5-Imino-1,2,4-thiadiazoles and quinazolines derivatives as glycogen synthase kinase 3β (GSK-3β) and phosphodiesterase 7 (PDE7) inhibitors were characterized for their ability to pass the blood–brain barrier (BBB) together with their human serum albumin (HSA) binding using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). To study the blood–brain barrier penetration, a parallel artificial membrane permeability assay (PAMPA) using a porcine brain lipid was employed. For the HPLAC investigation, HSA was previously covalently immobilized to the silica matrix of the HPLC column. This HSA-based column was used to characterize the high affinity binding sites of 5-imino-1,2,4-thiadiazoles and quinazolines derivatives to HSA. Displacement experiments in the presence of increasing concentrations of competitors known to bind selectively to the main binding sites of HSA were carried out to determine their possible binding site. The same drug–protein system was studied by CD.The analysed compounds were able to pass BBB, they present good drug-like properties and they showed a high affinity to HSA. Competition experiments showed an anticooperative interaction at sites I and II, and an independent binding at bilirubin binding site on HSA.
