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Medchemcomm. 2017 Aug 1;8(8):1659-1667. doi: 10.1039/C7MD00247E. Epub 2017 Jun 22.

Bitopic fluorescent antagonists of the A2A adenosine receptor based on pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine functionalized congeners.

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Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bldg. 8A, Rm. B1A-19, Bethesda, MD 20892-0810, USA.
Inserm, CHU Lille, UMR-S 1172 - JPArc - Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer, Univ. Lille, F-59000 Lille, France.
Department of Pharmaceutical Chemistry, Telangana University, Nizamabad, Telangana, India 503322.
Department of Pharmacology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, USA.
Unitat de Farmacologia, Departament Patologia i Terapeutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, 08907 L'Hospitalet de Llobregat, Spain.


A pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine antagonist of the A2A adenosine receptor (AR) was functionalized as amine congeners, fluorescent conjugates and a sulfonate, and the A2AAR binding modes were predicted computationally. The optimal n-butyl spacer was incorporated into the following A2AAR-selective (Ki, nM) conjugates: BODIPY630/650 derivative 11 (MRS7396, 24.6) and AlexaFluor488 derivative 12 (MRS7416, 30.3). Flow cytometry of 12 in hA2AAR-expressing HEK-293 cells displayed saturable binding (low nonspecific) and inhibition by known A2AAR antagonists. Water-soluble sulfonate 13 was a highly potent (Ki = 6.2 nM) and selective A2AAR antagonist based on binding and functional assays. Docking and molecular dynamics simulations predicted the regions of interaction of the distal portions of these chain-extended ligands with the A2AAR. The BODIPY630/650 fluorophore of 11 was buried in a hydrophobic interhelical (TM1/TM7) region, while AlexaFluor488 of 12 associated with the hydrophilic extracellular loops. In conclusion, we have identified novel high affinity antagonist probes for A2AAR drug discovery and characterization.

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