Solubility is a key physicochemical property used in drug discovery and development. By definition, the solubility of a substance is the maximum amount of the substance that dissolves in a given volume of solvent, and it varies depending on the solvent and conditions.
In the framework of this thesis, we determined both thermodynamic and kinetic solubility of topoisomerase IIα inhibitors. Thermodynamic or equilibrium solubility refers to the solubility of a compound at equilibrium, where the dissolved compound is in balance with the undissolved substance. Kinetic solubility, on the other hand, is the maximum solubility of the most rapidly precipitating species of a compound, often measured using concentrated stock solutions in organic solvents. Measurements of thermodynamic solubility provide more reproducible and accurate data compared to kinetic solubility.
To measure solubility, we used the shake-flask method. First, a calibration curve was prepared in the concentration range of 1 to 100 μM. For thermodynamic solubility measurements, phosphate buffer was added to the solid sample, while for kinetic solubility measurements, a concentrated stock solution of the sample in DMSO was diluted with phosphate buffer. The samples were then shaken for 24 hours at 37 ˚C. After 24 hours, the samples were centrifuged for 10 minutes at 18,000 rpm, the supernatant was diluted with diluent, and the samples were analyzed using UHPLC coupled with a UV/Vis detector.
The quality of calibration curves was evaluated using the regression coefficient, with a threshold set at R² ≥ 0.999. To verify method accuracy, we also used control samples at concentrations of 2, 20, and 60 μM. We found that the largest errors occurred at the lowest concentrations.
Some compounds degraded either during thermodynamic or kinetic solubility determination. For certain compounds, additional tests were carried out to assess their stability at different shaking time points and by replacing phosphate buffer with 0.1% trifluoroacetic acid. We found that shaking time did not affect compound stability, but compounds were more soluble in acidic medium. In phosphate buffer, the compound did not dissolve and only degradation products were observed, while with the addition of trifluoroacetic acid the compound dissolved, and its peak could therefore be detected. To confirm degradation, further analyses with LC-MS and HRMS were performed for one of the compounds, revealing that the compound degraded to a minor extent. The resulting degradation products were water-soluble and could therefore be reliably quantified in the supernatant.
|
