4.4. Thermodynamic Solubility (Shake Flask Method)

Compounds that showed activity in the enzymatic assay, 1, 2, 9a, 9e, 10a–e, were added into 1.5 mL Eppendorf tubes prior to the addition of pH 7.4 phosphate buffer to obtain a nominal concentration of 1 mg/mL. The samples were sonicated for 10 min and shaken overnight at 800 rpm (FB15013 TopMix, Fischer Scientific, Waltham, MA, USA) at room temperature. A first phase separation of supernatant and undissolved solid was performed by centrifugation at 5000 rpm for 20 min (Biofuge primo R). The supernatant was then transferred into Eppendorf tubes for a second centrifugation. An adequate dilution was made using the particle-free supernatant and the concentration quantified by LC-HRMS using a 4-point calibration curve (Agilent 1260 Infinity II).