Clofazimine pKa Determination by Potentiometry and Spectrophotometry: Reverse Cosolvent Dependence as an Indicator of the Presence of Dimers in Aqueous Solutions

The weakly basic antibiotic and anti-inflammatory drug, clofazimine (CFZ), was first described in 1957. It has been used therapeutically, most notably in the treatment of leprosy. Howe v e r , the compound is extremely insoluble in aqueous media, and, indeed, there is poor consensus about what its intrinsic solubility is since the reported values range from 0.04 to 11 ng/mL. To understand the speciation and solubilization of CFZ as a function of pH, it is of paramount importance to know the true aqueous pKa. However, there is also poor consensus about the value of the pKa (reported measured values range from 6.08 to 9.11). In the present study, we report the determination of the CFZ ionization constant using two independent techniques. A state-of-the-art potentiometric analysis was performed, drawing on titration data in methanol-water solutions (46-75 wt % MeOH) of CFZ, using the bias-reducing consensus of two different procedures of extrapolating the apparent psKa values to zero cosol v e n t to approximate the true aqueous pKa as 9.43 +/- 0.12 (25 degrees C, I = 0.15 M reference ionic strength). In parallel, spectrophotometric U V / vis titration data were acquired (250-600 nm at different pH) in 10 mM HEPES buffer solutions containing up to 54 wt % MeOH. The alternating least squares (ALS) method was used in the analysis of the absorbance-pH spectra. Uncharacteristically, the cosol v e n t UV/vis data in our study showed reverse cosol v e n t dependence (apparent pKa values increased with increasing cosolvent) which could be explained by a dimerization of the free base. The analysis of UV/vis data obtained from 54 wt % MeOH-water solution containing 20 mu M CFZ yielded the apparent pKa 9.51 +/- 0.17 (I approximate to 0.005 M). To assess whether self-assembly of CFZ was energetically feasible, density functional theory (DFT) calculations were used to study the putative CFZ dimers in aqueous and methanol media. The DFT-optimized geometries and infrared spectra of CFZ dimers using water and methanol as solvents were calculated and analyzed . Based on the lack of negative frequencies in calculated infrared spectra, it was confirmed that optimized geometries correspond to the true energetic minima. Visual analysis of optimized structures indicates the presence of stacking interactions between two CFZ molecules. The protonation site (the imine nitrogen atom) was determined by 1H NMR spectroscopy.

Automated summary

It is of great importance to understand the speciation and solubilization of the weakly basic antibiotic and anti-inflammatory drug, clofazimine (CFZ), as a function of pH. In this study, the ionization constant and pKa value of CFZ were determined using potentiometric and spectrophotometric methods. Additionally, density functional theory calculations confirmed the possibility of CFZ dimer formation in aqueous and methanol media.