The reagents and solvents were purchased from commercial sources (Fisher and Sigma-Aldrich) and used directly. Analytical thin-layer chromatography was performed on ready-to-use plates with silica gel 60 (Merck, F254). Flash column chromatography was performed over silica gel (grade 60, 230–400 mesh) on Teledyne Isco CombiFlash purification system. Final compounds were purified on preparative reversed phase high-pressure liquid chromatography (RP-HPLC) and was performed on Agilent 1260 Series system with Agilent 1260 Infinity II Variable Wavelength Detector (G7114A, UV = 254 nM) and a Waters BEH C18 (130Å, 5 μm, 10 mm X 250 mm) column. Final compounds were eluted using a gradient 95% water (with 0.1% formic acid) and 5% acetonitrile (with 0.1% formic acid) at a flow rate of 4 mL/min over 30 min.
NMR spectra were acquired on a Bruker AV500 instrument (500 MHz for 1H-NMR, 126 MHz for 13C-NMR). TLC-MS were acquired using Advion CMS-L MS. The Agilent 1260 Infinity II Variable Wavelength Detector (G7114A, UV = 254 nM) and an Agilent ZORBAX RR SB-C18 (80Å, 3.5 μm, 4.6 x 150 mm) at a flow rate of 1 mL/min using a solvent system of 100% water with 0.1% TFA to 40 or 60% methanol over 20min were used to assess purity of final compounds. All the purity of target compounds showed >95% in RP-HPLC. FP was monitored on a BMG CLARIOstar microplate reader.
NMR spectra were acquired on a Bruker AV500 instrument (500 MHz for 1H-NMR, 126 MHz for 13C-NMR). TLC-MS were acquired using Advion CMS-L MS. The Agilent 1260 Infinity II Variable Wavelength Detector (G7114A, UV = 254 nM) and an Agilent ZORBAX RR SB-C18 (80Å, 3.5 μm, 4.6 x 150 mm) at a flow rate of 1 mL/min using a solvent system of 100% water with 0.1% TFA to 40 or 60% methanol over 20min were used to assess purity of final compounds. All the purity of target compounds showed >95% in RP-HPLC. FP was monitored on a BMG CLARIOstar microplate reader.