410 Chem. Pharm. Bull. 64, 410–419 (2016) Vol. 64, No. 5 Regular Article

Design and Synthesis of a Piperidinone Scaffold as an through Kappa- Receptor: Structure–Activity Relationship Study of Matrine

Hiroyoshi Teramoto, Takayasu Yamauchi,* Yasushi Terado, Sanae Odagiri, Shigeru Sasaki, and Kimio Higashiyama Institute of Medicinal Chemistry, Hoshi University; Ebara, Shinagawa, Tokyo 142–8501, Japan. Received November 30, 2015; accepted February 8, 2016

The matrine-type 4-dimethylamino-1-pentanoylpiperidine (3a) has an antinociceptive effect through its impact on the κ- (KOR). Derivatives of 3a were synthesized by altering its amide and tertiary amine groups, and were evaluated for their antinociceptive effects. The results indicated that the distance between these groups on 3a was optimal for the antinociceptive effect. The effects obtained with compounds 8 and 9 indicated that the relative configuration of the 3- and 4-substituents influenced the effect mediated through the KOR. Key words antinociception; kappa opioid receptor (KOR); matrine; piperidone; acetic acid-induced abdominal contraction test; mouse

Narcotic such as are administered for studies for 1 and 2 and their antinociceptive activity using pain relief to cancer patients. Most analgesics are modifications of the A–D ring systems. The SAR studies µ-opioid receptor (MOR) agonists and have adverse effects showed that the amide group, tertiary amine group, and C- such as addiction,1) respiratory depression,2) and constipa- ring of 1 and 2 were important in determining their activity. tion.3,4) Although stimulation of the κ-opioid receptor (KOR) 4-Dimethylamino-1-pentanoylpiperidine (3) was identified as results in significant analgesia, KOR agonists do not suffer a lead compound through the antinociceptive effect of 1.12–14) from the same adverse effects as MOR agonists. Many KOR We hypothesized that the level of the antinociceptive effect agonists, including ethylketocyclazocine, U-50,488H, and nal- would be affected by the distance between the amide and furafine (TRK-820), have been developed and investigated for amino groups, which are important structural components their analgesic, anti-inflammatory and antipruritic activity5,6) of compound 3. To investigate this, model compounds (4–6) (Fig. 1). However, these agonists suffer from dose-limiting were designed to study the effect of the spatial relationship , sedation, and effects.7,8) Conse- between the amide and amine groups on the agonistic activity quently, the development of a KOR agonist that does not cause (Fig. 2). We also identified that compound 7, 3-Bn analogue of adverse effects is important. compound 3 with a trans-configuration, has antinociceptive We previously reported that (+)-matrine (1) and (+)-allo- effects through KOR which exhibited higher antinociceptive matrine (2), typical matrine-type lupine alkaloids produced by Sophora Leguminosae, have antinociceptive properties identi- cal to those of .9) The effects of 1 were mediated mainly through activation of the KOR and partially through the MOR, and those of 2 were mediated only through the KOR.10) Furthermore, we found that neither 1 nor 2 provided the activation in the guanosine-5′-O-(3-[35S] thio) trisphosphate ([35S]GTP γS) binding assay with the membranes of spinal cord, indicating that the supraspinal antinociceptive actions induced by 1 and 2 were not caused by direct stimulation of the KOR.11) Although intracerebroventricular pretreatment with an antiserum against A (1–17) did not af- fect the antinociceptive effect induced by subcutaneous (s.c.) treatment of 1 and 2, the antinociceptive effect was greatly attenuated by intrathecal (i.t.) pretreatment with an antiserum against (1–17) in mice. This suggested that the antinociceptive effect induced by s.c. treatment of 1 and 2 occurred without binding to the KOR in the ventricles of the brain, in where might stimulate the descending dynorphiner- gic neuron and production of dynorphin in the spinal cord. Because the pharmacological mechanism of action and chemical structures of 1 and 2 differ from conventional KOR Fig. 1. Structure of Conventional κ-Opioid Receptor Agonists and agonists, we performed structure–activity relationship (SAR) (+)-Matrine Derivatives

* To whom correspondence should be addressed. e-mail: [email protected] © 2016 The Pharmaceutical Society of Japan Vol. 64, No. 5 (2016) Chem. Pharm. Bull. 411

activity than compound 3.15) Because compound 7 exists as a rotamer by amide group, and we anticipated that the anti- nociceptive effects could be different from the rotamers. To investigate this, we designed and synthesized compounds 8 and 9 with the carbonyl group of the amide incorporated into piperidine. A SAR study was carried out to clarify the effect of the positional relationships between the benzyl and car- bonyl group for the rotamers. The antinociceptive effects of the analogues of compound 3 (4–6, 8, 9) were evaluated using acetic acid-induced abdominal contraction tests in mice.

Synthesis The synthetic routes for compounds 4a and b–6a and b are shown in Chart 1. Compounds 4a and b were prepared with pentanoyl chloride or benzoyl chloride from 1-methylpi- Fig. 2. Structure of Lead Compound 3 Derivatives Converted Amine peridine (10). The syntheses of 5a and b began with N-ben- and Amide Position zylation of commercially available 4-carboxamidopiperidine

Reagents and reaction conditions: (a) RCOCl, DMAP, Et3N, CH2Cl2, 0°C, 94–97%; (b) BnBr, NaHCO3, toluene, reflux, 57%; (c) LiAlH4, Et2O, reflux; (d) HCHO, HCO2H, 100°C, two steps, 83%; (e) Pd(OH)2, H2, EtOH, rt; (f) RCOCl, Et3N, CH2Cl2, rt, two steps, 72–91%; (g) (EtO)2POCH2CN, K2CO3, THF, reflux, 94%; (h) NaBH4, MeOH, Pyridine, reflux, 79%; (i) LiAlH4, THF, 0°C; (j) HCHO, HCO2H, 100°C, two steps, 61%. Chart 1

Reagents and reaction conditions: (a) PhCHO, piperidine, benzene, reflux; (b) Pd–C, H2, MeOH, rt; (c) HCl aq, EtOAc, rt, three steps, 99%; (d) Me2NH, Ti(Oi-Pr)4, THF, reflux, then NaCNBH4, AcOH, rt, 72%; (e) 1-iodopentane, t-BuOK, THF, rt, 74% (dr=2 : 1); (f) t-BuOK, THF, 0°C, 42%. Chart 2 412 Chem. Pharm. Bull. Vol. 64, No. 5 (2016)

Reagents and reaction conditions: (a) Me2NH, benzene, reflux, 92%; (b) PtO2, H2, MeOH, rt, 94%; (c) 1-bromopentane, KOH, DMSO, rt, 66%; (d) L-selectride, toluene, −78°C, 99%; (e) DIAD, DPPA, Ph3P, THF, 0°C to rt; (f) Pd–C, H2, HCHO, MeOH, two steps, 69%; (g) 1-iodopentane, t-BuOK, THF, rt, 59%. Chart 3

(11). Amide 12 was converted to the amine by reduction with 16) LiAlH4, and treated with HCHO and HCO2H to yield 13. After debenzylation using Pd(OH)2, acylation of the second- ary amine gave the target compounds 5a and b. Compounds 6a and b were synthesized from 1-benzyl-4-piperidone (14) using the Horner–Wadsworth–Emmons reaction to give olefin 17) 15. Reduction of 15 using NaBH4, followed by reduction of the nitrile and N,N-dimethylation, afforded tertiary amine 18. After debenzylation, acylation of the secondary amine gave the target compounds 6a and b. The synthetic routes for cis- and trans-8a and b are shown in Chart 2. The β-keto lactam 19 was prepared from β-alanine according to an established procedure.18,19) Condensation of 19 with benzaldehyde, followed by hydrogenation with Pd–C and treatment under acidic conditions gave the 3-benzyl derivative (20).20) Reductive amination of 20 using dimethylamine and

NaCNBH3 afforded cis-8a as a single diastereomer. Treatment Fig. 3. ORTEP Drawing of cis-9a of cis-8a with potassium tert-butoxide produced the diaste- reomers cis-8b and trans-8b, which could be separated. The tion by Pd(OH)2 in the presence of HCHO, gave (−)-trans-8a. relative configurations of these diastereomers were assigned Compound (−)-trans-8b was obtained by N-pentylation25) be- using coupling constants from 1H-NMR data. The results sug- fore debenzylation. gested that cis-8a could be epimerized to trans-8a under basic Biological Assay The antinociceptive effects of com- conditions. Therefore, epimerization of cis-8a to trans-8a was pounds 4a, b–6a and b, 8a and b, and 9a and b were evaluat- attempted using potassium tert-butoxide. ed in acetic acid-induced abdominal contraction assays (writh- Chart 3 shows the synthetic routes used to prepare cis- and ing tests). The results for 4a and b–6a and b are shown in Fig. trans-9a and b. Ketone 21 was prepared from 19 according 4A. Compared with 3a and b, the effects of 5a and b and 6a an established procedure.19) To prepare cis-9a, enaminone 22 and b were greatly attenuated, while the effects of 4a and b was obtained from ketone 21 and then reduced with PtO2. were similar to those of 3a and b. To investigate if the effects The relative configuration of cis-9a was confirmed by X-ray of 3a and b and 4a and b were mediated through the KOR, crystallography (Fig. 3). To prepare trans-9a and b, ketone 21 we attempted to antagonize these effects by pretreatment with was stereoselectively reduced with L-selectride to yield alcohol the KOR antagonist (norBNI) (Fig. 4B). 23,21) which was submitted to a Mitsunobu reaction using di- Pretreatment with norBNI resulted in a large dampening of phenylphosphoryl azide (DPPA), diisopropyl azodicarboxylate the effect of 3a, but the effect of 4a and b was not dampened. (DIAD), and triphenylphosphine (TPP).22) The prepared cis-9a These results show that the distance between the amide and and trans-9a were converted to cis-9b and trans-9b, respec- amine groups is important for antinociceptive effects through tively, by N-pentylation. the KOR, which is similar effects to what was found for 1 and In order to investigate the antinociception between racemic 2.10) and chiral compounds, (−)-trans-8a and b were synthesized The results for cis- and trans-8a and b, 9a and b are shown by alternative plan as shown in Chart 4. Diastereoselective in Fig. 5A. Compounds cis-8a, trans-8a and trans-9a and b 23,24) benzylation of 24, which was derived from L-aspartic showed high antinociceptive effects compared with all the acid, provided 25 with a 3,4-trans configuration. Deprotection other compounds tested. Since trans-9a showed the highest of the N-Boc group under acidic conditions, and hydrogena- activity, it was selected to investigate the selectivity of opioid Vol. 64, No. 5 (2016) Chem. Pharm. Bull. 413

Reagents and reaction conditions: (a) BnBr, LDA, THF, −78°C to rt, 52%; (b) HCl aq, EtOAc, rt, 78%; (c) Pd(OH)2, H2, HCHO, MeOH, rt, 58–71%; (d) 1-bromopentane, KOH, DMSO, rt, 66%. Chart 4

Fig. 4. The Antinociceptive Effects of s.c. Administration of Compounds 3a and b–6a and b (30 mg/kg, s.c.) in the Writhing Test in Mice 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. Each column represents the mean±S.E.M. of 10 mice in each group. (A) ** p<0.01 versus the compound 3a. ## p<0.01, ### p<0.001 versus the compound 3b. (B) The blockage of antinociceptive effects of 3a and b and 4a and b (100 mg/kg, s.c.) by pretreatment of KOR antagonist nor-BNI (3.2 mg/kg, s.c.) at 4 h before administration test drug. ** p<0.01, *** p<0.001 versus the compounds 3a and b alone. 414 Chem. Pharm. Bull. Vol. 64, No. 5 (2016)

Fig. 5. (A) The Antinociceptive Effects of s.c. Administration of Compounds 8 and 9 (30 mg/kg) in the Writhing Test in Mice; (B) The Blockage of Antinociceptive Effects of trans-9a (30 mg/kg, s.c.) by Pretreatment of KOR Antagonist nor-BNI (3.2 mg/kg, s.c.) at 4 h before Administration Test Drug; (C) The Antinociceptive Effects of Racemic and Optical Active trans-8a and b (30 mg/kg, s.c.) (A) Each column represents the mean±S.E.M. of 10 mice in each group. (B) *** p<0.001 versus the compound trans-9a alone. receptor by a writhing test. Its effect was evidently dampened Experimental with norBNI pretreatment (Fig. 5B). Comparison of the effects Chemistry 1 H- (400 MHz) and 13C-NMR (100 MHz) of these compounds indicated that the relative configuration spectra were obtained on a Bruker AVIII-400 instrument, of the dimethylamino and benzyl groups was more important and chemical shifts are reported in ppm on the δ-scale from than the positional relationship between the carbonyl group internal tetramethylsilane. MS spectra were measured with a on the amide and the benzyl group in determining the anti- JEOL JMS D-600 and JMS T-100LP spectrometer by using nociceptive effect. Therefore, the structural differences by the the chemical ionization (CI) with isobutene, the electron im- rotamers might not be as large as we expected. pact (EI) methods, and electrospray ionization (ESI) methods. Evaluation of the antinociceptive effect of (−)-trans-8a and All melting points were measured with a Yanagimoto Micro b showed it had a similar or inferior activity compared with melting point apparatus without collection. IR spectra were re- the racemic mixture of (±)-trans-8a and b (Fig. 5C). There- corded on Perkin-Elmer Spectrum Two. Optical rotation were fore, the (+)-enantiomer would probably have a similar antino- taken with a JASCO-DIP-370 polarimeter at room temperature ciceptive effect to (−)-trans-8a and b. (rt). Column chromatography was performed on Silica gel 60 In summary, the antinociceptive properties through the (100–210 µm, Kanto Chemical Co., Inc.). The X-ray diffraction KOR depended on the distance between the amide and amine analysis were carried out on Rigaku RAXIS RAPID. group. The relative configurations of the dimethylamino and 4-N-Methyl-1-N-pentanoylpiperazine (4a) benzyl groups in the (+)-matrine derivatives was important Pentanoyl chloride (3.61 g, 30.0 mmol), Et3N (6.06 g, for high antinociceptive activity. These results will be useful 59.9 mmol) and N,N-dimethyl-4-aminopyridine (DMAP) for development of analgesics through KOR. (0.24 g, 1.99 mmol) were added to a solution of 10 (2.00 g,

20.0 mmol) in CH2Cl2 (19 mL) at 0°C under the nitrogen at- mosphere. The reaction mixture was stirred for 2 h, poured Vol. 64, No. 5 (2016) Chem. Pharm. Bull. 415 on 10% aqueous NaOH and extracted with CH2Cl2. The com- rated. The resulting residue was used in the subsequent reac- bined organic layer was dried over anhydrous Na2SO4. After tion without further purification. Et3N (0.67 mL, 4.83 mmol) filtration, the solvent was evaporated, and the residue was and pentanoyl chloride (0.29 mL, 2.43 mmol) were added to a purified by chromatography on SiO2 (CH2Cl2–MeOH=12 : 1) solution of the residue in CH2Cl2 (6 mL) at 0°C. After being to give 4a (3.47 g, 18.8 mmol, 94%) as a light yellow oil. stirred at rt for 10 h, the reaction mixture was alkalized with 1 H-NMR (CDCl3) δ: 0.93 (3H, t, J=7.3 Hz), 1.37 (2H, sext, 10% aqueous NaOH, and extracted with CH2Cl2. The or- J=7.3 Hz), 1.55–1.67 (2H, m), 2.29–2.40 (6H, m), 2.30 (3H, ganic layer was dried over anhydrous Na2SO4 and evaporated. 13 s), 3.48 (2H, t, J=5.0 Hz), 3.63 (2H, t, J=5.0 Hz). C-NMR The result residue was purified by chromatography on SiO2 (CDCl3) δ: 12.9, 21.5, 26.4, 31.9, 40.4, 44.5, 45.0, 53.8, 54.3, (CH2Cl2–MeOH–28% NH4OH=80 : 9 : 1) to yield 5a (317 mg, −1 1 170.3. IR (film) cm : 1640. High resolution (HR)-MS (EI) 1.40 mmol, 86%) as a light yellow oil. H-NMR (CDCl3) δ: + m/z: Found 184.1598 (Calcd for C10H20N2O (M ) 184.1575). 0.93 (3H, t, J=7.3 Hz), 0.98–1.18 (2H, m), 1.29–1.43 (2H, m), 1-N-Benzoyl-4-N-methylpiperazine (4b) 1.54–1.96 (5H, m), 2.10 (2H, d, J=6.6 Hz), 2.20 (6H, s), 2.32 Prepared according to procedure for the preparation of (2H, t, J=7.6 Hz), 2.54 (1H, dt, J=13.2, 2.9 Hz), 2.99 (1H, dt, 1 4a, 97% yield; H-NMR (CDCl3) δ: 2.33 (3H, s), 2.32–2.46 J=13.2, 2.9 Hz), 3.85 (1H, br d, J=13.2 Hz), 4.62 (1H, br d, 13 13 (4H, br), 3.43 (2H, br), 3.79 (2H, br), 7.40 (5H, br). C-NMR J=13.2 Hz). C-NMR (CDCl3) δ: 12.9, 21.5, 26.5, 29.4, 30.3, −1 (CDCl3) δ: 41.3, 45.3, 46.9, 54.2, 126.6, 128.0, 128.9, 135.1, 32.0, 33.3, 40.6, 44.7, 44.9, 64.7, 170.1. IR (film) cm : 1630. −1 + 169.4. IR (film) cm : 1620. HR-MS (EI) m/z: Found 204.1253 HR-MS (EI) m/z: Found 226.2027 (Calcd for C13H26N2O (M ) + (Calcd for C12H16N2O (M ) 204.1262). 226.2045). 1-N-Benzyl-4-piperidinecarboxamide (12) 1-N-Benzoyl-4-(N,N-dimethylaminomethyl)piperidine (5b) Benzyl bromide (20.4 mL, 23.5 g, 172 mmol) was added to Prepared according to procedure for the preparation of 5a, 1 a suspension of 11 (20.0 g, 156 mmol) and NaHCO3 (23.5 g, 91% yield; H-NMR (C6D6, 80°C) δ: 0.97 (2H, dq, J=13.0, 280 mmol) in toluene (320 mL). After being stirred at reflux for 4.3 Hz), 1.31–1.45 (1H, m), 1.51 (2H, br d, J=13.0 Hz), 1.89 5 h, the reaction mixture was filtrated. The solid was recrystal- (2H, d, J=7.1 Hz), 2.04 (6H, s), 2.61 (2H, dt, J=12.6, 2.8 Hz), 13 lized with n-hexane–EtOAc to yield 12 (19.3 g, 88.4 mmol, 4.16 (2H, br d, J=12.6 Hz), 7.04–7.42 (5H, m). C-NMR (C6D6, 1 57%) as a white solid. H-NMR (CD3OD) δ: 1.62–1.80 (4H, 80°C) δ: 31.1, 34.9, 45.1, 45.9, 65.9, 127.4, 128.4, 129.3, 137.7, m), 2.03 (2H, dt, J=12.0, 4.0 Hz), 2.20 (1H, m), 2.93 (2H, br), 169.7. IR (film) cm−1: 1600, 1625. HR-MS (EI) m/z: Found 13 + 3.51 (2H, s), 7.23–7.33 (5H, m). C-NMR (CD3OD) δ: 29.5, 246.1702 (Calcd for C15H22N2O (M ) 246.1732). 43.4, 53.9, 64.1, 128.4, 129.3, 130.7, 138.3, 180.8. IR (KBr) (1-N-Benzylpiperidin-4-ylidene)acetonitrile (15) cm−1: 1590, 1640. HR-MS (EI) m/z: Found 218.1401 (Calcd for Potassium carbonate (3.65 g, 26.4 mmol) was added to + C13H18N2O (M ) 218.1419). a solution of diethyl cyanomethanephosphonate (4.97 mL, 1-N-Benzyl-4-(N,N-dimethylaminomethyl)piperidine (13) 31.7 mmol) in tetrahydrofuran (THF) (6 mL) under nitrogen

To a stirred suspension of LiAlH4 (630 mg, 16.5 mmol) in atmosphere. The reaction mixture was stirred at rt for 15 min, Et2O (25 mL) was added dropwise 12 (2.40 g, 11.0 mmol) in warmed to reflux for 20 min. After being cooled to rt, 14 Et2O (10 mL) at rt under nitrogen atmosphere. The reaction (5.00 g, 26.4 mmol) was added and refluxed for 12 h. The reac- mixture was stirred at reflux for 8 h, and 10% aqueous NaOH tion mixture was alkalized with 10% aqueous potassium car- was added. After filtration through Celite pad, the aqueous bonate and extracted with ethyl acetate. The combined organic layer was extracted with Et2O, and the combined organic layer was dried over anhydrous Na2SO4 and evaporated. The layer was dried over anhydrous Na2SO4 and the solvent was resulting residue was purified by chromatography on SiO2 (n- evaporated. The resulting residue was used in the subsequent hexane–EtOAc=1 : 1) to yield 15 (5.18 g, 24.4 mmol, 94%) as a 1 reaction without further purification. Ninety percent formic white solid. mp 85°C. H-NMR (CDCl3) δ: 2.37–2.41 (2H, m), acid (5.06 g, 110 mmol) and 36% formaldehyde (4.59 mL, 2.51–2.66 (6H, m), 3.56 (2H, s), 5.11 (1H, s), 7.26–7.38 (5H, m). 13 1.65 g, 55.0 mmol) were added to the residue. After being C-NMR (CDCl3) δ: 32.5, 34.9, 53.5, 62.1, 92.9, 116.5, 127.1, stirred at 100°C for 14 h, the reaction mixture was evaporated 128.2, 137.9, 164.9. IR (KBr) cm−1: 1600, 1630, 2220. HR-MS + to remove formic acid and formaldehyde, alkalized with 10% (EI) m/z: Found 212.1343 (Calcd for C14H16N2 (M ) 212.1313). aqueous NaOH and extracted with CH2Cl2. The combined (1-N-Benzylpiperidin-4-yl)acetonitrile (16) organic layer was dried over anhydrous Na2SO4 and evapo- Sodium borohydride (236 mg, 6.25 mmol) was added to a rated. The residue was purified by chromatography on SiO2 solution of 15 (1.06 g, 5.00 mmol) in pyridine–MeOH (10 mL; (CH2Cl2–MeOH–28% NH4OH=60 : 9 : 1) to yield 13 (2.26 g, 3 : 1) and heated at 120°C for 2 h. After being cooled to rt, 1 9.17 mmol, 83%) as a light yellow oil. H-NMR (CDCl3) δ: H2O was added and the solvent was removed, then the residue 1.22 (2H, ddt, J=12.7, 11.5, 3.6 Hz), 1.38–1.52 (1H, m), 1.70 was filtered with Et2O. After being alkalized with 10% aque- (2H, dm, J=12.7 Hz), 1.94 (2H, dt, J=11.5, 2.3 Hz), 2.10 (2H, d, ous NaOH, the reaction mixture was filtrated and extracted

J=7.1 Hz), 2.18 (6H, s), 2.88 (2H, br d, J=11.5 Hz), 3.49 (2H, s), with Et2O. The combined organic layer was dried over an- 13 7.20–7.32 (5H, m). C-NMR (CDCl3) δ: 30.3, 33.4, 45.4, 53.1, hydrous Na2SO4, and evaporated. The resulting residue was −1 62.9, 65.7, 126.2, 127.5, 128.4, 138.0. IR (film) cm : 1630. purified by chromatography on SiO2 (n-hexane–EtOAc=1 : 3) + HR-MS (EI) m/z: Found 232.1934 (Calcd for C15H24N2 (M ) to yield 16 (0.85 g, 3.95 mmol, 79%) as a light yellow oil. 1 232.1939). H-NMR (CDCl3) δ: 1.43 (2H, dt, J=11.7, 3.4 Hz), 1.57–1.78 4-(N,N-Dimethylaminomethyl)-1-N-pentanoylpiperidine (5a) (3H, m), 1.99 (2H, dt, J=11.7, 2.3 Hz), 2.28 (2H, d, J=6.4 Hz),

Pd(OH)2 (80 mg, 20% w/w) was added to a solution of 13 2.91 (2H, br d, J=11.7 Hz), 3.50 (2H, s), 7.24–7.35 (5H, m). 13 (400 mg, 1.62 mmol) in EtOH (6 mL). The reaction mixture C-NMR (CDCl3) δ: 23.5, 31.2, 32.7, 52.6, 62.7, 118.2, 126.6, was stirred at rt under hydrogen atmosphere (1 atm) for 24 h. 127.8, 128.6, 138.0. IR (film) cm−1: 1630, 2250. HR-MS (EI) + The suspension was filtered off, and the solvent was evapo- m/z: Found 214.1492 (Calcd for C14H18N2 (M ) 214.1470). 416 Chem. Pharm. Bull. Vol. 64, No. 5 (2016)

1-N-Benzyl-4-(2-N,N-dimethylaminoethyl)piperidine (18) (CDCl3) δ: 31.4, 36.2, 38.5, 59.1, 126.8, 128.5, 128.6, 129.6, −1 To a stirred suspension of LiAlH4 (2.66 g, 70.1 mmol) in 139.0, 171.1, 204.9. IR (KBr) cm : 1480, 1590, 1660, 3370. THF (40 mL) was added dropwise 16 (10.0 g, 46.7 mmol) at MS (ESI+) m/z: 204 (M+H+, base peak). HR-MS (ESI+) m/z: + 0°C under nitrogen atmosphere and stirred at 0°C for 2 h. Found 204.0982 (Calcd for C12H14NO2 (M+H ) 204.1025). After being quenched with 10% aqueous NaOH, the reaction cis-3-Benzyl-4-N,N-dimethylaminopiperidin-2-one (cis-8a) mixture was filtrated with EtOAc. The combined organic layer Dimethylamine (4.50 mL, 9.00 mmol, 2 M in MeOH) and was dried over anhydrous Na2SO4 and evaporated to give titanium tetraisopropoxide (0.68 mL, 2.30 mmol) were added crude product. The resulting residue was used in the subse- to a solution of 21 (203 mg, 1.00 mmol) in THF (2 mL). quent reaction without further purification. 90% Formic acid After being stirred at reflux for 18 h, acetic acid (0.90 mL, (21.5 g, 467 mmol) and 36% formaldehyde (19.5 mL, 233 mmol) 15.8 mmol) and sodium cyanoborohydride (189 mg, 3.00 mmol) was added to the crude, and the reaction mixture was stirred were added at rt. After being stirred for 1 h, the reaction at 100°C for 14 h. After being evaporated, the reaction mixture mixture was alkalized with 1 M aqueous NaOH and filtered. was alkalized with 10% NaOH and extracted with CH2Cl2. The filtrate was extracted with CHCl3 and evaporated. The The combined organic layer was dried over anhydrous Na2SO4 resulting residue was purified by chromatography on SiO2 and evaporated. The resulting residue was purified by chro- (CHCl3–MeOH=20 : 1) to yield cis-8a (166 mg, 0.72 mmol, 1 matography on SiO2 (CHCl3–MeOH–28% NH4OH=60 : 9 : 1) 72%) as a light yellow solid. mp 103–104°C. H-NMR (CDCl3) to yield 18 (6.95 g, 28.2 mmol, 61%) as a light yellow oil. δ: 1.74–1.89 (2H, m), 2.26 (6H, s), 2.61 (1H, dt, J=8.9, 6.2 Hz), 1 H-NMR (CDCl3) δ: 1.22–1.43 (5H, m), 1.62–1.66 (2H, dm, 2.73 (1H, q, J=6.2 Hz), 2.98 (1H, dd, J=13.7, 6.2 Hz), 3.12 (1H, J=9.2 Hz), 1.89–1.98 (2H, m), 2.20 (6H, s), 2.23 (2H, t, dd, J=13.7, 6.2 Hz), 3.23 (1H, dddd, J=12.3, 10.5, 5.8, 1.7 Hz), J=7.6 Hz), 2.85 (2H, br d, J=11.5 Hz), 3.47 (2H, s), 7.19–7.31 3.29–3.35 (1H, m), 5.88 (1H, br), 7.16–7.20 (1H, m), 7.25–7.32 13 13 (5H, m). C-NMR (CDCl3) δ: 32.0, 33.4, 34.1, 45.1, 53.3, 56.8, (4H, m). C-NMR (CDCl3) δ: 21.6, 32.8, 40.2, 42.9, 47.2, 61.1, 63.0, 126.3, 127.5, 128.5, 138.2. IR (film) cm−1: 1630. HR-MS 126.6, 128.8, 130.3, 141.8, 176.2. IR (KBr) cm−1: 1460, 1630, + + (EI) m/z: Found 246.2081 (Calcd for C16H26N2 (M ) 246.2096). 1670, 3290. MS (ESI+) m/z: 233 (M+H , base peak). HR-MS + 4-(2-N,N-Dimethylaminoethyl)-1-N-pentanoylpiperidine (6a) (ESI+) m/z: Found 233.1611 (Calcd for C14H21N2O (M+H ) Prepared according to procedure for the preparation of 5a, 233.1654). 1 72% yield; H-NMR (C6D6, 80°C) δ: 0.93 (3H, t, J=7.3 Hz), cis- and trans-3-Benzyl-4-N,N-dimethylamino-1-N- 1.01–1.19 (2H, m), 1.25–1.45 (4H, m), 1.48–1.77 (5H, m), 2.21 pentylpiperidin-2-one (cis-8b and trans-8b) (6H, s), 2.25–2.34 (4H, m), 2.53 (1H, dt, J=13.2, 2.9 Hz), 2.99 1-Iodopentane (171 mg, 0.86 mmol) and t-BuOK (0.72 mL, (1H, dt, J=13.2, 2.9 Hz), 3.83 (1H, br d, J=13.2 Hz), 4.60 (1H, 0.72 mmol, 1 M in THF) were added to a solution of cis-8a 13 br d, J=13.2 Hz). C-NMR (C6D6, 80°C) δ: 12.7, 21.3, 26.2, (166 mg, 0.72 mmol) in THF (2 mL). After being stirred at 30.8, 31.6, 31.7, 32.8, 33.0, 40.5, 44.2, 44.6, 55.6, 169.6. IR rt for 30 min, the reaction mixture was quenched with brine −1 (film) cm : 1620. HR-MS (EI) m/z: Found 240.2188 (Calcd for and extracted with CHCl3. The combined organic layer was + C14H28N2O (M ) 240.2201). dried over anhydrous Na2SO4 and evaporated. The resulting 1-N-Benzoyl-4-(2-N,N-dimethylaminoethyl)piperidine (6b) residue was purified by chromatography on SiO2 (CHCl3– Prepared according to procedure for the preparation of MeOH=30 : 1) to yield cis-8b (93 mg, 0.32 mmol, 45%) and 1 5a, 76% yield; H-NMR (C6D6, 80°C) δ: 0.90–1.03 (2H, m), trans-8b (61 mg, 0.21 mmol, 29%) perspective as a light yel- 1 1.18–1.38 (5H, m), 2.09 (2H, t, J=8.0 Hz), 2.08 (6H, s), 2.56 low oil. cis-8b: H-NMR (CDCl3) δ: 0.90 (3H, t, J=7.3 Hz), (2H, dt, J=13.2, 2.9 Hz), 4.14 (2H, br), 7.08–7.16 (5H, m). 1.24–1.37 (4H, m), 1.52 (2H, quin, J=7.3 Hz), 1.72–1.89 13 C-NMR (C6D6, 80°C) δ: 32.8, 34.5, 34.6, 45.3, 45.5, 57.3, (2H, m), 2.22 (6H, s), 2.58 (1H, dt, J=9.1, 6.2 Hz), 2.74 (1H, 127.5, 128.3, 129.2, 137.8, 169.6. IR (film) cm−1: 1580, 1625. q, J=6.2 Hz), 2.96 (1H, dd, J=13.7, 6.2 Hz), 3.10 (1H, dd, + HR-MS (EI) m/z: Found 260.1904 (Calcd for C16H24N2O (M ) J=13.7, 6.2 Hz), 3.17–3.26 (3H, m), 3.45 (1H, dt, J=13.4, 260.1889). 7.6 Hz), 7.15–7.19 (1H, m), 7.23–7.27 (2H, m), 7.30–7.32 (2H, 13 3-Benzylpiperidine-2,4-dione (21) m). C-NMR (CDCl3) δ: 13.9, 19.8, 22.3, 26.6, 28.9, 34.1, Benzaldehyde (2.00 mL, 19.6 mmol) and piperidine (2.00 mL, 40.4, 45.0, 46.6, 47.3, 59.3, 125.8, 127.8, 130.0, 139.6, 170.8. 20.2 mmol) were added to a solution of 20 (4.00 g, 18.8 mmol) IR (film) cm−1: 1450, 1640. MS (ESI+) m/z: 303 (M+H+, in benzene (50 mL). After being stirred at reflux for 4 h, the re- base peak). HR-MS (ESI+) m/z: Found 303.2460 (Calcd for + 1 action mixture was cooled until rt and evaporated. The residue C19H31N2O (M+H ) 303.2436). trans-8b: H-NMR (CDCl3) was dissolved in MeOH (30 mL) and Pd–C (1.0 g, 10% (w/w)) δ: 0.88 (3H, t, J=7.2 Hz), 1.17–1.23 (2H, m), 1.27–1.34 (2H, was added. After being stirred under hydrogen atmosphere m), 1.42–1.51 (2H, m), 1.59 (1H, ddt, J=13.1, 10.4, 4.9 Hz), (4 atm) at rt for 20 h, the reaction mixture was filtrated and 1.84–1.90 (1H, m), 2.22 (6H, s), 2.36–2.42 (1H, m), 2.69 (1H, concentrated to give brown oil. Six molar HCl (50 mL) was dt, J=9.1, 5.0 Hz), 2.90–2.97 (1H, m), 3.05–3.19 (3H, m), 3.32 added to a solution of the residual oil in EtOAc (50 mL). After (1H, dd, J=13.2, 5.0 Hz), 3.54 (1H, ddd, J=13.2, 8.5, 6.7 Hz), 13 being stirred at rt for 3 h, the reaction mixture was evapo- 7.15–7.19 (1H, m), 7.21–7.27 (4H, m). C-NMR (CDCl3) δ: rated, alkalized with NaHCO3 aq, and extracted with CHCl3. 14.0, 20.0, 22.5, 26.8, 29.0, 34.3, 40.6, 45.2, 46.7, 47.4, 59.5, −1 The combined organic layer was dried over anhydrous Na2SO4 125.9, 127.9, 130.1, 139.8, 171.0. IR (film) cm : 1600, 1640. and evaporated. The resulting residue was purified by chroma- MS (EI) m/z: 302 (M+, base peak). HR-MS (EI) m/z: Found + tography on SiO2 (CHCl3–MeOH=20 : 1) to yield 21 (3.80 g, 302.2364 (Calcd for C19H30N2O (M ) 302.2358). 18.7 mmol, 99%) as a white solid. mp 158–160°C. 1H-NMR trans-3-Benzyl-4-N,N-dimethylaminopiperidin-2-one

(CDCl3) δ: 2.33 (1H, ddd, J=17.3, 9.3, 5.8 Hz), 2.50 (1H, dt, (trans-8a) J=17.3, 5.3 Hz), 3.03 (1H, ddd, J=13.3, 9.3, 5.3 Hz), 3.27–3.40 t-BuOK (3.18 mL, 3.18 mmol, 1 M in THF) was added (3H, m), 3.42 (1H, t, J=5.3 Hz), 7.16–7.24 (5H, m). 13C-NMR dropwise to a solution of cis-8a (368 mg, 1.59 mmol) in THF Vol. 64, No. 5 (2016) Chem. Pharm. Bull. 417

(20 mL) at rt under nitrogen atmosphere. After being stirred 2.33 (1H, dd, J=17.3, 11.3 Hz), 2.57–2.65 (2H, m), 2.68 (1H, for 21 h, the reaction mixture was quenched with H2O and dd, J=12.5, 2.6 Hz), 2.99 (1H, dd, J=13.8, 2.4 Hz), 3.23–3.37 13 extracted with CHCl3. The combined organic layer was dried (2H, m), 6.99–7.17 (5H, m). C-NMR (C6D6) δ: 14.2, 22.8, over anhydrous Na2SO4 and evaporated. The residue was 27.3, 29.4, 30.0, 34.9, 37.9, 43.0, 47.8, 47.8, 62.7, 126.3, 128.7, −1 purified by chromatography on SiO2 (CHCl3–MeOH=10 : 1) 129.4, 141.4, 167.1. IR (film) cm : 1600, 1650. MS (EI) m/z: to yield trans-8a (154 mg, 0.66 mmol, 42%) as a yellow oil. 302 (M+), 98 (base peak). HR-MS (EI) m/z: Found 302.2352 1 + H-NMR (CDCl3) δ: 1.63 (1H, ddt, J=13.2, 10.1, 4.9 Hz), (Calcd for C19H30N2O (M ) 302.2358). 1.86–1.92 (1H, m), 2.23 (6H, s), 2.39–2.45 (1H, m), 2.72 (1H, cis-5-Benzyl-4-hydroxypiperidin-2-one (24) dt, J=9.4, 5.1 Hz), 2.92–2.99 (1H, m), 3.12 (1H, dd, J=13.3, L-Selectride (3.92 mL, 3.92 mmol, 1 M in THF) was added 5.1 Hz), 3.24–3.30 (2H, m), 5.89 (1H, br), 7.17–7.29 (5H, m). dropwise to a solution of 22 (200 mg, 0.98 mmol) in toluene 13 C-NMR (CDCl3) δ: 19.5, 33.7, 39.3, 40.7, 46.4, 59.2, 126.0, (10 mL) at –78°C under nitrogen atmosphere. After being 128.1, 130.0, 139.5, 174.0. IR (film) cm−1: 1600, 1660. MS stirred for 18 h, the reaction mixture was quenched with + (EI) m/z: 232 (M ), 84 (base peak). HR-MS (EI) m/z: Found saturated aqueous NH4Cl and extracted with EtOAc. The + 232.1557 (Calcd for C14H20N2O (M ) 232.1575). combined organic layer was dried over anhydrous Na2SO4 5-Benzyl-4-N,N-dimethylamino-5,6-dihydro-1H-pyridin-2- and evaporated. The resulting residue was purified by chro- one (23) matography on SiO2 (CHCl3–MeOH–28% NH4OH=100 : 9 : 1) Dimethylamine (4.5 mL, 9.0 mmol, 2 M in MeOH) was added to yield 24 (198 mg, 0.96 mmol, 99%) as a white solid. mp 1 to a solution of 22 (305 mg, 1.50 mmol) in benzene (10 mL). 183–185°C. H-NMR (CDCl3) δ: 1.93 (1H, d, J=3.6 Hz), After being stirred at reflux for 18 h, the reaction mixture was 2.10–2.18 (1H, m), 2.48 (1H, dd, J=18.0, 3.1 Hz), 2.55 (1H, dd, evaporated. The resulting residue was purified by chroma- J=18.0, 3.8 Hz), 2.67 (1H, dd, J=13.6, 7.6 Hz), 2.82 (1H, dd, tography on SiO2 (CHCl3–MeOH=10 : 1) to yield 23 (298 mg, J=13.6, 7.7 Hz), 3.14 (1H, ddd, J=11.5, 5.3, 3.6 Hz), 3.40 (1H, 1.29 mmol, 86%) as a light yellow solid. mp 184–187°C. t, J=11.5 Hz), 4.07 (1H, br), 5.76 (1H, br), 7.19–7.26 (3H, m), 1 13 H-NMR (CDCl3) δ: 2.67–2.70 (1H, m), 2.81 (1H, dd, J=13.9, 7.30–7.34 (2H, m). C-NMR (CDCl3) δ: 35.5, 39.6, 40.3, 65.7, 4.1 Hz), 2.93 (1H, dd, J=13.9, 10.8 Hz), 2.96 (6H, s), 3.08 (1H, 126.7, 128.8, 129.1, 139.2, 170.9. IR (KBr) cm−1: 1430, 1660, ddd, J=12.3, 4.7, 1.7 Hz), 3.35 (1H, dd, J=12.3, 3.8 Hz), 4.69 3200, 3300. MS (ESI+) m/z: 206 (M+H+), 180 (base peak). 13 (1H, s), 4.98 (1H, br), 7.15–7.38 (5H, m). C-NMR (CDCl3) HR-MS (ESI+) m/z: Found 206.1168 (Calcd for C12H16NO2 δ: 35.3, 36.8, 38.9, 41.2, 87.7, 126.3, 128.4, 128.8, 138.7, 161.7, (M+H+) 206.1181). 170.3. IR (film) cm−1: 1580, 1600, 1640. MS (EI) m/z: 230 trans-5-Benzyl-4-N,N-dimethylaminopiperidin-2-one (M+, base peak). HR-MS (EI) m/z: Found 230.1410 (Calcd for (trans-9a) + C14H18N2O (M ) 230.1419). Diisopropyl carboxylate (0.58 mL, 2.92 mmol) was added cis-5-Benzyl-4-N,N-dimethylaminopiperidin-2-one (cis-9a) to a solution of triphenylphosphine (1.10 g, 4.16 mmol) in THF

PtO2 (50 mg) was added to a solution of 23 (184 mg, (30 mL) at 0°C under nitrogen atmosphere. After being stirred 0.80 mmol) in MeOH (20 mL). After being stirred at rt for 12 h for 20 min, the solution of 24 (600 mg, 2.92 mmol) in THF under hydrogen atmosphere (6.8 atm), the reaction mixture (10 mL) and diphenylphosphoryl azide (1.34 mL, 6.21 mmol) was filtrated through Celite pad and evaporated. The resulting were added dropwise. After being stirred at 0°C for 3 h, the residue was purified by chromatography on SiO2 (CHCl3– reaction mixture was warmed to rt for 30 min, quenched with MeOH–28% NH4OH=90 : 9 : 1) to yield cis-9a (174 mg, H2O, and extracted with EtOAc. The combined organic layer 1 0.75 mmol, 94%) as a yellow solid. mp 150–152°C. H-NMR was dried over anhydrous Na2SO4 and evaporated. Pd–C (C6D6) δ: 1.76–1.84 (2H, m), 1.92 (6H, s), 2.29 (1H, dd, J=17.3, (430 mg, 10% (w/w)) was added to a solution of the resulting 10.9 Hz), 2.36 (1H, dd, J=13.8, 11.4 Hz), 2.54 (1H, dd, J=17.3, residue in MeOH (30 mL). After being stirred under hydro- 6.4 Hz), 2.59 (1H, dd, J=12.7, 3.4 Hz), 2.84 (1H, ddd, J=12.7, gen atmosphere (1 atm) for 4 h, 36% formaldehyde (11.3 mL, 3.9, 2.6 Hz), 2.91 (1H, dd, J=13.8, 2.4 Hz), 6.99–7.15 (5H, m), 132 mmol) was added to the reaction mixture and it was 13 7.87 (1H, br). C-NMR (C6D6) δ: 29.7, 34.4, 37.2, 42.1, 43.0, stirred further for 18 h. Palladium catalyst was removed by 62.4, 126.2, 128.7, 129.4, 141.3, 171.6. IR (KBr) cm−1: 1660. filtration and the filtrate was evaporated. The resulting residue + MS (EI) m/z: 232 (M , base peak). HR-MS (EI) m/z: Found was purified by chromatography on SiO2 (CHCl3–MeOH–28% + 232.1568 (Calcd for C14H20N2O (M ) 232.1575). NH4OH=180 : 9 : 1) to yield trans-9a (470 mg, 2.02 mmol, 1 cis-5-Benzyl-4-N,N-dimethylamino-1-N-pentylpiperidin-2- 69%) as a white solid. mp 146–147°C. H-NMR (CDCl3) δ: one (cis-9b) 2.04–2.16 (1H, m), 2.32 (6H, s), 2.33–2.42 (2H, m), 2.49 (1H, The solution of cis-9a (122 mg, 0.52 mmol) in dimethyl dd, J=17.2, 5.5 Hz), 2.73 (1H, dt, J=9.8, 5.5 Hz), 2.89–2.94 sulfoxide (DMSO) (8 mL) was added to a suspension of pow- (1H, m), 3.14 (1H, ddd, J=12.0, 4.8, 4.0 Hz), 3.20 (1H, dd, der potassium hydroxide (56 mg, 1.00 mmol) in DMSO (3 mL) J=13.9, 3.7 Hz), 5.74 (1H, br), 7.15–7.17 (2H, m), 7.20–7.23 (1H, 13 under nitrogen atmosphere. After being stirred at rt for 5 min, m), 7.28–7.31 (2H, m). C-NMR (CDCl3) δ: 28.4, 29.1, 29.8, 1-bromopentane (0.10 mL, 0.78 mmol) was added. After being 36.4, 38.4, 40.1, 44.8, 61.4, 126.5, 128.7, 129.1, 139.6, 172.8. −1 stirred for 1 h, the reaction mixture was quenched with H2O IR (KBr) cm : 1040, 1500, 1670, 3190. MS (ESI+) m/z: 233 + and extracted with CHCl3. The combined organic layer was (M+H ), 162 (base peak). HR-MS (ESI+) m/z: Found 233.1621 + dried over anhydrous Na2SO4 and evaporated. The resulting (Calcd for C14H21N2O (M+H ) 233.1654). residue was purified by chromatography on SiO2 (CHCl3– trans-5-Benzyl-4-N,N-dimethylamino-1-N-pentylpiperidin-2- MeOH–28% NH4OH=200 : 9 : 1) to yield cis-9b (114 mg, one (trans-9b) 1 0.38 mmol, 73%) as a light yellow oil. H-NMR (C6D6) δ: Prepared according to procedure for the preparation of cis- 1 0.87 (3H, t, J=7.2 Hz), 1.11–1.47 (6H, m), 1.83–1.88 (1H, m), 8b, 59% yield; H-NMR (CDCl3) δ: 0.85 (3H, t, J=7.2 Hz), 1.93–1.96 (1H, m), 1.96 (6H, s), 2.27 (1H, dd, J=13.8, 11.8 Hz), 1.15–1.33 (4H, m), 1.38–1.46 (2H, m), 2.06–2.16 (1H, m), 2.30 418 Chem. Pharm. Bull. Vol. 64, No. 5 (2016)

(6H, s), 2.34–2.42 (2H, m), 2.50 (1H, dd, J=17.0, 5.5 Hz), 2.67 363 (base peak). HR-MS (EI) m/z: Found 454.2970 (Calcd for + (1H, dt, J=9.8, 5.5 Hz), 2.92 (1H, dd, J=12.3, 9.6 Hz), 3.07 (1H, C31H38N2O (M ) 454.2984). dd, J=12.3, 5.0 Hz), 3.14–3.21 (1H, m), 3.29 (1H, dt, J=13.4, (3R,4S)-3-Benzyl-4-N,N-dimethylamino-1-N-pentyl- 7.6 Hz), 7.17–7.24 (3H, m), 7.29–7.33 (2H, m). 13C-NMR piperidin-2-one ((−)-trans-8b)

(CDCl3) δ: 14.1, 22.5, 26.9, 29.1, 36.5, 39.0, 40.1, 47.0, 50.5, Prepared according to procedure for the preparation of cis- −1 25 61.5, 126.4, 128.6, 129.1, 139.7, 169.6. IR (film) cm : 1040, 9b, 58% yield; [α]D −67.5 (c=0.38, CHCl3). 1180, 1450, 1650. MS (ESI+) m/z: 303 (M+H+, base peak). Acetic Acid-Induced Abdominal Contraction Assay

HR-MS (ESI+) m/z: Found 303.2452 (Calcd for C19H31N2O (Writhing Test) Evaluation for antinociceptive effects were (M+H+) 303.2436). carried out by acetic acid writhing test using male ICR mice (3R,4S)-3-Benzyl-4-N,N-dibenzylaminopiperidin-2-one (27) (Tokyo Laboratory Animals Science, Tokyo, Japan) weighing Six molar HCl (5 mL) was added to a solution of 26 approximately 25–35 g (5–6 weeks old). Mice had free access (245 mg, 0.51 mmol) in EtOAc (5 mL). After being stirred at to food and water in an animal room that was maintained rt for 3 h, the reaction mixture was diluted with H2O and at 24±1°C with a 12 h light–dark cycle (lights on 8:00 a.m.). extracted with CHCl3. The combined organic layer was dried Each mice were injected i.p. with 0.7% acetic acid in a vol- over anhydrous Na2SO4 and evaporated. The resulting residue ume of 10 mL/kg 30 min after administration of the test drug was purified by chromatography on SiO2 (CHCl3–MeOH–28% dissolved in saline. After 10 min mice were observed for an NH4OH=600 : 9 : 1) to yield 27 (152 mg, 0.40 mmol, 78%) as a additional 10 min during which abdominal contractions were 25 light yellow solid. mp 45–48°C. [α]D +36.8 (c=0.92, CHCl3). counted. The % antinociception was calculated from the mean 1 H-NMR (CDCl3) δ: 1.64 (1H, dq, J=11.8, 4.6 Hz), 2.03–2.07 number of contractions in each test group and control group (1H, m), 2.65 (1H, dt, J=11.9, 3.0 Hz), 2.78–2.83 (1H, m), (% antinociception=[(mean control responses−test responses)/ 2.91–2.98 (2H, m), 3.12–3.20 (1H, m), 3.25 (1H, dd, J=13.4, (mean control responses)]×100). The statistical significance 4.6 Hz), 3.42 (2H, d, J=13.6 Hz), 3.82 (2H, d, J=13.6 Hz), 6.17 of differences between groups was assessed with analysis of (1H, br), 6.64–6.66 (2H, m), 7.01–7.10 (3H, m), 7.20–7.36 (10H, variance followed by the Bonferroni–Dunn test or Student t- 13 m). C-NMR (CDCl3) δ: 21.0, 34.1, 39.0, 46.6, 53.9, 54.8, test. 125.7, 127.0, 127.7, 128.2, 129.0, 129.7, 138.8, 139.5, 175.1. IR (film) cm−1: 1600, 1660. MS (EI) m/z: 384 (M+), 236 (base Acknowledgments This work was MEXT-Supported Pro- peak). HR-MS (EI) m/z: Found 384.2191 (Calcd for C26H28N2O gram for the Strategic Research Foundation at Private Univer- (M+) 384.2202). sities, 2014–2018 (Grant No. S1411019). (3R,4S)-3-Benzyl-4-N,N-dimethylaminopiperidin-2-one ((−)-trans-8a) Conflict of Interest The authors declare no conflict of

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