To a solution of the isocyanate of Example 4E (1.60 g, 8.33 mmol) in CH2Cl2 (40 mL) was added 4 Å molecular sieves (˜0.5 g), followed by diisopropylethylamine (1.74 mL, 9.99 mmol) and methyl 2-amino-3-[(tert-butyloxycarbonyl)amino]-propanoate hydrochloride (2.33 g, 9.16 mmol) and the resulting mixture was stirred at rt for 1 h. DBU (1.50 mL, 9.99 mmol) was added and the mixture stirred at rt overnight. Additional DBU (0.37 mL, 2.47 mmol) was then added and the mixture stirred at rt for 5 h. The mixture was then filtered and the solid washed with CH2Cl2. The filtrate was washed with water and brine, dried (MgSO4), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, CH2Cl2/CH3OH, 98:2 to 80:20 gradient) to afford the title compound (2.71 g) as a beige foam. 1H NMR (CDCl3) δ 1.44 (s, 9H), 2.28, 2.29 (s, 3H), 3.56-3.60 (m, 1H), 3.68-3.78 (m, 1H), 4.32-4.35 (m, 1H), 5.02, 5.08 (br s, 1H), 6.63 (br s, 1H), 7.20, 7.25 (d, J=8.3 Hz, 1H), 7.62, 7.63 (d, J=8.3 Hz, 1H); HPLC a) column: Phenominex C18 4.6×50 mm, 4 min gradient, 10% MeOH/90% H2O/0.1% TFA to 90% MeOH/10% H2O/0.1% TFA, 1 min hold, 4 mL/min, UV detection at 220 nm, 3.00 min retention time; HPLC b) column: Shimadzu Shim-Pack VP-ODS C18 4.6×50 mm, 4 min gradient, 10% MeOH/90% H2O/0.1% TFA to 90% MeOH/10% H2O/0.1% TFA, 1 min hold, 4 mL/min, UV detection at 220 nm, 2.89 min retention time (99%); HPLC c) column: Daicel Chiralcel OD 4.6×250 mm, Isocratic 25% isopropanol/hexanes, 30 min, 1 mL/min, UV detection at 220 nm, 22.26 min retention time (99%); MS (ES) m/z 377 [M−H]−.
