Evaluating 7‑Aminopyrazolo[4,3‑d]pyrimidines as Human A₁ and A₃ Adenosine Receptor Antagonists

This study aimed to improve the adenosine receptor (AR) subtype selectivity of the previously reported 7-benzylamino-1-methyl-3-phenyl-pyrazolo-[3,4-d]-pyridazine analogue PPZ I, a nanomolar dual nonselective adenosine hA₁/hA₃ receptor antagonist (K_i = 11 nM hA₁R/13 nM hA₃R). To this end, 31 novel derivatives bearing the pyrazolo-[4,3-d]-pyrimidine scaffold were designed and synthesized, incorporating either 7-benzylamino or 7-phenylamino substituents. Binding affinities were determined using a bioluminescence resonance energy transfer assay against hA₁R and hA₃R, alongside functional characterization of antagonistic activity and selectivity across all four AR subtypes. These studies identified compounds 9 and 40 as the most promising derivatives in the series. Compound 9 exhibited approximately 4-fold selectivity for hA₁R over hA₃R, whereas compound 40 showed a 10-fold preference for hA₃R over hA₁R. Both compounds displayed low-affinity binding to the two hA₂R subtypes, which was more significant against hA_2AR. Overall, compounds 9 and 40 exhibited up to 3- and 6-fold selectivity for hA₁R and hA₃R, respectively, over the other hAR subtypes. Studies of the binding profile of 9 and 40 inside the orthosteric binding site of hA₁R and hA₃R showed that the pyrazole ring forming hydrogen bonding interactions with N^6.55 was complemented using molecular dynamics simulations and relative binding free energy calculations and validated through site-directed mutagenesis. The findings of this study will lead to the design and synthesis of more effective compounds against hARs that may have a therapeutic effect against human diseases, such as glaucoma and asthma.