General procedure for the synthesis of ethyl (2S,3S)-3-(((S)-1-(isopentylamino)-4-methyl-1-oxopentan-2-yl)carbamoyl)ethylene oxide-2-carboxylate from rel-(2R,3R)-3-(ethoxycarbonyl)oxirane-2-carboxylate and N1-isopentyl-L-leucinamide under nitrogen protection: under nitrogen protection (2S,3S)-3-((S)-3-(isopentylamino)-1-(isopentylamino)oxirane-2-carboxylic acid (25g, 0.16mol) and (S)-1-(isopentylamino)-1-(isopentylamino)-1-(isopentylamino)-1-(isopentylamino)-1-(isopentylamino)-1-(isopentylamino)-1-(isopentylamino)carbamoyl)ethylene oxide-2-carboxylic acid was prepared. -(ethoxycarbonyl)oxirane-2-carboxylic acid (25 g, 0.16 mol) and (S)-1-(isoamylamino)-2-amino-4-methyl-1-oxopentane hydrochloride (38 g, 0.16 mol) were dissolved in dichloromethane (750 mL) and cooled to 0 °C (ice water bath). Subsequently 2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (65 g, 0.17 mol) and diisopropylethylamine (DIPEA, 56 mL, 0.31 mol) were added. The reaction mixture was stirred at 0 °C for 1 h. After removing the ice bath, the reaction mixture was continued to be stirred for 2 h at room temperature. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane (750 mL), washed sequentially with saturated sodium bicarbonate solution (2 times) and saturated sodium chloride solution (1 time), the organic phase was dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a yellow viscous oily crude product. The crude product was purified by a silica gel column (60 mm × 300 mm), eluting with a gradient of 10% to 50% ethyl acetate in hexane solution. The combined eluents (2.5 L) were collected and concentrated under reduced pressure to give E64d (53 g, 100% yield) as a white solid. Recrystallization of E64d (36 g) in methyl tert-butyl ether in 1% ethanol (MTBE, 535 mL) afforded white short needle-like crystals of E64d (25 g, 69% yield) with a melting point of 123-124 °C.1H NMR (500 MHz, CDCl3) δ 6.68 (d, J=8.4 Hz, 1H), 6.08 (br s, 1H), 6.08 (br s, 1H), 6.08 (br s, 1H), 6.08 (br s, 1H), and 6.08 (br s, 1H). 4.36-4.40 (m, 1H), 4.21-4.28 (m, 2H), 3.67 (d, J=1.9 Hz, 1H), 3.46 (d, J=1.9 Hz, 1H), 3.19-3.30 (m, 2H), 1.49-1.65 (m, 4H), 1.38 (q, J=7.4 Hz, 2H), 1.30 (t, J= 7.0 Hz, 3H), 0.89-0.93 (m, 12H); ESI MS m/z 343.2 (M+H)+; elemental analysis (C17H30N2O5) is in accordance with the theoretical values; HPLC purity is in accordance with the requirements.
