3100 mass spectrometer

All commercial materials (Aldrich, Chemimpex, and GL Biochem) were used without further purification. All solvents were reagent grade or HPLC grade (RCI or DUKSAN). Anhydrous tetrahydrofuran (THF) was freshly distilled from sodium and benzophenone. Dry dichloromethane (CH₂Cl₂) was distilled from calcium hydride (CaH₂). All separations involved a mobile phase of 0.1% TFA (v/v) in acetonitrile (solvent A) and 0.1% TFA (v/v) in water (Solvent B). HPLC separations were performed with a Waters HPLC system equipped with a photodiode array detector (Waters 2996) using a Vydac 218TP™ C18 column (5 μm, 300 Å, 4.6 × 250 mm) at a flow rate of 0.6 mL/min for analytical HPLC and XBridge™ Prep C18 OBD™ column (10 μm, 300 Å, 30 × 250 mm) at a flow rate of 15 mL/min for preparative HPLC. Low-resolution mass spectral analyses were performed with a Waters 3100 mass spectrometer.

NMR spectra were acquired in DMSO-d₆ on a Bruker Avance III HD spectrometer (Bruker, Billerica, MA, USA) operating at 600 MHz for protons and equipped with an inverse TCI cryo probe enhanced for ¹H, ¹³C, and ²H. All NMR spectra were acquired at 298 K, in 3 mm solvent-matched Shigemi tubes using standard pulse programs for proton, carbon, HSQC, HMBC, COSY, and ROESY, with gradient selection and adiabatic versions where applicable. ¹H/¹³C chemical shifts were referenced to the residual solvent peak (DMSO-d₆: δ_H = 2.50, δ_C = 39.51). UHPLC-ESI-HRMS was performed using an Acquity I-class UPLC with an Acquity UPLC C18 column (1.7 µm, 2.1 mm × 100 mm), coupled to a Vion IMS QToF and a PDA detector (all from Waters, Milford, MA, USA). ESI+ ionization was used. The gradient extended over 12 min, increasing from 10% to 90% acetonitrile (LiChrosolv^®, Supelco, Bellefonte, PA, USA) with 0.1% formic acid (Sigma-Aldrich, Steinheim, Germany) in Milli-Q^® H₂O, with a flow rate of 0.45 mL/min. A Waters UNIFI 1.8.2 Scientific Information System was used to process and analyze the data. The preparative HPLC system consisted of a 600 HPLC pump, a 3100 mass spectrometer, a 2996 photo diode array detector, and a 2767 sample manager (all from Waters). The system was controlled with MassLynx version 4.1.

UPLC-ESI-HR-MS/MS-analysis of the samples was done using an Acquity I-class UPLC (Waters, Milford, MA, United States) coupled to a PDA detector and Vion^® IMS QToF (Waters), UPLC-ESI-IMS-MS analysis was done using the same Vion^® IMS QToF in IMS (ion mobility spectrometry)-mode. For the purification of compounds via preparative HPLC a 600 HPLC pump, a 3,100 mass spectrometer, a 2,996 photo diode array detector and a 2,767 sample manager were used with as election of columns (all Waters). NMR spectra were recorded using a Bruker Avance III HD spectrometer (Bruker, Billerica, MA, United States) operating at 599.90 MHz for ¹H and 150.86 MHz for ¹³C. For the readout of bioassays in 96-well plates a 1,420 Multilabel Counter VICTOR3^™ (Perkin Elmer, Waltham, MA, United States) was used.

A preparative HPLC-system (Waters) with a 600 HPLC pump, a 2996 photo diode array detector, a 3100 mass spectrometer and a 2767 sample manager was used to isolate the two compounds. MassLynx version 4.1 was used to control the system. The mobile phases consisted of A; Milli-Q water and B; acetonitrile (Prepsolv^®, Merck), both containing 0.1% formic acid (v/v), and flow rate was set to 6 mL/min. Atlantis Prep dC18 column (10 µm, 10 mm × 250 mm) (Waters) was used for the initial separation of the two compounds with gradient 10–88% acetonitrile over 13 min. XSelect CSH Prep Fluoro-Phenyl column (5 µm, 10 mm × 250 mm) (Waters) was used for final purification of 1, gradient 10–76% acetonitrile over 10 min. For the final purification of 2, XSelect CSH Phenyl-Hexyl prep column (5 µm, 10 mm × 250 mm) (Waters) was used with gradient 10–54% acetonitrile over 11 min.

Initially, the purification of 1 and 2 was done by preparative HPLC-MS using a 600 HPLC pump, a 3100 mass spectrometer, a 2996 photo diode array detector, and a 2767 sample manager (Waters). For infusion of the eluents into the ESI-quadrupole-MS, a 515 HPLC pump (Waters) and a flow splitter were used and 80% methanol in ddH₂O (v/v) acidified with 0.2% formic acid (Sigma) as make-up solution at a flow rate of 0.7 mL/min. The columns used for isolation were a Sunfire RP-18 preparative column (10 µm, 10 mm × 250 mm) and XSelect CSH preparative fluoro-phenyl column (5 µm, 10 mm × 250mm), both columns were purchased from Waters. The mobile phases for the gradients were A (ddH₂O with 0.1% (v/v) formic acid) and B (acetonitrile with 0.1% (v/v) formic acid), flow rate was set to 6 mL/min. Acetonitrile (Prepsolv^®, Merck, Darmstad, Germany) and formic acid (33015, Sigma) were purchased in appropriate quality, ddH₂O was produced with the in-house Milli-Q^® system. The collected fractions were reduced to dryness at 40 °C in vacuo and freeze drying using an 8L laboratory freeze dryer (Labconco, Fort Scott, KS, USA).