Improvement of cold tolerance by selective A1 adenosine receptor antagonists in rats

Previously we have shown that the improvement of cold tolerance by theophylline is due to antagonism at adenosine receptors rather than inhibition of phosphodiesterase. Since theophylline is a nonselective adenosine receptor antagonist for both A1 and A2 receptors, the present study investigated the adenosine receptor subtype involved in theophylline's action. Acute systemic injection of selective A1 receptor antagonists (1,3-dialkyl-8-aryl or 1,3-dialkyl-8-cyclopentyl xanthine derivatives) significantly increased both the total and maximal heat production as well as cold tolerance. In contrast, injection of a relatively selective A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (compound No. 19), failed to significantly alter the thermogenic response of the rat under cold exposure. Further, the relative effectiveness of these compounds in increasing total thermogenesis was positively correlated with their potency in blocking the A1 adenosine receptor (r = .52, p less than 0.01), but not in A2 adenosine receptor (r = .20, p less than 0.2). It is likely that the thermally beneficial effects of adenosine A1 antagonists are due to their attenuation of the inhibitory effects of endogenously released adenosine on lipolysis and glucose utilization, resulting in increased substrate mobilization and utilization for enhanced thermogenesis.     

Synthesis, biological studies and molecular modeling investigation of 1,3-dimethyl-2,4-dioxo-6-methyl-8-(substituted) 1,2,3,4-tetrahydro [1,2,4]-triazolo [3,4-f]-purines as potential adenosine receptor antagonists

A new series of potential adenosine receptor antagonists with a [1,2,4]-triazolo-[3,4-f]-purine structure have been synthesized, and their affinities at the four adenosine receptor subtypes (A1, A2A, A2B and A3) have been evaluated. The design was based on the demonstrated approach to novel A3 adenosine receptor antagonists of adding a third ring to the xanthine structure. Unfortunately, all the synthesized compounds were completely inactive at all four adenosine receptor subtypes independently of their substitutions. Preliminary molecular modeling investigation has demonstrated that only a low degree of steric and electrostatic complementarity has been observed for all the new synthesized triazolo-purines with respect to other structurally related A3 receptor antagonists. This analysis yielded valuable information about structure-activity relationships and further design of potential adenosine receptor antagonists.     

1,3-Dialkyl-8-(p-sulfophenyl)xanthines: potent water-soluble antagonists for A1- and A2-adenosine receptors

A series of 8-(substituted phenyl) derivatives of theophylline and other 1,3-dialkylxanthines were evaluated for potency and selectivity as antagonists at A1- and A2-adenosine receptors in brain tissue. Theophylline has a similar potency (Ki = 14 microM) at both A1 and A2 receptors. 8-Phenyltheophylline is 25-35-fold more potent as an adenosine receptor antagonist than theophylline, while 8-phenylcaffeine is only 2-3-fold more potent than caffeine. A p-hydroxyaryl substituent enhances the potency of 8-phenyltheophylline as an adenosine antagonist. p-Carboxy- and p-sulfoaryl substituents reduce potency of 8-phenyltheophylline, yielding water-soluble adenosine antagonists, which are some 2-5-fold more potent than theophylline at adenosine receptors. None of the 8-(substituted phenyl)theophyllines are particularly selective as antagonists toward A1- and A2-adenosine receptors. 1,3-Dipropyl-8-phenylxanthine represents a potent and somewhat selective A1-receptor antagonist about 23-fold more potent at A1 receptors than at A2 receptors. A p-hydroxyaryl substituent further enhances potency of the 1,3-dipropyl-8-phenylxanthine at both A1 and A2 receptors. The 8-(2-amino-4-chlorophenyl)-1,3-dipropylxanthine is a very potent and selective antagonist for A1 receptors, being nearly 400-fold more potent at A1 than at A2 receptors. The water-soluble 8-(p-sulfophenyl)- and 8-(p-carboxyphenyl)-1,3-propylxanthines no longer exhibit marked selectivity. Both compounds are much more potent as adenosine antagonists than theophylline. The striking selectivity of 1-isoamyl-3-isobutylxanthine as an A1 antagonist is retained in the 8-phenyl derivative but is virtually lost in the 8-p-sulfophenyl derivative.     

Effects of single and long-term theophylline treatment on the threshold of mechanical nociception: contribution of adenosine A1 and alpha2-adrenoceptors

The effects of single and long-term treatment with theophylline as well as the influence of adenosine A1 receptor agonist cyclopentyladenosine (CPA) and a-adrenergic receptor antagonists prazosin and yohimbine were assessed in the paw pressure test in rats. Both single (37.5 and 75 mg/kg) and long-term (75 mg/kg/day, 14 days i.p.) theophylline treatments exerted antinociceptive effect by increasing the mechanical pain threshold. Single treatment of theophylline (75 mg/kg) antagonized the antinociceptive effect of CPA (0.1 mg/kg); CPA (0.1 mg/kg) abolished the theophylline-induced antinociception. Chronic treatment with theophylline did not change the antinociceptive effect of CPA, while CPA decreased the theophylline antinociception. Yohimbine (0.5 mg/kg), an a 2-adrenoceptor antagonist, diminished the antinociception of a single dose (75 mg/kg) of theophylline, whereas prazocin, an a 1-adrenoceptor antagonist, did not affect it. These results suggest that adenosine A1 and a 2-adrenoceptors take part in the antinociception induced by a single dose of theophylline. The antinociception induced by chronic theophylline treatment probably has a more complex mechanism in which the involvement of adenosine A1.     

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.     

Adenosine sensory transduction pathways contribute to activation of the sensory irritation response to inspired irritant vapors.

The molecular mechanisms through which sensory irritants stimulate nasal trigeminal nerves are poorly understood. The current study was aimed at evaluating the potential contribution of purinergic sensory transduction pathways in this process. Aerosols of 4-36 mM adenosine 5'-triphosphate (ATP) and adenosine both acted as sensory irritants. Large dose capsaicin pretreatment to induce degeneration of transient receptor potential vanilloid type-1 (TRPV1)-expressing C fibers greatly reduced, but did not abolish, the sensory irritation response to ATP aerosol and was without effect on the response to adenosine aerosol, indicating that ATP acts largely on capsaicin-sensitive (primarily C fibers) and adenosine acts on capsaicin-insensitive (primarily Adelta fibers) nerves. The response to adenosine was diminished by pretreatment with the broad-based adenosine receptor antagonist theophylline (20 mg/kg) and A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.1 mg/kg), providing evidence that adenosine stimulates capsaicin-insensitive nerves via the A1 receptor. The sensory irritation responses to 275 ppm styrene and 110 ppm acetic acid vapors were significantly reduced by theophylline pretreatment suggesting a role for adenosine signaling pathways in activation of the sensory irritant response by these vapors. If sensory nerves are activated by mediators that are released from injured airway mucosal cells, then nasal sensory nerve activation may be a reflection of irritant-induced alterations in airway cell integrity.     

A new class of adenosine receptors in brain. Characterization by 2-chloro[3H]adenosine binding

Micromolar concentrations of adenosine and its analogs have profound depressant effects on neuronal firing and synaptic transmission in many brain areas. Using the adenosine agonist 2-chloro[3H]adenosine (Cl[3H]Ado), we have identified a distinct class of micromolar-affinity adenosine binding sites in rat forebrain membranes. Specific Cl[3H]Ado binding was reversible and saturable with an apparent KD of 9.1 microM and a Bmax of 61 pmoles/mg protein. The present studies were conducted using washed brain membrane fractions not treated with adenosine deaminase. Specific Cl[3H]Ado binding under these conditions was insensitive to (-)-N6-(R-phenylisopropyl)adenosine ((-)PIA) and treatment with 3 mM N-ethylmaleimide, unlike high-affinity A1 adenosine receptor binding. Treatment of membranes with adenosine deaminase revealed an additional population of binding sites sensitive to (-)PIA. Inhibition of Cl[3H]Ado binding by adenosine analogs exhibited an order of potency ClAdo greater than 5'-N-ethylcarboxamide adenosine (NECA) greater than (-)PIA which differs from that of both A1 and A2 adenosine receptors. The potent A1 and A2 receptor antagonist 8-phenyltheophylline had no significant effect on binding up to 10 microM. Specific binding, however, was inhibited by the adenosine antagonists 8(p-sulfophenyl)theophylline, isobutylmethylxanthine, theophylline, and caffeine. Micromolar Cl[3H]Ado binding was highly selective for adenosine agonists and antagonists. These results suggest that the micromolar-affinity Cl[3H]Ado binding sites may represent a novel central purinergic receptor, distinct from the A1 and A2 adenosine receptors involved in the regulation of adenylate cyclase.     

A steric and electrostatic comparison of three models for the agonist/antagonist binding site on the adenosine A1 receptor.

Several models have been described in the literature to explain the similarity of interaction of adenosine receptor agonists and antagonists with the binding site of the receptor. Besides the superposition of the nitrogen atoms of adenosine and xanthine (the "standard" model), two other models have been described: one in which xanthine is rotated around its "horizontal" axis before superposition ("flipped") and one in which the adenosine N6-region and the xanthine C8-region are superimposed ("N6-C8"). In this study we compared the steric and electrostatic properties of these models. The flipped model tends to show higher percentages of overlap for the positive electrostatic potentials and the N6-C8 model yielded predominantly a slightly higher overlap for the negative electrostatic potentials, although these differences were rather small. Since the N6-region in adenosine and the C8-region in xanthine are coinciding in this model, the N6-C8 model yielded a much larger overlap in van der Waals volume than the other two models. The N6-C8 model seems therefore to be the more probable model, also because the interactive groups point in the same direction for both adenosine and xanthine analogues. We determined the geometries of both the adenosine N6-substituents and the xanthine 8-substituents in earlier studies. The N6-C8 model causes a coincidence of these separately determined conformations.     

Non-xanthine heterocycles: activity as antagonists of A1- and A2-adenosine receptors

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-ethylcarboxamidoadenosine with A2-adenosine receptors in human platelet and rat phenochromocytoma 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 beta-carboline-3-ethyl carboxylate. The phenylimidazoline clonidine had no activity, whereas a related dihydroxyphenylimidazoline was a weak non-competitive adenosine antagonist.     

Involvement of A(3) receptors in the potentiation by adenosine of the inhibitory effect of theophylline on human eosinophil degranulation: possible novel mechanism of the anti-inflammatory action of theophylline

The current use of theophylline in asthma is based on both the bronchodilatory and the anti-inflammatory effects. The exact mechanism of these actions is still controversial and may include the inhibition of adenosine 3',5'-monophosphate phosphodiesterase enzyme (PDE) and antagonism of adenosine receptors. In this study, the mechanism of the anti-inflammatory action was investigated by studying the inhibition by theophylline of complement C5a (C5a)-induced degranulation of human eosinophils and its interaction with adenosine. Theophylline (10-1000 microM) inhibited C5a-induced release of eosinophil peroxidase (EPO) in a concentration-dependent manner with an IC(50) of 233.5 microM and a maximal inhibition of 90.3 +/- 3.0%. In contrast, the PDE4 inhibitor rolipram (up to 50 microM) had no effect. The adenosine A(3) receptor agonist N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine (IB-MECA) also inhibited release (IC(50) = 7.5 microM), but neither adenosine itself nor the selective A(1) and A(2) agonists and antagonists had any significant effect, even at 100 microM. The inhibition produced by clinically relevant concentration of theophylline (50 microM) was potentiated by ineffective concentrations of exogenous adenosine and additive to that produced by IB-MECA. The potent and selective A(3) antagonist MRS 1220, but not the A(1) or A(2) antagonists, significantly reversed the inhibitory effect of theophylline. These results suggest that therapeutic concentrations of theophylline inhibit human eosinophil partly by acting as an A(3) agonist. Together with the potentiation of theophylline action by adenosine, perhaps via the A(3) receptors, these novel actions may, at least in part, contribute to the mechanism of the anti-inflammatory action of this drug in vivo.     

The effect of adenosine on blood pressure variability in sinoaortic denervated rats is mediated by adenosine A2a-Receptor

It is known that adenosine decreases blood pressure (BP) level as well as blood pressure variability (BPV). However, there is little information about the effect of adenosine on BPV. With a computerized analytic system for BP and heart rate (HR) that could sample the data continuously in conscious, freely moving rats, we studied the effects of different agonists and antagonists of adenosine receptors on BPV in sinoaortic denervated (SAD) rats. It was found that both adenosine and 5'-N-cyclopropyl-carboxamidoadenosine (CPCA, a selective adenosine A,-receptor agonist) decreased BPV. whereas N6-cyclopentyladenosine (CPA, a selective adenosine A1-receptor agonist) had no significant effect on BPV. When the rats were pretreated with theophylline (the nonselective adenosine-receptor antagonist), the inhibitory effects of adenosine as well as CPCA on BPV were abolished. Furthermore, it was found that 8-(3-chlorostyryl)caffeine (CSC, a selective adenosine A2a-receptor antagonist), also could prevent such an effect on BPV of CPCA. By itself, however, neither theophylline nor CSC had any influence on BPV. These results suggest that the effect of adenosine on BPV is mediated by adenosine A2a-receptor.     

Release of endogenous adenosine does not mediate electrophysiological responses to morphine in the hippocampus in vitro

Previous studies have shown that adenosine receptor antagonists can block some of the effects of morphine and that morphine can enhance the release of adenosine. The possibility that some of the effects of morphine in the hippocampus are exerted through the release of adenosine was investigated using electrophysiological responses in vitro. Although both morphine and the adenosine receptor antagonist theophylline increase population spike responses, they appear to do so by independent mechanisms. Also, while adenosine significantly decreases the amplitude of the field excitatory postsynaptic potential (fEPSP) and theophylline increases this response (by blocking the effects of endogenous adenosine), morphine had no significant effect on the fEPSP. No significant interactions between morphine and theophylline were observed using either fEPSPs or the population spike response. These results suggest that, either morphine does not stimulate the release of adenosine in slices of hippocampus under normal electrophysio-logical recording conditions, or that the concentrations of adenosine, released by morphine, are not physiologically relevant in the hippocampus in vitro.     

The receptor mechanism mediating the contractile response to adenosine on lung parenchymal strips from actively sensitised,allergen-challenged Brown Norway rats

Parenchymal strips prepared from lungs removed from actively sensitised Brown Norway rats challenged with allergen show hyperresponsiveness to adenosine. The response is mast cell mediated and a preliminary pharmacological analysis suggested the involvement of a receptor (or receptors) that could not be classified as any of the known adenosine receptor subtypes. We present a further analysis of the response. Male Brown Norway (BN) rats, actively sensitised to ovalbumin (OA), were challenged intratracheally with OA and killed 3 h later to provide parenchymal strip preparations. The augmented contractile responses to adenosine were partially blocked by the 5-HT receptor antagonist, methysergide, or the A₁ receptor antagonist, DPCPX, and abolished in the presence of both antagonists. Responses to high concentrations of the A₁ receptor agonist, CPA were, like those to adenosine, augmented on tissues from allergen-challenged animals and blocked by a combination of methysergide and DPCPX. The A₃ receptor agonist, Cl-IB-MECA, did not contract the tissue, but partially blocked the response to adenosine. A combination of Cl-IB-MECA and methysergide induced a similar degree of blockade to that seen with either drug given alone. Combination of Cl-IB-MECA and/or methysergide with DPCPX abolished the response to adenosine. The effects of the A₃ receptor agonist, inosine, were augmented on tissues from allergen-challenged animals and markedly inhibited by disodium cromoglycate, methysergide or Cl-IB-MECA. Responses to adenosine were abolished when parenchymal strips were taken from rats pretreated 48 h previously with pertussis toxin. 8-SPT, CGS 15943, XAC, MRS 1754, DPCPX and theophylline, at concentrations which inhibit the A₁ A_2A and/or A_2B receptors but have negligible affinity for the rat A₃ receptor, inhibited responses to adenosine, but high concentrations were required and blockade was incomplete. MRS 1523 and MRS 1191, which are antagonists at the rat A₃ receptor, had no effect on the response to adenosine. The present results support and clarify our earlier conclusion that an atypical receptor mechanism mediates contraction of the parenchymal strip prepared from the lungs of actively sensitised BN rats challenged with allergen to adenosine. The response arises from a combined effect of adenosine on the A₁ receptor and a receptor with similarities to the A₃ receptor, but where Cl-IB-MECA behaves as an antagonist and MRS 1523 and MRS 1191 are inactive at concentrations that substantially exceed their affinities for the rat A₃ receptor.A part of this work was presented to the British Pharmacological Society in January 2003     

Release of endogenous adenosine does not mediate electrophysiological responses to morphine in the hippocampus in vitro

Previous studies have shown that adenosine receptor antagonists can block some of the effects of morphine and that morphine can enhance the release of adenosine. The possibility that some of the effects of morphine in the hippocampus are exerted through the release of adenosine was investigated using electrophysiological responses in vitro. Although both morphine and the adenosine receptor antagonist theophylline increase population spike responses, they appear to do so by independent mechanisms. Also, while adenosine significantly decreases the amplitude of the field excitatory postsynaptic potential (fEPSP) and theophylline increases this response (by blocking the effects of endogenous adenosine), morphine had no significant effect on the fEPSP. No significant interactions between morphine and theophylline were observed using either fEPSPs or the population spike response. These results suggest that, either morphine does not stimulate the release of adenosine in slices of hippocampus under normal electrophysio-logical recording conditions, or that the concentrations of adenosine, released by morphine, are not physiologically relevant in the hippocampus in vitro.     

1,2,4-Triazolo[1,5-a]quinoxaline as a versatile tool for the design of selective human A3 adenosine receptor antagonists: synthesis, biological evaluation, and molecular modeling studies of 2-(hetero)aryl- and 2-carboxy-substituted derivatives

A number of 4-oxo-substituted 1,2,4-triazolo[1,5-a]quinoxaline derivatives bearing at position-2 the claimed (hetero)aryl moiety (compounds 1-15) but also a carboxylate group (16-28, 32-36) or a hydrogen atom (29-31) were designed as human A3 (hA3) adenosine receptor (AR) antagonists. This study produced some interesting compounds and among them the 2-(4-methoxyphenyl)-1,2,4-triazolo[1,5-a]quinoxalin-4-one (8), which can be considered one of the most potent and selective hA3 adenosine receptor antagonists reported till now. Moreover, as a new finding, replacement of the classical 2-(hetero)aryl moiety with a 2-carboxylate function (compounds 16-28 and 32-36) maintained good hA3 AR binding activity but, most importantly and interestingly, produced a large increase in hA3 versus hA1 selectivity. A receptor-based SAR analysis provided new interesting insights about the steric and electrostatic requirements that are important for the anchoring of these derivatives at the hA3 receptor recognition site, thus highlighting the versatility of the triazoloquinoxaline scaffold for obtaining potent and selective hA3 AR antagonists.     

Antagonist pharmacology of adenosine A2B receptors from rat,guinea pig and dog

We have sought evidence for species differences between adenosine A2B receptors by comparing the potencies of eight adenosine receptor antagonists, representing four different chemical classes, at the native adenosine A2B receptors which mediate relaxation of smooth muscle from rat colon, guinea pig aorta and dog saphenous vein. In all three assays, the antagonists caused parallel rightward shifts in the concentration-response curves to NECA and there was no depression of the maximum responses. There were highly significant correlations between the pKB values on each of the three receptors. However, the pKB values of 8-SPT (8-p-(sulphophenyl)theophylline), XAC (8-[-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine), CGS 15943 (9-chloro-2,2-(furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine) and CGH 2473 N-[4-(3,4-dichloro-phenyl)-5-pyridin-4-yl-thiazol-2-yl]-acetamide) for the dog receptor exceeded by at least 0.5 log units the pKB values at the rat and guinea pig sites. Our data indicate species differences between the rat and guinea pig adenosine A2B receptors on the one hand and the dog adenosine A2B receptor on the other with respect to antagonist pharmacology.     

Effects of adenosine receptor agonists and antagonists in a genetic animal model of primary paroxysmal dystonia

1. Recent studies have shown beneficial effects of an adenosine A(2A) receptor agonist in dt(sz) mutant hamsters, an animal model of paroxysmal dystonia, in which stress and consumption of coffee can precipitate dystonic attacks. This prompted us to examine the effects of adenosine receptor agonists and antagonists on severity of dystonia in dt(sz) hamsters in more detail. 2. The non-selective adenosine A(1)/A(2A) receptor antagonists, caffeine (10 - 20 mg kg(-1) i.p.) and theophylline (10 - 30 mg kg(-1) s.c.), worsened the dystonia in dt(sz) hamsters. 3. Aggravation of dystonia was also caused by the selective adenosine A(1)/A(2A) antagonist CGS 15943 (9-chloro2-2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine) at a dose of 30 mg kg(-1) i.p. and by the adenosine A(1) antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine; 20 - 30 mg kg(-1) i.p.), while the A(2) antagonist DMPX (3,7-dimethyl-1-propargylxanthine; 2 - 4 mg kg(-1) i.p.) and the highly selective A(2A) antagonist ZM 241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol; 2 - 5 mg kg(-1) i.p.) failed to exert any effects on dystonia. 4. In contrast to the antagonists, both the adenosine A(1) receptor agonist CPA (N(6)-cyclopentyladenosine; 0.1 - 1.0 mg kg(-1) i.p.) and the A(2A) agonist CGS 21680 (2p-(2carboxyethylphen-ethylamino-5'-N-ethylcarboxamindoadenosine; 0.1 - 2.0 mg kg(-1) i.p.) exerted a striking improvement of dystonia. 5. These data suggest that the precipitating effects of methylxanthines are, at least in part, related to their adenosine receptor antagonistic action. 6. Although adenosine receptor agonists can be regarded as interesting candidates for the therapy of paroxysmal dystonia, adverse effects may limit the therapeutic potential of adenosine A(1) agonists, while beneficial effects of the adenosine A(2A) agonist CGS 21680 were already found at well tolerated doses.     

Synthesis, CoMFA analysis, and receptor docking of 3,5-diacyl-2, 4-dialkylpyridine derivatives as selective A3 adenosine receptor antagonists

3,5-Diacyl-2,4-dialkyl-6-phenylpyridine derivatives have been found to be selective antagonists at both human and rat A3 adenosine receptors (Li et al. J. Med. Chem. 1998, 41, 3186-3201). In the present study, ring-constrained, fluoro, hydroxy, and other derivatives in this series have been synthesized and tested for affinity at adenosine receptors in radioligand binding assays. Ki values at recombinant human and rat A3 adenosine receptors were determined using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyladenosine). Selectivity for A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors, and structure-activity relationships at various positions of the pyridine ring (the 3- and 5-acyl substituents and the 2- and 4-alkyl substituents) were probed. At the 5-position inclusion of a beta-fluoroethyl (7) or a gamma-fluoropropyl ester (26) was favorable for human A3 receptor affinity, resulting in Ki values of 4.2 and 9.7 nM, respectively, while the pentafluoropropyl analogue was clearly less potent at human A3 receptors. At the 2-, 3-, and 4-positions, fluoro or hydroxy substitution failed to enhance potency and selectivity at human A3 receptors. Several analogues were nearly equipotent at rat and human A3 receptors. To further define the pharmacophore conformationally, a lactam, a lactone, and thiolactones were tested in adenosine receptor binding. The most potent analogue in this group was compound 34, in which a thiolactone was formed between 3- and 4-positions and which had a Ki value of 248 nM at human A3 receptors. Using affinity data and a general pharmacophore model for A3 adenosine receptor antagonists recently proposed, we applied comparative molecular field analysis (CoMFA) to obtain a three-dimensional quantitative structure-activity relationship for pyridine derivatives, having good predictability (r2pred = 0.873) for compounds in the test set. A rhodopsin-based model of the human A3 receptor was built, and the pyridine reference ligand 2,3,4, 5-tetraethyl-6-phenyl-pyridine-3-thiocarboxylate-5-carboxylate (MRS 1476) was docked in the putative ligand binding site. Interactions between receptor transmembrane domains and the steric and the electrostatic contour plots obtained from the CoMFA analysis were analyzed.     

Thiazole and thiadiazole analogues as a novel class of adenosine receptor antagonists

Novel classes of heterocyclic compounds as adenosine antagonists were developed based on a template approach. Structure-affinity relationships revealed insights for extended knowledge of the receptor-ligand interaction. We replaced the bicyclic heterocyclic ring system of earlier described isoquinoline and quinazoline adenosine A(3) receptor ligands by several monocyclic rings and investigated the influence thereof on adenosine receptor affinity. The thiazole or thiadiazole derivatives seemed most promising, so we continued our investigations with these two classes of compounds. The large difference between a pyridine and isoquinoline ring in binding adenosine A(1) and A(3) receptors showed the importance of the second ring of the isoquinoline ligands. We prepared several N-[4-(2-pyridyl)thiazol-2-yl]benzamides, and these compounds showed adenosine affinities in the micromolar range. Most surprising in the series of the N-[4-(2-pyridyl)thiazol-2-yl]amides were the retained adenosine affinities by introduction of a cylopentanamide instead of the benzamide. A second series of compounds, the thiadiazolobenzamide series of compounds, revealed potent and selective adenosine receptor antagonists, especially N-(3-phenyl-1,2,4-thiadiazol-5-yl)-4-hydroxybenzamide (LUF5437, 8h) showing a K(i) value of 7 nM at the adenosine A(1) receptor and N-(3-phenyl-1,2,4-thiadiazol-5-yl)-4-methoxybenzamide (LUF5417, 8e) with a K(i) value of 82 nM at the adenosine A(3) receptor. 4-Hydroxybenzamide 8h is the most potent adenosine A(1) receptor antagonist of this new class of compounds. Structure--affinity relationships showed the existence of a steric restriction at the para-position of the benzamide ring for binding adenosine A(1) and A(3) receptors. The electronic nature of the 4-substituents played an important role in binding the adenosine A(3) receptor. Cis- and trans-4-substituted cyclohexyl derivatives were made next to the 4-substituted benzamide analogues. We used them to study the proposed specific interaction between the adenosine A(1) receptor and the 4-hydroxy group of this class of thiadiazolo compounds, as well as a suggested special role for the 4-methoxy group in binding the A(3) receptor. Both the adenosine A(1) and A(3) receptor slightly preferred the trans-analogues over the cis-analogues, while all compounds showed low affinities at the adenosine A(2A) receptor. Our investigations provided the potent and highly selective adenosine A(1) antagonist N-(3-phenyl-1,2,4-thiadiazol-5-yl)-trans-4-hydroxycyclohexanamide (VUF5472, 8m) showing a K(i) value of 20 nM. A third series of compounds was formed by urea analogues, N-substituted with thiazolo and thiadiazolo heterocycles. The SAR of this class of compounds was not commensurate with the SAR of the previously described quinazoline urea. On the basis of these findings we suggest the existence of a special interaction between adenosine receptors and a region of high electron density positioned between the thia(dia)zole ring and phenyl(pyridyl) ring. Molecular electrostatic potential contour plots showed that for this reason the ligands need either a thiadiazole ring instead of a thiazole or a 2-pyridyl group instead of a phenyl. The derived novel classes of antagonists will be useful for a better understanding of the molecular recognition at the adenosine receptors.     

Effects of a novel, selective, sigma1-ligand, MS-377, on phencyclidine-induced behaviour

We have reported previously the cloning and partial characterization of a chick A1 adenosine receptor expressed in the heart. We report herein the cloning of a chick A3 adenosine receptor and a comprehensive characterization of both the A1 and A3 receptors expressed in human embryonic kidney 293 cells. [125I]N6-(p-aminobenzyl)adenosine bound to both receptors with similar affinities and was used in competition studies. Although the selectivities of both agonists and antagonists were less than in other species, two antagonists, 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynal-(+/-)-dihydropyridine-3,5-dicarboxylate and 3,6-dichloro-2'-(isopropyloxy)-4'-methylflavone), were at least partially selective for A3 receptors while one antagonist [C8-(N-methylisopropyl)amine-N6-(5'endohydroxy)endonorboman-2-yl-9-methyladenine] was selective for A1 receptors. While both receptors coupled to the inhibition of adenylyl cyclase, we were unable to detect coupling of either receptor to phospholipase C or D.