To a mixture of (14-tert-butoxycarbonylamino-18-hydroxy-2,15-dioxo-3,16-diaza-tricyclo[14.3.0.04,6]nonadec-7-ene-4-carboxylic acid (150 mg, 0.32 mmol; prepared in Ex. 25, step 4) in 3 mL of DMSO was added potassium t-butoxide (91 mg, 0.81 mmol). The mixture was stirred at rt for 5 min, and then 2-fluoro-pyridine (39 mg, 0.40 mmoL) was added and the mixture was stirred at rt overnight. The reaction was quenched by adding 10 mL of water, and then the pH was adjusted to 4 using 0.1 N HCL. It was then extracted with ethyl acetate, dried, concentrated in vacuo to obtain a yellow oil. This crude product was purified by flash chromatography on silica gel eluting with 10% methanol in ethyl acetate to isolate 100 mg (56%) of 14-tert-butoxycarbonylamino-2,15-dioxo-18-(pyridin-2-yloxy)-3,16-diaza-tricyclo[14.3.0.04,6]nonadec-7-ene-4-carboxylic acid as a white solid. 1H NMR (500 MHz, MeOD) δ 1.24-1.53 (m, 6H), 1.36 (s, 9H), 1.55-1.68 (m, 3H), 1.73-1.90 (m, 1H), 2.02-2.11 (m, 1H), 2.16 (q, J=8.65 Hz, 1H), 2.35-2.45 (m, 1 H), 2.51 (dd, J=7.63, 2.75 Hz, 2H), 4.06-4.14 (m, 1H), 4.27 (d, J=11.29 Hz, 1 H), 4.33 (s, 1H), 4.58 (t, J=7.93 Hz, 1H), 5.46-5.57 (m, 2H), 5.70 (s, 1H), 6.79 (d, J=8.54 Hz, 1H), 6.93-7.02 (m, 1H), 7.68 (t, J=7.63 Hz, 1H), 8.16 (d, J=4.85 Hz, 1 H). LC-MS (YMC Xterra ODS S7 micrometer column: 3.0×50 mm length. Gradient: 100% Solvent A/0% Solvent B to 0% Solvent A/100% Solvent B. Gradient time: 4 min. Hold time: 1 min. Flow rate: 4 mL/min. Detector Wavelength: 220 nm. Solvent A: 10% MeOH/90% H2O/0.1% TFA. Solvent B: 10% H2O/90% MeOH/0.1% TFA.)(Retention time: 2.90 min), MS m/z 543 (M++1).
