Discovery of a High Affinity Adenosine A/A Receptor Antagonist with a Novel 7-Amino-pyrazolo[3,4-]pyridazine Scaffold.

Here we describe the design and synthesis of pyrazolo[3,4-]pyridazines as adenosine receptor (AR) ligands. We demonstrate that the introduction of a 3-phenyl group, together with a 7-benzylamino and 1-methyl group at the pyrazolopyridazine scaffold, generated the antagonist compound , which displayed 21 nM affinity and a residence time of ∼60 min, for the human AR, 55 nM affinity and a residence time of ∼73 min, for the human AR and 1.7 μΜ affinity for the human AR while not being toxic. Strikingly, the 2-methyl analog of , , had no significant affinity. Docking calculations and molecular dynamics simulations of the ligands inside the orthosteric binding area suggested that the 2-methyl group in hinders the formation of hydrogen bonding interactions with N which are considered critical for the stabilization inside the orthosteric binding cavity. We, therefore, demonstrate that is a novel scaffold for the development of high affinity AR ligands. From the mutagenesis experiments the biggest effect was observed for the Y271A mutation which caused an ∼10-fold reduction in the binding affinity of .