Development of Covalent, Clickable Probes for Adenosine A1 and A3 Receptors.

Adenosine receptors are attractive therapeutic targets for multiple conditions, including ischemia-reperfusion injury and neuropathic pain. Adenosine receptor drug discovery efforts would be facilitated by the development of appropriate tools to assist in target validation and direct receptor visualization in different native environments. We report the development of the first bifunctional (chemoreactive and clickable) ligands for the adenosine A1 receptor (A1R) and adenosine A3 receptor (A3R) based on an orthosteric antagonist xanthine-based scaffold and on an existing structure-activity relationship. Bifunctional ligands were functional antagonists with nanomolar affinity and irreversible binding at the A1R and A3R. In-depth pharmacological profiling of these bifunctional ligands showed moderate selectivity over A2A and A2B adenosine receptors. Once bound to the receptor, ligands were successfully "clicked" with a cyanine-5 fluorophore containing the complementary "click" partner, enabling receptor detection. These bifunctional ligands are expected to aid in the understanding of A1R and A3R localization and trafficking in native cells and living systems.

Adenosine A2AR/A1R Antagonists Enabling Additional H3R Antagonism for the Treatment of Parkinson's Disease.

Adenosine A1/A2A receptors (A1R/A2AR) represent targets in nondopaminergic treatment of motor disorders such as Parkinson's disease (PD). As an innovative strategy, multitargeting ligands (MTLs) were developed to achieve comprehensive PD therapies simultaneously addressing comorbid symptoms such as sleep disruption. Recognizing the wake-promoting capacity of histamine H3 receptor (H3R) antagonists in combination with the "caffeine-like effects" of A1R/A2AR antagonists, we designed A1R/A2AR/H3R MTLs, where a piperidino-/pyrrolidino(propyloxy)phenyl H3R pharmacophore was introduced with overlap into an adenosine antagonist arylindenopyrimidine core. These MTLs showed distinct receptor binding profiles with overall nanomolar H3R affinities (Ki < 55 nM). Compound 4 (ST-2001, Ki (A1R) = 11.5 nM, Ki (A2AR) = 7.25 nM) and 12 (ST-1992, Ki (A1R) = 11.2 nM, Ki (A2AR) = 4.01 nM) were evaluated in vivo. l-DOPA-induced dyskinesia was improved after administration of compound 4 (1 mg kg-1, i.p. rats). Compound 12 (2 mg kg-1, p.o. mice) increased wakefulness representing novel pharmacological tools for PD therapy.

Development and Application of Subtype-Selective Fluorescent Antagonists for the Study of the Human Adenosine A1 Receptor in Living Cells.

The adenosine A1 receptor (A1AR) is a G-protein-coupled receptor (GPCR) that provides important therapeutic opportunities for a number of conditions including congestive heart failure, tachycardia, and neuropathic pain. The development of A1AR-selective fluorescent ligands will enhance our understanding of the subcellular mechanisms underlying A1AR pharmacology facilitating the development of more efficacious and selective therapies. Herein, we report the design, synthesis, and application of a novel series of A1AR-selective fluorescent probes based on 8-functionalized bicyclo[2.2.2]octylxanthine and 3-functionalized 8-(adamant-1-yl) xanthine scaffolds. These fluorescent conjugates allowed quantification of kinetic and equilibrium ligand binding parameters using NanoBRET and visualization of specific receptor distribution patterns in living cells by confocal imaging and total internal reflection fluorescence (TIRF) microscopy. As such, the novel A1AR-selective fluorescent antagonists described herein can be applied in conjunction with a series of fluorescence-based techniques to foster understanding of A1AR molecular pharmacology and signaling in living cells.

C2-substituted quinazolinone derivatives exhibit A1 and/or A2A adenosine receptor affinities in the low micromolar range.

Antagonists of the adenosine receptors (A1 and A2A subtypes) are widely researched as potential drug candidates for their role in Parkinson's disease-related cognitive deficits (A1 subtype), motor dysfunction (A2A subtype) and to exhibit neuroprotective properties (A2A subtype). Previously the benzo-α-pyrone based derivative, 3-phenyl-1H-2-benzopyran-1-one, was found to display both A1 and A2A adenosine receptor affinity in the low micromolar range. Prompted by this, the α-pyrone core was structurally modified to explore related benzoxazinone and quinazolinone homologues previously unknown as adenosine receptor antagonists. Overall, the C2-substituted quinazolinone analogues displayed superior A1 and A2A adenosine receptor affinity over their C2-substituted benzoxazinone homologues. The benzoxazinones were devoid of A2A adenosine receptor binding, with only two compounds displaying A1 adenosine receptor affinity. In turn, the quinazolinones displayed varying degrees of affinity (low micromolar range) towards the A1 and A2A adenosine receptor subtypes. The highest A1 adenosine receptor affinity and selectivity were favoured by methyl para-substitution of phenyl ring B (A1Ki = 2.50 µM). On the other hand, 3,4-dimethoxy substitution of phenyl ring B afforded the best A2A adenosine receptor binding (A2AKi = 2.81 µM) among the quinazolinones investigated. In conclusion, the quinazolinones are ideal lead compounds for further structural optimization to gain improved adenosine receptor affinity, which may find therapeutic relevance in Parkinson's disease-associated cognitive deficits and motor dysfunctions as well as exerting neuroprotective properties.

Synthesis and Structure Activity Relationships of Chalcone based Benzocycloalkanone Derivatives as Adenosine A1 and/or A2A Receptor Antagonists.

Adenosine A1 and/or A2A receptor antagonists hold promise for the potential treatment of neurological conditions, such as Parkinson's disease. Herein, a total of seventeen benzocycloalkanone derivatives were synthesised and evaluated for affinity towards adenosine receptors (A1 and A2A AR). The obtained results allowed for the conclusion that affinity and/or selectivity of the 2-benzylidene-1-indanone and -tetralone derivatives toward A1 and/or A2A ARs may be modulated by the nature of the substituents (either -OH, -OCH3 or morpholine) attached at position C4 of the 1-indanone core and C5 of the 1-tetralone core as well as the meta (C3') and/or para (C4') position(s) on ring B. Several compounds (2A: -B: , 3B: -C: and 4A: -B: ) possessed affinity for the A1 and/or A2A AR below 10 µM. Additionally, compounds 2A: , 3B: and 4A: were A1 AR antagonists. These results, once again, confirmed the importance of C4 methoxy-group substitution on ring A in combination with meta (C3') and/or para (C4') hydroxyl-group substitution ring B of the 2-benzylidene-1-indanone scaffold leading to drug-like compounds 1H: and 1J: with affinity in the nanomolar-range.

Synthesis and evaluation of methoxy substituted 2-benzoyl-1-benzofuran derivatives as lead compounds for the development adenosine A1 and/or A2A receptor antagonists.

A series of fourteen methoxy substituted 2-benzoyl-1-benzofuran derivatives were synthesised and their affinities determined for adenosine A1 and A2A receptors via radioligand binding assays to establish the structure activity relationships pertinent for A1 and A2A affinity. Compound 3j (6,7-dimethoxybenzofuran-2-yl)(3-methoxyphenyl)methanone exhibited A1 affinity (A1Ki (rat) = 6.880 µM) as well as A2A affinity (A2AKi (rat) = 0.5161 µM). Compounds 3a-b &3i-k exhibited selective affinity towards A1 with Ki values below 10 µM. The results indicate that C6,7-diOCH3 substitution on ring A in combination with meta (C3')-OCH3 substitution on ring B is beneficial for A1 and A2A affinity and activity. Compounds 3a-b &3j-k showed low cytotoxicity. Upon in vitro and in silico evaluation, compound 3j may be considered lead-like (i.e. a molecular entity suitable for optimization) and, thus, of value in the design of novel, potent and selective adenosine A1 and A2A receptor antagonists.

5-Substituted 2-benzylidene-1-tetralone analogues as A1 and/or A2A antagonists for the potential treatment of neurological conditions.

Adenosine A1 and A2A receptors are attracting great interest as drug targets for their role in cognitive and motor deficits, respectively. Antagonism of both these adenosine receptors may offer therapeutic benefits in complex neurological diseases, such as Alzheimer's and Parkinson's disease. The aim of this study was to explore the affinity and selectivity of 2-benzylidene-1-tetralone derivatives as adenosine A1 and A2A receptor antagonists. Several 5-hydroxy substituted 2-benzylidene-1-tetralone analogues with substituents on ring B were synthesized and assessed as antagonists of the adenosine A1 and A2A receptors via radioligand binding assays. The results indicated that hydroxy substitution in the meta and para position of phenyl ring B, displayed the highest selectivity and affinity for the adenosine A1 receptor with Ki values in the low micromolar range. Replacement of ring B with a 2-amino-pyrimidine moiety led to compound 12 with an increase of affinity and selectivity for the adenosine A2A receptor. These substitution patterns led to enhanced adenosine A1 and A2A receptor binding affinity. The para-substituted 5-hydroxy analogue 3 behaved as an adenosine A1 receptor antagonists in a GTP shift assay performed with rat whole brain membranes expressing adenosine A1 receptors. In conclusion, compounds 3 and 12, showed the best adenosine A1 and A2A receptor affinity respectively, and therefore represent novel adenosine receptor antagonists that may have potential with further structural modifications as drug candidates for neurological disorders.

Imidazo[1,2-α]pyridines possess adenosine A1 receptor affinity for the potential treatment of cognition in neurological disorders.

Previous research has shown that bicyclic 6:5-fused heteroaromatic compounds with two N-atoms have variable degrees of adenosine A1 receptor antagonistic activity. Prompted by this imidazo[1,2-α]pyridine analogues were synthesized and evaluated for their adenosine A1 and A2A receptor affinity via radioligand binding studies and subjected to a GTP shift assay to determine its adenosine A1 receptor agonistic or antagonistic functionality. Imidazo[1,2-α]pyridine, the parent scaffold, was found devoid of affinity for the adenosine A1 and A2A receptors. The influence of substitution on position C2 showed no improvement for either adenosine A1 or A2A receptor affinity. The addition of an amino or a cyclohexylamino group to position C3 also showed no improvement of adenosine A1 or A2A receptor affinity. Surprisingly para-substitution on the phenyl ring at position C2 in combination with a cyclohexylamino group at position C3 led to adenosine A1 receptor affinity in the low micromolar range with compound 4d showing: (1) the highest affinity for the adenosine A1 receptor with a Ki value of 2.06µM and (2) adenosine A1 receptor antagonistic properties. This pilot study concludes that para-substituted 3-cyclohexylamino-2-phenyl-imidazo[1,2-α]pyridine analogues represent an interesting scaffold to investigate further structure-activity relationships in the design of novel imidazo[1,2-α]pyridine-based adenosine A1 receptor antagonists for the treatment of neurodegenerative disorders.

Synthesis and Pharmacological Evaluation of Identified and Putative Metabolites of the A1 Adenosine Receptor Antagonist 8-Cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine (CPFPX).

The A1 adenosine receptor (A1 AR) antagonist [18 F]8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine ([18 F]CPFPX), used in imaging human brain A1 ARs by positron emission tomography (PET), is stable in the brain, but rapidly undergoes transformation into one major (3-(3-fluoropropyl)-8-(3-oxocyclopenten-1-yl)-1-propylxanthine, M1) and several minor metabolites in blood. This report describes the synthesis of putative metabolites of CPFPX as standards for the identification of those metabolites. Analysis by (radio)HPLC revealed that extracts of human liver microsomes incubated with no-carrier-added (n.c.a.)[18 F]CPFPX contain the major metabolite, M1, as well as radioactive metabolites corresponding to derivatives functionalized at the cyclopentyl moiety, but no N1-despropyl species or metabolites resulting from functionalization of the N3-fluoropropyl chain. The putative metabolites were found to displace the binding of [3 H]CPFPX to the A1 AR in pig brain cortex at Ki values between 1.9 and 380 nm and the binding of [3 H]ZM241385 to the A2A AR in pig striatum at Ki values >180 nm. One metabolite, a derivative functionalized at the ω-position of the N1-propyl chain, showed high affinity (Ki 2 nm) to and very good selectivity (>9000) for the A1 AR.

Design and synthesis of novel, potent and selective hypoxanthine analogs as adenosine A1 receptor antagonists and their biological evaluation.

Multipronged approach was used to synthesize a library of diverse C-8 cyclopentyl hypoxanthine analogs from a common intermediate III. Several potent and selective compounds were identified and evaluated for pharmacokinetic (PK) properties in Wistar rats. One of the compounds 14 with acceptable PK parameters was selected for testing in in vivo primary acute diuresis model. The compound demonstrated significant diuretic activity in this model.

The role of 5-arylalkylamino- and 5-piperazino- moieties on the 7-aminopyrazolo[4,3-d]pyrimidine core in affecting adenosine A1 and A2A receptor affinity and selectivity profiles.

New 7-amino-2-phenylpyrazolo[4,3-d]pyrimidine derivatives, substituted at the 5-position with aryl(alkyl)amino- and 4-substituted-piperazin-1-yl- moieties, were synthesized with the aim of targeting human (h) adenosine A1 and/or A2A receptor subtypes. On the whole, the novel derivatives 1-24 shared scarce or no affinities for the off-target hA2B and hA3 ARs. The 5-(4-hydroxyphenethylamino)- derivative 12 showed both good affinity (Ki = 150 nM) and the best selectivity for the hA2A AR while the 5-benzylamino-substituted 5 displayed the best combined hA2A (Ki = 123 nM) and A1 AR affinity (Ki = 25 nM). The 5-phenethylamino moiety (compound 6) achieved nanomolar affinity (Ki = 11 nM) and good selectivity for the hA1 AR. The 5-(N4-substituted-piperazin-1-yl) derivatives 15-24 bind the hA1 AR subtype with affinities falling in the high nanomolar range. A structure-based molecular modeling study was conducted to rationalize the experimental binding data from a molecular point of view using both molecular docking studies and Interaction Energy Fingerprints (IEFs) analysis.[Formula: see text].

Synthesis and evaluation of N-substituted 2-amino-4,5-diarylpyrimidines as selective adenosine A1 receptor antagonists.

We report the synthesis and biological evaluation of new 2-amino-4,5-diarylpyrimidines as selective antagonists at the adenosine A1 receptor. The scaffold they are based upon is a deaza variation of a previously reported collection of 3-amino-5,6-diaryl-1,2,4-triazines, members of which had a subnanomolar affinity but limited selectivity over the A2A subtype. Initially, similar structure-affinity relationships at the 5-aryl ring were established, and then emphasis was put on increasing selectivity at the hA1AR by introducing substituents on the N2-position, all the while maintaining a nanomolar affinity. Compound 3z, bearing a trans 4-hydroxycyclohexyl substituent, was identified as a potent (Ki(hA1AR) = 7.7 nM) and selective (Ki(hA2AAR) = 1389 nM) antagonist at the human adenosine A1 receptor. Computational docking was effected at the A1 and A2A subtypes, rationalizing the effect of the 4-hydroxycyclohexyl substituent on selectivity, in relation with the nature of the substituent on the 5-position of the pyrimidine.

Discovery of 1,3-diethyl-7-methyl-8-(phenoxymethyl)-xanthine derivatives as novel adenosine A1 and A2A receptor antagonists.

Based on a previous report that a series of 8-(phenoxymethyl)-xanthines may be promising leads for the design of A1 adenosine receptor antagonists, selected novel and known 1,3-diethyl-7-methyl-8-(phenoxymethyl)-xanthine and 1,3,7-trimethyl-8-(phenoxymethyl)-xanthine analogs were synthesized and evaluated for their A1 and A2A adenosine receptor affinity. Generally, the study compounds exhibited affinity for both the A1 and A2A adenosine receptors. Replacement of the 1,3-dimethyl-substition with a 1,3-diethyl-substition pattern increased A1 and A2A binding affinity. Overall it was found that para-substitution on the phenoxymethyl side-chain of the 1,3-diethyl-xanthines decreased A1 affinity except for the 4-Br analog (4f) exhibiting the best A1 affinity in the submicromolar range. On the other hand A2A affinity for the 1,3-diethyl-xanthines were increased with para-substitution and the 4-OCH3 (4b) analog showed the best A2A affinity with a Ki value of 237nM. The 1,3-diethyl-substituted analogs (4a, and 4f) behaved as A1 adenosine receptor antagonists in GTP shift assays performed with rat whole brain membranes expressing A1 adenosine receptors. This study concludes that para-substituted 1,3-diethyl-7-methyl-8-(phenoxymethyl)-xanthine analogs represent novel A1 and A2A adenosine receptor antagonists that are appropriate for the design of therapies for neurodegenerative disorders such as Parkinson's and Alzheimer's disease.

2-Aminopyrimidines as dual adenosine A1/A2A antagonists.

In this study thirteen 2-aminopyrimidine derivatives were synthesised and screened as potential antagonists of adenosine A1 and A2A receptors in order to further investigate the structure activity relationships of this class of compounds. 4-(5-Methylfuran-2-yl)-6-[3-(piperidine-1-carbonyl)phenyl]pyrimidin-2-amine (8m) was identified as a compound with high affinities for both receptors, with an A2AKi value of 6.34 nM and an A1Ki value of 9.54 nM. The effect of selected compounds on the viability of cultured cells was assessed and preliminary results indicate low cytotoxicity. In vivo efficacy at A2A receptors was illustrated for compounds 8k and 8m since these compounds attenuated haloperidol-induced catalepsy in rats. A molecular docking study revealed that the interactions between the synthesised compounds and the adenosine A2A binding site most likely involve Phe168 and Asn253, interactions which are similar for structurally related adenosine A2A receptor antagonists.

1,3,7-Triethyl-substituted xanthines--possess nanomolar affinity for the adenosine A1 receptor.

Adenosine A1 receptors are attracting great interest as drug targets for their role in cognitive deficits. Antagonism of the adenosine A1 receptor may offer therapeutic benefits in complex neurological diseases, such as Alzheimer's and Parkinson's disease. The aim of this study was to discover potential selective adenosine A1 receptor antagonists. Several analogs of 8-(3-phenylpropyl)xanthines (3), 8-(2-phenylethyl)xanthines (4) and 8-(phenoxymethyl)xanthines (5) were synthesized and assessed as antagonists of the adenosine A1 and A2A receptors via radioligand binding assays. The results indicated that the 1,3,7-triethyl-substituted analogs (3d, 4d, and 5d), among each series, displayed the highest affinity for the adenosine A1 receptor with Ki values in the nanomolar range. This ethyl-substitution pattern was previously unknown to enhance adenosine A1 receptor binding affinity. The 1,3,7-triethyl-substituted analogs (3d, 4d, and 5d) behaved as adenosine A1 receptor antagonists in GTP shift assays performed with either rat cortical or whole brain membranes expressing adenosine A1 receptors. Further, in vivo evaluation of 3d showed reversal of adenosine A1 receptor agonist-induced hypolocomotion. In conclusion, the most potent evaluated compound, 8-(3-phenylpropyl)-1,3,7-triethylxanthine (3d), showed both in vitro and in vivo activity, and therefore represent a novel adenosine A1 receptor antagonist that may have potential as a drug candidate for dementia disorders.

PQ-69, a novel and selective adenosine A1 receptor antagonist with inverse agonist activity.

Potent and selective adenosine A1 receptor (A1AR) antagonists with favourable pharmacokinetic properties used as novel diuretics and antihypertensives are desirable. Thus, we designed and synthesized a series of novel 4-alkylamino substitution-2-arylpyrazolo[4,3-c]quinolin-3-one derivatives. The aim of the present study is to characterize the biological profiles of the optimized compound, PQ-69. In vitro binding assay revealed a K i value of 0.96 nM for PQ-69 in cloned hA1 receptor, which was 217-fold more selective compared with hA2A receptors and >1,000-fold selectivity for hA1 over hA3 receptor. The results obtained from [(35)S]-GTPγS binding and cAMP concentration assays indicated that PQ-69 might be an A1AR antagonist with inverse agonist activity. In addition, PQ-69 displayed highly inhibitory activities on isolated guinea pig contraction (pA2 value of 8.99) induced by an A1AR agonist, 2-chloro-N6-cyclopentyl adenosine. Systemic administration of PQ-69 (0.03, 0.3, 3 mg/kg) increased urine flow and sodium excretion in normal rats. Furthermore, PQ-69 displayed better metabolic stability in vitro and longer terminal elimination half-life (t 1/2) in vivo compared with 1,3-dipropyl-8-cyclopentylxanthine. These findings suggest that PQ-69 exhibits potent antagonist effects on A1AR in vitro, ex vivo and in vivo, it might be a useful research tool for investigating A1AR function, and it could be developed as a potential therapeutic agent.

7-Amino-2-phenylpyrazolo[4,3-d]pyrimidine derivatives: structural investigations at the 5-position to target human A1 and A(2A) adenosine receptors. Molecular modeling and pharmacological studies.

In previous research, several 7-amino-2-arylpyrazolo[4,3-d]pyrimidine derivatives were identified as highly potent and selective antagonists at the human A3 adenosine receptor. Structure-activity relationship studies highlighted that affinity and selectivity depended on the nature of the substituents at the 5- and 7-positions of the pyrazolo[4,3-d]pyrimidine scaffold. In particular, small lipophilic residues at the 5-position and a free amino group at position 7 afforded compounds able to bind all four human (h) adenosine receptors. Hence, to shift affinity toward the hA1 and/or hA(2A) subtypes, alkyl and arylalkyl chains of different length were appended at position 5 of the 2-phenylpyrazolo[4,3-d]pyrimidin-7-amine. Among the new compounds, a dual hA1/hA(2A) receptor antagonist was identified, namely the 5-(3-phenylpropyl) derivative 25, which shows high affinity both at human A1 (K(i) = 5.31 nM) and A(2A) (K(i) = 55 nM) receptors. We also obtained some potent and selective antagonists for the A1 receptor, such as the 5-(3-arylpropyl)-substituted compounds 26-31, whose affinities fall in the low nanomolar range (K(i) = 0.15-18 nM). Through an in silico receptor-driven approach, the obtained binding data were rationalized and the molecular bases of the hA1 and hA(2A) AR affinity and selectivity of derivatives 25-31 are explained.

Combining on-chip synthesis of a focused combinatorial library with computational target prediction reveals imidazopyridine GPCR ligands.

Using the example of the Ugi three-component reaction we report a fast and efficient microfluidic-assisted entry into the imidazopyridine scaffold, where building block prioritization was coupled to a new computational method for predicting ligand-target associations. We identified an innovative GPCR-modulating combinatorial chemotype featuring ligand-efficient adenosine A1/2B and adrenergic α1A/B receptor antagonists. Our results suggest the tight integration of microfluidics-assisted synthesis with computer-based target prediction as a viable approach to rapidly generate bioactivity-focused combinatorial compound libraries with high success rates.

Design and characterization of optimized adenosine A2A/A1 receptor antagonists for the treatment of Parkinson's disease.

The design and characterization of two, dual adenosine A(2A)/A(1) receptor antagonists in several animal models of Parkinson's disease is described. Compound 1 was previously reported as a potential treatment for Parkinson's disease. Further characterization of 1 revealed that it was metabolized to reactive intermediates that caused the genotoxicity of 1 in the Ames and mouse lymphoma L51784 assays. The identification of the metabolites enabled the preparation of two optimized compounds 13 and 14 that were devoid of the metabolic liabilities associated with 1. Compounds 13 and 14 are potent dual A(2A)/A(1) receptor antagonists that have excellent activity, after oral administration, across a number of animal models of Parkinson's disease including mouse and rat models of haloperidol-induced catalepsy, mouse and rat models of reserpine-induced akinesia, and the rat 6-hydroxydopamine (6-OHDA) lesion model of drug-induced rotation.

Structure-activity relationships of 2-amino-3-aroyl-4-[(4-arylpiperazin-1-yl)methyl]thiophenes. Part 2: Probing the influence of diverse substituents at the phenyl of the arylpiperazine moiety on allosteric enhancer activity at the A1 adenosine receptor.

In a preliminary article, we reported the potent allosteric enhancer activity at the A(1) adenosine receptor of a small series of 2-amino-3-(4-chlorobenzoyl)-4-[4-(aryl)piperazin-1-yl)methyl]thiophene derivatives bearing electron-withdrawing or electron-releasing groups at the para-position of the phenylpiperazine moiety. In the present study, we report the development of the compounds previously studied by modifying both the number and position of substituents on the phenylpiperazine moiety, aimed at establishing a structure-activity relationship identifying additional compounds with improved activity. The nature and the position of substituents on the phenyl ring tethered to the piperazine seemed to exert a fundamental influence on the allosteric enhancer activity, with the 3,4-difluoro 4i, 3-chloro-4-fluoro 4o, and 4-trifluoromethoxy 4ak derivatives being the most active compounds in binding (saturation and competition experiments) and functional cAMP studies. This study shows that it is also possible to obtain a good separation between allosteric enhancement and antagonistic activity at the A(1) adenosine receptor.