Combiflash purification system

Starting materials, reagents, and solvents were purchased from commercial vendors unless otherwise noted. ¹H NMR spectra were measured on a Varian 400 MHz NMR instrument. The chemical shifts were reported as δ ppm relative to TMS using residual solvent peak as the reference unless otherwise noted. The following abbreviations were used to express the multiplicities: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet; br = broad. High-performed liquid chromatography (HPLC) was carried out on GILSON GX-281 using Waters C18 5μM, 4.6*50mm and Waters Prep C18 5μM, 19*150mm reverse phase columns, eluted with a gradient system of 5:95 to 95:5 acetonitrile:water with a buffer consisting of 0.05% TFA. Mass spectra (MS) were performed on HPLC/MSD using electrospray ionization (ESI) for detection. All reactions were monitored by thin layer chromatography (TLC) carried out on Merck silica gel plates (0.25 mm thick, 60F254), visualized by using UV (254 nm) or dyes such as KMnO₄, p-Anisaldehyde and CAM (Cerium Ammonium Molybdate or Hanessian's Stain). Silica gel chromatography was carried out on a Teledyne ISCO CombiFlash purification system using pre-packed silica gel columns (12g~330g sizes). All compounds used for biological assays are greater than 95% purity based on NMR and HPLC by absorbance at 220 nm and 254 nm wavelengths.

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.