Multiwavelength UV-metric and pH-metric determination of the dissociation constants of the hypoxia-inducible factor prolyl hydroxylase inhibitor Roxadustat

Abstract The dissociation constant p K a of the Roxadustat with UV-VIS spectra, molar absorption coefficients and protonation equilibria are the key physicochemical parameters influencing many biopharmaceutical characteristics. Roxadustat is an orally bioavailable, hypoxia-inducible factor prolyl hydroxylase inhibitor with potential anti-anemic activity. It belongs to Active Pharmacenutical Ingredients, which have acidic/basic functionalities, their ionization state is controlled by solution pH and acid dissociation constants. Nonlinear regression of the pH-spectra with programs REACTLAB and SQUAD84 and of the pH-titration curve with ESAB determined four multiple consecutive dissociation constants with the protonation scheme. A sparingly soluble neutral molecule LH 3 of Roxadustat was dissociated to the soluble anions LH 2 − , LH 2− and L 3− or protonated to the cation LH 4 + in an aqueous medium. The graph of molar absorption coefficients of variously protonated species according to wavelength shows that the spectra of two anions LH 2 − and LH 2− are nearly the same in colour. The Roxadustat spectrum exhibited five sharp isosbestic points, which were related to the LH 2− /L 3− equilibrium. Four consecutive thermodynamic dissociation constants were estimated using UV-metric data p K T a1  = 3.60(04), p K T a2  = 5.62(14), p K T a3  = 7.66(16), p K T a4  = 9.08(02) at 25 °C and p K T a1  = 3.60(04), p K T a2  = 5.73(10), p K T a3  = 7.52(10), p K T a4  = 8.99(02) at 37 °C and using pH-metric data p K T a1  = 4.33(09), p K T a2  = 6.57(11), p K T a3  = 8.88(05), p K T a4  = 9.03(04) at 25 °C and p K T a1  = 4.25(09), p K T a2  = 6.49(10), p K T a3  = 8.80(06), p K T a4  = 9.00(05) at 37 °C The positive values of the enthalpy Δ H 0 showed that the dissociation process is endothermic and the positive values of the Gibbs free energy Δ G 0 at 25 °C indicated that the dissociation process was not spontaneous, which also was confirmed by a negative value of the entropy Δ S 0 . Four macro-dissociation constants of Roxadustat and six protonation locations were predicted by MARVIN.