To a mixture of 3-(6-methylpyridin-2-yl)-4-(trifluoromethyl)isoxazole-5-carboxylic acid (100 mg, 0.367 mmol) and DIEA (0.128 mL, 0.735 mmol) in DMF (3 mL) was added BOP—Cl (103 mg, 0.404 mmol). After 15 minutes, (3S)-ethyl 1-(2-hydroxy-2-(4-((Z)—N′-hydroxycarbamimidoyl)phenyl)ethyl)piperidine-3-carboxylate, (refer to Preparation 14B) (136 mg, 0.404 mmol) was added. The reaction mixture was stirred at room temperature. After 1 hour, 1M TBAF/THF (0.367 mL, 0.367 mmol) was added and the reaction mixture was stirred overnight. The reaction mixture was diluted with ethyl acetate and washed with saturated NaCl. The organic layer was dried with MgSO4, filtered, and concentrated. The resulting material was purified by on a silica gel cartridge using an EtOAc/hexanes gradient. The peak with the desired mass was isolated by LCMS. This residue was dissolved in MeCN and treated with 6N HCl overnight. The reaction mixture was filtered and purified by HPLC. HPLC conditions: PHENOMENEX® Luna C18 5 micron column (250×30 mm); 25-100% CH3CN/water (0.1% TFA); 25 minute gradient; 30 mL/min. Isolated fractions with correct mass were freeze-dried overnight to yield 6 mg of (3S)-1-(2-hydroxy-2-(4-(5-(3-(6-methylpyridin-2-yl)-4-(trifluoromethyl)isoxazol-5-yl)-1,2,4-oxadiazol-3-yl)phenyl)ethyl)piperidine-3-carboxylic acid as a TFA salt. 1H NMR (400 MHz, MeOH-d3) δ ppm 8.14 (2H, d, J=8.35 Hz), 7.81 (1H, t, J=7.69 Hz), 7.63 (3H, t, J=7.25 Hz), 7.39 (1H, d, J=7.91 Hz), 5.12-5.24 (1H, m), 3.93 (1H, d, J=12.74 Hz), 3.70-3.86 (1H, m), 3.57 (1H, d, J=11.86 Hz), 3.32-3.44 (1H, m), 3.24-3.32 (2H, m), 2.73-3.12 (2H, m), 2.48-2.56 (3H, m), 1.74-2.32 (2H, m), 1.41-1.65 (1H, m). MS (m+1)=544. HPLC Peak RT=2.96 minutes (Analytical Method A). Purity=90%.
