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  • A selected set of these new derivatives


    A selected set of these new derivatives was then used for functional assays at the human A1AR showing, as expected, an antagonist profile with IC50 values at nanomolar level. The same molecules showed also to be efficacious as nanomolar antagonists in a functional assay aimed at analyzing their ability to counteract the inhibition of the rhythmic contraction of mouse ileum tissue given by A1AR activation. In-silico pharmacokinetic evaluation suggests that the modulation of the chemical-physical profile of the molecules could take to a modification of their ability to enter CNS and to potentially give side effects at this level. The presented data confirm the N6,8,9-trisubstituted AMD3100 as a very promising scaffold for the development of potent and selective A1AR antagonists. The N6-cyclopentyl-9-methyl-8-phenyladenine (17), resulting the most active derivative of the series, could hence be used as a starting point for the development of new safe therapeutic agents for the treatment of intestinal diseases like constipation and postoperative ileus.
    Experimental section
    Acknowledgment This work was supported by the University of Camerino (Fondo di Ricerca di Ateneo and Progetto FAR FPI000042) and by a grant from the Ministry of Research (PRIN N° 2015E8EMCM_008, 2015).
    Introduction Adenosine monophosphate (AMP) is generated by the sequential hydrolysis of extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) by the ectonucleotidase CD39 (NTPDase1, ectonucleoside triphosphate diphosphohydrolase-1). AMP is hydrolyzed by CD73 (ecto – 5′ – nucleotidase) to adenosine, which in turn is rapidly metabolized and cleared from the circulation. CD73 and the adenosine receptors (A1R, A2AR, A2BR, A3R) are widely expressed, implicating adenosine signaling in a diverse range of physiological and pathophysiological processes. The signaling properties of extracellular AMP remain controversial. Over 30 years ago it was first reported that phosphorylated adenosine derivatives were low-affinity adenosine receptor agonists. In 2004 it was proposed that the G-protein-coupled-receptor (GPCR) orphan receptor GPR80/99 was a receptor for extracellular AMP. However, this was quickly discounted because of the endogenous expression of adenosine receptors on HEK293 cells used in the experiments. In 2011 the authors of the update on the classification of adenosine receptors noted “there [was] no good evidence that adenine nucleotides can act on adenosine receptors without being degraded to nucleosides first”. However, in 2013 AMP was shown to play a role in thermoregulation mediated by the A1R. This was supported by in vitro studies using HEK293 cells in which AMP activated the human A1R with equivalent potency to adenosine. The canonical ligand for all four receptors is adenosine, the potency of which varies according to receptor density and the type of response measured. The receptors are localized on the cell surface and belong to the GPCR family. Structural information of the GPCR family as a whole has expanded rapidly. Within the adenosine family of receptors the A2AR has been most thoroughly studied and the crystalline structure determined, which has enhanced the understanding of both orthosteric and allosteric binding sites. In addition a number of studies have demonstrated that adenosine receptors can exist as homodimers and heterodimers, which may impact GPCR function (reviewed in 7). The A2BR has been implicated in the pathophysiology of renal ischemic-reperfusion injury (IRI) by increasing post-ischemic blood flow and limiting TNF-α release from neutrophils. The A2BR is a Gs-coupled protein receptor and its activation increases intracellular adenylyl cyclase activity and cAMP levels. A2BR is also coupled to Gq proteins, which upon A2BR engagement activate phosphatidylinositol-phospholipase C, triggering a series of steps resulting in the opening of calcium channels. In many cells Gs coupling appears preferred. The A2BR is widely expressed throughout the body. However, because of the relatively low potency of adenosine for this receptor it may not be fully activated under physiological conditions but rather activated in pathological states, such as hypoxia, when the pericellular adenosine concentration is significantly elevated. Indeed the A2BR is upregulated on the renal vasculature following ischemia, mediated by the transcription factor HIF-1α, and this heightened expression is maintained for at least 4 weeks. The coordinated increase in the expression of A2BR and local concentration of adenosine in response to kidney ischemia implicates purinergic signaling in the pathophysiology of renal IRI. Indeed augmenting adenosine signaling, particularly via the A2BR, prior to acute kidney ischemia disease mitigates injury (reviewed in 13).