Discovery of a High Affinity Adenosine A1/A3 Receptor Antagonist with a Novel 7-Amino-pyrazolo[3,4-d]pyridazine Scaffold

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 similar to 60 min, for the human A(1)R, 55 nM affinity and a residence time of similar to 73 min, for the human A(3)R and 1.7 mu Mu 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 Y2717.46A mutation which caused an similar to 10-fold reduction in the binding affinity of 10b.

Automated summary

We describe the design and synthesis of pyrazolo[3,4-d]pyridazines as adenosine receptor (AR) ligands. Our results show that the compound 10b, with a 3-phenyl group, a 7-benzylamino group, and a 1-methyl group on the pyrazolopyridazine scaffold, exhibits high affinity for human A(1)R and A(3)R, as well as low toxicity. However, the 2-methyl analog, 15b, does not show significant affinity, possibly due to hindrance of critical hydrogen bonding interactions. This study demonstrates the potential of 10a as a novel scaffold for the development of high affinity AR ligands.