New heterocyclic A1/A3 adenosine receptor ligands through molecular simplification strategies

In this paper we report the synthesis of new A 1 /A 3 adenosine receptor antagonists designed as simplification products of the A 1 antagonists with pyrazolo[1′,5′:1,6]pyrimido [4,5-d]pyridazin-4(3H)-one scaffold previously developed by us. Notably, selective A 1 R antagonists are promising therapeutic agents in Alzheimer's disease and for the treatment of cognitive deficits, while A 3 R antagonists are potentially useful in the treatment of ischemia and certain types of cancer. Initial screening with NanoBRET competition binding assay revealed a number of products with pKi ≥5 for A 1 R and A 3 R. For some representative compounds the antagonist profiles, as well as their selectivity versus A 2A R and A 2B R, have been also validated by antagonizing NECA in cAMP accumulation. The most interesting compounds resulted the A 1 /A 3 mixed antagonist 3b (pKi = 6.41 and 6.29 for A 1 R and A 3 R respectively, pKb = 5.00 and 5.27 for the A 2a R and A 2b R) and the selective A 3 R antagonist 5c (pKi = 6.40, pKb values of 4.44, 6.17, 4.16, and 4.78 for the A 1 R, A 3 R, A 2a R and A 2b R, respectively). Furthermore, in silico simulations were carried out to study the molecular mechanism of the high affinity of 3b for A 1 /A 3 Rs as well as the selectivity of 5c for A 3 R over A 1 R. Overall, this work highlights new series of bicyclic small-molecules as valid candidates for further structural optimization towards the development of therapeutically relevant A 1 /A 3 adenosine receptor antagonists. • New A 1 /A 3 adenosine receptor antagonists. • Molecular Simplification Strategies. • Affinity measurement using a NanoBRET Binding Assay.