Discovery of a High Affinity Adenosine A(1)/A(3) Receptor Antagonist with a Novel 7-Amino-pyrazolo[3,4-d]pyridazine Scaffold

[Image: see text] Here we describe the design and synthesis of pyrazolo[3,4-d]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 10b, which displayed 21 nM affinity and a residence time of ∼60 min, for the human A(1)R, 55 nM affinity and a residence time of ∼73 min, for the human A(3)R and 1.7 μΜ affinity for the human A(2B)R while not being toxic. Strikingly, the 2-methyl analog of 10b, 15b, 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 15b hinders the formation of hydrogen bonding interactions with N(6.55) which are considered critical for the stabilization inside the orthosteric binding cavity. We, therefore, demonstrate that 10a is a novel scaffold for the development of high affinity AR ligands. From the mutagenesis experiments the biggest effect was observed for the Y271(7.46)A mutation which caused an ∼10-fold reduction in the binding affinity of 10b.