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Development of κ Opioid Receptor Agonists by Focusing on Phenyl Substituents of 4-Dimethylamino-3-phenylpiperidine Derivatives: Structure–Activity Relationship Study of Matrine Type Alkaloids
Hiroyoshi TeramotoTakayasu YamauchiShigeru SasakiKimio Higashiyama
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2016 Volume 64 Issue 5 Pages 420-431

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Abstract

A series of new κ opioid receptor (KOR) agonists were developed from the lead compound 4-dimethylamino-1-pentanoylpiperidine (3), a matrine-type alkaloid. Derivatives of 3 were synthesized with a variety of phenyl substituents and evaluated for their antinociceptive effects. Additionally, their selectivity for an opioid receptor was investigated for cis-4c and d, and trans-4g, all of which had high activity exerted through the KOR.

We previously reported that (+)-matrine (1) and (+)-allomatrine (2), a typical matrine type lupine alkaloid isolated from some Sophora plants (Leguminosae), have antinociceptive properties identical to those of pentazocine1) (Fig. 1). The effects of (+)-matrine are mediated mainly through activation of κ opioid receptors (KOR) and partially through μ receptors (MOR), while the effects of (+)-allomatrine are mediated only through activation of KOR.2) Because the skeleton of the matrine type alkaloids differs from those of conventional KOR agonists, such as ethylketocyclazocine,3) U-504884,5) and nalfurafine (TRK-820),6) they have the potential to be derivatized into novel KOR agonists that may not induce side effects such as dysphoria and psychotomimetic actions7,8) (Fig. 2). In fact, the structure–activity relationship (SAR) between the antinociceptive effects of these alkaloids and their structures is very interesting. During the development of new KOR agonists, compound 3 was identified as a lead compound by determining the essential structure required for the antinociceptive effects of (+)-matrine.911) When compound 3 was derivatized and tested, the antinociceptive effects of 4-dimethylamino-1-pentanoyl-3-phenylpiperidine (4a) were more potent than those of the lead compound 3. On the basis of the Topliss method,12) we designed and synthesized phenyl derivatives of 4a in an effort to further improve the activity. Herein, we report the synthesis of the derivatives of 4a and their pharmacological effects.

Fig. 1. Structure of (+)-Matrine Derivatives
Fig. 2. Structure of Conventional κ-Opioid Receptor Agonists

Synthesis

The synthetic route to the intermediates, cis- and trans-11ae, is shown in Chart 1. Hydrazones 7ae were prepared by condensation of commercially available 2,4,6-triisopropylbenzenesulfonyl hydrazide 5 and the respective acetophenones 6ae. Allylic alcohols 8ae were obtained through the Shapiro reaction with acrolein.13) Oxidation of 8ae with MnO2 yielded the respective dienones, followed by cyclization via a double aza-Michael reaction with benzylamine using microwave irradiation to give piperidones 9ae.13) Piperidones 9ae were converted to oximes 10ae and subsequently reduced with metal sodium in EtOH to yield the separable diastereomers cis- and trans-11ad, the 3,4-cis (matrine type) and 3,4-trans configuration compounds (allomatrine type). The relative configuration of these diastereomers were assigned by comparison to literature data and using the coupling constants from the 1H-NMR spectra.14) Amine 11e, which has a chloro group on the para position of the benzene ring, was obtained by reduction with LiAlH4 since dechlorination occurred during reduction with metal sodium.

Chart 1

Reagents and reaction conditions: (a) THF, rt, 67–81%; (b) n-BuLi, acrolein, THF, −78°C, 82–97%; (c) activated MnO2, CH2Cl2, rt; (d) BnNH2, MeCN, 100°C, MW, two steps, 67–92%; (e) NH2OH–HCl, AcONa, EtOH, H2O, reflux, 95–99%; (f) Na, EtOH, reflux, 56–85%; (g) LiAlH4, THF, reflux, 59–85%.

An alternative synthetic route for 11f and g was designed because only a trace of the expected allyl alcohol was obtained by the Shapiro reaction (Chart 2). Amidation of carboxylic acids 12f and g and amine 13 in the presence of 1-ethy-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 4-dimethylaminopyridine (DMAP) yielded amides 14f and g, which was followed by Dieckmann condensation15) with NaOEt to obtain β-keto lactam 15f and g.

Chart 2

Reagents and reaction conditions: (a) EDC, DMAP, CH2Cl2, 0°C, 99%; (b) NaOEt, toluene, reflux; (c) NH2OH–HCl, AcONa, EtOH, H2O, reflux; (d) LiAlH4, THF, reflux, three steps, 17–30%.

Separable amines cis- and trans-11f and g were obtained by reduction of oximes 16f and g, which were oximated from the ketones 15f and g. The relative configuration of cis- and trans-11f and g were also assigned from the coupling constant in the 1H-NMR spectra.

As shown in Chart 3, the final synthetic steps involved treatment with HCHO and HCO2H to afford the dimethylated amines cis- and trans-17ag. After debenzylation, the target compounds cis- and trans-4ag were obtained by N-acylation. To avoid dechlorination by reduction, debenzylation of the precursors cis- and trans-4eg having the chloro group were carried out by using 1-chloroethyl chloroformate and MeOH.16)

Chart 3

Reagents and reaction conditions: (a) 36% HCHO, HCO2H, reflux, 41–99%; (b) 10% Pd–C, HCO2NH4, MeOH, reflux; (c) 1-chloroethyl chloroformate, CH2Cl2, rt, then MeOH, reflux; (d) BuCOCl, Et3N, CH2Cl2, rt, two steps, 12–76%.

Biological Evaluation

To evaluate the antinociceptive potencies of the compounds, the writhing test was performed via subcutaneous (s.c.) administration of cis- and trans-4ag in mice. A single s.c. administration of all synthesized compounds produced a dose-dependent antinociceptive effect. The ED50 values of cis- and trans-4ag are shown in Table 1. In the writhing test, the antinociceptive effects of cis-4c, d and trans-4g were strikingly different compared with those of pentazocine, which is a well-known selective KOR agonist. Common to matrine-type and allomatrine-type derivatives, generally the compounds with a substituent at the ortho or meta position on the benzene ring had higher antinociception, except for the compound having the fluoro substituent (4d). For example, cis-4f and g had higher activity than cis-4e, the activities of the trans compounds were also the same as those of the cis compounds. From the results, we speculated that steric and electronic effects were more important than the type of substituent in determining the compound’s potency. This observation may be explained by the restriction of free rotation of the benzene ring containing a substituent caused by steric hindrance between the benzene ring and dimethylamino moiety. Further studies are required to confirm this hypothesis.

Table 1. ED50 Value in mg/kg of cis-4ag and trans-4ag for 30–40 Mice in Each Group
RED50 in mg/kg
Pentazocine3.80
14.70
cis-4aH5.47
cis-4b4′-Me8.07
cis-4c3′-Me0.91
cis-4d4′-F0.97
cis-4e4′-Cl6.44
cis-4f3′-Cl4.18
cis-4g2′-Cl2.21
25.89
trans-4aH4.80
trans-4b4′-Me8.33
trans-4c3′-Me5.78
trans-4d4′-F1.52
trans-4e4′-Cl4.92
trans-4f3′-Cl3.59
trans-4g2′-Cl0.60

Next, we investigated the opioid receptor selectivity of cis-4c, d and trans-4g, the three compounds with the highest antinociception, as shown in Fig. 3. The assessment of the selectivity was carried out using the writhing test with the MOR antagonist β-funaltrexamine (β-FNA), the δ-opioid receptor (DOR) antagonist naltrindole (NTI), or the KOR antagonist nor-BNI (norbinaltorphimine) as control compounds. In the presence of the synthesized compounds, there were scarcely any changes in the antinociceptive effects with β-FNA and NTI, but their effects were violently antagonized with nor-BNI. We assumed that the effects of cis-4c and d, matrine-type compounds, were only slightly antagonized by β-FNA because the antinociceptive effects of (+)-matrine (1) are partially mediated through MOR. The selectivity results were in agreement with what was expected from the known behavior of the control antagonists.

Fig. 3. The Blockage of Antinociceptive Effect of cis-4c, d and trans-4g (3 mg/kg, s.c.) after Pretreatment with MOR Antagonist β-FNA (40 mg/kg, s.c.), DOR Antagonist NTI (3.2 mg/kg, s.c.) or KOR Antagonist nor-BNI (3.2 mg/kg, s.c.) at 24 h, 15 min and 4 h before Administration of the Test Drug, Respectively, in the Acetic Acid-Induced Abdominal Constriction Assay in Mice1,2)

Each column represents the mean shown with the S.E.M. for 10 mice in each group. * p<0.05 (cis-4c), ** p<0.01 (cis-4d or trans-4g) versus the compound alone.

In summary, we succeeded in synthesizing the 4-dimethylamino-3-phenylpiperidine derivatives cis- and trans-4ag. The phenyl derivatives cis-4c, d and trans-4g showed high antinociceptive effects mediated through the activation of KOR. The results are valuable in providing a starting point toward the development of a novel KOR selective agonist.

Experimental

Chemistry

1H- (400 MHz) and 13C-NMR (100 MHz) spectra were obtained on a Bruker AVIII-400 instrument, and chemical sifts are reported in ppm on the δ-scale from internal tetramethylsilane. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), septet (sept), multiplet (m) or broad (br), with coupling constants (J) in hertz (Hz). MS spectra were measured with a JEOL JMS D-600 and JMS T-100LP spectrometer by using the chemical ionization (CI) with isobutene, the electron impact (EI) methods, and electrospray ionization (ESI) methods. All melting points were measured with a Yanagimoto Micro melting point apparatus without collection. IR spectra were recorded on Perkin-Elmer Spectrum Two. Microwave irradiation were carried out on Biotage Initiator. Column chromatography was performed on Silica gel 60 (100–210 µm, Kanto Chemical Co., Inc.).

Acetophenone (2,4,6-Triisopropylbenzene)sulfonylhydrazone (7a)

Acetophenone (2.00 g, 16.7 mmol) was added to a solution of 2,4,6-triisopropylbenzenesulfonyl hydrazide (5.00 g, 16.7 mmol) in tetrahydrofuran (THF). After being stirred at room temperature (rt) for 3 h, the solvent was evaporated. The resulting residue was purified by recrystallization with EtOH to yield 7a (5.40 g, 13.5 mmol, 81%) as a colorless solid. All spectroscopic and analytical data were consistent with the literature.17)

4′-Methylacetophenone (2,4,6-Triisopropylbenzene)sulfonylhydrazone (7b)

Prepared According to Procedure for the Preparation of 7a

Yield: 67%, colorless solid, mp 145–147°C. 1H-NMR (CDCl3) δ: 1.24 (6H, d, J=6.9 Hz), 1.30 (12H, d, J=6.8 Hz), 2.15 (3H, s), 2.32 (3H, s), 2.88 (1H, sept, J=6.9 Hz), 4.33 (2H, sept, J=6.8 Hz), 7.10 (2H, d, J=8.1 Hz), 7.16 (2H, s), 7.52 (2H, d, J=8.1 Hz), 7.84 (1H, br). 13C-NMR (CDCl3) δ: 13.0, 21.2, 24.8, 24.8, 30.0, 34.2, 123.7, 126.2, 128.8, 131.4, 134.6, 139.5, 150.7, 151.3, 153.3. IR (KBr) cm−1: 813, 1167, 1332, 1599, 2965, 3240. MS (ESI+) m/z: 415 (M+H+, base peak). High resolution (HR)-MS (ESI+) m/z: Found 415.2432 (Calcd for C24H35N2O2S (M+H+) 415.2419).

3′-Methylacetophenone (2,4,6-Triisopropylbenzene)sulfonylhydrazone (7c)

Prepared According to Procedure for the Preparation of 7a

Yield: 76%, colorless solid, mp 159–161°C. 1H-NMR (CDCl3) δ: 1.24 (6H, d, J=6.9 Hz), 1.30 (12H, d, J=6.8 Hz), 2.16 (3H, s), 2.31 (3H, s), 2.89 (1H, sept, J=6.9 Hz), 4.32 (2H, sept, J=6.8 Hz), 7.13–7.21 (4H, m), 7.39 (1H, d, J=7.7 Hz), 7.49 (1H, s), 7.71 (1H, br). 13C-NMR (CDCl3) δ: 13.1, 21.3, 23.5, 24.8, 29.9, 34.2, 123.4, 123.8, 126.8, 128.0, 130.1, 131.4, 137.2, 137.8, 150.7, 151.3, 153.3. IR (KBr) cm−1: 907, 1166, 1331, 1600, 2964, 3244. MS (ESI+) m/z: 415 (M+H+, base peak). HR-MS (ESI+) m/z: Found 415.2444 (Calcd for C24H35N2O2S (M+H+) 415.2419).

4′-Fluoroacetophenone (2,4,6-Triisopropylbenzene)sulfonylhydrazone (7d)

Prepared According to Procedure for the Preparation of 7a

Yield: 70%, colorless solid, mp 177–179°C. 1H-NMR (CDCl3) δ: 1.24 (6H, d, J=6.9 Hz), 1,29 (12H, d, J=6.8 Hz), 2.15 (3H, s), 2.89 (1H, sept, J=6.9 Hz), 4.29 (2H, sept, J=6.8 Hz), 6.96–7.01 (2H, m), 7.10 (2H, s), 7.59–7.63 (2H, m). 13C-NMR (CDCl3) δ: 13.2, 23.5, 24.8, 29.9, 34.1, 115.0 (d, J=21.7 Hz), 123.8, 128.1 (d, J=8.4 Hz), 131.3, 133.6 (d, J=3.2 Hz), 149.6, 151.4, 153.4, 163.4 (d, J=249.5 Hz). IR (KBr) cm−1: 836, 1169, 1511, 1600, 2959, 3241. MS (ESI+) m/z: 419 (M+H+, base peak). HR-MS (ESI+) m/z: Found 419.2200 (Calcd for C23H32FN2O2S (M+H+) 419.2169).

4′-Chloroacetophenone (2,4,6-Triisopropylbenzene)sulfonylhydrazone (7e)

Prepared According to Procedure for the Preparation of 7a

Yield: 71%, colorless solid. All spectroscopic and analytical data were consistent with the literature.18)

2-Phenyl-1,4-pentadien-3-ol (8a)

A solution of n-BuLi (3.44 mL, 5.50 mmol, 1.6 M, in n-hexane) was added dropwise to a solution of 7a (1.00 g, 2.50 mmol) in THF (10 mL) at −78°C under the nitrogen atmosphere. The reaction mixture was stirred at −78°C for 30 min, then warmed to 0°C and further stirred for 15 min. Acrolein (578 mg, 9.30 mmol) was added to the reaction mixture at −78°C. After being stirred for 1 h, the reaction mixture was quenched with saturated aqueous NaHCO3 and extracted with EtOAc. The combined organic layer was dried over anhydrous Na2SO4 and evaporated. The resulting residue was purified by chromatography on SiO2 (n-hexane–EtOAc=8 : 1) to yield the 8a (345 mg, 2.16 mmol, 86%) as a colorless oil. All spectroscopic and analytical data were consistent with the literature.19)

2-(4-Methylphenyl)-1,4-pentadien-3-ol (8b)

Prepared According to Procedure for the Preparation of 8a

Yield: 82%, colorless oil. 1H-NMR (CDCl3) δ: 2.35 (3H, s), 5.12 (1H, d, J=5.4 Hz), 5.18 (1H, dt, J=10.4, 1.4 Hz), 5.31–5.36 (2H, m), 5.37 (1H, br), 5.96 (1H, ddd, J=17.2, 10.4, 5.4 Hz), 7.15 (2H, d, J=8.1 Hz), 7.33 (2H, d, J=8.1 Hz). 13C-NMR (CDCl3) δ: 21.1, 74.5, 112.9, 115.9, 126.8, 129.0, 136.3, 137.5, 139.0, 149.7. IR (film) cm−1: 920, 1513, 3025, 3389. MS (EI) m/z: 174 (M+) 119 (base peak). HR-MS (EI) m/z: Found 174.1022 (Calcd for C12H14O (M+) 174.1045).

2-(3-Methylphenyl)-1,4-pentadien-3-ol (8c)

Prepared According to Procedure for the Preparation of 8a

Yield: 86%, colorless oil. 1H-NMR (CDCl3) δ: 2.36 (3H, s), 5.12 (1H, br), 5.19 (1H, dt, J=10.4, 1.4 Hz), 5.34 (1H, dt, J=17.2, 1.4 Hz), 5.37–5.38 (2H, m), 5.97 (1H, ddd, J=17.2, 10.4, 5.7 Hz), 7.10–7.13 (1H, m), 7.22–7.24 (3H, m). 13C-NMR (CDCl3) δ: 21.3, 74.2, 113.2, 115.6, 123.9, 127.5, 128.0, 128.3, 137.7, 138.9, 139.3, 149.9. IR (film) cm−1: 1487, 1602, 3396. MS (CI) m/z: 175 (M+H+), 157 (base peak). HR-MS (CI) m/z: Found 175.1137 (Calcd for C12H15O (M+H+) 175.1123).

2-(4-Fluorophenyl)-1,4-pentadien-3-ol (8d)

Prepared According to Procedure for the Preparation of 8a

Yield: 82%, colorless oil. 1H-NMR (CDCl3) δ: 5.06 (1H, d, J=5.0 Hz), 5.17 (1H, dt, J=10.4, 1.3 Hz), 5.31 (1H, dt, J=17.2, 1.4 Hz), 5.35 (1H, s), 5.38 (1H, s), 5.92 (1H, ddd, J=17.2, 10.4, 5.8 Hz), 6.98–7.03 (2H, m), 7.37–7.42 (2H, m). 13C-NMR (CDCl3) δ: 74.4, 113.5, 114.9 (d, J=21.3 Hz), 115.8, 128.5 (d, J=7.7 Hz), 135.3 (d, J=3.2 Hz), 138.7, 148.7, 162.2 (d, J=246.5 Hz). IR (film) cm−1: 1509, 1599, 1686, 3440. MS (ESI−) m/z: 177 (M−H+), 61 (base peak). HR-MS (ESI−) m/z: Found 177.0716 (Calcd for C11H10FO (M−H+) 177.0716).

2-(4-Chlorophenyl)-1,4-pentadien-3-ol (8e)

Prepared According to Procedure for the Preparation of 8a

Yield: 97%, colorless oil. 1H-NMR (CDCl3) δ: 5.08 (1H, d, J=5.7 Hz), 5.19 (1H, dt, J=10.4, 1.4 Hz), 5.32 (1H, dt, J=17.2, 1.4 Hz), 5.39 (1H, br), 5.43 (1H, t, J=1.1 Hz), 5.93 (1H, ddd, J=17.2, 10.4, 5.7 Hz), 7.30 (2H, d, J=8.7 Hz), 7.37 (2H, d, J=8.7 Hz). 13C-NMR (CDCl3) δ: 74.5, 114.2, 116.2, 128.3, 128.4, 133.5, 137.7, 138.6, 148.6. IR (film) cm−1: 1012, 1091, 1491, 3376. MS (ESI−) m/z: 193 (M−H+), 113 (base peak). HR-MS (ESI−) m/z: Found 193.0415 (Calcd for C11H10ClO (M−H+) 193.0420).

1-N-Benzyl-3-phenyl-4-piperidone (9a)

Activated MnO2 (4.00 g, 46.0 mmol) was added portionwise to a solution of 8a (250 mg, 1.56 mmol) in CH2Cl2 (15 mL). After being stirred at rt for 4 h, the reaction mixture was filtered through Celite pad and evaporated. The resulting residue was used in the subsequent reaction without further purification. Benzylamine (212 mg, 1.99 mmol) was added to a solution of the residue in CH3CN (10 mL). After being irradiated to microwave (100°C for 1 h), the reaction mixture was poured on H2O and extracted with CHCl3. The combined organic layer was dried over anhydrous Na2SO4 and evaporated. The resulting residue was purified by chromatography on SiO2 (n-hexane–EtOAc=2 : 1) to yield 9a (310 mg, 1.17 mmol, 75% over two steps) as a light yellow oil. All spectroscopic and analytical data were consistent with the literature.13)

1-N-Benzyl-3-(4-methylphenyl)-4-piperidone (9b)

Prepared According to Procedure for Preparation of 9a

Yield: 70% over two steps, light yellow oil. 1H-NMR (CDCl3) δ: 2.32 (3H, s), 2.46–2.56 (1H, m), 2.62–2.71 (2H, m), 2.76 (1H, dd, J=11.4, 10.0 Hz), 3.05–3.10 (1H, m), 3.17 (1H, ddd, J=11.4, 5.7, 2.4 Hz), 3.66 (2H, s), 3.78 (1H, dd, J=10.0, 5.7 Hz), 7.09–7.14 (4H, m), 7.26–7.29 (1H, m), 7.31–7.38 (4H, m). 13C-NMR (CDCl3) δ: 21.1, 40.7, 53.5, 56.0, 59.8, 61.9, 127.3, 128.4, 128.7, 128.9, 129.1, 133.6, 136.8, 137.9, 208.4. IR (film) cm−1: 1351, 1453, 1718, 2802. MS (ESI+) m/z: 280 (M+H+, base peak). HR-MS (ESI+) m/z: Found 280.1724 (Calcd for C19H22NO (M+H+) 280.1701).

1-N-Benzyl-3-(3-methylphenyl)-4-piperidone (9c)

Prepared According to Procedure for the Preparation of 9a

Yield: 67% over two steps, light yellow oil. 1H-NMR (CDCl3) δ: 2.32 (3H, s), 2.48–2.55 (1H, m), 2.61–2.71 (2H, m), 2.78 (1H, dd, J=11.4, 9.7 Hz), 3.02–3.09 (1H, m), 3.15 (1H, ddd, J=11.4, 5.7, 2.3 Hz), 3.65 (2H, s), 3.76 (1H, dd, J=9.7, 5.7 Hz), 7.01–7.07 (3H, m), 7.20 (1H, t, J=7.6 Hz), 7.22–7.28 (1H, m), 7.31–7.38 (4H, m). 13C-NMR (CDCl3) δ: 21.4, 40.7, 53.5, 56.3, 59.6, 61.9, 125.9, 127.3, 127.9, 128.2, 128.4, 128.9, 129.6, 136.6, 137.8, 137.9, 208.2. IR (film) cm−1: 1454, 1715, 2915. MS (ESI+) m/z: 280 (M+H+, base peak). HR-MS (ESI+) m/z: Found 280.1670 (Calcd for C19H22NO (M+H+) 280.1701).

1-N-Benzyl-3-(4-fluorophenyl)-4-piperidone (9d)

Prepared According to Procedure for the Preparation of 9a

Yield: 92% over two steps, light yellow oil. 1H-NMR (CDCl3) δ: 2.47–2.56 (1H, m), 2.63–2.76 (3H, m), 3.07–3.11 (1H, m), 3.17 (1H, ddd, J=11.4, 5.6, 2.4 Hz), 3.67 (2H, s), 3.80 (1H, dd, J=9.8, 5.6 Hz), 6.97–7.03 (2H, m), 7.17–7.20 (2H, m), 7.28–7.39 (5H, m). 13C-NMR (CDCl3) δ: 40.6, 53.3, 55.4, 59.7, 61.8, 115.2 (d, J=21.2 Hz), 127.4, 128.4, 129.0, 130.4 (d, J=7.9 Hz), 132.3, 137.4, 161.9 (d, J=245.5 Hz), 207.8. IR (film) cm−1: 1511, 1602, 1717. MS (ESI+) m/z: 284 (M+H+), 91 (base peak). HR-MS (ESI+) m/z: Found 284.1474 (Calcd for C18H19FNO (M+H+) 284.1451).

1-N-Benzyl-3-(4-chlorophenyl)-4-piperidone (9e)

Prepared According to Procedure for the Preparation of 9a

Yield: 67% over two steps, light yellow oil. 1H-NMR (CDCl3) δ: 2.46–2.55 (1H, m), 2.57–2.70 (2H, m), 2.74 (1H, dd, J=11.4, 9.8 Hz), 3.07–3.10 (1H, m), 3.16 (1H, ddd, J=11.4, 5.7, 2.4 Hz), 3.66 (2H, s), 3.79 (1H, dd, J=9.8, 5.7 Hz), 7.15 (2H, d, J=8.4 Hz), 7.26–7.30 (3H, m), 7.32–7.38 (4H, m). 13C-NMR (CDCl3) δ: 40.7, 53.4, 55.7, 59.6, 61.9, 127.5, 128.4, 128.5, 128.9, 130.3, 133.1, 135.1, 137.8, 207.7. IR (film) cm−1: 1492, 1717, 2806. MS (ESI+) m/z: 300 (M+H+, base peak). HR-MS (ESI+) m/z: Found 300.1171 (Calcd for C18H19ClNO (M+H+) 300.1155).

1-N-Benzyl-3-phenyl-4-hydroxyiminopiperidine (10a)

NaOAc (1.94 g, 23.7 mmol) and NH2OH–HCl (1.65 g, 23.7 mmol) were added to a solution of 9a (630 mg, 2.37 mmol) in EtOH–H2O 1 : 1 (20 mL). After being stirred at reflux for 3 h, the reaction mixture was alkalized with saturated aqueous Na2CO3 and extracted with CHCl3. The combined organic layer was dried over anhydrous Na2SO4 and evaporated to yield 10a (650 mg, 2.32 mmol, 98%) as a brown oil and was used without further purification. 1H-NMR shows double signal set (isomer A and B, ratio 2 : 1). All spectroscopic and analytical data were consistent with the literature.13)

1-N-Benzyl-4-hydroxyimino-3-(4-methylphenyl)piperidine (10b)

Prepared According to Procedure for the Preparation of 10a

Yield: 99%, brown oil. 1H-NMR shows a doubled signal set (isomer A and B, ratio 2 : 1). 1H-NMR (CDCl3) δ: 2.15–2.23 (2H of isomer B, m), 2.31 (3H of isomer A, s), 2.32 (3H of isomer B, s), 2.40–2.54 (3H; 2H of isomer A, m, 1H of isomer B, m), 2.71–2.80 (3H; 2H of isomer A, m, 1H of isomer B, m), 2.84–2.96 (2H of isomer A, m), 3.04–3.08 (1H of isomer B, m), 3.32 (1H of isomer B, dt, J=12.0, 1.8 Hz), 3.54 (2H of isomer B, s), 3.56 (1H of isomer A, d, J=13.2 Hz), 3.60 (1H of isomer A, d, J=13.2 Hz), 3.64 (1H of isomer A, dd, J=8.4, 4.7 Hz), 4.64 (1H of isomer B, d, J=3.8 Hz), 7.10 (2H of isomer B, d, J=8.0 Hz), 7.11 (2H of isomer A, d, J=8.0 Hz), 7.21 (2H of isomer A, d, J=8.0 Hz), 7.27–7.36 (10H, 5H of isomer A, m, 5H of isomer B, m), 7.45 (2H of isomer B, d, J=8.0 Hz). 13C-NMR (CDCl3), (isomer A) δ: 21.0, 23.5, 46.6, 52.5, 58.9, 62.6, 127.2, 128.3, 128.5, 128.9, 129.1, 136.0, 136.3, 137.9, 159.7; (isomer B) δ: 21.0, 29.2, 37.5, 53.7, 56.1, 62.8, 127.2, 128.2, 128.3, 128.9, 129.1, 135.8, 137.2, 138.2, 159.0. IR (film) cm−1: 951, 1453, 1513, 3277. MS (ESI+) m/z: 295 (M+H+, base peak). HR-MS (ESI+) m/z: Found 295.1827 (Calcd for C19H23N2O (M+H+) 295.1810).

1-N-Benzyl-4-hydroxyimino-3-(3-methylphenyl)piperidine (10c)

Prepared According to Procedure for the Preparation of 10a

Yield: 99%, brown oil. 1H-NMR shows a doubled signal set (isomer A and B, ratio 2 : 1). 1H-NMR (CDCl3) δ: 2.20–2.25 (2H of isomer B, m), 2.32 (6H, 3H of isomer A, s, 3H of isomer B, s), 2.39 (1H of isomer A, dd, J=12.0, 4.4 Hz), 2.47–2.61 (3H, 2H of isomer A, m, 1H of isomer B, m), 2.70–2.74 (1H of isomer A, m), 2.83–2.89 (3H, 2H of isomer A, m, 1H of isomer B, m), 3.06–3.10 (1H of isomer B, m), 3.34 (1H of isomer B, dt, J=12.0, 1.9 Hz), 3.50 (1H of isomer B, d, J=13.0 Hz), 3.56 (1H of isomer A, d, J=13.3 Hz), 3.60 (1H of isomer A, d, J=13.3 Hz), 3.61–3.65 (2H, 1H of isomer A, m, 1H of isomer B, m), 4.64 (1H of isomer B, d, J=4.2 Hz), 7.01–7.05 (2H, 1H of isomer A, m, 1H of isomer B, m), 7.12–7.21 (5H, 2H of isomer A, m, 3H of isomer B, m), 7.24–7.37 (11H, 6H of isomer A, m, 5H of isomer B, m). 13C-NMR (CDCl3), (isomer A) δ: 21.4, 23.5, 46.9, 52.6, 58.7, 62.6, 125.7, 127.2, 127.5, 128.1, 128.3, 128.7, 129.2, 137.8, 137.9, 138.9, 159.6; (isomer B) δ: 21.5, 29.3, 37.8, 54.0, 55.8, 62.8, 125.4, 126.9, 127.1, 127.2, 128.2, 129.0, 129.4, 137.7, 138.3, 140.0, 159.0. IR (film) cm−1: 1454, 1716, 2924. MS (ESI+) m/z: 295 (M+H+, base peak). HR-MS (ESI+) m/z: Found 295.1784 (Calcd for C19H23N2O (M+H+) 295.1810).

1-N-Benzyl-3-(4-fluorophenyl)-4-hydroxyiminopiperidine (10d)

Prepared According to Procedure for the Preparation of 10a

Yield: 95%, brown oil. 1H-NMR shows a doubled signal set (isomer A and B, ratio 2 : 1). 1H-NMR (CDCl3) δ: 2.15–2.23 (2H of isomer B, m), 2.39–2.56 (4H, 2H of isomer A, m, 2H of isomer B, m), 2.68–2.80 (2H of isomer A, m), 2.82–2.88 (2H of isomer A, m), 3.04–3.08 (1H of isomer B, m), 3.25–3.28 (1H of isomer B, m), 3.52 (1H of isomer B, d, J=13.0 Hz), 3.56 (1H of isomer B, d, J=13.0 Hz), 3.58 (2H of isomer A, s), 3.65 (1H of isomer A, dd, J=8.0, 4.6 Hz), 4.61 (1H of isomer B, d, J=3.9 Hz), 6.93–6.99 (4H, 2H of isomer A, m, 2H of isomer B, m), 7.24–7.34 (12H, 7H of isomer A, m, 5H of isomer B, m), 7.54 (2H of isomer B, dd, J=8.5, 5.6 Hz). 13C-NMR (CDCl3), (isomer A) δ: 23.4, 46.0, 52.3, 58.6, 62.5, 114.9 (d, J=21.0 Hz), 127.3, 128.3, 129.2, 130.1 (d, J=7.8 Hz), 134.7 (d, J=3.3 Hz), 137.3, 159.0, 161.6 (d, J=245.0 Hz); (isomer B) δ: 29.0, 37.1, 53.6, 56.0, 62.7, 114.8 (d, J=20.8 Hz), 127.2, 128.2, 129.0, 130.0 (d, J=7.8 Hz), 136.0 (d, J=3.0 Hz), 138.0, 158.3, 161.4 (d, J=244.4 Hz). IR (film) cm−1: 1509, 1601, 3277. MS (ESI+) m/z: 299 (M+H+, base peak). HR-MS (ESI+) m/z: Found 299.1581 (Calcd for C18H20FN2O (M+H+) 299.1560).

1-N-Benzyl-3-(4-chlorophenyl)-4-hydroxyiminopiperidine (10e)

Prepared According to Procedure for the Preparation of 10a

Yield: 99%, brown oil. 1H-NMR shows a doubled signal set (isomer A and B, ratio 2 : 1). 1H-NMR (CDCl3) δ: 2.16–2.25 (2H of isomer B, m), 2.39–2.45 (1H of isomer B, m), 2.53–2.61 (2H of isomer A, m), 2.67–2.71(1H of isomer A, m), 2.76–2.86 (4H, 3H of isomer A, m, 1H of isomer B, m), 3.05–3.09 (1H of isomer B, m), 3.27 (1H of isomer B, dt, J=12.0, 1.7 Hz), 3.52 (1H of isomer B, d, J=12.9 Hz), 3.57 (1H of isomer B, d, J=12.9 Hz), 3.58 (2H of isomer A, s), 3.64 (1H of isomer A, dd, J=7.2, 5.0 Hz), 4.63 (1H of isomer B, d, J=3.8 Hz), 7.23–7.34 (16H, 9H of isomer A, m, 7H of isomer B, m), 7.51 (2H of isomer B, d, J=8.4 Hz). 13C-NMR (CDCl3), (isomer A) δ: 23.4, 46.3, 52.4, 58.5, 62.6, 127.3, 128.3, 128.3, 129.2, 130.0, 132.5, 137.4, 137.6, 159.4; (isomer B) δ: 29.1. 37.3, 53.7, 55.9, 62.8, 127.3, 128.2, 128.3, 129.1, 129.9, 132.1, 138.0, 138.8, 158.5. IR (film) cm−1: 1091, 1493, 3270. MS (ESI+) m/z: 315 (M+H+, base peak). HR-MS (ESI+) m/z: Found 315.1252 (Calcd for C18H20ClN2O (M+H+) 315.1264).

cis- and trans-4-Amino-1-N-benzyl-3-phenylpiperidine (cis-, trans-11a)

Pieces of sodium metal (1.30 g, 56.5 mmol) was added portionwise to a solution of 10a (200 mg, 0.71 mmol) in EtOH (10 mL). After being stirred at reflux for 20 h, the reaction mixture was cooled until rt and acidified with 10% aqueous HCl and evaporated. The resulting residue was alkalized with 1 M aqueous NaOH and extracted with CHCl3. The organic layer was dried over anhydrous Na2SO4 and evaporated. The resulting residue was purified by chromatography on SiO2 (CHCl3–MeOH=10 : 1) to yield the primary amine (161 mg, 0.61 mmol, 85%, cistrans=2 : 3) perspective as a yellow oil. All spectroscopic and analytical data of cis-11a were consistent with the literature.13) trans-11a: 1H-NMR (CDCl3) δ: 1.43 (2H, br), 1.58 (1H, dq, J=12.8, 4.2 Hz), 1.95 (1H, ddd, J=12.8, 6.9, 2.6 Hz), 2.13 (1H, t, J=11.4 Hz), 2.16 (1H, dt, J=12.0, 2.6 Hz), 2.57 (1H, dt, J=10.8, 3.7 Hz), 2.86 (1H, dt, J=10.8, 4.2 Hz), 2.93 (1H, ddd, J=11.4, 3.7, 2.1 Hz), 2.96–3.02 (1H, m), 3.54 (2H, s), 7.21–7.25 (4H, m), 7.28–7.33 (6H, m). 13C-NMR (CDCl3) δ: 34.6, 52.8, 53.0, 53.6, 59.8, 62.8, 126.9, 127.0, 128.2, 128.2, 128.6, 129.1, 138.2, 141.6. IR (film) cm−1: 1450, 1500, 2920. MS (CI) m/z: 267 (M+H+, base peak). HR-MS (CI) m/z: Found 267.1884 (Calcd for C18H23N2 (M+H+) 267.1861).

cis- and trans-4-Amino-1-N-benzyl-3-(4-methylphenyl)piperidine (cis-, trans-11b)

Prepared According to Procedure for the Preparation of 11a

Yield: 56% (cistrans=2 : 3). cis-11b: yellow oil. 1H-NMR (CDCl3) δ: 1.21 (2H, br), 1.66–1.73 (1H, m), 1.91–1.97 (1H, m), 2.31 (3H, s), 2.47–2.55 (2H, m), 2.68–2.77 (2H, m), 3.02–3.06 (1H, m), 3.18–3.21 (1H, m), 3.54 (1H, d, J=13.1 Hz), 3.60 (1H, d, J=13.1 Hz), 7.11 (2H, d, J=7.9 Hz), 7.19–7.24 (3H, m), 7.28–7.36 (4H, m). 13C-NMR (CDCl3) δ: 20.9, 32.7, 46.0, 48.7, 49.6, 52.5, 63.5, 126.9, 128.2, 128.3, 128.9, 129.1, 135.9, 138.5, 138.8. IR (film) cm−1: 810, 1453, 1513, 2919. MS (ESI+) m/z: 281 (M+H+, base peak). HR-MS (ESI+) m/z: Found 281.2036 (Calcd for C19H25N2 (M+H+) 281.2018). trans-11b: yellow oil. 1H-NMR (CDCl3) δ: 1.37 (2H, br), 1.56 (1H, dq, J=12.7, 4.2 Hz), 1.93 (1H, ddd, J=12.7, 6.9, 2.6 Hz), 2.09 (1H, t, J=11.4 Hz), 2.14 (1H, dt, J=12.0, 2.6 Hz), 2.31 (3H, s), 2.52 (1H, dt, J=10.8, 3.8 Hz), 2.82 (1H, dt, J=10.8, 4.2 Hz), 2.90 (1H, ddd, J=11.4, 3.8, 2.1 Hz), 2.95–3.00 (1H, m), 3.53 (2H, s), 7.09–7.13 (4H, m), 7.20–7.31 (5H, m). 13C-NMR (100 MHz, CDCl3) δ: 21.0, 34.6, 52.3, 53.0, 53.6, 60.0, 62.9, 126.9, 128.0, 128.2, 129.1, 129.3, 136.4, 138.2, 138.5. IR (film) cm−1: 811, 1513, 2797, 2918. MS (ESI+) m/z: 281 (M+H+), 264 (base peak). HR-MS (ESI+) m/z: Found 281.2024 (Calcd for C19H25N2 (M+H+) 281.2018).

cis- and trans-4-Amino-1-N-benzyl-3-(3-methylphenyl)piperidine (cis-, trans-11c)

Prepared According to Procedure for the Preparation of 11a

Yield: 72% (cistrans=2 : 3). cis-11c: yellow oil. 1H-NMR (CDCl3) δ: 1.31 (2H, br), 1.68–1.74 (1H, m), 1.93–1.99 (1H, m), 2.33 (3H, s), 2.50–2.58 (2H, m), 2.70–2.79 (2H, m), 3.03–3.07 (1H, m), 3.20–3.23 (1H, m), 3.56 (1H, d, J=13.1 Hz), 3.61 (1H, d, J=13.1 Hz), 7.03 (1H, d, J=7.4 Hz), 7.10–7.12 (2H, m), 7.17–7.25 (2H, m), 7.29–7.37 (4H, m). 13C-NMR (CDCl3) δ: 21.3, 32.5, 46.1, 48.4, 49.4, 52.3, 63.3, 125.2, 126.7, 126.9, 127.9, 128.0, 128.9, 129.0, 137.5, 138.4, 141.7. IR (film) cm−1: 699, 1453, 1492, 2918. MS (ESI+) m/z: 281 (M+H+, base peak). HR-MS (ESI+) m/z: Found 281.2000 (Calcd for C19H25N2 (M+H+) 281.2018). trans-11c: yellow oil. 1H-NMR (CDCl3) δ: 1.57 (1H, dq, J=12.7, 4.1 Hz), 1.73 (2H, br), 1.93 (1H, ddd, J=12.7, 6.8, 2.6 Hz), 2.10 (1H, t, J=11.4 Hz), 2.14 (1H, dt, J=12.0, 2.6 Hz), 2.31 (3H, s), 2.53 (1H, dt, J=10.8, 3.7 Hz), 2.83 (1H, dt, J=10.8, 4.1 Hz), 2.90 (1H, ddd, J=11.4, 3.7, 2.1 Hz), 2.94–2.99 (1H, m), 3.52 (2H, s), 6.99–7.03 (3H, m), 7.18 (1H, t, J=7.8 Hz), 7.21–7.24 (1H, m), 7.26–7.31 (4H, m). 13C-NMR (CDCl3) δ: 21.4, 34.3, 52.4, 52.9, 53.5, 59.7, 62.8, 125.1, 126.9, 127.5, 128.1, 128.4, 128.9, 129.0, 138.1, 138.1, 141.3. IR (film) cm−1: 704, 1454, 2918. MS (ESI+) m/z: 281 (M+H+), 264 (base peak). HR-MS (ESI+) m/z: Found 281.1982 (Calcd for C19H25N2 (M+H+) 281.2018).

cis- and trans-4-Amino-1-N-benzyl-3-(4-fluorophenyl)piperidine (cis-, trans-11d)

Prepared According to Procedure for the Preparation of 11a

Yield: 68% (cistrans=1 : 1). cis-11d: yellow oil. 1H-NMR (CDCl3) δ: 1.65–1.71 (1H, m), 1.87–1.95 (1H, m), 2.51–2.57 (2H, m), 2.66 (1H, dd, J=10.8, 3.0 Hz), 2.77 (1H, t, J=9.6 Hz), 3.02–3.06 (1H, m), 3.16–3.20 (1H, m), 3.55 (1H, d, J=13.1 Hz), 3.59 (1H, d, J=13.1 Hz), 6.97–7.01 (2H, m), 7.22–7.36 (7H, m). 13C-NMR (CDCl3) δ: 32.3, 45.5, 49.7, 53.5, 56.7, 63.4, 115.0 (d, J=11.1 Hz), 127.1, 128.3, 129.1, 130.1, 137.3, 138.3, 161.6 (d, J=244.8 Hz). IR (film) cm−1: 1222, 1509, 2933. MS (ESI+) m/z: 285 (M+H+), 267 (base peak). HR-MS (ESI+) m/z: Found 285.1770 (Calcd for C18H22FN2 (M+H+) 285.1767). trans-11d: yellow oil. 1H-NMR (CDCl3) δ: 1.59 (1H, dq, J=12.5, 4.1 Hz), 1.92–1.96 (1H, m), 2.06–2.18 (2H, m), 2.57 (1H, dt, J=10.8, 3.7 Hz), 2.81 (1H, dt, J=10.8, 4.1 Hz), 2.90 (1H, ddd, J=11.4, 3.7, 2.1 Hz), 2.97–3.00 (1H, m), 3.54 (2H, s), 6.97–7.02 (2H, m), 7.16–7.22 (2H, m), 7.23–7.31 (5H, m). 13C-NMR (CDCl3) δ: 34.4, 51.6, 52.9, 53.8, 59.9, 62.9, 115.6 (d, J=21.1 Hz), 127.2, 128.4, 129.2, 129.6 (d, J=7.7 Hz), 137.2 (d, J=3.1 Hz), 138.1, 161.9 (d, J=244.9 Hz). IR (film) cm−1: 1509, 1603, 2802, 2934. MS (ESI+) m/z: 285 (M+H+), 250 (base peak). HR-MS (ESI+) m/z: Found 285.1775 (Calcd for C18H22FN2 (M+H+) 285.1767).

cis- and trans-4-Amino-1-N-benzyl-3-(4-chlorophenyl)piperidine (cis-, trans-11e)

To a stirred suspension of LiAlH4 (281 mg, 7.43 mmol) in THF (20 mL) was added dropwise a solution of 10e (778 mg, 2.48 mmol) in THF (10 mL) at rt under nitrogen atmosphere. After being stirred at reflux for 1.5 h, the reaction mixture was quenched with H2O and filtrated through Celite pad and extracted with CHCl3. The residue was treated with THF and refluxed for 30 min. After filtration, the combined organic layers were dried over anhydrous Na2SO4 and evaporated. The resulting residue was purified by chromatography on SiO2 (CHCl3–MeOH=10 : 1) to yield the primary amine (581 mg, 1.94 mmol, 78%, cistrans=4 : 1). cis-11e: yellow oil. 1H-NMR (CDCl3) δ: 1.39 (2H, br), 1.64–1.71 (1H, m), 1.87–1.93 (1H, m), 2.50–2.56 (2H, m), 2.66 (1H, dd, J=10.8, 2.9 Hz), 2.78 (1H, t, J=9.8 Hz), 3.01–3.05 (1H, m), 3.17–3.20 (1H, m), 3.55 (1H, d, J=13.1 Hz), 3.59 (1H, d, J=13.1 Hz), 7.22–7.36 (9H, m). 13C-NMR (CDCl3) δ: 32.5, 45.7, 49.0, 49.5, 52.7, 63.3, 126.9, 128.1, 128.2, 128.9, 129.9, 132.0, 138.3, 140.1. IR (film) cm−1: 1090, 1491, 2808, 2933. MS (ESI+) m/z: 301 (M+H+, base peak). HR-MS (ESI+) m/z: Found 301.1472 (Calcd for C18H22ClN2 (M+H+) 301.1472). trans-11e: yellow oil. 1H-NMR (CDCl3) δ: 1.46 (2H, br), 1.58 (1H, dq, J=12.7, 4.2 Hz), 1.93 (1H, ddd, J=12.7, 6.9, 2.6 Hz), 2.08 (1H, t, J=11.4 Hz), 2.15 (1H, dt, J=12.0, 2.6 Hz), 2.55 (1H, dt, J=10.8, 3.7 Hz), 2.82 (1H, dt, J=10.8, 4.2 Hz), 2.88 (1H, ddd, J=11.4, 3.7, 2.1 Hz), 2.96–3.00 (1H, m), 3.53 (2H, s), 7.16 (2H, d, J=8.4 Hz), 7.23–7.31 (7H, m). 13C-NMR (CDCl3) δ: 34.6, 52.0, 52.9, 53.6, 59.7, 62.8, 127.0, 128.2, 128.7, 129.0, 129.5, 132.5, 138.1, 140.1. IR (film) cm−1: 1090, 1492, 2799, 2932. MS (ESI+) m/z: 301 (M+H+, base peak). HR-MS (ESI+) m/z: Found 301.1447 (Calcd for C18H22ClN2 (M+H+) 301.1472).

3-{N-Benzyl-N-[2-(3-chlorophenyl)-acetyl]-amino}-propionic Acid Ethyl Ester (14f)

3-Chlorophenylacetic acid (2.47 g, 14.5 mmol) and ethyl 3-(N-benzylamino)propionate (3.00 g, 14.5 mmol) and DMAP (2.65 g, 21.8 mmol) were dissolved in CH2Cl2 (150 mL) at 0°C under nitrogen atmosphere. After being stirred for 1 h, EDC (3.31 g, 17.4 mmol) was added to the reaction mixture and stirred for 15 h. The reaction mixture was washed with saturated aqueous KHSO4 and dried over anhydrous Na2SO4 and evaporated. The resulting residue was purified by chromatography on SiO2 (n-hexane–EtOAc=2 : 1) to yield 14f (5.18 g, 14.4 mmol, 99%) as a light yellow oil. The NMR spectra of 14f showed a mixture of two rotamers. The ratio of two rotamers was 13 : 7. 1H-NMR (CDCl3) δ: 1.22 (3H of rotamer A, t, J=7.2 Hz), 1.25 (3H of rotamer B, t, J=7.1 Hz), 2.43 (2H of rotamer B, t, J=7.2 Hz), 2.64 (2H of rotamer A, t, J=6.9 Hz), 3.56 (2H of rotamer B, t, J=7.2 Hz), 3.64–3.68 (4H of rotamer A, m), 3.84 (2H of rotamer B, s), 4.06–4.15 (4H, 2H of rotamer A, m, 2H of rotamer B, m), 4.60 (2H of rotamer A, s), 4.62 (2H of rotamer B, s), 7.07–7.39 (18H, 9H of rotamer A, m, 9H of rotamer B, m). 13C-NMR (CDCl3) δ: 14.1, 32.6, 33.3, 40.2, 40.2, 42.8, 43.3, 48.1, 52.5, 60.5, 60.9, 126.1, 127.0, 127.0, 127.1, 127.4, 127.7, 127.9, 128.6, 128.9, 128.9, 129.0, 129.7, 129.8, 134.3, 134.4, 136.5, 136.7, 136.9, 137.1, 170.4, 170.9, 171.9. IR (film) cm−1: 1190, 1450, 1650, 1730. MS (ESI+) m/z: 382 (M+Na+, base peak). HR-MS (ESI+) m/z: Found 382.1234 (Calcd for C20H22ClNNaO3 (M+Na+) 382.1186).

3-{N-Benzyl-N-[2-(2-chlorophenyl)-acetyl]-amino}-propionic Acid Ethyl Ester (14g)

Prepared According to Procedure for the Preparation of 14f

Yield: 99%. Light yellow oil. The NMR spectra of 14g showed a mixture of two rotamers. The ratio of two rotamer was 13 : 7. 1H-NMR (CDCl3) δ: 1.23 (3H of rotamer A, t, J=7.1 Hz), 1.25 (3H of rotamer B, t, J=7.2 Hz), 2.53 (2H of rotamer B, t, J=7.4 Hz), 2.66 (2H of rotamer A, t, J=6.9 Hz), 3.63 (2H of rotamer B, t, J=7.4 Hz), 3.67 (2H of rotamer A, t, J=6.9 Hz), 3.80 (2H of rotamer A, s), 3.93 (2H of rotamer B, s), 4.07–4.16 (4H, 2H of rotamer A, m, 2H of rotamer B, m), 4.65 (2H of rotamer B, s), 4.66 (2H of rotamer A, s), 7.18–7.40 (18H, 9H of rotamer A, m, 9H of rotamer B, m). 13C-NMR (CDCl3) δ: 14.1, 32.6, 33.4, 38.0, 38.2, 42.8, 43.2, 48.4, 52.5, 60.5, 60.9, 126.3, 126.9, 126.9, 127.4, 127.6, 128.0, 128.3, 128.4, 128.5, 128.9, 129.3, 129.4, 130.9, 131.1, 133.3, 133.4, 134.0, 134.0, 136.6, 137.3, 170.1, 170.5, 170.9, 172.0. IR (film) cm−1: 1375, 1445, 1651, 1731. MS (ESI+) m/z: 360 (M+H+, base peak). HR-MS (ESI+) m/z: Found 360.1415 (Calcd for C20H23ClNO3 (M+H+) 360.1367).

1-N-Benzyl-3-(3-chlorophenyl)-2,4-piperidinedione (15f)

To a stirred suspension of NaOEt (2.85 g, 41.8 mmol) in toluene (200 mL) was slowly added a solution of 14f (5.00 g, 13.9 mmol) in toluene (40 mL) at rt under nitrogen atmosphere. After being stirred at reflux for 4 h, the reaction mixture was acidified with 1 M aqueous HCl and extracted with EtOAc. The combined organic layer was dried over anhydrous Na2SO4 and evaporated to give 15f and was used without further purification. For measurement of spectroscopic data, 15f mixture was purified by chromatography on SiO2 (n-hexane–EtOAc=2 : 3) to gain a colorless solid, mp 118–120°C. 1H-NMR (CDCl3) δ: 2.64–2.67 (2H, m), 3.45–3.56 (2H, m), 4.53 (1H, s), 4.72 (1H, d, J=14.5 Hz), 4.79 (1H, d, J=14.5 Hz), 7.07–7.09 (1H, m), 7.18–7.19 (1H, m), 7.28–7.40 (7H, m). 13C-NMR (CDCl3) δ: 37.7, 41.6, 50.7, 63.7, 127.1, 128.1, 128.2, 128.3, 128.6, 128.9, 130.0, 134.2, 134.7, 136.1, 167.0, 202.7. IR (KBr) cm−1: 1381, 1479, 1603, 1661. MS (ESI+) m/z: 314 (M+H+, base peak). HR-MS (ESI+) m/z: Found 314.0976 (Calcd for C18H17ClNO2 (M+H+) 314.0948).

1-N-Benzyl-3-(2-chlorophenyl)-2,4-piperidinedione (15g)

Prepared According to Procedure for the Preparation of 15f

Colorless solid, mp 163–166°C. 1H-NMR (CDCl3) δ: 2.71–2.83 (2H, m), 3.48–3.55 (1H, m), 3.71 (1H, ddd, J=13.5, 8.6, 5.1 Hz), 4.61 (1H, d, J=14.6 Hz), 4.65 (1H, s), 4.90 (1H, d, J=14.6 Hz), 7.23–7.25 (1H, m), 7.29–7.39 (7H, m), 7.41–7.43 (1H, m). 13C-NMR (CDCl3) δ: 38.59, 41.71, 50.8, 62.9, 127.0, 127.9, 128.4, 128.9, 129.4, 129.7, 132.3, 133.2, 133.8, 136.3, 166.5, 201.8. IR (KBr) cm−1: 1380, 1480, 1596, 1670. MS (ESI+) m/z: 314 (M+H+, base peak). HR-MS (ESI+) m/z: Found 314.0981 (Calcd for C18H17ClNO2 (M+H+) 314.0948).

1-N-Benzyl-3-(3-chlorophenyl)-4-hydroxyimino-2-piperidone (16f)

Prepared According to Procedure for the Preparation of 10a

Brown oil. 1H-NMR shows a doubled signal set (isomer A and B, ratio 4 : 1). 1H-NMR (CDCl3) δ: 2.56 (1H of isomer B, dt, J=15.0, 5.0 Hz), 2.61–2.69 (2H, 1H of isomer A, dt, J=18.5, 4.5, 1H of isomer B, m), 2.81 (1H of isomer A, ddd, J=18.5, 9.5, 6.9 Hz), 3.11–3.24 (2H of isomer A, m), 3.31 (1H of isomer B, ddd, J=12.4, 9.4, 5.0 Hz), 3.41 (1H of isomer B, dt, J=12.4, 5.2 Hz), 4.55 (1H of isomer B, d, J=14.5 Hz), 4.62 (1H of isomer A, s), 4.68 (2H of isomer A, s), 4.83 (1H of isomer B, d, J=14.5 Hz), 5.33 (1H of isomer B, s), 7.16–7.19 (1H of isomer A, m), 7.23–7.37 (17H, 8H of isomer A, m, 9H of isomer B, m). 13C-NMR (CDCl3), (isomer A) δ: 23.9, 42.1, 50.4, 53.2, 125.5, 127.4, 127.8, 128.0, 128.2, 128.7, 130.0, 134.7, 136.2, 136.9, 154.5, 169.0; (isomer B) δ: 26.9, 44.4, 47.4, 50.7, 126.3, 127.5, 127.7, 128.1, 128.7, 128.7, 129.9, 134.4, 136.2, 138.0, 153.4, 167.9. IR (film) cm−1: 972, 1485, 1635, 3215. MS (ESI+) m/z: 351 (M+Na+, base peak). HR-MS (ESI+) m/z: Found 351.0900 (Calcd for C18H17ClN2NaO2 (M+Na+) 351.0876).

1-N-Benzyl-3-(2-chlorophenyl)-4-hydroxyimino-2-piperidone (16g)

Prepared According to Procedure for the Preparation of 10a

Brown oil. 1H-NMR shows a single isomer. 1H-NMR (CDCl3) δ: 2.87 (2H, t, J=6.0 Hz), 3.39–3.54 (2H, m), 4.57 (1H, d, J=14.6 Hz), 4.74 (1H, s), 4.84 (1H, d, J=14.6 Hz), 7.20–7.42 (9H, m). 13C-NMR (CDCl3) δ: 24.2, 42.6, 50.7, 51.8, 126.7, 127.7, 128.3, 128.7, 128.9, 129.7, 132.6, 133.9, 134.0, 136.5, 155.3, 167.8. IR (film) cm−1: 754, 1446, 1643, 3300. MS (ESI+) m/z: 329 (M+H+, base peak). HR-MS (ESI+) m/z: Found 329.1102 (Calcd for C18H18ClN2O2 (M+H+) 329.1057).

cis- and trans-4-Amino-1-N-benzyl-3-(3-chlorophenyl)piperidine (cis-, trans-11f)

Prepared According to Procedure for the Preparation of 11e

Yield: 30% over three steps (cistrans=3 : 1). cis-11f: yellow oil. 1H-NMR (CDCl3) δ: 1.34 (2H, br), 1.65–1.72 (1H, m), 1.89–1.94 (1H, m), 2.54–2.67 (3H, m), 2.79 (1H, t, J=9.9 Hz), 3.02–3.06 (1H, m), 3.20–3.22 (1H, m), 3.57 (2H, s), 7.19–7.26 (4H, m), 7.30–7.37 (5H, m). 13C-NMR (CDCl3) δ: 18.4, 32.3, 46.0, 49.7, 57.6, 63.3, 126.6, 126.9, 127.0, 128.2, 128.9, 129.1, 129.3, 134.0, 138.3, 143.7. IR (film) cm−1: 698, 1453, 1595, 2931. MS (ESI+) m/z: 301 (M+H+, base peak). HR-MS (ESI+) m/z: Found 301.1506 (Calcd for C18H22ClN2 (M+H+) 301.1472). trans-11f: yellow oil. 1H-NMR (CDCl3) δ: 1.54–1.63 (1H, m), 1.94 (1H, ddd, J=12.9, 6.9, 2.6 Hz), 2.10 (1H, t, J=11.4 Hz), 2.16 (1H, dt, J=12.0, 2.6 Hz), 2.56 (1H, dt, J=10.8, 3.8 Hz), 2.84 (1H, dt, J=10.8, 4.2 Hz), 2.90 (1H, ddd, J=11.4, 3.8, 2.1 Hz), 2.96–3.01 (1H, m), 3.54 (2H, s), 7.09–7.12 (1H, dt, J=6.9, 1.7 Hz), 7.20–7.26 (4H, m), 7.28–7.31 (4H, m). 13C-NMR (CDCl3) δ: 34.5, 52.3, 52.9, 53.5, 59.5, 62.8, 126.5, 127.0, 127.1, 128.1, 128.2, 129.1, 129.9, 134.4, 138.0, 143.7. IR (film) cm−1: 698, 1453, 1595, 2932. MS (ESI+) m/z: 301 (M+H+, base peak). HR-MS (ESI+) m/z: Found 301.1505 (Calcd for C18H22ClN2 (M+H+) 301.1472).

cis- and trans-4-Amino-1-N-benzyl-3-(2-chlorophenyl)piperidine (cis-, trans-11g)

Prepared According to Procedure for the Preparation of 11e

Yield: 17% over three steps (cistrans=3 : 2). cis-11g: yellow oil. 1H-NMR (CDCl3) δ: 1.53 (2H, br), 1.59–1.69 (1H, m), 1.96–2.00 (2H, m), 2.18 (1H, dt, J=12.0, 2.5 Hz), 2.90–2.95 (2H, m), 2.97–3.02 (1H, m), 3.27–3.29 (1H, m), 3.56 (2H, s), 7.15 (1H, ddd, J=7.9, 6.9, 2.1 Hz), 7.22–7.33 (7H, m), 7.37 (1H, dd, J=7.9, 1.1 Hz). 13C-NMR (CDCl3) δ: 29.7, 32.3, 42.6, 48.6, 51.8, 63.5, 126.5, 127.0, 127.5, 128.2, 129.1, 129.2, 129.7, 134.3, 138.3, 139.2. IR (film) cm−1: 749, 1473, 2925. MS (ESI+) m/z: 301 (M+H+, base peak). HR-MS (ESI+) m/z: Found 301.1507 (Calcd for C18H22ClN2 (M+H+) 301.1472). trans-11g: yellow oil. 1H-NMR (CDCl3) δ: 1.59 (1H, dq, J=12.8, 4.2 Hz), 1.95 (1H, ddd, J=12.8, 6.8, 2.7 Hz), 2.10–2.20 (2H, m), 2.57 (1H, dt, J=10.8, 3.7 Hz), 2.86 (1H, dt, J=10.8, 4.2 Hz), 2.93 (1H, ddd, J=11.4, 3.7, 2.1 Hz), 2.97–3.02 (1H, m), 3.54 (2H, s), 7.21–7.33 (9H, m). 13C-NMR (CDCl3) δ: 34.6, 47.6, 52.8, 53.2, 58.7, 62.5, 127.0, 127.1, 127.7, 128.2, 128.3, 129.0, 129.3, 129.9, 138.1, 138.8. IR (film) cm−1: 753, 1474, 2927. MS (ESI+) m/z: 301 (M+H+, base peak). HR-MS (ESI+) m/z: Found 301.1511 (Calcd for C18H22ClN2 (M+H+) 301.1472).

cis-1-N-Benzyl-4-N,N-dimethylamino-3-phenylpiperidine (cis-17a)

Thirty-seven percent HCHO (0.63 mL, 7.50 mmol, in aqueous solution) and HCO2H (0.79 mL, 15.0 mmol) were added to cis-11a (200 mg, 0.75 mmol). After being stirred at reflux for 24 h, the reaction mixture was alkalized with 1 M aqueous NaOH and extracted with CHCl3. The combined organic layer was dried over anhydrous Na2SO4 and evaporated to yield cis-17a (205 mg, 0.70 mmol, 93%) as a brown oil and was used without further purification. 1H-NMR (CDCl3) δ: 1.68–1.72 (1H, m), 1.93–2.08 (2H, m), 2.11 (6H, s), 2.35–2.42 (2H, m), 2.98 (1H, dt, J=11.4, 2.3 Hz), 3.05–3.08 (1H, m), 3.15 (1H, d, J=2.7 Hz), 3.42 (1H, d, J=13.3 Hz), 3.47 (1H, d, J=13.3 Hz), 7.18–7.22 (2H, m), 7.23–7.24 (1H, m), 7.25–7.30 (5H, m), 7.77 (2H, d, J=7.2 Hz). 13C-NMR (CDCl3) δ: 25.0, 43.0, 43.7, 53.3, 59.1, 63.1, 66.6, 126.1, 126.8, 127.6, 128.1, 128.8, 130.6, 138.9, 143.0. IR (film) cm−1: 1460, 1500, 2760, 2940. MS (EI) m/z: 294 (M+H+), 91 (base peak). HR-MS (EI) m/z: Found 294.2072 (Calcd for C20H26N2 (M+H+) 294.2096).

cis-1-N-Benzyl-4-N,N-dimethylamino-3-(4-methylphenyl)piperidine (cis-17b)

Prepared According to Procedure for the Preparation of cis-17a

Yield: 99%. Brown oil. 1H-NMR (CDCl3) δ: 1.66–1.70 (1H, m), 1.94–2.07 (2H, m), 2.12 (6H, s), 2.28–2.36 (1H, m), 2.32 (3H, s), 2.39 (1H, dd, J=11.4, 3.4 Hz), 2.97 (1H, d, J=11.4 Hz), 3.04 (1H, d, J=10.0 Hz), 3.11 (1H, br), 3.39 (1H, d, J=13.2 Hz), 3.48 (1H, d, J=13.2 Hz), 7.10 (2H, d, J=7.8 Hz), 7.18–7.28 (5H, m), 7.66 (2H, d, J=7.8 Hz). 13C-NMR (CDCl3) δ: 21.0, 25.1, 43.2, 43.3, 53.2, 59.3, 63.1, 66.7, 126.8, 128.1, 128.3, 128.8, 130.4, 135.4, 138.9, 140.0. IR (film) cm−1: 1453, 1512, 2764, 2940. MS (ESI+) m/z: 309 (M+H+), 264 (base peak). HR-MS (ESI+) m/z: Found 309.2351 (Calcd for C21H29N2 (M+H+) 309.2331).

cis-1-N-Benzyl-4-N,N-dimethylamino-3-(3-methylphenyl)piperidine (cis-17c)

Prepared According to Procedure for the Preparation of cis-17a

Yield: 99%. Brown oil. 1H-NMR (CDCl3) δ: 1.68–1.72 (1H, m), 1.96–2.10 (2H, m), 2.12 (6H, s), 2.34–2.37 (1H, m), 2.36 (3H, s), 2.40 (1H, dd, J=11.4, 3.6 Hz), 2.97 (1H, dt, J=11.4, 2.3 Hz), 3.05–3.11 (2H, m), 3.41 (1H, d, J=13.2 Hz), 3.47 (1H, d, J=13.2 Hz), 7.03 (1H, d, J=7.5 Hz), 7.15–7.23 (2H, m), 7.25–7.33 (4H, m), 7.56 (1H, d, J=7.8 Hz), 7.63 (1H, s). 13C-NMR (CDCl3) δ: 21.5, 24.8, 43.0, 43.5, 53.1, 58.9, 62.9, 66.5, 126.6, 126.7, 127.2, 127.5, 127.9, 128.6, 131.2, 136.5, 138.8, 142.7. IR (film) cm−1: 1453, 2765, 2940. MS (ESI+) m/z: 309 (M+H+), 264 (base peak). HR-MS (ESI+) m/z: Found 309.2309 (Calcd for C21H29N2 (M+H+) 309.2331).

cis-1-N-Benzyl-4-N,N-dimethylamino-3-(4-fluorophenyl)piperidine (cis-17d)

Prepared According to Procedure for the Preparation of cis-17a

Yield: 97%. Brown oil. 1H-NMR (CDCl3) δ: 1.69–1.73 (1H, m), 1.91 (1H, dq, J=12.4, 4.1 Hz), 2.07 (1H, dt, J=11.6, 2.9 Hz), 2.12 (6H, s), 2.29–2.34 (1H, m), 2.37 (1H, dd, J=11.5, 3.6 Hz), 2.94 (1H, dt, J=11.5, 2.3 Hz), 3.05–3.10 (1H, m), 3.13 (1H, br), 3.44 (2H, s), 6.95–6.99 (2H, m), 7.20–7.29 (5H, m), 7.73–7.77 (2H, m). 13C-NMR (CDCl3) δ: 24.7, 42.7, 43.0, 53.4, 58.9, 63.0, 66.5, 114.2 (d, J=20.5 Hz), 126.8, 128.1, 128.8, 131.9 (d, J=7.4 Hz), 138.5 (d, J=3.3 Hz), 138.7, 161.5 (d, J=244.0 Hz). IR (film) cm−1: 1453, 1507, 2767, 2940. MS (ESI+) m/z: 313 (M+H+), 250 (base peak). HR-MS (ESI+) m/z: Found 313.2112 (Calcd for C20H26FN2 (M+H+) 313.2080).

cis-1-N-Benzyl-3-(4-chlorophenyl)-4-N,N-dimethylaminopiperidine (cis-17e)

Prepared According to Procedure for the Preparation of cis-17a

Yield: 99%. Brown oil. 1H-NMR (CDCl3) δ: 1.75–1.78 (1H, m), 2.02 (1H, dq, J=12.4, 4.0 Hz), 2.14 (1H, dt, J=11.6, 2.5 Hz), 2.23 (6H, s), 2.46 (1H, dd, J=11.6, 3.5 Hz), 2.93–2.98 (2H, m), 3.14–3.17 (1H, m), 3.36 (1H, br), 3.47 (2H, s), 7.23–7.31 (7H, m), 7.85 (2H, d, J=8.5 Hz). 13C-NMR (CDCl3) δ: 22.8, 41.2, 41.6, 52.7, 58.8, 62.8, 65.8, 127.1, 128.3, 128.3, 128.9, 132.0, 133.0, 138.1, 139.7. IR (film) cm−1: 1490, 2767, 2941. MS (ESI+) m/z: 329 (M+H+, base peak). HR-MS (ESI+) m/z: Found 329.1793 (Calcd for C20H26ClN2 (M+H+) 329.1785).

cis-1-N-Benzyl-3-(3-chlorophenyl)-4-N,N-dimethylaminopiperidine (cis-17f)

Prepared According to Procedure for the Preparation of cis-17a

Yield: 97%. Brown oil. 1H-NMR (CDCl3) δ: 1.68–1.73 (1H, m), 1.91 (1H, dq, J=12.4, 4.1 Hz), 2.05–2.11 (1H, m), 2.13 (6H, s), 2.31–2.39 (2H, m), 2.93 (1H, dt, J=11.5, 2.3 Hz), 3.07–3.11 (2H, m), 3.42 (1H, d, J=13.2 Hz), 3.47 (1H, d, J=13.2 Hz), 7.19–7.33 (8H, m), 7.53–7.55 (1H, m). 13C-NMR (CDCl3) δ: 24.5, 42.9, 42.9, 53.1, 58.4, 62.8, 66.3, 126.1, 126.8, 128.0, 128.5, 128.7, 128.7, 130.6, 133.2, 138.5, 144.8. IR (film) cm−1: 698, 1473, 1595, 2769, 2940. MS (ESI+) m/z: 329 (M+H+, base peak). HR-MS (ESI+) m/z: Found 329.1816 (Calcd for C20H26ClN2 (M+H+) 329.1785).

cis-1-N-Benzyl-3-(2-chlorophenyl)-4-N,N-dimethylaminopiperidine (cis-17g)

Prepared According to Procedure for the Preparation of cis-17a

Yield: 99%. Brown oil. 1H-NMR (CDCl3) δ: 1.84–1.88 (1H, m), 2.12 (6H, s), 2.13–2.19 (2H, m), 2.36–2.44 (2H, m), 2.86 (1H, dt, J=11.6, 2.6 Hz), 3.00–3.01 (1H, m), 3.41 (1H, d, J=13.6 Hz), 3.46 (1H, d, J=13.6 Hz), 3.71–3.74 (1H, m), 7.12–7.22 (7H, m), 7.25 (1H, dt, J=7.6, 1.5 Hz), 7.32 (1H, dd, J=7.9, 1.5 Hz). 13C-NMR (CDCl3) δ: 22.1, 30.0, 38.0, 53.4, 54.3, 58.5, 63.4, 126.3, 126.9, 127.2, 127.9, 128.2, 129.3, 129.5, 134.7, 138.1, 139.8. IR (film) cm−1: 740, 1471, 2767, 2949. MS (ESI+) m/z: 329 (M+H+, base peak). HR-MS (ESI+) m/z: Found 329.1833 (Calcd for C20H26ClN2 (M+H+) 329.1785).

trans-1-N-Benzyl-4-N,N-dimethylamino-3-phenylpiperidine (trans-17a)

Prepared According to Procedure for the Preparation of cis-17a

Yield: 92%. Brown oil. 1H-NMR (CDCl3) δ: 1.68 (1H, dq, J=12.1, 3.7 Hz), 1.80–1.83 (1H, m), 1.96–2.12 (2H, m), 2.16 (6H, s), 2.75 (1H, dt, J=11.2, 2.8 Hz), 2.90–2.99 (2H, m), 3.02–3.05 (1H, m), 3.48 (1H, d, J=13.2 Hz), 3.52 (1H, d, J=13.2 Hz), 7.18–7.23 (4H, m), 7.25–7.29 (6H, m). 13C-NMR (CDCl3) δ: 22.9, 40.3, 46.6, 53.2, 62.1, 62.9, 64.9, 126.4, 127.0, 127.9, 128.2, 128.3, 129.1, 138.1, 142.8. IR (film) cm−1: 1450, 2920. MS (CI) m/z: 295 (M+H+), 116 (base peak). HR-MS (CI) m/z: Found 295.2163 (Calcd for C20H27N2 (M+H+) 295.2174).

trans-1-N-Benzyl-4-N,N-dimethylamino-3-(4-methylphenyl)piperidine (trans-17b)

Prepared According to Procedure for the Preparation of cis-17a

Yield: 99%. Brown oil. 1H-NMR (CDCl3) δ: 1.66 (1H, dq, J=12.4, 3.8 Hz), 1.78–1.82 (1H, m), 2.00–2.06 (2H, m), 2.16 (6H, s), 2.29 (3H, s), 2.72 (1H, dt, J=11.1, 3.3 Hz), 2.88–2.95 (2H, m), 3.03 (1H, d, J=11.0 Hz), 3.47 (1H, d, J=13.3 Hz), 3.51 (1H, d, J=13.3 Hz), 7.06–7.11 (4H, m), 7.19–7.28 (5H, m). 13C-NMR (CDCl3) δ: 21.1, 22.7, 40.3, 46.2, 53.2, 62.2, 62.9, 64.9, 126.9, 127.6, 128.1, 129.0, 129.1, 135.7, 138.1, 139.8. IR (film) cm−1: 1038, 1453, 1514, 2776, 2935. MS (ESI+) m/z: 309 (M+H+), 264 (base peak). HR-MS (ESI+) m/z: Found 309.2343 (Calcd for C21H29N2 (M+H+) 309.2331).

trans-1-N-Benzyl-4-N,N-dimethylamino-3-(3-methylphenyl)piperidine (trans-17c)

Prepared According to Procedure for the Preparation of cis-17a

Yield: 94%. Brown oil. 1H-NMR (CDCl3) δ: 1.68 (1H, dq, J=12.4, 3.9 Hz), 1.82 (1H, ddd, J=12.4, 6.3, 2.9 Hz), 2.01–2.08 (2H, m), 2.17 (6H, s), 2.32 (3H, s), 2.76 (1H, dt, J=11.1, 3.9 Hz), 2.88–2.95 (2H, m), 3.02–3.05 (1H, m), 3.48 (1H, d, J=13.2 Hz), 3.52 (1H, d, J=13.2 Hz), 6.99–7.01 (3H, m), 7.16 (1H, t, J=7.7 Hz), 7.21–7.26 (1H, m), 7.29–7.30 (4H, m). 13C-NMR (CDCl3) δ: 21.5, 22.8, 40.4, 46.6, 53.3, 62.1, 62.9, 64.9, 124.9, 127.0, 127.3, 128.2, 128.2, 128.6, 129.1, 137.7, 138.2, 142.8. IR (film) cm−1: 1454, 2777, 2935. MS (ESI+) m/z: 309 (M+H+), 264 (base peak). HR-MS (ESI+) m/z: Found 309.2313 (Calcd for C21H29N2 (M+H+) 309.2331).

trans-1-Benzyl-4-N,N-dimethylamino-3-(4-fluorophenyl)piperidine (trans-17d)

Prepared According to Procedure for the Preparation of cis-17a

Yield: 74%. Brown oil. 1H-NMR (CDCl3) δ: 1.66 (1H, dq, J=12.4, 4.0 Hz), 1.82 (1H, ddd, J=12.4, 6.5, 3.0 Hz), 1.98–2.07 (2H, m), 2.16 (6H, s), 2.69 (1H, dt, J=11.2, 3.8 Hz), 2.87–2.97 (2H, m), 3.02–3.06 (1H, m), 3.48 (1H, d, J=13.1 Hz), 3.52 (1H, d, J=13.1 Hz), 6.94–6.98 (2H, m), 7.14–7.18 (2H, m), 7.23–7.33 (5H, m). 13C-NMR (CDCl3) δ: 22.6, 40.4, 46.1, 53.3, 62.3, 63.0, 65.2, 115.3 (d, J=21.1 Hz), 127.2, 128.3, 129.2, 129.3, 138.2, 138.5 (d, J=3.3 Hz), 161.5 (d, J=243.8 Hz). IR (film) cm−1: 1453, 1509, 2779, 2935. MS (ESI+) m/z: 313 (M+H+), 268 (base peak). HR-MS (ESI+) m/z: Found 313.2124 (Calcd for C20H26FN2 (M+H+) 313.2080).

trans-1-N-Benzyl-3-(4-chlorophenyl)-4-N,N-dimethylaminopiperidine (trans-17e)

Prepared According to Procedure for the Preparation of cis-17a

Yield: 77%. Brown oil. 1H-NMR (CDCl3) δ: 1.66 (1H, dq, J=12.4, 3.9 Hz), 1.82 (1H, ddd, J=12.4, 6.4, 2.7 Hz), 2.00 (1H, t, J=11.0 Hz), 2.04 (1H, dt, J=11.8, 2.7 Hz), 2.16 (6H, s), 2.70 (1H, dt, J=11.0, 3.8 Hz), 2.86–2.96 (2H, m), 3.03–3.06 (1H, m), 3.48 (1H, d, J=13.4 Hz), 3.52 (1H, d, J=13.4 Hz), 7.13 (2H, d, J=8.4 Hz), 7.24 (2H, d, J=8.4 Hz), 7.24–7.32 (5H, m). 13C-NMR (CDCl3) δ: 22.3, 40.3, 46.2, 53.2, 61.9, 62.9, 65.0, 127.0, 128.2, 128.5, 129.1, 129.1, 131.9, 138.0, 141.3. IR (film) cm−1: 1091, 1492, 2780, 2936. MS (ESI+) m/z: 329 (M+H+, base peak). HR-MS (ESI+) m/z: Found 329.1818 (Calcd for C20H26ClN2 (M+H+) 329.1785).

trans-1-N-Benzyl-3-(3-chlorophenyl)-4-N,N-dimethylaminopiperidine (trans-17f)

Prepared According to Procedure for the Preparation of cis-17a

Yield: 92%. Brown oil. 1H-NMR (CDCl3) δ: 1.66 (1H, dq, J=12.4, 3.8 Hz), 1.82 (1H, ddd, J=12.4, 6.0, 2.9 Hz), 1.99–2.08 (2H, m), 2.17 (6H, s), 2.71 (1H, dt, J=11.3, 3.8 Hz), 2.87–2.96 (2H, m), 3.04 (1H, ddd, J=11.0, 6.0, 3.8 Hz), 3.50 (2H, s), 7.08 (1H, dt, J=7.3, 1.5 Hz), 7.14–7.20 (4H, m), 7.22–7.31 (4H, m). 13C-NMR (CDCl3) δ: 22.4, 40.3, 46.5, 53.1, 61.7, 62.8, 64.9, 126.1, 126.6, 127.0, 127.9, 128.2, 129.1, 129.6, 134.0, 138.0, 144.9. IR (film) cm−1: 698, 1453, 2781, 2935. MS (ESI+) m/z: 329 (M+H+, base peak). HR-MS (ESI+) m/z: Found 329.1795 (Calcd for C20H26ClN2 (M+H+) 329.1785).

trans-1-Benzyl-3-(2-chlorophenyl)-4-N,N-dimethylaminopiperidine (trans-17g)

Prepared According to Procedure for the Preparation of cis-17a

Yield: 41%. Brown oil. 1H-NMR (CDCl3) δ: 1.72 (1H, dq, J=12.1, 3.9 Hz), 1.82–1.92 (2H, m), 2.08 (1H, dt, J=11.4, 2.7 Hz), 2.18 (6H, s), 2.78–2.83 (1H, m), 2.94 (1H, ddd, J=11.3, 3.5, 2.1 Hz), 3.05 (1H, ddd, J=11.4, 5.9, 3.1 Hz), 3.51 (1H, d, J=13.3 Hz), 3.56 (1H, d, J=13.3 Hz), 3.56–3.59 (1H, m), 7.11 (1H, ddd, J=7.9, 7.2, 1.8 Hz), 7.19–7.33 (8H, m). 13C-NMR (CDCl3) δ: 22.0, 40.3, 41.9, 52.9, 60.5, 62.5, 64.0, 126.6, 126.9, 127.2, 128.1, 128.2, 128.9, 129.5, 134.1, 138.0, 139.5. IR (film) cm−1: 750, 1036, 1453, 2780, 2936. MS (ESI+) m/z: 329 (M+H+), 284 (base peak). HR-MS (ESI+) m/z: Found 329.1833 (Calcd for C20H26ClN2 (M+H+) 329.1785).

cis-4-N,N-Dimethylamino-1-N-pentanoyl-3-phenylpiperidine (cis-4a)

Pd–C (10 mg, 10% (w/w)) and HCO2NH4 (69 mg, 1.09 mmol) were added to a solution of cis-17a (50 mg, 0.17 mmol) in MeOH (3 mL). After being stirred at reflux for 4 h, the reaction mixture was filtrated through Celite pad and poured on H2O and extracted with CHCl3. The combined organic layer was dried over anhydrous Na2SO4 and evaporated. The resulting residue was used in the subsequent reaction without further purification. Et3N (53 µL, 0.60 mmol) and BuCOCl (36 µL, 0.30 mmol) were added to a solution of crude in CH2Cl2 (3 mL). After being stirred at rt for 18 h, the reaction mixture was alkalized with 1 M aqueous NaOH and extracted with CHCl3. The combined organic layer was dried over anhydrous Na2SO4 and evaporated. The resulting residue was purified by chromatography on SiO2 (CHCl3–MeOH=10 : 1) to yield cis-4a (20 mg, 0.07 mmol, 41% over two steps) as a light yellow oil. 1H-NMR (C6D6, 80°C) δ: 0.81 (3H, t, J=6.9 Hz), 1.18–1.27 (3H, m), 1.44 (1H, dd, J=13.3, 3.3 Hz), 1.52–1.59 (3H, m), 1.75–1.90 (3H, m), 1.94 (6H, s), 2.24 (1H, dt, J=11.7, 4.5 Hz), 2.51–2.56 (1H, m), 2.88 (1H, m), 2.93 (1H, dd, J=13.4, 3.7 Hz), 7.04 (1H, tt, J=7.4, 1.5 Hz), 7.12 (2H, t, J=7.4 Hz), 7.31–7.32 (2H, m). 13C-NMR (C6D6, 60°C) δ: 14.0, 22.7, 25.7, 27.5, 30.1, 32.6, 43.0, 59.8, 66.3, 78.1, 126.6, 128.2, 130.0, 141.9, 171.0. IR (film) cm−1: 1050, 1450, 1640, 2960. MS (EI) m/z: 288 (M+), 84 (base peak). HR-MS (EI) m/z: Found 288.2209 (Calcd for C18H28N2O (M+) 288.2201).

cis-4-N,N-Dimethylamino-3-(4-methylphenyl)-1-N-pentanoylpiperidine (cis-4b)

Prepared According to Procedure for the Preparation of cis-4a

Yield: 27% over two steps. Light yellow oil. 1H-NMR (C6D6, 80°C) δ: 0.81 (3H, t, J=6.9 Hz), 1.22–1.36 (4H, m), 1.44–1.46 (1H, m), 1.57–1.58 (2H, m), 1.80–1.90 (3H, m), 1.96 (6H, s), 2.10 (3H, s), 2.25–2.27 (1H, m), 2.53 (1H, br), 2.90–2.96 (2H, m), 6.96 (2H, d, J=7.8 Hz), 7.24 (2H, d, J=7.8 Hz). 13C-NMR (C6D6, 60°C) δ: 14.0, 20.9, 22.8, 25.5, 27.6, 30.2, 32.7, 42.9, 50.6, 66.4, 77.9, 129.1, 130.0, 136.0, 138.8, 171.1. IR (film) cm−1: 1439, 1513, 1644, 2956. MS (ESI+) m/z: 303 (M+H+, base peak). HR-MS (ESI+) m/z: Found 303.2467 (Calcd for C19H31N2O (M+H+) 303.2436).

cis-4-N,N-Dimethylamino-3-(3-methylphenyl)-1-N-pentanoylpiperidine (cis-4c)

Prepared According to Procedure for the Preparation of cis-4a

Yield: 26% over two steps. Light yellow oil. 1H-NMR (C6D6, 80°C) δ: 0.81 (3H, t, J=7.3 Hz), 1.18–1.26 (3H, m), 1.44–1.60 (4H, m), 1.80–1.92 (3H, m), 1.97 (6H, s), 2.17 (3H, s), 2.24 (1H, dt, J=11.6, 4.5 Hz), 2.55–2.58 (1H, m), 2.87 (1H, m), 2.97 (1H, dd, J=13.5, 3.7 Hz), 6.89 (1H, d, J=7.6 Hz), 7.06 (1H, t, J=7.9 Hz), 7.16–7.21 (2H, m). 13C-NMR (C6D6, 60°C) δ: 13.9, 21.5, 22.8, 25.7, 27.5, 30.1, 32.6, 43.0, 50.9, 66.4, 78.0, 127.1, 127.4, 128.2, 130.8, 137.5, 141.9, 171.0. IR (film) cm−1: 1439, 1644, 2956. MS (ESI+) m/z: 303 (M+H+, base peak). HR-MS (ESI+) m/z: Found 303.2423 (Calcd for C19H31N2O (M+H+) 303.2436).

cis-4-N,N-Dimethylamino-3-(4-fluorophenyl)-1-N-pentanoylpiperidine (cis-4d)

Prepared According to Procedure for the Preparation of cis-4a

Yield: 21% over two steps. Light yellow oil. 1H-NMR (C6D6, 80°C) δ: 0.82 (3H, t, J=7.3 Hz), 1.19–1.28 (2H, m), 1.37–1.41 (3H, m), 1.51–1.71 (4H, m), 1.92 (6H, s), 1.95–1.97 (1H, m), 2.14 (1H, dt, J=11.7, 4.5 Hz), 2.46–2.52 (1H, m), 2.78 (1H, br), 2.84 (1H, dd, J=13.5, 3.4 Hz), 6.80 (2H, t, J=8.7 Hz), 7.19 (2H, br). 13C-NMR (C6D6, 60°C) δ: 14.0, 22.8, 25.4, 27.6, 30.2, 32.7, 42.9, 50.7, 66.4, 77.8, 115.0 (d, J=20.6 Hz), 131.5 (d, J=7.4 Hz), 137.4, 162.2 (d, J=245.0 Hz), 171.1. IR (film) cm−1: 1439, 1509, 1643, 2769, 2932. MS (ESI+) m/z: 307 (M+H+, base peak). HR-MS (ESI+) m/z: Found 307.2219 (Calcd for C18H28FN2O (M+H+) 307.2186).

trans-4-N,N-Dimethylamino-1-N-pentanoyl-3-phenylpiperidine (trans-4a)

Prepared According to Procedure for the Preparation of cis-4a

Yield: 43% over two steps. Light yellow oil. 1H-NMR (C6D6, 80°C) δ: 0.88 (3H, t, J=7.4 Hz), 1.21–1.39 (5H, m), 1.52 (1H, ddd, J=12.8, 6.1, 2.9 Hz), 1.67–1.75 (2H, m), 1.96 (6H, s), 2.12–2.17 (2H, m), 2.44 (1H, br), 2.59–2.67 (3H, m), 7.04–7.07 (3H, m), 7.14–7.18 (2H, m). 13C-NMR (C6D6, 60°C) δ: 14.0, 22.9, 27.8, 30.1, 32.9, 40.1, 48.1, 53.1, 65.5, 77.7, 126.8, 128.1, 128.6, 142.1, 170.4. IR (film) cm−1: 1060, 1440, 1640, 2930. MS (EI) m/z: 288 (M+), 104 (base peak). HR-MS (EI) m/z: Found 288.2183 (Calcd for C18H28N2O (M+) 288.2201).

trans-4-N,N-Dimethylamino-3-(4-methylphenyl)-1-N-pentanoylpiperidine (trans-4b)

Prepared According to Procedure for the Preparation of cis-4a

Yield: 20% over two steps. Light yellow oil. 1H-NMR (C6D6, 80°C) δ: 0.89 (3H, t, J=7.4 Hz), 1.23–1.39 (5H, m), 1.54–1.57 (1H, m), 1.68–1.75 (2H, m), 1.99 (6H, s), 2.14 (3H, s), 2.14–2.18 (2H, m), 2.46 (1H, br), 2.57–2.68 (3H, m), 6.99 (4H, br). 13C-NMR (C6D6, 60°C) δ: 14.0, 20.9, 22.9, 27.9, 30.1, 33.0, 40.2, 47.5, 52.8, 65.6, 77.6, 127.9, 129.4, 136.1, 139.1, 170.4. IR (film) cm−1: 1438, 1645, 2929. MS (ESI+) m/z: 303 (M+H+, base peak). HR-MS (ESI+) m/z: Found 303.2451 (Calcd for C19H31N2O (M+H+) 303.2436).

trans-4-N,N-Dimethylamino-3-(3-methylphenyl)-1-N-pentanoylpiperidine (trans-4c)

Prepared According to Procedure for the Preparation of cis-4a

Yield: 63% over two steps. Light yellow oil. 1H-NMR (C6D6, 80°C) δ: 0.88 (3H, t, J=7.3 Hz), 1.16–1.39 (5H, m), 1.54 (1H, ddd, J=12.9, 6.2, 3.1 Hz), 1.68–1.75 (2H, m), 1.99 (6H, s), 2.14–2.16 (2H, m), 2.17 (3H, s), 2.46 (1H, br), 2.57–2.70 (3H, m), 6.89–6.94 (3H, m), 7.11 (1H, t, J=7.5 Hz). 13C-NMR (C6D6, 60°C) δ: 14.0, 21.5, 22.8, 27.9, 30.0, 33.0, 40.2, 45.1, 52.9, 65.5, 78.3, 125.2, 127.6, 128.5, 129.0, 137.9, 142.1, 170.5. IR (film) cm−1: 1439, 1644, 2931. MS (ESI+) m/z: 303 (M+H+, base peak). HR-MS (ESI+) m/z: Found 303.2413 (Calcd for C19H31N2O (M+H+) 303.2436).

trans-4-N,N-Dimethylamino-3-(4-fluorophenyl)-1-N-pentanoylpiperidine (trans-4d)

Prepared According to Procedure for the Preparation of cis-4a

Yield: 76% over two steps. Light yellow oil. 1H-NMR (C6D6, 80°C) δ: 0.89 (3H, t, J=7.3 Hz), 1.14–1.23 (2H, m), 1.31–1.40 (2H, m), 1.44–1.51 (2H, m), 1.68–1.75 (2H, m), 1.93 (6H, s), 2.15 (2H, t, J=7.5 Hz), 2.49–2.51 (4H, m), 6.80–6.88 (4H, m). 13C-NMR (C6D6, 60°C) δ: 14.1, 22.9, 27.9, 33.0, 40.2, 40.3, 48.9, 52.8, 65.5, 78.5, 115.4 (d, J=21.0 Hz), 129.7 (d, J=7.6 Hz), 137.9 (d, J=3.2 Hz), 162.1 (d, J=244.0 Hz), 170.7. IR (film) cm−1: 1439, 1510, 1643, 2868, 2932. MS (ESI+) m/z: 307 (M+H+), 160 (base peak). HR-MS (ESI+) m/z: Found 307.2218 (Calcd for C18H28FN2O (M+H+) 307.2186).

cis-3-(4-Chlorophenyl)-4-N,N-dimethylamino-1-N-pentanoylpiperidine (cis-4e)

1-Chloroethyl chloroformate (75 µL, 0.69 mmol) was added to a solution of cis-17e in CH2Cl2 (4 mL). After being stirred at rt for 2 h, the reaction mixture was evaporated and alkalized with 1 M aqueous NaOH and extracted with CHCl3. The combined organic layer was dried over anhydrous Na2SO4 and evaporated. The resulting residue was briefly purified by chromatography on SiO2 (CHCl3–MeOH=40 : 1) to give brown oil. The oil was dissolved in methanol (15 mL) and stirred at reflux for 1 h. The reaction mixture was evaporated, the resulting residue was used in the subsequent reaction without further purification. Et3N (0.18 mL, 1.26 mmol) and BuCOCl (76 µL, 0.63 mmol) were added to a solution of the residue in CH2Cl2 (3 mL). After being stirred at rt for 24 h, the reaction mixture was alkalized with 1 M aqueous NaOH and extracted with CHCl3. The combined organic layer was dried over anhydrous Na2SO4 and evaporated. The resulting residue was purified by chromatography on SiO2 (CHCl3–MeOH=10 : 1) to yield cis-4e (30 mg, 0.09 mmol, 15% over three steps) as light yellow oil. 1H-NMR (C6D6, 80°C) δ: 0.82 (3H, t, J=7.2 Hz), 1.20–1.35 (5H, m), 1.50–1.62 (4H, m), 1.89 (6H, s), 1.95–1.96 (1H, m), 2.20 (1H, br), 2.47 (1H, br), 2.81 (2H, br), 7.10 (2H, d, J=8.2 Hz), 7.14 (2H, br). 13C-NMR (C6D6, 60°C) δ: 13.9, 22.7, 24.9, 27.5, 30.1, 32.7, 42.6, 50.6, 66.2, 77.7, 128.5, 131.4, 132.9, 139.9, 171.1. IR (film) cm−1: 1093, 1445, 1643, 2957. MS (ESI+) m/z: 323 (M+H+, base peak). HR-MS (ESI+) m/z: Found 323.1925 (Calcd for C18H28ClN2O (M+H+) 323.1890).

cis-3-(3-Chlorophenyl)-4-N,N-dimethyl-1-N-pentanoylpiperidine (cis-4f)

Prepared According to Procedure for the Preparation of cis-4e

Yield: 27% over three steps. Light yellow oil. 1H-NMR (C6D6, 80°C) δ: 0.83 (3H, t, J=7.3 Hz), 1.15–1.48 (4H, m), 1.57–1.63 (3H, m), 1.89 (6H, s), 1.90–1.94 (3H, m), 2.11 (1H, dt, J=11.6, 4.5 Hz), 2.45–2.51 (1H, m), 2.74–2.81 (2H, m), 6.87 (1H, t, J=7.9 Hz), 7.02–7.05 (1H, m), 7.20–7.22 (1H, m), 7.45 (1H, s). 13C-NMR (C6D6, 60°C) δ: 13.9, 22.8, 27.5, 30.1, 32.7, 33.8, 42.8, 53.5, 66.3, 77.6, 126.9, 129.2, 129.4, 130.3, 134.3, 143.8, 171.1. IR (film) cm−1: 1435, 1643, 2956. MS (ESI+) m/z: 323 (M+H+, base peak). HR-MS (ESI+) m/z: Found 323.1935 (Calcd for C18H28ClN2O (M+H+) 323.1890).

cis-3-(2-Chlorophenyl)-4-N,N-dimethyl-1-N-pentanoylpiperidine (cis-4g)

Prepared According to Procedure for the Preparation of cis-4e

Yield: 25% over three steps. Light yellow oil. 1H-NMR (C6D6, 80°C) δ: 0.76 (3H, t, J=7.3 Hz), 1.13–1.18 (2H, m), 1.31–1.55 (5H, m), 1.82–1.91 (3H, m), 2.03 (6H, s), 2.28 (1H, br), 2.54 (1H, m), 3.02–3.05 (1H, m), 3.61 (1H, dd, J=8.6, 3.7 Hz), 6.78 (1H, dt, J=7.7, 1.5 Hz), 6.86 (1H, dt, J=7.8, 1.3 Hz), 7.21 (1H, dd, J=7.8, 1.5 Hz), 7.53 (1H, d, J=7.7 Hz). 13C-NMR (C6D6, 60°C) δ: 13.9, 22.8, 27.2, 27.5, 30.2, 39.8, 43.2, 49.3, 65.2, 77.7, 126.4, 127.9, 129.9, 131.9, 134.6, 138.9, 170.8. IR (film) cm−1: 1037, 1440, 1650, 2957. MS (ESI+) m/z: 323 (M+H+, base peak). HR-MS (ESI+) m/z: Found 323.1936 (Calcd for C18H28ClN2O (M+H+) 323.1890).

trans-3-(4-Chlorophenyl)-4-N,N-dimethylamino-1-N-pentanoylpiperidine (trans-4e)

Prepared According to Procedure for the Preparation of cis-4e

Yield: 13% over three steps. Light yellow oil. 1H-NMR (C6D6, 80°C) δ: 0.89 (3H, t, J=7.5 Hz), 1.31–1.40 (5H, m), 1.43–1.49 (1H, m), 1.68–1.75 (2H, m), 1.92 (6H, s), 2.15 (2H, t, J=7.5 Hz), 2.43–2.49 (4H, m), 6.80 (2H, d, J=8.5 Hz), 7.12 (2H, d, J=8.5 Hz). 13C-NMR (C6D6, 60°C) δ: 14.0, 22.9, 27.8, 30.1, 32.9, 40.0, 46.6, 51.6, 65.5, 77.7, 128.8, 129.5, 132.6, 140.5, 170.4. IR (film) cm−1: 1189, 1454, 1643, 2956. MS (ESI+) m/z: 323 (M+H+, base peak). HR-MS (ESI+) m/z: Found 323.1937 (Calcd for C18H28ClN2O (M+H+) 323.1890).

trans-3-(3-Chlorophenyl)-4-N,N-dimethyl-1-N-pentanoylpiperidine (trans-4f)

Prepared According to Procedure for the Preparation of cis-4e

Yield: 18% over three steps. Light yellow oil. 1H-NMR (C6D6, 80°C) δ: 0.89 (3H, t, J=7.4 Hz), 1.14–1.19 (1H, m), 1.30–1.47 (5H, m), 1.66–1.74 (2H, m), 1.90 (6H, s), 2.13 (2H, t, J=7.4 Hz), 2.40–2.51 (4H, m), 6.81 (1H, d, J=7.7 Hz), 6.89 (1H, t, J=7.7 Hz), 7.04 (1H, d, J=7.7 Hz), 7.16 (1H, s). 13C-NMR (C6D6, 60°C) δ: 14.0, 22.9, 27.8, 30.1, 32.9, 40.0, 46.2, 50.9, 65.4, 77.7, 118.9, 126.4, 127.1, 129.9, 134.7, 144.3, 170.5. IR (film) cm−1: 1454, 1643, 2957. MS (ESI+) m/z: 323 (M+H+, base peak). HR-MS (ESI+) m/z: Found 323.1902 (Calcd for C18H28ClN2O (M+H+) 323.1890).

trans-3-(2-Chlorophenyl)-4-N,N-dimethyl-1-N-pentanoylpiperidine (trans-4g)

Prepared According to Procedure for the Preparation of cis-4e

Yield: 12% over three steps. Light yellow oil. 1H-NMR (C6D6, 80°C) δ: 0.89 (3H, t, J=7.4 Hz), 1.24–1.40 (5H, m), 1.52 (1H, ddd, J=12.9, 6.4, 2.9 Hz), 1.70–1.78 (2H, m), 1.97 (6H, s), 2.14–2.22 (1H, m), 2.27–2.36 (2H, m), 2.49 (1H, m), 2.71 (1H, dt, J=11.1, 3.5 Hz), 3.32 (1H, dt, J=10.9, 4.2 Hz), 6.79 (1H, dt, J=7.6, 1.7 Hz), 6.96 (1H, dt, J=7.6, 1.2 Hz), 7.02 (1H, dd, J=7.9, 1.7 Hz), 7.20 (1H, dd, J=7.9, 1.2 Hz). 13C-NMR (C6D6, 60°C) δ: 14.0, 22.9, 27.8, 30.1, 33.0, 40.1, 44.0, 51.6, 64.5, 77.7, 126.9, 127.9, 128.9, 130.0, 134.6, 138.9, 170.6. IR (film) cm−1: 1035, 1438, 1644, 2931. MS (ESI+) m/z: 323 (M+H+, base peak). HR-MS (ESI+) m/z: Found 323.1934 (Calcd for C18H28ClN2O (M+H+) 323.1890).

Acetic Acid-Induced Abdominal Contraction Assay (Writhing Test)1,2)

Evaluation for antinociceptive effects were carried out by acetic acid writhing test using male ICR mice (Tokyo Laboratory Animals Science, Tokyo, Japan) weighing approximately 25–35 g (5–6 weeks old). Mice had free access to food and water in an animal room that was maintained at 24±1°C with a 12 h light–dark cycle (lights on 8:00 a.m.). Each mouse was injected i.p. with 0.7% acetic acid in a volume of 10 mL/kg 30 min after administration of the test drug dissolved in saline. After 10 min mice were observed for an additional 10 min during which abdominal contractions were counted. The % antinociception was calculated from the mean number of contractions in each test group and control group (% antinociception=[(mean control responses−test responses)/(mean control responses)]×100). ED50 values for each compound were determined by linear regression techniques. The statistical significance of differences between groups was assessed with analysis of variance followed by the Bonferroni–Dunnett test.

Acknowledgment

This work was MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2014–2018 (Grant No. S1411019).

Conflict of Interest

The authors declare no conflict of interest.

References
 
© 2016 The Pharmaceutical Society of Japan
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