Synthesis and Characterization of New Ligands for the Purinergic P1 Receptors

Adenosine (Ado) is a naturally occurring nucleoside, involved in the regulation of many physiological and pathophysiological processes through the activation of four specific cell membrane receptors (AA1R, AA2AR, AA2BR, and AA3R), which belong to the superfamily of G protein-coupled receptors. Ado receptors (ARs) are responsible for the wide variety of effects produced by Ado throughout several organ systems. As they are ubiquitously expressed in the body and are often coexpressed in the same tissue, medicinal chemistry research has been focused, since their first identification, on the discovery of selective ARs agonists and antagonists, to exploit the therapeutic potential, but also to improve the knowledge about the complicate physiological role of Ado and its receptors. Among the four AR subtypes, AA3R is the most enigmatic one, and little is still known about its physiological pleiotropic effects. Hence, with the aim at finding new potent and selective agonists for AA3R, disubstituted 5'-N-methylcarboxamidoAdo (MECA) derivatives bearing a methyl group in the N6-position and an aralkynyl substituent in the C2-position were designed, synthesized, and tested both in binding and functional assays. The new compounds showed affinity in the sub-nanomolar range, and very high selectivity for the target subtype, resulting among the most potent and selective AA3R ligands reported so far. Functional studies, carried out by performing a new Eu-GTP functional assay, which avoids the use of any radiolabelled compound, displayed that all the new molecules behave as AA3R full agonists. Afterwards, the attention has been focused on the importance of AA2AR as novel target for the non-dopaminergic treatment of Parkinson's disease (PD). Starting from the observation that three 9-ethyladenine derivatives, bearing in the C8-position a bromine atom, an ethoxy group, or a furyl ring ameliorate motor deficits in rat models of PD, new series of molecules were designed and synthesized with the aim at finding new potential tools suitable for in vivo studies on rat models of PD. Firstly, 9-ethyladenine derivatives bearing halogens, alkoxy groups, and aromatic rings in the C8-position have been prepared together with the three known above mentioned molecules, which were used as reference compounds. Binding studies, performed both at rat and human ARs, showed that the three reference 9-ethyladenine derivatives were the most active AA2AR antagonists at human, but not at rat receptors. In fact, the newly synthesized 8-iodo, 8-methoxy, 8-phenethoxy, and 8-trifluoromethyl derivatives exhibited higher affinity at rat than the reference compounds, thus resulting as potential new tools suitable for in vivo studies on rat models of PD. Secondly, a new series of C2/C8-disubstituted 9-ethyladenines was designed on the basis of the results obtained with the C8-substituted derivatives and taking into account previous observations demonstrating that the presence of alkoxy groups in the C2-position of 9-ethyladenine favours the interaction with AA2AR. Biological studies revealed that the most active compounds were those bearing a phenethoxy group in the C2-position. These molecules showed affinity at AA2AR in the low nanomolar range and good AA2AR selectivity in particular vs the AA3R subtype, the 8-bromo and 8-iodo derivatives being the most active.