Molecular probes for extracellular adenosine receptors

Abstract

Derivatives of adenosine receptor agonists (N6-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 (1985)]. The "functionalized congener" approach has been extended to the synthesis of spectroscopic and other probes for adenosine receptors that retain high affinity (Ki approximately 10(-9)-10(-8) M) in A1-receptor binding. The probes have been synthesized from an antagonist xanthine amine congener (XAC) and an adenosine amine congener (ADAC). [3H]ADAC has been synthesized and found to bind highly specifically to A1-adenosine receptors of rat and calf cerebral cortical membranes with KD 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 19F nuclear magnetic resonance spectroscopy. Potential applications of the spectroscopic probes in characterization of adenosine receptors are discussed.

Fig. 1

Structures and sites of tritiation of the functionalized congeners which are potent adenosine receptor antagonist (1, XAC) and agonist (2, ADAC) analogs.

Fig. 2

Saturation of [³H]ADAC binding to cerebral cortex membranes from rat (A) and calf (B). Specific (●— ●) and nonspecific (○— ○) binding was determined for 120 min at 37°. Values are means of a typical experiment done in triplicate. Right panels: Scatchards plots of the same data. K_D (nM) and B_max (pmol/mg protein) were 1.4 and 0.57, respectively, for rat and 0.34 and 0.64, respectively, for calf cerebral cortex membranes.

Fig. 3

Inhibition of [³H]ADAC binding to rat cerebral cortex membranes by adenosine agonists and antagonists. Binding of 1 nM [³H]ADAC was measured for 120 min at 37°. Values are from a typical experiment done in triplicate. Slope factors were 0.98 for ADAC (●); 0.94 for R-P1A (◆); 1.01 for XAC (■); 0.98 for NECA (▼); 1.02 for1,3-dipropyl-8-(p-sulfophenyl)xanthine (▲); 0.80 for 8-(p-sulfophenyl)theophylline (○); and 0.94 for theophylline (□).

Fig. 4

Effect of adenosine analogs on adenylate cyclase activity of rat pheochromocytoma (PC12) cell (A) and human platelet (B) membranes. Adenylate cyclase was measured for 10 min at 37°. Values are means of a typical experiment done in triplicate. The EC₅₀ values were 100 and 240 nm for NECA (●— ●) and 800 and 980 nM for ADAC (■— ■) in PC12 and platelet membranes, respectively.

Fig. 5

Structures of molecular probes for adenosine receptors derived from the functionalized congeners ADAC and XAC (Fig. 1) through coupling at the primary amino group.

Fig. 6

Multistep syntheses of several molecular probes containing an extended chain linking the reporter group to an adenosine receptor antagonist (A) or agonist (B).

Fig. 7

Schematic representation of binding of functionalized congeners at a receptor site. (A) A subset of the surface of the drug is exposed to the medium. (B) An analog containing a chain on the exposed portion is synthesized. This “functionalized congener” contains a chemical functional group ( ), such as an amine or carboxylic acid, for the covalent attachment to carrier molecules. (C) The presence of a bulky group close to the pharmacophore may prevent binding. (D) Attachment of a bulky group through the functionalized chain. The attached moiety (carrier) may enhance affinity due to an energetically favorable interaction, either specific or nonspecific, at a distal (accessory) site. At adenosine receptors the presence of a positively-charged ammonium group is associated with high affinity, possibly reflecting a distal electrostatic interaction.