Probes for narcotic receptor mediated phenomena. 48. C7- and C8-Substituted 5-phenylmorphan opioids from diastereoselective alkylation
The exploration of the effect of substituents at C7 and C8 of the 5-phenylmorphans on their affinity for opioid receptors was enabled by our recently introduced \xe2\x80\x9cone pot\xe2\x80\x9d diastereoselective synthesis that provided C7-oxo, hydroxy and alkyl substituents, C8-alkyl substituted 5-phenylmorphans, and compounds that had a new cyclohexane ring that includes the C7 and C8 carbon atoms of the 5-phenylmorphan. The affinity of the 5-phenylmorphans for opioid receptors is increased by a C8-methyl substituent, compared with its C7 analogue. The affinity of the newly synthesized compounds is generally for the \xce\xbc-opioid receptor, rather than the \xce\xb4- or \xce\xba-receptors. Addition of a new cyclohexane ring to the C7 and C8 positions on the cyclohexane ring of the 5-phenylmorphans enhances \xce\xbc-receptor affinity, bringing the Ki to the subnanomolar level. Unexpectedly, the N-methyl substituted compounds generally had higher affinity than comparable N-phenethyl-substituted relatives. The configurations of two compounds were determined by single-crystal X-ray crystallographic analyses.
1. Introduction<!>2.1. Chemistry<!>2.2. Opioid Receptor Affinity<!>3. Conclusions<!>4.1. Materials and methods<!>4.2.1 General procedure of debenzylation<!>4.2.2. 5-(3-Methoxyphenyl)-8-methyl-2-azabicyclo[3.3.1]nonane (6a)<!>4.2.3. 5-(3-Methoxyphenyl)-7-methyl-2-azabicyclo[3.3.1]nonane (6b)<!>4.2.4. 5-(3-Methoxyphenyl)-7-phenyl-2-azabicyclo[3.3.1]nonane (6c)<!>4.2.5. 5-(3-Methoxyphenyl)-7,8-cyclohexyl-2-azabicyclo[3.3.1]nonane (6d)<!>4.2.6. 5-(3-Methoxyphenyl)-2-azabicyclo[3.3.1]nonan-7-one (9a)<!>4.3. General procedure of N-methylation<!>4.4. General procedure of N-phenethylation<!>4.5. General procedure of O-demethylation<!>4.5.1. 3-(2-Methyl-8-methyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol (7a)<!>4.5.2. 3-(2-Phenethyl-8-methyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol (8a)<!>4.5.3. 3-(2-Methyl-7-methyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol (7b)<!>4.5.4. 3-(2-Phenethyl-7-methyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol (8b)<!>4.5.5. 3-(2-Methyl-7-phenyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol (7c)<!>4.5.6. 3-(2-Phenethyl-7-phenyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol (8c)<!>4.5.7. 3-(2-Methyldecahydro-1,5-methanobenzo[c]azocin-5(2H)-yl)phenol (7d)<!>4.5.8. 3-(2-Phenethyldecahydro-1,5-methanobenzo[c]azocin-5(2H)-yl)phenol (8d)<!>4.5.9. 5-(3-Methoxyphenyl)-2-methyl-2-azabicyclo[3.3.1]nonan-7-one (10a)<!>4.6. 5-(3-Methoxyphenyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-7-one (10b)<!>4.6.1. 5-(3-Hydroxyphenyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-7-one (10c)<!>4.6.2 5-(3-Hydroxyphenyl)-2-methyl-2-azabicyclo[3.3.1]nonan-7-ol (11a)<!>4.6.3. 5-(3-Hydroxyphenyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-7-ol (11b)<!>4.7.1. Cell culture and membrane preparation for binding assays<!>4.7.2 Opioid binding assays<!>4.8. X-ray crystal data on compounds 11a and 7b<!>4.8.1. Compound 11a<!>4.8.2. Compound 7b
<p>We have examined the effects of substituents in various positions on the cyclohexane ring of the 5-phenylmorphan structure[1] because we have found that some substituents in specific positions in that structure can have a remarkable effect on opioid receptor affinity and efficacy. For example, the (-)-C9S-OH enantiomer 1 was found to have subnanomolar affinity for the μ-opioid receptor as well as moderate affinity for the δ-receptor, and it was considerably more potent than morphine as an analgesic.[2].</p><p>Other C9-analogs also had high affinity for the μ-receptor.[2] With the hope of finding additional compounds that had affinity for both μ- and δ-opioid receptors, we thought that it would be of interest to examine the effects of substituents in the C7- and C8 positions of the 5-phenylmorphan nucleus; the C8 position is comparable to the C9 position in its spatial distance from the nitrogen atom. Since compounds with both μ- and δ- opioid receptor affinity have been said to have enhanced properties or fewer side-effects than those that act at only μ-receptors[3-8]_ENREF_3_ENREF_3_ENREF_4 we thought it would be of interest to determine whether compounds with substituents at C8 act similarly to compounds with substituents at C9, or whether compounds with substituents a further distance from that nitrogen atom, at, say, C7, would improve receptor affinity and efficacy. We needed, then, a synthetic path to the C8 and C7 substituted 5-phenylmorphans, and we discovered one. We have previously reported this "one pot" diastereoselective synthesis that provided C7-oxo, hydroxy and alkyl substituents, as well as C8-alkyl substituted 5-phenylmorphans, as well as compounds that had a new cyclohexane ring that includes the C7 and C8 carbon atoms of the 5-phenylmorphan.[9] The use of this synthetic pathway has allowed us to prepare N-derivatives other than the N-methyl and N-benzyl compounds that were noted in our initial paper. We now report on compounds that have been synthesized and evaluated in opioid receptor binding assays.</p><!><p>The C-7 and C-8 substituted 5-phenylmorphans were synthesized in a 3-step procedure based on highly distereoselective acid-catalyzed ene-imine cyclization that has been previously reported by our group.[9] The reported cyclization protocol always gave C7-trans and C8-cis isomers (relative to the piperidine ring) as a single product without any minor isomers. Thus, the cyclohexane annulated compound 7d and 8d also were formed as a single product with the same stereochemistry. As we noted previously, [9] a possible mechanism that explained the obtained data suggested 6-membered Re-face C-C bond formation and an intramolecular hydride shift as key aspects of the highly diastereoselective cyclization that gave trans selectivity at C7 and cis selectivity at C8.</p><p>Alkylation of the known tetrahydropyridine 2 with a substituted allyl bromide generated a quaternary carbon center. Acid-catalyzed ene-imine cyclization of the alkylated intermediate 3, followed by in-situ reduction of resulting cyclized imine 4, gave the desired N-benzyl-5-phenylmorphans 5a-d[9] in good yields (Scheme 1). The N-Bn compounds 5a-d were converted into their secondary amines 6 by Pd/C catalyzed hydrogenolysis (Scheme 2). The amines were alkylated to their N-methyl relatives by reductive alkylation with formalin and Pd/C, or N-alkylated with phenethyl bromide to obtain the N-phenethyl derivatives. Finally, the OMe group was deprotected to yield the desired phenolic products 7 and 8.</p><p>The relative stereochemistry between the piperidine ring and the C7-methyl and C7-hydroxyl substituent in the cyclohexane ring was determined by X-ray crystallographic analyses (Fig. 1).[9] With a C7α-methyl substituent the relative stereochemistry of the C7 moiety in racemic 7b and the piperidine ring, as shown in Fig. 1, was trans (C1R*, C5S*and C7S*), and with the C7β-hydroxyl it was cis (C1S*, C5R*, and C7S*). For the synthesis of 7-oxo and hydroxy derivatives, N-benzyl-7-oxo-5-phenylmorphan 9 was prepared by the reported procedure.[10] Debenzylation of 9, then N-alkylation followed by O-demethylation gave the desired C7-oxo compound 10c. The C7-hydroxy derivatives 11a-b were obtained by NaBH4 reduction, followed by O-demethylation (Scheme 3). Unlike the C7-methyl or phenyl substituted compounds (7b-c, 8b-c) that were trans-oriented relative to the piperidine ring, reduction of C7-oxo compounds (10a-b) gave C7β-(cis)-hydroxy derivatives 11a-b (Fig. 1). The configuration of compound 11a, prepared from the enantiopure ketone 10a by optical resolution using a chiral acid, was determined by X-ray crystallography. The absolute configuration of the compound 11a was found to be C1S, C5R, and C7S.</p><!><p>As noted in Table 1, with the exception of one pair of compounds, the 7β-OH compounds 11a and 11b, the N-methyl substituted derivatives were found to have greater affinity for the μ-opioid receptor than their N-phenethyl-substituted relatives. This is very unusual among the 5-phenylmorphans where compounds with an N-phenethyl substituent almost invariably have higher affinity than the N-methyl relative for the μ-receptor.[1] The higher affinity of the N-phenethyl analogues also holds in the pyridin-6-ol series of compounds introduced by Hutchison et al.[11], and with oxide-bridged phenylmorphans.[12-14].</p><p>The C8-methyl compounds were found to have higher μ-opioid affinity than the comparable C7-methyl compounds in both the N-methyl and the N-phenethyl-substituted phenylmorphans. (7a vs 7b, and 8a vs 8b). An oxygen atom in the C7β position (11a and 11b) and the C7-ketone (10c) in the cyclohexane ring of these phenylmorphans reduced affinity to the μ-receptor, unlike the hydroxyl group at C9 which greatly increased affinity.[2] The N-phenethyl compounds with a C8-methyl (8a, Ki = 2.2 nM) or a compound with an additional cyclohexane ring at the C7-C8 position (8d, Ki = 4 nM) had greater, or nearly equivalent affinity, for the μ-receptor as morphine (Table 1).</p><p>A substituent at C8 appears to confer higher affinity for opioid receptors than that same substituent at C7. The C7-methyl isomers 7b and 8b had less affinity for all of the opioid receptors than the C8-methyl compounds 7a and 8a. Most surprising was the affinity of 7d, the N-methyl relative of 8d; it was found to have subnanomolar affinity for the μ-receptor (Ki = 0.9 nM). The observed ca. 4-fold difference in affinity of the N-methyl-substituted 7d and the N-phenethyl-substituted 8d, though not extreme, is very unusual in the 5-phenylmorphan series. The additional cyclohexane ring has apparently caused a substantial change in the ability of N-methyl substituted phenylmorphan-type compounds to bind to the μ-receptor. None of the compounds in Table 1 had appreciable affinity for δ-opioid receptors, and only 8c, the C7-phenyl compound in the N-phenethyl series, had good affinity for κ-receptors (Ki = 4 nM).</p><!><p>We have found that, in general, the affinity of the 5-phenylmorphans for opioid receptors is increased by a C8-methyl substituent, compared with its C7 analogue. The affinity is mostly for the μ-opioid receptor, rather than δ- or κ-receptors, and an additional cyclohexane ring added to the C7 and C8 positions on the azabicyclo ring system (3-(2-methyl-7,8-cyclohexyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol, 7d) considerably enhances μ-receptor affinity, lowering the Ki to the subnanomolar level. Further, when a substituent other than an hydroxyl group appears at either C7 or C8 in the 5- phenylmorphans, the N-methyl substituted isomer was found to have higher μ-affinity than its N-phenethyl relative.</p><!><p>Compounds 2,[9] 5a-d,[9] and 9[10] were prepared as previously described. n-BuLi was titrated and THF was dried over molecular sieves before use. Other chemicals and solvents were used directly without further purification. Chemicals were purchased from Sigma-Aldrich Inc. or Alfa Aesar. Melting points were determined on a Buchi B-545 instrument and are uncorrected. Thin layer chromatography (TLC) analyses were carried out on Analtech silica gel GHLF 0.25 mm plates using various concentrations of CHCl2/MeOH containing 1% NH4OH or of EtOAc:n-hexane. Visualization was accomplished under UV light or by staining in an iodine chamber. Flash column chromatography was performed with Fluka silica gel 60 (mesh 220-400) using CHCl3: MeOH: NH4OH (90: 9: 1). Proton and carbon nuclear magnetic resonance (1H- and 13C-NMR) spectra were recorded in CDCl3 with CHCl3 (ä 7.24), as an internal standard, on a Varian Gemini-300 spectrometer and a Bruker DMX500 wide-bore spectrometer ((proton frequency 500.13 MHz) running XWINNMR v3.1, carbon 125.757) with the values given in ppm and J (Hz) assignments of 1H resonance coupling. Mass spectra (HRMS) were recorded on a VG 7070E spectrometer or a JEOL SX102a mass spectrometer. IR spectra were recorded on a Beckman IR 4230 spectrometer. Atlantic Microlabs, Inc., Norcross, GA, or Micro-Analysis, Inc., Wilmington, DE, performed elemental analyses, and the results were within ±0.4% of the theoretical values.</p><!><p>To a reaction flask, an N-Bn compound, Pd/C (10 wt %), AcOH (0.1 mL), and MeOH (5 mL/1 mmol of N-Bn) were added. After the vessel was evacuated and refilled with hydrogen gas 3 times, the reaction mixture was stirred at 60 °C under a balloon pressure of hydrogen gas. After 6 h, the mixture was filtered through celite pad to remove the catalyst, and the solvent was evaporated. The residue was dissolved in CH2Cl2 (100 mL) and it was washed with 2 N NaOH. The aqueous layer was extracted twice with CH2Cl2 (100 mL each). The combined organics were washed with brine, dried over Na2SO4, filtered, and evaporated. The crude product was purified by column chromatography.</p><!><p>Using the general procedure, the N-Bn compound 5a (1.0 g, 3.0 mmol) was converted into secondary amine 6a (0.76 g, > 99 %). 1H NMR (500 MHz, CDCl3): ä 7.26-7.23 (t, J = 8.0 Hz, 1H), 6.96-6.91 (m, 2H), 6.74-6.73 (dd, J = 1.5, 8.0 Hz, 1H), 3.80 (s, 3H), 3.43-3.37 (dt, J = 5.0, 12.5 Hz, 1H), 3.02 (br s, 1H), 2.95-2.91 (m, 2H), 2.16-1.99 (m, 3H), 1.88-1.65 (m, 6H), 1.07-1.06 (d, J = 5.0 Hz, 3H); 13C NMR (126 MHz, CDCl3): ä 159.6, 154.0, 129.1, 117.3, 111.4, 110.2, 55.2, 52.5, 42.4, 39.8, 38.5, 38.1, 35.9, 35.3, 31.5, 19.3; IR cm-1 (neat): 2926.8, 1607.4, 1582.2, 1486.3, 1453.1, 1430.9, 1291.9, 1268.0, 1049.1; HRMS calcd for C16H24NO (M+H)+: 246.1858, found: 246.1866.</p><!><p>Using the general procedure, N-Bn compound 5b (1.0 g, 3.0 mmol) was converted into secondary amine 6b (0.75 g, > 99 %). 1H NMR (500 MHz, CDCl3): ä 7.35-7.24 (m, 1H), 6.95-6.88 (m, 2H), 6.75-6.74 (d, J = 6.0 Hz, 1H), 3.81 (s, 3H), 3.55-3.46 (m, 2H), 3.05-3.02 (d, J = 7.5 Hz, 2H), 2.95-2.90 (m, 1H), 2.51-2.40 (m, 1H), 2.15-2.00 (m, 4H), 1.84-1.79 (m, 2H), 1.32-1.17 (m, 2H), 0.90-0.89 (d, J = 6.5 Hz, 3H); 13C NMR (126 MHz, CDCl3): ä 159.6, 153.8, 129.3, 117.2, 111.4, 110.4, 55.4, 48.6, 48.5, 48.3, 42.5, 41.1, 37.7, 36.3, 28.6, 24.6; IR cm-1 (neat): 2950, 2920, 2867, 2836, 1606, 1582, 1485, 1456, 1430, 1285, 1256, 1169, 1050; HRMS calcd for C16H24NO: 246.1858 (M+H)+, found: 246.1859.</p><!><p>Using the general procedure, N-Bn compound 5c (1.5 g, 3.77 mmol) was converted into secondary amine 6c (1.2 g, > 99 %). 1H NMR (500 MHz, CDCl3): ä 7.25-7.14 (m, 6H), 6.94-6.86 (m, 3H), 6.72-6.68 (m, 1H), 3.82 (br s, 1H), 3.73 (s, 3H), 3.69-3.58 (m, 1H), 3.29-3.25 (m, 1H), 2.44-2.42 (d, J = 11.0 Hz, 1H), 2.35-2.29 (t, J = 15.0 Hz, 2H), 2.20-2.19 (d, J = 8.5 Hz, 2H), 2.01-1.95 (m, 2H), 1.87-1.47 (m, 2H); 13C NMR (126 MHz, CDCl3): ä 159.6, 151.9, 145.9, 129.4, 128.5, 126.9, 126.4, 116.9, 111.2, 110.8, 55.1, 53.5, 51.4, 48.3, 47.1, 41.1, 39.1, 36.7, 35.6; IR cm-1 (neat): 3327.0, 3057.7, 3025.7, 2914.1, 2843.7, 1599.7, 1581.1, 1491.8, 1451.8, 1255.4, 1160.8, 1050.8, 1032.5; HRMS calcd for C21H26NO: 308.2014 (M+H)+, found: 308.2015.</p><!><p>Using the general procedure, N-Bn compound 5d (0.9 g, 2.4 mmol) was converted into secondary amine 6d (0.63 g, 93 %). 1H NMR (500 MHz, CDCl3): ä 7.37 (br s, 1H), 7.10-7.07 (t, J = 8.0 Hz, 1H), 6.77-6.72 (m, 2H), 6.59-6.57 (d, J = 8.0 Hz, 1H), 3.63 (s, 3H), 3.37-3.31 (dt, J = 5.5, 12.5 Hz, 1H), 3.30 (br s, 1H), 3.07-3.03 (dd, J = 7.5, 12.0, Hz, 1H), 2.24-2.21 (d, J = 12.5 Hz, 1H), 1.97-1.71 (m, 7H), 1.58-1.48 (m, 3H), 1.22-1.11 (m, 4H), 0.71-0.65 (m, 1H); 13C NMR (126 MHz, CDCl3): ä 159.3, 151.9, 129.0, 116.8, 111.0, 110.3, 54.9, 52.5, 46.6, 45.6, 40.8, 38.4, 37.2, 35.5, 35.3, 35.2, 29.9, 26.6, 26.2; IR cm-1 (neat): 2921.7, 2848.3, 1601.1, 1581.8, 1485.9, 1446.1, 1429.8, 1253.4, 1168.6, 1046.6; HRMS calcd for C19H28NO: 286.2171 (M+H)+, found: 286.2170.</p><!><p>Using the general procedure, N-Bn compound 9 (4 g, 0.012 mol) was converted into secondary amine 9a (3.0 g, > 99 %, pale yellow oil). 1H NMR (500 MHz, CDCl3): ä 7.24-7.20 (t, J = 8.5 Hz, 1H), 6.88-6.87 (d, J = 1.0 Hz, 1H), 6.82 (s, 1H), 6.73-6.71 (d, J = 8.0 Hz, 1H), 3.74 (s, 3H), 3.71-3.70 (m, 1H), 2.89-2.87 (d, J = 7.5 Hz, 2H), 2.77-2.74 (d, J = 17.0 Hz, 1H), 2.54 (m, 2H), 2.43-2.40 (d, J = 16.5 Hz, 1H), 2.31-2.09 (m, 2H), 1.85-1.73 (m, 2H); 13C NMR (126 MHz, CDCl3): ä 210.3, 159.7, 150.3, 129.6, 116.8, 111.2, 111.0, 55.2, 53.2, 49.4, 47.4, 39.2, 39.1, 38.9, 37.4.</p><!><p>To a reaction flask, amine (1.1 g, 4.5 mmol), Pd/C (0.11 g, 10 wt %), AcOH (0.05 mL), formalin (37 % in water, 0.34 mL, 4.5 mmol), and MeOH (20 mL) were added. After the vessel was evacuated 3 × using H2 gas, the reaction mixture was stirred at room temperature under a balloon pressure of H2 gas. After 3 h, the mixture was filtered through a celite pad to remove the catalyst, and the solvent was evaporated. The residue was dissolved in CH2Cl2 (20 mL) and it was washed with 2 N NaOH. The aqueous layer was extracted with CH2Cl2 (20 mL × 2). The combined organics were washed with brine, dried over Na2SO4, filtered, and evaporated. The crude product was purified by column chromatography.</p><!><p>Amine (1.4 g, 5.7 mmol), K2CO3 (2.4 g, 17.1 mmol), KI (0.23 g, 1.4 mmol), and phenethyl bromide (1.2 mL, 8.6 mmol) were dissolved in anhyd acetonitrile (50 mL), and the resulting mixture was refluxed overnight. After additional phenethyl bromide (0.4 mL, 2.8 mmol) was added, the mixture was refluxed for another 4 h. The reaction was cooled to room temperature and filtered through a celite pad, and then the solvent was evaporated. The crude product was purified by column chromatography.</p><!><p>The methoxy compound was dissolved in 48 % HBr (4 mL per 1 mmol of methoxy compound), and the resulting mixture was refluxed for 2.5 h. After the reaction was completed, the solution was cooled to − 78 °C, and then concd NH4OH was slowly added to pH 9. The product was extracted with CH2Cl2 (20 mL × 3). The combined organics were washed with brine, dried over Na2SO4, filtered, and evaporated. The crude product was purified by column chromatography.</p><!><p>Using the general procedures, secondary amine 6a (0.35 g, 1.43 mmol) was N-methylated (0.34 g, 92 %). 1H NMR (500 MHz, CDCl3): ä 7.24-7.21 (t, J = 8.0 Hz, 1H), 6.91-6.86 (m, 2H), 6.72-6.70 (dd, J = 2.0, 8.0 Hz, 1H), 3.79 (s, 3H), 2.86-2.82 (m, 1H), 2.66-2.58 (m, 2H), 2.46 (m, 3H), 2.36-2.32 (m, 1H), 2.07-1.80 (m, 4H), 1.49-1.43 (m, 4H), 1.00-0.99 (d, J = 6.5 Hz, 3H); 13C NMR (126 MHz, CDCl3): ä 159.6, 155.4, 129.2, 117.7, 111.7, 110.1, 60.7, 55.3, 49.5, 47.3, 40.7, 37.6, 36.6, 34.5, 34.3, 28.1, 19.4.; IR cm-1 (neat): 2929.4, 2867.7, 2785.6, 1608.0, 1582.0, 1484.3, 1451.2, 1290.7, 1264.1, 1047.5; HRMS calcd for C17H26NO: 260.2014 (M+H)+, found: 260.2000.</p><p>The compound in this N-methyl compound (0.20 g, 0.77 mmol) was then O-demethylated to give 7a using the general procedure (0.20 g, > 99 %). 1H NMR (500 MHz, CDCl3): ä 7.16-7.13 (t, J = 8.0 Hz, 1H), 6.85-6.83 (d, J = 8.0 Hz, 1H), 6.76-6.75 (d, J = 4.0 Hz, 1H), 6.63-6.61 (d, J = 8.0 Hz, 1H), 3.02-2.97 (m, 1H), 2.77 (br s, 1H), 2.68-2.63 (m, 1H), 2.51 (m, 3H), 2.33-2.29 (m, 1H), 1.99-1.81 (m, 4H), 1.58-1.50 (m, 4H), 1.07-1.06 (d, J = 6.5 Hz, 3H); 13C NMR (126 MHz, CDCl3): ä 156.3, 155.0, 129.4, 117.0, 113.0, 112.8, 60.6, 49.7, 46.6, 40.2, 37.6, 36.1, 35.3, 34.4, 29.1, 19.8; IR cm-1 (neat): 3049.9, 2929.4, 2868.7, 1598.6, 1583.2, 1450.4, 1273.4, 1166.3; HRMS calcd for C16H24NO: 246.1858 (M+H)+, found: 246.1855. Compound 7a was converted into its HBr salt and crystallized from EtOH/EtOAc (0.20 g); mp 200.4-201.8 °C. Anal. Calcd. for C16H24NOBr: C, 58.90; H, 7.41; N, 4.29. Found: C, 58.62; H, 7.20; N, 4.21.</p><!><p>Using the general procedure, secondary amine 6a (0.25 g, 1.02 mmol) was converted into the N-phenethyl compound (0.22 g, 62 %). 1H NMR (500 MHz, CDCl3): ä 7.27-7.16 (m, 6H), 6.91-6.90 (d, J = 7.5 Hz, 1H), 6.87 (s, 1H), 6.71-6.69 (d, J = 8.0 Hz, 1H), 3.77 (s, 3H), 3.02-2.98 (m, 1H), 2.86-2.69 (m, 6H), 2.32-2.29 (dd, J = 4.0, 15.0 Hz, 1H), 2.04-1.78 (m, 4H), 1.51-1.44 (m, 4H), 0.93-0.92 (d, J = 6.5 Hz, 3H); 13C NMR (126 MHz, CDCl3): ä 159.6, 155.4, 141.1, 129.2, 129.0, 128.4, 125.9, 117.7, 111.8, 110.1, 61.3, 59.3, 55.3, 46.3, 40.6, 37.6, 36.6, 35.3, 34.8, 34.7, 28.4, 19.4; IR cm-1 (neat): 3026.4, 2929.7, 2865.4, 1606.8, 1582.4, 1485.3, 1453.7, 1291.2, 1265.4, 1050.5; HRMS calcd for C24H32NO: 350.2484 (M+H)+, found: 350.2491.</p><p>The compound (0.15 g, 0.43 mmol) was O-demethylated using the general procedure to give 8a (0.15 g, > 99 %). 1H NMR (500 MHz, CDCl3): ä 7.26-7.11 (m, 6H), 6.84-6.79 (m, 2H), 6.66-6.60 (m, 2H), 3.04-3.00 (m, 1H), 2.88-2.72 (m, 6H), 2.28-2.25 (d, J = 13.0 Hz, 1H), 1.96-1.79 (m, 4H), 1.50-1.46 (m, 4H), 0.98-0.97 (d, J = 6.0 Hz, 3H); 13C NMR (126 MHz, CDCl3): ä 156.1, 155.2, 140.9, 129.5, 129.0, 128.5, 126.1, 117.2, 113.1, 112.9, 60.9, 59.1, 46.8, 40.3, 37.6, 36.3, 35.7, 34.8, 34.4, 29.0, 19.7; IR cm-1 (neat): 2929.6, 1584.9, 1453.1, 1364.3, 1453.1, 1364.3, 1272.6, 1163.9; HRMS calcd for C23H30NO: 336.2327 (M+H)+; found: 336.2317. Compound 8a was converted into its HBr salt and crystallized from EtOH/EtOAc (0.14 g); mp 258.0-259.6 °C. Anal. Calcd. for C23H30NOBr + 0.25 H2O: C, 65.67; H, 7.31; N, 3.33. Found: C, 65.65; H, 7.10; N, 3.33.</p><!><p>Using the general procedure, secondary amine 6b (0.30 g, 1.22 mmol) was converted into the N-methyl derivative (0.195 g, 61 %). 1H NMR (500 MHz, CDCl3): ä 7.30-7.27 (t, J = 8.0 Hz, 1H), 7.00-6.99 (d, J = 7.5 Hz, 1H), 6.96 (s, 1H), 6.78-6.77 (d, J = 7.5 Hz, 1H), 3.84 (s, 3H), 3.14 (br s, 1H), 3.07-3.01 (dt, J = 12.5, 4.5 Hz, 1H), 2.92-2.89 (dd, J = 11.5, 7.5, 1H), 2.51 (s, 3H), 2.30-2.21 (m, 5H), 2.03-1.96 (m, 1H), 1.89-1.87 (d, J = 12.0 Hz, 1H), 1.24-1.19 (t, J = 13.5, 1H), 1.03-0.98 (t, J = 13.5 Hz, 1H), 0.91-0.89 (d, J = 5.5 Hz, 3H); 13C NMR (126 MHz, CDCl3): ä 159.6, 153.4, 129.2, 117.3, 111.4, 110.4, 55.2, 54.9, 51.2, 47.8, 43.2, 39.2, 37.2, 35.7, 33.4, 28.8, 24.6; IR cm-1 (neat): 2950, 2923, 2837, 1608, 1583, 1486, 1456, 1431, 1379, 1291, 1258, 1171, 1053; HRMS calcd for C17H26NO: 260.2014 (M+H)+, found: 260.2014.</p><p>The compound (0.16 g, 0.62 mmol) was O-demethylated to yield 7b (0.15 g, > 99 %). 1H NMR (500 MHz, CDCl3): ä 10.17 (br s, 1H), 7.13-7.10 (t, J = 8.0 Hz, 1H), 6.79-6.77 (d, J = 8.0 Hz, 1H), 6.74 (s, 1H), 6.61-6.59 (d, J = 9.5 Hz, 1H), 3.16 (br s, 1H), 3.04-2.90 (m, 2H), 2.46 (s, 3H), 2.25-2.15 (m, 3H), 2.07-1.94 (m, 3H), 1.80-1.77 (d, J = 12.5 Hz, 1H), 1.18-1.13 (t, J = 13.0 Hz, 1H), 1.01-0.97 (t, J = 12.0 Hz, 1H), 0.85-0.84 (d, J = 5.5 Hz, 3H); 13C NMR (126 MHz, CDCl3): ä 157.4, 152.7, 129.3, 115.8, 113.5, 112.8, 54.9, 51.1, 47.8, 42.6, 38.2, 36.4, 35.3, 33.2, 28.7, 24.8; IR cm-1 (neat): 3047.7, 2949.2, 2922.5, 2866.3, 1597.7, 1581.9, 1482.0, 1448.0, 1378.8, 1366.3, 1278.0, 1246.2, 1186.1, 1137.0; HRMS calcd for C16H24NO: 246.1858 (M+H)+, found: 246.1862. The title compound 7b was converted into its HBr salt and crystallized from EtOH/EtOAc (0.17 g); mp 213.5-214.7 °C. Anal. Calcd. for C16H24NOBr: C, 58.90; H, 7.41; N, 4.29. Found: C, 58.71; H, 7.57; N, 4.16. The relative configuration of 7b was determined by X-ray crystallography (Fig. 1, C1 = R, C5 = S, and C7 = S).</p><!><p>Using the general procedure, secondary amine 6b (0.25 g, 1.02 mmol) was converted into the N-phenethyl derivative (0.18 g, 50 %). 1H NMR (500 MHz, CDCl3): ä 7.26-7.19 (m, 6H), 6.94-6.92 (d, J = 8.0 Hz, 1H), 6.89 (s, 1H), 6.71-6.69 (d, J = 7.5 Hz, 1H), 3.76 (s, 3H), 3.21 (br s, 1H), 3.10-2.93 (m, 2H), 2.80-2.72 (m, 4H), 2.21-2.00 (m, 5H), 1.96-1.90 (m, 1H), 1.81-1.79 (d, J = 12.5 Hz, 1H), 1.16-1.11 (t, J = 12.5 Hz, 1H), 0.95-0.90 (t, J = 13.0 Hz, 1H), 0.83-0.81 (d, J = 5.5 Hz, 3H); 13C NMR (126 MHz, CDCl3): ä 159.6, 153.7, 140.9, 129.2, 128.9, 128.4, 126.1, 117.3, 111.5, 110.4, 58.1, 55.2, 53.0, 49.7, 48.0, 39.0, 37.3, 36.3, 35.8, 34.7, 28.8, 24.7; IR cm-1 (neat): 3026.1, 2948.7, 2923.9, 1607.0, 1582.6, 1485.7, 1455.2, 1431.4, 1291.3, 1258.5, 1170.7, 1052.6; HRMS calcd for C24H32NO: 350.2484 (M+H)+, found: 350.2488.</p><p>The compound (0.15 g, 0.43 mmol) was O-demethylated to yield 8b (0.14 g, 97 %). 1H NMR (500 MHz, CDCl3 w/0.1 mL of CD3OD): ä 7.24-7.14 (m, 6H), 6.86-6.85 (m, 2H), 6.69-6.68 (d, J = 8.0 Hz, 1H), 4.78 (br s, 1H), 3.28 (br s, 1H), 3.20-2.99 (m, 2H), 2.83-2.77 (m, 4H), 2.23-2.09 (m, 5H), 1.96-1.85 (m, 2H), 1.21-1.15 (t, J = 13.0 Hz, 1H). 1.03-0.97 (t, J = 12.5 Hz, 1H), 0.87-0.85 (d, J = 5.5 Hz, 3H); 13C NMR ä (126 MHz, CDCl3 w/0.1 mL of CD3OD): ä 156.6, 152.9, 139.9, 129.1, 128.5, 128.3, 126.0, 115.9, 112.6, 111.8, 57.6, 52.7, 52.6, 49.2, 48.8, 38.2, 36.5, 35.6, 33.7, 33.6, 28.6; IR cm-1 (neat): 3026.4, 2948.5, 2923.9, 2865.7, 1738.6, 1598.9, 1583.0, 1496.0, 1482.6, 1454.3, 1366.1, 1276.0, 1229.9, 1217.1; HRMS calcd for C23H30NO: 336.2327 (M+H)+, found: 336.2339. The title compound 18d was converted into its HBr salt and crystallized from EtOH/EtOAc (0.16 g); mp 232.0-233.2 °C. Anal. Calcd. for C23H30NOBr + 0.5 M H2O: C, 64.98; H, 7.40; N, 3.29. Found: C, 64.83; H, 7.40; N, 3.26.</p><!><p>Using the general procedure, secondary amine 6c (0.25 g, 0.81 mmol) was converted into the N-methyl derivative (0.25 g, 96 %). 1H NMR (500 MHz, CDCl3): ä 7.26-7.13 (m, 6H), 6.95-6.91 (m, 2H), 6.71-6.69 (d, J = 5.5, 1H), 3.83 (s, 3H), 3.74 (s, 3H), 3.47-3.42 (m, 1H), 3.18-3.10 (m, 2H), 2.95-2.89 (m, 1H), 2.48 (s, 3H), 2.41-2.38 (m, 1H), 2.28-2.15 (m, 3H), 2.00-1.97 (m, 2H), 1.70-1.64 (dt, J = 1.5, 13.5 Hz, 1H), 1.53-1.47 (dt, J = 3.0, 13.5 Hz, 2H); 13C NMR (126 MHz, CDCl3): ä 159.6, 153.1, 147.2, 129.3, 126.3, 117.3, 111.5, 110.5, 55.2, 54.8, 51.4, 47.0, 43.3, 40.2, 38.7, 37.2, 35.8, 32.6.; IR cm-1 (neat): 3059.5, 3026.1, 2924.6, 2844.4, 1599.5, 1581.8, 1486.4, 1431.3, 1288.7, 1248.1, 1165.1, 1050.9, 1027.9; HRMS calcd for C22H28NO: 322.2171 (M+H)+, found: 322.2170.</p><p>The compound (0.25 g, 0.77 mmol) was O-demethylated to yield 7c (0.23 g, 97 %). 1H NMR (500 MHz, CDCl3): ä 9.50 (br s, 1H), 7.26-7.06 (m, 6H), 6.77-6.74 (m, 2H), 6.59-6.57 (d, J = 9.5 Hz, 1H), 3.43-3.37 (m, 1H), 3.23 (br s, 1H), 3.15-3.09 (dt, J = 5.0, 12.0 Hz, 1H), 2.97-2.93 (m, 1H), 2.46 (s, 3H), 2.40-2.37 (d, J = 12.0 Hz, 1H), 2.25-2.20 (t, J = 7.5 Hz, 2H), 2.07-1.91 (m, 3H), 1.67-1.61 (t, J = 13.0 Hz, 1H), 1.54-1.48 (dt, J = 3.0, 13.0 Hz, 1H); 13C NMR (126 MHz, CDCl3): ä 157.3, 152.5, 146.9, 129.5, 128.6, 126.9, 126.4, 116.1, 113.6, 112.7, 54.7, 51.3, 46.8, 42.8, 40.2, 38.1, 36.5, 35.5, 32.1.; IR cm-1 (neat): 3060.4, 3027.3, 2934.6, 1598.0, 1582.1, 1492.4, 1451.8, 1376.6, 1279.4, 1245.5.; HRMS 308.2011 ((M+H+·); 308.2014 calcd for C21H26NO). HRMS calcd for C21H26NO: 308.2014 (M+H)+, found 308.2011. The title compound 7c was converted into its HBr salt and crystallized from MeOH/EtOAc (0.18 g); mp 268.8-270.1 °C. Anal. Calcd. for C21H26NOBr + 0.3 M H2O: C, 64.10; H, 7.07; N, 3.56. Found: C, 63.84; H, 6.73; N, 3.54.</p><!><p>Using the general procedure, secondary amine 6c (0.25 g, 0.81 mmol) was converted into its N-phenethyl derivative (0.19 g, 57 %). 1H NMR (500 MHz, CDCl3): ä 7.35-7.15 (m, 11H), 6.94-6.89 (m, 2H), 6.73-6.71 (dd, J = 8.0, 2.0 Hz, 1H), 3.76 (s, 3H), 3.53-3.44 (m, 2H), 3.22-3.20 (m, 2H), 2.95 (s, 2H), 2.43-2.27 (m, 4H), 2.21-1.92 (m, 4H), 1.73-1.57 (m, 2H); 13C NMR (126 MHz, CDCl3): ä 159.7, 152.5, 146.5, 139.6, 136.2, 129.4, 129.1, 128.8, 128.4, 127.0, 126.4, 118.3, 117.2, 111.5, 112.2, 110.8, 57.7, 55.3, 53.5, 49.6, 40.0, 37.3, 36.4, 36.2, 35.9, 33.5; IR cm-1 (neat): 3059.3, 3025.4, 2927.6, 2833.0, 1600.8, 1581.5, 1492.7, 1452.6, 1289.3, 1243.6, 1165.9, 1048.8, 1030.2; HRMS calcd for C29H34NO: 412.2640 (M+H)+, found 412.2632.</p><p>The compound (0.20 g, 0.49 mmol) was O-demethylated to yield 8c (0.15 g, 79 %). 1H NMR (500 MHz, CDCl3): ä 7.27-7.09 (m, 11H), 6.83-6.79 (m, 2H), 6.64-6.63 (dd, J = 7.5, 1.5 Hz, 1H), 3.43-3.41 (m, 2H), 3.14-3.12 (m, 2H), 2.88-2.84 (m, 3H), 2.37-2.21 (m, 3H), 2.14-1.88 (m, 4H), 1.68-1.50 (m, 2H); 13C NMR (126 MHz, CDCl3): ä 156.6, 152.9, 146.9, 140.1, 136.2, 129.6, 129.4, 129.0, 127.4, 127.0, 126.5, 116.7, 113.6, 112.7, 57.9, 52.9, 50.0, 46.9, 40.2, 38.0, 36.8, 36.1, 33.9, 33.0; IR cm-1 (neat): 3059.7, 3026.1, 2927.7, 2848.7, 1599.0, 1582.9, 1493.0, 1452.0, 1365.5, 1276.0, 1232.3, 1177.1; HRMS calcd for C28H32NO: 398.2484 (M+H)+, found 398.2488. The title compound 8c was converted into its HBr salt and crystallized from EtOAc/E2tO (0.10 g); mp 155.6-157.1 °C. Anal. Calcd. for C28H32NOBr + 1.2 M H2O: C, 67.29; H, 6.94; N, 2.80. Found: C, 66.95; H, 6.58; N, 2.76.</p><!><p>Using the general procedure, secondary amine 6d (0.22 g, 0.77 mmol) was converted into its N-methyl derivative (0.23 g, > 99 %). 1H NMR (500 MHz, CDCl3): ä 7.15-7.11 (t, J = 8.0 Hz, 1H), 6.77-6.71 (m, 2H), 6.63-6.62 (d, J = 6.0 Hz, 1H), 3.68 (s, 3H), 3.32-3.28 (m, 1H), 3.15 (br s, 1H), 2.92-2.87 (m, 1H), 2.68 (s, 3H), 2.46-2.44 (d, J = 13.5 Hz, 1H), 2.19-2.13 (m, 1H), 2.04-2.00 (m, 2H), 1.88-1.56 (m, 8H), 1.36-1.09 (m, 3H), 0.82-0.75 (m, 1H); 13C NMR (126 MHz, CDCl3): ä 159.6, 152.1, 129.4, 117.0, 111.3, 110.6, 62.7, 55.2, 49.5, 47.4, 47.3, 45.2, 35.7, 34.8, 34.6, 34.1, 30.0, 27.1, 26.8, 25.8; IR cm-1 (neat): 2925.3, 2851.4, 1601.4, 1582.5, 1486.0, 1448.2, 1256.1, 1050.4; HRMS calcd for C20H30NO: 300.2327 (M+H)+, found 300.2319.</p><p>The compound (0.18 g, 0.60 mmol) was O-demethylated to yield 7d (0.17 g, > 99 %). 1H NMR (500 MHz, CDCl3): ä 8.00 (br s, 1H), 7.10-7.06 (t, J = 8.5 Hz, 1H), 6.75 (s, 2H), 6.63-6.61 (d, J = 7.5 Hz, 1H), 3.02-2.97 (m, 1H), 2.76 (br s, 1H), 2.67-2.62 (m, 1H), 2.48 (m, 3H), 2.26-2.23 (d, J = 14.5 Hz, 1H), 1.99-1.81 (m, 3H), 1.78-1.73 (m, 2H), 1.64-1.50 (m, 5H), 1.28-1.02 (m, 4H), 0.82-0.75 (m, 1H); 13C NMR (126 MHz, CDCl3): ä 156.9, 154.2, 129.3, 116.3, 113.3, 112.9, 60.8, 49.5, 48.1, 46.3, 45.8, 36.9, 36.3, 35.8, 35.3, 35.1, 31.1, 27.6, 26.6; IR cm-1 (neat): 3044.3, 2920.2, 2849.1, 1597.4, 1583.2, 1446.1, 1273.1, 1246.2.; HRMS 286.2171 ((M+H+·); 286.2171 calcd for C19H28NO). HRMS calcd for C19H28NO: 286.2171 (M+H)+, found 286.2171. Compound 7d was converted into its HBr salt and crystallized from EtOH (0.14 g); mp 223.8-225.3 oC. Anal. Calcd. for C16H24NOBr + 0.5 M H2O: C, 60.83; H, 7.79; N, 3.73. Found: C, 60.85; H, 8.02; N, 3.48.</p><!><p>Using the general procedure, secondary amine 6d (0.22 g, 1.02 mmol) was converted into its N-phenethyl derivative (0.29 g, 97 %). 1H NMR (500 MHz, CDCl3): ä 7.37-7.26 (m, 6H), 7.01-6.99 (d, J = 7.5 Hz, 1H), 6.97 (s, 1H), 3.86 (s, 3H), 3.08-3.04 (m, 1H), 2.94-2.80 (m, 7H), 2.42-2.39 (m, 1H), 2.21-2.06 (m, 2H), 1.99-1.56 (m, 7H), 1.45-1.18 (m, 4H), 1.01-0.97 (t, J = 10.5 Hz, 2H); 13C NMR (126 MHz, CDCl3): ä 159.6, 155.4, 141.1, 129.0, 128.3, 125.9, 117.6, 111.7, 110.0, 61.4, 59.4, 55.2, 49.9, 49.8, 48.9, 48.7, 46.1, 37.4, 35.8, 34.8, 34.4, 30.4, 27.2, 26.6; IR cm-1 (neat): 3026.3, 2970.6, 2920.7, 2848.5, 1602.1, 1581.8, 1485.3, 1450.3, 1366.0, 1230.0, 1216.9, 1051.2; HRMS calcd for C27H36NO: 390.2797 (M+H)+, found 390.2797.</p><p>The compound (0.20 g, 0.51 mmol) was O-demethylated to yield 8d (0.18 g, 94 %). 1H NMR (500 MHz, CDCl3): ä 7.30-7.13 (m, 6H), 6.85-6.84 (m, 2H), 6.70-6.68 (d, J = 9.0 Hz, 1H), 3.05-3.01 (m, 1H), 2.90-2.81 (m, 5H), 2.31-2.28 (d, J = 14.5 Hz, 1H), 2.08-1.89 (m, 3H), 1.83-1.52 (m, 6H), 1.44-1.38 (m, 1H), 1.31-1.12 (m, 4H), 1.02-0.97 (t, J = 11.0 Hz, 1H), 0.88-0.83 (m, 1H); 13C NMR (126 MHz, CDCl3): ä 156.3, 155.0, 140.8, 129.4, 129.1, 129.0, 128.6, 128.5, 126.1, 116.9, 113.0, 112.8, 61.0, 59.3, 49.9, 48.5, 46.7, 36.9, 36.3, 36.2, 35.7, 35.3, 35.0, 34.2, 30.8, 26.5; IR cm-1 (neat): 3456.5, 3026.5, 297.7, 2923.3, 2850.6, 1597.6, 1584.4, 1447.0, 1365.9, 1229.3, 1217.1, 1119.0; HRMS 376.2649 ((M+H+·); 376.2640 calcd for C26H34NO). HRMS calcd for C26H34NO: 376.2640 (M+H)+, found 376.2649. Compound 8d was converted into its HBr salt and crystallized from EtOH/EtOAc (0.14 g); mp 150.2-153.6 oC. Anal. Calcd. for C23H30NOBr + 0.5 M H2O: C, 67.13; H, 7.58; N, 3.01. Found: C, 67.04; H, 7.63; N, 2.82.</p><!><p>To a reaction flask, NBn compound 9 (4 g, 0.012 mol), Pd/C (0.4 g, 10 wt %), AcOH (1 mL), and MeOH (100 mL) were added. After the vessel was evacuated using H2 gas 3 ×, the reaction mixture was stirred at 60 °C under balloon pressure of H2 gas. After 6 h, the mixture was filtered through a celite pad to remove the catalyst, and the solvent was evaporated. The residue was dissolved in CH2Cl2 (100 mL) and was washed with 2 N NaOH; the aqueous layer was extracted with CH2Cl2 (100 mL × 2). The combined organics were washed with brine, dried over Na2SO4, filtered, and evaporated. The crude product was purified by column chromatography to give the secondary amine as a pale yellow oil (3.0 g, > 99 %). 1H NMR (500 MHz, CDCl3): ä 7.24-7.20 (t, J = 8.5 Hz, 1H), 6.88-6.87 (d, J = 1.0 Hz, 1H), 6.82 (s, 1H), 6.73-6.71 (d, J = 8.0 Hz, 1H), 3.74 (s, 3H), 3.71-3.70 (m, 1H), 2.89-2.87 (d, J = 7.5 Hz, 2H), 2.77-2.74 (d, J = 17.0 Hz, 1H), 2.54 (m, 2H), 2.43-2.40 (d, J = 16.5 Hz, 1H), 2.31-2.09 (m, 2H), 1.85-1.73 (m, 2H); 13C NMR (126 MHz, CDCl3): ä 210.3, 159.7, 150.3, 129.6, 116.8, 111.2, 111.0, 55.2, 53.2, 49.4, 47.4, 39.2, 39.1, 38.9, 37.4.</p><p>The secondary amine (1.1 g, 4.5 mmol) was methylated using Pd/C (0.11 g, 10 wt %), AcOH (0.05 mL), formalin (37 % in water, 0.34 mL, 4.5 mmol), and MeOH (20 mL) to give 0.69 g (59 %) of 10a as a colorless oil. 1H NMR (500 MHz, CDCl3): ä 7.28-7.24 (t, J = 8.0 Hz, 1H), 6.92-6.91 (d, J = 7.5 Hz, 1H), 6.87 (s, 1H), 6.77-6.75 (dd, J = 8.0, 2.0 Hz, 1H), 3.78 (s, 3H), 3.45 (br s, 1H), 2.94-2.90 (dd, J = 2.0, 17.0 Hz, 1H), 2.78-2.68 (m, 2H), 2.46-2.43 (m, 2H), 2.39 (s, 3H), 2.34-2.31 (m, 1H), 2.20-2.13 (m, 2H), 1.96-1.86 (m, 2H); 13C NMR (126 MHz, CDCl3): ä 210.5, 159.9, 150.2, 129.8, 117.1, 111.4, 111.3, 56.1, 55.4, 53.3, 47.6, 42.8, 38.8, 38.3, 38.0.</p><!><p>The secondary amine (1.4 g, 5.7 mmol), obtained by N-debenzylation of 9, was converted to its N-phenethyl derivative 10b by the general procedure (1.3 g, 67 %, colorless oil). 1H NMR (500 MHz, CDCl3): ä 7.31-7.21 (m, 6H), 6.96-6.95 (d, J = 8.0 Hz, 1H), 6.91 (s, 1H), 6.80-6.79 (d, J = 8.0 Hz, 1H), 3.82 (s, 3H), 3.61 (br s, 1H), 2.93-2.70 (m, 7H), 2.49-2.34 (m, 3H), 2.30-2.14 (m, 2H), 2.05-1.89 (m, 2H); 13C NMR (126 MHz, CDCl3): ä 210.5, 159.9, 150.2, 140.3, 129.7, 128.9, 128.5, 126.3, 117.1, 111.4, 111.2, 57.1, 55.4, 54.7, 53.4, 45.7, 39.3, 38.2, 37.8, 34.7; IR cm-1 (neat): 2930, 2811, 1694, 1597, 1494, 1462, 1358, 1251, 1278, 1045; HRMS calcd for C23H28NO2: 350.2120 (M+H)+, found 350.2115.</p><!><p>Using the general procedure for O-demethylation, compound 10c was obtained as a pale yellow oil (69 mg, 72 %). 1H NMR (500 MHz, CDCl3): ä 7.28-7.14 (m, 6H), 6.85-6.83 (d, J = 7.5 Hz, 1H), 6.80 (s, 1H), 6.70-6.69 (d, J = 8.0 Hz, 1H), 3.64 (br s, 3H), 2.95-2.72 (m, 7H), 2.46-2.34 (m, 3H), 2.22-2.14 (m, 2H), 2.00-1.94 (dt, J = 13.0, 4.5 Hz, 1H), 1.88-1.86 (d, J = 18.0 Hz, 1H); 13C NMR (126 MHz, CDCl3): ä 211.2, 156.7, 150.0, 139.8, 129.9, 128.8, 128.6, 126.4, 116.5, 114.0, 112.5, 57.0, 54.6, 53.3, 45.9, 39.2, 39.1, 37.7, 37.4, 34.2; IR cm-1 (neat): 3054, 2944, 2816, 1699, 1600, 1495, 1446, 1265, 1180, 1122; HRMS calcd for C22H26NO2: 336.1964 (M+H)+, found 336.1923. Anal. Calcd. for C22H25NO2: C, 78.83; H, 7.52; N, 4.18. Found: C, 78.57; H, 7.54; N, 4.00. Compound 10c was converted into its HBr salt and crystallized from EtOH/EtOAc (64 mg), mp (HBr salt): 244.0 ∼ 244.3 °C.</p><!><p>To a stirred solution of the enantiopure ketone 10a (15 mg, 0.044 mmol) in MeOH (3 mL) was added NaBH4 (12.2 mg, 0.32 mmol) portionwise at 0 °C. The resulting mixture was warmed up to room temperature and stirred for 12 h. The reaction was quenched with H2O and the product was extracted with CH2Cl2 (20 mL × 3). The combined organics were washed with brine, dried over Na2SO4, filtered, and evaporated. The crude product was purified by preparative TLC. 1H NMR (500 MHz, CDCl3): ä 7.30-7.28 (t, J = 7.5 Hz, 1H), 6.92-6.90 (d, J = 7.0 Hz, 1H), 6.86 (s, 1H), 6.77-6.75 (dd, J = 2.0, 8.0 Hz, 1H), 4.15-4.13 (t, J = 4.0 Hz, 1H), 3.84 (s, 3H), 3.12 (br s, 1H), 2.73-2.61 (m, 3H), 2.42 (s, 3H), 2.38-2.35 (d, J = 12.5 Hz, 1H), 2.27-2.25 (d, J = 14.0 Hz, 1H), 2.09-2.06 (d, J = 14.5 Hz, 1H), 1.96-1.92 (dt, J = 13.5, 4.0 Hz, 1H), 1.81-1.77 (dd, J = 10.0, 14.0 Hz, 1H), 1.72-1.69 (d, J = 13.0 Hz, 1H), 1.61-1.56 (dt, J = 3.5, 14.0 Hz, 1H).</p><p>Compound 11a was obtained as a colorless oil using the general procedure for O-demethylation (12 mg, 80 % over two steps). 1H NMR (500 MHz, CDCl3 w/one drop of CD3OD): ä 7.12-7.09 (t, J = 8.0 Hz, 1H), 6.72(s, 2H), 6.62-6.61 (d, J = 7.5 Hz, 1H), 4.03-4.01 (t, J = 5.5 Hz, 1H), 3.10 (br s, 1H), 2.67- 2.61 (m, 2H), 2.30 (s, 3H), 2.23-2.19 (m, 1H), 2.08-1.94 (m, 2H), 1.87-1.80 (m, 3H), 1.70-1.60 (m, 2H); 13C NMR (126 MHz, CDCl3 w/one drop of CD3OD): ä 156.9, 154.6, 129.5, 115.9, 112.9, 112.0, 67.0, 55.4, 46.9, 46.2, 43.1, 38.0, 34.2, 33.2, 32.0; IR cm-1 (neat): 3246, 2938, 1585, 1480, 1449, 1374, 1279, 1194, 1063, 1048; HRMS calcd for C15H22NO2: 248.1651 (M+H)+, found 248.1655. Compound 11a was converted into its HBr salt and crystallized in EtOAc with few drops of MeOH. The absolute configuration of 11a was determined by X-ray crystallography (Fig 1, C1 = S, C5 = R, and C7 = S). Anal. Calcd. for C15H22NO2Br: C, 54.89; H, 6.76; N, 4.27. Found: C, 54.66; H, 6.78; N, 4.08.</p><!><p>To a stirred solution of the ketone 10b (0.17 g, 0.49 mmol) in MeOH (5 mL) was added NaBH4 (1.8 g, 49 mmol) portionwise at 0 °C. The resulting mixture was warmed up to room temperature and stirred for 12 h. The reaction was quenched with water and the product was extracted with CH2Cl2 (5 mL × 3). The combined organics were washed with brine, dried over Na2SO4, filtered, and evaporated. The crude product was purified by column chromatography to give a colorless oil (0.13 g, 76 %). 1H NMR (500 MHz, CDCl3): ä 7.29-7.24 (m, 6H), 6.92-6.90 (d, J = 7.5 Hz, 1H), 6.88 (s, 1H), 6.78-6.76 (dd, J = 8.0, 1.5 Hz, 1H), 4.13 (br s, 1H), 3.83 (s, 3H), 3.27 (br s, 1H), 2.85-2.70 (m, 7H), 2.43-2.40 (dd, J = 12.5, 2.0 Hz, 1H), 2.31-2.29 (d, J = 14.0 Hz, 1H), 2.09-2.06 (m, 1H), 1.99-1.96 (m, 1H), 1.76-1.69 (m, 2H), 1.60-1.55 (dt, J = 3.5, 14.0 Hz, 1H); 13C NMR (126 MHz, CDCl3): ä 159.7, 155.4, 139.9, 129.5, 128.7, 128.6, 126.3, 117.1, 111.2, 110.3, 68.0, 59.0, 55.2, 55.0, 47.9, 45.0, 37.5, 35.5, 34.5, 34.3, 32.8.; IR cm-1 (neat): 3362, 3026, 2935, 2832, 1602, 1582, 1486, 1453, 1430, 1370, 1292, 1259, 1159, 1132, 1051; HRMS calcd for C23H30NO2: 352.2277 (M+H)+, found 352.2236.</p><p>Compound 11b was obtained as a pale brown solid using the general procedure for O-demethylation (70 mg, 66 %). 1H NMR (500 MHz, CDCl3): ä 7.38-7.21 (m, 6H), 7.21 (s, 1H), 6.80-6.77 (m, 2H), 4.27 (br s, 1H), 3.35 (br s, 1H), 2.96-2.75 (m, 7H), 2.54-2.52 (d, 12.0 Hz, 1H), 2.43-2.41 (d, J = 13.5 Hz, 1H), 2.19-2.16 (d, J = 14.5 Hz, 1H), 2.08-2.06 (d, J = 11.5 Hz, 1H), 1.82-1.73 (m, 2H), 1.64-1.61 (d, J = 4.0 Hz, 1H); 13C NMR (126 MHz, CDCl3): ä 157.4, 155.0, 139.7, 129.6, 128.8, 128.7, 126.5, 115.0, 113.5, 112.5, 68.3, 59.0, 55.3, 48.2, 45.1, 37.5, 35.5, 34.4, 34.1, 32.5; IR cm-1 (neat): 3245, 3026, 2929, 1600, 1585, 1496, 1453, 1370, 1277, 1131; HRMS calcd for C22H28NO2: 338.2120 (M+H)+, found 338.2111. Compound 11b was converted into its HBr salt and crystallized from EtOAc (64 mg), mp 227.4-229.2 °C. Anal. Calcd. for C22H28NO2Br: C, 63.20; H, 6.75; N, 3.35. Found: C, 62.86; H, 6.70; N, 3.26.</p><!><p>As noted previously,[1, 15, 16] the recombinant CHO cells (hMOR-CHO, hDOR-CHO and hKOR-CHO) were produced by stable transfection with the respective human opioid receptor cDNA, and were provided by Dr. Larry Toll (SRI International, CA). The cells were grown on plastic flasks in DMEM (100%) (hDOR-CHO and hKOR-CHO) or DMEM/F-12 (50%/50%) medium (hMOR-CHO) containing 10% FBS, and G-418 (0.10-0.2 mg/mL) under 95% air/5% CO2 at 37 °C. Cell monolayers were harvested and frozen at -80 °C.</p><!><p>We used [3H][D-Ala2 -MePhe4,Gly-ol5]enkephalin ([3H]DAMGO, SA=44-48 Ci/mmol) to label MOR, [3H][D-Ala2,D-Leu5]enkephalin ([3H]DADLE, SA=40-50 Ci/mmol) to label DOR and [3H](-)-U69,593 (SA=50 Ci/mmol) to label KOR binding sites. On the day of the assay, cell pellets were thawed on ice for 15 min then homogenized with a polytron in 10 mL/pellet of ice-cold 10 mM Tris-HCl, pH 7.4. Membranes were then centrifuged at 30,000 × g for 10 minutes, resuspended in 10 ml/pellet ice-cold 10mM Tris-HCl, pH 7.4 and again centrifuged 30,000 × g for 10 min. Membranes were then resuspended in 25 °C 50 mM Tris-HCl, pH 7.4 (∼100 mL/pellet hMOR-CHO, 50 mL/pellet hDOR-CHO and 120 mL/pellet hKOR-CHO). All assays took place in 50 mM Tris-HCl, pH 7.4, with a protease inhibitor cocktail [bacitracin (100 μg/mL), bestatin (10 μg/mL), leupeptin (4 μg/mL) and chymostatin (2 μg/mL)], in a final assay volume of 1.0 mL. All drug dilution curves were made up with buffer containing 1 mg/mL BSA. Nonspecific binding was determined using 20 μM levallorphan ([3H]DAMGO and [3H]DADLE) and 1 μM (-)-U69,593 (for [3H]U69,593 binding). [3H]Radioligands were used at ∼ 2 nM concentrations. Triplicate samples were filtered with Brandel Cell Harvesters (Biomedical Research & Development Inc., Gaithersburg, MD), over Whatman GF/B filters, after a 2 h incubation at 25 °C. The filters were punched into 24-well plates to which was added 0.6 mL of LSC-cocktail (Cytoscint). Samples were counted, after an overnight extraction, in a Trilux liquid scintillation counter at 44% efficiency. Opioid binding assays had ∼30 μg protein per assay tube. Inhibition curves were generated by displacing a single concentration of radioligand by 10 concentrations of drug. The pooled data of three experiments (typically 30 data points) are fit to the two-parameter logistic equation for the best-fit estimates of the IC50 and N values: Y=100/(1+([INHIBITOR]/IC50)N), where "Y" is the percent of control value. Ki values for test drugs are calculated according to the standard equation: Ki = IC50/(1+[radioligand]/Kd]). For the [3H]radioligands, the following Kd values (nM±SD, n=3) were used in the Ki calculation: [3H]DAMGO (0.93±0.04), [3H]DADLE (1.9±0.3) and [3H](-)-U69,593 (11±0.6). The corresponding Bmax values were (fmol/mg protein ± SD, n=3): [3H]DAMGO (1912±68), [3H]DADLE (3655±391) and [3H](-)-U69,593 (3320±364).</p><!><p>Single-crystal X-ray diffraction data on compounds 11a and 7b were collected using MoKα radiation and a Bruker APEX-2 CCD area detector. Crystals were prepared for data collection by coating with high viscosity microscope oil. The oil-coated crystal was mounted on a micro-mesh mount (Mitergen, Inc.) and transferred to the diffractometer. The structures were solved by direct methods and refined by full-matrix least squares on F2 values using the programs found in the SHELXTL suite (Bruker, SHELXTL v6.10, 2000, Bruker AXS Inc., Madison, WI). Corrections were applied for Lorentz, polarization, and absorption effects. Parameters refined included atomic coordinates and anisotropic thermal parameters for all non-hydrogen atoms. Hydrogen atoms on carbons were included using a riding model [coordinate shifts of C applied to H atoms] with C-H distance set at 0.96 Å. Complete.</p><p>Atomic coordinates for 11a and 7b have been deposited with the Cambridge Crystallographic Data Centre (deposition numbers CCDC 933697, and CCDC 933698, respectively). Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK [fax: +44(0)-1223-336033 or e-mail: deposit@ccdc.cam.ac.uk.</p><!><p>A 0.288 × 0.209 × 0.062 mm3 crystal was prepared for data collection and a data set collected at 296°K. The crystal was monoclinic in space group P 21, with unit cell dimensions a = 8.0575(6), b = 6.1223(5), c = 15.2147(14) Å, and β = 102.892(3)°. Data was 99.3% complete to 25.00° θ (∼ 0.83 Å) with an average redundancy of 3.97. The final anisotropic full matrix least-squares refinement on F2 with 181 variables converged at R1 = 6.29%, for the observed data and wR2 = 18.85% for all data.</p><!><p>A 0.258 × 0.184 × 0.058 mm3 crystal was prepared for data collection and a data set collected at 100°K. The crystal was orthorhombic in space group P b c a, with unit cell dimensions a = 19.330(2), b = 8.4573(9), and c = 19.3738(19) Å. Data was 99.5% complete to 25.00° θ (∼ 0.83 Å) with an average redundancy of 8.30. The final anisotropic full matrix least-squares refinement on F2 with 183 variables converged at R1 = 5.00%, for the observed data and wR2 = 16.25% for all data.</p>