A novel VIVO–pyrimidinone complex: synthesis, solution speciation and human serum protein binding

Abstract

The pyrimidinones mhcpe, 2-methyl-3H-5-hydroxy-6-carboxy-4-pyrimidinone ethyl ester (mhcpe, 1), 2,3-dimethyl-5-benzyloxy-6-carboxy-4-pyrimidinone ethyl ester (dbcpe, 2) and N-methyl-2,3-dimethyl-5-hydroxy-6-carboxyamido-4-pyrimidinone (N-MeHOPY, 3), are synthesized and their structures determined by single crystal X-ray diffraction. The acid–base properties of 1 are studied by potentiometric and spectrophotometric methods, the pK_a values being 1.14 and 6.35. DFT calculations were carried out to determine the most stable structure for each of the H₂L⁺, HL and L⁻ forms (HL = mhcpe) and assign the groups involved in the protonation–deprotonation processes. The mhcpe⁻ ligand forms stable complexes with V^IVO²⁺ in the pH range 2 to 10, and potentiometry, EPR and UV-Vis techniques are used to identify and characterize the V^IVO–mhcpe species formed. The results are consistent with the formation of V^IVO, (V^IVO)L, (V^IVO)L₂, (V^IVO)₂L₂H₋₂, (V^IVO)L₂H₋₁, (V^IVO)₂L₂H₋₃, (V^IVO)LH₋₂ species and V^IVO-hydrolysis products. Calculations indicate that the global binding ability of mhcpe towards V^IVO²⁺ is similar to that of maltol (Hmaltol = 3-hydroxy-2-methyl-4H-pyran-4-one) and lower than that of 1,2-dimethyl-3-hydroxy-4-pyridinone (Hdhp). The interaction of V^IVO-complexes with human plasma proteins (transferrin and albumin) is studied by circular dichroism (CD), EPR and ⁵¹V NMR spectroscopy. V^IVO–mhcpe–protein ternary complexes are formed in both cases. The binding of V^IVO²⁺ to transferrin (hTF) in the presence of mhcpe involves mainly (V^IVO)₁(hTF)(mhcpe)₁, (V^IVO)₂(hTF)(mhcpe)₁ and (V^IVO)₂(hTF)(mhcpe)₂ species, bound at the Fe^III binding sites, and the corresponding conditional formation constants are determined. Under the conditions expected to prevail in human blood serum, CD data indicate that the V^IVO–mhcpe complexes mainly bind to hTF; the formation of V^IVO–hTF–mhcpe complexes occurs in the presence of Fe^III as well, distinct EPR signals being clearly obtained for Fe^III–hTF and to V^IVO–hTF–mhcpe species. Thus this study indicates that transferrin plays the major role in the transport of V^IVO–mhcpe complexes under blood plasma conditions in the form of ternary V^IV–ligand–protein complexes.