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

Abstract

In this paper we report the synthesis of new A₁/A₃ adenosine receptor antagonists designed as simplification products of the A₁ antagonists with pyrazolo[1′,5′:1,6]pyrimido [4,5-d]pyridazin-4(3H)-one scaffold previously developed by us. Notably, selective A₁R antagonists are promising therapeutic agents in Alzheimer's disease and for the treatment of cognitive deficits, while A₃R 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 A₁R and A₃R. For some representative compounds the antagonist profiles, as well as their selectivity versus A_2AR and A_2BR, have been also validated by antagonizing NECA in cAMP accumulation. The most interesting compounds resulted the A₁/A₃ mixed antagonist 3b (pKi = 6.41 and 6.29 for A₁R and A₃R respectively, pKb = 5.00 and 5.27 for the A_2aR and A_2bR) and the selective A₃R antagonist 5c (pKi = 6.40, pKb values of 4.44, 6.17, 4.16, and 4.78 for the A₁R, A₃R, A_2aR and A_2bR, respectively). Furthermore, in silico simulations were carried out to study the molecular mechanism of the high affinity of 3b for A₁/A₃Rs as well as the selectivity of 5c for A₃R over A₁R. Overall, this work highlights new series of bicyclic small-molecules as valid candidates for further structural optimization towards the development of therapeutically relevant A₁/A₃ adenosine receptor antagonists.