Inhibition of human tumour cell proliferation by analogues of adenosine

The effects of adenosine and several structural analogues of adenosine upon thymidine incorporation into human tumour cells and rat cervical lymphocytes were investigated. The analogue NECA, which has equal specificity for the A₁ and A₂ receptor, had the most inhibitory effect on lymphocyte proliferation while the A₁ agonists had limited effects, suggesting that these cells possess principally A₂ adenosine receptors. In the case of human tumour cells, however, the most inhibitory effect on proliferation was obtained with the A₁-specific analogues. The general order of inhibitory effects of adenosine analogues on thymidine incorporation in human tumour cells was: S-ENBA>CPA=R-PIA>S-PIA>NECA. These findings suggest that in the cells presently studied the A₁ adenosine receptor predominates. Removal of exogenous adenosine by growth in the presence of adenosine deaminase inhibited thymidine incorporation. The effect of adenosine removal lends further support to the proposal that adenosine has some, as yet unidentified, regulatory role in the control of human tumour cell proliferation. © 1997 John Wiley & Sons, Ltd.     

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 A_2A 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 A_2A receptors. The influence of substitution on position C2 showed no improvement for either adenosine A₁ or A_2A receptor affinity. The addition of an amino or a cyclohexylamino group to position C3 also showed no improvement of adenosine A₁ or A_2A 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 K_i value of 2.06 µM 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.     

Deaza-analogues of Adenosine and 2-Chloroadenosine as Agonists of Adenosine Receptors

Abstract

A variety of adenosine analogues have been recently evaluated in order Lo find more potent and selective agonists on adenosine receptors. The most potent adenosine analogues acting on A₁ receptor, a high affinity receptor inhibitory to adenylate cyclase, are N⁶-substituted compounds. So ⁶-cyclohexyladenosine (CHA) and ⁶-L-phenylisopropyladenosine (L-PIA) are extremely potent agonists on A₂ receptor, whereas they are relatively weak agonists on A receptor, a lower affinity receptor which is stirnulatory to cyclase, and they have no effect on the adenosine P site.     

A Functional Screening of Adenosine Analogues at the Adenosine A2B Receptor: A Search for Potent Agonists

Abstract

Various adenosine analogues were tested at the adenosine A_2B receptor. Agonist potencies were determined by measuring the cyclic AMP production in Chinese Hamster Ovary cells expressing human A_2B receptors. 5′-.N-Substituted carboxamidoadenosines were most potent. 5′-N-Ethylcarboxamidoadenosine (NECA) was most active with an ECso value of 3.1 μM. Other ribose modified derivatives displayed low to negligible activity. Potency was reduced by substitution on the exocyclic amino function (N⁶) of the purine ring system. The most active N⁶-substituted derivative N⁶-methyl-NECA was 5 fold less potent than NECA. C8-and most C2-substituted analogues were virtually inactive. 1-Deaza-analogues had a reduced potency, 3-and 7-deazaanalogues were not active.     

Strategically Functionalized Adenosines: Agonists for Adenosine Receptors

Abstract

The syntheses of three classes of adenosine analogues involving cyclosubstitution at the 6-position and functionalization at the 2-position are reported. The target molecules synthesized are stable with respect to hydrolytic deamination by mammalian adenosine deaminase, and, because of major structural changes at the 2- and 6-positions, these compounds are expected to be poor phosphorylation substrates for the kinases. Adenosine receptor binding data reveal that several of the compounds synthesized show excellent A₁ receptor affinity and A₂/A₁ selectivity.     

Design,synthesis, and binding of homologated truncated 4′-thioadenosine derivatives at the human A3 adenosine receptors

We synthesized homologated truncated 4′-thioadenosine analogues 3 in which a methylene (CH₂) group was inserted in place of the glycosidic bond of a potent and selective A₃ adenosine receptor antagonist 2. The analogues were designed to induce maximum binding interaction in the binding site of the A₃ adenosine receptor. However, all homologated nucleosides were devoid of binding affinity at all subtypes of adenosine receptors, indicating that free rotation through the single bond allowed the compound to adopt an indefinite number of conformations, disrupting the favorable binding interaction essential for receptor recognition.     

Epac is required for exogenous and endogenous stimulation of adenosine A<Subscript>2B</Subscript> receptor for inhibition of angiotensin II-induced collagen synthesis and myofibroblast differentiation

Angiotensin II (Ang II) plays an important role on the pathogenesis of cardiac fibrosis. Prolong and overstimulation of angiotensin II type 1 receptor with Ang II-induced collagen synthesis and myofibroblast differentiation in cardiac fibroblasts, leading to cardiac fibrosis. Although adenosine and its analogues are known to have cardioprotective effects, the mechanistic by which adenosine A₂ receptors (A₂Rs) inhibit Ang II-induced cardiac fibrosis is not clearly understood. In the present study, we examined the effects of exogenous adenosine and endogenous adenosine on Ang II-induced collagen and myofibroblast differentiation determined by α-smooth muscle action (α-SMA) overexpression and their underlying signal transduction. Elevation of endogenous adenosine levels resulted in the inhibition of Ang II-induced collagen type I and III and α-SMA synthesis in cardiac fibroblasts. Moreover, treatment with exogenous adenosine which selectively stimulated A₂Rs also suppressed Ang II-induced collagen synthesis and α-SMA production. These antifibrotic effects of both endogenous and exogenous adenosines are mediated through the A_2B receptor (A_2BR) subtype. Stimulation of A_2BR exhibited antifibrotic effects via the cAMP-dependent and Epac-dependent pathways. Our results provide new mechanistic insights regarding the role for cAMP and Epac on A_2BR-mediated antifibrotic effects. Thus, A_2BR is one of the potential therapeutic targets against cardiac fibrosis.     

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

Antagonists of the adenosine receptors (A₁ and A_2A subtypes) are widely researched as potential drug candidates for their role in Parkinson’s disease-related cognitive deficits (A₁ subtype), motor dysfunction (A_2A subtype) and to exhibit neuroprotective properties (A_2A subtype). Previously the benzo-α-pyrone based derivative, 3-phenyl-1H-2-benzopyran-1-one, was found to display both A₁ and A_2A 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 A_2A adenosine receptor affinity over their C2-substituted benzoxazinone homologues. The benzoxazinones were devoid of A_2A 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 A_2A adenosine receptor subtypes. The highest A₁ adenosine receptor affinity and selectivity were favoured by methyl para-substitution of phenyl ring B (A₁K_i = 2.50 μM). On the other hand, 3,4-dimethoxy substitution of phenyl ring B afforded the best A_2A adenosine receptor binding (A_2AK_i = 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.     

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

Adenosine A₁ and A_2A 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 A_2A 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 A_2A 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 K_i 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 A_2A receptor. These substitution patterns led to enhanced adenosine A₁ and A_2A 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 A_2A receptor affinity respectively, and therefore represent novel adenosine receptor antagonists that may have potential with further structural modifications as drug candidates for neurological disorders.     

Synthesis of hybrid analogues of caffeine and eudistomin D and its affinity for adenosine receptors

Four bis-N-n-propyl analogues (3–6) in the uracil ring of two hybrid molecules (1 and 2) of caffeine and eudistomin D, a β-carboline alkaloid from a marine tunicate, were synthesized, and their affinity and selectivity for adenosine receptors A₁, A_2A, and A₃ were examined. All the compounds (3–6) showed better potency as adenosine receptor ligands than caffeine. Bis-N-n-propylation (3 and 4, respectively) of the uracil ring in 1 and 2 resulted in higher affinity for A₁ and A_2A adenosine receptors. Furthermore, it was found that a compound (5) possessing a n-propyloxy group at C-7 in compound 3 with a nitrogen at the β-position of the pyridine ring (β-N type) enhanced remarkably affinity for adenosine receptor A₃ subtype, while n-propyloxy substitution (compound 6) at C-5 in compound 4 with a nitrogen at the δ-position of the pyridine ring (δ-N type) reduced affinity for all the adenosine receptor, A₁, A_2A, and A₃. Among all the compounds (1–6) examined, compound 5 showed the most potent affinity for adenosine receptor A₃ subtype (K_i value, 0.00382 μM).     

5′‐N‐ethylcarboxamide induces IL‐6 expression via MAPKs and NF‐κB activation through Akt,Ca2+/PKC,cAMP signaling pathways in mouse embryonic stem cells

Many studies suggest that adenosine modulates cell responses in a wide array of tissues through potent and selective regulation of cytokine production. This study examined the effects of adenosine on interleukin (IL)‐6 expression and its related signal pathways in mouse embryonic stem (ES) cells. In this study, the adenosine analogue 5′‐N‐ethylcarboxamide (NECA) increased IL‐6 protein expression level. Mouse ES cells expressed the A₁, A_2A, A_2B, and A₃ adenosine receptors (ARs), whose expression levels were increased by NECA and NECA‐induced increase of IL‐6 mRNA expression or secretion level was inhibited by the non‐specific AR inhibitor, caffeine. NECA increased Akt and protein kinase C (PKC) phosphorylation, intracellular Ca²⁺ and cyclic adenosine monophosphate (cAMP) levels, which were blocked by caffeine. On the other hand, NECA‐induced IL‐6 secretion was partially inhibited by Akt inhibitor, bisindolylmaleimide I (PKC inhibitor), SQ 22536 (adenylate cyclate inhibitor) and completely blocked by the 3 inhibitor combination treatment. In addition, NECA increased mitogen activated protein kinase' (MAPK) phosphorylation, which were partially inhibited by the Akt inhibitor, bisindolylmaleimide I, and SQ 22536 and completely blocked by the 3 inhibitor combination treatment. NECA‐induced increases of IL‐6 protein expression and secretion levels were inhibited by MAPK inhibition. NECA‐induced increase of nuclear factor (NF)‐κB phosphorylation was inhibited by MAPK inhibitors. NECA also increased cAMP response element‐binding protein (CREB) phosphorylation, which was blocked by MAPK or NF‐κB inhibitors. Indeed, NECA‐induced increase of IL‐6 protein expression and secretion was blocked by NF‐κB inhibitors. In conclusion, NECA stimulated IL‐6 expression via MAPK and NF‐κB activation through Akt, Ca²⁺/PKC, and cAMP signaling pathways in mouse ES cells. J. Cell. Physiol. 219: 752–759, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of Adenosine Receptor Subtypes on Hippocampal Extracellular Serotonin Level and Serotonin Reuptake Activity

Abstract: To clarify the effects of adenosine receptor subtypes (A₁, A₂, and A₃) on hippocampal serotoninergic function, hippocampal extracellular serotonin (5-HT) levels were determined by in vivo microdialysis in freely moving rats under various conditions. Both adenosine and an adenosine A₁ receptor agonist, 2-chloro-N⁶-cyclopentyladenosine, decreased extracellular 5-HT levels, whereas an adenosine A₁ receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT), and caffeine increased these levels. A selective A_2A receptor agonist (CGS-21680), an adenosine A₂ receptor agonist (PD-125944), an adenosine A₂ receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), and an adenosine A₃ receptor agonist, N⁶-2-(4-aminophenyl)ethyladenosine (APNEA), did not affect extracellular 5-HT levels. When the adenosine A₁ receptor was blocked by CPT, the hippocampal extracellular 5-HT level was increased by adenosine, CGS-21680, and PD-125944, and decreased by caffeine, DMPX, and APNEA. When both adenosine A₁ and A₂ receptors were blocked by CPT and DMPX, the extracellular 5-HT level was decreased by adenosine, caffeine, and APNEA. The hippocampal extracellular 5-HT level was not affected by administration of APNEA alone, but was decreased by this agent when the adenosine A₁ receptor was blocked, irrespective of whether the adenosine A₂ receptor was functional. These inhibitory effects of adenosine, caffeine, and APNEA on extracellular 5-HT levels, during both adenosine A₁ and A₂ receptor blockade, were inhibited by selective 5-HT reuptake inhibitors. These results indicate that the stimulatory effects of the adenosine A₂ receptor and the inhibitory effects of the A₃ receptor on hippocampal extracellular 5-HT levels are masked by the inhibitory effects of the adenosine A₁ receptor.     

SYNTHESIS OF 3′-UREIDOADENOSINE ANALOGUES AND THEIR BINDING AFFINITY TO THE A3 ADENOSINE RECEPTOR

Novel 3′-ureidoadenosine analogues were synthesized from 1,2:5,6-di-O-isopropylidene-D-glucose in order to lead to stronger hydrogen bonding at the A₃ adenosine receptor than the corresponding 3′-aminoadenosine derivatives. However, all synthesized 3′-ureidoadenosine analogues have lost their binding affinities to the all subtypes of adenosine receptors, indicating that bulky 3′-urea moiety led to conformational distortion.     

Endogenous Expression of Adenosine A<Subscript>1</Subscript>, A<Subscript>2 </Subscript>and A<Subscript>3</Subscript> Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [³H]DPCPX, selective A₁R antagonist, revealed a single binding site with a K _D = 9.4 ± 1.4 nM and B _max = 62.7 ± 8.6 fmol/mg protein. Binding of [³H]DPCPX in intact cell revealed a K _D = 17.7 ± 1.3 nM and B _max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [³H]ZM241385 binding experiments revealed a single binding site population of receptors with K _D = 16.5 ± 1.3 nM and B _max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K _D = 4.7 ± 0.6 nM and B _max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A_2A receptor in C6 cells. A₁, A_2A, A_2B and A₃ adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A₁R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A₁ and A₂ receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.     

BDNF-induced presynaptic facilitation of GABAergic transmission in the hippocampus of young adults is dependent of TrkB and adenosine A<Subscript>2A</Subscript> receptors

Brain-derived neurotrophic factor (BDNF) and adenosine are widely recognized as neuromodulators of glutamatergic transmission in the adult brain. Most BDNF actions upon excitatory plasticity phenomena are under control of adenosine A_2A receptors (A_2ARs). Concerning gamma-aminobutyric acid (GABA)-mediated transmission, the available information refers to the control of GABA transporters. We now focused on the influence of BDNF and the interplay with adenosine on phasic GABAergic transmission. To assess this, we evaluated evoked and spontaneous synaptic currents recorded from CA1 pyramidal cells in acute hippocampal slices from adult rat brains (6 to 10 weeks old). BDNF (10–100 ng/mL) increased miniature inhibitory postsynaptic current (mIPSC) frequency, but not amplitude, as well as increased the amplitude of inhibitory postsynaptic currents (IPSCs) evoked by afferent stimulation. The facilitatory action of BDNF upon GABAergic transmission was lost in the presence of a Trk inhibitor (K252a, 200 nM), but not upon p75^NTR blockade (anti-p75^NTR IgG, 50 μg/mL). Moreover, the facilitatory action of BDNF onto GABAergic transmission was also prevented upon A_2AR antagonism (SCH 58261, 50 nM). We conclude that BDNF facilitates GABAergic signaling at the adult hippocampus via a presynaptic mechanism that depends on TrkB and adenosine A_2AR activation.     

Role of extracellular cysteine residues in the adenosine A<Subscript>2A</Subscript> receptor

The G protein-coupled A_2A adenosine receptor represents an important drug target. Crystal structures and modeling studies indicated that three disulfide bonds are formed between ECL1 and ECL2 (I, Cys71^2.69-Cys159^45.43; II, Cys74^3.22-Cys146^45.30, and III, Cys77^3.25-Cys166^45.50). However, the A_2BAR subtype appears to require only disulfide bond III for proper function. In this study, each of the three disulfide bonds in the A_2AAR was disrupted by mutation of one of the cysteine residues to serine. The mutant receptors were stably expressed in Chinese hamster ovary cells and analyzed in cyclic adenosine monophosphate (cAMP) accumulation and radioligand binding studies using structurally diverse agonists: adenosine, NECA, CGS21680, and PSB-15826. Results were rationalized by molecular modeling. The observed effects were dependent on the investigated agonist. Loss of disulfide bond I led to a widening of the orthosteric binding pocket resulting in a strong reduction in the potency of adenosine, but not of NECA or 2-substituted nucleosides. Disruption of disulfide bond II led to a significant reduction in the agonists’ efficacy indicating its importance for receptor activation. Disulfide bond III disruption reduced potency and affinity of the small adenosine agonists and NECA, but not of the larger 2-substituted agonists. While all the three disulfide bonds were essential for high potency or efficacy of adenosine, structural modification of the nucleoside could rescue affinity or efficacy at the mutant receptors. At present, it cannot be excluded that formation of the extracellular disulfide bonds in the A_2AAR is dynamic. This might add another level of G protein-coupled receptor (GPCR) modulation, in particular for the cysteine-rich A_2A and A_2BARs.     

Excitatory and Inhibitory Effects of A1 and A2A Adenosine Receptor Activation on the Electrically Evoked [3H]Acetylcholine Release from Different Areas of the Rat Hippocampus

Abstract: The modulation by adenosine analogues and endogenous adenosine of the electrically evoked release of [³H]acetylcholine ([³H]ACh) was compared in subslices of the three areas of the rat hippocampus (CA1, CA3, and dentate gyrus). The mixed A₁/A₂ agonist 2-chloroadenosine (CADO; 2–10 µM) inhibited, in a concentration-dependent manner, the release of [³H]ACh from the three hippocampal areas, being more potent in the CA1 and CA3 areas than in the dentate gyrus. The inhibitory effect of CADO (5 µM) on [³H]ACh release was prevented by the A₁ antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM) in the three hippocampal areas and was converted in an excitatory effect in the CA3 and dentate gyrus areas. The A_2A agonist CGS-21680 (30 nM) produced a greater increase of the evoked release of [³H]ACh in the CA3 than in the dentate gyrus areas, whereas no consistent effect was found in the CA1 area or in the whole hippocampal slice. The excitatory effect of CGS-21680 (30 nM) in the CA3 area was prevented by the adenosine receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 µM). Both adenosine deaminase (2 U/ml) and DPCPX (250 nM) increased the evoked release of [³H]ACh in the CA1 and CA3 areas but not in the dentate gyrus. The amplitude of the effect of DPCPX and adenosine deaminase was similar in the CA1 area, but in the CA3 area DPCPX produced a greater effect than adenosine deaminase. It is concluded that the electrically evoked release of [³H]ACh in the three areas of the rat hippocampus can be differentially modulated by adenosine. In the CA1 area, only A₁ inhibitory receptors modulate ACh release, whereas in the CA3 area, both A_2A excitatory and A₁ inhibitory adenosine receptors modulate ACh release. In the dentate gyrus, both A₁ inhibitory and A_2A excitatory adenosine receptors are present, but endogenous adenosine does not activate them.     

Neuroprotection by adenosine in the brain: From A<Subscript>1</Subscript> receptor activation to A<Subscript>2A</Subscript> receptor blockade

Adenosine is a neuromodulator that operates via the most abundant inhibitory adenosine A₁ receptors (A₁Rs) and the less abundant, but widespread, facilitatory A_2ARs. It is commonly assumed that A₁Rs play a key role in neuroprotection since they decrease glutamate release and hyperpolarize neurons. In fact, A₁R activation at the onset of neuronal injury attenuates brain damage, whereas its blockade exacerbates damage in adult animals. However, there is a down-regulation of central A₁Rs in chronic noxious situations. In contrast, A_2ARs are up-regulated in noxious brain conditions and their blockade confers robust brain neuroprotection in adult animals. The brain neuroprotective effect of A_2AR antagonists is maintained in chronic noxious brain conditions without observable peripheral effects, thus justifying the interest of A_2AR antagonists as novel protective agents in neurodegenerative diseases such as Parkinsons and Alzheimers disease, ischemic brain damage and epilepsy. The greater interest of A_2AR blockade compared to A₁R activation does not mean that A₁R activation is irrelevant for a neuroprotective strategy. In fact, it is proposed that coupling A_2AR antagonists with strategies aimed at bursting the levels of extracellular adenosine (by inhibiting adenosine kinase) to activate A₁Rs might constitute the more robust brain neuroprotective strategy based on the adenosine neuromodulatory system. This strategy should be useful in adult animals and especially in the elderly (where brain pathologies are prevalent) but is not valid for fetus or newborns where the impact of adenosine receptors on brain damage is different.     

Synthesis of a New Series of 1H‐Imidazol‐1‐yl Substituted 8‐Phenylxanthines as Adenosine Receptor Ligands

A new series of 1H‐imidazol‐1‐yl substituted 8‐phenylxanthine analogs has been synthesized to study the effects of the imidazole group on the binding affinity of compounds for adenosine receptors. Competition binding studies of these compounds were carried out in vitro with human cloned receptors using [³H]DPCPX and [³H]ZM 241385 as radioligands at A₁ and A_2A adenosine receptors, respectively. The effect of the substitution pattern of the (imidazolyl)alkoxy group on various positions of the phenyl ring at C(8) was also studied. The xanthine derivatives displayed varying degrees of affinity and selectivity towards A₁ and A_2A receptor subtypes despite a common but variedly substituted Ar C(8).