Structure–Activity Relationship Study of CYM51010, an Agonist for the µ–δ Opioid Receptor Heterodimer

Abstract

Although opioid analgesics are indispensable in treating pain, these drugs are accompanied by life-threatening side effects. While clinically relevant opioid drugs target the µ opioid receptor (MOR), a heterodimer between the MOR and the δ opioid receptor (DOR) has emerged as another target to develop safer analgesics. Although some heterodimer-preferring agonists have been reported so far, it is still difficult to activate the MOR/DOR heterodimer selectively in the presence of MOR or DOR monomers/homodimers. To gain insights to develop selective agonists for MOR/DOR, herein we prepared analogs of CYM51010, one of the reported heterodimer-preferring agonists, and collected structure–activity relationship information. We found that the ethoxycarbonyl group was needed for the activity for the heterodimer, although this group could be substituted with functional groups with similar sizes, such as an ethoxycarbonyl group. As for the acetylaminophenyl group, not a type of substituent, but rather a substituent located at a specific position (para-position) was essential for the activity. Changing the linker length between the acetylaminophenyl group and the piperidine moiety also had deleterious effects on the activity. On the other hand, the substitution of the acetylamino group with a trifluoroacetylamino group and the substitution of the phenethyl group with a benzyl group diminished the activities for the monomers/homodimers while keeping the activity for MOR/DOR, which enhanced the selectivity. Our findings herein will play an important role in developing selective agonists for MOR/DOR and for elucidating the physiological roles of this heterodimer in analgesic processes and in the establishment of side effects.

Introduction

The opioid receptor is a member of the G protein-coupled receptor (GPCR) family which is subdivided into three types: µ, δ, and κ opioid receptors (MOR, DOR, and KOR, respectively). Clinically relevant narcotic analgesics like morphine and fentanyl exert their antinociceptive activity by binding to and activating MOR.^1,2) Today, opioid drugs are indispensable in controlling intractable pain. However, chronic treatment leads to the development of analgesic tolerance and physical dependence. Rewarding effects and respiratory depression are also other serious adverse effects,³⁾ which drive many people to overdose death.⁴⁾ To gain safer, side-effect-free analgesics, many efforts have been undertaken, including the identification of G protein-biased MOR agonists,⁵⁾ targeting particular splice variants of MOR,^6,7) and the usage of the agonists for DOR,^8,9) KOR,^10,11) or both of them (i.e., dual agonists).¹²⁾ Development of ligands for a heterodimer composed of MOR and DOR (MOR/DOR) described hereafter is also one of these strategies.

The modulatory roles of DOR toward MOR have been suggested for decades. For example, enhancement of morphine analgesia by the injection of leucine enkephalin, an endogenous DOR agonist, was reported as early as 1979,¹³⁾ and that the development of analgesic tolerance and dependence for morphine is attenuated in the absence of DOR was reported in the late 1990s.^14,15) Various GPCRs are now known to interact with each other to form homo- and heterodimers,^16,17) and these phenomena are discussed in association with the formation of a heterodimer between MOR and DOR. MOR/DOR in cells exogenously expressing both protomers and in endogenous mouse tissue has been detected by means of co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays.^18,19) In addition, some evidence shows that this heterodimer exists in the dorsal root ganglion neuron which plays a pivotal role in pain transmission.^20,21) Although the X-ray crystal structure or cryo-EM structure of MOR/DOR has not been reported so far, a computational analysis suggests that the dimer interface is mainly composed of transmembrane 1 and 2, and helix 8 of the two protomers.²²⁾

The first-generation ligands for MOR/DOR were designed by bridging MOR agonist and DOR antagonist by a linker moiety^23,24) (Fig. 1(A)). One of these bivalent series, MDAN-21,²³⁾ showed potent antinociceptive activity without developing antinociceptive tolerance and physical dependence. Although this compound is far beyond the rule-of-five area proposed by Lipinski,²⁵⁾ the result shed light on the possibility that an ideal medicine for pain could be developed by targeting MOR/DOR. A later high-throughput screening project afforded a more druglike compound CYM51010 (Fig. 1(B)) whose activity is biased toward MOR/DOR compared with monomeric or homodimeric MOR or DOR.²⁶⁾ CYM51010 showed antinociceptive activity as potent as morphine with a lesser extent of antinociceptive tolerance and attenuated neuropathic pain which are difficult to treat by using clinically relevant opioids.^26–29) In a later study, MP135 (Fig. 1(C)), a G protein-biased and a more selective agonist for MOR/DOR compared to CYM51010, was identified from a library composed of carfentanyl amides which are analogous to CYM51010.³⁰⁾ Although MP135 showed antinociceptive activity as expected, it retained respiratory depression and rewarding effects which are unwanted side effects of conventional opioid drugs. Since MP135 retains activity for MOR, these effects could be attributed to the activation of MOR. However, the possibility that a safer analgesic medicine may be obtained by developing MOR/DOR agonists needs more investigation. Concerning this point, some hypotheses have suggested that the lower antinociceptive tolerance of these compounds is attributed to the inhibition of the endocytosis of the heterodimer³¹⁾ and that the change of the side effects profile is attributed to the change of the downstream signal transduction pathway that occurs upon heterodimerization.^18,20,32) In contrast, other research suggested that the MOR/DOR heterodimer is increased in pathological conditions like morphine dependence,³³⁾ plays a role in developing morphine tolerance,³⁴⁾ and, indeed, has pronociceptive effects.^34,35) From these insights, antagonists for MOR/DOR are suggested to be beneficial in treating pain.³⁵⁾ To gain maximal benefit from MOR/DOR-targeting compounds in treating pain conditions, more knowledge about this heterodimer, especially in vivo, is urgently needed. However, the shortage of research tools hampers the progress in this field. Some means are available to interfere with the heterodimer which include a selective antibody,³³⁾ dimerization-interfering peptide,³⁴⁾ and a specific antagonist which is less druglike due to the bivalent and peptidic nature³⁶⁾ (Fig. 1(D)). However, the means to selectively activate the MOR/DOR heterodimer in the presence of monomer and homodimer is lacking at present. In this study, with the aim to develop selective agonists for MOR/DOR, we synthesized a series of CYM51010 analogs and collected structure–activity relationship information to elucidate the key structure of CYM51010.

Fig. 1. Chemical Structures of the Previously Reported Ligands for MOR/DOR

Results and Discussion

In-House Experiment to Compare the Activity/Selectivity of CYM51010 and MP135

To determine the starting point for the structural development of MOR/DOR selective agonists, we first compared the activities of known monovalent heterodimer-preferring agonists CYM51010 and MP135 for the cells coexpressing MOR and DOR and the cells expressing either MOR or DOR alone. In these assays, we used the CellKey™-based assay system as in our previous reports^37,38) which detects the behavioral changes of the cells upon agonist treatment as the changes of electrical impedance of the cells.³⁹⁾ Although MP135 was reported to be more potent and selective than CYM51010 in the [³⁵S]GTPγS binding assays,³⁰⁾ they were not so different in our CellKey™-based assays (Table 1). The concepts of ligand efficiency and lipophilic efficiency state that the increase in molecular weight or lipophilicity without elaboration of the affinity/activity should be avoided.^40–42) Hence, considering the drug-likeness parameters of the two compounds, we chose CYM51010, with the smaller molecular weight, as the starting point for the structural development.

Table 1. Comparison of the Activity and the Drug-Likeness Parameters between CYM51010 and MP135

Compound	Molecular weight	CLogP	MOR/DOR		MOR		DOR
			EC50 (µM)	Emax (%)	EC50 (µM)	Emax (%)	EC50 (µM)	Emax (%)
CYM51010	409	4.04	0.0457	100	0.0457	113	0.288	103
MP135	516	5.14	0.0460	97.8	0.112	93.8	1.25	93.1

The CLogP values were calculated by using ChemDraw Professional version 22.2. The E_max values were calculated as the percentage of maximum response caused by CYM51010 (for MOR/DOR), DAMGO (for MOR), and SNC80 (for DOR).

Below we describe the preparation of CYM51010 analogs whose three regions were sequentially modified: the ester moiety (northern part), the acetylaminobenzyl moiety (western part), and the phenethyl moiety (eastern part) (Fig. 2).

Fig 2. Three Modified Parts of CYM51010

Chemistry

Preparation of CYM51010

CYM51010 used in the above-described pharmacological evaluation was prepared as shown in Chart 1. First, an S_N2 reaction between phenethyl bromide (1) and the enolate prepared from 2 was performed to afford compound 3. N-tert-Butoxycarbonyl group deprotection of 3 and the subsequent reductive amination reaction with aldehyde 4 gave the desired CYM51010. MP135 was synthesized according to the literature procedure.³⁰⁾

Chart 1. Preparation of CYM51010

Boc: tert-butoxycarbonyl; THF: tetrahydrofuran; TFA: trifluoroacetic acid.

Modification of the Northern Part

We first designed compounds in which the ethoxycarbonyl group of CYM51010 was replaced with a hydrogen or a hydroxy group (compounds 10 and 12 in Chart 2). A commercially available compound 5 was alkylated with a Grignard reagent to afford 6. Dehydration of this compound under acidic conditions provided a regioisomeric mixture of alkenes 7 and 8. Simultaneous deprotection of the N-benzyl group and reduction of the double bonds gave 9, and the subsequent reductive amination gave the designed compound 10. Compound 12 was prepared from compound 6 by two similar steps.

Chart 2. Substitution of the Ethoxycarbonyl Moiety with a Hydrogen (Compound 10) or a Hydroxy Group (Compound 12)

The numbers of the compounds used in the subsequent biological evaluations are underlined.

We also prepared compounds in which the ethoxycarbonyl group is replaced with other functional groups (Chart 3). A two-step modification of the N-substituent of compound 3 (synthesized in Chart 1) gave 15, and the Bechamp reduction gave 16. The ethoxycarbonyl moiety of 16 was reduced to afford 17, and both the amino and the hydroxy groups of this compound were acetylated to give 18. The O-acetyl group was selectively hydrolyzed to provide 19, and a Swern oxidation gave the intermediate 20. After rough purification, this aldehyde was further converted by two sequential modifications. A nucleophilic addition with a Grignard reagent gave alcohol 21, which was further oxidized to afford ketone 22. Alternatively, a Wittig reaction of 20 gave Z-olefin 23 which was reduced to yield 24.

Chart 3. Synthesis of CYM51010 Analogs 18, 19, and 21–24

The numbers of the compounds used in the subsequent biological evaluations are underlined. DMAP: 4-dimethylaminopyridine; DMP: Dess–Martin periodinane.

Modification of the Western Part: Length of the Linker and Substitution Pattern on the Benzene Ring

For the acetylaminobenzyl group of CYM51010, we modified the linker length between the piperidine and the benzene rings and the substitution pattern of the disubstituted benzene ring. The structures of the designed compounds are shown in Fig. 3.

Fig. 3. Structures of the Designed Compounds

Compounds 25 were prepared by the sequential reaction of S_NAr reactions of fluoronitrobenzene with compound 13 (synthesized in Chart 3), Bechamp reduction, and acetylation reactions (Chart 4 (A)). Compounds 26 were synthesized by S_N2 reactions of nitrobenzyl bromide with compound 13, and similar functional group modifications as for compounds 25 (Chart 4 (B)). Compounds 27o and 27p were prepared by conjugated additions of 13 to o- or p-nitrostyrenes, and the subsequent functional group modifications (Chart 4 (C)). In the case of the meta-counterpart, the first conjugated addition reaction did not proceed as expected due to the low reactivity of m-nitrostyrene. Hence, mesylate 39 was prepared by 5 steps from m-nitrophenylacetic acid (34) and compound 27m was synthesized by an S_N2 reaction of mesylate 39 with amine 13.

Chart 4. Modification of the Substitution Pattern of the Benzene Ring and the Linker Length

The numbers of the compounds used in the subsequent biological evaluations are underlined. DBU: diazabicyclo[5.4.0]-7-undecene; Ms = methanesulfonyl.

Next, the acetylamino moiety of CYM51010 was diversified into other functional groups (Chart 5). First, the secondary amide was methylated with methyl iodide to give 40a, albeit in low yield. Replacement of the acetylamino group with a methoxycarbonyl, a methoxy, or a benzyloxy group was accomplished by replacing the aldehyde in the reductive amination reactions to afford compounds 40b–d. Compound 40d was further converted by hydrogenolysis to 40e. Aniline 16 in Chart 3 was mesylated to give sulfonamide 40f or sulfonimide 40g, depending on the timing of the addition of the reagents (see experimental section for details). Finally, acylation of 16 with acid anhydride or acid chloride gave 40h–j.

Chart 5. Modification of the Acetylamino Group of CYM51010

The numbers of the compounds used in the subsequent biological evaluations are underlined.

Modification of the Eastern Part

Diversification of the phenethyl moiety of CYM51010 is depicted in Chart 6. This was accomplished by treating the enolate derived from compound 2 (in Chart 1) with various alkylating reagents, and modifying the substituents on the nitrogen atoms of the resultant compounds 42a–d in a similar manner as before. Compound 44e was prepared directly from 2 without the alkylation step.

Chart 6. Modification of the Phenethyl Group of CYM51010

The numbers of the compounds used in the subsequent biological evaluations are underlined.

Biological Evaluation

As the first screening, we determined the activities of the prepared compounds for MOR/DOR, MOR, and DOR at a concentration of 10 µM and compared them with those of CYM51010 (Figs. 4–7). The size of the substituents in the northern part seems to be correlated with the activities not only for the heterodimer but also for each monomer (compounds 10, 12, 19, and 18, in order). A substituent size as large as the ethoxycarbonyl group is enough (compounds 21–24), and the oxygen atoms are not necessary for the activity (compounds 23 and 24). At this stage of structural development, no enhancement of activity or selectivity over and above CYM51010 was observed.

Fig. 4. Relationships between the Structure of the Northern Part and Activity for (A) MOR/DOR, (B) MOR, and (C) DOR

The concentration of each of the compounds was 10 µM.

Next, we determined the effects of modifications in the western part (the substitution pattern on the benzene ring and the length of the linker moiety, Fig. 5). Both the shorter (25o, 25m, and 25p) and the longer linkers (27o, 27m, and 27p) showed deleterious effects on the activity for MOR/DOR. Change of the substitution pattern from para to ortho or meta diminished the activity for DOR (26o and 26m), while retaining the activity for MOR and slightly decreasing the activity for MOR/DOR. None of these modifications led to the enhancement of activity or selectivity for MOR/DOR.

Fig. 5. Effects of the Substitution Pattern on the Benzene Ring and the Linker Length on the Activity for (A) MOR/DOR, (B) MOR, and (C) DOR

The concentration of each compound was 10 µM.

Fixing the substitution pattern to para and the linker length to n = 1 which are identical with CYM51010, we determined the effects of the substituent on the benzene ring in the western part (Fig. 6). Methylation of the secondary amide of CYM51010 (compound 40a) diminished the activity for MOR/DOR. The methyl ester 40b without an active proton also showed weaker activity than CYM51010. These results imply that a hydrogen bond donor would be required for the activity. This observation is consistent with the results that the sulfonamide 40f with a hydrogen bond donor showed higher activity than sulfonimide 40g which lacked a hydrogen bond donor. Substitution of the acetyl group of CYM51010 by the trifluoroacetyl group (i.e., compound 40h) increased the activity for MOR/DOR while slightly diminishing the activity for MOR and retaining the activity for DOR. Substitution to a trichloroacetyl or benzoyl group (40i and 40j, respectively) did not enhance the activity for the MOR/DOR. While substitution of the acetylamino group of CYM51010 with hydroxy or benzyloxy groups (40e and 40d, respectively) diminished the activity for MOR/DOR, substitution with a methoxy group (i.e., compound 40c) retained the activity, albeit in the absence of a hydrogen bond donor. Some beneficial effects of downsizing the acetyl group may have compensated for the absence of a hydrogen bond donor. Compound 40c showed a weaker activity for DOR and seemed to be more selective for MOR/DOR than CYM51010.

Fig. 6. Effects of the Substituents on the Benzene Ring on the Activity for (A) MOR/DOR, (B) MOR, and (C) DOR

The concentration of each compound was 10 μM.

Finally, we determined the effects of the structure of the eastern part on the activity (Fig. 7). Compounds 44e and 44a showed much lower activity than CYM51010, meaning that the phenylalkyl groups are necessary for the activity. Compound 44b retained the activity for MOR/DOR while slightly diminishing the activity for MOR and DOR. Compound 44c showed a trend toward greater selectivity for MOR/DOR. As for compound 44d, although the activity for MOR was diminished while that for MOR/DOR was retained, that for DOR was also retained. Hence, from the phenylalkyl series, compounds 44b and 44c were selected for further evaluation (Fig. 8).

Fig. 7. Effects of the Structure of the Phenethyl Moiety of CYM51010 on the Activity for (A) MOR/DOR, (B) MOR, and (C) DOR

The concentration of the compounds was 10 µM.

Fig. 8. Concentration-Dependent Activities of the Selected Compounds on (A) MOR/DOR, (B) MOR, and (C) DOR

As CYM51010 worked as a full agonist for all three types of receptors in the previous assays (Figs. 4–7), in this concentration-dependent assay, we selected this compound as the standard.

To gain more insight into the selectivity between the heterodimer and the monomers/homodimers, we intended to determine the E_max and EC₅₀ values of the selected compounds 40h, 40c, 44b, and 44c. Unfortunately, the concentration–response curves of these compounds did not reach a plateau at the maximal concentration of the assay range (10⁻⁵ M), making it impossible to determine these values. Hence, we treated the receptor-expressing cells with varying concentrations of the compounds and compared the responses with those evoked by CYM51010 (Fig. 8). For MOR/DOR, compound 44c showed a trend toward exceeding the maximal response caused by CYM51010. At this stage of the investigation, we cannot determine whether the E_max value of 44c is greater than CYM51010 or whether this result is attributed to the experimental variations. The other three compounds displayed similar potency and efficacy for the heterodimer as did CYM51010, although 40c seems to be less effective than the other compounds (compare the activities at 10⁻⁶ M). As for the activities for MOR or DOR monomer/homodimer, compounds 40c and 44c retained the activity for the MOR and the DOR, respectively. Hence, the selectivities of these compounds for the heterodimer will not be improved from the parental CYM51010. By contrast, compound 40h showed reduced activity toward the MOR and the DOR. The activity of 44b for MOR was also remarkably reduced, although the activity toward DOR was not very different. These results suggest that introducing the trifluoroacetylaminobenzyl group at the western part and the benzyl group on the eastern part will direct future studies to obtain selective agonists for the heterodimer.

Conclusion

A selective agonist for MOR/DOR is urgently needed to elucidate the role of this heterodimer in the analgesic processes and the establishment of unwanted side effects in vivo. To achieve this goal, in this study, we synthesized CYM51010 analogs whose three parts were modified and performed structure–activity relationship studies. In the northern part, a functional group as large as an ethoxycarbonyl group was indispensable for the activity, while for the western part, both the para-substituted acetylaminophenyl group and the methylene linker between the acetylaminophenyl group and the piperidine moiety were required. Substitution of the acetylamino group in the western part with a trifluoroacetylamino group and substitution of the phenethyl group in the eastern part with a benzyl group diminished the activities for MOR and DOR monomers/homodimers while retaining the activity for the heterodimer, which will enhance the selectivity. The information obtained herein provides clues to design more selective agonists for MOR/DOR in future studies.

Experimental

Organic Synthesis

General Remarks

The reagents and solvents were obtained from commercial suppliers and used without further purification. IR spectra were recorded with a JASCO FT/IR-460Plus spectrometer. ¹H- and ¹³C-NMR spectra were recorded with an Agilent Technologies VXR-400 NMR spectrometer. Chemical shifts are reported as δ values (ppm) referenced to tetramethylsilane. Mass spectra were obtained with a JMS-AX505HA, JMS-700 MStation, or JMS-T100LP spectrometer by applying the electrospray ionization (ESI). Saturated aqueous sodium hydrogen carbonate solution added to quench the reactions was added until the pH of the water layer was adjusted to 9. The reactions were monitored by TLC on Merck silica gel (Art. 5715). Column chromatography was performed by using silica gel unless otherwise noted. Silica gels were purchased from Fuji Silysia (CHROMATOREX^® PSQ 60B (60 µm) and CHROMATOREX^® NH-DM2035). All the reactions were performed under an argon atmosphere.

Preparation of CYM51010

1-(tert-Butyl) 4-Ethyl 4-Phenethylpiperidine-1,4-dicarboxylate (3)

To a flame-dried flask were added tetrahydrofuran (135 mL), n-butyl lithium (1.57 M in n-hexane, 16.6 mL, 23.3 mmol) and diisopropylamine (3.6 mL, 23 mmol), and the mixture was stirred at −78 °C for 15 min. Then the mixture was warmed up to 0 °C and stirred for 30 min. After cooling down to −78 °C, 1-(tert-butyl) 4-ethyl piperidine-1,4-dicarboxylate (2, 5.8 mL, 23 mmol) was added, and the mixture was stirred at this temperature for 15 min and then at 0 °C for 30 min. Then the mixture was cooled down to −78 °C and (2-bromoethyl)benzene (1, 3.00 mL, 22.2 mmol) was added. The mixture was stirred at this temperature for 1 h, and then at 0 °C for 14 h while allowing the mixture to warm to ambient temperature. Then saturated aqueous ammonium chloride was added, and the mixture was extracted with ethyl acetate three times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (n-hexane/diethyl ether, 4 : 1) gave the title compound (6.41 g, 17.8 mmol, 80.2%) as a pale brown oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.30 (t, J = 7.1 Hz, 3H), 1.36–1.51 (m, 2H), 1.45 (s, 9H), 1.78–1.87 (m, 2H), 2.12–2.22 (m, 2H), 2.48–2.56 (m, 2H), 2.92 (br t, 2H), 3.89 (br s, 2H), 4.20 (q, J = 7.1 Hz, 2H), 7.11–7.21 (m, 3H), 7.24–7.30 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz,) δ: 14.4, 28.5, 30.5, 33.4, 41.3, 42.2, 45.6, 60.6, 79.4, 126.0, 128.3, 128.4, 141.7, 154.9, 175.3; IR (neat) cm⁻¹: 2976, 1725, 1695, 1423, 1365, 1249, 1175, 1094, 1029, 971, 867, 762, 700; ESI-MS m/z: 384.2139 (Calcd for C₂₁H₃₁NNaO₄: 384.2151).

Ethyl 1-(4-Acetamidobenzyl)-4-phenethylpiperidine-4-carboxylate (CYM51010)

To a solution of 3 (6.41 g, 17.8 mmol) in dichloromethane (45 mL) was added trifluoroacetic acid (45 mL) dropwise at 0 °C. After stirring at ambient temperature for 4 h, the mixture was concentrated under reduced pressure. The residue was taken up with dichloromethane and washed with saturated aqueous sodium hydrogen carbonate solution. The aqueous layer was then extracted with dichloromethane six times, and the combined organic layers were washed with brine twice, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was used for the next reaction without further purification. To this residue were added dichloromethane (100 mL), N-(4-formylphenyl)acetamide (4, 4.97 g, 30.5 mmol), acetic acid (1.7 mL, 31 mmol), and sodium tri(acetoxy)borohydride (6.47 g, 30.5 mmol). The mixture was stirred at ambient temperature for 12 h. Then, saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with dichloromethane three times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (n-hexane/ethyl acetate, 1 : 4) gave the title compound (5.90 g, 14.4 mmol, 81.1% for 2 steps) as a colorless solid. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.28 (t, J = 7.1 Hz, 3H), 1.47–1.59 (m, 2H), 1.77–1.85 (m, 2H), 2.07 (t, J = 10.1 Hz, 2H), 2.12–2.24 (m, 4H), 2.46–2.55 (m, 2H), 2.69 (br d, 2H), 3.40 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 4.83 (s, 2H), 7.09–7.19 (m, 3H), 7.21–7.29 (m, 4H), 7.43 (d, J = 8.4 Hz, 2H), 7.58 (br s, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.5, 24.6, 30.7, 33.6, 42.3, 45.5, 51.2, 60.4, 62.9, 119.9, 126.0, 128.4, 128.5, 129.8, 134.6, 136.9, 142.1, 168.5, 176.0; IR (film) cm⁻¹: 3583, 2926, 1723, 1667, 1604, 1538, 1410, 1369, 1315, 1179, 1027; ESI-MS m/z: 409.2490 (Calcd for C₂₅H₃₃N₂O₃: 409.2491).

Structural Diversification of the Northern Part

1-Benzyl-4-phenethylpiperidin-4-ol (6)

To a solution of 1-benzyl-4-piperidone (5, 300 mg, 1.59 mmol) in tetrahydrofuran (4 mL) was added phenethylmagnesium bromide (1.0 M in tetrahydrofuran, 8.4 mL, 8.4 mmol) dropwise at 0 °C, and the mixture was stirred at ambient temperature for 2 h. Then, saturated aqueous sodium hydrogen carbonate solution was added, and the precipitated solid was removed by filtration through a Celite pad. The solution was extracted with chloroform six times, and the combined organic layers were washed with brine (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (n-hexane/ethyl acetate, 1 : 1) gave the title compound (254 mg, 0.861 mmol, 54.1%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.56–1.68 (m, 2H), 1.68–1.82 (m, 4H), 2.35 (ddd, J = 2.9, 11.3, 11.3 Hz, 2H), 2.60–2.68 (m, 2H), 2.68–2.75 (m, 2H), 3.52 (s, 2H), 7.14–7.22 (m, 3H), 7.22–7.36 (m, 7H), an exchangeable OH proton was not observed; ¹³C-NMR (CDCl₃, 100 MH) δ: 29.6, 37.2, 45.0, 49.7, 63.4, 69.7, 126.0, 127.3, 128.5, 128.6, 128.7, 129.5, 138.6, 142.7; IR (film) cm⁻¹: 3025, 2938, 1601, 1494, 1454, 1072, 736, 699; ESI-MS m/z: 296.2015 (Calcd for C₂₀H₂₆NO: 296.2014).

1-Benzyl-4-(2-phenylethylidene)piperidine (7) and 1-Benzyl-4-phenethyl-1,2,3,6-tetrahydropyridine (8)

To a solution of 6 (141 mg, 0.521 mmol) in toluene (20 mL) was added p-toluenesulfonic acid monohydrate (149 mg, 0.781 mmol), and the mixture was refluxed for 14 h while using a Soxhlet extractor filled with molecular sieves 4 Å. After cooling to ambient temperature, saturated aqueous sodium hydrogen carbonate solution was added. The mixture was extracted with dichloromethane four times, and the combined organic layers were washed with brine (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (chloroform/methanol, 15 : 1) gave the title compound (141 mg, 0.509 mmol, 97.7%) as a brown oil as a mixture of isomers. Analytical samples were prepared by separating the small portion of the mixture. Compound 7: ¹H-NMR (CDCl₃, 400 MHz) δ: 2.20–2.30 (m, 2H), 2.34–2.42 (m, 2H), 2.42–2.52 (m, 4H), 3.35 (d, J = 7.4 Hz, 2H), 3.52 (s, 2H), 5.32 (t, J = 7.4 Hz, 1H), 7.14–7.21 (m, 3H), 7.22–7.37 (m, 7H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 28.2, 33.3, 36.0, 54.5, 55.2, 63.0, 120.9, 125.7, 126.9, 128.1, 128.2, 128.3, 128.4, 129.1, 137.1, 141.5; IR (neat) cm⁻¹: 3025, 2899, 2796, 1492, 1453, 1359, 1126, 737, 697; ESI-MS m/z: 278.1895 (Calcd for C₂₀H₂₄N: 278.1908). Compound 8: ¹H-NMR (CDCl₃, 400 MHz) δ: 2.08–2.17 (m, 2H), 2.22–2.31 (m, 2H), 2.56 (dd, J = 5.7, 5.7 Hz, 2H), 2.66–2.77 (m, 2H), 2.92–3.00 (m, 2H), 3.57 (s, 2H), 5.38–5.43 (m, 1H), 7.14–7.22 (m, 3H), 7.24–7.38 (m, 7H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 29.3, 33.9, 38.7, 49.8, 52.8, 62.7, 119.1, 125.7, 126.9, 128.1, 128.2, 128.3, 129.1, 135.9, 138.3, 142.2; IR (neat) cm⁻¹: 3025, 2922, 1495, 1454, 1364, 1119, 1028, 824, 739, 698; ESI-MS m/z: 278.1895 (Calcd for C₂₀H₂₄N: 278.1908).

4-Phenethylpiperidine (9)

To a solution of the mixture of isomers 7 and 8 (71.7 mg, 0.259 mmol) in methanol (4 mL) was added activated charcoal. After stirring at ambient temperature for 2 h, activated charcoal was removed by filtration through a pad of Celite, and the filtrate was concentrated under reduced pressure. The residue was then solved in methanol (4 mL). To this solution was added palladium hydroxide on activated carbon (palladium: 20%, 3.6 mg), and the mixture was hydrogenated catalytically under a hydrogen atmosphere at ambient temperature. After stirring for 19 h, the catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated under reduced pressure. Column chromatography (NH silica gel, chloroform/methanol, 15 : 1) gave the title compound (48.0 mg, 0.254 mmol, 98.1%) as a yellow amorphous. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.08–1.21 (m, 1H), 1.50–1.62 (m, 2H), 1.62–1.78 (m, 4H), 2.50–2.67 (m, 4H), 3.06 (ddd, J = 3.2, 3.2, 12.1 Hz, 2H), 7.13–7.21 (m, 3H), 7.24–7.31 (m, 2H), an exchangeable NH proton was not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 33.1, 33.2, 35.8, 39.1, 46.6, 125.9, 128.6, 128.7, 142.9; IR (neat) cm⁻¹: 2921, 2852, 1454, 1273, 699, 440; ESI-MS m/z: 190.1598 (Calcd for C₁₃H₂₀N: 190.1595).

N-(4-((4-Phenethylpiperidin-1-yl)methyl)phenyl)acetamide (10)

To a solution of 9 (89.3 mg, 0.472 mmol) in dichloromethane (4 mL), were added N-(4-formylphenyl)acetamide (4, 115 mg, 0.708 mmol), acetic acid (40.5 µL, 0.708 mmol), and sodium tri(acetoxy)borohydride (150 mg, 0.708 mmol). The mixture was stirred at ambient temperature for 6 h. Then, saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with dichloromethane four times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (n-hexane/ethyl acetate, 1 : 4) gave the title compound (60.6 mg, 0.180 mmol, 38.2%) as a colorless solid. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.20–1.34 (m, 3H), 1.49–1.76 (m, 4H), 1.84–1.96 (m, 2H), 2.17 (s, 3H), 2.57–2.65 (m, 2H), 2.85 (d, J = 11.4 Hz, 2H), 3.43 (s, 2H), 7.09–7.20 (m, 4H), 7.22–7.33 (m, 4H), 7.42 (d, J = 8.4 Hz, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 24.5, 32.2, 33.0, 35.2, 38.3, 53.7, 62.9, 119.6, 125.5, 128.25, 128.28, 129.8, 134.4, 136.6, 142.7, 168.2; IR (film) cm⁻¹: 3301, 2924, 1666, 1604, 1540, 1453, 1410, 1369, 1317, 1103, 698; ESI-MS m/z: 337.2264 (Calcd for C₂₂H₂₉N₂O: 337.2279).

4-Phenethylpiperidin-4-ol (11)

To a solution of 6 (150 mg, 0.508 mmol) in methanol (4 mL) was added palladium hydroxide on activated carbon (palladium: 20%, 7.2 mg), and the mixture was hydrogenated catalytically under a hydrogen atmosphere at ambient temperature. After stirring for 18 h, the catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated under reduced pressure. Column chromatography (NH silica gel, chloroform/methanol, 15 : 1) gave the title compound (104 mg, 0.507 mmol, 99.9%) as a yellow solid. mp: 76.6–78.9 °C (decomp.); ¹H-NMR (CDCl₃, 400 MHz) δ: 1.50–1.71 (m, 4H), 1.75–1.83 (m, 2H), 2.68–2.76 (m, 2H), 2.82–2.91 (m, 2H), 2.92–3.02 (m, 2H), 3.47 (s, 1H), 7.15–7.23 (m, 3H), 7.25–7.32 (m, 2H), an exchangeable NH proton was not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 29.0, 37.9, 42.4, 45.0, 69.8, 125.7, 128.2, 128.3, 142.4; IR (neat) cm⁻¹: 2939, 1602, 1454, 1275, 982, 833, 754, 700; ESI-MS m/z: 206.1540 (Calcd for C₁₃H₂₀NO: 206.1544).

N-(4-((4-Hydroxy-4-phenethylpiperidin-1-yl)methyl)phenyl)acetamide (12)

This compound was prepared according to a similar procedure as compound 10, starting from compound 11. Column chromatography (n-hexane/ethyl acetate, 1 : 4) gave the title compound (76.0 mg, 0.216 mmol, 78.5%) as a colorless solid. mp: 169.0–170.3 °C (decomp.); ¹H-NMR (CDCl₃, 400 MHz) δ: 1.57–1.83 (m, 6H), 2.16 (s, 3H), 2.36 (ddd, J = 2.5, 11.5, 11.5 Hz, 2H), 2.59–2.67 (m, 2H), 2.67–2.75 (m, 2H), 3.50 (s, 2H), 7.19 (d, J = 7.6 Hz, 3H), 7.26 (d, J = 7.9 Hz, 4H), 7.36 (br s, 1H), 7.44 (d, J = 7.9 Hz, 2H), an exchangeable OH proton was not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 24.8, 29.5, 37.1, 37.2, 45.0, 45.1, 49.5, 62.8, 69.7, 120.0, 126.0, 128.62, 128.66, 128.7, 130.1, 137.1, 142.7, 168.6; IR (neat) cm⁻¹: 3307, 2918, 1663, 1605, 1546, 1320, 1078, 727; ESI-MS m/z: 353.2212 (Calcd for C₂₂H₂₉N₂O₂: 353.2229).

Ethyl 4-Phenethylpiperidine-4-carboxylate (13)

To a solution of 3 (6.31 g, 17.5 mmol) in dichloromethane (40 mL) was slowly added trifluoroacetic acid (40 mL) dropwise at 0 °C. After stirring for 4 h while allowing the mixture to warm up, the mixture was concentrated under reduced pressure. The residue was taken up with dichloromethane and washed with saturated aqueous sodium hydrogen carbonate solution. The aqueous layer was then extracted with dichloromethane six times, and the combined organic layers were washed with brine twice, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (chloroform/methanol, 15 : 1) gave the title compound (4.54 g, 17.4 mmol, 99.4%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.30 (t, J = 7.1 Hz, 3H), 1.44–1.56 (m, 2H), 1.78–1.88 (m, 2H), 2.00–2.38 (m, 2H), 2.48–2.56 (m, 2H), 2.67–2.87 (m, 2H), 2.98–3.08 (m, 2H), 4.20 (q, J = 7.1 Hz, 2H), 7.10–7.21 (m, 3H), 7.23–7.31 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 13.4, 29.4, 33.3, 41.7, 42.7, 44.7, 59.5, 125.0, 127.3, 127.5, 140.9, 174.7; IR (neat) cm⁻¹: 2958, 1723, 1604, 1496, 1454, 1261, 1174, 1027, 802, 700, 442; ESI-MS m/z: 262.1804 (Calcd for C₁₆H₂₄NO₂: 262.1807).

Ethyl 1-(4-Nitrobenzyl)-4-phenethylpiperidine-4-carboxylate (15)

This compound was prepared according to a similar procedure as compound 10, starting from compound 13 and using 4-nitrobenzaldehyde (14) instead of N-(4-formylphenyl)acetamide (4). Column chromatography (NH silica gel, n-hexane/ethyl acetate, 1 : 2) gave the title compound (76.0 mg, 0.576 mmol, 72.8%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.29 (t, J = 7.1 Hz, 3H), 1.50–1.65 (m, 2H), 1.78–1.88 (m, 2H), 2.00–2.28 (m, 4H), 2.47–2.57 (m, 2H), 2.63–2.75 (m, 2H), 3.54 (s, 2H), 4.19 (q, J = 7.1 Hz, 2H), 7.11–7.22 (m, 3H), 7.24–7.32 (m, 2H), 7.49 (d, J = 8.7 Hz, 2H), 8.14–8.20 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.4, 30.6, 33.5, 42.2, 45.2, 51.2, 60.4, 62.4, 123.5, 125.9, 128.2, 128.4, 129.4, 141.8, 146.7, 147.1, 175.7; IR (neat) cm⁻¹: 3577, 2926, 1722, 1604, 1520, 1344, 1178, 740; ESI-MS m/z: 397.2116 (Calcd for C₂₃H₂₉N₂O₄: 397.2127).

Ethyl 1-(4-Aminobenzyl)-4-phenethylpiperidine-4-carboxylate (16)

To a solution of 15 (204 mg, 0.513 mmol) in a mixture of ethanol and water (1 : 1, 2.6 mL) were added iron powder (115 mg, 2.05 mmol) and ammonium hydrochloride (220 mg, 4.11 mmol). The mixture was refluxed for 3 h. After cooling to an ambient temperature, saturated aqueous sodium hydrogen carbonate solution was added, and iron powder was removed by filtration through a Celite pad. Then the mixture was extracted with chloroform four times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (chloroform/methanol, 30 : 1) gave the title compound (116 mg, 0.316 mmol, 61.6%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.28 (t, J = 7.1 Hz, 3H), 1.47–1.68 (m, 2H), 1.77–1.85 (m, 2H), 2.00–2.10 (m, 2H), 2.15–2.25 (m, 2H), 2.46–2.54 (m, 2H), 2.67–2.75 (m, 2H), 3.35 (s, 2H), 3.42–3.82 (br s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 6.61–6.66 (m, 2H), 7.05–7.20 (m, 4H), 7.23–7.29 (m, 3H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.4, 30.6, 33.5, 42.2, 45.4, 50.9, 60.3, 62.9, 114.9, 125.9, 128.1, 128.3, 128.4, 130.4, 142.0, 145.4, 175.9; IR (KBr) cm⁻¹: 3463, 3374, 2924, 1711, 1625, 1519, 1291, 1201, 1175, 700; ESI-MS m/z: 367.2397 (Calcd for C₂₃H₃₁N₂O₂: 367.2386).

(1-(4-Aminobenzyl)-4-phenethylpiperidin-4-yl)methanol (17)

To a suspension of lithium aluminum hydride (571 mg, 15.1 mmol) in tetrahydrofuran (25 mL) was added a solution of 16 (1.84 g, 5.02 mmol) in tetrahydrofuran (25 mL) dropwise at 0 °C. After stirring at this temperature for 2 h, sodium sulfate decahydrate was added and mixture was stirred at ambient temperature for 13 h. The resultant solid was removed by filtration through a pad of Celite, and the filtrate was concentrated under reduced pressure. Column chromatography (chloroform/methanol/ammonia solution (25% in water), 150 : 10 : 1) gave the title compound (1.55 g, 4.80 mmol, 95.6%) as a yellow solid. mp: 149.7–153.1 °C (decomp.); ¹H-NMR (CDCl₃, 400 MHz) δ: 1.40–1.64 (m, 4H), 1.64–1.76 (m, 2H), 2.30–2.50 (m, 4H), 2.52–2.60 (m, 2H), 3.40 (s, 2H), 3.53 (s, 2H), 3.60 (br s, 1H), 6.64 (d, J = 8.3 Hz, 2H), 7.09 (d, J = 8.3 Hz, 2H), 7.14–7.22 (m, 3H), 7.26–7.31 (m, 2H), two exchangeable NH protons were not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 29.5, 29.8, 31.7, 35.6, 48.9, 62.8, 67.4, 114.8, 125.6, 127.7, 128.2, 128.3, 130.4, 143.0, 145.3; IR (film) cm⁻¹: 2226, 1621, 1517, 1454, 1288, 1049, 755, 700: ESI-MS m/z: 353.2282 (Calcd for C₂₁H₂₉N₂O: 325.2279).

(1-(4-Acetamidobenzyl)-4-phenethylpiperidin-4-yl)methyl Acetate (18)

To a solution of 17 (685 mg, 2.11 mmol) in dichloromethane (21 mL) were added acetic anhydride (400 µL, 4.22 mmol), triethylamine (586 µL, 4.22 mmol) and 4-dimethylaminopyridine (7.7 mg, 0.063 mmol), and the mixture was stirred at ambient temperature for 30 min. Then saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with dichloromethane three times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (chloroform/methanol, 15 : 1) gave the title compound (787 mg, 1.92 mmol, 91.2%) as a white amorphous. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.49–1.63 (m, 3H), 1.63–1.75 (m, 4H), 2.07 (s, 3H), 2.17 (s, 3H), 2.35–2.47 (m, 4H), 2.49–2.60 (m, 2H), 3.46 (s, 2H), 4.03 (s, 2H), 7.11–7.23 (m, 3H), 7.24–7.34 (m, 4H), 7.43 (d, J = 8.3 Hz, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 20.9, 24.5, 29.4, 32.2, 34.3, 37.7, 49.0, 62.7, 77.2, 119.7, 125.7, 128.2, 128.3, 129.6, 134.3, 136.7, 142.6, 168.2, 171.2; IR (film) cm⁻¹: 2934, 1736, 1667, 1603, 1537, 1368, 1238, 1039, 755; ESI-MS m/z: 409.2472 (Calcd for C₂₅H₃₃N₂O₃ 409.2491).

N-(4-((4-(Hydroxymethyl)-4-phenethylpiperidin-1-yl)methyl)phenyl)acetamide (19)

To a solution of 18 (117 mg, 0.286 mmol) in tetrahydrofuran (0.6 mL) was added a solution of lithium hydroxide monohydrate (16.1 mg, 0.384 mmol) in water (0.6 mL). The resultant mixture was stirred at ambient temperature for 14 h. Then saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with chloroform four times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (chloroform/methanol, 10 : 1) gave the title compound (99.4 mg, 0.272 mmol, 95.1%) as a white solid. mp: 65.6–70.2 °C (decomp.); ¹H-NMR (CDCl₃, 400 MHz) δ: 1.45–1.63 (m, 4H), 1.65–1.77 (m, 2H), 2.17 (s, 3H), 2.31–2.50 (m, 4H), 2.51–2.62 (m, 2H), 4.04 (s, 2H), 3.53 (s, 2H), 7.12 (br s, 1H), 7.15–7.22 (m, 3H), 7.24–7.32 (m, 4H), 7.43 (d, J = 8.2 Hz, 2H), an exchangeable OH proton was not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 24.4, 29.5, 31.9, 35.6, 36.7, 49.1, 62.8, 67.4, 119.7, 125.6, 128.2, 128.3, 129.8, 134.3, 136.7, 143.0, 168.3; IR (film) cm⁻¹: 2927, 1667, 1604, 1542, 1318, 1047, 909, 730; ESI-MS m/z: 367.2375 (Calcd for C₂₃H₃₁N₂O₂: 367.2385).

N-(4-((4-(1-Hydroxybutyl)-4-phenethylpiperidin-1-yl)methyl)phenyl)acetamide (21)

A solution of oxalyl chloride (0.28 mL, 3.2 mmol) in dichloromethane (7 mL) was cooled to −78 °C. Dimethyl sulfoxide (0.28 mL, 4.1 mmol) was added to this solution, and the resultant mixture was stirred at this temperature for 20 min. Then a solution of 19 (240 mg, 0.655 mmol) in dichloromethane (4 mL) was added dropwise, and the mixture was stirred at −78 °C for 2 h. Then triethylamine (1.1 mL, 8.1 mmol) was added, and the mixture was stirred at ambient temperature for 2 h. After adding saturated aqueous sodium hydrogen carbonate solution, and the mixture was extracted with dchloromethane four times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Rough Purification with column chromatography (chloroform/methanol, 30 : 1) gave the aldehyde 20 with some impurity as a yellow amorphous material. To this amorphous material in tetrahydrofuran (6 mL) was added propylmagnesium bromide (1.01 M in tetrahydrofuran, 1.7 mL, 1.7 mmol) dropwise at 0 °C. The mixture was stirred at ambient temperature for 1 h. Then saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with chloroform six times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (chloroform/methanol/ammonia solution (25% in water), 100 : 10 : 1) gave the title compound (44.5 mg, 0.109 mmol, 16.6% from compound 19) as a yellow oil. Simultaneously, compound 19 (58.0 mg, 0.158 mmol, 24.1%) was recovered as a colorless solid which was regenerated by the reduction of compound 20. ¹H-NMR (CDCl₃, 400 MHz) δ: 0.96 (t, J = 7.0 Hz, 3H), 1.20–1.50 (m, 3H), 1.50–1.59 (m, 2H), 1.59–1.86 (m, 6H), 2.16 (s, 3H), 2.21–2.34 (m, 2H), 2.45–2.56 (m, 1H), 2.56–2.75 (m, 3H), 3.46 (s, 2H), 7.15–7.25 (m, 4H), 7.23–7.34 (m, 3H), 7.43 (d, J = 8.3 Hz, 2H), two exchangeable OH and NH protons were not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.1, 20.2, 24.5, 29.9, 31.2, 33.1, 34.2, 37.8, 49.1, 62.7, 76.8, 119.7, 125.6, 128.2, 128.3, 129.8, 134.4, 136.6, 143.3, 168.1; IR (film) cm⁻¹: 2953, 1668, 1604, 1540, 1410, 1316, 755, 699; ESI-MS m/z: 409.2837 (Calcd for C₂₆H₃₇N₂O₂: 409.2855).

N-(4-((4-Butyryl-4-phenethylpiperidin-1-yl)methyl)phenyl)acetamide (22)

To a solution of 21 (44.5 mg, 0.108 mmol) in dichloromethane (1 mL) was added Dess–Martin periodinane (139 mg, 0.326 mmol) at 0 °C, and the mixture was stirred at ambient temperature for 1 h. Then saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with dichloromethane four times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (chloroform/methanol, 15 : 1) gave the title compound (19.1 mg, 0.0470 mmol, 43.6%) as a brown oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 0.93 (t, J = 7.3 Hz, 3H), 1.25 (m, 3H), 1.50–1.71 (m, 4H), 1.72–2.00 (m, 2H), 2.00–2.15 (m, 2H), 2.17 (s, 3H), 2.32–2.40 (m, 2H), 2.43 (t, J = 7.3 Hz, 2H), 2.56–2.75 (m, 2H), 3.42 (s, 2H), 7.06–7.21 (m, 3H), 7.21–7.32 (m, 4H), 7.37–7.48 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 13.9, 17.0, 24.5, 29.6, 30.2, 32.1, 39.0, 50.1, 50.5, 62.4, 119.6, 125.9, 128.1, 128.4, 129.9, 131.0, 137.0, 141.7, 168.3, 214.0; IR (neat) cm⁻¹: 2929, 1696, 1603, 1537, 1369, 1315, 754, 700; ESI-MS m/z: 407.2687 (Calcd for C₂₆H₃₅N₂O₂: 407.2698).

(Z)-N-(4-((4-(But-1-en-1-yl)-4-phenethylpiperidin-1-yl)methyl)phenyl)acetamide (23)

The crude aldehyde 20 was prepared from alcohol 19 (127 mg, 0.346 mmol) according to the procedure in compound 21. To a mixture of (n-butyl)triphenylphosphonium bromide (593 mg, 1.54 mmol) in tetrahydrofuran (6 mL) was added n-butyllithium (1.53 M in n-hexane, 0.95 mL, 1.5 mmol) dropwise at 0 °C. The mixture was stirred at this temperature for 30 min and then cooled to −78 °C. To this mixture, a solution of the roughly purified aldehyde 20 in tetrahydrofuran (4 mL) was added dropwise. After stirring at ambient temperature for 1 h, saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with chloroform four times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (silica gel, chloroform/methanol/ ammonia solution (25% in water), 100 : 10 : 1, then NH silica gel, n-hexane/ethyl acetate 1 : 2) gave the title compound (56.9 mg, 0.146 mmol, 42.2%) as a yellow amorphous. ¹H-NMR (CDCl₃, 400 MHz) δ: 0.99 (t, J = 7.4 Hz, 3H), 1.55–1.98 (m, 6H), 2.13 (ddq, J = 1.6, 7.4, 7.4 Hz, 2H), 2.19 (s, 3H), 2.31–2.51 (m, 2H), 2.51–2.64 (m, 2H), 2.89 (m, 2H), 3.69 (s, 2H), 5.11 (d, J = 12.1 Hz, 1H), 5.47 (dt, J = 7.4, 12.1 Hz, 1H), 7.087.20 (m, 3H), 7.227.30 (m, 2H), 7.41 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 8.2 Hz, 2H), an exchangeable NH proton was not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.1, 22.2, 24.4, 30.5, 37.3, 38.4, 44.0, 50.2, 62.8, 119.7, 125.5, 128.2, 128.3, 129.7, 133.3, 134.1, 134.1, 136.7, 143.3, 168.3; IR (neat) cm⁻¹: 2931, 1666, 1604, 1540, 1410, 1316, 1105, 755; ESI-MS m/z: 391.2766 (Calcd for C₂₆H₃₅N₂O: 391.2749).

N-(4-((4-Butyl-4-phenethylpiperidin-1-yl)methyl)phenyl)acetamide (24)

To a solution of 23 (52.3 mg, 0.134 mmol) in methanol (2 mL) was added palladium on carbon (palladium: 10%, wetted with approx. 50% water, 16.1 mg), and the mixture was hydrogenated catalytically under a hydrogen atmosphere at ambient temperature. After stirring for 22 h, the catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated under reduced pressure. Column chromatography (n-hexane/ethyl acetate, 1 : 1) gave the title compound (26.2 mg, 0.0668 mmol, 49.8%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 0.92 (t, J = 7.1 Hz, 3H), 1.12–1.40 (m, 6H), 1.42–1.52 (m, 4H), 1.52–1.68 (m, 2H), 2.15 (s, 3H), 2.32–2.43 (m, 4H), 2.43–2.58 (m, 2H), 3.45 (s, 2H), 7.13–7.21 (m, 3H), 7.22–7.32 (m, 3H), 7.36–7.50 (m, 3H), an exchangeable NH proton was not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.1, 23.5, 24.5, 24.9, 25.8, 29.4, 33.2, 35.2, 49.3, 62.9, 64.6, 119.6, 125.5, 128.2, 128.3, 129.7, 134.4, 136.7, 143.3, 168.2; IR (neat) cm⁻¹: 2928, 1666, 1604, 1541, 1410, 1369, 1317, 731, 698; ESI-MS m/z: 393.2907 (Calcd for C₂₆H₃₇N₂O: 393.2905).

Structural Diversification of the Western Part

Ethyl 1-(2-Nitrophenyl)-4-phenethylpiperidine-4-carboxylate (28o)

To a suspension of potassium carbonate (63.5 mg, 0.459 mmol) in acetonitrile (7 mL) were added 13 (199 mg, 0.765 mmol) and 2-fluoronitrobenzene (115 mg, 2.05 mmol). The mixture was refluxed for 3 h. After cooling to ambient temperature, water was added to the mixture, and the mixture was extracted with dichloromethane four times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (NH silica gel, n-hexane/ethyl acetate 10 : 1) gave the title compound (99.0 mg, 0.259 mmol, 33.8%) as a yellow oil. ¹H-NMR (400 MHz, CDCl₃) δ: 1.30 (t, J = 7.1 Hz, 3H), 1.66–1.78 (m, 2H), 1.84–1.94 (m, 2H), 2.31 (br d, J = 13.1 Hz, 2H), 2.50–2.59 (m, 2H), 2.89 (ddd, J = 2.0, 12.0, 12.0 Hz, 2H), 3.13–3.22 (m, 2H), 4.21 (q, J = 7.1 Hz, 2H), 6.97–7.03 (m, 1H), 7.09–7.22 (m, 4H), 7.24–7.31 (m, 2H), 7.41–7.48 (m, 1H), 7.76 (dd, J = 1.6, 8.1 Hz, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.4, 29.6, 33.6, 42.4, 45.1, 49.7, 60.5, 120.9, 121.2, 125.9, 128.2, 128.4, 128.5, 131.3, 133.4, 141.6, 146.5, 175.4; IR (film) cm⁻¹: 2926, 1722, 1603, 1519, 1455, 1386, 1341, 1295, 1181, 1026, 848, 750, 700; ESI-MS m/z: 383.1967 (Calcd for C₂₂H₂₇N₂O₄: 383.1970).

Ethyl 1-(3-Nitrophenyl)-4-phenethylpiperidine-4-carboxylate (28m)

To a suspension of potassium carbonate (163 mg, 1.17 mmol) in N,N-dimethylformamide (9 mL) were added 13 (514 mg, 1.97 mmol) and 3-fluoronitrobenzene (210 µL, 1.96 mmol). The mixture was stirred at 100 °C for 41 h. The same work-up and purification processes as 28o gave the title compound (81.7 mg, 0.214 mmol, 10.8%) as a yellow oil. ¹H-NMR (400 MHz, CDCl₃) δ: 1.31 (t, J = 7.1 Hz, 3H), 1.60–1.71 (m, 2H), 1.84–1.92 (m, 2H), 2.34 (br d, J = 13.2 Hz, 2H), 2.51–2.60 (m, 2H), 2.98 (ddd, J = 2.7, 12.1, 12.1 Hz, 2H), 3.60 (ddd, J = 3.6, 3.6, 13.0 Hz, 2H), 4.21 (q, J = 7.1 Hz, 2H), 7.11–7.22 (m, 4H), 7.24–7.31 (m, 2H), 7.34 (dd, J = 8.2, 8.2 Hz, 1H), 7.59–7.65 (m, 1H), 7.70 (dd, J = 2.3, 2.3 Hz, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.3, 30.4, 32.9, 42.0, 45.1, 46.3, 60.6, 109.7, 113.2, 121.3, 126.0, 128.2, 128.4, 129.6, 141.5, 149.2, 151.8, 175.1; IR (film) cm⁻¹: 2926, 1724, 1616, 1527, 1455, 1347, 1184, 857, 737, 700; ESI-MS m/z: 383.1972 (Calcd for C₂₂H₂₇N₂O₄: 383.1970).

Ethyl 1-(4-Nitrophenyl)-4-phenethylpiperidine-4-carboxylate (28p)

This compound was prepared according to a similar procedure as compound 28m. The title compound was obtained (144 mg, 0.376 mmol, 49.0%) as a yellow solid. mp: 112.7–112.9 °C; ¹H-NMR (400 MHz, CDCl₃) δ: 1.31 (t, J = 7.1 Hz, 3H), 1.56–1.66 (m, 2H), 1.83–1.92 (m, 2H), 2.33 (br d, J = 13.3 Hz, 2H), 2.51–2.59 (m, 2H), 3.06–3.18 (m, 2H), 3.77 (ddd, J = 3.6, 3.6, 13.6 Hz, 2H), 4.22 (q, J = 7.1 Hz, 2H), 6.78–6.83 (m, 2H), 7.11–7.22 (m, 3H), 7.24–7.31 (m, 2H), 8.08–8.15 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.3, 30.4, 32.8, 42.0, 44.9, 45.3, 60.8, 112.4, 126.0, 126.1, 128.2, 128.4, 137.9, 141.3, 154.6, 175.0; IR (film) cm⁻¹: 2965, 1721, 1600, 1484, 1324, 1262, 1190, 1112, 902, 828, 752, 695; ESI-MS m/z: 383.1965 (Calcd for C₂₂H₂₇N₂O₄: 383.1970).

Ethyl 1-(2-Aminophenyl)-4-phenethylpiperidine-4-carboxylate (29o)

This compound was prepared according to a similar procedure as compound 16. Column chromatography (n-hexane/ethyl acetate 2 : 1) gave the title compound (68.5 mg, 0.194 mmol, 75.1%) as a brown solid. mp: 53.0–56.6 °C (decomp.); ¹H-NMR (CDCl₃, 400 MHz) δ: 1.31 (t, J = 7.1 Hz, 3H), 1.60–1.73 (m, 2H), 1.85–1.95 (m, 2H), 2.34 (br d, J = 13.2 Hz, 2H), 2.51–2.61 (m, 2H), 2.71 (br t, J = 10.8 Hz, 2H), 2.97–3.07 (m, 2H), 3.95 (br s, 2H), 4.22 (q, J = 7.1 Hz, 2H), 6.69–6.77 (m, 2H), 6.88–7.01 (m, 2H), 7.13–7.23 (m, 3H), 7.24–7.33 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.7, 30.9, 34.80, 34.81, 45.5, 49.4, 60.7, 115.3, 118.8, 120.1, 124.7, 126.2, 128.6, 128.7, 140.2, 141.7, 142.2, 176.1; IR (film) cm⁻¹: 3433, 2950, 1721, 1608, 1499, 1455, 1385, 1267, 1178, 1094, 1027, 928, 745; ESI-MS m/z: 353.2235 (Calcd for C₂₂H₂₉N₂O₂: 353.2229).

Ethyl 1-(3-Aminophenyl)-4-phenethylpiperidine-4-carboxylate (29m)

This compound was prepared according to a similar procedure as compound 16. Column chromatography (n-hexane/ethyl acetate 3 : 1) gave the title compound (35.9 mg, 0.102 mmol, 51.5%) as a brown oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.29 (t, J = 7.1 Hz, 3H), 1.57–1.71 (m, 2H), 1.80–1.91 (m, 2H), 2.29 (br d, J = 13.2 Hz, 2H), 2.49–2.60 (m, 2H), 2.84 (ddd, J = 2.6, 12.0, 12.0 Hz, 2H), 3.46 (ddd, J = 3.7, 3.7, 12.9 Hz, 2H), 3.58 (br s, 2H), 4.19 (q, J = 7.1 Hz, 2H), 6.16–6.22 (m, 1H), 6.25 (dd, J = 2.2, 2.2 Hz, 1H), 6.33–6.40 (m, 1H), 7.02 (dd, J = 8.0, 8.0 Hz, 1H), 7.11–7.21 (m, 3H), 7.23–7.31 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.3, 30.5, 33.2, 42.0, 45.2, 47.0, 60.4, 103.1, 106.7, 107.1, 125.9, 128.2, 128.3, 129.8, 141.8, 147.2, 152.6, 175.5; IR (film) cm⁻¹: 2952, 1720, 1603, 1498, 1455, 1182, 1026, 965, 699; ESI-MS m/z: 353.2230 (Calcd for C₂₂H₂₉N₂O₂: 353.2229).

Ethyl 1-(4-Aminophenyl)-4-phenethylpiperidine-4-carboxylate (29p)

This compound was prepared according to a similar procedure as compound 16. Column chromatography (n-hexane/ethyl acetate 2 : 1) gave the title compound (86.3 mg, 0.245 mmol, 89.8%) as a black oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.29 (t, J = 7.1 Hz, 3H), 1.63–1.75 (m, 2H), 1.82–1.91 (m, 2H), 2.32 (br d, J = 13.3 Hz, 2H), 2.51–2.59 (m, 2H), 2.73 (ddd, J = 2.4, 11.8, 11.8 Hz, 2H), 3.29 (ddd, J = 3.6, 3.6, 12.6 Hz, 2H), 3.41 (br s, 2H), 4.22 (q, J = 7.1 Hz, 2H), 6.61–6.67 (m, 2H), 6.78–6.85 (m, 2H), 7.12–7.22 (m, 3H), 7.23–7.31 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.4, 30.5, 33.6, 42.1, 45.1, 49.0, 60.4, 116.1, 118.9, 125.9, 128.2, 128.4, 139.9, 141.8, 144.9, 175.6; IR (film) cm⁻¹: 3359, 2951, 1721, 1624, 1513, 1455, 1386, 1180, 1095, 1026, 825, 700; ESI-MS m/z: 353.2229 (Calcd for C₂₂H₂₉N₂O₂: 353.2229).

Ethyl 1-(2-Acetamidophenyl)-4-phenethylpiperidine-4-carboxylate (25o)

This compound was prepared according to a similar procedure as compound 18. Column chromatography (n-hexane/ethyl acetate 2 : 1) gave the title compound (40.2 mg, 0.102 mmol, 91.1%) as a colorless solid. mp: 90.2–93.0 °C (decomp.); ¹H-NMR (CDCl₃, 400 MHz) δ: 1.33 (t, J = 7.1 Hz, 3H), 1.61–1.72 (m, 2H), 1.89–1.97 (m, 2H), 2.20 (s, 3H), 2.39 (br d, J = 13.4 Hz, 2H), 2.53–2.61 (m, 2H), 2.71–2.81 (m, 2H), 2.85–2.94 (m, 2H), 4.24 (q, J = 7.1 Hz, 2H), 7.00–7.06 (m, 1H), 7.07–7.23 (m, 4H), 7.23–7.32 (m, 2H), 8.33 (br d, J = 7.8 Hz, 1H), 8.43 (br s, 1H), an exchangeable NH proton was not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.4, 24.9, 30.5, 34.5, 42.1, 45.0, 50.2, 60.6, 119.3, 120.2, 123.5, 125.2, 126.0, 128.2, 128.4, 133.3, 141.5, 141.6, 168.0, 175.4; IR (neat) cm⁻¹: 2952, 1723, 1689, 1590, 1516, 1448, 1178, 758, 700; ESI-MS m/z: 395.2324 (Calcd for C₂₄H₃₁N₂O₃: 395.2334).

Ethyl 1-(3-Acetamidophenyl)-4-phenethylpiperidine-4-carboxylate (25m)

This compound was prepared according to a similar procedure as compound 18. Column chromatography (n-hexane/ethyl acetate 1 : 1) gave the title compound (32.3 mg, 0.0820 mmol, 100%) as a yellow solid. mp: 90.4–91.3 °C (decomp.); ¹H-NMR (CDCl₃, 400 MHz) δ: 1.29 (t, J = 7.1 Hz, 3H), 1.59–1.70 (m, 2H), 1.80–1.91 (m, 2H), 2.14 (s, 3H), 2.29 (br d, J = 13.1 Hz, 2H), 2.49–2.59 (m, 2H), 2.82–2.93 (m, 2H), 3.50 (ddd, J = 3.8, 3.8, 13.2 Hz, 2H), 4.19 (q, J = 7.1 Hz, 2H), 6.67 (br d, J = 8.2 Hz, 1H), 6.82 (br d, J = 7.7 Hz, 1H), 7.12–7.21 (m, 4H), 7.22–7.32 (m, 4H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.3, 24.6, 30.4, 33.1, 42.0, 45.2, 46.8, 60.5, 107.7, 110.6, 112.1, 125.9, 128.2, 128.3, 129.3, 138.7, 141.7, 152.0, 168.3, 175.4; IR (neat) cm⁻¹: 2927, 1723, 1610, 1550, 1495, 1371, 1257, 1182, 1026, 910, 732, 699; ESI-MS m/z: 395.2323 (Calcd for C₂₄H₃₁N₂O₃: 395.2334).

Ethyl 1-(4-Acetamidophenyl)-4-phenethylpiperidine-4-carboxylate (25p)

This compound was prepared according to a similar procedure as compound 18. Column chromatography (n-hexane/ethyl acetate 1 : 1) gave the title compound (49.5 mg, 0.125 mmol, 95.9%) as a colorless solid. mp: 97.1–99.9 °C (decomp.); ¹H-NMR (CDCl₃, 400 MHz) δ: 1.29 (t, J = 7.1 Hz, 3H), 1.61–1.74 (m, 2H), 1.83–1.91 (m, 2H), 2.13 (s, 3H), 2.31 (br d, J = 13.3 Hz, 2H), 2.51–2.59 (m, 2H), 2.77–2.88 (m, 2H), 3.43 (ddd, J = 3.7, 3.7, 13.0 Hz, 2H), 4.19 (q, J = 7.1 Hz, 2H), 6.84–6.91 (m, 2H), 7.12–7.23 (m, 4H), 7.24–7.31 (m, 2H), 7.32–7.38 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.3, 24.3, 30.5, 33.2, 42.0, 45.1, 47.5, 60.5, 116.9, 121.4, 125.9, 128.2, 128.4, 130.0, 141.7, 148.6, 168.1, 175.4; IR (film) cm⁻¹: 2952, 1723, 1660, 1601, 1514, 1371, 1318, 1181, 1025, 825, 700; ESI-MS m/z: 395.2321 (Calcd for C₂₄H₃₁N₂O₃: 395.2334).

Ethyl 1-(2-Nitrobenzyl)-4-phenethylpiperidine-4-carboxylate (30o)

To a solution of 13 (103 mg, 0.395 mmol) in 1,4-dioxane (2 mL) were added 2-nitrobenzyl bromide (127 mg, 0.588 mmol) and aqueous sodium hydroxide solution (0.5 M, 1.2 mL). After stirring at ambient temperature for 1 h, aqueous sodium hydroxide solution (0.5 M, 0.3 mL) was added to the mixture. The mixture was further stirred for 1 h at this temperature and then extracted with ethyl acetate three times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (n-hexane/ethyl acetate 6 : 1 then chloroform/methanol 20 : 1) gave the title compound (144 mg, 0.364 mmol, 92.1%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.29 (t, J = 7.1 Hz, 3H), 1.43–1.60 (m, 2H), 1.76–1.85 (m, 2H), 2.10–2.22 (m, 4H), 2.45–2.53 (m, 2H), 2.58–2.67 (m, 2H), 3.74 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 7.10–7.20 (m, 3H), 7.23–7.30 (m, 2H), 7.35–7.41 (m, 1H), 7.53 (ddd, J = 1.3, 7.3, 7.7 Hz, 1H), 7.56–7.61 (m, 1H), 7.79 (dd, J = 1.2, 8.1 Hz, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.4, 30.6, 33.6, 42.2, 45.2, 51.2, 59.2, 60.3, 124.3, 125.8, 127.8, 128.2, 128.4, 130.8, 132.2, 134.2, 141.9, 149.8, 175.7; IR (neat) cm⁻¹: 2924, 1722, 1524, 1345, 1177, 1028, 859, 792, 730, 699; ESI-MS m/z: 397.2122 (Calcd for C₂₃H₂₉N₂O₄: 397.2127).

Ethyl 1-(3-Nitrobenzyl)-4-phenethylpiperidine-4-carboxylate (30m)

This compound was prepared according to a similar procedure as compound 30o. Column chromatography (n-hexane/ethyl acetate, 10 : 1 to 4 : 1) gave the title compound (839 mg, 0.212 mmol, 85.8%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.29 (t, J = 7.1 Hz, 3H), 1.50–1.60 (m, 2H), 1.80–1.88 (m, 2H), 2.10–2.27 (m, 4H), 2.47–2.56 (m, 2H), 2.66–2.75 (m, 2H), 3.54 (s, 2H), 4.19 (q, J = 7.1 Hz, 2H), 7.10–7.21 (m, 3H), 7.24–7.33 (m, 2H), 7.47 (dd, J = 7.9, 7.9 Hz, 1H), 7.65 (d, J = 7.6 Hz, 1H), 8.07–8.14 (m, 1H), 8.18–8.22 (m, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.7, 30.8, 33.8, 42.5, 45.5, 51.4, 60.6, 62.6, 122.3, 123.9, 126.1, 128.5, 128.6, 129.3, 135.2, 141.4, 142.1, 148.6, 175.9; IR (neat) cm⁻¹: 2925, 1723, 1530, 1455, 1349, 1179, 1029, 807, 733, 700; ESI-MS m/z: 397.2118 (Calcd for C₂₃H₂₉N₂O₄: 397.2127).

Ethyl 1-(2-Aminobenzyl)-4-phenethylpiperidine-4-carboxylate (31o)

This compound was prepared according to a similar procedure as compound 16. Column chromatography (chloroform/methanol, 100 : 0 to 100 : 1) gave the title compound (113 mg, 0.309 mmol, 93.6%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.30 (t, J = 7.1 Hz, 3H), 1.41–1.70 (m, 2H), 1.77–1.86 (m, 2H), 2.02–2.12 (m, 2H), 2.15–2.24 (m, 2H), 2.45–2.55 (m, 2H), 2.62–2.73 (m, 2H), 3.47 (s, 2H), 4.19 (q, J = 7.1 Hz, 2H), 4.38–5.10 (br s, 2H), 6.60–6.69 (m, 2H), 6.92–6.98 (m, 1H), 7.08 (ddd, J = 1.4, 7.6, 7.6 Hz, 1H), 7.11–7.21 (m, 2H), 7.23–7.31 (m, 3H); ¹³C-NMR (CDCl_3, 100 MHz) δ: 14.4, 30.6, 33.8, 42.0, 45.5, 50.7, 60.4, 62.3, 115.4, 117.5, 122.6, 125.9, 128.25, 128.26, 128.4, 130.4, 142.0, 147.0, 175.8; IR (neat) cm⁻¹: 3433, 2951, 1723, 1615, 1496, 1461, 1179, 1029, 749, 699; ESI-MS m/z: 367.2367 (Calcd for C₂₃H₃₁N₂O₂: 367.2386).

Ethyl 1-(3-Aminobenzyl)-4-phenethylpiperidine-4-carboxylate (31m)

This compound was prepared according to a similar procedure as compound 24. Column chromatography (chloroform/methanol, 10 : 1) gave the title compound (281 mg, 0.767 mmol, 40.8%) as a yellow solid. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.28 (t, J = 7.1 Hz, 3H), 1.49–1.62 (m, 2H), 1.77–1.87 (m, 2H), 2.02–2.13 (m, 2H), 2.15–2.28 (m, 2H), 2.46–2.55 (m, 2H), 2.47–2.78 (m, 2H), 3.18–3.90 (br s, 2H), 3.36 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 6.46–6.60 (m, 1H), 6.65–6.71 (m, 2H), 7.00–7.20 (m, 4H), 7.22–7.31 (m, 2H); ¹³C-NMR (CDCl_3, 100 MHz) δ: 14.6, 30.8, 33.7, 42.4, 45.5, 51.4, 60.5, 63.6, 114.0, 115.9, 119.7, 126.0, 128.4, 128.5, 129.2, 139.9, 142.2, 146.5, 176.0; IR (KBr) cm⁻¹: 3392, 2923, 1719, 1605, 1465, 1299, 1178, 697; ESI-MS m/z: 367.2375 (Calcd for C₂₃H₃₁N₂O₂: 367.2386).

Ethyl 1-(2-Acetamidobenzyl)-4-phenethylpiperidine-4-carboxylate (26o)

This compound was prepared according to a similar procedure as compound 18. Column chromatography (n-hexane/ethyl acetate, 2 : 1 to 1 : 1) gave the title compound (62.9 mg, 0.154 mmol, 40.8%) as a colorless oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.31 (t, J = 7.1 Hz, 3H), 1.45–1.55 (m, 2H), 1.65–1.90 (m, 4H), 2.11–2.34 (m, 5H), 2.48–2.56 (m, 2H), 2.69–2.79 (m, 2H), 3.56 (s, 2H), 4.21 (q, J = 7.1 Hz, 2H), 6.94–7.01 (m, 1H), 7.02–7.08 (m, 1H), 7.11–7.22 (m, 3H), 7.24–7.31 (m, 4H), 8.25 (d, J = 8.1 Hz, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.4, 24.9, 30.6, 34.0, 42.2, 45.3, 50.5, 60.6, 62.4, 120.5, 123.1, 124.9, 126.0, 128.2, 128.4, 128.5, 129.7, 138.8, 141.6, 168.0, 175.3; IR (neat) cm⁻¹: 2920, 1723, 1691, 1591, 1536, 1452, 1179, 757; ESI-MS m/z: 409.2471 (Calcd for C₂₅H₃₃N₂O₃: 409.2491).

Ethyl 1-(3-Acetamidobenzyl)-4-phenethylpiperidine-4-carboxylate (26m)

This compound was prepared according to a similar procedure as compound 18. Column chromatography (n-hexane them chloroform/methanol 10 : 1) gave the title compound (78.4 mg, 0.192 mmol, 83.1%) as a colorless oil. ¹H-NMR (CD₃OD, 400 MHz) δ: 1.27 (t, J = 7.1 Hz, 3H), 1.52–1.63 (m, 2H), 1.76–1.84 (m, 2H), 2.12 (s, 3H), 2.15–2.28 (m, 4H), 2.45–2.53 (m, 2H), 2.76–2.85 (m, 2H), 3.54 (s, 2H), 4.15 (q, J = 7.1 Hz, 2H), 7.15–7.17 (m, 5H), 7.21–7.30 (m, 3H), 7.43–7.48 (m, 1H), 7.54 (br s, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.6, 24.8, 30.8, 33.7, 42.4, 45.5, 51.4, 60.6, 63.3, 118.9, 120.6, 125.3, 126.1, 128.5, 128.6, 129.0, 138.1, 139.8, 142.2, 168.6, 176.1; IR (neat) cm⁻¹: 2926, 1723, 1668, 1613, 1556, 1489, 1443, 1370, 1179, 1028, 699; ESI-MS m/z: 409.2478 (Calcd for C₂₅H₃₃N₂O₃: 409.2491).

Ethyl 1-(2-Nitrophenethyl)-4-phenethylpiperidine-4-carboxylate (32o)

o-Nitrostyrene used in this reaction is prepared according to a literature procedure.⁴³⁾ To a solution of 13 (100 mg, 0.382 mmol) in acetonitrile (4 mL) were added 1,8-diazabicyclo[5.4.0]-7-undecene (64.0 mg, 0.420 mmol) and o-nitrostyrene (62.7 mg, 0.420 mmol). The mixture was refluxed for 23 h. After cooling to ambient temperature, saturated aqueous sodium hydrogen carbonate solution was added to the mixture, and the mixture was extracted with dichloromethane four times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (n-hexane/ethyl acetate 5 : 1) gave the title compound (118 mg, 0.288 mmol, 75.3%) as a yellow oil. ¹H-NMR (400 MHz, CDCl₃) δ: 1.29 (t, J = 7.1 Hz, 3H), 1.50–1.66 (m, 2H), 1.78–1.88 (m, 2H), 2.14–2.29 (m, 4H), 2.48–2.56 (m, 2H), 2.57–2.66 (m, 2H), 2.75–2.85 (m, 2H), 3.04–3.13 (m, 2H), 4.18 (q, J = 7.1 Hz, 2H), 7.11–7.21 (m, 3H), 7.24–7.30 (m, 2H), 7.31–7.40 (m, 2H), 7.51 (ddd, J = 1.2, 7.5, 7.5 Hz, 1H), 7.88 (dd, J = 1.2, 8.0 Hz, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.3, 30.5, 30.6, 33.5, 42.2, 45.2, 51.0, 59.3, 60.3, 124.6, 125.8, 127.1, 128.2, 128.3, 132.4, 132.8, 135.5, 141.9, 149.6, 175.7; IR (film) cm⁻¹: 2949, 1723, 1608, 1525, 1455, 1349, 1177, 1092, 1028, 862, 742, 700; ESI-MS m/z: 411.2269 (Calcd for C₂₄H₃₁N₂O₄: 411.2283).

Ethyl 1-(4-Nitrophenethyl)-4-phenethylpiperidine-4-carboxylate (32p)

This compound was prepared according to a similar procedure as compound 32o. p-Nitrostyrene used in this reaction is prepared according to a literature procedure.⁴³⁾ Column chromatography (n-hexane/ethyl acetate 5 : 1) gave the title compound (113 mg, 0.276 mmol, 70.5%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.29 (t, J = 7.1 Hz, 3H), 1.50–1.66 (m, 2H), 1.78–1.88 (m, 2H), 2.14–2.29 (m, 4H), 2.48–2.56 (m, 2H), 2.57–2.66 (m, 2H), 2.75–2.85 (m, 2H), 3.04–3.13 (m, 2H), 4.18 (q, J = 7.1 Hz, 2H), 7.11–7.21 (m, 3H), 7.23–7.31 (m, 2H), 7.32–7.39 (m, 2H), 8.10–8.17 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.3, 30.5, 30.6, 33.5, 42.2, 45.2, 51.0, 59.3, 60.3, 123.5, 125.9, 128.2, 128.3, 129.4, 141.8, 146.4, 148.4, 175.7; IR (film) cm⁻¹: 2949, 1723, 1608, 1525, 1455, 1349, 1177, 1092, 1028, 862, 742, 700; ESI-MS m/z: 411.2277 (Calcd for C₂₄H₃₁N₂O₄: 411.2283).

Ethyl 1-(2-Aminophenethyl)-4-phenethylpiperidine-4-carboxylate (33o)

This compound was prepared according to a similar procedure as compound 16. Column chromatography (n-hexane/ethyl acetate 3 : 1) gave the title compound (83.1 mg, 0.219 mmol, 97.6%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.29 (t, J = 7.1 Hz, 3H), 1.50–1.64 (m, 2H), 1.79–1.88 (m, 2H), 2.12–2.30 (m, 4H), 2.47–2.61 (m, 4H), 2.67–2.75 (m, 2H), 2.82–2.93 (m, 2H), 4.19 (q, J = 7.1 Hz, 2H), 6.63–6.72 (m, 2H), 6.96–7.06 (m, 2H), 7.11–7.21 (m, 3H), 7.24–7.31 (m, 2H), two exchangeable NH protons were not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.3, 30.1, 30.5, 33.6, 42.2, 45.2, 51.3, 58.8, 60.3, 115.7, 118.4, 125.7, 125.8, 127.2, 128.2, 128.3, 130.1, 141.8, 145.1, 175.6; IR (film) cm⁻¹: 2949, 1723, 1623, 1497, 1455, 1178, 1027, 749, 700; ESI-MS m/z: 381.2545 (Calcd for C₂₄H₃₃N₂O₂: 381.2542).

Ethyl 1-(4-Aminophenethyl)-4-phenethylpiperidine-4-carboxylate (33p)

This compound was prepared according to a similar procedure as compound 16. Column chromatography (n-hexane/ethyl acetate 1 : 3) gave the title compound (85.3 mg, 0.224 mmol, 98.9%) as a yellow solid. mp: 49.2–52.2 °C (decomp.); ¹H-NMR (CDCl₃, 400 MHz) δ: 1.29 (t, J = 7.1 Hz, 3H), 1.52–1.65 (m, 2H), 1.77–1.88 (m, 2H), 2.12 (br t, J = 11.3 Hz, 2H), 2.24 (br d, J = 13.2 Hz, 2H), 2.43–2.60 (m, 4H), 2.62–2.76 (m, 2H), 2.76–2.90 (m, 2H), 3.54 (br s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 6.59–6.67 (m, 2H), 6.94–7.02 (m, 2H), 7.11–7.21 (m, 3H), 7.23–7.31 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.4, 30.5, 32.8, 33.5, 42.3, 45.3, 51.1, 60.3, 61.2, 115.2, 125.8, 128.2, 128.3, 129.4, 130.3, 141.9, 144.4, 175.7; IR (film) cm⁻¹: 3371, 2926, 1722, 1625, 1516, 1454, 1178, 751, 700; ESI-MS m/z: 381.2526 (Calcd for C₂₄H₃₃N₂O₂: 381.2542).

Ethyl 1-(2-Acetamidophenethyl)-4-phenethylpiperidine-4-carboxylate (27o)

This compound was prepared according to a similar procedure as compound 18. Column chromatography (n-hexane/ethyl acetate 1 : 1) gave the title compound (32.3 mg, 0.0765 mmol, 95.3%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.29 (t, J = 7.1 Hz, 3H), 1.51–1.69 (m, 2H), 1.77–1.90 (m, 2H), 2.21 (s, 3H), 2.16–2.36 (m, 4H), 2.48–2.57 (m, 2H), 2.54–2.69 (m, 2H), 2.70–2.79 (m, 2H), 2.77–2.94 (m, 2H), 4.19 (q, J = 7.1 Hz, 2H), 6.98–7.35 (m, 8H), 7.94 (br d, J = 8.0 Hz, 1H), 10.26 (br s, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.3, 24.6, 30.5, 31.4, 33.4, 42.1, 44.9, 52.0, 60.5, 60.6, 123.4, 124.4, 126.0, 126.9, 128.1, 128.4, 129.9, 132.9, 136.7, 141.5, 168.3, 175.2; IR (neat) cm⁻¹: 2929, 1723, 1589, 1530, 1452, 1369, 1298, 1178, 754, 700; ESI-MS m/z: 423.2626 (Calcd for C₂₆H₃₅N₂O₃: 423.2647).

Ethyl 1-(4-Acetamidophenethyl)-4-phenethylpiperidine-4-carboxylate (27p)

This compound was prepared according to a similar procedure as compound 18. Column chromatography (n-hexane/ethyl acetate 3 : 1) gave the title compound (75.1 mg, 0.178 mmol, 99.4%) as a yellow solid. mp: 72.6–73.7 °C (decomp.); ¹H-NMR (CDCl₃, 400 MHz) δ: 1.29 (t, J = 7.1 Hz, 3H), 1.52–1.63 (m, 2H), 1.79–1.86 (m, 2H), 2.08–2.20 (m, 2H), 2.16 (s, 3H), 2.24 (br d, J = 13.3 Hz, 2H), 2.48–2.57 (m, 4H), 2.72–2.88 (m, 4H), 4.18 (q, J = 7.1 Hz, 2H), 7.09–7.21 (m, 5H), 7.24–7.30 (m, 2H), 7.37–7.42 (m, 2H), an exchangeable NH proton was not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.3, 24.4, 30.5, 33.0, 33.4, 42.2, 45.3, 51.1, 60.3, 60.6, 120.0, 125.8, 128.2, 128.3, 129.0, 135.9, 136.3, 141.8, 168.4, 175.7; IR (film) cm⁻¹: 2930, 1724, 1537, 1371, 1318, 1179, 1027, 911, 822, 733, 700; ESI-MS m/z: 423.2641 (Calcd for C₂₆H₃₅N₂O₃: 423.2647).

2-(3-Nitrophenyl)ethan-1-ol (35)

To a solution of 3-nitrophenylacetic acid (34, 150 mg, 0.828 mmol) in tetrahydrofuran (4 mL) was added borane–tetrahydrofuran complex (0.90 M in tetrahydrofuran, 1.8 mL, 1.6 mmol) at 0 °C. The mixture was stirred at ambient temperature for 1 h. Then, saturated aqueous ammonium chloride solution was added, and the mixture was extracted with dichloromethane four times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (n-hexane/ethyl acetate 1 : 3) gave the title compound (77.4 mg, 0.565 mmol, 92.5%) as a yellow solid. The spectral data were consistent with the literature.⁴⁴⁾

2-(3-Aminophenyl)ethan-1-ol (36)

This compound was prepared according to a similar procedure as compound 16. Column chromatography (n-hexane/ethyl acetate, 1 : 4) gave the title compound (76.0 mg, 0.216 mmol, 94.4%) as a yellow oil. The spectral data were consistent with the literature.⁴⁵⁾

3-Acetamidophenethyl Acetate (37)

This compound was prepared according to a similar procedure as compound 18. Column chromatography (n-hexane/ethyl acetate, 1 : 3) gave the title compound (127 mg, 0.575 mmol, 100%) as a brown oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 2.04 (s, 3H), 2.17 (s, 3H), 2.91 (t, J = 7.0 Hz, 2H), 4.27 (t, J = 7.0 Hz, 2H), 6.96 (br d, J = 7.5 Hz, 1H), 7.21–7.26 (m, 2H), 7.33 (br d, J = 8.2 Hz, 1H), 7.42 (br s, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 20.9, 24.5, 34.9, 64.7, 118.0, 120.2, 124.7, 129.0, 138.0, 138.7, 168.4, 171.0; IR (neat) cm⁻¹: 3304, 1735, 1669, 1613, 1555, 1489, 1441, 1368, 1239, 1035; ESI-MS m/z: 244.0942 (Calcd for C₁₂H₁₅NNaO₃: 244.0949).

N-(3-(2-Hydroxyethyl)phenyl)acetamide (38)

This compound was prepared according to a similar procedure as compound 19. Column chromatography (chloroform/methanol, 15 : 1) gave the title compound (73.7 mg, 0.412 mmol, 87.6%) as a colorless solid. mp: 106.8–107.8 °C (decomp.); ¹H-NMR (CDCl₃, 400 MHz) δ: 2.17 (s, 3H), 2.85 (t, J = 6.5 Hz, 2H), 3.86 (t, J = 6.5 Hz, 2H), 6.98 (br d, J = 7.4 Hz, 1H), 7.20 (br s, 1H), 7.23–7.30 (m, 1H), 7.34 (br d, J = 8.1 Hz, 1H), 7.41(br s, 1H), an exchangeable OH proton was not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 24.6, 39.1, 63.4, 118.0, 120.4, 125.0, 129.1, 129.2, 139.6, 168.2; IR (neat) cm⁻¹: 3302, 2924, 1667, 1613, 1556, 1488, 1441, 1043; ESI-MS m/z: 202.0840 (Calcd for C₁₀H₁₃NNaO₂: 202.0844).

3-Acetamidophenethyl Methanesulfonate (39)

To a mixture of 38 (64.8 mg, 0.362 mmol) and triethylamine (76 µL, 0.54 mmol) in dichloromethane (3 mL) was added methanesulfonyl chloride (31 µL, 0.40 mmol) at 0 °C. After stirring for 16 h at ambient temperature, saturated aqueous sodium hydrogen carbonate solution was added to the mixture, and the mixture was extracted with dichloromethane four times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (n-hexane/ethyl acetate 1 : 2) gave the title compound (72.8 mg, 0.283 mmol, 78.3%) as a colorless oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 2.17 (s, 3H), 2.88 (s, 3H), 3.03 (t, J = 6.7 Hz, 2H), 4.41 (t, J = 6.7 Hz, 2H), 6.98 (br d, J = 7.4 Hz, 1H), 7.23–7.30 (m, 3H), 7.48 (br s, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 24.3, 35.2, 37.0, 69.7, 118.1, 120.0, 124.5, 128.9, 137.0, 137.9, 168.0; IR (neat) cm⁻¹: 3304, 1668, 1593, 1556, 1488, 1442, 1347, 1170, 954, 800, 697; ESI-MS m/z: 280.0635 (Calcd for C₁₁H₁₅NNaO₄S: 280.0619).

Ethyl 1-(3-Acetamidophenethyl)-4-phenethylpiperidine-4-carboxylate (27m)

To a solution of 13 (137 mg, 0.524 mmol) in acetonitrile (5 mL) were added potassium carbonate (109 mg, 0.786 mmol) and 39 (67.4 mg, 0.262 mmol). The mixture was refluxed for 24 h. After cooling to ambient temperature, saturated aqueous sodium hydrogen carbonate solution was added to the mixture, and the mixture was extracted with dichloromethane four times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (chloroform/methanol 15 : 1) gave the title compound (94.5 mg, 0.224 mmol, 85.5%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.29 (t, J = 7.1 Hz, 3H), 1.53–1.63 (m, 2H), 1.79–1.87 (m, 2H), 2.09–2.21 (m, 2H), 2.16 (s, 3H), 2.25 (br d, J = 13.2 Hz, 2H), 2.47–2.61 (m, 4H), 2.74–2.87 (m, 4H), 4.19 (q, J = 7.1 Hz, 2H), 6.94 (br d, J = 7.5 Hz, 2H), 7.08–7.33 (m, 7H), 7.36 (br s, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.0, 24.2, 30.2, 33.1, 33.2, 41.9, 44.9, 50.7, 60.0, 60.1, 117.2, 119.7, 124.3, 125.5, 127.8, 128.0, 128.5, 137.6, 141.0, 141.5, 168.0, 175.4; IR (film) cm⁻¹: 2950, 1724, 1613, 1556, 1489, 1371, 1178, 1027, 732, 699;ESI-MS m/z: 423.2645 (Calcd for C₂₆H₃₅N₂O₃: 423.2647).

Ethyl 1-(4-(N-Methylacetamido)benzyl)-4-phenethylpiperidine-4-carboxylate (40a)

To a solution of CYM51010 (86.5 mg, 0.212 mmol) in N,N-dimethylformamide (2 mL), was added sodium hydride (ca. 60%, 17.1 mg, 0.424 mmol) at ambient temperature. After cooling to 0 °C, methyl iodide (26 µL, 0.42 mmol) was added to the mixture, and the mixture was stirred at this temperature for 17 h while allowing the mixture to warm to ambient temperature. Then water was added, and the mixture was extracted with dichloromethane. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (ethyl acetate/methanol 50 : 1) gave the title compound (6.6 mg, 0.016 mmol, 7.4%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.21–1.36 (m, 3H), 1.51–1.61 (m, 2H), 1.80–1.93 (m, 5H), 2.12 (br, t, J = 11.4 Hz, 2H), 2.22 (br d, J = 13.2 Hz, 2H), 2.47–2.55 (m, 2H), 2.72 (br d, J = 11.9 Hz, 2H), 3.25 (s, 3H), 3.47 (s, 2H), 4.19 (q, J = 7.1 Hz, 2H), 7.09–7.21 (m, 5H), 7.23–7.30 (m, 2H), 7.35 (d, J = 8.1 Hz, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.6, 22.6, 30.7, 33.7, 37.3, 42.4, 45.4, 51.3, 60.5, 62.8, 126.0, 127.0, 128.4, 128.6, 130.4, 138.4, 142.0, 143.5, 170.8, 175.9; IR (film) cm⁻¹: 3583, 2923, 1722, 1622, 1605, 1512, 1454, 1373, 1297, 1209, 1177, 1020, 974, 699; ESI-MS m/z: 445.2447 (Calcd for C₂₆H₃₄N₂O₃Na: 445.24671).

Ethyl 1-(4-(Methoxycarbonyl)benzyl)-4-phenethylpiperidine-4-carboxylate (40b)

This compound was prepared according to a similar procedure as compound 10, starting from compound 13 and using methyl 4-formylbenzoate (41b) instead of N-(4-formylphenyl)acetamide (4). Column chromatography (n-hexane/ethyl acetate 6 : 1) gave the title compound (97.7 mg, 0.239 mmol, 76.5%) as a pale yellow solid. mp: 45.4–45.8 °C; ¹H-NMR (CDCl₃, 400 MHz) δ: 1.28 (t, J = 7.1 Hz, 3H), 1.49–1.60 (m, 2H), 1.78–1.86 (m, 2H), 2.11 (br t, J = 11.4 Hz, 2H), 2.20 (br d, J = 13.2 Hz, 2H), 2.46–2.55 (m, 2H), 2.64–2.75 (br d, J = 11.8 Hz, 2H), 3.49 (s, 2H), 3.89 (s, 3H), 4.18 (q, J = 7.1 Hz, 2H), 7.09–7.19 (m, 3H), 7.22–7.30 (m, 2H), 7.38 (d, J = 8.2 Hz, 2H) 7.98 (d, J = 8.2 Hz, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.5, 30.7, 33.7, 42.4, 45.4, 51.4, 52.1, 60.5, 63.1, 126.0, 128.4, 128.5, 128.99, 129.01, 129.7, 142.1, 144.4, 167.2, 175.9; IR (film) cm⁻¹: 2951, 2803, 1723, 1610, 1435, 1278, 1177, 1111, 1020, 754; ESI-MS m/z: 410.2348 (Calcd for C₂₅H₃₂N₂O₄: 410.2331).

Ethyl 1-(4-Methoxybenzyl)-4-phenethylpiperidine-4-carboxylate (40c)

This compound was prepared according to a similar procedure as compound 10, starting from compound 13 and using p-anisaldehyde (41c) instead of N-(4-formylphenyl)acetamide (4). Column chromatography (n-hexane/ethyl acetate 4 : 1) gave the title compound (344 mg, 0.903 mmol, 71.0%) as a colorless solid. mp: 90.2–90.6 °C; ¹H-NMR (CDCl₃, 400 MHz) δ: 1.28 (t, J = 7.1 Hz, 3H), 1.48–1.58 (m, 2H), 1.77–1.85 (m, 2H), 2.06 (br t, J = 11.8 Hz, 2H), 2.19 (br d, J = 13.2 Hz, 2H), 2.45–2.55 (m, 2H), 2.66–2.74 (m, 2H), 3.39 (s, 2H), 3.78 (s, 3H), 4.18 (q, J = 7.1 Hz, 2H), 6.84 (d, J = 8.7 Hz, 2H), 7.10–7.32 (m, 7H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.5, 30.7, 33.7, 42.4, 45.5, 51.1, 55.3, 60.4, 62.9, 113.6, 126.0, 128.4, 128.5, 130.4, 130.6, 142.1, 158.8, 176.0; IR (film) cm⁻¹: 3424, 2964, 2929, 2798, 1721, 1512, 1465, 1241, 1173, 1031, 747, 703, 504; ESI-MS m/z: 382.2380 (Calcd for C₂₄H₃₂NO₃: 382.2382).

Ethyl 1-(4-(Benzyloxy)benzyl)-4-phenethylpiperidine-4-carboxylate (40d)

This compound was prepared according to a similar procedure as compound 10, starting from compound 13 and using p-(benzyloxy)benzaldehyde (41d) instead of N-(4-formylphenyl)acetamide (4). Column chromatography (chloroform/methanol 50 : 1) gave the title compound (257 mg, 0.562 mmol, 73.3%) as a pale brown oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.18 (br t, J = 7.1 Hz, 3H), 1.47–1.60 (br m, 2H), 1.75–1.86 (m, 2H), 2.06 (br t, J = 11.4 Hz, 2H), 2.19 (br d, J = 6.7 Hz, 2H), 2.44–2.55 (m, 2H), 2.71 (br d, J = 11.8, 2H), 3.39 (s, 2H), 4.17 (q, J = 7.1 Hz, 2H), 4.03 (s, 2H), 6.91 (d, J = 4.3 Hz, 2H), 7.08–7.46 (m, 12H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.5, 30.7, 33.6, 42.3, 45.5, 51.1, 60.4, 62.8, 70.1, 114.6, 126.0, 127.6, 128.0, 128.4, 128.5, 128.6, 130.5, 130.8, 137.2, 142.1, 158.0, 175.9; IR (film) cm⁻¹: 2925, 1723, 1510, 1454, 1240, 1175, 1027, 698; ESI-MS m/z: 471.2681 (Calcd for C₃₀H₃₆NO₃: 458.2695).

Ethyl 1-(4-Hydroxybenzyl)-4-phenethylpiperidine-4-carboxylate (40e)

This compound was prepared according to a similar procedure as compound 24, starting from compound 40d and using ethanol as the solvent. Column chromatography (chloroform/methanol 50 : 1) gave the title compound (11.5 mg, 0.0313 mmol, 14.4%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.28 (br t, J = 7.1 Hz, 3H), 1.51–1.62 (m, 2H), 1.75–1.85 (m, 2H), 2.11 (br t, J = 11.5 Hz, 2H), 2.21 (br d, J = 13.3 Hz, 2H), 2.44–2.53 (m, 2H), 2.81 (br d, J = 11.8 Hz, 2H), 3.39 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 6.60 (d, J = 4.2 Hz, 2H), 7.04–7.19 (m, 5H), 7.22–7.30 (m, 2H), an exchangeable OH proton was not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.5, 30.7, 33.3, 42.5, 45.5, 51.1, 60.6, 62.9, 115.4, 126.0, 128.4, 128.5, 131.1, 142.0, 155.6, 176.0, two coincidental carbon signals are overlapping in the aromatic region; IR (film) cm⁻¹: 3583, 2926, 1722, 1613, 1516, 1454, 1216, 1178, 771; ESI-MS m/z: 368.2211 (Calcd for C₂₃H₃₀NO₃: 368.2226).

Ethyl 1-(4-(Methylsulfonamido)benzyl)-4-phenethylpiperidine-4-carboxylate (40f)

A solution of 16 (100 mg, 0.273 mmol) in dichloromethane (3.4 mL) was cooled to –10 °C, and to this solution were added triethylamine (41 µL, 0.30 mmol) and methanesulfonyl chloride (23 µL, 0.30 mmol). After stirring at –10 °C for 16 h, triethylamine (41 µL, 0.30 mmol) and methanesulfonyl chloride (23 µL, 0.30 mmol) were added to the mixture. The mixture was further stirred for 6 h at this temperature. Then water was added to the mixture, and the mixture was extracted with dichloromethane three times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (chloroform/methanol, 50 : 1) gave the title compound (29.7 mg, 0.0669 mmol, 24.5%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.28 (t, J = 7.1 Hz, 3H), 1.49–1.59 (br m, 2H), 1.77–1.86 (br m, 2H), 2.09 (br t, J = 11.3 Hz, 2H), 2.20 (br d, J = 13.3 Hz, 2H), 2.45–2.55 (m, 2H), 2.66–2.75 (br m, 2H), 3.00 (s, 3H), 3.43 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 7.10–7.20 (m, 5H), 7.22–7.33 (m, 4H), an exchangeable NH proton was not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.5, 30.7, 33.6, 39.3, 42.4, 45.4, 51.2, 60.5, 62.7, 121.0, 126.0, 128.4, 128.5, 130.5, 135.7, 135.9, 142.0, 176.0; IR (film) cm⁻¹: 3583, 3255, 3026, 2927, 2854, 2809, 1722, 1614, 1510, 1455, 1393, 1369, 1333, 1214, 1179, 1155, 1105, 1072, 1028, 973, 914, 858, 750, 700; ESI-MS m/z: 445.2148 (Calcd for C₂₄H₃₃N₂O₄S: 445.2161).

Ethyl-1-(4-(N-(methylsulfonyl)methylsulfonamido)benzyl)-4-phenethylpiperidine-4-carboxylate (40g)

A solution of 16 (100 mg, 0.273 mmol) in dichloromethane (3.4 mL) was cooled to –10 °C, and to this solution were added triethylamine (96 µL, 0.68 mmol) and methanesulfonyl chloride (53 µL, 0.68 mmol). After stirring at –10 °C for 4 h, water was added to the mixture. The mixture was extracted with dichloromethane three times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (NH silica gel, n-hexane/ethyl acetate, 1 : 1) gave the title compound (94.0 mg, 0.180 mmol, 66.0%) as a colorless oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.28 (t, J = 7.1 Hz, 3H), 1.49–1.65 (m, 2H), 1.78–1.87 (m, 2H), 2.11 (br t, J = 11.5 Hz, 2H), 2.20 (br d, J = 13.2 Hz, 2H), 2.47–2.55 (m, 2H), 2.70 (br d, J = 12.0 Hz, 2H), 3.40 (s, 6H), 3.47 (s, 2H), 4.19 (q, J = 7.1 Hz, 2H), 7.11–7.20 (m, 3H), 7.25–7.32 (m, 4H), 7.39–7.45 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.6, 30.7, 33.7, 42.4, 42.8, 45.4, 51.4, 60.5, 62.7, 126.0, 128.4, 128.5, 130.2, 130.5, 132.1, 142.0, 142.1, 175.9; IR (film) cm⁻¹: 2934, 2805, 1721, 1369, 1162, 978, 907, 761; ESI-MS m/z: 523.1915 (Calcd for C₂₄H₃₅N₂O₆S₂: 523.1937).

Ethyl 4-Phenethyl-1-(4-(2,2,2-trifluoroacetamido)benzyl)piperidine-4-carboxylate (40h)

This compound was prepared according to a similar procedure as compound 18, starting from compound 16 and using trifluoroacetic anhydride instead of acetic anhydride. Column chromatography (chloroform/methanol, 20 : 1) gave the title compound (60.5 mg, 0.129 mmol, 60.0%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.28 (t, J = 7.1 Hz, 3H), 1.46–1.60 (m, 2H), 1.76–1.87 (br m, 2H), 2.09 (br t, J = 11.5 Hz, 2H), 2.20 (br d, J = 13.2 Hz, 2H), 2.45–2.56 (m, 2H), 2.69 (br d, J = 11.4 Hz, 2H), 3.43 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 7.08–7.20 (m, 3H), 7.22–7.38 (m, 4H), 7.47–7.60 (m, 2H), 8.24 (br s, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.5, 30.7, 33.6, 42.3, 45.4, 51.2, 60.5, 62.7, 115.8 (q, J = 286.7 Hz), 120.4, 126.0, 128.4, 128.5, 130.0, 134.1, 137.0, 142.0, 154.8 (q, J = 37.2 Hz), 176.0; ¹⁹F-NMR (CDCl₃, 300 MHz) δ: −75.6; IR (neat) cm⁻¹: 3305, 2950, 1724, 1550, 1155; ESI-MS m/z: 471.2209 (Calcd for C₂₅H₃₀F₃N₂O₃: 471.2230).

Ethyl 4-Phenethyl-1-(4-(2,2,2-trichloroacetamido)benzyl)piperidine-4-carboxylate (40i)

This compound was prepared according to a similar procedure as compound 18, starting from compound 16, without 4-dimethylaminopyridine, and using trichloroacetic anhydride instead of acetic anhydride. Column chromatography (n-hexane/ethyl acetate, 4 : 1) gave the title compound (56.0 mg, 0.110 mmol, 40.2%) as a brown oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.28 (t, J = 7.1 Hz, 3H), 1.48–1.60 (m, 2H), 1.78–1.85 (m, 2H), 2.09 (br t, J = 11.4 Hz, 2H), 2.20 (br d, J = 13.2 Hz, 2H), 2.46–2.54 (m, 2H), 2.69 (br d, J = 11.9 Hz, 2H), 3.44 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 7.09–7.21 (m, 3H), 7.24–7.30 (m, 2H), 7.34 (d, J = 8.5 Hz, 2H), 7.51 (d, J = 8.5 Hz, 2H), 8.37 (br s, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.5, 30.7, 33.6, 42.3, 45.4, 51.2, 60.5, 62.8, 93.0, 120.3, 126.0, 128.4, 128.5, 130.0, 134.9, 136.7, 142.0, 159.3, 175.9; IR (film) cm⁻¹: 3583, 2925, 1720, 1601, 1524, 1179, 1027; ESI-MS m/z: 511.1297 (Calcd for C₂₅H₃₀Cl₃N₂O₃: 511.1322).

Ethyl 1-(4-Benzamidobenzyl)-4-phenethylpiperidine-4-carboxylate (40j)

This compound was prepared according to a similar procedure as compound 18, starting from compound 16 and using benzoyl chloride instead of acetic anhydride. Column chromatography (n-hexane/ethyl acetate, 1 : 1) gave the title compound (114 mg, 0.243 mmol, 49.3%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.22–1.34 (m, 3H), 1.50–1.61 (m, 2H), 1.71–1.86 (m, 2H), 2.02–2.15 (m, 2H), 2.17–2.24 (m, 2H), 2.47–2.54 (m, 2H), 2.68–2.77 (m, 2H), 3.45 (s, 2H), 4.19 (q, J = 7.1 Hz, 2H), 7.10–7.20 (m, 3H), 7.23–7.35 (m, 4H), 7.45–7.61 (m, 5H), 7.80 (s, 1H), 7.84–7.91 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.6, 30.7, 33.5, 42.3, 45.4, 51.1, 60.5, 62.7, 120.2, 126.0, 127.2, 128.1, 128.4, 128.5, 130.0, 131.8, 134.1, 135.0, 136.9, 141.9, 165.7, 175.8; IR (film) cm⁻¹: 3308, 2949, 1723, 1654, 1603, 1530, 1410, 1319, 1266, 1028, 909, 839, 700;ESI-MS m/z: 471.2659 (Calcd for C₃₀H₃₅N₂O₃: 471.2648).

Structural Diversification of the Eastern Part

1-(tert-Butyl) 4-Ethyl 4-Ethylpiperidine-1,4-dicarboxylate (42a)

This compound was prepared according to a similar procedure as compound 3, using ethyl iodide instead of (2-bromoethyl)benzene. Column chromatography (n-hexane/ethyl acetate, 10 : 1) gave the title compound (199 mg, 0.648 mmol, 41.8%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 0.81 (t, J = 7.6 Hz, 3H), 1.26 (t, J = 7.1 Hz, 3H), 1.28–1.38 (m, 2H), 1.44 (s, 9H), 1.54 (q, J = 7.6 Hz, 2H), 2.09 (br d, J = 13.0 Hz, 2H), 2.77–2.95 (m, 2H), 3.75–3.97 (m, 2H), 4.17 (q, J = 7.1 Hz, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 8.2, 14.2, 28.4, 32.9, 33.0, 41.2, 45.8, 60.3, 79.3, 154.8, 175.4; IR (film) cm⁻¹: 2972, 1726, 1697, 1422, 1365, 1247, 1174, 1105, 1027; ESI-MS m/z: 308.1825 (Calcd for C₁₅H₂₇NNaO₄: 308.1837).

1-(tert-Butyl) 4-Ethyl 4-Benzylpiperidine-1,4-dicarboxylate (42b)

This compound was prepared according to a similar procedure as compound 3, using benzyl bromide instead of (2-bromoethyl)benzene. Column chromatography (n-hexane/ethyl acetate, 19 : 1 to 4 : 1) gave the title compound (2.98 g, 8.56 mmol, 55.1%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.18 (t, J = 7.2 Hz, 3H), 1.37–1.50 (m, 2H), 1.44 (s, 9H), 2.08 (br d, J = 13.0 Hz, 2H), 2.73–2.88 (m, 2H), 2.81 (s, 2H), 3.93 (br s, 2H), 4.10 (q, J = 7.2 Hz, 2H), 7.02–7.07 (m, 2H), 7.17–7.29 (m, 3H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.2, 28.5, 33.4, 41.3, 46.7, 47.3, 60.6, 79.5, 126.9, 128.2, 130.0, 136.4, 155.0, 174.9; IR (film) cm⁻¹: 2977, 1727, 1694, 1423, 1365, 1175, 1083, 702; ESI-MS m/z: 370.2010 (Calcd for C₂₀H₂₉NNaO₄: 370.1994).

1-(tert-Butyl) 4-Ethyl 4-(3-Phenylpropyl)piperidine-1,4-dicarboxylate (42c)

This compound was prepared according to a similar procedure as compound 3, using (3-bromopropyl)benzene instead of (2-bromoethyl)benzene. Column chromatography (n-hexane/ethyl acetate, 3 : 1) gave the title compound (346 mg, 0.947 mmol, 77.7%) as a colorless oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.22 (t, J = 7.1 Hz, 3H), 1.26–1.38 (m, 2H), 1.43 (s, 9H), 1.48–1.61 (m, 4H), 2.07 (br d, J = 13.2 Hz, 2H), 2.50–2.63 (m, 2H), 2.72–3.00 (m, 2H), 3.83 (br s, 2H), 4.14 (q, J = 7.1 Hz, 2H), 7.10–7.21 (m, 3H), 7.23–7.31 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.2, 25.5, 28.4, 33.3, 35.9, 39.7, 41.3, 45.4, 60.4, 79.3, 125.8, 128.2, 128.3, 141.8, 154.8, 175.4; IR (film) cm⁻¹: 2975, 1725, 1695, 1422, 1365, 1248, 1174, 1095, 699; ESI-MS m/z: 398.2292 (Calcd for C₂₂H₃₃NNaO₄: 398.2307).

1-(tert-Butyl) 4-Ethyl 4-(4-Phenylbutyl)piperidine-1,4-dicarboxylate (42d)

This compound was prepared according to a similar procedure as compound 3, using (4-bromobutyl)benzene instead of (2-bromoethyl)benzene. Column chromatography (n-hexane/ethyl acetate, 3 : 1) gave the title compound (534 mg, 1.37 mmol, 67.3%) as a pale yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.22 (t, J = 7.1 Hz, 3H), 1.16–1.37 (m, 4H), 1.44 (s, 9H), 1.46–1.62 (m, 4H), 2.07 (br d, J = 13.2 Hz, 2H), 2.58 (t, J = 7.6 Hz, 2H), 2.78–2.94 (m, 2H), 3.85 (br s, 2H), 4.13 (q, J = 7.1 Hz, 2H), 7.11–7.20 (m, 3H), 7.27–7.29 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.5, 23.7, 28.7, 31.9, 33.3, 35.9, 40.4, 41.9, 45.8, 60.7, 79.6, 125.9, 128.5, 128.6, 142.5, 155.1, 175.8; IR (film) cm⁻¹: 2934, 1725, 1695, 1422, 1365, 1248, 1174, 1097, 1027, 866, 747, 699; ESI-MS m/z: 412.2464 (Calcd for C₂₃H₃₅NNaO₄: 412.2463).

Ethyl 4-Ethylpiperidine-4-carboxylate (43a)

To a solution of 42a (43.0 mg, 0.151 mmol) in dichloromethane (1 mL) was slowly added trifluoroacetic acid (1 mL) at 0 °C. After stirring at ambient temperature for 3 h, the mixture was concentrated under reduced pressure. To the residue was added saturated aqueous sodium hydrogen carbonate solution, and the mixture was extracted with dichloromethane four times. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography (chloroform/methanol, 15 : 1) gave the title compound (26.6 mg, 0.144 mmol, 95.2%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 0.82 (t, J = 7.6 Hz, 3H), 1.27 (t, J = 7.1 Hz, 3H), 1.46–1.56 (m, 2H), 1.57 (q, J = 7.6 Hz, 2H), 2.19 (d, J = 13.7 Hz, 2H), 2.69–2.81 (m, 2H), 3.05–3.15 (m, 2H), 4.18 (q, J = 7.1 Hz, 2H), an exchangeable NH proton was not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 8.1, 14.3, 33.5, 33.8, 43.6, 45.9, 60.2, 175.8; IR (film) cm⁻¹: 2966, 1725, 1538, 1448, 1151, 1025, 932, 812; ESI-MS m/z: 186.1489 (Calcd for C₁₀H₂₀NO₂: 186.1494).

Ethyl 4-Benzylpiperidine-4-carboxylate (43b)

This compound was prepared according to a similar procedure as compound 43a. The obtained crude product (96.8 mg, 0.391 mmol, 89.1%, yellow oil) was used for the next reaction without purification. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.18 (t, J = 7.2 Hz, 3H), 1.38–1.51 (m, 2H), 2.06–2.16 (m, 2H), 2.58–2.68 (m, 2H), 2.82 (s, 2H), 2.93–3.01 (m, 2H), 4.09 (q, J = 7.2 Hz, 2H), 7.02–7.07 (m, 2H), 7.13–7.31 (m, 3H), an exchangeable NH proton was not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.2, 34.5, 44.0, 47.2, 47.5, 60.4, 126.7, 128.0, 130.1, 136.7, 175.5; IR (film) cm⁻¹:2938, 1723, 1446, 1177, 1028, 742, 702; ESI-MS m/z: 248.1640 (Calcd for C₁₅H₂₂NO₂: 248.1651).

Ethyl 4-(3-Phenylpropyl)piperidine-4-carboxylate (43c)

This compound was prepared according to a similar procedure as compound 43a. The obtained crude product (182 mg, 0.663 mmol, 100%, yellow oil) was used for the next reaction without purification. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.22 (t, J = 7.1 Hz, 3H), 1.28–1.41 (m, 2H), 1.47–1.61 (m, 4H), 2.00–2.18 (m, 3H), 2.49–2.71 (m, 4H), 2.91 (ddd, J = 3.8, 3.8, 12.9 Hz, 2H), 4.19 (q, J = 7.1 Hz, 2H), 7.10–7.21 (m, 3H), 7.22–7.31 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.2, 25.4, 34.5, 36.0, 40.3, 43.8, 45.5, 60.2, 125.7, 128.2, 128.3, 141.9, 175.9; IR (neat) cm⁻¹: 2939, 1723, 1495, 1453, 1278, 1171, 1027, 749, 700; ESI-MS m/z: 276.1955 (Calcd for C₁₇H₂₆NO₂: 276.1963).

Ethyl 4-(4-Phenylbutyl)piperidine-4-carboxylate (43d)

This compound was prepared according to a similar procedure as compound 43a. Column chromatography (chloroform/methanol, 10 : 1) gave the title compound (111 mg, 0.385 mmol, 100%) as a yellow oil. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.22 (t, J = 7.1 Hz, 3H), 1.48–1.71 (m, 6H), 2.24 (br d, J = 13.8 Hz, 2H), 2.58 (t, J = 7.6 Hz, 2H), 2.58 (t, J = 7.6 Hz, 2H), 2.75–2.90 (m, 2H), 3.20 (br d, J = 12.8 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H), 4.71 (br s, 1H), 7.09–7.20 (m, 3H), 7.21–7.30 (m, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.5, 23.5, 31.3, 31.7, 35.8, 40.6, 42.3, 44.9, 61.3, 126.0, 128.60, 128.63, 142.4, 174.9; IR (neat) cm⁻¹: 2935, 1723, 1681, 1454, 1201, 1023, 832, 799, 721, 699; ESI-MS m/z: 290.2130 (Calcd for C₁₈H₂₈NO₂: 290.2120).

Ethyl 1-(4-Acetamidobenzyl)-4-ethylpiperidine-4-carboxylate (44a)

This compound was prepared according to a similar procedure as compound 10. Column chromatography (n-hexane/ethyl acetate, 1 : 10) gave the title compound (66.3 mg, 0.200 mmol, 30.3%) as a colorless solid. mp: 84.5–88.6 °C (decomp.); ¹H-NMR (CDCl₃, 400 MHz) δ: 0.79 (t, J = 7.6 Hz, 3H), 1.25 (t, J = 7.1 Hz, 3H), 1.38–1.50 (m, 2H), 1.53 (q, J = 7.6 Hz, 2H), 1.97–2.16 (m, 4H), 2.22 (s, 3H), 2.63–2.77 (m, 2H), 3.42 (s, 2H), 4.15 (q, J = 7.1 Hz, 2H), 7.17 (br s, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.3, 22.4, 28.2, 32.8, 33.4, 45.1, 51.1, 60.1, 62.6, 119.6, 119.7, 129.8, 136.8, 168.1, 176.1; IR (film) cm⁻¹: 2965, 1725, 1605, 1539, 1370, 1316, 1154, 1022, 753; ESI-MS m/z: 333.2181 (Calcd for C₁₉H₂₉N₂O₃: 333.2178).

Ethyl 1-(4-Acetamidobenzyl)-4-benzylpiperidine-4-carboxylate (44b)

This compound was prepared according to a similar procedure as compound 10. Column chromatography (n-hexane/ethyl acetate, 1 : 4) gave the title compound (128 mg, 0.326 mmol, 95.6%) as a colorless solid. mp: 140.8–142.3 °C; ¹H-NMR (CDCl₃, 400 MHz) δ: 1.16 (t, J = 7.1 Hz, 3H), 1.50–1.62 (m, 2H), 1.92–2.05 (m, 2H), 2.05–2.18 (m, 2H), 2.08 (s, 3H), 2.66–2.77 (m, 2H), 2.80 (s, 2H), 3.38 (s, 2H), 4.07 (q, J = 7.1 Hz, 2H), 6.95–7.05 (m, 2H), 7.14–7.30 (m, 5H), 7.38–7.48 (m, 2H), 7.65 (br s, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.3, 24.6, 33.5, 46.7, 47.1, 51.2, 60.4, 62.9, 119.9, 126.7, 128.1, 129.8, 130.0, 134.5, 136.9, 137.0, 168.6, 175.6; IR (film) cm⁻¹: 3294, 1728, 1665, 1601, 1542, 1409, 1312, 1178, 700; ESI-MS m/z: 395.2326 (Calcd for C ₂₄H₃₁N₂O₃: 395.2335).

Ethyl 1-(4-Acetamidobenzyl)-4-(3-phenylpropyl)piperidine-4-carboxylate (44c)

This compound was prepared according to a similar procedure as compound 10. Column chromatography (n-hexane/ethyl acetate, 2 : 1) gave the title compound (182 mg, 0.431 mmol, 52.0%) as a colorless solid. mp: 117.0–117.6 °C (decomp.); ¹H-NMR (CDCl₃, 400 MHz) δ: 1.21 (t, J = 7.1 Hz, 3H), 1.37–1.50 (m, 2H), 1.50–1.57 (m, 2H), 1.62 (s, 2H), 1.93–2.05 (m, 2H), 2.10 (br d, J = 13.3 Hz, 2H), 2.16 (s, 3H), 2.49–2.60 (m, 2H), 2.60–2.72 (m, 2H), 3.37 (s, 2H), 4.12 (q, J = 7.1 Hz, 2H), 7.09–7.19 (m, 4H), 7.20–7.32 (m, 4H), 7.41 (d, J = 8.2 Hz, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 13.9, 24.1, 25.3, 33.2, 35.7, 39.6, 44.9, 50.8, 59.9, 62.5, 119.4, 125.4, 127.9, 128.0, 129.3, 134.1, 136.5, 141.6, 168.1, 175.7; IR (film) cm⁻¹: 2939, 1723, 1604, 1537, 1370, 1315, 1176, 1028, 752, 700; ESI-MS m/z: 423.2636 (Calcd for C₂₆H₃₅N₂O₃: 423.2647).

Ethyl 1-(4-Acetamidobenzyl)-4-(4-phenylbutyl)piperidine-4-carboxylate (44d)

This compound was prepared according to a similar procedure as compound 10. Column chromatography (n-hexane/ethyl acetate, 1 : 1) gave the title compound (142 mg, 0.326 mmol, 79.4%) as a colorless amorphous. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.15–1.29 (m, 2H), 1.21 (t, J = 7.1 Hz, 3H), 1.39–1.65 (m, 6H), 1.96–2.15 (m, 4H), 2.16 (s, 3H), 2.56 (t, J = 7.7 Hz, 2H), 2.62–2.72 (m, 2H), 3.39 (s, 2H), 4.12 (q, J = 7.1 Hz, 2H), 7.11–7.19 (m, 3H), 7.21–7.34 (m, 5H), 7.42 (d, J = 8.3 Hz, 2H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.5, 23.9, 24.8, 29.9, 32.0, 33.8, 35.9, 45.5, 51.4, 60.4, 63.1, 119.9, 125.9, 128.5, 128.6, 130.1, 134.6, 137.0, 142.7, 168.5, 176.4; IR (film) cm⁻¹: 2934, 1724, 1668, 1604, 1538, 1410, 1369, 1315, 1208, 1174, 1027, 732, 699; ESI-MS m/z: 437.2795 (Calcd for C₂₇H₃₇N₂O₃: 437.2804).

Ethyl Piperidine-4-Carboxylate (43e)

This compound was prepared according to a similar procedure as compound 43a. The obtained crude product (320 mg, 2.04 mmol, 100%, yellow oil) was used for the next reaction without purification. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.26 (t, J = 7.1 Hz, 3H), 1.84–1.99 (m, 2H), 2.02–2.16 (m, 2H), 2.50–2.60 (m, 1H), 2.82–2.99 (m, 2H), 3.18–3.36 (m, 2H), 4.16 (q, J = 7.1 Hz, 2H), an exchangeable NH proton was not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.1, 25.6, 38.9, 43.3, 60.9, 173.1; IR (neat) cm⁻¹: 2946, 1729, 1449, 1281, 1185, 1044, 861, 720; ESI-MS m/z: 158.1178 (Calcd for C₈H₁₆NO₂: 158.1181).

Ethyl 1-(4-Acetamidobenzyl)piperidine-4-carboxylate (44e)

This compound was prepared according to a similar procedure as compound 10. Column chromatography (chloroform/methanol, 15 : 1) gave the title compound (521 mg, 1.71 mmol, 83.8%) as a colorless solid. mp: 92.8–96.4 °C (decomp.); ¹H-NMR (CDCl₃, 400 MHz) δ: 1.24 (t, J = 7.1 Hz, 3H), 1.53–1.81 (m, 2H), 1.81–1.92 (m, 2H), 1.95–2.09 (m, 2H), 2.16 (s, 3H), 2.20–2.32 (m, 1H), 2.82 (br d, J = 11.6 Hz, 2H), 3.44 (s, 2H), 4.12 (q, J = 7.1 Hz, 2H), 7.23–7.28 (m, 2H), 7.42 (d, J = 8.3 Hz, 2H), an exchangeable NH proton was not observed; ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.1, 24.5, 28.2, 41.1, 52.8, 60.2, 62.6, 119.7, 129.6, 134.3, 136.7, 168.2, 175.2; IR (film) cm⁻¹: 2945, 1729, 1668, 1604, 1540, 1370, 1317, 1048; ESI-MS m/z: 327.1685 (Calcd for C₁₇H₂₄N₂NaO₃: 327.1684).

Biological Evaluation

Cell Culture

Human embryonic kidney (HEK)293 cells stably expressing the opioid receptors were constructed in our previous reports.^38,46,47) In MOR and DOR co-expressing cells, MOR and DOR vectors were transfected in a 1 : 1 ratio and their expression rates on cell surface was confirmed by immunocytochemical staining (Supplementary Fig. 1) and functional assay using CellKey™ system, based on maximal cellular responses by either a MOR agonist DAMGO or a DOR agonist SNC80 in cells expressing MOR, DOR or MOR/DOR (Supplementary Fig. 2). HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and penicillin (100 U mL⁻¹) and streptomycin (100 µg mL⁻¹) at 37°C in a humidified incubator (5% CO₂). Geneticin (flag-MOR: 0.7 mg mL⁻¹) or hygromycin (T7-DOR: 0.25 mg mL⁻¹, Halotag-MOR/T7-DOR: 0.1 mg mL^–1) was added to the culture medium to maintain the stable cell lines.

CellKey™ Assay

The CellKey™ assay has been described previously.⁴⁶⁾ Briefly, CellKey™ buffer was prepared by adding 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 0.1% bovine serum albumin to Hanks’s balanced salt solution (1.3 mM CaCl₂·2H₂O, 0.81 mM MgSO₄, 5.4 mM KCl, 0.44 mM KH₂PO₄, 4.2 mM NaHCO₃, 136.9 mM NaCl, 0.34 mM Na₂HPO₄ and 5.6 mM D-glucose). At day 1, the cells were seeded onto poly-D-lysine- coated CellKey™ 96-well microplates at a density as follows: flag-MOR, 6 × 10⁴ cells per well; T7-DOR, 7 × 10⁴ cells per well; Halotag-MOR/T7-DOR, 5 × 10⁴ cells per well. At day 2, the cells were washed with CellKey™ buffer, and incubated in this buffer at ambient temperature for 30 min. After monitoring the impedance baseline for 5 min, the compounds were added, and impedance currents within 30 min were measured. The values in Figs. 4–8 were calculated by dividing the changes of impedance by the compounds (max–min) by the changes of impedance by vehicle treatment (max–min). DAMGO and SNC80 were purchased from Sigma-Aldrich (Cat. No. E7384 and S2812, respectively).

Acknowledgments

This work was supported by JSPS KAKENHI Grant Number: 21K06584 for YU.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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