Abstract
Sixtyβeight new substituted pyrazolo[3,4βb]pyridine derivatives were synthesized and tested for enriching a library of active A~1~ adenosine receptor (AR) antagonists belonging to the same class. These compounds were also used as an external test set to check the reliability of a 3D QSAR model recently reported by us. To investigate the binding mode of pyrazolopyridine derivatives, a model of the bovine A~1~AR (bA~1~AR) was developed by a novel homology modeling approach and used to evaluate the main interactions of the ligands with the receptor through docking studies. Results suggest important interactions of the ligands mainly with L3.33(88), T3.36(91), Q3.37(92) and H6.52(251), in agreement with mutagenesis data. The racemic mixture of the most active compound was separated into the corresponding enantiomers which showed a bA~1~AR affinity in the nanomolar range, with the __R__enantiomer sevenfold more active than the __S__enantiomer, according to results derived from calculations on the receptor model. Analysis of the bovine/human A~1~AR affinity profile of ligands supported the hypothesis that such receptors should be characterized by a different size of their binding site, responsible for the different affinity of the antagonists.