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

Abstract

(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 ₃AR and MW < 500 and other favorable physicochemical properties. E _max (maximal efficacy in a mouse chronic constriction injury pain model) of previously characterized A ₃AR 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 ₃AR, 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 ₃AR 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 ₃AR agonists for chronic pain treatment.