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Activation of the Gi protein-coupled A3 adenosine receptor (AR) is associated with anticancer, antiischemic and anti-inflammatory effects. hA3 AR antagonists are being examined as promising agents for the treatment of glaucoma.1 Fluorescent probes are useful tools to investigate specific subcellular components in cells, tissues and organisms. In high throughput screening, fluorescent ligands represents a safer, more powerful and more versatile alternative to radioligands.2 We have recently reported a series of pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine (PTP) based fluorophore-conjugated hA3 AR antagonists.3 Unfortunately, none of them reached affinities in the nM range. Here we report a novel series of PTP based fluorophore-conjugated hA3 AR antagonist with an additional pharmacophoric group at the 5-position, in order to obtain more potent hA3 AR antagonists. References 1. Müller, C.E.; Jacobson, K.A. Recent developments in adenosine receptor ligands and their potential as novel drugs. Biochim. Biophys. Acta 2011, 1808, 1290-1308. 2. Kuder,K.; Kiec-Kononowicz, K. Fluorescent GPCR ligands as new tools in pharmacology. Curr. Med. Chem. 2008, 15, 2132–2143. 3. Kozma, E.; Kumar, T.S.; Federico, S.; Phan, K.; Balasubramanian, R.; Gao, Z.G.; Paoletta, S.; Moro, S.; Spalluto, G.; Jacobson, K.A. Novel fluorescent antagonist as a molecular probe in A3 adenosine receptor binding assays using flow cytometry. Biochem. Pharmacol. 2012, 83, 1552-1561.

Fluorescent probes are useful tools to investigate specific subcellular components in cells, tissues and organisms. A classic application of fluorescent ligands of a specific GPCR (G-protein coupled receptors) is the investigation of the receptor location, and, if their binding is reversible, it could provide pharmacological information such as affinity and proximity between interacting molecules. In cultured cell systems, methods based on fluorescence have permitted to observe the receptor cycling and the formation of oligomeric receptor complexes. In high throughput screening, fluorescent ligands represents a safer, more powerful and more versatile alternative to radioligands. [1] The introduction of the bulky fluorophore into a small molecule ligand could lead to deep modifications not only from a physicochemical point of view but also in its pharmacological properties. [2] In order to allow a correct interaction of the pharmacophore with its receptor, it is usually separated from the fluorophore by a linker or a spacer, which should be attached at a specific position that doesn't interfere with the ligand activity. Length and chemical nature of this spacer are optimised in order to obtain the desired biological activity. Also, the choice of the fluorescent molecule to link to the ligand is very important. [1,3] We decided to develop a fluorescent-ligand for the A3AR (A3 adenosine receptor) subtype using the pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine (PTP) as active scaffold at the hA3AR. [4] PTP bears a alkyldiamino spacer at the 5 position which possess a terminal amino group that could be functionalized with the fluorescein isothiocyanate (FITC). FITC represent a useful fluorescent tool because shows a significant extinction coefficent, high quantum yields, and water solubility. [3] The homology model of the hA3AR based on the crystal structure of the hA2AAR was used to perform the docking studies of conjugated derivatives (Prof. Stefano Moro, University of Padua) in order to evaluate what interactions occurs inside the binding pocket. References [1] R.J. Middleton, B. Kellam. Fluorophore-tagged GPCR ligands. Curr. Opin. Chem. Biol. 2005, 9, 517-525. [2] R.P. Haughland. Introduction to fluorescence techniques. Handbook of Fluorescent Probes and Research Products. Chapter 1. 2002 Eugene, Oregon: Molecular Probes. [3] N. Baindur, D.J. Triggle. Concepts and progress in the development and utilization of receptor-specific fluorescent ligands. Med. Res. Rev. 1994, 14, 591-664. [4] S.L. Cheong, S. Federico, G. Venkatesan, et al. The A3 adenosine receptor as multifaceted therapeutic target: pharmacology, medicinal chemistry and in silico approaches. Med. Res. Rev. In press: DOI 10.1002/med.20254