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

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 (0.10C0.2 mg/mL) less than 95% air flow/5% CO2 at 37 C. carboxylic acid (()?13 and ()?14). Conversion of ()?13 and ()?14 to the racemic mixture of and esters (()?15) was carried out using the procedure of Kaiser et al.7 Separation of the racemic isomer ()?16 from ()?15 was achieved under reflux conditions using one equivalent of NaOH in EtOH-H2O. The esters were more stable to the alkaline conditions and required 2 equivalents of NaOH for hydrolysis to the racemic acid ()?14, with concentrated HCl utilized for neutralization. Open in a separate Eriocitrin window Plan 3 Synthesis of racemic isomer; (e) NaOH, EtOH, H2O, reflux, 70%. The commercially available racemic isomer ()?13 was resolved6, 11 while shown in Plan 4. The 1and 1mixture of enantiomers ()?13) was separated using (+)- dehydroabietylamine. The 1salt (17) that created was purified by recrystallization from aqueous methanol. Foundation hydrolysis of the salt 17 gave the desired (?)?13 (salt formation with (+)-dehydroabietylamine was recovered and the base was reacted with one equivalent of quinine in EtOAc. The 1salt (18) that created was recrystallized from EtOAc; foundation hydrolysis offered (+)?13 (racemate) was carried out similarly (Scheme 5)6, 11 to give (?)?14 (series (Plan 6 top, (?)? and (+)? 29 through 32), and another set of four amines in the 1series (Plan 6 bottom, (?)? and (+)?29 through 32). Open in a separate window Plan 6 Synthesis of the and compounds (+)?29 and (?)?30 had high affinity for the -receptor (isomer (+)?29 (Fig. 2, form (+)?31 (stereochemistry in (+)?31 needs to be avoided in the design of a higher afffinity ligand. Between the two stereoisomers ((+)?29 and (?)?30) in the 1stereoisomers ((+)?31, and (?)?32). Also, it was observed that when the stereochemistry of the NPCM side-chain is definitely invariant, the (1((((Hz) projects of 1H resonance coupling. The high resolution electrospray ionization (ESI) mass spectra were obtained on a Waters LCT Leading time-of-flight (TOF) mass spectrometer. Thin-layer chromatography (TLC) was performed on 0.25 mm Analtech GHLF silica gel. Adobe flash column chromatography was performed with Bodman silica gel LC 60 A. Enantiomeric purity was assessed by HPLC (Shimadzu LC-6A having a Shimadzu SPD-6AV UM detector (at 254 nm) using Daicel’s Chiralcel OD and OJ column (250 4.6 mm). Elemental analyses were performed by Atlantic Microlabs Inc., Norcross, GA, and were within 0.4% for C, H, and N. The elemental analysis, 1H NMR, and 13C NMR were used to confirm 95% purity. General procedure for syntheses of (= 12.6 Hz), 6.08 (d, 1H, = 12.6 Hz), 3.11 (m, 2H), 1.19 (d, 12H, Eriocitrin = 6.6 Hz); 13C NMR (CDCl3) 173.45, 137.26, 130.05, 129.55, 129.04, 127.94, 127.29, 46.19, 19.20. The diisopropylammonium (= 4.5 Hz), 7.58 (d, 1H, = 1.8 Hz), 7.33C7.38 (m, 3H), 7.07 (d, 1H, = 12.6 Hz), 5.97 (d, 1H, = 12.6 Hz); 13C NMR (CDCl3) 171.16, 164.00, 134.60, 130.15, 129.60, 128.32, 118.85. (1.0, MeOH); 1H NMR (CDCl3) 7.71 (m, 1H), 7.51C7.57 (m, 2H), 7.33C7.42 (m, 3H), 7.24C7.30 (m, 1H), 6.86C6.96 (m, 3H), 6.76 (dd, 1H, = 8.4, 2.4 Hz), 4.70 (br-d, 1H, = 124 Hz), 3.90C4.05 (m, 2H), 3.81 (s, 3H), 1.90C2.30 (m, 7H), 1.50C 1.80 (m, 3H); 13C NMR (CDCl3) 166.69, 166.27, 159.60, 153.23, 153.00, 142.22, 142.05, 135.51, 129.42, 129.34, 128.73, 127.71, 118.45, 117.93, 117.17, 117.08, 111.52, 111.44, 110.41, 55.16, 49.26, 46.78, 42.44, 39.89, 38.86, 38.53, 36.95, 36.43, 36.20, 35.55, 34.95, 34.71, 31.84, 29.17, 20.45, 20.32; HRMS (TOF MS Sera+) calcd for C24H28NO2 (M+H)+ 362.2120, found: 362.2117. (1.0, MeOH); 1H NMR (CDCl3): 7.71 (dd, 1H, = 8.1, 2.4 Hz), 4.70 (br-d, 1H, = 124 Hz), 3.90C4.05 (m, 2H), 3.81 (s, 3H), 1.90C 2.30 (m, 7H), 1.50C1.80 (m, 3H); 13C NMR (CDCl3) 166.65, 166.23, 159.58, 153.19, 152.97, 142.17, 142.00, 135.48, 129.39, 129.31, 128.70, 127.68, 118.43, Rabbit Polyclonal to SEPT6 117.91, 117.14, 117.05, 111.49, 111.41, 110.39, 55.13, 49.22, 46.75, 42.40, 39.86, 38.84, 38.50, 36.93, 36.41, 36.17, 35.54, 34.92, 34.69, 31.82, 29.14, 20.44, 20.29; HRMS (TOF MS Sera+) calcd for C24H28NO2 (M+H)+ 362.2120, found 362.2117. General procedure for syntheses of phenolic amides ((10.42, MeOH); 1H NMR (CDCl3) 7.72 (m, 1H), 7.48C7.55 (m, Eriocitrin Eriocitrin 2H), 7.28C7.44 (m, 4H), 7.15C7.22 (m, 1H), 6.81C6.97 (m, 3H), 6.75 (dd, 1H, = 8.4, 2.4 Hz), 4.70 (br-d, 1H, = 124 Hz), 3.75C4.05 (m, 2H), 1.40C2.25 (m, 10H); HRMS.