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1.  Functionalized Congeners of 1,4-Dihydropyridines as Antagonist Molecular Probes for A3 Adenosine Receptors 
Bioconjugate chemistry  1999;10(4):667-677.
4-Phenylethynyl-6-phenyl-1,4-dihydropyridine derivatives are selective antagonists at human A3 adenosine receptors, with Ki values in a radioligand binding assay vs [125I]AB-MECA [N6-(4-amino-3-iodobenzyl)-5′-N-methylcarbamoyl-adenosine] in the submicromolar range. In this study, functionalized congeners of 1,4-dihydropyridines were designed as chemically reactive adenosine A3 antagonists, for the purpose of synthesizing molecular probes for this receptor subtype. Selectivity of the new analogues for cloned human A3 adenosine receptors was determined in radioligand binding in comparison to binding at rat brain A1 and A2A receptors. Benzyl ester groups at the 3- and/or 5-positions and phenyl groups at the 2- and/or 6-positions were introduced as potential sites for chain attachment. Structure–activity analysis at A3 adenosine receptors indicated that 3,5-dibenzyl esters, but not 2,6-diphenyl groups, are tolerated in binding. Ring substitution of the 5-benzyl ester with a 4-fluorosulfonyl group provided enhanced A3 receptor affinity resulting in a Ki value of 2.42 nM; however, a long-chain derivative containing terminal amine functionalization at the 4-position of the 5-benzyl ester showed only moderate affinity. This sulfonyl fluoride derivative appeared to bind irreversibly to the human A3 receptor (1 h incubation at 100 nM resulting in the loss of 56% of the specific radioligand binding sites), while the binding of other potent dihydropyridines and other antagonists was generally reversible. At the 3-position of the dihydropyridine ring, an amine-functionalized chain attached at the 4-position of a benzyl ester provided higher A3 receptor affinity than the corresponding 5-position isomer. This amine congener was also used as an intermediate in the synthesis of a biotin conjugate, which bound to A3 receptors with a Ki value of 0.60 μM.
PMCID: PMC3446815  PMID: 10411465
2.  Structure–Activity Relationships of 9-Alkyladenine and Ribose-Modified Adenosine Derivatives at Rat A3 Adenosine Receptors† 
Journal of medicinal chemistry  1995;38(10):1720-1735.
9-Alkyladenine derivatives and ribose-modified N6-benzyladenosine derivatives were synthesized in an effort to identify selective ligands for the rat A3 adenosine receptor and leads for the development of antagonists. The derivatives contained structural features previously determined to be important for A3 selectivity in adenosine derivatives, such as an N6-(3-iodobenzyl) moiety, and were further substituted at the 2-position with halo, amino, or thio groups. Affinity was determined in radioligand binding assays at rat brain A3 receptors stably expressed in Chinese hamster ovary (CHO) cells, using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5′-(N-methyluronamide)), and at rat brain A1 and A2a receptors using [3H]-N6-PIA ((R)-N6-phenylisopropyladenosine) and [3H]CGS 21680 (2-[[[4-(2-carboxyethyl)-phenyl]ethyl]amino]-5′-(N-ethylcarbamoyl)adenosine), respectively. A series of N6-(3-iodobenzyl) 2-amino derivatives indicated that a small 2-alkylamino group, e.g., methylamino, was favored at A3 receptors. N6-(3-Iodobenzyl)-9-methyl-2-(methylthio)adenine was 61-fold more potent than the corresponding 2-methoxy ether at A3 receptors and of comparable affinity at A1 and A2a receptors, resulting in a 3–6-fold selectivity for A3 receptors. A pair of chiral N6-(3-iodobenzyl) 9-(2,3-dihydroxypropyl) derivatives showed stereoselectivity, with the R-enantiomer favored at A3 receptors by 5.7-fold. 2-Chloro-9-(β-d-erythrofuranosyl)-N6-(3-iodobenzyl)adenine had a Ki value at A3 receptors of 0.28 µM. 2-Chloro-9-[2-amino-2,3-dideoxy-β-d-5-(methylcarbamoyl)-arabinofuranosyl]-N6-(3-iodobenzyl)adenine was moderately selective for A1 and A3 vs A2a receptors. A 3′-deoxy analogue of a highly A3-selective adenosine derivative retained selectivity in binding and was a full agonist in the inhibition of adenylyl cyclase mediated via cloned rat A3 receptors expressed in CHO cells. The 3′-OH and 4′-CH2OH groups of adenosine are not required for activation at A3 receptors. A number of 2′,3′-dideoxyadenosines and 9-acyclic-substituted adenines appear to inhibit adenylyl cyclase at the allosteric “P” site.
PMCID: PMC3445626  PMID: 7752196
3.  Pharmacological Characterization of Novel A3 Adenosine Receptor-selective Antagonists 
Neuropharmacology  1997;36(9):1157-1165.
The effects of putative A3 adenosine receptor antagonists of three diverse chemical classes (the flavonoid MRS 1067, the 6-phenyl-1,4-dihydropyridines MRS 1097 and MRS 1191, and the triazoloquinazo-line MRS 1220) were characterized in receptor binding and functional assays. MRS1067, MRS 1191 and MRS 1220 were found to be competitive in saturation binding studies using the agonist radioligand [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide) at cloned human brain A3 receptors expressed in HEK-293 cells. Antagonism was demonstrated in functional assays consisting of agonist-induced inhibition of adenylate cyclase and the stimulation of binding of [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP-γ-S) to the associated G-proteins. MRS 1220 and MRS 1191, with KB values of 1.7 and 92 nM, respectively, proved to be highly selective for human A3 receptor vs human A1 receptor-mediated effects on adenylate cyclase. In addition, MRS 1220 reversed the effect of A3 agonist-elicited inhibition of tumor necrosis factor-α formation in the human macrophage U-937 cell line, with an IC50 value of 0.3 μM. Published by Elsevier Science Ltd.
PMCID: PMC3433714  PMID: 9364471
Dihydropyridine; flavonoid; triazoloquinazoline; adenylate cyclase; tumor necrosis factor; guanine nucleotides; adenosine A3 receptor; adenosine
4.  Allosteric Modulation of A3 Adenosine Receptors by a Series of 3-(2-Pyridinyl)isoquinoline Derivatives 
Molecular pharmacology  2001;60(5):1057-1063.
Allosteric modulators of A1 and A2A adenosine receptors have been described; however, for the A3 adenosine receptor, neither an allosteric site nor a compound with allosteric effects has been described. In this study, the allosteric modulation of human A3 adenosine receptors by a series of 3-(2-pyridinyl)isoquinoline derivatives was investigated by examining their effects on the dissociation of the agonist radioligand, [125I]N6-(4-amino-3-iodobenzyl)-5′ -N-methylcarboxamidoadenosine (I-AB-MECA), from the receptor. Several 3-(2-pyridinyl)isoquinoline derivatives, including VUF5455, VUF8502, VUF8504, and VUF8507, slowed the dissociation of the agonist radioligand [125I]I-AB-MECA in a concentration-dependent manner, suggesting an allosteric interaction. These compounds had no effect on the dissociation of the radiolabeled antagonist [3H]PSB-11 from the A3 adenosine receptor, suggesting a selective enhancement of agonist binding. By comparison, compounds of similar structure (VUF8501, VUF8503, VUF8505), the classical adenosine receptor antagonist CGS15943 and the A1 receptor allosteric enhancer PD81723 did not significantly influence the dissociation rate of [125I]I-AB-MECA. The effect of agonist on forskolin-induced cAMP production was significantly enhanced by VUF5455. When the subtype-selectivity of the allosteric enhancement was tested the compounds had no effect on the dissociation of either [3H]N6-[(R)-phenylisopropyl]adenosine from the A1 adenosine receptor or [3H]CGS21680 from the A2A adenosine receptor. Probing of structure-activity relationships suggested that a carbonyl group is essential for allosterism but preferred only for competitive antagonism. The presence of a 7-methyl group decreased the competitive binding affinity without a major loss of the allosteric enhancing activity, suggesting that the structural requirements for allosteric enhancement might be distinct from those for competitive antagonism.
PMCID: PMC3953614  PMID: 11641434
5.  Structure–Activity Relationships of 1,3-Dialkylxanthine Derivatives at Rat A3 Adenosine Receptors 
Journal of medicinal chemistry  1994;37(20):3373-3382.
1,3-Dialkylxanthine analogues containing carboxylic acid and other charged groups on 8-position substituents were synthesized. These derivatives were examined for affinity in radioligand binding assays at rat brain A3 adenosine receptors stably expressed in CHO cells using the new radioligand [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide), and at rat brain A1 and A2a receptors using [3H]PIA and [3H]CGS 21680, respectively. A synthetic strategy for introducing multiple carboxylate groups at the 8-position using iminodiacetic acid derivatives was explored. The presence of a sulfonate, a carboxylate, or multiple carboxylate groups did not result in a significant enhancement of affinity at rat A3 receptors, although as previously observed an anionic group tended to diminish potency at A1 and A2a receptors. The rat A3 receptor affinity was not highly dependent on the distance of a carboxylate group from the xanthine pharmacophore. 2-Thio vs 2-oxo substitution favored A3 potency, and 8-alkyl vs 8-aryl substitution favored A3 selectivity, although few derivatives were truly selective for rat A3 receptors. 1,3-Dimethyl-8-(3-carboxypropyl)-2-thioxanthine was 7-fold selective for A3 vs A2a receptors. 1,3,7-Trimethyl-8-(trans-2-carboxyvinyl)xanthine was somewhat selective for A3 vs A1 receptors. For 8-arylxanthines affinity at A3 receptors was enhanced by 1,3-dialkyl substituents, in the order dibutyl > dipropyl > diallyl.
PMCID: PMC3471218  PMID: 7932565
6.  2-Substitution of N6-Benzyladenosine-5′-uronamides Enhances Selectivity for A3 Adenosine Receptors 
Journal of medicinal chemistry  1994;37(21):3614-3621.
Adenosine derivatives bearing an N6-(3-iodobenzyl) group, reported to enhance the affinity of adenosine-5′-uronamide analogues as agonists at A3 adenosine receptors (J. Med. Chem. 1994, 37, 636–646), were synthesized starting from methyl β-d-ribofuranoside in 10 steps. Binding affinities at A1 and A2a receptors in rat brain membranes and at cloned rat A3 receptors from stably transfected CHO cells were compared. N6-(3-Iodobenzyl)adenosine was 2-fold selective for A3 vs A1 or A2a receptors; thus it is the first monosubstituted adenosine analogue having any A3 selectivity. The effects of 2-substitution in combination with modifications at the N6- and 5′-positions were explored. 2-Chloro-N6-(3-iodobenzyl)adenosine had a Ki value of 1.4 nM and moderate selectivity for A3 receptors. 2-Chloro-N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide, which displayed a Ki value of 0.33 nM, was selective for A3 vs A1 and A2a receptors by 2500- and 1400-fold, respectively. It was 46,000-fold selective for A3 receptors vs the Na+-independent adenosine transporter, as indicated in displacement of [3H]N6-(4-nitrobenzyl)-thioinosine binding in rat brain membranes. In a functional assay in CHO cells, it inhibited adenylate cyclase via rat A3 receptors with an IC50 of 67 nM. 2-(Methylthio)-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide and 2-(methylamino)-N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide were less potent, but nearly as selective for A3 receptors. Thus, 2-substitution (both small and sterically bulky) is well-tolerated at A3 receptors, and its A3 affinity-enhancing effects are additive with effects of uronamides at the 5′-position and a 3-iodobenzyl group at the N6-position.
PMCID: PMC3468333  PMID: 7932588
7.  Sulfur-Containing 1,3-Dialkylxanthine Derivatives as Selective Antagonists at A1-Adenosine Receptors 
Journal of medicinal chemistry  1989;32(8):1873-1879.
Sulfur-containing analogues of 8-substituted xanthines were prepared in an effort to increase selectivity or potency as antagonists at adenosine receptors. Either cyclopentyl or various aryl substituents were utilized at the 8-position, because of the association of these groups with high potency at A1-adenosine receptors. Sulfur was incorporated on the purine ring at positions 2 and/or 6, in the 8-position substituent in the form of 2- or 3-thienyl groups, or via thienyl groups separated from an 8-aryl substituent through an amide-containing chain. The feasibility of using the thienyl group as a prosthetic group for selective iodination via its Hg2+ derivative was explored. Receptor selectivity was determined in binding assays using membrane homogenates from rat cortex [[3H]-N6-(phenylisopropyl) adenosine as radioligand] or striatum [[3H]-5′-(N-ethylcarbamoyl)adenosine as radioligand] for A1- and A2-adenosine receptors, respectively. Generally, 2-thio-8-cycloalkylxanthines were at least as A1 selective as the corresponding oxygen analogue. 2-Thio-8-aryl derivatives tended to be more potent at A2 receptors than the oxygen analogue. 8-[4-[(Carboxymethyl)oxy]phenyl]-1,3-dipropyl-2-thioxanthine ethyl ester was >740-fold A1 selective.
PMCID: PMC3479653  PMID: 2754711
8.  Functionalized Congeners of Adenosine: Preparation of Analogues with High Affinity for A1-Adenosine Receptors 
Journal of medicinal chemistry  1985;28(9):1341-1346.
A series of functionalized congeners of adenosine based on N6-phenyladenosine, a potent A1-adenosine receptor agonist, was synthesized. Derivatives of the various congeners should be useful as receptor and histochemical probes and for the preparation of radioligands and affinity columns or as targeted drugs. N6-[4-(Carboxymethyl)phenyl]adenosine served as the starting point for synthesis of the methyl ester, the methyl amide, the ethyl glycinate, and various substituted anilides. One of the latter, N6-[4-[[[4-(carbomethoxymethyl)anilino]carbonyl]methyl]phenyl]adenosine, served as the starting point for the synthesis of another series of congeners including the methyl amide, the hydrazide, and the aminoethyl amide. The terminal amino function of the last congener was acylated to provide further analogues. The various congeners were potent competitive antagonists of binding of N6-[3H]cyclohexyladenosine to A1-adenosine receptors in rat cerebral cortical membranes. The affinity of the congener for the A1 receptor was highly dependent on the nature of the spacer group and the terminal moiety with Ki values ranging 1–100 nM. A biotinylated analogue had a Ki value of 11 nM. A conjugate derived from the Bolton–Hunter reagent had a Ki value of 4.5 nM. The most potent congener contained a terminal [(aminoethyl)amino]carbonyl function and had a Ki value of less than 1 nM.
PMCID: PMC3469267  PMID: 2993623
Biochemical Pharmacology  1987;36(10):1697-1707.
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 ~ 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.
PMCID: PMC3388543  PMID: 3036153
10.  Modulation of adenosine receptor affinity and intrinsic efficacy in adenine nucleosides substituted at the 2-position 
Bioorganic & medicinal chemistry  2004;12(11):2995-3007.
We studied the structural determinants of binding affinity and efficacy of adenosine receptor (AR) agonists. Substituents at the 2-position of adenosine were combined with N6-substitutions known to enhance human A3AR affinity. Selectivity of binding of the analogues and their functional effects on cAMP production were studied using recombinant human A1, A2A, A2B, and A3ARs. Mainly sterically small substituents at the 2-position modulated both the affinity and intrinsic efficacy at all subtypes. The 2-cyano group decreased hA3AR affinity and efficacy in the cases of N6-(3-iodobenzyl) and N6-(trans-2-phenyl-1-cyclopropyl), for which a full A3AR agonist was converted into a selective antagonist; the 2-cyano-N6-methyl analogue was a full A3AR agonist. The combination of N6-benzyl and various 2-substitutions (chloro, trifluoromethyl, and cyano) resulted in reduced efficacy at the A1AR. The environment surrounding the 2-position within the putative A3AR binding site was explored using rhodopsin-based homology modeling and ligand docking.
PMCID: PMC3463103  PMID: 15142558
Purines; Cyclic AMP; Binding; Antagonists; Agonists; GPCR; Molecular modeling
11.  2-Substituted adenosine derivatives: affinity and efficacy at four subtypes of human adenosine receptors 
Biochemical pharmacology  2004;68(10):1985-1993.
The affinity and efficacy at four subtypes (A1, A2A, A2B and A3) of human adenosine receptors (ARs) of a wide range of 2-substituted adenosine derivatives were evaluated using radioligand binding assays and a cyclic AMP functional assay in intact CHO cells stably expressing these receptors. Similar to previous studies of the N6-position, several 2-substituents were found to be critical structural determinants for the A3AR activation. The following adenosine 2-ethers were moderately potent partial agonists (Ki, nM): benzyl (117), 3-chlorobenzyl (72), 2-(3-chlorophenyl)ethyl (41), and 2-(2-naphthyl)ethyl (130). The following adenosine 2-ethers were A3AR antagonists: 2,2-diphenylethyl, 2-(2-norbornan)ethyl, R- and S-2-phenylbutyl, and 2-(2-chlorophenyl)ethyl. 2-(S-2-Phenylbutyloxy)a-denosine as an A3AR antagonist right-shifted the concentration–response curve for the inhibition by NECA of cyclic AMP accumulation with a KB value of 212 nM, which is similar to its binding affinity (Ki = 175 nM). These 2-substituted adenosine derivatives were generally less potent at the A1AR in comparison to the A3AR, but fully efficacious, with binding Ki values over 100 nM. The 2-phenylethyl moiety resulted in higher A3AR affinity (Ki in nM) when linked to the 2-position of adenosine through an ether group (54), than when linked through an amine (310) or thioether (1960). 2-[2-(l-Naphthyl)ethyloxy]adenosine (Ki = 3.8 nM) was found to be the most potent and selective (>50-fold) A2A agonist in this series. Mixed A2A/A3AR agonists have been identified. Interestingly, although most of these compounds were extremely weak at the A2BAR, 2-[2-(2-naphthyl)ethyloxy]adenosine (EC50 = 1.4 µM) and 2-[2-(2-thienyl)-ethyloxy]adenosine (EC50 = 1.8 (M) were found to be relatively potent A2B agonists, although less potent than NECA (EC50 = 140 nM).
PMCID: PMC3408601  PMID: 15476669
Adenosine receptors; Purines; Nucleosides; GPCR; Efficacy; Structure–activity relationships
12.  A New High Affinity, Iodinated Adenosine Receptor Antagonist as a Radioligand/Photoaffinity Crosslinking Probe 
Molecular pharmacology  1987;32(1):184-188.
A new high affinity antagonist photoaffinity crosslinking radioligand has been synthesized for use in studying adenosine receptors. This compound, PAPAXAC (8-[-4-[[[[[2-(4-aminophenyl-acetylamino)ethyl]amino]carbonyl]-methyl]oxy]phenyl]-1,3-di-propylxanthine), has been labeled with 125I by a chloramine T method. The radioligand [125I]PAPAXAC binds to A1 adenosine receptors from bovine cerebral cortex with high affinity (KD= 0.1 nM), appropriate stereoselectivity, and A1 adenosine receptor specificity. Binding is not perturbed by guanine nucleotides. Adenylate cyclase assays document that PAPAXAC is an antagonist capable of completely blocking the ability of N6-R-phenyl-2-propyladenosine (R-PIA) to inhibit adenylate cyclase activity via A1 adenosine receptors. [125IPAPAXAC can be incorporated covalently into a peptide of Mr = 40,000 using the heterobifunctional crosslinking agent N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate. Covalent labeling can be inhibited with adenosine receptor ligands to demonstrate a potency series of R-PIA > S-PIA > NECA ≫ IBMX. Guanine nucleotides do not decrease covalent incorporation. These results suggest that antagonists such as [125I]PAPAXAC recognize the same A1 adenosine receptor-binding subunit as agonists, such as [125I]AZPNEA, which labels a similar Mr peptide with the same pharmacological potency series. This new antagonist photoaffinity crosslinking probe/radioligand should be of great utility in the molecular characterization of A1 adenosine receptors.
PMCID: PMC3478951  PMID: 3614192
Dietary flavonoids have varied effects on animal cells, such as inhibition of platelet binding and aggregation, inhibition of inflammation, and anticancer properties, but the mechanisms of these effects remain largely unexplained. Adenosine receptors are involved in the homeostasis of the immune, cardiovascular, and central nervous systems, and adenosine agonists/antagonists exert many similar effects. The affinity of flavonoids and other phytochemicals to adenosine receptors suggests that a wide range of natural substances in the diet may potentially block the effects of endogenous adenosine. We used competitive radioligand binding assays to screen flavonoid libraries for affinity and a computational CoMFA analysis of flavonoids to compare steric and electrostatic requirements for ligand recognition at three subtypes of adenosine receptors. Flavone derivatives, such as galangin, were found to bind to three subtypes of adenosine receptors in the μM range. Pentamethylmorin (Ki 2.65 μM) was 14- to 17-fold selective for human A3 receptors than for A1 and A2A receptors. An isoflavone, genistein, was found to bind to A1 receptors. Aurones, such as hispidol (Ki 350 nM) are selective A1 receptor antagonists, and, like genistein, are present in soy. The flavones, chemically optimized for receptor binding, have led to the antagonist, MRS 1067 (3,6-dichloro-2′-(isopropoxy)-4′-methylflavone), which is 200-fold more selective for human A3 than A1 receptors. Adenosine receptor antagonism, therefore, may be important In the spectrum of biological activities reported for the flavonoids.
PMCID: PMC3429336  PMID: 12083460
We recently reported that 2-substitution of N6-benzyladenosine-5'-uronamides greatly enhances selectivity of agonists for rat A3 adenosine receptors J. Med. Chem. 1994, 37, 3614–3621). Specifically, 2-Chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (2-CI-IB-MECA), which displayed a K1 value of 0.33 nM, is the most selective for A3 receptors yet reported with selectivity versus A1 and A2a receptors of 2500- and 1400-fold, respectively. In order to obtain pharmacological tools for the study of A3 adenosine receptors, two routes for radiolabeling of 2-CI-IB-MECA through incorporation of tritium at the 5'-methylamido group were compared. One route formed a 2',3'-protected nucleoside 5'-carboxylic acid (9), which was condensed with methylamine and deprotected. The more efficient synthesis started from D-ribose and provided 2-CI-IB-MECA (12) in six steps with an overall yield of 5.6 %. Tritium was introduced in the penultimate step by heating N6-(3-iodobenzyl)-2-chloro-2',3'-di-O-acetyl-5'-(methoxycarbonyl)adenosine (17) with [3H]methylamine in methanol at 60 °C for 2 h. The specific activity of [3H]2-CI-IB-MECA was 29 Ci/mmol with a radiochemical purity of 99%.
PMCID: PMC3572746  PMID: 23598401
Adenosine Derivatives; Radioligands; Adenosine Receptors; Tritium; Nucleosides
The objective of this study was to create constitutively active mutant human A3 adenosine receptors (ARs) using single amino acid replacements, based on findings from other G protein-coupled receptors. A3 ARs mutated in transmembrane helical domains (TMs) 1, 3, 6, and 7 were expressed in COS-7 cells and subjected to agonist radioligand binding and phospholipase C (PLC) and adenylyl cyclase (AC) assays. Three mutant receptors, A229E in TM6 and R108A and R108K in the DRY motif of TM3, were found to be constitutively active in both functional assays. The potency of the A3 agonist Cl-IB-MECA (2–chloro-N6-(3–iodobenzyl)adenosine-5′-N-methyluronamide) in PLC activation was enhanced by at least an order of magnitude over wild type (EC50 951 nM) in R108A and A229E mutant receptors. Cl-IB-MECA was much less potent (>10-fold) in C88F, Y109F and Y282F mutants or inactive following double mutation of the DRY motif. The degree of constitutive activation was more pronounced for the AC signaling pathway than for the PLC signaling pathway. The results indicated that specific locations within the TMs proximal to the cytosolic region were responsible for constraining the receptor in a G protein-uncoupled conformation.
PMCID: PMC3626079  PMID: 11396942
purines; G protein-coupled receptor; phospholipase C; adenylyl cyclase; radioligand binding; nucleosides
A newly synthesized, chemically reactive adenosine derivative, N6-(3-isothiocyanatobenzyl)adenosine-5'-N-methyluronamide, was found to bind selectively to A3 receptors. Ki values for this isothiocyanate derivative in competition binding at rat brain A1, A2a, and A3 receptors were 145, 272 and 10.0 nM, respectively. A preincubation with this derivative resulted in irreversible inhibition of radioligand binding at rat A3 receptors in membranes of transfected CHO cells or RBL-2H3 mast cells, but not at rat A1 or A2a receptors. The loss of binding sites for 0.1 nM [125I]N6-(4-aminobenzyl)adenosine-5'-N-methyluronamide, a high affinity A3 receptor radioligand, in transfected CHO cell membranes was concentration-dependent with an IC50 of 50 nM. No change was observed in the Kd value of the remaining A3 receptor sites. The inhibition was also insensitive to theophylline (1 mM), consistent with the pharmacology of rat A3 receptors. Structurally similar adenosine analogues lacking the chemically reactive isothiocyanate group failed to irreversibly inhibit A3-binding.
PMCID: PMC3425636  PMID: 8074705
17.  Novel selective antagonist radioligands for the pharmacological study of A2B adenosine receptors 
Purinergic Signalling  2006;2(4):583-588.
The adenosine A2B receptor is the least well characterized of the four adenosine subtypes due to the lack of potent and selective agonists and antagonists. Despite the widespread distribution of A2B receptor mRNA, little information is available with regard to their function. The characterization of A2B receptors, through radioligand binding studies, has been performed, until now, by using low-affinity and non-selective antagonists like 1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX),(4-(2-[7-amino-2-(2-furyl)-[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl)-phenol ([3H]ZM 241385) and 3-(3,4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propyl-xanthine ([125I]ABOPX). Recently, high-affinity radioligands for A2B receptors, [N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide ([3H]MRS 1754), N-(2-(2-Phenyl-6-[4-(2,2,3,3-tetratritrio-3-phenylpropyl)-piperazine-1-carbonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl)-acetamide ([3H]OSIP339391) and N-benzo[1,3]dioxol-5-yl-2-[5-(1,3-dipropyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide] ([3H]MRE 2029F20), have been introduced. This minireview offers an overview of these recently developed radioligands and the most important applications of drugs towards A2B receptors.
PMCID: PMC2096665  PMID: 18404460
A2B adenosine receptors; novel and selective antagonist radioligands; pharmacological studies
18.  Agonist Derived Molecular Probes for A2A Adenosine Receptors 
The adenosine agonist 2-(4-(2-carboxyethyl)phenylethylamino)-5′-N-ethylcarboxamidoadenosine (CGS21680) was recently reported to be selective for the A2A adenosine receptor subtype, which mediates its hypotensive action. To investigate structurelactivity relationships at a distal site, CGS21680 was derivatized using a functionalized congener approach. The carboxylic group of CGS21680 has been esterified to form a methyl ester, which was then treated with ethylenediamine to produce an amine congener. The amine congener was an intermediate for acylation reactions, in which the reactive acyl species contained a reported group, or the precursor for such. For radioiodination, derivatives of p-hydroxyphenylpropionic, 2-thiophenylacetic, and p-aminophenylacetic acids were prepared. The latter derivative (PAPA-APEC) was iodinated electrophilically using [125I]iodide resulting in a radioligand which was used for studies of competition of binding to striatal A, adenosine receptors in bovine brain. A biotin conjugate and an aryl sulfonate were at least 350-fold selective for A, receptors. For spectroscopic detection, a derivative of the stable free radical tetramethyl-1-piperidinyloxy (TEMPO) was prepared. For irreversible inhibition of receptors, meta- and para-phenylenediisothiocyanate groups were incorporated in the analogs. We have demonstrated that binding at A2A receptors is relatively insensitive to distal structural changes at the 2-position, and we report high affinity molecular probes for receptor characterization by radioactive, spectroscopic and affinity labelling methodology.
PMCID: PMC3157953  PMID: 2561548
19.  Pyrazolo-triazolo-pyrimidines as adenosine receptor antagonists: Effect of the N-5 bond type on the affinity and selectivity at the four adenosine receptor subtypes 
Purinergic Signalling  2007;4(1):39-46.
In the last few years, many efforts have been made to search for potent and selective human A3 adenosine antagonists. In particular, one of the most promising human A3 adenosine receptor antagonists is represented by the pyrazolo-triazolo-pyrimidine family. This class of compounds has been strongly investigated from the point of view of structure-activity relationships. In particular, it has been observed that fundamental requisites for having both potency and selectivity at the human A3 adenosine receptors are the presence of a small substituent at the N8 position and an unsubstitued phenyl carbamoyl moiety at the N5 position. In this study, we report the role of the N5-bond type on the affinity and selectivity at the four adenosine receptor subtypes. The observed structure-activity relationships of this class of antagonists are also exhaustively rationalized using the recently published ligand-based homology modeling approach.
PMCID: PMC2245997  PMID: 18368532
Adenosine receptors; Antagonist binding; Ligand-based homology modeling; Molecular modeling
20.  Molecular modelling study of 2-phenylethynyladenosine (PEAdo) derivatives as highly selective A3 adenosine receptor ligands 
Purinergic Signalling  2006;2(4):589-594.
A series of 2-phenylethynyladenosine (PEAdo) derivatives substituted in the N6- and 4′position was synthesised and the new derivatives were tested at the four human adenosine receptors stably transfected into Chinese hamster ovary (CHO) cells, using radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assay (A2B). Binding studies showed that the presence of a phenyl ethynyl group in the 2 position of adenosine favoured the interaction with A3 receptors, resulting in compounds endowed with high affinity and selectivity for the A3 subtype. Additional substitution of the N6- and 4′position increases both A3 affinity and selectivity. The results showed that the new compounds have a good affinity for the A3 receptor and in particular, the N6-methoxy-2-phenylethynyl-5′N-methylcarboxamidoadenosine, with a Ki at A3 of 1.9 nM and a selectivity A1/A3 and A2A/A3 of 4,800- and 8,600-fold, respectively. Therefore, it is one of the most potent and selective agonists at the human A3 adenosine receptor subtype reported so far. Furthermore, functional assays of inhibition of 10 μM forskolin-stimulated cAMP production via the adenosine A3 receptor revealed that the new trisubstituted adenosine derivatives behave as full agonist of this receptor subtype. Docking analysis of these compounds was performed at a homology model of the human A3 receptor based on the bovine rhodopsin crystal structure as template, and the results are in accordance with the biological data.
PMCID: PMC2096654  PMID: 18404461
adenosine; adenosine agonists; adenosine receptors; agonists; G-protein-coupled receptors; homology modelling; signal transduction
21.  Structure-activity relationships and molecular modeling of 1,2,4-triazoles as adenosine receptor antagonists 
ACS medicinal chemistry letters  2012;3(9):715-720.
The structure-activity relationship (SAR) for a novel class of 1,2,4-triazole antagonists of the human A2A adenosine receptor (hA2AAR) was explored. Thirty-three analogs of a ligand that was discovered in a structure-based virtual screen against the hA2AAR were tested in hA1, A2A, and A3 radioligand binding assays and in functional assays for the A2BAR subtype. As a series of closely related analogs of the initial lead, 1, did not display improved binding affinity or selectivity, molecular docking was used to guide the selection of more distantly related molecules. This resulted in the discovery of 32, a hA2AAR antagonist (Ki 200 nM) with high ligand efficiency. In the light of the SAR for the 1,2,4-triazole scaffold, we also investigated the binding mode of these compounds based on docking to several A2AAR crystal structures.
PMCID: PMC3549271  PMID: 23342198
1,2,4-triazole; A2A adenosine receptor; antagonist; molecular docking; structure-activity relationship
22.  Cl-IB-MECA Reduces Ischemia/Reperfusion Injury in Mice by Activating the A3 Adenosine Receptor 
We used pharmacological agents and genetic methods to determine whether the potent A3 adenosine receptor (AR) agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5’-N-methylcarboxamide (Cl-IB-MECA) protects against myocardial ischemia/reperfusion injury in mice via the A3AR or via interactions with other AR subtypes. Pretreating wildtype (WT) mice with Cl-IB-MECA reduced myocardial infarct size induced by 30 min of coronary occlusion and 24 h of reperfusion at doses (30 and 100 µg/kg) that concomitantly reduced blood pressure and stimulated systemic histamine release. The A3AR-selective antagonist MRS 1523, but not the A2AAR antagonist ZM 241385, blocked the reduction in infarct size provided by Cl-IB-MECA, suggesting a mechanism involving the A3AR. To further examine the selectivity of Cl-IB-MECA, we assessed its cardioprotective effectiveness in A3AR gene ‘knock-out’ (A3KO) mice. Cl-IB-MECA did not reduce myocardial infarct size in A3KO mice in vivo and did not protect isolated perfused hearts obtained from A3KO mice from injury induced by global ischemia and reperfusion. Additional studies using WT mice treated with compound 48/80 to deplete mast cell contents excluded the possibility that Cl-IB-MECA was cardioprotective by releasing mediators from mast cells. These data demonstrate that Cl-IB-MECA protects against myocardial ischemia/reperfusion injury in mice principally by activating the A3AR.
PMCID: PMC2430759  PMID: 16985166
Biochemical pharmacology  1988;37(4):655-664.
A variety of non-xanthine heterocycles were found to be antagonists of binding of [3H]phenylisopropyladenosine to rat brain A1-adenosine receptors and of activation of adenylate cyclase via interaction of N-ethylcarboxarnidoadenosine with A2-adenosine receptors in human platelet and rat pheochromocytoma cell membranes. The pyrazolopyridines tracazolate, cartazolate and etazolate were several fold more potent than theophylline at both A1- and A2-adenosine receptors. The pyrazolopyridines, however, were still many fold less potent than 8-phenyltheophylline and other 8-phenyl-1,3-dialkylxanthines. A structurally related N6-substituted 9-methyladenine was also a potent adenosine antagonist with selectivity for A1 receptors. None of several aryl-substituted heterocycles, including a thiazolopyrimidine, imidazopyridines, benzimidazoles, a pyrazoloquinoline, a mesoionic xanthine analog and a triazolopyridazine exhibited the high potency typical of 8-phenyl-1,3-dialkylxanthines. A furyl-substituted triazoloquinazoline was very potent at both A1 and A2 receptors. A pteridin-2,4-dione, 1,3-dipropyllumazine, was somewhat less potent than theophylline at A1- and A2-adenosine receptors, whereas 1,3-dimethyllumazine was much less potent. A benzopteridin-2,4-dione, alloxazine, was somewhat more potent than theophylline. Other heterocycles with antagonist activity were the dibenzazepine carbamazepine and β-carboline-3-ethyl carboxylate. The phenylimidazoline clonidine had no activity, whereas a related dihydroxyphenylimidazoline was a weak non-competitive adenosine antagonist.
PMCID: PMC3445624  PMID: 2829919
Journal of receptor research  1992;12(2):149-169.
The adenosine agonist [3H]CGS21680 (2-[4-[[2-carboxyethyl]phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine) bound to A2 receptors in human striatal membranes with a Kd of 17.8±1.1 nM and a Bmax of 313± 10 fmol/mg protein. The addition of 100 μM GTP diminished both the affinity of agonist radioligand for A2 adenosine binding sites and the total binding, resulting in Kd and Bmax values of 28.6±1.0 nM and 185± 22 fmol/mg of protein. Adenosine ligands competed for [3H]CGS21680 with the expected potency order. The adenosine antagonist [3H]XAC (8-[4-[[[[(2-aminoethyl)-amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine), although A1-selective in the rat, binds to human striatal A2 receptors with high affinity. 25 nM CPX (8-cyclopentyl-1,3-dipropylxanthine), an A1-selective antagonist, was added to the incubation medium and effectively eliminated 91% of [3H]XAC (1 nM) binding to human A1 receptors, yet preserved 90% of binding to A2 receptors. [3H]XAC exhibited saturable, specific binding (50% of total) to A2 sites with a Kd of 2.98±0.54 nM and a Bmax of 0.71±0.23 pmol/mg protein (25°C, non-specific binding defined with 100 μM NECA). The potency order for antagonists against 1 nM [3H]XAC was CGS15943A > XAC ≈ PD115,199 > PAPA-XAC > CPX > HTQZ ≈ XCC ≈ CP-66,713 > theophylline ≈ caffeine, indicative of an A2-type binding site. A2a-receptors were found to be present in the human cortex, albeit at a much lower density than in the striatum. Photoaffinity labeling using 125I-PAPA-APEC revealed a molecular weight of 45K, but proteolytic cleavage was observed, resulting in fragments of MW 43K and 37K. In the absence of proteolytic inhibitors the 37K fragment, which still bound 125I-PAPA-APEC, was predominant.
PMCID: PMC3429337  PMID: 1583620
25.  Putative role of the adenosine A3 receptor in the antiproliferative action of N6-(2-isopentenyl)adenosine 
Purinergic Signalling  2011;7(4):453-462.
We tested a panel of naturally occurring nucleosides for their affinity towards adenosine receptors. Both N6-(2-isopentenyl)adenosine (IPA) and racemic zeatin riboside were shown to be selective human adenosine A3 receptor (hA3R) ligands with affinities in the high nanomolar range (Ki values of 159 and 649 nM, respectively). These values were comparable to the observed Ki value of adenosine on hA3R, which was 847 nM in the same radioligand binding assay. IPA also bound with micromolar affinity to the rat A3R. In a functional assay in Chinese hamster ovary cells transfected with hA3R, IPA and zeatin riboside inhibited forskolin-induced cAMP formation at micromolar potencies. The effect of IPA could be blocked by the A3R antagonist VUF5574. Both IPA and reference A3R agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (Cl-IB-MECA) have known antitumor effects. We demonstrated strong and highly similar antiproliferative effects of IPA and Cl-IB-MECA on human and rat tumor cell lines LNCaP and N1S1. Importantly, the antiproliferative effect of low concentrations of IPA on LNCaP cells could be fully blocked by the selective A3R antagonist MRS1523. At higher concentrations, IPA appeared to inhibit cell growth by an A3R-independent mechanism, as was previously reported for other A3R agonists. We used HPLC to investigate the presence of endogenous IPA in rat muscle tissue, but we could not detect the compound. In conclusion, the antiproliferative effects of the naturally occurring nucleoside IPA are at least in part mediated by the A3R.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-011-9244-9) contains supplementary material, which is available to authorized users.
PMCID: PMC3224645  PMID: 21720785
Adenosine A3 receptor; N6-(2-isopentenyl)adenosine (IPA); Antitumor agent; Modified nucleoside; HPLC; Zeatin riboside

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