POTENT DUAL ACTING N6-SUBSTITUTED-5ʹ-C-ETHYL-TETRAZOLYL-ADENOSINE DERIVATIVES: SYNTHESIS, BINDING, FUNCTIONAL ASSAYS AND ANTINOCICEPTIVE EFFECTS IN MICE

POTENT DUAL ACTING N6-SUBSTITUTED-5ʹ-C-ETHYL-TETRAZOLYL-ADENOSINE DERIVATIVES: SYNTHESIS, BINDING, FUNCTIONAL ASSAYS AND ANTINOCICEPTIVE EFFECTS IN MICE Background Adenosine, the natural ligand of P1 receptors, is implicated in the control of many physiological and pathological conditions such as inflammation, cardiovascular and central nervous system (CNS) diseases.1,2 P1 receptors belong to the large family of G protein-coupled receptors (GPCRs) and are represented by four subtypes: A1, A2A, A2B and A3 adenosine receptors (ARs). Substitutions at both purine and sugar moiety of adenosine results on AR ligands endowed with different affinity and selectivity at the four AR subtypes. Aim The aim of this work was to study a series of N6-substituted-5ʹ-C-(ethyl-tetrazol-2-yl)-adenosine and 2- chloroadenosine derivatives as novel, highly potent dual acting human (h) A1AR and A3AR ligands, potentially useful in the treatment of pain and other diseases. Combining the analgesic effects of both A1 and A3AR agonists in only one molecule might be advantageous in terms of reducing side effects and synergizing the antinociceptive activities. Moreover, based on the fact that A3AR represents the AR subtype with the higher species differences for both affinity and efficacy, some selected compounds were also evaluated for affinity and efficacy at rat (r) A3AR. Methods The novel compounds were tested for affinity at the human recombinant ARs, stably transfected into Chinese hamster ovary (CHO) cells, utilizing radioligand binding assays (A1, A2A, and A3) or an adenylyl cyclase activity assay (A2B). Selected compounds were also tested at the recombinant rA3AR, stably transfected into CHO cells utilizing radioligand binding assays. All compounds were additionally tested for their functional effects on hA1, hA2A, and hA3 ARs by determination of adenylyl cyclase activity. Molecular docking calculations were carried out using homology models of the hA1- and hA3ARs. The analgesic activity was evaluated using the formalin test. Results This study reports for the first time that potent dual acting N6-substituted-5′-C-(ethyl-tetrazol-2-yl)- adenosine and 2-chloroadenosine derivatives showed strikingly different efficacy at human and rat A3ARs, acting as antagonists at hA3A and as agonists at rA3AR. The combination of a 5′-C-ethyl- tetrazol-2-yl moiety with a small cycloalkyl or 3-halobenzyl group at N6-position in adenosine derivatives provided very potent dual A1 and A3 ligands at both human and rat A1 and A3ARs (Ki values at both receptor subtypes in the subnanomolar range).The N6-(3-halobenzyl)-5′-C-(ethyl-tetrazol-2-yl)-adenosine derivatives exhibited increased binding affinity at the hA1AR upon changing the substituent on the N6 benzyl moiety from F to Cl, Br and I and they were almost equipotent at hA3AR. The molecular bases of the hA1- and hA3AR recognition and activation of this series of adenosine derivatives were explained through an in silico receptor-driven approach. The most potent antinocifensive activity was obtained with N6-methyl-5ʹ-C-(ethyl-tetrazol-2-yl)-adenosine, which at 0.5 mg/kg reduced both the first and the second phases of formalin test. A combination of this compound (0.3 mg/kg) with the A3AR agonist 2-Cl- IBMECA (1 mg/kg) reduced completely the nocifensive behavior in both the first and the second phases of formalin test.3The antinociceptive effect of this compound was reverted by MRS1334, a selective A3 antagonist, demonstrating that both A1 and A3 agonistic effects contributed to analgesic behavior. Conclusion This study demonstrates that a combination of an A1agonist and an A3AR agonist shows a highly potent analgesic activity in mice. Therefore, combining both A1 and A3AR agonistic activity in one single molecule, such as the 5ʹ-C-tetrazolyl-N6-substituted-adenosine derivatives, could have the advantages of a single molecule activating two AR pathways both leading to benefit in pain. References (1) Jacobson, K. A.; Muller, C. E. Medicinal chemistry of adenosine, P2Y and P2X receptors.Neuropharmacology 2016, 104, 31-49. (2) Beamer, E.; Goloncser, F.; Horvath, G.; Beko, K.; Otrokocsi, L.; Kovanyi, B.; Sperlagh, B. Purinergic mechanisms in neuroinflammation: An update from molecules to behavior. Neuropharmacology 2016, 104, 94-10. (3) Petrelli, R.; Scortichini, M.; Kachler, S.; Boccella, S.; Cerchia, C.; Torquati, I.; Del Bello, F.; Salvemini, D.; Novellino, E.; Luongo, L.; Maione, S.; Jacobson, K.A.; Lavecchia, A.; Klotz, K.-N- ;Cappellacci, L. Exploring the Role of N6-Substituents in Potent Dual Acting 5'-C-Ethyl-tetrazolyl- adenosine Derivatives: Synthesis, Binding, Functional Assay and Antinociceptive Effects in Mice. J. Med. Chem. 2017, DOI: 10.1021/acs.jmedchem.7b00291