Reaction Steps
Proline (3S,5R) methyl-7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (B7a) (35.2 mg, 102 μmol) in DMF (0.7 ml) was treated with (S)-2-(tert-butoxycarbonylamino)-3,3-dimethylbutanoic acid (33.3 mg, 144 μmol, 1.2 eq), 1-hydroxybenzotriazole (HOBT, 16.5 mg, 122 μmol, 1.2 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 63.3 mg, 330 μmol, 3.2 eq), and N,N-Diisopropylethylamine (DIEA, 86 μA, 68 μmol, 4 eq) for three days at room temp. The reaction was quenched with 1M histamine in water (381 μl, 3.7 eq) and blown to dryness with N2 gas. The residue was extracted with ethyl acetate (2×10 ml) and 0.5 N hydrochloric acid (2×5 ml). The organics were washed with 1M potassium carbonate in water (2×2 ml), dried over sodium sulfate, filtered and concentrated. The product was purified on silica (ethyl acetate/hexane gradient) to give (3S,5R)-methyl-2-((S)-2-(tert-butoxycarbonylamino)-3,3-dimethylbutanoyl)-7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (C7a) (36.9 mg, 70.6 μmol). 1H NMR (CDCl3) δ 1.07 (s, 9H), 1.39 (s, 9H), 1.60 (s, 1H), 2.10 (m, 1H), 2.45 (m, 1H), 2.71 (s, 2H), 3.60 (m, 2H), 3.75 (s, 3H), 3.99 (d, 1H), 4.13 (d, 1H), 4.30 (d, 1H), 4.60 (t, 1H), 5.08 (d, 1H), 7.15 (m, 1H), 7.29 (m, 2H), 7.60 (m, 2H); LCMS for C26H36N3O6Cl 521.23 [M], found ES+ 522.23 [M+H]+ and 422.23 [M+H-Boc]+ at 3.5 minutes retention time, using 10-90% acetonitrile/water (with 0.1% formic acid buffer) gradient over 5 minutes. The minor diastereomer (3S,5S)-methyl 7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (B7b) (12.4 mg, 36 μmol) was reacted similarly to give (3S,5S)-methyl-2-((S)-2-(tert-butoxycarbonylamino)-3,3-dimethylbutanoyl)-7-(3-chloro phenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (C7b) (9.4 mg, 18 μmol, 50%). LCMS for C26H36N3O6Cl 521.23 [M], found ES+ 522.23 [M+H]+ and 422.23 [M+H-Boc]+ at 3.5 minutes retention time, using 10-90% acetonitrile/water (with 0.1% formic acid buffer) gradient over 5 minutes.
