In Vivo Phenotypic Screening for Treating Chronic Neuropathic Pain: Modification of C2-Arylethynyl Group of Conformationally Constrained A(3) Adenosine Receptor Agonists

[Image: see text] (N)-Methanocarba adenosine 5′-methyluronamides containing 2-arylethynyl groups were synthesized as A(3) adenosine receptor (AR) agonists and screened in vivo (po) for reduction of neuropathic pain. A small N(6)-methyl group maintained binding affinity, with human > mouse A(3)AR and MW < 500 and other favorable physicochemical properties. E(max) (maximal efficacy in a mouse chronic constriction injury pain model) of previously characterized A(3)AR agonist, 2-(3,4-difluorophenylethynyl)-N(6)-(3-chlorobenzyl) derivative 6a, MRS5698, was surpassed. More efficacious analogues (in vivo) contained the following C2-arylethynyl groups: pyrazin-2-yl 23 (binding K(i), hA(3)AR, nM 1.8), fur-2-yl 27 (0.6), thien-2-yl 32 (0.6) and its 5-chloro 33, MRS5980 (0.7) and 5-bromo 34 (0.4) equivalents, and physiologically unstable ferrocene 36, MRS5979 (2.7). 33 and 36 displayed particularly long in vivo duration (>3 h). Selected analogues were docked to an A(3)AR homology model to explore the environment of receptor-bound C2 and N(6) groups. Various analogues bound with μM affinity at off-target biogenic amine (M(2), 5HT(2A), β(3), 5HT(2B), 5HT(2C), and α(2C)) or other receptors. Thus, we have expanded the structural range of orally active A(3)AR agonists for chronic pain treatment.