Molecular probes for extracellular adenosine receptors

Abstract

Derivatives of adenosine receptor agonists (N 6 -phenyladenosines) and antagonists (1,3-dialkyl-8-phenylxanthines) bearing functionalized chains suitable for attachment to other molecules have been reported [Jacobson et al., J. med. Chem. 28, 1334 and 1341]. The "functionalized congener" approach has been extended to the synthesis of spectroscopic and other probes for adenosine receptors that retain high affinity (K i ~ 10 −9 −10 −8 M) in A 1 -receptor binding. The probes have been synthesized from an antagonist xanthine amine congener (XAC) and an adenosine amine congener (ADAC). [ 3 H]ADAC has been synthesized and found to bind highly specifically to A 1 -adenosine receptors of rat and calf cerebral cortical membranes with K D values of 1.4 and 0.34 nM respectively. The higher affinity in the bovine brain, seen also with many of the probes derived from ADAC and XAC, is associated with phenyl substituents. The spectroscopic probes contain a reporter group attached at a distal site of the functionalized chain. These bifunctional ligands may contain a spin label (e.g. the nitroxyl radical TEMPO) for electron spin resonance spectroscopy, or a fluorescent dye, including fluorescein and 4-nitrobenz-2-oxa-1,3-diazole (NBD), or labels for 19 F nuclear magnetic resonance spectroscopy. Potential applications of the spectroscopic probes in characterization of adenosine receptors are discussed."Functionalized congener" refers to a drug derivative that contains a chemical functional group, such as an amine or carboxylic acid, attached through a spacer chain to a position on the pharmacophore, which position is relatively insensitive to steric bulk. The congeners are designed as intermediates for the synthesis of covalent conjugates that retain binding properties at the receptor site for the drug. The aims of this approach are 2-fold: (1) to develop potential new pharmaceutical agents in which the activity of the primary pharmacophore (portion of drug molecule essential for biological activity) may be modulated through distal changes, and (2) to synthesize radioactive and non-radioactive molecular probes for the drug binding site. Since the strategy of this drug design allows groups to be placed at a distance from the binding site of the primary pharmacophore, it is possible to overcome constraints on the size of the attached group [1][2][3][4][5].Sites for functionalization of adenosine receptor ligands have been identified for both agonists (derivatives of N 6 -phenyladenosine) and antagonists (derivatives of 1,3-dialkyl-8-phenylxanthine . A common structure-activity relationship (SAR) feature in both agonist and antagonist series is that the presence of a distal amino group located on the attached chain in many cases results in increased affinity to adenosine receptors.In this study we have examined the parameters of binding of tritiated ADAC and have demonstrated the utility of both ADAC and XAC as synthetic precursors for receptor probes. Biotin-containing probes for adenosine receptors have been reported [6]. ...

Quantitative analysis of the formation and diffusion of A 1 -adenosine receptor-antagonist complexes in single living cells

“…The choice of XAC as the basis of this fluorescent probe was based on structure-activity relationships that show that substituents on the C8 position of the xanthine ring are tolerated for A 1 -AR binding (19,(24)(25)(26). The fluorophore BODIPY 630͞650 was chosen because it is pharmacologically inactive at the A 1 -AR and has photophysical properties suitable for single-cell applications (27).…”

2-[2-[4-[2-[2-[1,3-Dihydro-1,1-bis(4-hydroxyphenyl)-3-oxo-5-isobenzofuranthioureidyl]ethylaminocarbonyl]ethyl]phenyl] ethylamino]-5?-N-ethylcarboxamidoadenosine (FITC-APEC): A fluorescent ligand for A2a-adenosine receptors

“…APEC has a high potency as an A 2a -adenosine receptor agonist both in vitro and in vivo [16]. While the synthesis of fluorescent adenosine receptor ligands was reported previously [22], these derivatives are selective for A 1 -adenosine receptors and their usefulness for direct fluorescence measurement of receptors has not been described.…”

“…1) may restrict access to the receptor, thus yielding a moderate decrease in affinity. Another possibility is the detrimental influence on affinity of a negative charge on a chain, which is noted as a general phenomenon for adenosine receptor ligands [22].…”

Functionalized Congeners of Tyrosine-Based P2X7 Receptor Antagonists: Probing Multiple Sites for Linking and Dimerization

“…Such functionalized congeners have been used effectively for the structural investigations of other purine receptors (18,19,31), including adenosine A 1 , A 2A , and A 3 receptors. Suitable sites and chemical approaches to cross-linking these antagonists have been found on the tyrosyl side chain (R 2 ) and on the CR-substituent (R 3 ).…”