (td, J = 12.0, 2.4 Hz, 1H), 1.85–1.79 (m, 2H), 1.41 (ddd, J = 24.6, 12.0, 4.2 Hz, 1H). δ(Z-isomer) 7.49–7.46 (m, 1H), 7.43–7.35 (m, 3H), 7.33–7.30 (m, 1H), 7.23 (t, J = 7.8 Hz, 3H), 7.20–7.16 (m, 1H), 7.14 (d, J = 7.8 Hz, 1H), 7.10–7.09 (m, 2H), 7.07–7.02 (m, 1H), 3.55 (tt, J = 12.0, 3.6 Hz, 1H), 2.92–2.84 (m, 2H), 2.66–2.64 (m, 2H), 2.44 (s, 3H), 2.43–2.41 (m, 2H), 1.97–1.88 (m, 2H), 1.78–1.73 (m, 2H), 1.70–1.65 (m, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 168.14, 168.09, 140.2, 140.1, 137.4, 137.2, 137.0, 136.9, 131.4, 131.2, 130.8, 130.5, 130.3, 130.1, 130.04, 129.96, 129.7, 129.6, 129.5, 128.6, 128.5, 128.4, 128.2, 127.9, 127.2, 127.1, 126.9, 126.8, 126.6, 126.5, 126.0, 125.8, 60.4, 60.2, 59.0, 54.8, 53.1, 53.0, 52.9, 33.82, 33.77, 32.34, 32.32, 30.6, 29.8, 29.3, 19.2, 18.9; two diastereomers, not distinguished, several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₇H₃₀ClN₂O, 433.2041; found, 433.2042.

N-(2,6-Dimethyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]propionamide (13)

Oil: 144.3 mg (99%). IR (ATR): 1654 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.26 (t, J = 7.8 Hz, 2H), 7.19–7.13 (m, 4H), 7.09 (d, J = 7.8 Hz, 2H), 4.20 (tt, J = 12.0, 3.6 Hz, 1H), 2.99–2.97 (m, 2H), 2.76–2.73 (m, 2H), 2.55–2.53 (m, 2H), 2.21 (s, 6H), 2.11 (td, J = 12.0, 1.8 Hz, 2H), 1.99–1.97 (m, 2H), 1.80 (q, J = 7.8 Hz, 2H), 1.42 (ddd, J = 24.6, 12.0, 3.6 Hz, 2H), 1.02 (t, J = 7.8 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 174.2, 140.3, 138.5, 137.3, 128.7, 128.6, 128.3, 127.9, 126.0, 60.5, 55.7, 53.2, 33.8, 30.4, 27.9, 19.0, 9.3. HRMS: m/z [M + H]⁺ calcd for C₂₄H₃₃N₂O, 365.2587; found, 365.2587.

N-(2,6-Dimethylphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]hexanamide (14)

Oil; yield: 148.5 mg (91%). IR (ATR): 1652 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.26 (t, J = 7.8 Hz, 2H), 7.16–7.13 (m, 4H), 7.09 (d, J = 7.8 Hz, 2H), 4.20 (tt, J = 12.0, 3.6 Hz, 1H), 2.99–2.97 (m, 2H), 2.76–2.73 (m, 2H), 2.55–2.53 (m, 2H), 2.21 (s, 6H), 2.11 (td, J = 12.0, 1.8 Hz, 2H), 1.98–1.96 (m, 2H), 1.78 (t, J = 7.8 Hz, 2H), 1.55 (dt, J = 15.0, 7.8 Hz, 2H), 1.42 (ddd, J = 24.6, 12.0, 3.6 Hz, 2H), 1.25–1.19 (m, 2H), 1.18–1.13 (m, 2H), 0.83 (t, J = 7.8 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 173.5, 140.3, 138.5, 137.3, 128.7, 128.6, 128.3, 127.9, 126.0, 60.4, 55.7, 53.2, 34.5, 33.8, 31.6, 30.4, 24.6, 22.5, 19.1, 13.9. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₇H₃₉N₂O, 407.3057; found, 407.3057.

N-(2,6-Dimethyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]-2-furancarboxamide (15)

White solid: 195.4 mg (61%): MP: 130–131 °C. IR (ATR): 1641 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.36 (dd, J = 1.8, 0.6 Hz, 1H), 7.28–7.26 (m, 2H), 7.21 (t, J = 7.8 Hz, 1H), 7.19–7.17 (m, 3H), 7.11 (d, J = 7.8 Hz, 2H), 6.13 (dd, J = 3.6, 1.8 Hz, 1H), 5.28 (dd, J = 3.6, 1.8 Hz, 1H), 4.33 (tt, J = 12.0, 3.6 Hz, 1H), 3.04–3.02 (m, 2H), 2.78–2.75 (m, 2H), 2.59–2.56 (m, 2H), 2.20 (s, 6H), 2.17 (td, J = 12.0, 1.8 Hz, 2H), 2.06–2.04 (m, 2H), 1.60 (ddd, J = 24.0, 12.0, 3.6 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 159.4, 147.4, 144.4, 140.3, 138.4, 138.1, 128.8, 128.64, 128.56, 128.4, 126.0, 114.5, 111.1, 60.4, 56.8, 53.2, 33.8, 30.1, 19.0. HRMS: m/z [M + H]⁺ calcd for C₂₆H₃₁N₂O₂, 403.2380; found, 403.2379.

2-Fluoro-N-(2,6-dimethylphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]benzamide (16)

Solid; yield: 98.6 mg (57%); E/Z = 8.1:1; MP: 81–82 °C. IR (ATR): 1635 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ(E-isomer) 7.29–7.27 (m, 2H), 7.20–7.18 (m, 3H), 7.14–7.10 (m, 1H), 7.07 (td, J = 7.2, 1.2 Hz, 1H), 6.97 (dd, J = 7.2, 6.6 Hz, 1H), 6.90 (d, J = 7.2 Hz, 2H), 6.88–6.83 (m, 2H), 4.27 (tt, J = 12.0, 3.6 Hz, 1H), 3.06–3.04 (m, 2H), 2.79–2.76 (m, 2H), 2.60–2.57 (m, 2H), 2.31 (s, 6H), 2.17 (td, J = 12.0, 1.8 Hz, 2H), 2.08–2.06 (m, 2H), 1.68 (ddd, J = 24.6, 12.6, 3.6 Hz, 2H). δ(Z-isomer) 7.48 (td, J = 7.2, 1.2 Hz, 1H), 7.45–7.42 (m, 1H), 7.23 (t, J = 7.2 Hz, 3H), 7.17–7.15 (m, 3H), 7.14–7.10 (m, 4H), 3.74 (tt, J = 12.0, 3.6 Hz, 1H), 2.90–2.88 (m, 2H), 2.67–2.64 (m, 2H), 2.43–2.41 (m, 2H), 2.35 (s, 6H), 1.84 (m, 4H), 1.60–1.57 (m, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 167.3, 166.5, 158.7 (d, ¹J_C–F = 248.4 Hz), 140.3, 140.1, 138.1, 137.6, 136.1, 131.0, 130.9, 130.5, 130.4, 128.7, 128.5, 128.4, 128.3, 127.8, 127.8, 127.8, 126.0, 125.8, 125.7, 124.7, 123.0 (d, ¹J_C–F = 4.2 Hz), 116.1, 115.9, 115.8, 115.6, 60.4, 60.1, 59.5, 57.4, 53.2, 53.1, 33.7, 30.1, 30.0, 19.4, 19.4; two diastereomers, not distinguished, some C–F couplings are not described and several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₈H₃₂FN₂O, 431.2493; found, 431.2494.

2-Chloro-N-(2,6-dimethylphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]benzamide (17)

Yellow solid; yield: 121.2 mg (68%); E/Z = 2.3:1; MP 125–126 °C. IR (ATR): 1644 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ(E-isomer) 7.29–7.27 (m, 2H), 7.19–7.18 (m, 3H), 7.16–7.12 (m, 1H), 7.06 (td, J = 7.2, 1.2 Hz, 1H), 6.98 (dd, J = 8.4, 7.2 Hz, 1H), 6.92 (d, J = 7.2 Hz, 2H), 6.87 (td, J = 7.2, 1.2 Hz, 1H), 6.83 (dd, J = 7.2, 1.2 Hz, 1H), 4.35 (tt, J = 12.0, 3.6 Hz, 1H), 3.05–3.04 (m, 2H), 2.80–2.77 (m, 2H), 2.61–2.58 (m, 2H), 2.34 (s, 6H), 2.20 (t, J = 12.0 Hz, 2H), 2.14–2.12 (m, 2H), 1.63–1.56 (m, 2H). δ(Z-isomer) 7.49–7.47 (m, 1H), 7.45–7.44 (m, 1H), 7.39–7.37 (m, 2H), 7.32–7.29 (m, 1H), 7.23 (t, J = 7.2 Hz, 2H), 7.16–7.12 (m, 3H), 7.10–7.09 (m, 2H), 3.67 (tt, J = 12.0, 3.6 Hz, 1H), 2.92–2.87 (m, 2H), 2.66–2.63 (m, 2H), 2.44 (s, 3H), 2.43–2.40 (m, 2H), 2.35 (s, 3H), 2.04–2.03 (m, 1H), 1.82–1.76 (m, 2H), 1.72–1.56 (m, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 168.0, 167.9, 140.3, 140.1, 138.0, 137.9, 137.4, 136.9, 136.4, 135.8, 131.5, 130.3, 130.2, 130.1, 130.04, 130.00, 129.6, 129.5, 129.0, 128.72, 128.67, 128.5, 128.4, 128.3, 127.9, 127.8, 127.7, 127.2, 127.0, 126.1, 126.0, 125.3, 60.4, 60.1, 59.9, 56.9, 53.2, 53.1, 33.8, 33.7, 31.1, 30.8, 30.1, 19.9, 19.6, 19.5; two diastereomers, not distinguished, several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₈H₃₂ClN₂O, 447.2198; found, 447.2198.

2-Methyl-N-(2,6-dimethylphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]benzamide (18)

White solid; yield: 100.1 mg (59%); E/Z = 2.4:1; MP: 119–120 °C. IR (ATR): 1636 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ(E-isomer) 7.28 (t, J = 7.8 Hz, 3H), 7.21–7.19 (m, 3H), 7.10–7.08 (m, 2H), 6.91 (d, J = 7.8 Hz, 2H), 6.75 (t, J = 7.8 Hz, 1H), 6.72 (dd, J = 7.8, 1.8 Hz, 1H), 4.32 (tt, J = 12.0, 4.0 Hz, 1H), 3.14–3.10 (m, 2H), 2.83 (m, 2H), 2.66–2.63 (m, 2H), 2.48 (s, 3H), 2.27 (s, 6H), 2.27–2.25 (m, 2H), 2.10–2.09 (m, 2H), 1.74–1.72 (m, 2H). δ(Z-isomer) 7.38 (d, J = 7.8 Hz, 1H), 7.32 (td, J = 7.8, 1.2 Hz, 1H), 7.23 (t, J = 7.8 Hz, 2H), 7.17–7.12 (m, 3H), 7.02 (td, J = 7.8, 1.2 Hz, 3H), 6.97 (dd, J = 7.8, 6.6 Hz, 2H), 3.75 (tt, J = 12.0, 4.0 Hz, 1H), 2.88–2.86 (m, 2H), 2.6–2.63 (m, 2H), 2.49 (s, 3H), 2.43–2.40 (m, 2H), 2.38 (s, 6H), 1.74–1.72 (m, 6H). ¹³C NMR (150 MHz, CDCl₃): δ 170.9, 170.8, 140.0, 138.7, 137.6, 137.4, 137.1, 136.5, 136.2, 135.6, 134.6, 130.8, 128.83, 128.81, 128.7, 128.5, 128.43, 128.37, 128.3, 127.6, 126.15, 126.06, 126.0, 125.6, 125.4, 124.1, 60.2, 60.1, 59.3, 56.7, 53.2, 53.0, 33.7, 33.5, 33.4, 29.9, 29.8, 20.3, 19.8, 19.6; two diastereomers, not distinguished, several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₉H₃₅N₂O, 427.2744; found, 427.2745.

2-Bromo-N-(2,6-dimethylphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]benzamide (19)

White solid; yield: 90.2 mg (46%); E/Z = 2.0:1; MP: 140–142 °C. IR (ATR): 1642 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ(E-isomer) 7.27 (t, J = 7.8 Hz, 2H), 7.19–7.17 (m, 3H), 7.16–7.11 (m, 2H), 7.00–6.96 (m, 2H), 6.92 (d, J = 7.8 Hz, 2H), 6.79 (dd, J = 7.8, 1.2 Hz, 1H), 4.35 (tt, J = 12.0, 3.6 Hz, 1H), 3.06–3.04 (m, 2H), 2.79–2.77 (m, 2H), 2.60–2.58 (m, 2H), 2.35 (s, 6H), 2.20 (t, J = 12.0 Hz, 2H), 2.15–2.13 (m, 2H), 1.63–1.52 (m, 2H). δ(Z-isomer) 7.66 (d, J = 7.8 Hz, 1H), 7.47 (dd, J = 7.8, 1.2 Hz, 2H), 7.44–7.42 (m, 2H), 7.31–7.29 (m, 1H), 7.25–7.23 (m, 2H), 7.09 (d, J = 7.8 Hz, 2H), 6.90 (d, J = 7.8 Hz, 2H), 3.69 (tt, J = 12.0, 3.6 Hz, 1H), 2.93–2.86 (m, 2H), 2.66–2.63 (m, 2H), 2.48 (s, 3H), 2.43–2.40 (m, 2H), 2.35 (s, 3H), 2.10–2.07 (m, 1H), 1.79 (ddd, J = 24.0, 12.0, 2.4 Hz, 2H), 1.71 (td, J = 12.0, 2.4 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 168.6, 168.5, 140.3, 140.1, 139.5, 138.0, 137.9, 137.4, 136.9, 135.7, 133.5, 133.2, 130.2, 129.7, 129.1, 128.72, 128.67, 128.5, 128.4, 128.3, 127.9, 127.8, 127.7, 127.5, 127.2, 126.1, 126.0, 125.8, 120.6, 119.3, 60.4, 60.2, 60.0, 56.9, 53.2, 53.1, 33.8, 30.9, 30.2, 20.1, 19.9, 19.4; two diastereomers, not distinguished. HRMS: m/z [M + H]⁺ calcd for C₂₈H₃₂BrN₂O, 491.1693; found, 491.1693.

2-Iodo-N-(2,6-dimethylphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]benzamide (20)

White solid; yield: 40.9 mg (19%); E/Z = 2.2:1; MP 143–145 °C. IR (ATR): 1640 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ(E-isomer) 7.29–7.27 (m, 2H), 7.20–7.17 (m, 3H), 7.16–7.08 (m, 3H), 6.92 (d, J = 7.2 Hz, 2H), 6.80 (td, J = 7.2, 1.8 Hz, 1H), 6.74 (dd, J = 7.2, 1.8 Hz, 1H), 4.34 (tt, J = 12.0, 3.6 Hz, 1H), 3.06–3.04 (m, 2H), 2.79–2.77 (m, 2H), 2.60–2.58 (m, 2H), 2.35 (s, 6H), 2.24–2.10 (m, 4H), 1.62–1.58 (m, 2H). δ(Z-isomer) 7.91 (dd, J = 7.8, 1.2 Hz, 1H), 7.77 (dd, J = 7.8, 1.2 Hz, 2H), 7.44 (td, J = 7.8, 1.2 Hz, 1H), 7.40 (dd, J = 7.8, 1.2 Hz, 1H), 7.24 (t, J = 7.8 Hz, 2H), 7.16–7.08 (m, 1H), 6.99 (dd, J = 7.8, 1.2 Hz, 2H), 6.93–6.90 (m, 2H), 3.69 (tt, J = 12.0, 3.6 Hz, 1H), 2.93–2.86 (m, 2H), 2.67–2.64 (m, 2H), 2.55 (s, 3H), 2.43–2.40 (m, 2H), 2.35 (s, 3H), 1.81–1.69 (m, 5H), 1.48 (ddd, J = 24.0, 12.0, 3.6 Hz, 1H). ¹³C NMR (150 MHz, CDCl₃): δ 170.0, 169.6, 143.6, 140.44, 140.34, 140.1, 139.9, 138.1, 138.0, 137.8, 136.9, 135.8, 130.1, 129.8, 129.2, 128.7, 128.6, 128.4, 128.3, 128.04, 127.99, 127.9, 127.7, 127.2, 126.9, 126.4, 126.1, 126.0, 94.6, 92.9, 60.4, 60.2, 60.0, 57.0, 53.2, 53.1, 33.8, 31.4, 30.6, 30.3, 20.3, 19.4; two diastereomers, not distinguished, several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₈H₃₂IN₂O, 539.1554; found, 539.1554.

2,6-Difluoro-N-(2,6-dimethylphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]benzamide (21)

White solid; yield: 117.5 mg (65%); E/Z = 2.4:1; MP 131–132 °C. IR (ATR): 1652 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ(E-isomer) 7.27 (t, J = 7.8 Hz, 1H), 7.20–7.17 (m, 3H), 7.16–7.12 (m, 1H), 7.07 (tt, J = 7.8, 7.2 Hz, 1H), 6.98 (dd, J = 7.8, 7.2 Hz, 1H), 6.92 (d, J = 7.8 Hz, 2H), 6.66 (t, J = 7.8 Hz, 2H), 4.27 (tt, J = 12.0, 3.6 Hz, 1H), 3.06–3.04 (m, 2H), 2.79–2.76 (m, 2H), 2.60–2.57 (m, 2H), 2.30 (s, 6H), 2.17 (td, J = 12.0, 1.8 Hz, 2H), 2.15–2.12 (m, 2H), 1.67 (ddd, J = 24.0, 12.0, 3.6 Hz, 2H). δ(Z-isomer) 7.40 (tt, J = 7.8, 7.2 Hz, 1H), 7.25–7.23 (m, 3H), 7.20–7.17 (m, 1H), 7.16–7.12 (m, 2H), 7.11–7.10 (m, 2H), 7.02 (dd, J = 7.8, 7.2 Hz, 2H), 3.69 (tt, J = 12.0, 3.6 Hz, 1H), 2.92–2.91 (m, 2H), 2.67–2.65 (m, 2H), 2.44–2.42 (m, 2H), 2.35 (s, 6H), 1.90–1.87 (m, 2H), 1.80 (td, J = 12.0, 1.8 Hz, 2H), 1.61 (ddd, J = 24.0, 12.0, 3.6 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 163.0, 161.5, 159.8, 159.7, 158.11, 158.05, 158.0, 140.4, 140.1, 138.3, 137.2, 136.8, 135.8, 130.8, 130.23, 130.16, 130.1, 128.8, 128.7, 128.6, 128.4, 128.1, 128.0, 127.9, 126.1, 126.0, 112.00, 111.98, 111.9, 111.8, 111.24, 111.22, 111.10, 111.07, 60.4, 60.2, 60.0, 57.8, 53.2, 53.1, 33.8, 31.2, 30.0, 19.3, 19.1, 19.1; two diastereomers, not distinguished, C–F couplings are not described and several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₈H₃₁F₂N₂O, 449.2399; found, 449.2399.

N-(2-Chloro-6-methyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]propionamide (22)

Oil; yield: 105.8 mg (69%). IR (ATR): 1659 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.31 (t, J = 4.8 Hz, 1H), 7.27–7.25 (m, 2H), 7.19 (d, J = 4.8 Hz, 2H), 7.20–7.16 (m, 3H), 4.28 (tt, J = 12.0, 3.6 Hz, 1H), 3.01–2.96 (m, 2H), 2.76–2.73 (m, 2H), 2.56–2.53 (m, 2H), 2.30 (s, 3H), 2.16–2.08 (m, 3H), 1.97–1.94 (m, 1H), 1.89 (dq, J = 16.2, 7.2 Hz, 1H), 1.78 (dq, J = 16.2, 7.2 Hz, 1H), 1.51 (ddd, J = 24.0, 12.0, 3.6 Hz, 1H), 1.43 (ddd, J = 24.0, 12.0, 3.6 Hz, 1H), 1.04 (t, J = 7.2 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 174.0, 140.3, 139.9, 137.2, 135.4, 129.6, 129.0, 128.6, 128.3, 127.9, 126.0, 60.4, 55.9, 53.2, 53.1, 33.8, 30.8, 29.7, 27.8, 19.4, 9.2. HRMS: m/z [M + H]⁺ calcd for C₂₃H₃₀ClN₂O, 385.2041; found, 385.2042.

N-(2-Chloro-6-methyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]-2-furancarboxamide (23)

White solid; 165.0 mg (67%); MP: 108–109 °C. IR (ATR): 1646 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.32–7.30 (m, 2H), 7.28–7.25 (m, 2H), 7.24 (t, J = 7.8 Hz, 1H), 7.20–7.17 (m, 4H), 6.18 (dd, J = 3.6, 1.8 Hz, 1H), 5.70 (dd, J = 3.6, 0.6 Hz, 1H), 4.38 (tt, J = 12.0, 3.6 Hz, 1H), 3.06–3.01 (m, 2H), 2.78–2.76 (m, 2H), 2.59–2.56 (m, 2H), 2.30 (s, 3H), 2.21–2.14 (m, 2H), 2.03–2.00 (m, 1H), 1.71 (td, J = 12.0, 3.6 Hz, 1H), 1.65 (ddd, J = 24.0, 12.0, 3.6 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 159.4, 147.4, 144.4, 140.5, 140.3, 137.3, 135.8, 129.4, 129.3, 128.6, 128.3, 127.9, 126.0, 114.7, 111.0, 60.4, 57.2, 53.2, 53.1, 33.8, 30.3, 29.5, 19.4. HRMS: m/z [M + H]⁺ calcd for C₂₅H₂₈ClN₂O₂, 432.1834; found, 423.1834. Chiral HPLC separation: less polar: [α]²⁰_D +13.81 (c 0.215, CHCl₃) as 98.8% ee; more polar: [α]²⁰_D −8.81 (c 0.110, CHCl₃) as 99.1% ee. [CHIRALPAK IG, ethanol/hexane = 3:7].

4-Chloro-N-(2-chloro-6-methylphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]benzamide (24)

White solid; yield: 60.6 mg (32%); MP: 145 °C. IR (ATR): 1645 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.30 (dt, J = 9.0, 2.4 Hz, 2H), 7.29–7.27 (m, 2H), 7.20–7.12 (m, 4H), 7.10 (dt, J = 9.0, 2.4 Hz, 2H), 7.06 (t, J = 7.8 Hz, 1H), 7.01 (d, J = 7.8 Hz, 1H), 4.23 (tt, J = 12.0, 3.6 Hz, 1H), 3.06–3.04 (m, 2H), 2.80–2.77 (m, 2H), 2.60–2.57 (m, 2H), 2.28 (s, 3H), 2.16 (ddd, J = 19.2, 12.0, 1.8 Hz, 2H), 2.04–1.98 (m, 2H), 1.86–1.77 (m, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 170.4, 140.3, 139.4, 138.2, 135.7, 135.4, 135.0, 129.5, 129.0, 128.7, 128.44, 128.37, 127.9, 127.8, 126.0, 60.4, 57.8, 53.2, 33.8, 30.0, 29.7, 19.6; several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₇H₂₉Cl₂N₂O, 467.1651; found, 467.1652. Chiral HPLC separation: less polar: [α]²⁰_D −48.6 (c 0.085, CHCl₃) as >99.9% ee; more polar: [α]²⁰_D +48.4 (c 0.080, CHCl₃) as 99% ee. [CHIRALPAK IG, 2-propanol/hexane = 5:5].

N-(2-Ethyl-6-methylphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]acetamide (25)

White solid; yield: 139.6 mg (98%); MP: 71–72 °C. IR (ATR): 1637 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.26 (t, J = 7.8 Hz, 2H), 7.22 (t, J = 7.8 Hz, 1H), 7.17 (td, J = 7.8, 1.2 Hz, 4H), 7.10 (dd, J = 7.8, 1.2 Hz, 1H), 4.16 (tt, J = 12.0, 3.6 Hz, 1H), 2.98–2.96 (m, 2H), 2.75–2.73 (m, 2H), 2.58 (q, J = 7.8 Hz, 2H), 2.55–2.52 (m, 2H), 2.23 (s, 3H), 2.11 (tt, J = 12.0, 3.6 Hz, 2H), 1.99–1.94 (m, 2H), 1.68 (s, 3H), 1.42 (ddd, J = 24.0, 12.0, 3.6 Hz, 2H), 1.24 (t, J = 7.8 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 171.5, 142.8, 140.5, 138.5, 137.1, 128.8, 128.7, 128.5, 128.4, 126.6, 126.1, 60.6, 56.0, 53.4, 53.3, 33.9, 30.5, 24.2, 23.3, 19.2, 14.3; several signals overlapped. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₃₃N₂O, 365.2587; found, 365.2588.

N-(2-Ethyl-6-methyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]propionamide (26)

Oil; yield: 77.9 mg (52%). IR (ATR): 1652 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.26 (t, J = 7.2 Hz, 2H), 7.22 (t, J = 7.2 Hz, 1H), 7.17 (td, J = 7.2, 1.8 Hz, 4H), 7.10 (dd, J = 7.2, 1.8 Hz, 1H), 4.18 (tt, J = 12.0, 3.6 Hz, 1H), 2.98–2.96 (m, 2H), 2.75–2.73 (m, 2H), 2.61–2.51 (m, 4H), 2.21 (s, 3H), 2.11 (tt, J = 12.0, 3.0 Hz, 2H), 1.99–1.93 (m, 2H), 1.81 (ddd, J = 15.0, 7.2, 3.0 Hz, 2H), 1.41 (ddd, J = 24.0, 12.0, 3.0 Hz, 2H), 1.23 (t, J = 7.2 Hz, 3H), 1.02 (t, J = 7.2 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 174.4, 142.7, 140.3, 137.9, 137.0, 128.6, 128.5, 128.3, 128.1, 126.3, 126.0, 60.4, 55.8, 53.23, 53.18, 33.8, 30.3, 27.9, 24.0, 19.1, 14.1, 9.3; several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₅H₃₅N₂O, 379.2744; found, 379.2745.

N-(2-Methylphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]butyramide (27)

Oil; yield: 136.6 mg (87%). IR (ATR): 1651 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.26 (t, J = 7.8 Hz, 2H), 7.22 (t, J = 7.8 Hz, 1H), 7.17 (td, J = 7.8, 1.2 Hz, 4H), 7.10 (dd, J = 7.8, 1.2 Hz, 1H), 4.18 (tt, J = 12.0, 3.6 Hz, 1H), 2.98–2.96 (m, 2H), 2.75–2.72 (m, 2H), 2.61–2.51 (m, 4H), 2.21 (s, 3H), 2.11 (tt, J = 12.0, 3.6 Hz, 2H), 1.98–1.92 (m, 2H), 1.81–1.72 (m, 2H), 1.62–1.53 (m, 2H), 1.40 (ddd, J = 24.0, 12.0, 3.6 Hz, 2H), 1.24 (t, J = 7.8 Hz, 3H), 0.82 (t, J = 7.8 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 173.5, 142.7, 140.3, 137.8, 137.0, 128.6, 128.5, 128.3, 128.1, 126.3, 126.0, 60.4, 55.8, 53.22, 53.17, 36.5, 33.7, 30.3, 23.9, 19.1, 18.2, 14.0, 13.9; several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₆H₃₇N₂O, 393.2900; found, 393.2901.

N-(2-Ethyl-6-methyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]cyclopropanecarboxamide (28)

Oil; yield: 155.5 mg (quant.). IR (ATR): 1646 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.26 (t, J = 7.8 Hz, 2H), 7.21 (J = 7.8 Hz, 1H), 7.17 (td, J = 7.8, 1.2 Hz, 4H), 7.11 (dd, J = 7.8, 1.2 Hz, 1H), 4.19 (tt, J = 12.0, 3.6 Hz, 1H), 2.98–2.96 (m, 2H), 2.75–2.72 (m, 2H), 2.66 (dt, J = 22.8, 7.8 Hz, 1H), 2.58 (dt, J = 22.8, 7.8 Hz, 1H), 2.55–2.52 (m, 2H), 2.27 (s, 3H), 2.09 (tt, J = 12.0, 3.0 Hz, 2H), 1.99–1.93 (m, 2H), 1.43 (ddt, J = 24.0, 12.0, 3.6 Hz, 2H), 1.23 (t, J = 7.8 Hz, 3H), 1.08 (tt, J = 7.8, 3.6 Hz, 1H), 0.95 (dd, J = 3.6, 2.4 Hz, 2H), 0.57–0.49 (m, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 174.1, 143.5, 140.3, 137.9, 137.8, 128.6, 128.4, 128.3, 128.1, 126.5, 126.0, 60.4, 56.0, 53.25, 53.21, 33.8, 30.4, 24.5, 19.2, 14.4, 12.5, 8.2, 7.8; several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₆H₃₅N₂O, 391.2744; found, 391.2744.

N-(2-Ethyl-6-methyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]cyclopentanecarboxamide (29)

Oil: 144.1 mg (88%). IR (ATR): 1646 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.25 (t, J = 7.8 Hz, 2H), 7.22 (t, J = 7.8 Hz, 1H), 7.17 (t, J = 7.8 Hz, 4H), 7.10 (dd, J = 7.8, 1.2 Hz, 1H), 4.18 (tt, J = 12.0, 3.6 Hz, 1H), 2.98–2.96 (m, 2H), 2.75–2.72 (m, 2H), 2.62–2.52 (m, 4H), 2.22 (quin, J = 8.4 Hz, 1H), 2.22 (s, 3H), 2.10 (ddt, J = 12.0, 3.6, 1.8 Hz, 2H), 1.97–1.90 (m, 2H), 1.79–1.68 (m, 4H), 1.58–1.50 (m, 2H), 1.43–1.35 (m, 4H), 1.24 (t, J = 7.8 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 178.1, 142.9, 140.4, 138.1, 137.2, 128.6, 128.3, 128.0, 126.0, 60.4, 55.9, 53.3, 53.2, 42.6, 33.7, 31.5, 31.4, 30.4, 26.4, 24.0, 19.4, 13.9; several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₈H₃₉N₂O, 419.3057; found, 419.3058.

N-(2-Ethyl-6-methyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]cyclohexanecarboxamide (30)

White solid; yield: 127.1 mg (74%). MP 88–90 °C. IR (ATR): 1642 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.25 (t, J = 7.8 Hz, 2H), 7.22 (t, J = 7.8 Hz, 1H), 7.19 (dd, J = 7.8, 1.2 Hz, 1H), 7.16 (dd, J = 7.8, 1.2 Hz, 3H), 7.10 (d, J = 7.8 Hz, 1H), 4.16 (tt, J = 12.0, 3.6 Hz, 1H), 2.98–2.95 (m, 2H), 2.75–2.72 (m, 2H), 2.57 (q, J = 7.8 Hz, 2H), 2.54–2.51 (m, 2H), 2.22 (s, 3H), 2.09 (ddd, J = 21.0, 12.0, 2.4 Hz, 2H), 1.97–1.94 (m, 1H), 1.88–1.85 (m, 1H), 1.80 (tt, J = 12.0, 3.6 Hz, 1H), 1.65–1.63 (m, 2H), 1.58–1.46 (m, 5H), 1.44–1.34 (m, 2H), 1.24 (t, J = 7.8 Hz, 3H), 1.17 (ddt, J = 25.2, 13.2, 3.6 Hz, 1H), 0.92 (ddt, J = 25.2, 13.2, 3.6 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 177.3, 142.9, 140.4, 137.9, 137.1, 128.6, 128.4, 128.3, 127.9, 126.1, 126.0, 60.4, 55.8, 53.3, 53.2, 42.9, 33.7, 30.3, 30.2, 29.8, 29.7, 25.6, 25.55, 25.52, 23.9, 19.5, 14.0. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₉H₄₁N₂O, 433.3213; found, 433.3214. Chiral HPLC separation: less polar: [α]²⁰_D +4.15 (c 0.250, CHCl₃) as 99% ee; more polar: [α]²⁰_D −4.32 (c 0.080, CHCl₃) as 96% ee. [CHIRALPAK IF, ethanol/hexane = 5:95].

N-(2-Ethyl-6-methyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]-2-furancarboxamide (31)

White solid; 144.5 mg (87%). MP: 141–142 °C. IR (ATR): 1630 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.35 (d, J = 1.8 Hz, 1H), 7.29–7.25 (m, 3H), 7.20–7.17 (m, 4H), 7.12 (d, J = 7.2 Hz, 1H), 6.12 (dd, J = 3.6, 1.8 Hz, 1H), 5.27 (dd, J = 3.6, 1.8 Hz, 1H), 4.31 (tt, J = 12.0, 3.6 Hz, 1H), 3.03–3.01 (m, 2H), 2.78–2.75 (m, 2H), 2.61–2.51 (m, 4H), 2.21 (s, 3H), 2.17 (td, J = 12.0, 1.8 Hz, 2H), 2.06–2.01 (m, 2H), 1.59 (dtt, J = 24.0, 12.0, 3.6 Hz 2H), 1.07 (t, J = 7.8 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 159.6, 147.4, 144.4, 143.5, 140.3, 137.9, 137.8, 128.73, 128.67, 128.6, 128.3, 126.5, 126.0, 114.8, 111.0, 60.4, 57.0, 53.2, 33.8, 30.1, 30.0, 24.1, 19.1, 14.0; several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₇H₃₃N₂O₂, 417.2537; found, 417.2537. Chiral HPLC separation: less polar: [α]²⁰_D +4.50 (c 0.03, CHCl₃) as 98% ee; more polar: [α]²⁰_D −4.43 (c 0.15, CHCl₃) as 97% ee. [CHIRALPAK IG, ethanol/hexane = 3:7].

N-(2-Ethyl-6-methyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]-3-furancarboxamide (32)

White solid; 251.8 mg (63%). MP 39–40 °C. IR (ATR): 1622 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.31–7.25 (m, 3H), 7.21–7.17 (m, 4H), 7.14 (t, J = 1.8 Hz, 1H), 7.13 (d, J = 7.2 Hz, 1H), 6.36 (dd, J = 1.2, 0.6 Hz, 1H), 6.25 (dd, J = 1.8, 0.6 Hz, 1H), 4.29 (tt, J = 12.0, 3.6 Hz, 1H), 3.03–3.02 (m, 2H), 2.77–2.75 (m, 2H), 2.60–2.52 (m, 4H), 2.20 (s, 3H), 2.18–2.14 (m, 2H), 2.06–2.03 (m, 2H), 1.60–1.54 (m, 2H), 1.10 (t, J = 7.2 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 163.5, 144.7, 143.5, 141.9, 140.3, 137.9, 129.0, 128.7, 128.6, 128.3, 126.6, 126.0, 122.6, 111.3, 60.4, 56.9, 53.23, 53.20, 33.7, 30.1, 24.1, 19.1, 13.9; several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₇H₃₃N₂O₂, 417.2464; found, 417.2538. Chiral HPLC separation: less polar: [α]²⁰_D +4.93 (c 0.12, MeOH) as 97% ee; more polar: [α]²⁰_D −5.81 (c 0.215, MeOH) as 97% ee. [CHIRALPAK IG, ethanol/hexane = 2:8].

N-(2-Ethyl-6-methyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]-2-thiophenecarboxamide (33)

White solid; 823.2 mg (85%). MP 50–51 °C. IR (ATR): 1608 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃) δ 7.31 (t, J = 7.8 Hz 1H), 7.28–7.25 (m, 2H), 7.25 (dd, J = 6.0, 1.2 Hz, 1H), 7.21 (d, J = 7.8 Hz, 1H), 7.19–7.17 (m, 3H), 7.13 (d, J = 7.8 Hz, 1H), 6.78–6.76 (m, 2H), 4.33 (tt, J = 12.0, 3.6 Hz, 1H), 3.03–3.01 (m, 2H), 2.77–2.75 (m, 2H), 2.62–2.53 (m, 4H), 2.23 (s, 3H), 2.16 (td, J = 12.0, 1.8 Hz, 2H), 2.06–2.01 (m, 2H), 1.58 (ddd, J = 24.0, 12.0, 3.6 Hz, 2H), 1.09 (t, J = 7.8 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 162.7, 143.9, 140.3, 138.7, 138.4, 137.8, 131.9, 130.9, 129.1, 128.8, 128.7, 128.3, 126.7, 126.6, 126.0, 60.4, 57.5, 53.3, 53.2, 33.8, 30.2, 30.1, 24.1, 19.2, 13.7. HRMS: m/z [M + H]⁺ calcd for C₂₇H₃₃N₂OS, 433.2308; found, 433.2303. Chiral HPLC separation: less polar: [α]²⁰_D +11.2 (c 0.060, MeOH) as 99% ee; more polar: [α]²⁰_D −10.8 (c 0.160, MeOH) as 99% ee. [CHIRALPAK IG, ethanol/hexane = 2:8].

4-Chloro-N-(2-ethyl-6-methylphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]benzamide (34)

White solid; yield: 118.2 mg (64%). MP: 141–143 °C. IR (ATR): 1635 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.26 (t, J = 7.2 Hz, 2H), 7.18 (dt, J = 9.0, 2.4 Hz, 4H), 7.16 (t, J = 2.4 Hz, 1H), 7.11 (t, J = 7.2 Hz, 1H), 7.07 (dt, J = 9.0, 2.4 Hz, 2H), 7.04 (d, J = 7.2 Hz, 1H), 6.97 (d, J = 7.2 Hz, 1H), 4.16 (tt, J = 12.0, 3.9 Hz, 1H), 3.05–3.03 (m, 2H), 2.78–2.76 (m, 2H), 2.63–2.48 (m, 4H), 2.28 (s, 3H), 2.14 (td, J = 12.0, 1.8 Hz, 2H), 1.99–1.92 (m, 2H), 1.75 (quin-d, J = 12.0, 3.6 Hz, 2H), 1.16 (t, J = 7.2 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 169.9, 142.3, 140.4, 139.0, 136.7, 135.6, 135.3, 129.0, 128.7, 128.5, 128.3, 128.2, 127.6, 126.1, 126.0, 60.3, 58.1, 53.3, 33.8, 29.8, 24.0, 19.6, 13.6. HRMS: m/z [M + H]⁺ calcd for C₂₉H₃₄ClN₂O, 461.2354; found, 461.2355. Chiral HPLC separation: less polar: [α]²⁰_D +9.76 (c 0.050, CHCl₃) as >99.9% ee; more polar: [α]²⁰_D −10.3 (c 0.060, CHCl₃) as 99% ee. [CHIRALPAK IA, 2-propanol/hexane = 3:7].

N-(2-Isopropyl-6-methyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]-2-furancarboxamide (35)

White solid; 106.0 mg (38%). MP: 94–95 °C. IR (ATR): 1626 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.33 (dd, J = 1.8, 0.6 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.27–7.25 (m, 2H), 7.21 (dd, J = 7.8, 1.2 Hz, 1H), 7.19–7.17 (m, 3H), 7.12 (ddd, J = 7.8, 1.8, 0.6 Hz, 1H), 6.12 (dd, J = 3.6, 1.8 Hz, 1H), 5.31 (dd, J = 3.6, 0.6 Hz, 1H), 4.24 (tt, J = 12.0, 3.6 Hz, 1H), 3.05–3.01 (m, 3H), 2.77–2.74 (m, 2H), 2.58–2.55 (m, 2H), 2.24 (s, 3H), 2.16 (tdd, J = 12.0, 6.0, 2.4 Hz, 2H), 2.08–2.01 (m, 2H), 1.64 (ddd, J = 24.0, 12.0, 3.6 Hz, 2H), 1.25 (d, J = 7.2 Hz, 3H), 0.82 (d, J = 7.2 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 159.6, 148.3, 147.4, 144.2, 140.5, 137.7, 137.1, 128.9, 128.7, 128.4, 128.3, 125.9, 124.9, 115.2, 110.9, 60.3, 57.6, 53.3, 53.2, 33.8, 30.00, 29.98, 28.2, 24.5, 24.2, 19.3. HRMS: m/z [M + H]⁺ calcd for C₂₈H₃₅N₂O₂, 431.2693; found, 431.2693. Chiral HPLC separation: less polar: [α]²⁰_D +44.48 (c 1.00, CHCl₃) as 99.9% ee; more polar: [α]²⁰_D −46.18 (c 1.00, CHCl₃) as 99.9% ee. [CHIRALPAK IG, ethanol/hexane = 3:7].

N-(2-Fluoro-6-methyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]propionamide (36)

Oil; yield: 77.4 mg (86%). IR (ATR): 1660 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.27–7.21 (m, 3H), 7.19–715 (m, 3H), 7.07 (d, J = 7.8 Hz, 1H), 6.98 (t, J = 7.8 Hz, 1H), 4.57 (tt, J = 12.0, 4.2 Hz, 1H), 3.01–2.97 (m, 2H), 2.75–2.71 (m, 2H), 2.55–2.52 (m, 2H), 2.26 (s, 3H), 2.15–2.11 (m, 2H), 2.03–2.01 (m, 1H), 1.92–1.79 (m, 3H), 1.50 (ddt, J = 24.0, 12.0, 4.2 Hz, 1H), 1.40 (ddd, J = 24.0, 12.0, 4.2 Hz, 1H), 1.03 (t, J = 7.8 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 174.0, 159.6 (d, ¹J_C–F = 245.7 Hz), 140.3, 140.0, 129.3 (d, ¹J_C–F = 8.7 Hz), 128.6, 128.3, 126.7, 126.6, 126.5, 126.0, 113.9 (d, ¹J_C–F = 21.6 Hz), 60.5, 54.7, 53.2, 53.1, 33.8, 30.0, 29.7, 27.7, 18.3, 9.2; several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₃H₃₀FN₂O, 369.2337; found, 369.2338.

N-(2-Chlorophenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]propionamide (37)

White solid; yield: 46.1 mg (31%). MP: 83–84 °C. IR (ATR): 1659 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.50–7.49 (m, 1H), 7.33–7.29 (m, 2H), 7.26 (t, J = 7.8 Hz, 2H), 7.21–7.19 (m, 1H), 7.17–7.15 (m, 3H), 4.66 (tt, J = 12.0, 4.2 Hz, 1H), 3.04–3.01 (m, 1H), 2.97–2.94 (m, 1H), 2.75–2.72 (m, 2H), 2.55–2.52 (m, 2H), 2.18 (td, J = 12.0, 1.8 Hz, 1H), 2.12 (td, J = 12.0, 1.8 Hz, 1H), 2.02–2.00 (m, 1H), 2.01 (dq, J = 16.8, 7.8 Hz, 1H), 1.93–1.90 (m, 1H), 1.86 (dq, J = 16.8, 7.8 Hz, 1H), 1.59 (ddd, J = 24.0, 12.0, 4.2 Hz, 1H), 1.26 (ddd, J = 24.0, 12.0, 4.2 Hz, 1H), 1.04 (t, J = 7.8 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 173.5, 140.2, 137.1, 135.1, 132.1, 130.7, 129.6, 128.6, 128.4, 127.7, 126.0, 60.5, 53.8, 53.13, 53.05, 33.8, 31.2, 29.1, 28.0, 9.3. HRMS: m/z [M + H]⁺ calcd for C₂₂H₂₈ClN₂O, 371.1885; found, 371.1886.

N-(2-Chloro-6-fluorophenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]propionamide (38)

Oil; yield: 28.8 mg (63%). IR (ATR): 1666 cm^–1 (CO). ¹H NMR (600 MHz, CDCl₃): δ 7.32–7.29 (m, 2H), 7.28–7.25 (m, 2H), 7.19–7.15 (m, 3H), 7.13–7.10 (m, 1H), 4.60 (tt, J = 12.0, 3.6 Hz, 1H), 3.02–2.98 (m, 2H), 2.76–2.73 (m, 2H), 2.55–2.53 (m, 2H), 2.13 (ddd, J = 24.0, 12.0, 3.6 Hz, 2H), 2.06–2.03 (m, 1H), 2.01–1.97 (m, 1H), 1.93 (dq, J = 16.2, 7.2 Hz, 1H), 1.87 (dq, J = 16.2, 7.2 Hz, 1H), 1.52 (ddt, J = 24.0, 12.0, 3.6 Hz, 1H), 1.42 (ddd, J = 24.0, 12.0, 3.6 Hz, 1H), 1.05 (t, J = 7.2 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 173.5, 160.3 (d, ¹J_C–F = 248.6 Hz), 140.2, 136.7, 130.1 (d, ¹J_C–F = 8.6 Hz), 128.6, 128.4, 126.4, 126.3, 126.1, 126.06, 126.04, 126.01, 115.4 (d, ¹J_C–F = 21.6 Hz), 60.4, 54.9, 53.2, 53.1, 33.8, 29.9, 29.6, 27.7, 9.1; some C–F couplings are not described and several signals overlapped. HRMS: m/z [M + H]⁺ calcd for C₂₂H₂₇ClFN₂O: 389.1790; found: 389.1791.

Single-Crystal X-ray Analysis

The crystal structures of compound 34 were obtained via single-crystal X-ray analysis. The typical crystal data for 34 are shown in the Supporting Information.

Stereochemical Stability of 8, 23, 24, and 30–35

The racemization of 8, 23, 24, and 30–35 at 37, 80, or 100 °C in toluene was examined as previously described.³⁵ The method for determining the ΔG^⧧ is shown in the Supporting Information.

In Vitro MOR Assays

MOR assays were conducted to determine the agonistic/antagonistic efficacy of the compounds against the MOR. A stable MOR-expressing cell strain (MOR-CHO) was generated by transfecting a human MOR (CAT no. RG202243, OriGene Technologies, Inc., Rockville, MD, USA) and a human Gα16 (CAT no. RC202243, OriGene Technologies, Inc.) into the CHO cell line (CCL-61, ATCC) using the 4D-Nucleofector system (Lonza, Basel, Switzerland). The intracellular calcium concentration was measured. Cells were seeded (5 × 10⁴ cells/well) into 96-well black clear bottom plates (Greiner, Frickenhausen, Germany) and cultured at 37 °C under 5.0% CO₂ conditions. After 24 h, the medium was replaced with component B (1× Hank’s balanced salt solution + 20 mM HEPES buffer, pH 7.4) with 2 mM probenecid, and a Ca²⁺ indicator dye (FLIPR Calcium 4 Assay kit R8141, Molecular Devices, San Jose, CA, USA) was added to the wells. After 1 h, the compounds investigated in component B were added to the wells.

To determine the MOR antagonistic effects of the test compounds, we examined the pretreatment effects of the compounds or the opioid antagonist naloxone on the fentanyl-mediated increase in calcium utilization. Nine concentrations (0.0000064, 0.000032, 0.00016, 0.0008, 0.004, 0.02, 0.1, 0.5, and 2.5 μM) of the test compounds and naloxone were added to the culture plates; after 10 min of incubation (at 37 °C), the effect of fentanyl (0.025 μM) was assessed. Fentanyl-analogue-induced fluorescence changes were measured via a Flexstation III and the SOFTMAX PRO software (Molecular Devices). The following measurement conditions were maintained: excitation wavelength: 485 nm, fluorescence wavelength: 525 nm, and cutoff value: 515 nm. The obtained values were calculated to determine the EC₅₀ or IC₅₀ values using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA).

In Vitro Competitive Radioligand Binding Assay

The in vitro binding affinities of the fentanyl analogues to MOR were screened at Eurofins Panlabs Discovery Services Taiwan, Ltd. (New Taipei City, Taiwan). Human recombinant MORs expressed in CHO-K1 cells were used in modified Tris-HCl buffer pH 7.4. An 11 μg aliquot was incubated with 0.6 nM [³H]-diprenorphine for 60 min at 25 °C (membrane proteins may vary from lot to lot, and the concentration can be adjusted if necessary.). Nonspecific binding was estimated in the presence of 10 μM naloxone. After incubation, the samples were rapidly filtrated through buffer-soaked glass fiber filters under vacuum and rinsed several times with an ice-cold buffer in a 48- or 96-sample cell harvester. The experimental conditions are outlined in Supporting Information Table S1. The residues were evaluated for radioactivity in a scintillation counter using a scintillation cocktail. The IC₅₀ values (the concentration causing half-maximal inhibition of control-specific binding) were computed from the displacement data using a nonlinear, least-squares regression analysis (MathIQ: ID Business Solutions, Ltd., Woking, UK). The concentration and the Eurofins Cerep’s historical value for the dissociation constant of the radioligand were deduced. The relative affinities (Ki) were derived from the IC₅₀ values using the Cheng–Prusoff equation.³⁶

In Vivo Ethopharmacological Experiments

All animal experiments were approved by the Experimental Animal Care and Use Committee of the National Institute of Neuroscience, National Center of Neurology and Psychiatry (#2021001R4). Specific pathogen-free, 4 to 5 week-old male mice were supplied by CLEA Japan (Tokyo, Japan). The mice were of the well-characterized outbred strain Jcl/ICR. At the commencement of the experiment, the mice weighed 20–25 g. The animals were housed in sterile and ventilated individual cages, exposed to 12/12 h light/dark cycles with dawn/dusk phases, and provided sterilized food and water ad libitum. The mice were segregated into groups of 12 and treated with either the enantiomer of compound 31, “(+)-31” (2 mg/kg), or naloxone (2 mg/kg) 30 min before morphine (10 mg/kg) administration. The control group was injected with saline, and all drugs were administered intraperitoneally. Test articles and comparators were reconstituted and diluted in 0.9% saline (OTSUKA, Tokyo, Japan). After allowing adaptation to the environment for 3 h, the locomotor activity under the influence of drugs was measured for 120 min using an animal-movement analysis system (ACTIMO-100 Bio Research Center, Tokyo, Japan).

Calculation of DFT-Based ECD

Compound aR-35′ was built on Spartan’20 (Wave function, Inc., Irvine, CA, USA) and subjected to a conformational search with molecular mechanics MMFF94,³⁷ with which the program is equipped, using the threshold at 40 kJ/mol from the global minimum conformer and successive conformer narrowing based on HF/3-21G (threshold: 40 kJ/mol) and ωB97X-D/6-31G* (threshold: 10 kJ/mol) to yield 23 stable conformers. Then, the free energies based on ωB97X-D/6-31G* and B3LYP/6-31G*³⁸ of the remaining conformers were calculated with vibrational analyses (Supporting Information Tables S2–S4). ECD spectral calculations were performed with CAM-B3LYP/def2-TZVP²⁹ for the stable conformers within 8 kJ/mol of free energy by either ωB97X-D/6-31G* or B3LYP/6-31G* (Supporting Information Table S5). The UV and ECD spectra were constructed based on the frequencies and oscillator and rotary strengths acquired by implementing the NORMDIST function in Microsoft Excel (Microsoft Corp., Redmond, WA, USA). The widths and signal intensities were adjusted to appropriately reproduce the spectra. The UV and ECD spectra of the individual conformers were averaged considering the Boltzmann distribution. The wavelengths of the UV and ECD spectra were calibrated to align with the experimental UV spectra (+27 nm), and the same value was used to correct the wavelength of the ECD spectra. The rotatory strength values of aR-35′ were multiplied by −1 to create the ECD spectra of compound aS-35′. The obtained oscillator and rotatory strengths and calculation details are described in Supporting Information Table S5.

Molecular Docking Studies

A 3D model structure of the target protein was engineered for the docking analysis of the ligand and MOR (Protein Data Bank: 5C1M).³² First, the chemical structure of compound 31 was generated, and a simplified molecular input line entry system (SMILES) file was created that was used as input in OpenBabel’s Confab method to generate 488 protein conformers.³⁹ After performing structural optimization calculations at the HF/STO-3G level using Gaussian 16,⁴⁰ the conformers that exceeded an energy threshold of 10 kJ/mol compared to the most stable conformer were excluded. The relaxed structures and the most stable conformer were classified into 50 clusters based on their root-mean-square deviation value (Supporting Information Table S6). The AutoDock Vina-based docking tool, integrated into Yet Another Scientific Artificial Reality Application (YASARA) structure, was used to perform rigid docking of the 50 conformers to the orthosteric site of the MOR.⁴¹ Candidate poses with docking scores <8.0 kcal/mol were excluded, yielding 23 binding poses.

MD Simulation

Membrane protein MD simulations were performed for the 23 candidate poses using the YASARA structure to validate their binding poses.⁴¹ The force field used was Amber14, which conducts 10 ns of MD simulations in the fast mode (captures snapshots at 250 ps intervals). Using the boundary elements method with the YASARA2 force field, the average binding energies were obtained from snapshots of the second half of the entire MD simulation (Supporting Information Table S7). Subsequently, MD simulations were performed for the highest binding energies for aS and aR and prolonged to 100 ns under the same conditions. Finally, pharmacophore analyses of the 100 ns MD trajectories were conducted using LigandScout.⁴²

Glossary

Abbreviations

Ac

acetyl

Ar

aryl

ATR

attenuated total reflection

CHO

Chinese hamster ovary

DMF

N,N-dimethylformamide

ECD

electronic circular dichroism

Et

ethyl

G

Gibbs free energy

HPLC

high-performance liquid chromatography

HRMS

high-resolution mass spectroscopy

i-Pr

isopropyl

IR

infrared spectroscopy

M

mol/L

Me

methyl

MRP

morphine

NLX

naloxone

n-Pr

n-propyl

N.C.

not calculated

NOESY

nuclear Overhauser effect spectroscopy

ppm

parts per million

NMR

nuclear magnetic resonance

PDB

Protein Data Bank

UV

ultraviolet

Tyr

tyrosine

Met

methionine

Ile

isoleucine

Trp

tryptophan

Val

valine

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c00935.

• NOESY spectra of compound 17, X-ray structural analysis of compound 34, stereochemical stability of compounds 8, 23, 24, and 30–35, in vitro MOR assays, in vitro competitive radioligand binding assay, calculation of DFT-based ECD spectra, docking study, MD simulation, ¹H, ¹³C, 2D NMR, and HRMS spectra, and HPLC chromatograms of target compounds (PDF)

• Predicted aR-31 binding configurations in the MOR (PDB)

• Predicted aS-31 binding configurations in the MOR (PDB)

• X-ray structural analysis of compound 34 (CIF)

• SMILES molecular formula strings (CSV)

Author Contributions

H.A., H.N., T.O., and H.T. contributed to the study conception and design. Material preparation and data collection and analysis were performed by H.A., R.T., S.K., T.T., H.S., H.T., K.N., and K.M. Pharmacological experiments were performed by M.F. and K.T. Calculations were performed by R.T., M.H., and T.T. The first draft of the manuscript was written by H.A. and H.T. All authors read and approved the final manuscript.

The authors declare no competing financial interest.