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

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