Synthesis, CoMFA analysis, and receptor docking of 3,5-diacyl-2,4-dialkylpyridine derivatives as selective A3 adenosine receptor antagonists

3,5-Diacyl-2,4-dialkyl-6-phenylpyridine derivatives have been found to be selective antagonists at both human and rat A(3) adenosine receptors (Li et al. J. Med. Chem. 1998, 41, 3186-3201), In the present study, ring-constrained, fluoro, hydroxy, and other derivatives in this series have been synthesized and tested for affinity at adenosine receptors in radioligand binding assays. K-i values at recombinant human and rat A(3) adenosine receptors were determined using [I-125]AB-MECA (N-6-(4-amino-3-iodobenzyl)-5 '-N-methylcarbamoyladenosine). Selectivity for A(3) adenosine receptors was determined vs radioligand binding at rat brain A(1) and A(2A) receptors, and structure-activity relationships at various positions of the pyridine ring (the 3- and 5-acyl substituents and the 2- and 4-alkyl substituents) were probed. At the 5-position inclusion of a beta-fluoroethyl (7) or a gamma-fluoropropyl ester (26) was favorable for human A(3) receptor affinity, resulting in K-i values of 4.2 and 9.7 nM, respectively, while the pentafluoropropyl analogue was clearly less potent at human A(3) receptors. At the 2-, 3-, and 4-positions, fluoro or hydroxy substitution failed to enhance potency and selectivity at human A(3) receptors. Several analogues were nearly equipotent at rat and human A(3) receptors. To further define the pharmacophore conformationally, a lactam, a lactone, and thiolactones were tested in adenosine receptor binding. The most potent analogue in this group was compound 34, in which a thiolactone was formed between 3- and 4-positions and which had a K-i value of 248 nM at human A(3) receptors. Using affinity data and a general pharmacophore model for A(3) adenosine receptor antagonists recently proposed, we applied comparative molecular field analysis (CoMFA) to obtain a three-dimensional quantitative structure-activity relationship for pyridine derivatives, having good predictability (r(pred)(2) = 0.873) for compounds in the test set. A rhodopsin-based model of the human A(3) receptor was built, and the pyridine reference ligand 2,3,4,5-tetraethyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS 1476) was docked in the putative ligand binding site. Interactions between receptor transmembrane domains and the steric and the electrostatic contour plots obtained from the CoMFA analysis were analyzed.