[ 3 H]-MRE 2029-F20, a selective antagonist radioligand for the human A 2B adenosine receptors Pier Giovanni Baraldi, a, * Mojgan Aghazadeh Tabrizi, a Delia Preti, a Andrea Bovero, a Francesca Fruttarolo, a Romeo Romagnoli, a Allan R. Moorman, c Stefania Gessi, b Stefania Merighi, b Katia Varani b and Pier Andrea Borea b a Dipartimento di Scienze Farmaceutiche, Universit a di Ferrara, 44100 Ferrara, Italy b Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia, Universit a di Ferrara, 44100 Ferrara, Italy c King Pharmaceuticals R & D, 4000 CentreGreen Way, Suite 300, Cary, NC 27513, USA Received 17 December 2003; revised 19 March 2004; accepted 29 March 2004 Available online Abstract—MRE 2029-F20 [N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1 pyrazol-3-yloxy]-acetamide] is a selective antagonist ligand of A 2B adenosine receptors. For use as a radioligand, 1,3-diallyl-xan- thine, the precursor of [ 3 H]-MRE 2029-F20, was synthesized, and tritiated on the allyl groups. [ 3 H]-MRE 2029-F20 bound to human A 2B receptors expressed in CHO cells showed a K D value of1.65 0.10 nM and B max value of36 4 fmol/mg protein. [ 3 H]- MRE2029-F20 represents a useful tool for the pharmacological characterization of human A 2B adenosine receptor subtype. Ó 2004 Elsevier Ltd. All rights reserved. The biological activity of adenosine occurs through the activation ofspecific receptors located on cellmem- branes and belonging to the extensive family of G-pro- tein coupled receptors. 1;2 Adenosine interacts with four pharmacologically distinct adenosine receptor subtypes designated A 1 ,A 2A ,A 2B , and A 3 . 1;3 The adenosine receptorsare associated with different messenger sys- tems:A 1 and A 3 mediate adenylate cyclase inhibition and the decrease of intracellular cyclic AMP levels. 4 A 2A and A 2B are positively coupled to adenylate cyclase via G s and mediate the stimulation of the adenylate cyclase activity and theincreaseof cAMP levels. However, coupling to phospholipase C via G q , resulting in mobi- lization of intracellular calcium, and direct coupling to calcium channels has also been described. 1;2 A 2B aden- osine receptors have been implicated in the regulation of mast cell secretion, 5 geneexpression, 6 cell growth, 7 intestinalfunction, 8 neurosecretion, 9 vasculartone, 10 and asthma. 2;11;12 While the A 1 , A 2A , and A 3 adenosine receptorshave been pharmacologically characterized through the use of highly potent and selective agonists and/or antagonists, the study ofthe A 2B receptor has been precluded due to the lack of selective ligands, 13–15 and the absence of an appropriate radioligand binding assay. Recently, [ 3 H]-ZM241385 has been proposed as a useful radioligand forstudying theA 2B adenosinereceptor subtype. 16 Building on this research,Jacobson and co-workers have reported some xanthine derivatives endowed with good affinity butlimited significant selectivity for the human A 2B adenosine receptor subtype. 17;18 An evolu- tion of this study led to the synthesis of 8-phenyl xan- thine carboxylicacid congeners.In particular,the derivative named MRS-1754, 19 ([N-(4-cyanophenyl)-2- [4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purine- 8-yl)phenoxy]-acetamide] proved to be the most potent and selective A 2B adenosine receptor antagonist yet re- ported.This result,led to the synthesis and character- ization of the tritium labeled form of MRS-1754 as the first radioligand for the A 2B receptor. 20 Very recently our group has reported the synthesis and evaluationof a seriesof 8-heterocyclic substituted Keywords:Selectiveantagonist;Adenosinereceptors;Radioligand; Pharmacological characterization. * Corresponding author. Tel.: +39-0532-291293; fax: +39-0532-291296; e-mail: pgb@dns.unife.it 0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2004.03.084 Bioorganic & Medicinal Chemistry Letters 14 (2004) 3607–3610