ABSTRACT
The structure-activity relationship (SAR) of 1,2,4-triazolo[1,5-a]-1,3,5-triazine derivatives related to ZM241385 as antagonists of the A(2A) adenosine receptor (AR) was explored through the synthesis of analogues substituted at the 5 position. The A(2A) AR X-ray structure was used to propose a structural basis for the activity and selectivity of the analogues and to direct the synthetic design strategy to provide access to solvent-exposed regions. Thus, we have identified a point of substitution for the attachment of solubilizing groups to enhance both aqueous solubility and physicochemical properties, maintaining potent interactions with the A(2A) AR and, in some cases, receptor subtype selectivity. Among the most potent and selective novel compounds were a long-chain ether-containing amine congener 20 (K(i) 11.5 nM) and its urethane-protected derivative 14 (K(i) 17.8 nM). Compounds 20 and 31 (K(i) 11.5 and 16.9 nM, respectively) were readily water-soluble up to 10 mM. The analogues were docked in the crystallographic structure of the hA(2A) AR and in a homology model of the hA(3) AR, and the per residue electrostatic and hydrophobic contributions to the binding were assessed and stabilizing factors were proposed.
Subject(s)
Adenosine A2 Receptor Antagonists/chemical synthesis , Triazines/chemical synthesis , Triazoles/chemical synthesis , Adenosine A2 Receptor Antagonists/chemistry , Adenosine A2 Receptor Antagonists/pharmacology , Animals , CHO Cells , Cricetinae , Cricetulus , Crystallography, X-Ray , HEK293 Cells , Humans , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Protein Binding , Protein Conformation , Radioligand Assay , Solubility , Static Electricity , Structure-Activity Relationship , Triazines/chemistry , Triazines/pharmacology , Triazoles/chemistry , Triazoles/pharmacology , WaterABSTRACT
The aim of this study was to examine in vitro, by means of kinetic analysis and molecular docking simulations, the effects of the flavone diosmetin and its flavanone analog hesperetin on CYP (cytochrome P450) 2C9-mediated drug metabolism. To this purpose, the conversion of diclofenac to 4'-hydroxydiclofenac by human liver microsomes was used as a model assay for assessing the CYP2C9 inhibitory activity of these two flavonoids. Kinetic analyses showed that diosmetin and hesperetin were reversible, dead-end inhibitors of 4'-hydroxydiclofenac formation; their mean K(i) (inhibitor dissociation constant) values were 1.71 ± 0.58 and 21.50 ± 3.62 µM, respectively. Diosmetin behaved as a competitive inhibitor, since it increased markedly the K(m) (substrate concentration yielding 50% of V(max)) of the reaction without affecting the V(max) (maximum velocity of reaction). Hesperetin modified markedly K(m) and to a lesser extent also modified V(max), thus acting as a mixed competitive-noncompetitive inhibitor. The results of molecular docking simulations were consistent with those of kinetic analysis, since they showed that the putative binding sites of both diosmetin and hesperetin coincided with the CYP2C9 substrate binding site. The demonstration that diosmetin and hesperetin inhibit CYP2C9-mediated diclofenac metabolism at low micromolar concentrations is of potential clinical relevance because CYP2C9 is responsible for the biotransformation of various therapeutically important drugs that have narrow therapeutic indexes.
