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 .