Human adenosine A3 receptor as selective target of new 2-aralkynyl-N6-methyl-MECA derivatives

Adenosine A3 receptor (AA3R) is involved in a variety of key physiological processes such as release of inflammatory mediators and inhibition of tumour necrosis factor-α production. Its activation is suggested to take part in immunosuppression and in the protection from brain and heart ischaemia. Recent studies suggest that AA3R agonists could be employed as therapeutic agents for the treatment of rheumatoid arthritis, dry eye disorders, asthma, as antiinflammatory agents, and in cancer therapy as cytostatic and chemoprotective compounds [1]. Hence, the design and synthesis of potent and selective AA3R agonists could help to provide tools for the further characterization of the physiopathological role of this receptor and for the development of new drugs. Our group has previously published the synthesis and biological evaluation of 2-(ar)alkynyl-N6-methoxyadenosine and corresponding 5′-N-methylcarboxamidoadenosine (MECA) derivatives, which demonstrated to possess different degrees of affinity and selectivity for the human AA3R [2]. In particular, some MECA derivatives bearing an aromatic ring directly linked to the 2-ethynyl group showed the best profile for this receptor subtype, with low nanomolar affinity and selectivity from 1,600- to 22,000-fold. As previously, we had demonstrated that also the introduction of a methyl group in N6-position of 2-phenylethynyladenosine favoured the interaction with the human AA3R [3], we designed and synthesised 2-aralkynyl-N6-methyl MECA analogues with the aim at improving AA3R affinity and selectivity. The synthesised compounds were evaluated in radioligand binding studies and in functional assays performed in CHO cells stably transfected with human ARs. Functional studies were carried out using a new Eu-GTP assay which exploits the unique fluorescence properties of lanthanide chelates and provides a powerful alternative to assays that utilise radioisotopes. Results proved that the new compounds possess sub-nanomolar AA3R affinity and remarkable selectivity versus the other AR subtypes. Functional studies demonstrated also that the new derivatives behave as full agonists resulting to be among the most potent and selective AA3R ligands reported so far [4]. A molecular modelling study was performed to analyse the particular pharmacological behaviour of the presented molecules for the four AR subtypes. In particular, we made an attempt to get a rationalisation of the high selectivities of the presented molecules for the human AA3R using the recently solved AA2AR crystal structure as template for homology modelling studies and by carrying out docking experiments at the four AR binding sites