Synthesis and anti-renal fibrosis activity of conformationally locked truncated 2-hexynyl-N⁶-substituted-(N)-methanocarba-nucleosides as A₃ adenosine receptor antagonists and partial agonists

Truncated N⁶-substituted-(N)-methanocarba-adenosine derivatives with 2-hexynyl substitution were synthesized to examine parallels with corresponding 4′-thioadenosines. Hydrophobic N⁶ and/or C2 substituents were tolerated in A₃AR binding, but only an unsubstituted 6-amino group with a C2-hexynyl group promoted high hA _2AAR affinity. A small hydrophobic alkyl (4b and 4c) or N ⁶-cycloalkyl group (4d) showed excellent binding affinity at the hA₃AR and was better than an unsubstituted free amino group (4a). A₃AR affinities of 3-halobenzylamine derivatives 4f-4i did not differ significantly, with K_i values of 7.8-16.0 nM. N⁶-Methyl derivative 4b (K_i = 4.9 nM) was a highly selective, low efficacy partial A₃AR agonist. All compounds were screened for renoprotective effects in human TGF-β1-stimulated mProx tubular cells, a kidney fibrosis model. Most compounds strongly inhibited TGF-β1-induced collagen I upregulation, and their A₃AR binding affinities were proportional to antifibrotic effects; 4b was most potent (IC₅₀ = 0.83 μM), indicating its potential as a good therapeutic candidate for treating renal fibrosis.