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 (Ki = 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 hA2AR. 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 N6.55 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.