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

The dissociation constant pK(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 LH3 of Roxadustat was dissociated to the soluble anions LH2-, LH2- and L3- or protonated to the cation LH4+ in an aqueous medium. The graph of molar absorption coefficients of variously protonated species according to wavelength shows that the spectra of two anions LH2- and LH2- are nearly the same in colour. The Roxadustat spectrum exhibited five sharp isosbestic points, which were related to the LH2-/L3- equilibrium. Four consecutive thermodynamic dissociation constants were estimated using UV-metric data pK(T)a1 = 3.60(04), pK(T)a2 = 5.62(14), pK(T)a3 = 7.66(16), pK(T)a4 = 9.08(02) at 25 degrees C and pK(T)a1 = 3.60(04), pK(T)a2 = 5.73(10), pK(T)a3 = 7.52(10), pK(T)a4 =8.99(02) at 37 degrees C and using pH-metric data pK(T)a4=-433(09), pK(T)a2 = 6 57(11), pK(T)a3 = 8.88(05), pKTa4 = 9.03(04) at 25 degrees C and pK(T)a1 = 4.25(09), pK(T)a2 = 6.49(10), pK(T)a3 = 8.80(06), pK(T)a4, = 9.00(05) at 37 degrees C The positive values of the enthalpy Delta H-0 showed that the dissociation process is endothermic and the positive values of the Gibbs free energy Delta G(0) at 25 degrees C indicated that the dissociation process was not spontaneous, which also was confirmed by a negative value of the entropy Delta S-0. Four macro-dissociation constants of Roxadustat and six protonation locations were predicted by MARVIN. (C) 2018 Elsevier B.V. All rights reserved.