Rat A3 Receptors Research Articles

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.

A3 Adenosine Receptors: Protective vs. Damaging Effects Identified Using Novel Agonists and Antagonists.

Investigation of the physiologic role of the A3 adenosine receptor has been facilitated by the availability of selective agonists and antagonists. Selective agonists include IB-MECA and the 2-chloro derivative Cl-IB-MECA. Selective antagonists have been identified and designed with the aid of molecular modeling among various nonpurine classes of heterocycles: flavonoids, 1,4-dihydropyridine derivatives, triazoloquinazolines, isoquinolines, and a triazolonaphthyridine. The dihydropyridine 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS 1191) is 1,300-fold selective for human A3 (Ki of 31 nM) vs. A1/A2A adenosine receptors and also 28-fold A3 selective in rat tissue (Ki of 1.42 mM). 9-Chloro-2-(2-furyl)-5-phenylacetylamino[1,2,4]-triazolo[1,5-c]quinazoline (MRS 1220) is useful as an A3 selective antagonist only in human tissue, with a Ki value of 0.65 nM. The pyridine derivative 5-propyl 2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate (MRS 1523) is a selective antagonist of both rat and human A3 receptors, with Ki values of 113 and 19 nM, respectively. Paradoxical effects of A3 agonists in the brain, heart and other tissues indicate that acute activation of A3 receptors at greater than 10 mM concentrations acts as a lethal input to cells, whereas low, nanomolar concentrations of A3 receptor agonists protect against apoptosis or ischemic damage. Adenosine A3 receptor agonists, antagonists, or both, may be useful in treating inflammatory conditions.

Synthesis and biological activity of a new series of N6-arylcarbamoyl, 2-(Ar)alkynyl-N6-arylcarbamoyl, and N6-carboxamido derivatives of adenosine-5'-N-ethyluronamide as A1 and A3 adenosine receptor agonists.

A new series of 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuranuronamide++ +-b earing N-arylureas or N-arylcarboxamido groups at the purine 6 position and N-arylureas combined with halogens or alkynyl chains at the 2 position have been synthesized and tested for affinity at A1 and A2A adenosine receptors in rat brain membranes and at cloned rat A3 receptors expressed in CHO cells. The derivatives contained the 5' substituent found in the potent, nonselective agonist 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuranuronamide++ + (NECA). While the carboxamido derivatives (9-13) showed affinity for A1 receptors, the urea derivatives (30-45) showed different degrees of affinity and selectivity for the A3 adenosine receptor subtype. In particular the derivative bearing a p-sulfonamidophenyl-urea at the 6 position, 31 showed a high affinity (Ki = 9 nM) and selectivity for the A3 receptors compared to that of the reference compound 1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-be ta-D-ribofuranuronamide (IB-MECA). Furthermore, the importance of the stereochemistry in the interaction of these ligands at the rat A3 adenosine receptors has been evaluated by introducing a chiral chain at the 6 position. The introduction of halogens or alkynyl chains at the purine 2 position of selected ureas did not give the expected enhancement of potency at A2A and/or A3 receptors but rather showed a dramatic reduction of A2A affinity, resulting in compounds with good A2A/A3 selectivity. For example, the 2-(3-hydroxy-3-phenyl-1-propyn-1-yl)-6-(4-methoxyphenylurea) derivative 61 showed the capability to bind simultaneously to A1 and A3 receptor subtypes, excluding the A2A receptor. Compound 31 was shown to be an agonist, 9-fold more potent than NECA, at A3 receptors in rat RBL-2H3 mast cell membranes through stimulation of binding of [35S]GTP-gamma-S.

Structure-activity relationships and molecular modeling of 3, 5-diacyl-2,4-dialkylpyridine derivatives as selective A3 adenosine receptor antagonists.

The structure-activity relationships of 6-phenyl-1,4-dihydropyridine derivatives as selective antagonists at human A3 adenosine receptors have been explored (Jiang et al. J. Med. Chem. 1997, 39, 4667-4675). In the present study, related pyridine derivatives have been synthesized and tested for affinity at adenosine receptors in radioligand binding assays. Ki values in the nanomolar range were observed for certain 3,5-diacyl-2,4-dialkyl-6-phenylpyridine derivatives in displacement of [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyladenosine) at recombinant human A3 adenosine receptors. Selectivity for A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors. 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. A 4-phenylethynyl group did not enhance A3 selectivity of pyridine derivatives, as it did for the 4-substituted dihydropyridines. At the 2- and 4-positions ethyl was favored over methyl. Also, unlike the dihydropyridines, a thioester group at the 3-position was favored over an ester for affinity at A3 adenosine receptors, and a 5-position benzyl ester decreased affinity. Small cycloalkyl groups at the 6-position of 4-phenylethynyl-1,4-dihydropyridines were favorable for high affinity at human A3 adenosine receptors, while in the pyridine series a 6-cyclopentyl group decreased affinity. 5-Ethyl 2, 4-diethyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate , 38, was highly potent at human A3 receptors, with a Ki value of 20 nM. A 4-propyl derivative, 39b, was selective and highly potent at both human and rat A3 receptors, with Ki values of 18.9 and 113 nM, respectively. A 6-(3-chlorophenyl) derivative, 44, displayed a Ki value of 7.94 nM at human A3 receptors and selectivity of 5200-fold. Molecular modeling, based on the steric and electrostatic alignment (SEAL) method, defined common pharmacophore elements for pyridine and dihydropyridine structures, e.g., the two ester groups and the 6-phenyl group. Moreover, a relationship between affinity and hydrophobicity was found for the pyridines.

Activation and Desensitization of Rat A3-Adenosine Receptors by Selective Adenosine Derivatives and Xanthine-7-Ribosides.

Strategy, Management and Health PolicyVenture Capital Enabling TechnologyPreclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, PharmacokineticsClinical Development Phases I-III Regulatory, Quality, ManufacturingPostmarketing Phase IV Xanthine and adenosine derivatives, known to bind to recombinant rat A3 adenosine receptors stably expressed in Chinese hamster ovary cells, were characterized in a functional assay consisting of activation of A3 receptor-stimulated binding of [35S]GTPγS in rat RBL-2H3 cell membranes. 1,3-Dibutylxanthine-7-riboside-5'-N-methylcarboxamide (DBXRM, 7b), previously shown to inhibit adenylyl cyclase via rat A3 receptors with full efficacy, appeared to be a partial agonist at the rat A3 receptor of RBL-2H3 cells. Full agonists, such as Cl-IB-MECA or I-AB-MECA, were more potent and effective than the partial agonist DBXRM in causing desensitization of rat A3 receptors, as indicated by loss of [35S]GTPγS binding. At A1 receptors, antagonism of agonist-elicited inhibition of rat adipocyte adenylyl cyclase was observed for several xanthine-7-riboside derivatives that had been shown to be full agonists at rat A3 receptors. A new xanthine riboside (3'-deoxyDBXRM, 7c) was synthesized and found to be a partial agonist at rat A3 receptors and an antagonist at rat A1 receptors. Thus, it is possible for the same compound to stimulate one adenosine receptor subtype (A3) and block another subtype (A1) within the same species.

Activation of multiple sites by adenosine analogues in the rat isolated aorta.

1. The presence of A2 receptors mediating relaxation in the rat isolated aorta has been previously demonstrated. However, agonist dependency of the degree of rightward shift elicited by 8-sulphophenyltheophylline (8-SPT) led to the suggestion that the population of receptors in this tissue is not a homogeneous one. In this study we have re-examined the effects of 8-SPT in the absence and presence of the NO synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester) and investigated antagonism of responses by the potent A2a receptor ligands PD 115,199 (N-[2-dimethylamino)ethyl]-N-methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3 dipropyl-1H-purin-8-yl)) benzene sulphonamidexanthine), ZM 241385 (4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-yl amino]ethyl)phenol), and CGS 21680 (2-[p-(2-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine). We have also investigated the antagonist effects of BWA1433 (1,3-dipropyl-8-(4-acrylate)phenylxanthine) which has been shown to have affinity at rat A3 receptors. 2. Adenosine, R-PIA (N6-R-phenylisopropyl adenosine), CPA (N6-cyclopentyladenosine) and NECA (5'-N-ethylcarboxamidoadenosine) all elicited relaxant responses in the phenylephrine pre-contracted rat isolated aorta with the following potency order (p[A50] values in parentheses): NECA (7.07 +/- 0.11) > R-PIA (5.65 +/- 0.10) > CPA (5.05 +/- 0.12) > adenosine (4.44 +/- 0.12). 3. 8-SPT (10-100 microM) caused parallel rightward shifts of the E/[A] curves to NECA (pKB = 5.23 +/- 0.16). A smaller rightward shift of E/[A] curves to CPA was observed (pA2 = 4.85 +/- 0.17). However, no significant shifts of E/[A] curves to either adenosine or R-PIA were observed. 4. In the absence of endothelium E/[A] curves to NECA and CPA were right-shifted compared to controls. However, removal of the endothelium did not produce a substantial shift of adenosine E/[A] curves, and E/[A] curves to R-PIA were unaffected by removal of the endothelium. 5. In the presence of L-NAME (100 microM) E/[A] curves to NECA and CPA were right-shifted. However, no further shift of the CPA E/[A] curve was obtained when 8-SPT (50 microM) was administered concomitantly. The locations of curves to R-PIA and adenosine were unaffected by L-NAME (100 microM). 6. In the presence of PD 115,199 (0.1 microM) a parallel rightward shift of NECA E/[A] curves was observed (pA2 = 7.50 +/- 0.19). PD 115,199 (0.1 and 1 microM) gave smaller rightward shifts of E/[A] curves to R-PIA and CPA, but E/[A] curves to adenosine were not significantly shifted in the presence of PD 115,199 (0.1 or 1 microM). 7. The presence of ZM 241385 (3 nM-0.3 microM) caused parallel rightwad shifts of NECA E/[A] curves (pKB = 8.73 +/- 0.11). No significant shifts of E/[A] curves to adenosine, CPA or R-PIA were observed in the presence of 0.1 microM ZM 241385. 8. CGS 21680 (1 microM) elicited a relaxant response equivalent to approximately 40% of the NECA maximum response. In the presence of this concentration of CGS 21680, E/[A] curves to NECA were right-shifted in excess of 2-log units, whereas E/[A] curves to R-PIA were not significantly shifted. 9. BWA1433 (100 microM) caused a small but significant right-shift of the E/[A] curve to R-PIA yielding a pA2 estimate of 4.1 IB-MECA (N6-(3-iodo-benzyl)adenosine-5(1)-N-methyl uronamide) elicited relaxant responses which were resistant to blockade by 8-SPT (p[A]50 = 5.26 +/- 0.13). 10. The results suggest that whereas relaxations to NECA (10 nM-1 microM) are mediated via adenosine A2a receptors, which are located at least in part on the endothelium, R-PIA and CPA may activate A2b receptors on the endothelium and an additional, as yet undefined site, which is likely to be located on the smooth muscle and which is not susceptible to blockade by 8-SPT, PD 115,199 or ZM 241385. This site is unlikely to be an A3 receptor since the very small shift obtained in the presence of BWA1433 (100 microM), and the low potency of IB-MECA is not consistent with the affin

KW‐3902, a selective high affinity antagonist for adenosine A1 receptors

1. We demonstrate that 8-(noradamantan-3-yl)-1,3-dipropylxanthine (KW-3902) is a very potent and selective adenosine A1 receptor antagonist, assessed by radioligand binding and cyclic AMP response in cells. 2. In rat forebrain adenosine A1 receptors labelled with [3H]-cyclohexyladenosine (CHA), KW-3902 had a Ki value of 0.19 nM, whereas it showed a Ki value of 170 nM in rat striatal A2A receptors labelled with [3H]-2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoad enosine (CGS21680), indicating 890 fold A1 receptor selectivity versus the A2A receptor. KW-3902 at 10 microM showed no effect on recombinant rat A3 receptors expressed on CHO cells. 3. Saturation studies with [3H]-KW-3902 revealed that it bound with high affinity (Kd = 77 pM) and limited capacity (Bmax = 470 fmol mg-1 of protein) to a single class of recognition sites. A high positive correlation was observed between the pharmacological profile of adenosine ligands inhibiting the binding of [3H]-KW-3902 and that of [3H]-CHA. 4. KW-3902 showed potent A1 antagonism against the inhibition of forskolin-induced cyclic AMP accumulation in DDT1 MF-2 cells by the A1-selective agonist, cyclopentyladenosine with a dissociation constant (KB value) of 0.34 nM. KW-3902 antagonized 5'-N-ethylcarboxamidoadenosine-elicited cyclic AMP accumulation via A2B receptors with a KB value of 52 nM. 5. KW-3902 exhibited marked species-dependent differences in the binding affinities. The highest affinity was for the rat A1 receptor (ki = 0.19 nM) and these values for guinea-pig and dog A1 receptors were 1.3 and 10 nM, respectively.

Interactions of flavonoids and other phytochemicals with adenosine receptors.

Flavone derivatives and other phytochemicals were found to bind to three subtypes of adenosine receptors in the micromolar range. Affinity was determined in radioligand binding assays at rat brain A1 and A2A receptors using [3H]-N6-PIA ([3H]-(R)-N6-phenylisopropyladenosine) and [3H]CGS21680 ([3H]-2-[[4-(2-carboxyethyl)phenyl]ethylamino]-5'- (N-ethylcarbamoyl)adenosine), respectively. Affinity was determined at cloned human and rat brain A3 receptors using [125I]-AB-MECA [N6-(4-amino-3-iodobenzyl)adenosine-5'-(N-methyluronamide)]. A structure-activity analysis indicated that the hydroxyl groups of naturally occurring flavones are not essential for affinity at adenosine receptors. Galangin, 14, displayed Ki values of 1 microM at both rat A1 and A2A receptors and 3 microM at human A3 receptors. Methylation but not acetylation of the hydroxyl groups of galangin enhanced A3 affinity. Pentamethylmorin, 20, appeared to bind with 14-17-fold selectivity for human A3 receptors vs rat A1 and A2A receptors, with a Ki value of 2.65 microM. Two flavone derivatives (14 and 15) showed 14-fold greater affinity at human vs rat A3 receptors. Reduction of the 2,3-olefinic bond, as in (+/-)-dihydroquercetin, or glycosidation, as in robinin, greatly diminished affinity. An isoflavone, genistein, also bound only very weakly at A3 receptors. alpha-Naphthoflavone had greater receptor affinity (0.79 microM at A1 receptors) than the beta-isomer. Other natural products of plant origin, including oxogalanthine lactam, hematoxylin, and arborinine were found to bind to A1 adenosine receptors with Ki values of 3-13 microM. These findings indicate that the flavones, flavonols, flavanones, and other phytochemicals may provide leads for the development of novel adenosine antagonists. The unexpected finding of considerable affinity of flavones at both rat and human A3 receptors may explain some of the previously observed biological effects of these compounds.

Derivatives of the triazoloquinazoline adenosine antagonist (CGS15943) are selective for the human A3 receptor subtype.

The adenosine antagonist 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS15943) binds to human A3 receptors with high affinity (Ki = 14 nM), while it lacks affinity at rat A3 receptors. Acylated derivatives of the 5-amino group and other modifications were prepared in an effort to provide A3 subtype selectivity. Affinity was determined in radioligand binding assays at rat brain A1 and A2A receptors using [3H]-(R)-PIA ([3H]-(R)-N6-(phenylisopropyl)-adenosine) and [3H]CGS 21680 ([3H]-2-[[4-(2-carboxy ethyl)phenyl]ethylaminol]-5'-(N- ethyl- carbamoyl)adenosine), respectively. Affinity was determined at cloned human A3 receptors using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5'-(N-methylcarbamoyl)adenosine). A series of straight chain alkyl amides demonstrated that the optimal chain length occurs with the 5-N-propionyl derivative, 3, which had a Ki value of 7.7 nM at human A3 receptors, and was 40- and 14-fold selective vs rat A1 and A2A receptors, respectively. The 5-N-benzoyl derivative, 10, displayed Ki values of 680 and 273 nM at rat A1 and A2A receptors, respectively, and 3.0 nM at human A3 receptors. A 5-N-phenylacetyl derivative, 12, was 470-fold selective for human A3 vs rat A1 receptors with a Ki value of 0.65 nM. A conjugate of Boc-gamma-aminobutyric acid was also prepared but was nonselective. Conversion of the 5-amino group of CGS15943 to an oxo function resulted in lower affinity but 15-fold selectivity for human A3 receptors.

A SURVEY OF NONXANTHINE DERIVATIVES AS ADENOSINE RECEPTOR LIGANDS.

The binding affinities at rat A1, A2a, and A3 adenosine receptors of a wide range of heterocyclic derivatives have been determined. Mono-, bi-, tricyclic and macrocyclic compounds were screened in binding assays, using either [3H]PIA or [3H]CGS 21680 in rat brain membranes or [125I]AB-MECA in CHO cells stably transfected with rat A3 receptors. Several new classes of adenosine antagonists (e.g. 5-oxoimidazopyrimidines and a pyrazoloquinazoline) were identified. Various sulfonylpiperazines, 11-hydroxytetrahydrocarbazolenine, 4H-pyrido[1,2-a]pyrimidinone, folic acid, and cytochalasin H and J bound to A3 receptors selectively. Moreover, cytochalasin A, which bound to A1 adenosine receptors with Ki value of 1.9 μM, inhibited adenylyl cyclase in rat adipocytes, but not via reversible A1 receptor binding.

Interaction of 1,4-dihydropyridine and pyridine derivatives with adenosine receptors: selectivity for A3 receptors.

1,4-Dihydropyridine and pyridine derivatives bound to three subtypes of adenosine receptors in the micromolar range. Affinity was determined in radioligand binding assays at rat brain A1 and A2A receptors using [3H]-(R)-PIA [[3H]-(R)-N6-(phenylisopropyl)adenosine] and [3H]CGS 21680 [[3H]-2-[[4-(2-carboxyethyl)phenyl]ethylamino]-5'-(N-ethylcarbamoyl++ +) adenosine], respectively. Affinity was determined at cloned human and rat A3 receptors using [125I]AB-MECA [N6-(4-amino-3-iodobenzyl)-5'-(N-methylcarbamoyl)adenosine]. Structure-activity analysis at adenosine receptors indicated that sterically bulky groups at the 4-, 5-, and 6-positions are tolerated. (R,S)-Nicardipine, 12, displayed Ki values of 19.6 and 63.8 microM at rat A1 and A2A receptors, respectively, and 3.25 microM at human A3 receptors. Similarly, (R)-niguldipine, 14, displayed Ki values of 41.3 and 1.90 microM at A1 and A3 receptors, respectively, and was inactive at A2A receptors. A preference for the R- vs the S-enantiomer was observed for several dihydropyridines at adenosine receptors, in contrast with the selectivity at L-type Ca2+ channels. A 4-trans-beta-styryl derivative, 24, with a Ki value of 0.670 microM at A3 receptors, was 24-fold selective vs A1 receptors (Ki = 16.1 microM) and 74-fold vs A2A receptors (Ki = 49.3 microM). The affinity of 24 at L-type Ca2+ channels, measured in rat brain membranes using [3H]isradipine, indicated a Ki value of 0.694 microM, and the compound is thus nonselective between A3 receptors and L-type Ca2+ channels. Inclusion of a 6-phenyl group enhanced A3 receptor selectivity: Compound 28 (MRS1097; 3,5-diethyl 2-methyl-6-phenyl-4-(trans-2-phenylvinyl)-1,4(R,S)-dihydro-pyridin e-3, 5-dicarboxylate) was 55-fold selective vs A1 receptors, 44-fold selective vs A2A receptors, and over 1000-fold selective vs L-type Ca2+ channels. In addition, compound 28 attenuated the A3 agonist-elicited inhibitory effect on adenylyl cyclase. Furthermore, whereas nicardipine, 12, displaced radioligand from the Na(+)-independent adenosine transporter with an apparent affinity of 5.36 +/- 1.51 microM, compound 28 displaced less than 10% of total binding at a concentration of 100 microM. Pyridine derivatives, when bearing a 4-alkyl but not a 4-phenyl group, maintained affinity for adenosine receptors. These findings indicate that the dihydropyridines may provide leads for the development of novel, selective A3 adenosine antagonists.

Novel N6-(substituted-phenylcarbamoyl)adenosine-5'-uronamides as potent agonists for A3 adenosine receptors.

A series of adenosine-5'-uronamide derivatives bearing N6-phenylurea groups have been synthesized and tested for their affinity at A1 and A2A adenosine receptors in rat brain membranes and at cloned rat A3 receptors from stably transfected CHO cells. Some N6-arylcarbamoyl derivatives, N6-((2-chlorophenyl)carbamoyl)-, N6-((3-chlorophenyl)carbamoyl)-, and N6-((4-methoxyphenyl)carbamoyl)adenosine-5'-ethyluronamide (4l-n), were found to have affinity at A3 receptors in the low nanomolar range (Ki values < 10 nM). In CHO cells stably transfected with the rat A3 receptor, compound 4n was found to be a full agonist in inhibiting adenylate cyclase activity. The present study represents the first example of N6-acyl-substituted adenosine analogs having high affinity at adenosine receptors and, in particular, at the A3 receptor subtype.

6-phenyl-1,4-dihydropyridine derivatives as potent and selective A3 adenosine receptor antagonists.

An approach to designing dihydropyridines that bind to adenosine receptors without binding to L-type calcium channels has been described. 1,4-Dihydropyridine derivatives substituted with beta-styryl or phenylethynyl groups at the 4-position and aryl groups at the 6-position were synthesized and found to be selective for human A3 receptors. Combinations of methyl, ethyl, and benzyl esters were included at the 3- and 5-positions. Affinity was determined in radioligand binding assays at rat brain A1 and A2A receptors using [3H]-(R)-PIA [[3H]-(R)-N6-(phenylisopropyl)adenosine] and [3H]CGS 21680 [[3H]-2-[[4-(2-carboxyethyl)phenyl]ethylamino]-5'- (N-ethylcarbamoyl)adenosine], respectively. Affinity was determined at cloned human and rat A3 receptors using [125I]AB-MECA [N6-(4-amino-3-iodobenzyl)-5'-(N-ethylcarbamoyl)-adenosine]. Structure-activity analysis indicated that substitution of the phenyl ring of the beta-styryl group but not of the 6-phenyl substituent was tolerated in A3 receptor selective agents. Replacement of the 6-phenyl ring with a 3-thienyl or 3-furyl group reduced the affinity at A3 receptors by 4- and 9-fold, respectively. A 5-benzyl ester 4-trans-beta-styryl derivative, 26, with a Ki value of 58.3 nM at A3 receptors, was > 1700-fold selective vs either A1 receptors or A2A receptors. Shifting the benzyl ester to the 3-position lowered the affinity at A3 receptors 3-fold. A 5-benzyl, 3-ethyl ester 4-phenylethynyl derivative, 28, displayed a Ki value of 31.4 nM at A3 receptors and 1300-fold selectivity vs A1 receptors. The isomeric 3-benzyl, 5-ethyl diester was > 600-fold selective for A3 receptors. Oxidation of 28 to the corresponding pyridine derivative reduced affinity at A3 receptors by 88-fold and slightly increased affinity at A1 receptors.

The in vitro pharmacology of ZM 241385, a potent, non‐xanthine, A2a selective adenosine receptor antagonist

1. This paper describes the in vitro pharmacology of ZM 241385 (4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin- 5-yl amino]ethyl) phenol), a novel non-xanthine adenosine receptor antagonist with selectivity for the A2a receptor subtype. 2. ZM 241385 had high affinity for A2a receptors. In rat phaeochromocytoma cell membranes, ZM 241385 displaced binding of tritiated 5'-N-ethylcarboxamidoadenosine (NECA) with a pIC50 of 9.52, (95% confidence limits, c.l., 9.02-10.02). In guinea-pig isolated Langendorff hearts, ZM 241385 antagonized vasodilatation of the coronary bed produced by 2-chloroadenosine (2-CADO) and 2-[p-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680) with pA2 values of 8.57 (c.l., 8.45-8.68) and 9.02 (c.l., 8.79-9.24) respectively. 3. ZM 241385 had low potency at A2b receptors and antagonized the relaxant effects of adenosine in the guinea-pig aorta with a pA2 of 7.06, (c.l., 6.92-7.19). 4. ZM 241385 had a low affinity at A1 receptors. In rat cerebral cortex membranes it displaced tritiated R-phenylisopropyladenosine (R-PIA) with a pIC50 of 5.69 (c.l., 5.57-5.81). ZM 241385 antagonized the bradycardic action of 2-CADO in guinea-pig atria with a pA2 of 5.95 (c.l., 5.72-6.18). 5. ZM 241385 had low affinity for A3 receptors. At cloned rat A3 receptors expressed in chinese hamster ovary cells, it displaced iodinated aminobenzyl-5'-N-methylcarboxamido adenosine (AB-MECA) with a pIC50 of 3.82 (c.l., 3.67-4.06). 6. ZM 241385 had no significant additional pharmacological effects on the isolated tissues used in these studies at concentrations three orders of magnitude greater than those which block A2a receptors. At 10 microM it displayed only minor inhibition of the bradycardic effects in guinea-pig atria to some concentrations of carbachol. At 10 microM, ZM 241385 had a small inhibitory effect on relaxant effects of isoprenaline in guinea-pig aortae but no effect on sodium nitrite-induced relaxation. ZM 241385(100 microM) was without effect on phenylephrine-induced tone in guinea-pig aortae.7. ZM 241385 (10 microM) had no inhibitory effect on rat hepatocyte phosphodiesterase types I, II, III and IV but caused a small inhibition of the calcium calmodulin-activated type I enzyme.8. ZM 241385 is the most selective adenosine A2a receptor antagonist yet described and is therefore a useful tool for characterization of responses mediated by A2 adenosine receptors.

Structure-activity relationships of 9-alkyladenine and ribose-modified adenosine derivatives at rat A3 adenosine receptors.

9-Alkyladenine derivatives and ribose-modified N6-benzyladenosine derivatives were synthesized in an effort to identify selective ligands for the rat A3 adenosine receptor and leads for the development of antagonists. The derivatives contained structural features previously determined to be important for A3 selectivity in adenosine derivatives, such as an N6-(3-iodobenzyl) moiety, and were further substituted at the 2-position with halo, amino, or thio groups. Affinity was determined in radioligand binding assays at rat brain A3 receptors stably expressed in Chinese hamster ovary (CHO) cells, using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5'-(N-methyluronamide)), and at rat brain A1 and A2a receptors using [3H]-N6-PIA ((R)-N6-phenylisopropyladenosine) and [3H]CGS 21680 (2-[[[4-(2-carboxyethyl)-phenyl]ethyl]amino]-5'- (N-ethylcarbamoyl)adenosine), respectively. A series of N6-(3-iodobenzyl) 2-amino derivatives indicated that a small 2-alkylamino group, e.g., methylamino, was favored at A3 receptors. N6-(3-Iodobenzyl)-9-methyl-2-(methylthio)adenine was 61-fold more potent than the corresponding 2-methoxy ether at A3 receptors and of comparable affinity at A1 and A2a receptors, resulting in a 3-6-fold selectivity for A3 receptors. A pair of chiral N6-(3-iodobenzyl) 9-(2,3-dihydroxypropyl) derivatives showed stereoselectivity, with the R-enantiomer favored at A3 receptors by 5.7-fold. 2-Chloro-9-(beta-D-erythrofuranosyl)-N6-(3-iodobenzyl)adenine had a Ki value at A3 receptors of 0.28 microM. 2-Chloro-9-[2-amino-2,3-dideoxy-beta-D-5-(methylcarbamoyl)- arabinofuranosyl]-N6-(3-iodobenzyl)adenine was moderately selective for A1 and A3 vs A2a receptors. A 3'-deoxy analogue of a highly A3-selective adenosine derivative retained selectivity in binding and was a full agonist in the inhibition of adenylyl cyclase mediated via cloned rat A3 receptors expressed in CHO cells. The 3'-OH and 4'-CH2OH groups of adenosine are not required for activation at A3 receptors. A number of 2',3'-dideoxyadenosines and 9-acyclic-substituted adenines appear to inhibit adenylyl cyclase at the allosteric "P" site.

G-protein-dependent activation of phospholipase C by adenosine A 3 receptors in rat brain

The recently cloned G protein-coupled adenosine A3 receptor has been proposed to play a role in the pathophysiology of cerebral ischemia. Because phospholipase C activation occurs as a very early response to brain ischemia, we evaluated the ability of A3- selective and nonselective adenosine analogues to elicit phosphoinositide hydrolysis. In myo-[3H]inositol-labeled rat striatal and hippocampal slices, A3 agonists stimulated formation of [3H]inositol phosphates in a concentration-dependent manner. In striatum, the potency order was 2-chloro-N6-(3-iodobenzyl)- adenosine-5'-N-methyluronamide > or = N6-(3-iodobenzyl)- adenosine-5'-N-methyluronamide >> N-methyl-1,3-di-n-butylxanthine-7-beta-D-ribofuronamide > or = 5'-N-ethylcarboxamidoadenosine > or = N6-2-(4-aminophenyl)-ethyladenosine > N6-(p-sulfophenyl)-adenosine = 1,3-dibutylxanthine-7- riboside, which is identical to the potency order in binding studies at cloned rat A3 receptors. Stimulation of phospholipase C activity was abolished by guanosine-5'-O-(2-thiodiphosphate), confirming the involvement of a G protein-coupled receptor. Activation of phospholipase C was higher in the striatum than in the hippocampus, consistent with A3 receptor densities. Stimulation of phospholipase C activity by adenosine analogues was only modestly antagonized by xanthine derivatives and at much higher concentrations than needed for blocking adenosine A1, A2A, and A2b receptors. In the presence of an A1/A2 antagonist, a selective A3 in rat striation. Thus, stimulation of phospholipase C activity agonist only weakly inhibited forskolin-stimulated adenylyl cyclase activity represents a principal transduction mechanism for A3 receptors in mammalian brain, and perhaps A3 receptor-mediated increases of inositol phosphates in the ischemic brain contribute to neurodegeneration by raising intracellular calcium levels.

Selective ligands for rat A3 adenosine receptors: structure-activity relationships of 1,3-dialkylxanthine 7-riboside derivatives.

1,3-Dibutylxanthine 7-riboside has been found to be a partial agonist at A3 adenosine receptors (van Galen et al. Mol. Pharmacol. 1994, 45, 1101-1111). 1,3-Dialkylxanthine 7-riboside analogues modified at the 1-, 3-, and 8-purine positions and at the ribose 5'-position were synthesized. The nucleoside analogues were examined for affinity in radioligand binding assays at rat brain A3 adenosine receptors stably expressed in CHO cells, using the radioligand [[125I]-4-amino-3-iodobenzyl]adenosine-5'-N-methyluronamide (AB-MECA). Affinity was assayed at rat brain A1 and A2a receptors using [3H]PIA and [3H]CGS 21680, respectively. The affinity of xanthine 7-ribosides at A3 receptors depended on the 1,3-dialkyl substituents in the order: Pent > or = Bu >> Hx > Pr approximately Me. 1,3-Dipentylxanthine 7-riboside was slightly selective for A3 receptors (2-fold vs A1 and 10-fold vs A2a). 8-Methoxy substitution was tolerated at A3 receptors. 2-Thio vs 2-oxo substitution increased potency at all three subtypes and slightly increased A3 vs A1 selectivity. The 5'-uronamide modification, which was previously found to enhance A3 selectivity in N6-benzyladenosine derivatives, was also incorporated into the xanthine 7-ribosides, with similar results. The affinity of 1,3-dialkylxanthine 7-riboside 5'-uronamides at A3 receptors depended on the N-alkyluronamide substituent in the order: MeNH > EtNH >> NH2 >> Me2N. Affinity of the 5'-uronamides at A3 receptors was dependent on the 1,3-dialkyl substitution in the order: Bu > Pent > Hex. 1,3-Dibutylxanthine 7-riboside 5'-N-methylcarboxamide, with a Ki value of 229 nM at A3 receptors, was 160-fold selective for rat A3 vs A1 receptors and > 400-fold selective vs A2a receptors. This derivative acted as a full agonist in the A3 receptor-mediated inhibition of adenylate cyclase.

2-Substitution of N6-benzyladenosine-5'-uronamides enhances selectivity for A3 adenosine receptors.

Adenosine derivatives bearing an N6-(3-iodobenzyl) group, reported to enhance the affinity of adenosine-5'-uronamide analogues as agonists at A3 adenosine receptors (J. Med. Chem. 1994, 37, 636-646), were synthesized starting from methyl beta-D-ribofuranoside in 10 steps. Binding affinities at A1 and A2a receptors in rat brain membranes and at cloned rat A3 receptors from stably transfected CHO cells were compared. N6-(3-Iodobenzyl)adenosine was 2-fold selective for A3 vs A1 or A2a receptors; thus it is the first monosubstituted adenosine analogue having any A3 selectivity. The effects of 2-substitution in combination with modifications at the N6- and 5'-positions were explored. 2-Chloro-N6-(3-iodobenzyl)adenosine had a Ki value of 1.4 nM and moderate selectivity for A3 receptors. 2-Chloro-N6-(3-iodobenzyl)adenosine- 5'-N-methyluronamide, which displayed a Ki value of 0.33 nM, was selective for A3 vs A1 and A2a receptors by 2500- and 1400-fold, respectively. It was 46,000-fold selective for A3 receptors vs the Na(+)-independent adenosine transporter, as indicated in displacement of [3H]N6-(4- nitrobenzyl)-thioinosine binding in rat brain membranes. In a functional assay in CHO cells, it inhibited adenylate cyclase via rat A3 receptors with an IC50 of 67 nM. 2-(Methylthio)-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide and 2-(methylamino)-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide were less potent, but nearly as selective for A3 receptors. Thus, 2-substitution (both small and sterically bulky) is well-tolerated at A3 receptors, and its A3 affinity-enhancing effects are additive with effects of uronamides at the 5'-position and a 3-iodobenzyl group at the N6-position.

Species Differences in Ligand Affinity at Central A3-Adenosine Receptors.

[Table: see text] Binding affinities of purine derivatives at A3 adenosine receptors in different species were compared. Binding was carried out using the novel high affinity agonist ligand [125I]AB-MECA (3-iodo-4-aminobenzyladenosine-5'-N-methyluronamide) in the presence of 1.0 μM XAC (8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine), an A1- and A2a-adenosine antagonist. XAC was added to eliminate binding to non-A3 receptors. In rat brain membranes [125I]AB-MECA exhibited saturable, specific binding with a Kd of 2.28 nM and a Bmax of 43 fmol/mg protein. The affinity of [125I]AB-MECA at the gerbil and rabbit brain A3-receptors was similar to the rat, suggesting that the affinity of this agonist is not highly species dependent. The affinity of various xanthine derivatives was measured in [125I]AB-MECA competition binding assays. Gerbil and rabbit brain A3-receptors were similar in the affinity of antagonists whose potency order in both species was: BWA522 ≥ CPX > XCC, XAC, SPX, BWA1433 > theophylline. The affinities of 8-arylxanthines at the rat, rabbit, and gerbil brain A3 receptors were considerably less than the previously reported affinities at cloned sheep and human A3 receptors. Species differences in agonist affinity were assessed by comparing Ki values at cloned rat brain A3 receptors expressed in CHO cells with cloned sheep and human A3 receptors. Human and rat brain A3 receptors were highly similar in the relative affinities of agonists, and sheep brain A3 receptors were unlike either human or rat A3 receptors in agonist affinity.

Structure-activity relationships of 1,3-dialkylxanthine derivatives at rat A3 adenosine receptors.

1,3-Dialkylxanthine analogues containing carboxylic acid and other charged groups on 8-position substituents were synthesized. These derivatives were examined for affinity in radioligand binding assays at rat brain A3 adenosine receptors stably expressed in CHO cells using the new radioligand [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide), and at rat brain A1 and A2a receptors using [3H]PIA and [3H]CGS 21680, respectively. A synthetic strategy for introducing multiple carboxylate groups at the 8-position using iminodiacetic acid derivatives was explored. The presence of a sulfonate, a carboxylate, or multiple carboxylate groups did not result in a significant enhancement of affinity at rat A3 receptors, although as previously observed an anionic group tended to diminish potency at A1 and A2a receptors. The rat A3 receptor affinity was not highly dependent on the distance of a carboxylate group from the xanthine pharmacophore. 2-Thio vs 2-oxo substitution favored A3 potency, and 8-alkyl vs 8-aryl substitution favored A3 selectivity, although few derivatives were truly selective for rat A3 receptors. 1,3-Dimethyl-8-(3-carboxypropyl)-2-thioxanthine was 7-fold selective for A3 vs A2a receptors. 1,3,7-Trimethyl-8-(trans-2-carboxyvinyl)xanthine was somewhat selective for A3 vs A1 receptors. For 8-arylxanthines affinity at A3 receptors was enhanced by 1,3-dialkyl substituents, in the order dibutyl > dipropyl > diallyl.