Hit-to-Lead Optimization of Heterocyclic Carbonyloxycarboximidamides as Selective Antagonists at Human Adenosine A3 Receptor

Antagonism of the human adenosine A₃ receptor (hA₃R) has potential therapeutic application. Alchemical relative binding free energy calculations of K18 and K32 suggested that the combination of a 3-(2,6-dichlorophenyl)-isoxazolyl group with 2-pyridinyl at the ends of a carbonyloxycarboximidamide group should improve hA₃R affinity. Of the 25 new analogues synthesized, 37 and 74 showed improved hA₃R affinity compared to K18 (and K32). This was further improved through the addition of a bromine group to the 2-pyridinyl at the 5-position, generating compound 39. Alchemical relative binding free energy calculations, mutagenesis studies and MD simulations supported the compounds’ binding pattern while suggesting that the bromine of 39 inserts deep into the hA₃R orthosteric pocket, so highlighting the importance of rigidification of the carbonyloxycarboximidamide moiety. MD simulations highlighted the importance of rigidification of the carbonyloxycarboximidamide, while suggesting that the bromine of 39 inserts deep into the hA₃R orthosteric pocket, which was supported through mutagenesis studies 39 also selectively antagonized endogenously expressed hA₃R in nonsmall cell lung carcinoma cells, while pharmacokinetic studies indicated low toxicity enabling in vivo evaluation. We therefore suggest that 39 has potential for further development as a high-affinity hA₃R antagonist.