2-Arylethynyl derivatives of (N)-methanocarba adenosine 5'-uronamides are selective A(3)AR (adenosine receptor) agonists. Here we substitute a 1,2,3-triazol-1-yl linker in place of the rigid, linear ethynyl group to eliminate its potential metabolic liability. Docking of nucleosides containing possible short linker moieties at the adenine C2 position using a hybrid molecular model of the A(3)AR (based on the A(2A)AR agonist-bound structure) correctly predicted that a triazole would maintain the A(3)AR selectivity, due to its ability to fit a narrow cleft in the receptor. The analogues with various N-6 and C2-aryltriazolyl substitution were synthesized and characterized in binding (K-i at hA(3)AR 0.3-12 nM) and in vivo to demonstrate efficacy in controlling chronic neuropathic pain (chronic constriction injury). Among N-6-methyl derivatives, a terminal pyrimidin-2-yl group in 9 (MRS7116) increased duration of action (36% pain protection at 3 h) in vivo. N-6-Ethyl 5-chlorothien-2-yl analogue 15 (MRS7126) preserved in vivo efficacy (85% protection at 1 h) with short duration. Larger N6 groups, e.g. 17 (MRS7138, > 90% protection at 1 and 3 h), greatly enhanced in vivo activity. Thus, we have combined structure-based methods and phenotypic screening to identify nucleoside derivatives having translational potential.

Multivalent dendrimeric conjugates of GPCR ligands may have increased potency or selectivity in comparison to monomeric ligands, a phenomenon that was tested in a model of cytoprotection in mouse HL-1 cardiomyocytes. Quantitative RT-PCR indicated high expression levels of endogenous A(1) and A(2A) adenosine receptors (ARs), but not of A(2B) and A(3)ARs. Activation of the heterologously expressed human A(3)AR in HL-1 cells by AR agonists significantly attenuated cell damage following 4h exposure to H(2)O(2) (750 microM) but not in untransfected cells. The A(3) agonist IB-MECA (EC(50) 3.8 microM) and the non-selective agonist NECA (EC(50) 3.9 microM) protected A(3) AR-transfected cells against H(2)O(2) in a concentration-dependent manner, as determined by lactate dehydrogenase release. A generation 5.5 PAMAM (polyamidoamine) dendrimeric conjugate of a N(6)-chain-functionalized adenosine agonist was synthesized and its mass indicated an average of 60 amide-linked nucleoside moieties out of 256 theoretical attachment sites. It non-selectively activated the A(3)AR to inhibit forskolin-stimulated cAMP formation (IC(50) 66nM) and, similarly, protected A(3)-transfected HL-1 cells from apoptosis-inducing H(2)O(2) with greater potency (IC(50) 35nM) than monomeric nucleosides. Thus, a PAMAM conjugate retained AR binding affinity and displayed greatly enhanced cardioprotective potency.

Background An approach to use multivalent dendrimer carriers for delivery of nucleoside signaling molecules to their cell surface G protein-coupled receptors (GPCRs) was recently introduced. Results A known adenosine receptor (AR) agonist was conjugated to polyamidoamine (PAMAM) dendrimer carriers for delivery of the intact covalent conjugate to on the cell surface. Depending on the linking moiety, multivalent conjugates of the N 6 -chain elongated functionalized congener ADAC ( N 6 -[4-[[[4-[[[(2-aminoethyl)amino]carbonyl]methyl]anilino]carbonyl]methyl]phenyl]-adenosine) achieved unanticipated high selectivity in binding to the cytoprotective human A 3 AR, a class A GPCR. The key to this selectivity of > 100-fold in both radioreceptor binding (K i app = 2.4 nM) and functional assays (EC 50 = 1.6 nM in inhibition of adenylate cyclase) was maintaining a free amino group (secondary) in an amide-linked chain. Attachment of neutral amide-linked chains or thiourea-containing chains preserved the moderate affinity and efficacy at the A 1 AR subtype, but there was no selectivity for the A 3 AR. Since residual amino groups on dendrimers are associated with cytotoxicity, the unreacted terminal positions of this A 3 AR-selective G2.5 dendrimer were present as carboxylate groups, which had the further benefit of increasing water-solubility. The A 3 AR selective G2.5 dendrimer was also visualized binding the membrane of cells expressing the A 3 receptor but did not bind cells that did not express the receptor. Conclusion This is the first example showing that it is feasible to modulate and even enhance the pharmacological profile of a ligand of a GPCR based on conjugation to a nanocarrier and the precise structure of the linking group, which was designed to interact with distal extracellular regions of the 7 transmembrane-spanning receptor. This ligand tool can now be used in pharmacological models of tissue rescue from ischemia and to probe the existence of A 3 AR dimers.