N6-substituted N-alkyladenosine-5'-uronamides: bifunctional ligands having recognition groups for A1 and A2 adenosine receptors

The coronary vasoactivity of N-ethyl-1'-deoxy-1'-(6-amino-9H-purin-9-yl)-beta-D-ribofuranuronamide (NECA, 1) is over 2 orders of magnitude greater than that of adenosine, and the vasoactivity of certain N6-substituted adenosines is as much as 1 order of magnitude greater. Such results suggest that a combination of appropriate modifications at N6 and C-5' might additively augment the agonist potency of adenosine. At low temperatures 1-deoxy-1-(6-chloro-9H-purin-9-yl)-2',3'-O-isopropylidene- beta-D-ribofuranosyl chloride (5), obtained in three steps from inosine, reacts with amines to yield uronamides. The subsequent reaction of such uronamides with amines at elevated temperatures displaces the purine 6-chloro group to yield, after deblocking, N-alkyl(or aryl)-N6-alk(ar)yl-adenosine-5'-uronamides. At the coronary artery A2 receptor the potency of N6-modified analogues of 1 is similar to that of the N6-substituted adenosine, rather than equal to or greater than 1. As agonists in the A2 receptor-mediated stimulation of adenylate cyclase in plasma membranes of PC12 pheochromocytoma cells or human platelets, N6-substituted analogues of 1 are intermediate between the high potency of 1 and the lower potency of the N6-substituted adenosines. At the A1 receptor of rat brain the potency of an N6-substituted analogue of 1 is often greater than that of the corresponding N6-substituted adenosine. At all four receptors, replacing the ethyl group of N-ethyl-N6-3-pentyladenosine-5'-uronamide by larger alkyl groups reduces potency; amides of secondary amines are inactive or have only marginal activity. Analogues of 1 containing a chiral center in the N6 substituent retain the stereoselectivity characteristic of each of the four receptors. Thus, at either A1 or A2 adenosine receptors, adenosine analogues interact with both the N6 and the C-5' receptor regions. However, the effects of N6 and C-5' modifications on potency are less than additive, evidence that the interaction of a substituent with its receptor region influences the interaction of other substituents with their respective receptor regions.     

Potent adenosine receptor antagonists that are selective for the A1 receptor subtype

The xanthines currently represent the most potent class of adenosine receptor antagonists. However, known derivatives of xanthine show little difference in antagonist potency between the two putative adenosine receptor subtypes, A1 and A2. We conducted a systematic study of xanthine structure-activity relationships that compared antagonist potency at the A1 receptor of adipocytes with potency at the A2 receptor of platelets. Since adenosine receptors are coupled to adenylate cyclase in these tissues, inhibition of adenylate cyclase via A1 receptors and stimulation via A2 receptors were used as models of receptor activation. Antagonist potency was quantitated by Schild analysis, which yields an estimate of affinity (Ki) for the drug-receptor interaction. Ki values of a series of xanthine analogues enabled us to identify structural modifications than enhanced antagonist selectivity for one receptor subtype over the other. We found that changes in the substituent at position 8 of the xanthine nucleus influenced antagonist potency at the A1 adenosine receptor more than at the A2 receptor. In particular, an 8-cyclohexyl or 8-cyclopentyl substituent promoted antagonist selectivity for the A1 receptor subtype. Thus, 1,3-dipropyl-8-cyclopentylxanthine had comparatively high affinity (Ki = 0.47 +/- 2 nM) at the A1 receptor, and was roughly 150-fold more potent as an antagonist of the A1- than of the A2-adenosine receptor subtype. In addition, the cycloalkylxanthines were relatively ineffective as inhibitors of cyclic nucleotide phosphodiesterases when used at concentrations that produced marked adenosine receptor antagonism.     

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.     

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.     

7-Deaza-2-phenyladenines: structure-activity relationships of potent A1 selective adenosine receptor antagonists

A series of derivatives of 7-deazapurines with varying substituents in the 2-, 6-, and 9-position was synthesized in an attempt to improve the adenosine receptor affinity and A1 or A2 selectivity. The adenosine receptor affinities were assessed by measuring the inhibition of [3H]-(R)-N6-(phenylisopropyl) adenosine (R-PIA) binding to rat brain A1 and inhibition of [3H]-5'-(N-ethylcarboxamido)adenosine (NECA) binding to rat striatum A2 adenosine receptors. A selected set of compounds representing the main structural variations was further examined in adenosine receptor coupled adenylate cyclase assays. All tested compounds antagonized the inhibition of adenylate cyclase elicited by interaction of R-PIA with A1 receptors in rat fat cell membranes and the activation of adenylate cyclase elicited by interaction of NECA with A2 receptors of pheochromocytoma PC12 cell membranes. The results indicate that 7-deazahypoxanthines have a potential for A2 selectivity, while all 7-deazaadenines are A1 selective. Introduction of a phenyl residue in the 2-position of 7-deazaadenines increases A1 activity tremendously. 2-(p-Chlorophenyl)-7,8-dimethyl-9-phenyl-7-deazaadenine (29) is potent and specific for the A1 receptors of rat brain (Ki = 122 nM), having no affinity for the A2 receptors of rat striatum. The compound has low activity at the A2 receptors of rat PC12 cell membranes where it appears to act as a noncompetitive inhibitor. A 1-phenylethyl substituent at the 9-position was found to be superior to a phenyl residue in terms of A1 affinity. The most potent A1 antagonist in the present series is the highly A1 selective (790-fold) (R)-7,8-dimethyl-2-phenyl-9-(1-phenylethyl)-7-deazaadenine (31, Ki = 4.7 nM), which is 30-35 times more potent at A1 receptors than its S enantiomer. The solubility of six of the potent 7-deaza-2-phenyladenines was determined by means of an A1 binding assay. Chloro substitution of the 2-phenyl ring appeared to improve the solubility as well as the solubility over A1 affinity ratio of 9-phenyl- and 9-(1-phenylethyl)-substituted 7-deazadenines.     

Analogues of caffeine and theophylline: effect of structural alterations on affinity at adenosine receptors

A variety of analogues of caffeine and theophylline in which the 1-,3-, and 7-methyl substituents have been replaced with n-propyl, allyl, propargyl, and isobutyl and, in a few cases, with chloroethyl, hydroxyethyl, or benzyl were assessed for potency and selectivity as antagonists at A1- and A2-adenosine receptors in brain tissue. Caffeine and theophylline are nonselective for these receptors. Nearly all of the 22 analogues of caffeine are more potent than caffeine itself at adenosine receptors. Replacement of the 1-methyl moiety with n-propyl, allyl, or propargyl substituent has little effect on potency at the A1 receptor while enhancing potency about 7- to 10-fold at the A2 receptor. 3,7-Di-methyl-1-propylxanthine is only slightly (1.4-fold) more potent than caffeine at the A1 receptor while being 10-fold more potent at the A2 receptor. 1,3-Di-n-propyl-7-methylxanthine is also selective for the A2 receptor, being 8-fold more potent than caffeine at the A1 receptor and 40-fold more potent at the A2 receptor. A number of other caffeine analogues including 3,7-dimethyl-1-n-propylxanthine, 7-allyl-1,3-dimethylxanthine, and 1,3-dimethyl-7-propargylxanthine are also somewhat selective for the A2 receptor. The most potent caffeine analogue was 1,3-di-n-propyl-7-propargylxanthine, which was about 100-fold more potent than caffeine at both A1 and A2 receptors. The 10 theophylline analogues were relatively nonselective except for the 1-ethyl analogue and the 1,3-diallyl analogue, which were selective for the A2 receptor, and the 1,3-di-n-propyl, 1,3-diisobutyl, and 1,3-dibenzyl analogues, which were somewhat selective for the A1 receptor. 1,3-Di-n-propylxanthine was 20-fold more potent than theophylline at the A1 receptor and 5-fold more potent at the A2 receptor.     

Methanocarba analogues of purine nucleosides as potent and selective adenosine receptor agonists

Adenosine receptor agonists have cardioprotective, cerebroprotective, and antiinflammatory properties. We report that a carbocyclic modification of the ribose moiety incorporating ring constraints is a general approach for the design of A(1) and A(3) receptor agonists having favorable pharmacodynamic properties. While simple carbocyclic substitution of adenosine agonists greatly diminishes potency, methanocarba-adenosine analogues have now defined the role of sugar puckering in stabilizing the active adenosine receptor-bound conformation and thereby have allowed identification of a favored isomer. In such analogues a fused cyclopropane moiety constrains the pseudosugar ring of the nucleoside to either a Northern (N) or Southern (S) conformation, as defined in the pseudorotational cycle. In binding assays at A(1), A(2A), and A(3) receptors, (N)-methanocarba-adenosine was of higher affinity than the (S)-analogue, particularly at the human A(3) receptor (N/S affinity ratio of 150). (N)-Methanocarba analogues of various N(6)-substituted adenosine derivatives, including cyclopentyl and 3-iodobenzyl, in which the parent compounds are potent agonists at either A(1) or A(3) receptors, respectively, were synthesized. The N(6)-cyclopentyl derivatives were A(1) receptor-selective and maintained high efficacy at recombinant human but not rat brain A(1) receptors, as indicated by stimulation of binding of [(35)S]GTP-gamma-S. The (N)-methanocarba-N(6)-(3-iodobenzyl)adenosine and its 2-chloro derivative had K(i) values of 4.1 and 2.2 nM at A(3) receptors, respectively, and were highly selective partial agonists. Partial agonism combined with high functional potency at A(3) receptors (EC(50) < 1 nM) may produce tissue selectivity. In conclusion, as for P2Y(1) receptors, at least three adenosine receptors favor the ribose (N)-conformation.     

2-(Arylalkylamino)adenosin-5'-uronamides: a new class of highly selective adenosine A2 receptor ligands

The synthesis and receptor-binding profiles at adenosine receptor subtypes for a series of 2-(arylalkylamino)-adenosin-5'-uronamides is described. Halogenated 2-phenethylamino analogues such as 3e show greater than 200-fold selectivity for the A2 receptor subtype on the basis of rat brain receptor binding. The general structure-activity relationship of this series of compounds is discussed both in terms of potency at A2 receptors as well as receptor subtype selectivity. It is possible to introduce a hydrophilic carboxyalkyl substituent to this series such as in CGS 21680A (3h) and still retain good potency and selectivity for A2 receptors. In addition, functional data in a perfused working rat heart model shows that these compounds possess full agonist properties at A2 receptors with 3h having a greater than 1500-fold separation between A2 (coronary vasodilatory) and A1 (negative chronotropic) receptor mediated events.     

Structure-activity relationships for N6-substituted adenosines at a brain A1-adenosine receptor with a comparison to an A2-adenosine receptor regulating coronary blood flow

A series of 145 N6-substituted adenosines have been screened as inhibitors of the binding of [3H]cyclohexyladenosine to an A1-adenosine receptor in rat brain membranes and the results compared to the potencies of these analogs in increasing coronary blood flow via activation of an A2-adenosine receptor. The A1 receptor shows greater stereoselectivity in the N6 region of the receptor towards asymmetric aralkyl substituents, and shows greater bulk tolerance in the N6 region of the receptor such that it retains affinity for certain N6-tertiary alkyladenosines and N6-cycloalkyladenosines that are inactive at the coronary A2 receptor. At the A1 receptor, the most potent analogs have either aliphatic N6-substituents with four or more methylene residues or have an N6-halophenyl substituent. At the A2 receptor, the most potent analogs have an N6-phenethyl or similar heteroarylethyl substituent. Certain sets or series of analogs appear useful for identifying the subtypes of adenosine receptors involved in physiological functions.     

Potent and orally bioavailable 8-bicyclo[2.2.2]octylxanthines as adenosine A1 receptor antagonists

In the search for a selective adenosine A1 receptor antagonist with greater aqueous solubility than the compounds currently in clinical trials as diuretics, a series of 1,4-substituted 8-cyclohexyl and 8-bicyclo[2.2.2]octylxanthines were investigated. The binding affinities of a variety of cyclohexyl and bicyclo[2.2.2]octylxanthines for the rat and human adenosine A1, A2A, A2B, and A3 receptors are presented. Bicyclo[2.2.2]octylxanthine 16 exhibited good pharmaceutical properties and in vivo activity in a rat diuresis model (ED50=0.3 mg/kg po). Optimization of the bridgehead substituent led to propionic acid 29 (BG9928), which retained high potency (hA1, Ki=7 nM) and selectivity for the adenosine A1 receptor (915-fold versus adenosine A2A receptor; 12-fold versus adenosine A2B receptor) with improved oral efficacy in the rat diuresis model (ED50=0.01 mg/kg) as well as high oral bioavailability in rat, dog, and cynomolgus monkey.     

1,3,8-trisubstituted xanthines. Effects of substitution pattern upon adenosine receptor A1/A2 affinity

A series of 11 8-substituted xanthines having three different substitution patterns on the 1- and 3-positions [pattern a (R1 = R3 = CH2CH2CH3), b (R1 = CH2CH2CH3, R3 = CH3), and c (R1 = CH3, R3 = CH2CH2CH3)] was prepared. These compounds were assessed for affinity and selectivity in binding to adenosine A1 and A2 receptors. Compounds with greatest affinity at the A1 receptor had the 1,3-substitution pattern a. With one exception, compounds with pattern a also exhibited the most potent binding at the A2 receptor; however, several compounds with pattern c were equipotent at the A2 receptor with those having pattern a. Additionally, the substituents on the 1- and 3-positions of these 8-substituted xanthines were equally important for determining maximum affinity to the A1 receptor, while the substituent at the 3-position is more important than the substituent at the 1-position for potency at the A2 receptor. As a result of this, it is possible to maximize selectivity for the A1 receptor by choice of the 1- and 3-position substituents. However, the R1/R3 substitution pattern required for maximum A1 selectivity is also dependent upon the substituent in the 8-position in a manner which is not fully understood.     

Synthesis and structure-activity relationships of 6-substituted 2',3'-dideoxypurine nucleosides as potential anti-human immunodeficiency virus agents

In order to study the structure-activity relationships of 2',3'-dideoxypurine nucleosides as potential anti-HIV agents, various 6-substituted purine analogues have been synthesized and examined in virus-infected and uninfected human peripheral blood mononuclear cells. N6-methyl-2',3'-dideoxyadenosine (D2MeA, 7a) was initially synthesized from adenosine via 2',3'-O-bisxanthate 3. As extension of this reaction to other N6-substituted compounds failed, a total synthetic method utilizing 2',3'-dideoxyribose derivative 9 was used for the synthesis of other purine nucleosides. An acid-stable derivative of N6-methyl-2',3'-dideoxyadenosine, 2'-fluoroarabinofuranosyl analogue 32 (D2MeFA), has been synthesized from the appropriate carbohydrate 24 by condensation with N6-methyladenine 23. Among these compounds, N6-methyl derivative (D2MeA) 7a proved to be one of the most potent antiviral agents. The order of potency for the 6-substituted compounds was NHMe greater than NH2 greater than Cl approximately N(Me)2 greater than SMe greater than OH approximately NHEt greater than SH greater than NHBn approximately H. The results suggest that a bulk tolerance effect at the 6-position of the 2',3'-dideoxypurine nucleoside may dictate the antiviral activity of these compounds. Acid-stable analogue 32 (D2MeFA) was found to be 20-fold less potent than the parent compound. Both D2MeA and D2MeFA were resistant to calf intestine adenosine deaminase. The presence of a fluorine atom in the carbohydrate moiety greatly increased stability to acid, making D2MeFA a potential orally active antiviral agent that could be useful for the treatment of retroviral infections in humans.     

Synthesis of new 2-phenyladenines and 2-phenylpteridines and biological evaluation as adenosine receptor ligands

Synthesis and biological assays of a series of 2-phenylpteridine derivatives are described to compare their affinities to adenosine receptors with those of the corresponding adenines, purposely prepared, and 8-azaadenines previously described. This study demonstrates that the enlargement of the five-membered ring of the adenine nucleus to a six-membered one is a modification that does not allow the molecules to maintain high activity towards adenosine receptors; in fact, pteridine derivatives did not show themselves to be good adenosine receptor ligands. On the contrary, N(6)-cycloalkyl- or N(6)-alkyl-2-phenyladenines showed a very high affinity and selectivity for A(1) adenosine receptors. We demonstrate also that the 9-benzyl substituent is crucial for conferring high affinity for A(3) receptors to molecules having a 2-phenyladenine-like nucleus.     

Evidence that adenosine receptors in the dog left atrium are not of the typical A1 or A2 adenosine receptor subtypes.

The adenosine receptors from the isolated dog left atrium were characterized using the non-selective agonists 5'-N-ethyl-carboxamidoadenosine (NECA) and adenosine, the A1-selective agonist N6-R-phenylisopropyladenosine (R-PIA), and the A2 adenosine receptor agonist C2-naphthylethoxyadenosine (NEA). The potency order of the agonists in the dog left atrium was NECA greater than adenosine greater than R-PIA = NEA. This potency order was the same as that found in the guinea pig aorta (A2) but different from that in the guinea pig left atrium (A1). In the guinea pig left atrium the potency order was NECA greater than R-PIA greater than adenosine much greater than NEA. The negative inotropic responses to NECA in the dog left atrium were antagonised by the non-selective antagonist 8-phenyltheophylline (8-PT) and the A1-selective antagonists 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and N6-endonorbornan-2-yl-9-methyladenine (N-0861), giving pKB values of 6.3, 7.3 and 5.1, respectively. These values are significantly different from those estimates determined in either the guinea pig left atrium or guinea pig aorta. The potency order of the agonists and the relatively low potencies of the A1-selective antagonists suggests that the adenosine receptors in the dog left atrium are not of the classical A1 adenosine receptor subclass and may instead be more closely related to the A2 adenosine receptor.     

Differential interactions of indolealkylamines with 5-hydroxytryptamine receptor subtypes

Affinities of drugs for 21 indolealkylamine derivatives, some with putative hallucinogenic activity, were determined at 5-HT1A, 5-HT2A and 5-HT2B recognition sites, using radioligand competition studies. Nearly all of the derivatives displayed greatest potency for the 5-HT2A receptor, labelled by [125I]R-(-)DOI in the cortex of the rat. Most derivatives displayed 2-10 times lower affinity at the HT2B receptor labelled by [3H]ketanserin in bovine cortex. Derivatives lacking ring substituents displayed lower affinities for all of the recognition sites, compared to derivatives substituted in the 4- or 5-position of the indole ring. The 4-hydroxylated derivatives displayed 25-380-fold selectivity for the 5-HT2A site, vs the 5-HT1A site, while the 5-substituted derivatives displayed approximately equal potency at the 5-HT1A and 5-HT2A sites. Affinity of all the compounds at the 5-HT2B site was greater than 300 nM. The 6-substituted derivatives displayed greater than micromolar affinities for all of the 5-HT recognition sites examined. The size of the N,N-dialkyl substituent was a secondary determinant of affinity, with groups larger than N,N-diisopropyl resulting in a marked reduction in affinity at both the 5-HT2A and 5-HT1A recognition sites. This study demonstrated that hallucinogenic 4-hydroxy-indolealkylamines, like psychotomimetic phenylisopropylamines, bind potently and selectively to the 5-HT2A recognition site, labelled by [125I]R-(-)DOI. This provides further evidence indicating that this recently described subtype of the 5-HT2 receptor may partially mediate the action of hallucinogenic agents.     

Agonist activity of 2- and 5'-substituted adenosine analogs and their N6-cycloalkyl derivatives at A1- and A2-adenosine receptors coupled to adenylate cyclase.

The activity of N6-cycloalkyl derivatives of adenosine, 2-chloroadenosine, 5'-chloroadenosine and N-ethylcarboximidoadenosine (NECA) and of 2-fluoroadenosine and 5-methylthioadenosines were compared at the A1-adenosine receptor inhibitory to adenylate cyclase in rat fat cell membranes and at the A2A-adenosine receptors stimulatory to adenylate cyclase in rat PC12 cell membranes. The N6-cycloalkyl derivatives in all cases were more potent (4- to 23-fold) than the parent compound at the A1 receptor, and were less potent (1.6- to 11-fold) than the parent compound at the A2A receptor. N6-Cyclopentyl-5'-chloroadenosine was the most selective agonist (900-fold) for the A1 receptor, while 2-fluoroadenosine was the only agonist with some selectivity (4.8-fold) for the A2A receptor. 5'-Methylthioadenosine was a weak agonist at both adenosine receptors. A 2-fluoro derivative of 5'-methylthioadenosine was somewhat more potent. Affinities of these analogs for inhibition of binding of radioligands to rat brain A1 and A2A receptors are presented.     

In vitro antiplatelet profile of FR171113, a novel non-peptide thrombin receptor antagonist

We have examined the potency of several adenosine receptor antagonists at adenosine A₁ and A_2A receptors using quantitative autoradiography and have compared the results with those of previous studies using the same radioligands in membrane preparations. The agonists [³H]cyclohexyladenosine and [³H]2-[p-(2-carbonylethyl)-phenylethylamino]-5′-N-ethylcarboxamido adenosine ([³H]CGS 21680) were used as radioligands for the two receptors. The results show that 1,3-dipropyl-8-cyclopentyl xanthine (DPCPX) is almost 1000-fold and 8-chloro-4-cyclohexyl-amino-1-(trifluoromethyl)[1,2,4]triazolo[4,3-a] quinoxaline (CP-68,247) about 300-fold more potent at adenosine A₁ receptors in cortex and striatum than at striatal adenosine A_2A receptors. Conversely, 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine (SCH 58261) is approximately 1000-fold and 4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-yl amino]ethyl)phenol (ZM 241,385) about 400-fold more potent at adenosine A_2A than at A₁ receptors. Caffeine and its metabolites did not show any selectivity. Other studied antagonists were non-selective or showed a modest (20- to 40-fold) adenosine A_2A receptor selectivity. Thus, only a few of the antagonists show such high selectivity that it is not offset by differences in drug distribution and levels of receptor subtype expression.     

Relative agonist potencies of C2-substituted analogues of adenosine: evidence for adenosine A2B receptors in the guinea pig aorta.

Nine C2-substituted adenosine analogues that are potent and selective for the A2-adenosine receptor were tested for their ability to induce relaxations of the guinea pig aorta. Compounds tested were 2-phenylethoxyadenosine (PEA), 2-phenylethoxy-5'-N-ethylcarboxamidoadenosine (PENECA), 2-cyclohexylethoxyadenosine (CEA), 2-fluorophenylethoxyadenosine (FPEA), 2-methoxyphenylethoxyadenosine (MPEA), 2-naphthylethoxyadenosine (NEA), 2-phenylaminoadenosine (CV-1808), 2-phenylethylaminoadenosine (PEAA) and 2-carboxyethylphenethylamino-5'-N-ethylcarboxamidoadenosine (CGS21680). The responses to these agents were compared to those of three standard adenosine receptor agonists, 5'-N-ethylcarboxamidoadenosine (NECA), N6-cyclohexyladenosine (CHA) and R-N6-phenylisopropyladenosine (R-PIA). The C2-ethoxyadenosine analogues were 30- to 140-fold less potent than NECA and the C2-amino-substituted analogues were 250 to 1000-fold less potent than NECA at inducing relaxations of the guinea pig aorta. All of the analogues were also less potent than the A1-selective agonist R-PIA. However, only responses to NECA were competitively antagonized by the non-selective adenosine receptor antagonist 8-phenyltheophylline (8-PT), pKB = 6.83 +/- 0.05. The results suggest that the C2-substituted analogues produce relaxations of the guinea pig aorta through a combination of actions at A2-adenosine receptors and at xanthine resistant sites. The lack of potency of these analogues at activating the xanthine sensitive A2-receptors in the guinea pig aorta suggests that these adenosine receptors may be of the A2b-subtype.     

Do adenosine A3 receptors exist?

1. It has been suggested that adenosine A1 receptors may be sub-divided into A1 and A3 types, based on the relative potencies of 5'-N-ethylcarboxamidoadenosine (NECA) and selected N6-substituted adenosine analogues. At A1 receptors (rat adipocytes) N6-phenylisopropyladenosine (PIA) was reported to be approx. 100-fold more potent than NECA, whereas the compounds were equipotent at A3 receptors (those in cardiac and neuronal preparations). 2. The study reported here has systematically evaluated this proposal, the rank orders of potency of NECA, R- and S-PIA, N6-cyclopentyladenosine (CPA) and N6-cyclohexyladenosine (CHA) being determined in rat adipocytes, guinea-pig ileum and rat and guinea-pig atria. 3. R-PIA, CHA and CPA were found to have consistent potencies relative to NECA across all 6 tissues, including rat adipocytes. The rank order was CPA greater than or equal to CHA, R-PIA greater than or equal to NECA greater than S-PIA. 4. We conclude that the relative potencies of these agonists do not support the concept that adenosine A1 receptors in rat adipocytes differ from those in neuronal and cardiac tissues.     

The affinity, selectivity and biological activity of telenzepine enantiomers

The binding of the enantiomers of telenzepine to muscarinic receptor subtypes present in guinea-pig cerebral cortex, myocardium and salivary glands has been examined. The (+) enantiomer is more potent in all assays and exhibits a greater selectivity than the (-) enantiomer for the different receptor subtypes. As a consequence, the enantiomeric potency ratio varies from ca. 400 (cortical 'M1' receptors) to ca. 50 (cardiac receptors). In functional assays in vitro in the rabbit vas deferens and rat atria, the affinity constants and enantiomeric potency ratios for the two isomers agree with those found for the appropriate muscarinic receptor subtype in binding assays. A high enantiomeric potency ratio, 180, is found in vivo for the ability of the telenzepine enantiomers to inhibit the production of lesions in the modified Shay rat preparation. The data are compatible with the blockade of M1 receptors by (+)-telenzepine being responsible for this action of telenzepine and would tend to exclude the possibility that the anti-ulcer action of telenzepine is mediated via a muscarinic or non-muscarinic action of the (-) enantiomer.