A solution of (2'S)-4-(N-tert-Butoxycarbonylpyrrolidin-2-yl)-4-oxo-butanoylglycylglycin methyl ester (15) (250mg, 0.72mmol) in dichloromethane (2ml) was treated with trifluoroacetic acid (1ml) in dichloromethane (1ml) and the resulting mixture stirred at room temperature for 1h. The solution was then concentrated to dryness in vacuo, then the residue was dissolved in tetrahydrofuran (2ml) and placed under an argon atomsphere. N-Methylmorpholine (160microl, 1.45mmol) was then added, and the mixture stirred for 5min, before being added to a stirred solution of N-tert-butoxycarbonyl-L-alanine (150mg, 0.80mmol) in dry tetrahydrofuran (2ml) that had been pre-treated with N-methylmorpholine (90microl, 0.83mmol) and ethyl chloroformate (80microl, 0.80mmol) at 0degC for 10min. The resulting mixture was stirred at 0degC for 30min., then at room temperature for 5.5h. Chloroform (50ml) was then added, and the solution washed sequentially with 1M hydrochloric acid (40ml), sodium bicarbonate (40ml), water (40ml), and saturated sodium chloride (40ml), then dried over magnesium sulphate and concentrated under reduced pressure. The residue was recrystallised from ethyl acetate to give (2'S)-4-(N-tert-butoxycarbonyl-L-alanylpyrrolidin-2-yl)-4-oxo-butanoylglyylglycine methyl ester (17) (0.17g, 57%) as a white solid, m.p. 124degC, [alpha]D -52deg (c 1.0; dichloromethane).

Proton Nmr deltaH (300MHz, CDCl3) 1.31(3H, d, J=7.0Hz, CH3), 1.40(9H, s, C(CH3)3), 2.22-1.91(4H, m, 2xCH2), 2.47(1H, ddd, J=6.0, 6.0, 16.0Hz, CHaHbCO), 2.60(1H, ddd, J=5.5, 8.0, 16.0Hz, CHaHbCO), 2.74-2.88(2H, m, CH2CO), 3.52-3.74(2H, m, CH2N), 3.72(3H, s, OCH3), 3.92-4.03(2H, m, CH2CO2Me), 4.40-4.49(1H, m, CHMe), 4.58(1H, dd, J=5.0, 9.0Hz, CHN), 5.29(1H, br. d, J=8.0Hz, NHBoc), 6.25(1H, br. s, NH); Vmax: 3342, 1708, 1642cm^-1; m/z (NH3, CI) 431(M+NH4⁺, 26%), 414(M+H⁺, 100%), 304(20%), 70(57%). Found[M+H]⁺ 414.2210, C19H32N3O7 requires 414.2240.

Preparation of (2'S)-4-(N-tert-Butoxycarbonyl-L-alanylpyrrolidin-2-yl)-4-oxo-butanoylglyyl-L-alanine Methyl Ester (18)

A solution of (2'S)-4-(N-tert-butoxycarbonylpyrrolidin-2-yl)-4-oxo-butanoylglycyl-L-alaine methyl ester (16) (0.50g, 1.40mmol) in dichloromethane (2ml) was treated with trifluoroacetic acid (1ml) in dichloromethane (1ml) and the resulting mixture stirred at room temperature for 1h. The solution was then concentrated to dryness in vacuo, then the residue was dissolved in dry N,N-dimethylformamide (4ml) and placed under an argon atomsphere. N-Methylmorpholine (0.35ml, 3.18mmol) was then added, and the mixture stirred for 1h, before being added to a stirred solution of N-tert-butoxycarbonyl-L-alanine (0.27g, 1.42mmol) in dry N,N-dimethylformamide (2ml) that had been pre-treated with N-methylmorpholine (0.15ml, 1.36mmol) and ethyl chloroformate (0.13ml, 1.36mmol) at 0degC for 30min. The resulting mixture was stirred at 0degC for 15min., then at room temperature for 5.5h. Water (50ml) was then added, and the mixture extracted with ethyl acetate (3x80ml). The combined extracts were washed sequentially with 1M hydrochloric acid (50ml), sodium bicarbonate (50ml), and saturated sodium chloride (3x50ml), then dried over magnesium sulphate and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (petroleum ether/ethyl acetate 1:3) to give (2'S)-4-(N-tert-butoxycarbonyl-L-alanylpyrrolidin-2-yl)-4-oxo-butanoylglyyl-L-alanine methyl ester (18) (0.16g, 27%) as a white solid, m.p. 126degC, [alpha]D -44deg (c 1.0; dichloromethane).

Proton Nmr deltaH (300MHz, CDCl3) 1.30(3H, d, J=7.0Hz, CH(CH3)NHBoc), 1.34(3H, d, J=7.0Hz, CH(CH3)CO2Me), 1.39(9H, s, OC(CH3)3), 1.86-2.18(4H, m, 2xCH3), 2.41(1H, ddd, J=6.0, 6.0, 15.5Hz, CHaHbCO), 2.57(1H, ddd, J=5.0, 8.5, 15.5Hz, CHaHbCO), 2.73(1H, ddd, J=5.5, 5.5, 18.0Hz, CHa'HbCO), 2.87(1H, ddd, J=5.5, 8.5, 18.0Hz, CHaHb'CO), 3.51-3.74(2H, m, CH2N), 3.70(3H, s, OCH3), 4.39-4.55(2H, m, 2xCHMe), 4.58 (1H, dd, J=5.0, 9.0Hz, CHN), 5.29(1H, br. d, J=8.0Hz, NHBoc), 6.25(1H, d, J=7.0Hz, NH); Vmax: 3429, 1729, 1704, 1674, 1644cm^-1; m/z (NH3, CI) 428(M+H⁺, 100%), 70(90%). Found [M+H]⁺ 428.2403, C20H34N3O7 requires 428.2396