pubs.acs.org/jmc Published on Web 10/19/2009 r 2009 American Chemical Society J. Med. Chem. 2009, 52, 7897–7900 7897 DOI: 10.1021/jm900754g Synthesis and Biological Evaluation of 2-Alkynyl-N 6 -methyl-5 0 -N-methylcarboxamidoadenosine Derivatives as Potent and Highly Selective Agonists for the Human Adenosine A 3 Receptor ‡ Rosaria Volpini, † Michela Buccioni, † Diego Dal Ben, † Catia Lambertucci, † Carmen Lammi, † Gabriella Marucci, † Anna T. Ramadori, † Karl-Norbert Klotz, § and Gloria Cristalli* ,† † Department of Chemical Sciences, University of Camerino, Via S. Agostino 1, I-62032 Camerino, Italy, and § Institut f € ur Pharmakologie und Toxikologie, Universit € at W€ urzburg, D-97078 W€ urzburg, Germany Received May 29, 2009 A new series of 2-aralkynyl-N 6 -methyl-MECAs 10-13 were synthesized and evaluated in radioligand binding studies and in a new Eu-GTP functional assay that provides a powerful alternative to radioisotope use. The new compounds possess high affinity and selectivity for the AA 3 R with N 6 -methyl-2-phenyl- ethynylMECA (10) showing a subnanomolar affinity and about 100000-fold selectivity vs AA 1 R and AA 2A R. Furthermore, the new nucleosides showed to be full agonists, the N 6 -methyl-2-(2-pyridinyl)- ethynylMECA (13) being the most potent in the series. Introduction Adenosine (Ado, Figure 1) is a naturally occurring nucleo- side having a role in an ample variety of physiological and pathophysiological processes through the activation of at least four G-protein-coupled receptors (P1) cloned and classi- fied as Ado A 1 ,A 2A ,A 2B , and A 3 receptors (AA 1 R, AA 2A R, AA 2B R, and AA 3 R, respectively). 1,2 In particular, AA 3 R is expressed in a broad range of tissues and couples to G i/o proteins leading to inhibition of adenylyl cyclase, increase of intracellular Ca 2þ concentration, and activation of phospho- inositide 3-kinase. 3 Since this subtype is involved in a variety of key physiological processes, experimental evidence suggests that AA 3 R agonists could be employed as therapeutic agents for the treatment of several pathologies. 4 Cristalli and co- workers have previously published the synthesis and biologi- cal activity of 2-aralkynyl-N 6 -methoxyAdo compounds and the corresponding 5 0 -N-methyl and 5 0 -N-ethylcarboxamido derivatives (2-aralkynyl-N 6 -methoxyMECAs and NECAs, respectively), which are endowed with good affinity and different degrees of selectivity for the human AA 3 R. 5-7 Among them, some MECA derivatives bearing an aromatic ring directly linked to the 2-ethynyl group showed the best profile for this receptor subtype (1-4, AA 3 R affinity in the low nanomolar range and selectivity ranging from 1600- to 22000-fold; for details, see Table 1). 8 Furthermore, in a previous work by the same authors it was demonstrated that the introduction of a methyl group in N 6 -position of 2-phenylethynylAdo (N 6 -methylPEAdo, Figure 1) 9 favored the interaction with the human AA 3 R, this compound having comparable AA 3 R affinity and higher selectivity than the corresponding N 6 -methoxy-2-phenylethynylAdo (N 6 -methoxy- PEAdo, Figure 1) (N 6 -methylPEAdo K i AA 3 R = 3.4 nM, selectivity AA 1 R/AA 3 R = 500 and AA 2A R/AA 3 R = 2500 vs N 6 -methoxyPEAdo, K i AA 3 R = 3.8 nM, selectivity AA 1 R/ AA 3 R=300 and AA 2A R/AA 3 R=1100). 8 Starting from these observations and to find highly potent and selective AA 3 R agonists, the synthesis of N 6 -methyl analogues of 1-4 was undertaken. All of the new 2-aralkynyl- N 6 -methyl-MECAs 10-13 were evaluated in radioligand binding studies and in functional assays. In particular, a novel time-resolved fluorometric (TRF) method, which exploits the unique fluorescence properties of lanthanide chelates and provides a powerful alternative to the assays that utilize radioisotopes, was set up to evaluate the potency of the new compounds. Results and Discussion Chemistry. The 2-alkynyl-N 6 -methylMECA derivatives 10-13 were synthesized starting from 2-iodo-N 6 -methylAdo (5), which was obtained from commercially available gua- nosine in four steps. 10 Protection of 2 0 - and 3 0 -hydroxyl groups of 5, using acetone and p-toluenesulfonic acid at room temperature for 16 h, gave 6, which was reacted with KMnO 4 in basic conditions to obtain the acid derivative 7. Treatment of 7 with thionyl chloride in the presence of dry DMF and subsequent reaction with methylamine at 0 °C for 1 h furnished the protected 2-iodo-N 6 -methylMECA 8. Figure 1. Structure of known potent and selective AA 3 R ligands. ‡ Contribution to celebrate the 100th anniversary of the Division of Medicinal Chemistry of the American Chemical Society. *To whom correspondence should be addressed. Phone: þ39-0737- 402252. Fax: þ39-0737-402295. E-mail: gloria.cristalli@unicam.it.