Exploring the Role of N⁶-Substituents in Potent Dual Acting 5'-C-Ethyltetrazolyladenosine Derivatives: Synthesis, Binding, Functional Assays, and Antinociceptive Effects in Mice ∇

Structural determinants of affinity of N⁶-substituted-5'-C-(ethyltetrazol-2-yl)adenosine and 2-chloroadenosine derivatives at adenosine receptor (AR) subtypes were studied with binding and molecular modeling. Small N⁶-cycloalkyl and 3-halobenzyl groups furnished potent dual acting A₁AR agonists and A₃AR antagonists. 4 was the most potent dual acting human (h) A₁AR agonist (K_i = 0.45 nM) and A₃AR antagonist (K_i = 0.31 nM) and highly selective versus A_2A; 11 and 26 were most potent at both h and rat (r) A₃AR. All N⁶-substituted-5'-C-(ethyltetrazol-2-yl)adenosine derivatives proved to be antagonists at hA₃AR but agonists at the rA₃AR. Analgesia of 11, 22, and 26 was evaluated in the mouse formalin test (A₃AR antagonist blocked and A₃AR agonist strongly potentiated). N⁶-Methyl-5'-C-(ethyltetrazol-2-yl)adenosine (22) was most potent, inhibiting both phases, as observed combining A₁AR and A₃AR agonists. This study demonstrated for the first time the advantages of a single molecule activating two AR pathways both leading to benefit in this acute pain model.