A 100 mL round bottom flask equipped with a magnetic stir bar was flushed with nitrogen and charged with 2.31 g (5 mmol, 1 eq) of 1-{3-[(R)-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)(3-hydroxyphenyl)-methyl]-benzoyl}-4-piperidinone (Example 136), 1.41 g (8.4 mmol, 1.68 eq) of ethyl 4-aminobutyrate hydrochloride, 0.47 g (8.4 mmol, 1.68 eq) of potassium hydroxide, and 10 mL of ethanol. The reaction was stirred vigorously at room temperature under nitrogen for 2.5 hours. A solution of 0.32 g (5 mmol, 1 eq) of sodium cyanoborohydride in 5 mL of ethanol was added and the reaction was stirred overnight. The reaction was quenched by careful addition of 1 mL of saturated aqueous NH4Cl solution and stirred vigorously for 30 minutes and concentrated in vacuo. The residue was partitioned between 50 mL dichloromethane and water, and the aqueous solution was adjusted to pH=8-9 using saturated sodium bicarbonate solution. The organic layer was separated and dried over sodium sulfate/magnesium sulfate and the solvent was removed in vacuo, leaving 2.81 g of crude product as a viscous, amber-colored oil. Chromatography of this material on a pre-packed 4×15 cm Biotage® silica gel cartridge eluting with dichloromethane/ethyl acetate with 2% ammonium hydroxide yielded 1.57 g (53%) of 4-(1-{3-[(R)-((2S,5R)-4-Allyl-2,5-dimethyl-piperazin-1-yl)-(3-hydroxy-phenyl)-methyl]-benzoyl}-piperidin-4-ylamino)-butyric acid ethyl ester as an off-white foam. Calculated for C34H48N4O4 0.1 CH2Cl2: %C, 69.98; H, 8.30; N, 9.57. Found: %C, 70.07; H, 8.39; N, 9.65. 1H NMR (300 MHz, CDCl3): {tilde over (□)}{tilde over (□)}1{tilde over (□)}1m, 1H), 7.38-7.23 (m, 3H), 7.11 (t, J=7.9 Hz, 1H), 6.68-6.56 (m, 1H), 6.65 (t, J=7.9 Hz, 2H), 5.93-5.80 (m, 1H), 5.21-5.14 (m, 3H), 4.52 (br s, 1H), 4.14 (q, J=7.2 Hz, 2H), 3.67 (m, 1H), 3.36 (br d, 1H), 2.98-2.78 (m, 4H), 2.73-2.55 (m, 5H), 2.46 (m, 1H), 2.37 (t, J=7.2 Hz, 2H), 2.12 (dd, J=1, 9.3 Hz, 1H), 1.92 (m, 1H), 1.79 (quint, J=7.1 Hz, 2H), 1.26 (t, J=7.1 Hz, 3H), 1.15 (br d, J=5.6 Hz, 3H), 0.98 (d, J=6.2 Hz, 3H).
