Exploring the Role of N 6 -Substituents in Potent Dual Acting 5'-C-Ethyltetrazolyladenosine Derivatives: Synthesis, Binding, Functional Assays, and Antinociceptive Effects in Mice ∇.

Structural determinants of affinity of N ⁶ -substituted-5'-C-(ethyltetrazol-2-yl)adenosine and 2-chloroadenosine derivatives at adenosine receptor (AR) subtypes were studied with binding and molecular modeling. Small N ⁶ -cycloalkyl and 3-halobenzyl groups furnished potent dual acting A ₁ AR agonists and A ₃ AR antagonists. 4 was the most potent dual acting human (h) A ₁ AR agonist (K _i = 0.45 nM) and A ₃ AR antagonist (K _i = 0.31 nM) and highly selective versus A _2A ; 11 and 26 were most potent at both h and rat (r) A ₃ AR. All N ⁶ -substituted-5'-C-(ethyltetrazol-2-yl)adenosine derivatives proved to be antagonists at hA ₃ AR but agonists at the rA ₃ AR. Analgesia of 11, 22, and 26 was evaluated in the mouse formalin test (A ₃ AR antagonist blocked and A ₃ AR agonist strongly potentiated). N ⁶ -Methyl-5'-C-(ethyltetrazol-2-yl)adenosine (22) was most potent, inhibiting both phases, as observed combining A ₁ AR and A ₃ AR 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.