Measurements of kinetic solubility

Using a 10 mM stock solution of each test compound in 100% DMSO, sample dilutions were prepared to a theoretical concentration of 200 μM both in PBS (pH 7.4, containing 2% DMSO) and in 100% DMSO. An aliquot of the 200 μM DMSO solution was then further diluted to 10 μM (n = 2 for each compound) and equilibrated at room temperature on an orbital shaker for 2 h. PBS dilutions were filtered using a Multiscreen HTS solubility filter plate (Millipore, Billerica, MA), and the filtrates were analysed by LC (1100 binary LC system, Agilent Technologies Ltd, Cheshire, UK) with a variable wavelength UV detector (Agilent Technologies Ltd) and a MS detector (Quattro Micro MS, Waters Ltd, Hertfordshire, UK). The concentration of a test compound in the PBS filtrate was determined by comparing its UV absorbance peak (λ = 254 nm) with that of the two DMSO dilutions. Mass spectrometry was used to confirm the presence of the expected molecular ion in the measured UV peak. The effective concentration range of the assays was 5 – 200 μM.