A facile and novel synthesis of N(2)-, C(6)-substituted pyrazolo[3,4-d]pyrimidine-4 carboxylate derivatives as adenosine receptor antagonists.

An efficient synthetic procedure was adopted to synthesize a series of new molecules containing the pyrazolo[3,4-d]pyrimidine (PP) scaffold, which have been evaluated as promising human adenosine receptor (AR) antagonists. The effect of substitutions at the N(2), C(4) and C(6) positions of PPs on the affinity and selectivity towards the adenosine receptors were explored. Most of the pyrazolo[3,4-d]pyrimidine-4-carboxylates displayed from moderate to good affinity at the human A3AR (hA3AR), as indicated by the low micromolar range of Ki values (Ki hA3AR = 0.7-34 µM). In particular, compounds 60 and 62 displayed good affinity at the hA3AR (60, Ki hA3AR = 2.2 µM and 62, Ki hA3AR = 2.9 µM) and selectivity towards the other AR subtypes (60, >46-fold selective and 62, >34-fold selective, respectively). In view of these results, these novel PP analogues were docked both in the crystallographic structure of the hA2AAR and in a homology model of the hA3AR in order to support the structure activity relationship (SAR) analysis. These preliminary results demonstrated that pyrazolo[3,4-d]pyrimidine can be considered a promising scaffold to obtain new molecules with potent hA3AR antagonist activity.

Novel 8-(p-substituted-phenyl/benzyl)xanthines with selectivity for the A2A adenosine receptor possess bronchospasmolytic activity.

A new series of 8-(p-substituted-phenyl/benzyl)xanthines has been synthesized and evaluated in vitro for adenosine receptor binding affinity and in vivo for bronchospasmolytic effects. It was observed that the nature of substituent at para-position of 8-phenyl/benzyl group on the xanthine scaffold remarkably affects the binding affinity and selectivity of xanthine derivatives for various adenosine receptor subtypes and also their bronchospasmolytic effects. Newly synthesized 8-phenylxanthines displayed potent binding affinity and significant selectivity for A2A receptors and also produced potent bronchospasmolytic effects. Replacement of phenyl ring with benzyl moiety at C8 of xanthine skeleton resulted in notable reduction in adenosine receptor affinity and broncholytic effects.

Adenosine receptors and cancer.

The A(1), A(2A), A(2B) and A(3) G-protein-coupled cell surface adenosine receptors (ARs) are found to be upregulated in various tumor cells. Activation of the receptors by specific ligands, agonists or antagonists, modulates tumor growth via a range of signaling pathways. The A(1)AR was found to play a role in preventing the development of glioblastomas. This antitumor effect of the A(1)AR is mediated via tumor-associated microglial cells. Activation of the A(2A)AR results in inhibition of the immune response to tumors via suppression of T regulatory cell function and inhibition of natural killer cell cytotoxicity and tumor-specific CD4+/CD8+ activity. Therefore, it is suggested that pharmacological inhibition of A(2A)AR activation by specific antagonists may enhance immunotherapeutics in cancer therapy. Activation of the A(2B)AR plays a role in the development of tumors via upregulation of the expression levels of angiogenic factors in microvascular endothelial cells. In contrast, it was evident that activation of A(2B)AR results in inhibition of ERK1/2 phosphorylation and MAP kinase activity, which are involved in tumor cell growth signals. Finally, A(3)AR was found to be highly expressed in tumor cells and tissues while low expression levels were noted in normal cells or adjacent tissue. Receptor expression in the tumor tissues was directly correlated to disease severity. The high receptor expression in the tumors was attributed to overexpression of NF-kappaB, known to act as an A(3)AR transcription factor. Interestingly, high A(3)AR expression levels were found in peripheral blood mononuclear cells (PBMCs) derived from tumor-bearing animals and cancer patients, reflecting receptor status in the tumors. A(3)AR agonists were found to induce tumor growth inhibition, both in vitro and in vivo, via modulation of the Wnt and the NF-kappaB signaling pathways. Taken together, A(3)ARs that are abundantly expressed in tumor cells may be targeted by specific A(3)AR agonists, leading to tumor growth inhibition. The unique characteristics of these A(3)AR agonists make them attractive as drug candidates.

Synthesis, biological and modeling studies of 1,3-di-n-propyl-2,4-dioxo-6-methyl-8-(substituted) 1,2,3,4-tetrahydro [1,2,4]-triazolo [3,4-f]-purines as adenosine receptor antagonists.

A new series of potential adenosine receptor antagonists with a [1,2,4]-triazolo-[3,4-f]-purine structure bearing at the 1 and 3 position n-propyl groups have been synthesized, and their affinities at the four human adenosine receptor subtypes (A(1), A(2A), A(2B) and A(3)) have been evaluated. In this case the presence of n-propyl groups seems to induce potency at the A(2A) and A(3) adenosine receptor subtypes as opposed to our previously reported series bearing methyl substituents at the 1 and 3 positions. In particular the non-acylated derivative 17 showed affinity at these two receptor subtypes in the micromolar range. Indeed, preliminary molecular modeling investigations according to the experimental binding data indicate a modest steric and electrostatic antagonist-receptor complementarity.

A2B adenosine receptor agonists: synthesis and biological evaluation of 2-phenylhydroxypropynyl adenosine and NECA derivatives.

In the search for agonists for the elusive A2B adenosine receptor subtypes, 2-phenylhydroxypropynyl-5'-N-methylcarboxamido adenosine (PHPMECA, 14), 2-phenylhydroxypropynyl-5'-N-propylcarboxamido adenosine (PHPPECA, 15), and N6-ethyl-2-phenylhydroxypropynyl-5'-N-ethylcarboxamidoadenosine (19) were synthesized on the basis that introduction of alkynyl chains in 2-position of adenosine derivatives resulted in reasonably good A2B potency compared to NECA [see N6-ethyl-2-phenylhydroxypropynyl adenosine (5) EC50 = 1,700 nM and 2-phenylhydroxypropynyl-5'-N-ethylcarboxamido adenosine (PHPNECA, 8) EC50 = 1,100 nM, respectively]. Radioligand binding studies and adenylyl cyclase assays, performed with recently cloned human A1, A2A, A2B, and A3 adenosine receptors, showed that these modifications produced a decrease in potency at A2B receptor, as well as a general reduction in affinity at the other receptor subtypes. On the other hand, the contemporary presence of an ethyl substituent in N6-position and of a 4'-ethylcarboxamido group in the same compounds led to (R,S)-N6-ethyl-2-phenylhydroxypropynyl-5'-N-ethylcarboxamidoadenosine and (S)-N6-ethyl-2-phenylhydroxypropynyl-5'-N-ethylcarboxamidoadenosine, which did not show the expected increase in potency at A2B subtype. Hence, (S)-2-phenylhydroxypropynyl-5'-N-ethylcarboxamidoadenosine [(S)-PHPNECA] with EC50 A2B = 220 nM remains the most potent agonist at A2B receptor reported so far.

Comparative pharmacology of human beta-adrenergic receptor subtypes--characterization of stably transfected receptors in CHO cells.

Although many beta1-receptor antagonists and beta2-receptor agonists have been used in pharmacotherapy for many years their pharmacological properties at all three known subtypes of beta-adrenergic receptors are not always well characterized. The aim of this study was, therefore, to provide comparative binding characteristics of agonists (epinephrine, norepinephrine, isoproterenol, fenoterol, salbutamol, salmeterol, terbutalin, formoterol, broxaterol) and antagonists (propranolol, alprenolol, atenolol, metoprolol, bisoprolol, carvedilol, pindolol, BRL 37344, CGP 20712, SR 59230A, CGP 12177, ICI 118551) at all three subtypes of human beta-adrenergic receptors in an identical cellular background. We generated Chinese hamster ovary (CHO) cells stably expressing the three beta-adrenergic receptor subtypes at comparable levels. We characterized these receptor subtypes and analyzed the affinity of routinely used drugs as well as experimental compounds in competition binding studies, using the non-selective antagonist 125I-cyanopindolol as a radioligand. Furthermore, we analyzed the beta-receptor-mediated adenylyl cyclase activity in isolated membranes from these cell lines. The results from our experiments show that all compounds exhibit distinct patterns of selectivity and activity at the three beta-receptor subtypes. In particular, a number of beta2- or beta3-receptor agonists that are inverse agonists at the other subtypes were identified. In addition, beta1-receptor antagonists with agonistic activity at beta2- and beta3-receptors were found. These specific mixtures of agonism, antagonism, and inverse agonism at different subtypes may have important implications for the therapeutic use of the respective compounds.

International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors.

Four adenosine receptors have been cloned and characterized from several mammalian species. The receptors are named adenosine A(1), A(2A), A(2B), and A(3). The A(2A) and A(2B) receptors preferably interact with members of the G(s) family of G proteins and the A(1) and A(3) receptors with G(i/o) proteins. However, other G protein interactions have also been described. Adenosine is the preferred endogenous agonist at all these receptors, but inosine can also activate the A(3) receptor. The levels of adenosine seen under basal conditions are sufficient to cause some activation of all the receptors, at least where they are abundantly expressed. Adenosine levels during, e.g., ischemia can activate all receptors even when expressed in low abundance. Accordingly, experiments with receptor antagonists and mice with targeted disruption of adenosine A(1), A(2A), and A(3) expression reveal roles for these receptors under physiological and particularly pathophysiological conditions. There are pharmacological tools that can be used to classify A(1), A(2A), and A(3) receptors but few drugs that interact selectively with A(2B) receptors. Testable models of the interaction of these drugs with their receptors have been generated by site-directed mutagenesis and homology-based modelling. Both agonists and antagonists are being developed as potential drugs.

7-Nitrobenzofurazan (NBD) derivatives of 5'-N-ethylcarboxamidoadenosine (NECA) as new fluorescent probes for human A(3) adenosine receptors.

New fluorescent ligands for adenosine receptors (ARs), obtained by the insertion, in the N(6) position of NECA, of NBD-moieties with linear alkyl spacers of increasing length, proved to possess a high affinity and selectivity for the A(3) subtype expressed in CHO cells. In fluorescence microscopy assays, compound 2d, the most active and selective for human A(3)-AR, permitted visualization and localization of this human receptor subtype, showing its potential suitability for internalization and trafficking studies in living cells.

Regulation of p42/p44 mitogen-activated protein kinase by the human adenosine A3 receptor in transfected CHO cells.

In this study we have investigated whether the human adenosine A3 receptor activates p42/p44 mitogen-activated protein kinase (MAPK) in transfected Chinese hamster ovary (CHO) cells (designated CHO-A3). The high affinity adenosine A3 receptor agonist IB-MECA (1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-beta-D-ribofuranuronamide) stimulated time (peak activation occurring after 5 min) and concentration-dependent (pEC50=9.0+/-0.2) increases in p42/p44 MAPK in CHO-A3 cells. Adenosine A3 receptor-mediated increases in p42/p44 MAPK were sensitive to pertussis toxin and the MAPK kinase 1 inhibitor PD 98059 (2'-amino-3'-methoxyflavone). The broad range protein tyrosine kinase inhibitor genistein and the phosphatidylinositol 3-kinase inhibitors wortmannin and LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) also blocked adenosine A3 receptor stimulation of p42/p44 MAPK. In contrast, inhibition of protein kinase C had no significant effect on adenosine A3 receptor-induced p42/p44 MAPK activation. IB-MECA (pEC50=10.1+/-0.2) also increased the expression of luciferase in CHO-A3 cells transiently transfected with a luciferase reporter gene containing the c-fos promoter. Furthermore, IB-MECA-induced increases in luciferase gene expression were sensitive to pertussis toxin, PD 98059, genistein, wortmannin and LY 294002. In conclusion, we have shown that the human adenosine A3 receptor stimulates p42/p44 MAPK and c-fos-mediated luciferase gene expression in transfected CHO cells.

7-Deazaadenines bearing polar substituents: structure-activity relationships of new A(1) and A(3) adenosine receptor antagonists.

A series of 28 new pyrrolo[2,3-d]pyrimidine-4-amines, pyrimido[4, 5-b]indole-4-amines, and tetrahydropyrimido[4,5-b]indole-4-amines was synthesized and their adenosine receptor affinity determined in radioligand binding assays at rat A(1) and A(2A) adenosine receptors (ARs). Selected compounds were additionally investigated in binding assays at recombinant A(3) ARs. The 2-phenyl residue in (R)-7-(1-methylbenzyl)-2-phenylpyrrolo[2,3-d]pyrimidine-4-amine (ADPEP, 1) and in the corresponding pyrimido[4,5-b]indole (APEPI, 3) could be bioisosterically replaced by heterocyclic rings, such as 2-thienyl and 4-pyridyl. The resulting compounds retained high affinity and selectivity for A(1) ARs. Judging from the investigation of selected compounds, it appears that they are also potent at human A(1) ARs and selective not only versus A(2A) ARs but also highly selective versus A(2B) and A(3) ARs. The p-pyridyl-substituted derivatives 11 and 27 (APPPI) may be interesting pharmacological tools due to their fluorescent properties. Pyrrolo[2,3-d]pyrimidine-4-amine derivatives which were simultaneously substituted at N7 and N(4), combining the substitution pattern of ADPEP (1) and DPEAP (2), showed very low affinity for A(1) ARs. This finding supports our previously published hypothesis of different binding modes for pyrrolopyrimidines, such as ADPEP (1) and DPEAP (2). DPEAP (2), a pyrrolo[2,3-d]pyrimidine-4-amine substituted at the amino group (N(4)), was found to exhibit high affinity for human A(3) ARs (K(i) = 28 nM), whereas N(4)-unsubstituted analogues were inactive. DPEAP (2) and related compounds provide new leads for the development of antagonists for the human A(3) AR.

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives as highly potent and selective human A(3) adenosine receptor antagonists: influence of the chain at the N(8) pyrazole nitrogen.

An enlarged series of pyrazolotriazolopyrimidines previously reported, in preliminary form (Baraldi et al. J. Med. Chem. 1999, 42, 4473-4478), as highly potent and selective human A(3) adenosine receptor antagonists is described. The synthesized compounds showed A(3) adenosine receptor affinity in the sub-nanomolar range and high levels of selectivity evaluated in radioligand binding assays at human A(1), A(2A), A(2B), and A(3) adenosine receptors. In particular, the effect of the chain at the N(8) pyrazole nitrogen was analyzed. This study allowed us to identify the derivative with the methyl group at the N(8) pyrazole combined with the 4-methoxyphenylcarbamoyl moiety at the N(5) position as the compound with the best binding profile in terms of both affinity and selectivity (hA(3) = 0.2 nM, hA(1)/hA(3) = 5485, hA(2A)/hA(3) = 6950, hA(2B)/hA(3) = 1305). All the compounds proved to be full antagonists in a specific functional model where the inhibition of cAMP generation by IB-MECA was measured in membranes of CHO cells stably transfected with the human A(3) receptor. The new compounds are among the most potent and selective A(3) antagonists so far described. The derivatives with higher affinity at human A(3) adenosine receptors proved to be antagonists, in the cAMP assay, capable of inhibiting the effect of IB-MECA with IC(50) values in the nanomolar range, with a trend strictly similar to that observed in the binding assay. Also a molecular modeling study was carried out, with the aim to identify possible pharmacophore maps. In fact, a sterically controlled structure-activity relationship was found for the N(8) pyrazole substituted derivatives, showing a correlation between the calculated molecular volume of pyrazolo[4,3-e]1,2, 4-triazolo[1,5-c]pyrimidine derivatives and their experimental K(i) values.

Adenosine receptors and their ligands.

The regulatory actions of adenosine are mediated via four subtypes of G protein-coupled receptors distinguished as A1, A2A, A2B and A3 receptors. Their presence on basically every cell makes them an interesting target for the pharmacological intervention in many pathophysiological situations. A large number of ligands have been synthesized over the last two decades and provide agonists and antagonists that are more or less selective for the known receptor subtypes. In addition, many radioligands are available in tritiated or radioiodinated form. The comparative pharmacological characterization of all four human adenosine receptor subtypes revealed that some of the compounds thought to be selective from data in other species have unexpected potencies at human receptors. As a result, compounds that exhibit high affinity to only one subtype are an exception. Although the selection of ligands is immense, it is less than satisfying for most subtypes of adenosine receptors.

Agonist-induced internalization and recycling of the human A(3) adenosine receptors: role in receptor desensitization and resensitization.

A(3) adenosine receptors have been proposed to play an important role in the pathophysiology of cerebral ischemia with a regimen-dependent nature of the therapeutic effects probably related to receptor desensitization and down-regulation. Here we studied the agonist-induced internalization of human A(3) adenosine receptors in transfected Chinese hamster ovary cells, and then we evaluated the relationship between internalization and signal desensitization and resensitization. Binding of N(6)-(4-amino-3-[(125)I]iodobenzyl)adenosine-5'-N-methyluronamide to membranes from Chinese hamster ovary cells stably transfected with the human A(3) adenosine receptor showed a profile typical of these receptors in other cell lines (K:(D) = 1.3+/-0.08 nM; B(max) = 400+/-28 fmol/mg of proteins). The iodinated agonist, bound at 4 degrees C to whole transfected cells, was internalized by increasing the temperature to 37 degrees C with a rate constant of 0.04+/-0.034 min(-1). Agonist-induced internalization of A(3) adenosine receptors was directly demonstrated by immunogold electron microscopy, which revealed the localization of these receptors in plasma membranes and intracellular vesicles. Moreover, short-term exposure of these cells to the agonist caused rapid desensitization as tested in adenylyl cyclase assays. Subsequent removal of the agonist led to restoration of the receptor function and recycling of the receptors to the cell surface. The rate constant of receptor recycling was 0.02+/-0.0017 min(-1). Blockade of internalization and recycling demonstrated that internalization did not affect signal desensitization, whereas recycling of internalized receptors was implicated in the signal resensitization.

[(3)H]MRE 3008F20: a novel antagonist radioligand for the pharmacological and biochemical characterization of human A(3) adenosine receptors.

The lack of a radiolabeled selective A(3) adenosine receptor antagonist is a major drawback for an adequate characterization of this receptor subtype. This paper describes the pharmacological and biochemical characterization of the tritiated form of a new potent A(3) adenosine receptor antagonist, the pyrazolo triazolo pyrimidine derivative [(3)H]5N-(4-methoxyphenylcarbamoyl)amino-8-propyl-2-(2-furyl )pyrazolo [4,3-e] -1,2,4- triazolo[1,5-c]pyrimidine ([(3)H]MRE 3008F20). [(3)H]MRE 3008F20 bound specifically to the human adenosine A(3) receptor expressed in CHO cells (hA(3)CHO), and saturation analysis revealed a single high affinity binding site, K(D) = 0.80 +/- 0.06 nM, with a B(max) = 300 +/- 33 fmol/mg protein. This new ligand displayed high selectivity (1294-, 165-, and 2471-fold) in binding assay to human A(3) versus A(1), A(2A), and A(2B) receptors, respectively, and binds to the rat A(3) receptors with a K(i) > 10 microM. The pharmacological profile of [(3)H]MRE 3008F20 binding to hA(3)CHO cells was evaluated using known adenosine receptor agonists and antagonists with a rank order of potency consistent with that typically found for interactions with the A(3) adenosine receptors. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency identical with that observed in binding experiments. Thermodynamic data indicated that [(3)H]MRE 3008F20 binding to hA(3)CHO is entropy- and enthalpy-driven in agreement with the typical behavior of other adenosine antagonists to A(1) and A(2A) receptors. These results show that [(3)H]MRE 3008F20 is the first antagonist radioligand with high affinity and selectivity for the human A(3) adenosine receptor and may be used to investigate the physiopathological role of A(3) adenosine receptors.

Activation of the A3 adenosine receptor affects cell cycle progression and cell growth.

The A3 adenosine receptor has been implicated in modulation of cell growth. As a first step to the characterization of the underlying mechanisms, we exposed Chinese hamster ovary (CHO) cells transfected with the human A3 receptor (A3R-CHO) to selective A3 receptor ligands. At micromolar concentrations, the A3 agonists N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) and its 2-chloro derivative Cl-IB-MECA reduced cell number, with no effects on either parental CHO cells (not expressing any adenosine receptor), or CHO cells transfected with the human A1 receptor. Cl-IB-MECA also reduced cell number in the human HEK293 cell line transfected with the human A3 receptor cDNA as opposed to the respective untransfected wild-type cells. In A3R-CHO, agonist-induced effects were antagonized by nanomolar concentrations of A3 antagonists, including the triazoloquinazoline derivative MRS 1220, the dihydropyridine derivative MRS 1191, and the triazolonaphthyridine derivative L-249,313. A3 agonist-induced effects were not due to modulation of cell adhesion, nor to necrosis or apoptosis. Growth curves revealed highly impeded growth, and flow-cytometric analysis showed markedly reduced bromodeoxyuridine incorporation into nuclei. The effect on cell cycle was completely antagonized by MRS1191. Hence, activation of the human A3 receptor in A3R-CHO results in markedly impaired cell cycle progression, suggesting an important role for this adenosine receptor subtype in cell cycle regulation and cell growth.

Water-soluble phosphate prodrugs of 1-propargyl-8-styrylxanthine derivatives, A(2A)-selective adenosine receptor antagonists.

Water-soluble prodrugs of potent, A(2A)-selective adenosine receptor (AR) antagonists were prepared. 8-(m-Bromostyryl)-3, 7-dimethyl-1-propargylxanthine (BS-DMPX, 11) and the analogous 8-(m-methoxystyryl)xanthine derivative (MS-DMPX, 5b) were used as starting points. It was found that polar functional groups suitable for the attachment of a prodrug moiety were tolerated on the styryl ring and even better on the 3-substituent. 8-(m-Hydroxystyryl)-DMPX (7) and 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-1-propargylxanthine (5e, MSX-2) were the most potent and A(2A)-selective compounds and were selected for prodrug formation. For the preparation of 5e a new ring-closure method was applied. Treatment of 6-amino-1-(3-hydroxypropyl)-5-(m-methoxycinnamoylamino)-3-propa rgylur acil with hexamethyldisilazane at high temperature resulted in higher yields of the target xanthine than the standard ring-closure procedure using sodium hydroxide. Phosphate prodrugs were prepared by classical phosphorylation using phosphorus oxychloride and alternatively by using a phosphoramidite method. Phosphates of the aliphatic alcohol 5e could be obtained by both methods in similar yields. The phenolic compound 7, however, could be phosphorylated only by using the phosphoramidite method. The disodium salts of the phosphate prodrugs exhibited high water solubility (8-(m-methoxystyryl)-7-methyl-3-[3-O-phosphatylpropyl]-1- propargylxan thine disodium salt, 9b: 17 mM, 9 mg/mL). Prodrug 9b was found to be stable in aqueous solution (pH 7) but readily cleaved by phosphatases to liberate 5e (MSX-2). Compound 5e showed high affinity for rat A(2A) AR (K(i) = 8 nM), human recombinant A(2A) AR (K(i) = 5 nM), and human native A(2A) AR (K(i) = 15 nM) and was highly selective versus rat A(1) AR (110-fold), human recombinant A(2A) AR (500-fold), human A(2B) AR (>2000-fold), and human A(3) AR (>2000-fold).

2-Substituted N-ethylcarboxamidoadenosine derivatives as high-affinity agonists at human A3 adenosine receptors.

A number of 2-substituted 5'-N-ethylcarboxamidoadenosine (NECA) derivatives was investigated for their affinity and selectivity at human A3 adenosine receptors. The compounds were tested in radioligand competition studies and modulation of adenylyl cyclase activity on membranes from CHO cell lines stably transfected with the four human adenosine receptor subtypes. In binding studies the most potent compound, 2-(3-hydroxy-3-phenyl)propyn-1-yl-NECA (PHPNECA), exhibited a subnanomolar affinity for A3 adenosine receptors with a Ki value of 0.4 nM. As opposed to the limited A3 selectivity of PHPNECA, a 100-fold selectivity compared to both A1 and A2A receptors was found for 2-(2-phenyl)ethynyl-NECA (PENECA; Ki 6 nM). The EC50 values for activation of adenylyl cyclase via A2A adenosine receptors were in good agreement with the respective Ki values from binding experiments. In contrast, IC50 values for A1 and A3 receptor-mediated inhibition of adenylyl cyclase were shifted to higher values compared to the respective affinities determined in radioligand competition studies. Similar discrepancies between binding and functional data have been observed for the inhibitory A1 adenosine receptor in previous studies. Therefore, the same A3 selectivity of PENECA compared to A1 receptors was found in binding and adenylyl cyclase inhibition whereas the selectivity compared to A2A receptors that was detected in ligand binding was obscured in the functional assay. The series of compounds presented in this study identifies 2-substitution of the purine system as a promising target for the development of A3-selective high-affinity ligands.