To a stirred solution of {4-[(2R,3R)-3-{[2-(2,3-dihydro-1-benzofuran-5-yl)-2-oxoethyl]thio}-1-(4-fluorophenyl)-4-oxoazetidin-2-yl]phenoxy}acetic acid (Method 2) (15.7 mg, 0.031 mmol) in DMF (2 ml, dry) was added N-methylmorpholine (10 μl, 0.091 mmol). TBTU (13.1 mg, 0.041 mmol) and additional DMF (2 ml) were added and the reaction mixture was stirred under at 30° C. for 25 minutes. Glycyl-3-cyclohexyl-D-alanine (7.1 mg, 0.031 mmol) (Method 7) was added and the reaction mixture was stirred for 2 hours. The formation of the ketone of the title compound was confirmed. M/z: 716.57 (M−1). Without further purification, methanol (6 ml) and sodium borohydride (16.1 mg, 0.043 mmol) were added and the reaction mixture was stirred for 20 minutes. Ammonium acetate (85.5 mg) was added. The methanol was removed under reduced pressure and the remaining DMF-solution was purified with preparative HPLC on a C8 column. A gradient from 20 to 55% MeCN in 0.1M NH4OAc was used as eluent. The pure fractions were collected and the MeCN was removed under reduced pressure. The residue was extracted between EtOAc and water, the water phase was acidified to pH 2 with KHSO4 (2M). The phases were separated and the organic phase was passed through a phase separator. The solvent was removed under reduced pressure. The residue was dissolved in ACN and water. After lyophilisation, the title compound was obtained. H-NMR (400 MHz, DMSO-d6): 0.69-1.67 (m, 13H), 2.76-2.91 (m, 2H), 2.98-311 (m, 2H), 3.75 (d, 2H), 4.15-4.25 (m, 2H), 4.40-4.47 (m, 2H), 4.49 (s, 2H), 4.55-4.64 (m, 1H), 5.00 (b, 1H), 5.54 (b, 1H), 6.61 (dd, 1H), 6.96 (d, 3H), 7.08-7.16 (m, 3H), 7.18-7.24 (m, 2H), 7.34 (d, 2H), 8.07 (d, 1H), 8.19 (t, 1H), 12.51 (b, 1H). M/z: 718.57 (M−1).
