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1.  Structure–Activity Relationships of 1,3-Dialkylxanthine Derivatives at Rat A3 Adenosine Receptors 
Journal of medicinal chemistry  1994;37(20):3373-3382.
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.
PMCID: PMC3471218  PMID: 7932565
2.  Xanthine Functionalized Congeners as Potent Ligands at A2-Adenosine Receptors†,‡ 
Journal of medicinal chemistry  1987;30(1):211-214.
Amide derivatives of a carboxylic acid congener of 1,3-dialkylxanthine, having a 4-[(carboxymethyl)oxy]phenyl substituent at the 8-position, have been synthesized in order to identify potent antagonists at A2-adenosine receptors stimulatory to adenylate cyclase in platelets. Distal structural features of amide-linked chains and the size of the 1,3-dialkyl groups have been varied. 1,3-Diethyl groups, more than 1,3-dimethyl or 1,3-dipropyl groups, favor A2 potency, even in the presence of extended chains attached at the 8-(p-substituted-phenyl) position. Polar groups, such as amines, on the chain simultaneously enhance water solubility and A2 potency. Among the most potent A2 ligands are an amine congener, 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]-1,3-diethylxanthine, and its D-lysyl conjugate, which have KB values of 21 and 23 nM, respectively, for the antagonism of N-ethyl-adenosine-5′-uronamide-stimulated adenylate cyclase activity in human platelet membranes. Strategies for the selection and tritiation of new radioligands for use in competitive binding assays at A2-adenosine receptors have been considered.
PMCID: PMC3433718  PMID: 3806597
3.  Structure–Activity Relationships of 9-Alkyladenine and Ribose-Modified Adenosine Derivatives at Rat A3 Adenosine Receptors† 
Journal of medicinal chemistry  1995;38(10):1720-1735.
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-(β-d-erythrofuranosyl)-N6-(3-iodobenzyl)adenine had a Ki value at A3 receptors of 0.28 µM. 2-Chloro-9-[2-amino-2,3-dideoxy-β-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.
PMCID: PMC3445626  PMID: 7752196
4.  Trifunctional Agents as a Design Strategy for Tailoring Ligand Properties: Irreversible Inhibitors of A1 Adenosine Receptors† 
Bioconjugate chemistry  1991;2(2):77-88.
The 1,3-phenylene diisothiocyanate conjugate of XAC (8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]-oxy]phenyl]-l,3-dipropylxanthine, a potent A1 selective adenosine antagonist) has been characterized as an irreversible inhibitor of A1 adenosine receptors. To further extend this work, a series of analogues were prepared containing a third substituent in the phenyl isothiocyanate ring, incorporated to modify the physiochemical or spectroscopic properties of the conjugate. Symmetrical trifunctional cross-linking reagents bearing two isothiocyanate groups were prepared as general intermediates for cross-linking functionalized congeners and receptors. Xanthine isothiocyanate derivatives containing hydrophilic, fluorescent, or reactive substituents, linked via an amide, thiourea, or methylene group in the 5-position, were synthesized and found to be irreversible inhibitors of A1 adenosine receptors. The effects of the 5-substituent on water solubility and on the A1/A2 selectivity ratio derived from binding assays in rat brain membranes were examined. Inhibition of binding of [3H]-N6-(2-phenylisopropyl)-adenosine and [3H]CGS21680 (2-[[2-[4-(2-carboxyethyl)phenyl]ethyl]amino]adenosine-5′-N-ethylcarboxamide) at central A1 and A2 adenosine receptors, respectively, was measured. A conjugate of XAC and 1,3,5-triisothiocyanatobenzene was 894-fold selective for A1 receptors. Reporter groups, such as fluorescent dyes and a spin-label, were included as chain substituents in the irreversibly binding analogues, which were designed for spectroscopic assays, histochemical characterization, and biochemical characterization of the receptor protein.
PMCID: PMC3427756  PMID: 1868116
5.  Search for New Purine- and Ribose-Modified Adenosine Analogues as Selective Agonists and Antagonists at Adenosine Receptors† 
Journal of medicinal chemistry  1995;38(7):1174-1188.
The binding affinities at rat A1, A2a, and A3 adenosine receptors of a wide range of derivatives of adenosine have been determined. Sites of modification include the purine moiety (1-, 3-, and 7-deaza; halo, alkyne, and amino substitutions at the 2- and 8-positions; and N6-CH2-ring, -hydrazino, and -hydroxylamino) and the ribose moiety (2′-, 3′-, and 5′-deoxy; 2′- and 3′-O-methyl; 2′-deoxy 2′-fluoro; 6′-thio; 5′-uronamide; carbocyclic; 4′- or 3′-methyl; and inversion of configuration). (−)- and (+)-5′-Noraristeromycin were 48- and 21-fold selective, respectively, for A2a vs A1 receptors. 2-Chloro-6′-thioadenosine displayed a Ki value of 20 nM at A2a receptors (15-fold selective vs A1). 2-Chloroadenin-9-yl(β-L-2′-deoxy-6′-thiolyxofuranoside) displayed a Ki value of 8 μM at A1 receptors and appeared to be an antagonist, on the basis of the absence of a GTP-induced shift in binding vs a radiolabeled antagonist (8-cyclopentyl-1,3-dipropylxanthine). 2-Chloro-2′-deoxyadenosine and 2-chloroadenin-9-yl(β-D-6′-thioarabinoside) were putative partial agonists at A1 receptors, with Ki values of 7.4 and 5.4 μM, respectively. The A2a selective agonist 2-(1-hexynyl)-5′-(N-ethylcarbamoyl)adenosine displayed a Ki value of 26 nM at A3 receptors. The 4′-methyl substitution of adenosine was poorly tolerated, yet when combined with other favorable modifications, potency was restored. Thus, N6-benzyl-4′-methyladenosine-5′-(N-methyluronamide) displayed a Ki value of 604 nM at A3 receptors and was 103- and 88-fold selective vs A1 and A2a receptors, respectively. This compound was a full agonist in the A3-mediated inhibition of adenylate cyclase in transfected CHO cells. The carbocyclic analogue of N6-(3-iodobenzyl)adenosine-5′-(N-methyluronamide) was 2-fold selective for A3 vs A1 receptors and was nearly inactive at A2a receptors.
PMCID: PMC3457658  PMID: 7707320
6.  2-Substituted adenosine derivatives: affinity and efficacy at four subtypes of human adenosine receptors 
Biochemical pharmacology  2004;68(10):1985-1993.
The affinity and efficacy at four subtypes (A1, A2A, A2B and A3) of human adenosine receptors (ARs) of a wide range of 2-substituted adenosine derivatives were evaluated using radioligand binding assays and a cyclic AMP functional assay in intact CHO cells stably expressing these receptors. Similar to previous studies of the N6-position, several 2-substituents were found to be critical structural determinants for the A3AR activation. The following adenosine 2-ethers were moderately potent partial agonists (Ki, nM): benzyl (117), 3-chlorobenzyl (72), 2-(3-chlorophenyl)ethyl (41), and 2-(2-naphthyl)ethyl (130). The following adenosine 2-ethers were A3AR antagonists: 2,2-diphenylethyl, 2-(2-norbornan)ethyl, R- and S-2-phenylbutyl, and 2-(2-chlorophenyl)ethyl. 2-(S-2-Phenylbutyloxy)a-denosine as an A3AR antagonist right-shifted the concentration–response curve for the inhibition by NECA of cyclic AMP accumulation with a KB value of 212 nM, which is similar to its binding affinity (Ki = 175 nM). These 2-substituted adenosine derivatives were generally less potent at the A1AR in comparison to the A3AR, but fully efficacious, with binding Ki values over 100 nM. The 2-phenylethyl moiety resulted in higher A3AR affinity (Ki in nM) when linked to the 2-position of adenosine through an ether group (54), than when linked through an amine (310) or thioether (1960). 2-[2-(l-Naphthyl)ethyloxy]adenosine (Ki = 3.8 nM) was found to be the most potent and selective (>50-fold) A2A agonist in this series. Mixed A2A/A3AR agonists have been identified. Interestingly, although most of these compounds were extremely weak at the A2BAR, 2-[2-(2-naphthyl)ethyloxy]adenosine (EC50 = 1.4 µM) and 2-[2-(2-thienyl)-ethyloxy]adenosine (EC50 = 1.8 (M) were found to be relatively potent A2B agonists, although less potent than NECA (EC50 = 140 nM).
doi:10.1016/j.bcp.2004.06.011
PMCID: PMC3408601  PMID: 15476669
Adenosine receptors; Purines; Nucleosides; GPCR; Efficacy; Structure–activity relationships
7.  Structure–Activity Relationships and Molecular Modeling of 3,5-Diacyl-2,4-dialkylpyridine Derivatives as Selective A3 Adenosine Receptor Antagonists 
Journal of medicinal chemistry  1998;41(17):3186-3201.
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.
doi:10.1021/jm980093j
PMCID: PMC3474377  PMID: 9703464
8.  Functionalized Congeners of 1,3-Dialkylxanthines: Preparation of Analogues with High Affinity for Adenosine Receptors 
Journal of medicinal chemistry  1985;28(9):1334-1340.
A series of functionalized congeners of 1,3-dialkylxanthines has been prepared as adenosine receptor antagonists. On the basis of the high potency of 8-(p-hydroxyphenyl)-1,3-dialkylxanthines, the parent compounds were 8-[4-[(carboxymethyl)oxy]phenyl] derivatives of theophylline and 1,3-dipropylxanthine. A series of analogues including esters of ethanol and N-hydroxysuccinimide, amides, a hydrazide, an acylurea, and anilides were prepared. The potency in blocking A1-adenosine receptors (inhibition of binding of N6-[3H]cyclohexyladenosine to brain membranes) and A2-adenosine receptors (inhibition of 2-chloroadenosine-elicited accumulations of cyclic AMP in brain slices) was markedly affected by structural changes distal to the primary pharmacophore (8-phenyl-1,3-dialkylxanthine). Potencies in the dipropyl series at the A1 receptor ranged from K1 values of 1.2 nM for a congener with a terminal amidoethyleneamine moiety to a K1 value of 58 nM for the parent carboxylic acid to a K1 of 96 nM for the bulky ureido congener. Certain congeners were up to 145-fold more active at A1 receptors than at A2 receptors. Various derivatives of the congeners should be useful as receptor probes and for radioidodination, avidin binding, and preparation of affinity columns.
PMCID: PMC3468300  PMID: 2993622
9.  N6-Substituted adenosine derivatives: selectivity, efficacy, and species differences at A3 adenosine receptors 
Biochemical pharmacology  2003;65(10):1675-1684.
The activation of the human A3 adenosine receptor (AR) by a wide range of N6-substituted adenosine derivatives was studied in intact CHO cells stably expressing this receptor. Selectivity of binding at rat and human ARs was also determined. Among N6-alkyl substitutions, small N6-alkyl groups were associated with selectivity for human A3ARs vs. rat A3ARs, and multiple points of branching were associated with decreased hA3AR efficacy. N6-Cycloalkyl-substituted adenosines were full (≤5 carbons) or partial (≥6 carbons) hA3AR agonists. N6-(endo-Norbornyl)adenosine 13 was the most selective for both rat and human A1ARs. Numerous N6-arylmethyl analogues, including substituted benzyl, tended to be more potent in binding to A1 and A3 vs. A2AARs (with variable degrees of partial to full A3AR agonisms). A chloro substituent decreased the efficacy depending on its position on the benzyl ring. The A3AR affinity and efficacy of N6-arylethyl adenosines depended highly on stereochemistry, steric bulk, and ring constraints. Stereoselectivity of binding was demonstrated for N6-(R-1-phenylethyl)adenosine vs. N6-(S-1-phenylethyl)adenosine, as well as for the N6-(1-phenyl-2-pentyl)adenosine, at the rat, but not human A3AR. Interestingly, DPMA, a potent agonist for the A2AAR (Ki = 4 nM), was demonstrated to be a moderately potent antagonist for the human A3AR (Ki = 106 nM). N6-[(1S,2R)-2-Phenyl-1-cyclopropyl]adenosine 48 was 1100-fold more potent in binding to human (Ki = 0.63 nM) than rat A3ARs. Dual acting A1/A3 agonists (N6-3-chlorobenzyl- 29, N6-(S-1-phenylethyl)- 39, and 2-chloro-N6-(R-phenylisopropyl)adenosine 53) might be useful for cardioprotection.
PMCID: PMC3142561  PMID: 12754103
Purines; Nucleosides; GPCR; Cyclic AMP; Receptor binding; Structure–activity relationships
10.  Partial Agonists for A3 Adenosine Receptors 
Selective agonists for A3 adenosine receptors (ARs) could potentially be therapeutic agents for a variety of disorders, including brain and heart ischemic conditions, while partial agonists may have advantages over full agonists as a result of an increased selectivity of action. A number of structural determinants for A3AR activation have recently been identified, including the N6-benzyl group, methanocarba substitution of ribose, 2-chloro and 2-fluoro substituents, various 2’- and 3’-substitutions and 4’-thio substitution of oxygen. The 2-chloro substitution of CPA and R-PIA led to A3 antagonism (CCPA) and partial agonism (Cl-R-PIA). 2-Chloroadenosine was a full agonist, while 2-fluoroadenosine was a partial agonist. Both 2’- and 3’- substitutions have a pronounced effect on its efficacy, although the effect of 2’-substitution was more dramatic. The 4-thio substitution of oxygen may also diminish efficacy, depending on other substitutions. Both N6-methyl and N6-benzyl groups may contribute to the A3 affinity and selectivity; however, an N6-benzyl group but not an N6-methyl group diminishes A3AR efficacy. N6-benzyl substituted adenosine derivatives have similar potency for human and rat A3ARS while N6-methyl substitution was preferable for the human A3AR. The combination of 2-chloro and N6-benzyl substitutions appeared to reduce efficacy further than either modification alone. The A2AAR agonist DPMA was shown to be an antagonist for the human A3AR. Thus, the efficacy of adenosine derivatives at the A3AR appears to be more sensitive to small structural changes than at other subtypes. Potent and selective partial agonists for the A3AR could be identified by screening known adenosine derivatives and by modifying adenosine and the adenosine derivatives.
PMCID: PMC3425644  PMID: 15078216
11.  Functionalized Congeners of 1,4-Dihydropyridines as Antagonist Molecular Probes for A3 Adenosine Receptors 
Bioconjugate chemistry  1999;10(4):667-677.
4-Phenylethynyl-6-phenyl-1,4-dihydropyridine derivatives are selective antagonists at human A3 adenosine receptors, with Ki values in a radioligand binding assay vs [125I]AB-MECA [N6-(4-amino-3-iodobenzyl)-5′-N-methylcarbamoyl-adenosine] in the submicromolar range. In this study, functionalized congeners of 1,4-dihydropyridines were designed as chemically reactive adenosine A3 antagonists, for the purpose of synthesizing molecular probes for this receptor subtype. Selectivity of the new analogues for cloned human A3 adenosine receptors was determined in radioligand binding in comparison to binding at rat brain A1 and A2A receptors. Benzyl ester groups at the 3- and/or 5-positions and phenyl groups at the 2- and/or 6-positions were introduced as potential sites for chain attachment. Structure–activity analysis at A3 adenosine receptors indicated that 3,5-dibenzyl esters, but not 2,6-diphenyl groups, are tolerated in binding. Ring substitution of the 5-benzyl ester with a 4-fluorosulfonyl group provided enhanced A3 receptor affinity resulting in a Ki value of 2.42 nM; however, a long-chain derivative containing terminal amine functionalization at the 4-position of the 5-benzyl ester showed only moderate affinity. This sulfonyl fluoride derivative appeared to bind irreversibly to the human A3 receptor (1 h incubation at 100 nM resulting in the loss of 56% of the specific radioligand binding sites), while the binding of other potent dihydropyridines and other antagonists was generally reversible. At the 3-position of the dihydropyridine ring, an amine-functionalized chain attached at the 4-position of a benzyl ester provided higher A3 receptor affinity than the corresponding 5-position isomer. This amine congener was also used as an intermediate in the synthesis of a biotin conjugate, which bound to A3 receptors with a Ki value of 0.60 μM.
doi:10.1021/bc9900136
PMCID: PMC3446815  PMID: 10411465
12.  8-SUBSTITUTED XANTHINES AS ANTAGONISTS AT A1- AND A2-ADENOSINE RECEPTORS 
Biochemical pharmacology  1988;37(19):3653-3661.
Two classes of 8-substituted analogs of theophylline (1,3-dialkylxanthines), having 8-cycloalkyl, 8-cycloalkenyl or 8-(para-substituted aryl) groups, were shown to be potent and, in some cases, receptor subtype selective antagonists at A1- and A2-adenosine receptors. New analogs based on a functionalized cogener approach and on classical medicinal chemical approaches were prepared. Affinity at A1-adenosine receptors was evaluated by inhibition of binding of [3H)N6-phenylisopropyladenosine to rat brain membranes. Activity at A2A-adenosine receptors was measured by the reversal of 5′-N-ethylcarboxamidoadenosine (NECA)-stimulated production of cyclic AMP in membranes from rat pheochromocytoma PC12 cells. Cycloalkenyl analogs containing rigid olefinic bonds differed greatly in potency from the saturated analogs. The selectivity of phenylsulfonamide analogs depended on distal structural features. Novel xanthine analogs include diamino-, thiol-, aldehyde, and halogen-substituted derivatives, peptide conjugates of 8-[4-[2-aminoethylaminocarbonylmethyloxy]phenyl]1,3-dipropylxanthine (XAC), and a hydroxyethylamide analog of XAC.
PMCID: PMC3469272  PMID: 3178879
13.  A Functionalized Congener Approach to Adenosine Receptor Antagonists: Amino Acid Conjugates of 1,3-Dipropylxanthine 
Molecular pharmacology  1986;29(2):126-133.
SUMMARY
1,3-Dipropyl-8-phenylxanthine, a synthetic analog of theophylline and a potent antagonist of adenosine at A1 and A2-adenosine receptors, has been attached covalently through a functionalized chain to amino acids and oligopeptides. The xanthine conjugates have been studied as competitive inhibitors of the specific binding of [3H]N6-cyclohexyladenosine to A1-receptors of rat cerebral cortical membranes and for inhibition of cyclic AMP accumulation elicited by 2-chloroadenosine in guinea pig brain slices through A2-receptors. A free amino group on the extended chain generally resulted in high potency at A1-receptors. The potency (in some cases extending into the subnanomolar range) and selectivity for A1-receptors (up to 200-fold) suggest that this approach can yield a versatile class of “functionalized congeners” of adenosine receptor antagonists in which distal modifications of the attached moiety (“carrier”) can serve also to improve pharmacodynamic and pharmacokinetic parameters. The water solubility in many of the more potent analogs has been enhanced by two orders of magnitude over that of simple, uncharged 8-phenyl xanthine derivatives. Analogs in which the carrier contains d-tyrosine have potential for development of iodinated radioligands for adenosine receptors. The functionalized congener approach is potentially applicable to other drugs and for development of prodrugs.
PMCID: PMC3459325  PMID: 3005825
14.  Allosteric Modulation of A3 Adenosine Receptors by a Series of 3-(2-Pyridinyl)isoquinoline Derivatives 
Molecular pharmacology  2001;60(5):1057-1063.
Allosteric modulators of A1 and A2A adenosine receptors have been described; however, for the A3 adenosine receptor, neither an allosteric site nor a compound with allosteric effects has been described. In this study, the allosteric modulation of human A3 adenosine receptors by a series of 3-(2-pyridinyl)isoquinoline derivatives was investigated by examining their effects on the dissociation of the agonist radioligand, [125I]N6-(4-amino-3-iodobenzyl)-5′ -N-methylcarboxamidoadenosine (I-AB-MECA), from the receptor. Several 3-(2-pyridinyl)isoquinoline derivatives, including VUF5455, VUF8502, VUF8504, and VUF8507, slowed the dissociation of the agonist radioligand [125I]I-AB-MECA in a concentration-dependent manner, suggesting an allosteric interaction. These compounds had no effect on the dissociation of the radiolabeled antagonist [3H]PSB-11 from the A3 adenosine receptor, suggesting a selective enhancement of agonist binding. By comparison, compounds of similar structure (VUF8501, VUF8503, VUF8505), the classical adenosine receptor antagonist CGS15943 and the A1 receptor allosteric enhancer PD81723 did not significantly influence the dissociation rate of [125I]I-AB-MECA. The effect of agonist on forskolin-induced cAMP production was significantly enhanced by VUF5455. When the subtype-selectivity of the allosteric enhancement was tested the compounds had no effect on the dissociation of either [3H]N6-[(R)-phenylisopropyl]adenosine from the A1 adenosine receptor or [3H]CGS21680 from the A2A adenosine receptor. Probing of structure-activity relationships suggested that a carbonyl group is essential for allosterism but preferred only for competitive antagonism. The presence of a 7-methyl group decreased the competitive binding affinity without a major loss of the allosteric enhancing activity, suggesting that the structural requirements for allosteric enhancement might be distinct from those for competitive antagonism.
PMCID: PMC3953614  PMID: 11641434
15.  Characterization of the locomotor depression produced by an A2-selective adenosine agonist 
FEBS letters  1990;261(1):67-70.
Adenosine analogs, such as N6-cyclohexyladenosine (CHA) that are selective for A1-adenosine receptors, and analogs, such as 5′-N-ethylcarboxamidoadenosine (NECA) that are active at both A1 and A2 receptors, cause a profound depression of locomotor activity in mice via a central mechanism. The depression is effectively reversed by non-selective adenosine antagonists such as theophylline. We report that 2-[(2-aminoethyl-amino)carbonylethylphenylethylamino]-5′-N-ethylcarboxamidoadenosine (APEC), an amine derivative of the A2-selective agonist, CGS21680, is a potent locomotor depressant in mice. The in vivo pharmacology is consistent with A2-selectivity at a central site of action. Two parameters indicative of locomotor activity, horizontal activity and total distance travelled, were measured using a computerized activity monitor. From dose-response curves it was found that APEC (ED50 16 μg/kg) is more potent than CHA (ED50 60 μg/kg) and less potent than NECA (ED50 2 μg/kg). The locomotor depression by APEC was reversible by theophylline, but not by the A1-selective antagonists 8-cyclopentyltheophylline (CPT) and 8-cyclopentyl-1,3-dipropyl-2-thioxanthine, nor by the peripheral antagonists 8-p-sulfophenyltheophylline (8-PST) and 1,3-dipropyl-8-p-sulfophenylxanthine. The locomotor activity depression elicited by NECA and CHA was reversed by A1-selective antagonists. These results suggest that the effects of APEC are due to stimulation of A2 adenosine receptors in the brain.
PMCID: PMC3469261  PMID: 2307237
Adenosine analog; Locomotor depression; Adenosine receptor
16.  Structural determinants of efficacy at A3 adenosine receptors: modification of the ribose moiety 
Biochemical pharmacology  2004;67(5):893-901.
We have found previously that structural features of adenosine derivatives, particularly at the N6- and 2-positions of adenine, determine the intrinsic efficacy as A3 adenosine receptor (AR) agonists. Here, we have probed this phenomenon with respect to the ribose moiety using a series of ribose-modified adenosine derivatives, examining binding affinity and activation of the human A3 AR expressed in CHO cells. Both 2′- and 3′-hydroxyl groups in the ribose moiety contribute to A3 AR binding and activation, with 2′-OH being more essential. Thus, the 2′-fluoro substitution eliminated both binding and activation, while a 3′-fluoro substitution led to only a partial reduction of potency and efficacy at the A3 AR. A 5′-uronamide group, known to restore full efficacy in other derivatives, failed to fully overcome the diminished efficacy of 3′-fluoro derivatives. The 4′-thio substitution, which generally enhanced A3 AR potency and selectivity, resulted in 5′-CH2OH analogues (10 and 12) which were partial agonists of the A3 AR. Interestingly, the shifting of the N6-(3-iodobenzyl)adenine moiety from the 1′- to 4′-position had a minor influence on A3 AR selectivity, but transformed 15 into a potent antagonist (16) (Ki = 4.3 nM). Compound 16 antagonized human A3 AR agonist-induced inhibition of cyclic AMP with a KB value of 3.0 nM. A novel apio analogue (20) of neplanocin A, was a full A3 AR agonist. The affinities of selected, novel analogues at rat ARs were examined, revealing species differences. In summary, critical structural determinants for human A3 AR activation have been identified, which should prove useful for further understanding the mechanism of receptor activation and development of more potent and selective full agonists, partial agonists and antagonists for A3 ARs.
PMCID: PMC3150582  PMID: 15104242
Nucleosides; A3 adenosine receptor agonist; A3 adenosine receptor antagonist; Adenylyl cyclase; Phospholipase C; Partial agonist
17.  Probing the Binding Site of the A1 Adenosine Receptor Reengineered for Orthogonal Recognition by Tailored Nucleosides 
Biochemistry  2007;46(25):7437-7448.
His272 (7.43) in the seventh transmembrane domain (TM7) of the human A3 adenosine receptor (AR) interacts with the 3′ position of nucleosides, based on selective affinity enhancement at a H272E mutant A3 AR (neoceptor) of 3′-ureido, but not 3′-OH, adenosine analogues. Here, mutation of the analogous H278 of the human A1 AR to Ala, Asp, Glu, or Leu enhanced the affinity of novel 2′- and 3′-ureido adenosine analogues, such as 10 (N6-cyclopentyl-3′-ureido-3′-deoxyadenosine), by >100-fold, while decreasing the affinity or potency of adenosine and other 3′-OH adenosine analogues. His278 mutant receptors produced a similar enhancement regardless of the charge character of the substituted residue, implicating steric rather than electrostatic factors in the gain of function, a hypothesis supported by rhodopsin-based molecular modeling. It was also demonstrated that this interaction was orientationally specific; i.e., mutations at the neighboring Thr277 did not enhance the affinity for a series of 2′- and 3′-ureido nucleosides. Additionally, H-bonding groups placed on substituents at the N6 or 5′ position demonstrated no enhancement in the mutant receptors. These reengineered human A1 ARs revealed orthogonality similar to that of the A3 but not the A2A AR, in which mutation of the corresponding residue, His278, to Asp did not enhance nucleoside affinity. Functionally, the H278D A1 AR was detectable only in a measure of membrane potential and not in calcium mobilization. This neoceptor approach should be useful for the validation of molecular modeling and the dissection of promiscuous GPCR signaling.
doi:10.1021/bi7001828
PMCID: PMC3140710  PMID: 17542617
18.  NON-XANTHINE HETEROCYCLES: ACTIVITY AS ANTAGONISTS OF A1- AND A2-ADENOSINE RECEPTORS 
Biochemical pharmacology  1988;37(4):655-664.
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-ethylcarboxarnidoadenosine with A2-adenosine receptors in human platelet and rat pheochromocytoma 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 β-carboline-3-ethyl carboxylate. The phenylimidazoline clonidine had no activity, whereas a related dihydroxyphenylimidazoline was a weak non-competitive adenosine antagonist.
PMCID: PMC3445624  PMID: 2829919
19.  125I-4-(2-[7-Amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)phenol, a High Affinity Antagonist Radioligand Selective for the A2a Adenosine Receptor 
Molecular pharmacology  1995;48(6):970-974.
SUMMARY
The A2a adenosine receptor (AR) mediates several important physiological effects of adenosine, including vasodilation and inhibition of platelet aggregation. Until recently, no antagonist radioligand of sufficient selectivity or affinity was available. We describe the synthesis and characterization by radioligand binding of 125I-4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}-{1,3,5}triazin-5-yl-amino]ethyl)phenol (125I-ZM241385) in membranes from two cell types that express A2a ARs. Membranes from Chinese hamster ovary (CHO) cells expressing a recombinant canine A2a AR bound 125I-ZM241385 with high affinity, and agonist competition experiments with 2-(p-carboxyethyl)-phenylamino-5′-N-carboxamidoadenosine, 5′-N-ethylcarboxamidoadenosine, and (−)-N6-[(R)-phenylisopropyl]adenosine revealed a potency order characteristic of an A2a AR binding site. Membranes from bovine striatum, which contain a native A2a AR, also bound 125I-ZM241385 with similarly high affinity and also displayed a pharmacological profile for displacement of radioligand binding that was consistent with that of an A2a AR. Also, under conditions in which 125I-ZM241385 bound with high affinity to a recombinant rat A2a AR expressed in CHO cells, no specific binding was detectable in membranes from CHO cells expressing functional rat A1, A2b, or A3 ARs, indicating that over the range of concentrations used in radioligand binding assays, 125I-ZM241385 is a highly selective antagonist radioligand for study of A2a ARs within a given species.
PMCID: PMC3479638  PMID: 8848012
20.  Functionalized Congeners of Adenosine: Preparation of Analogues with High Affinity for A1-Adenosine Receptors 
Journal of medicinal chemistry  1985;28(9):1341-1346.
A series of functionalized congeners of adenosine based on N6-phenyladenosine, a potent A1-adenosine receptor agonist, was synthesized. Derivatives of the various congeners should be useful as receptor and histochemical probes and for the preparation of radioligands and affinity columns or as targeted drugs. N6-[4-(Carboxymethyl)phenyl]adenosine served as the starting point for synthesis of the methyl ester, the methyl amide, the ethyl glycinate, and various substituted anilides. One of the latter, N6-[4-[[[4-(carbomethoxymethyl)anilino]carbonyl]methyl]phenyl]adenosine, served as the starting point for the synthesis of another series of congeners including the methyl amide, the hydrazide, and the aminoethyl amide. The terminal amino function of the last congener was acylated to provide further analogues. The various congeners were potent competitive antagonists of binding of N6-[3H]cyclohexyladenosine to A1-adenosine receptors in rat cerebral cortical membranes. The affinity of the congener for the A1 receptor was highly dependent on the nature of the spacer group and the terminal moiety with Ki values ranging 1–100 nM. A biotinylated analogue had a Ki value of 11 nM. A conjugate derived from the Bolton–Hunter reagent had a Ki value of 4.5 nM. The most potent congener contained a terminal [(aminoethyl)amino]carbonyl function and had a Ki value of less than 1 nM.
PMCID: PMC3469267  PMID: 2993623
21.  A Binding Site Model and Structure-Activity Relationships for the Rat A3 Adenosine Receptor 
Molecular pharmacology  1994;45(6):1101-1111.
SUMMARY
A novel adenosine receptor, the A3 receptor, has recently been cloned. We have systematically investigated the hitherto largely unexplored structure-activity relationships (SARs) for binding at A3 receptors, using 125I-N6-2-(4-aminophenyl)ethyladenosine as a radioligand and membranes from Chinese hamster ovary cells stably transfected with the rat A3-cDNA. As is the case for A1 and A2a, receptors, substitutions at the N6 and 5′ positions of adenosine, the prototypic agonist ligand, may yield fairly potent compounds. However, the highest affinity and A3 selectivity is found for N6,5′-disubstituted compounds, in contrast to A1 and A2a receptors. Thus, N6-benzyladenosine-5′-N-ethylcarboxamide is highly potent (Ki, 6.8 nM) and moderately selective (13- and 14-fold versus A1 and A2a). The N6 region of the A3 receptor also appears to tolerate hydrophilic substitutions, in sharp contrast to the other subtypes. Potencies of N6,5′-disubstituted compounds in inhibition of adenylate cyclase via A3 receptors parallel their high affinity in the binding assay. None of the typical xanthine or nonxanthine (A1/A2) antagonists tested show any appreciable affinity for rat A3 receptors. 1,3-Dialkylxanthines did not antagonize the A3 agonist-induced inhibition of adenylate cyclase. A His residue in helix 6 that is absent in A3 receptors but present in A1/A2 receptors may be causal in this respect. In a molecular model for the rat A3 receptor, this mutation, together with an increased bulkiness of residues surrounding the ligand, make antagonist binding unfavorable when compared with a previously developed A1 receptor model. Second, this A3 receptor model predicted similarities with A1 and A2 receptors in the binding requirements for the ribose moiety and that xanthine-7-ribosides would bind to rat A3 receptors. This hypothesis was supported experimentally by the moderate affinity (Ki 6 μM) of 7-riboside of 1,3-dibutylxanthine, which appears to be a partial agonist at rat A3 receptors. The model presented here, which is consistent with the detailed SAR found in this study, may serve to suggest future chemical modification, site-directed mutagenesis, and SAR studies to further define essential characteristics of the ligand-receptor interaction and to develop even more potent and selective A3 receptor ligands.
PMCID: PMC3479652  PMID: 8022403
22.  Methanocarba Analogues of Purine Nucleosides as Potent and Selective Adenosine Receptor Agonists 
Journal of medicinal chemistry  2000;43(11):2196-2203.
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 A1 and A3 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 A1, A2A, and A3 receptors, (N)-methanocarba-adenosine was of higher affinity than the (S)-analogue, particularly at the human A3 receptor (N/S affinity ratio of 150). (N)-Methanocarba analogues of various N6-substituted adenosine derivatives, including cyclopentyl and 3-iodobenzyl, in which the parent compounds are potent agonists at either A1 or A3 receptors, respectively, were synthesized. The N6-cyclopentyl derivatives were A1 receptor-selective and maintained high efficacy at recombinant human but not rat brain A1 receptors, as indicated by stimulation of binding of [35S]GTP-γ-S. The (N)-methanocarba-N6-(3-iodobenzyl)adenosine and its 2-chloro derivative had Ki values of 4.1 and 2.2 nM at A3 receptors, respectively, and were highly selective partial agonists. Partial agonism combined with high functional potency at A3 receptors (EC50 < 1 nM) may produce tissue selectivity. In conclusion, as for P2Y1 receptors, at least three adenosine receptors favor the ribose (N)-conformation.
PMCID: PMC3471159  PMID: 10841798
23.  Activity of Novel Adenine Nucleotide Derivatives as Agonists and Antagonists at Recombinant Rat P2X Receptors 
Drug development research  2000;49(4):253-259.
Strategy, Management and Health PolicyVenture Capital Enabling TechnologyPreclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, PharmacokineticsClinical Development Phases I-III Regulatory, Quality, ManufacturingPostmarketing Phase IV
The effects of structural modifications of adenine nucleotides previously shown to enhance either agonist (2-thioether groups) or antagonist (additional phosphate moieties at the 3′- or 2′-position) properties at P2Y1 receptors were examined at recombinant rat P2X1, P2X2, P2X3, and P2X4 receptors expressed in Xenopus oocytes. The potency of P2Y1 agonists HT-AMP (2-(hexylthio)adenosine-5′-monophosphate) and PAPET (2-[2-(4-aminophenyl)ethylthio]adenosine-5′-triphosphate) was examined at P2X receptors. Both nucleotides showed a preference for the Group I (α,β-meATP-sensitive, fast-inactivating) P2X sub-units. HT-AMP was 5-fold more potent than ATP at P2X3 receptors and a partial agonist at all except P2X2 receptors, at which it was a full agonist. The efficacy of HT-AMP was as low as 23% at P2X4 receptors. PAPET was a weak partial agonist at rat P2X4 receptors and a nearly full agonist at the other subtypes. At rat P2X3 receptors, PAPET was more potent than any other known agonist (EC50 = 17 ± 3 nM). MRS 2179 (N6-methyl-2′-deoxyadenosine 3′, 5-bisphosphate, a potent P2Y1 receptor antagonist) inhibited ATP-evoked responses at rat P2X1 receptors with an IC50 value of 1.15 ± 0.21 μM. MRS 2179 was a weak antagonist at rat P2X3 receptors, with an IC50 value of 12.9 ± 0.1 μM, and was inactive at rat P2X2 and P2X4 receptors. Thus, MRS 2179 was 11-fold and 130-fold selective for P2Y1 receptors vs. P2X1 and P2X3 receptors, respectively. MRS 2209, the corresponding 3′-deoxy-2′-phosphate isomer, was inactive at rat P2X1 receptors, thus demonstrating its greater selectivity as a P2Y1 receptor antagonist. Various adenine bisphosphates in the family of MRS 2179 containing modifications of either the adenine (P2Y1 antagonists with 2- and 6-substitutions), the phosphate (a 3′,5′-cyclic diphosphate, inactive at P2Y1 receptors), or the ribose moieties (antagonist carbocyclic analogue), were inactive at both rat P2X1 and P2X3 receptors. An anhydrohexitol derivative (MRS 2269) and an acyclic derivative (MRS 2286), proved to be selective antagonists at P2Y1 receptors, since they were inactive as agonist or antagonist at P2X1 and P2X3 receptors.
doi:10.1002/1098-2299(200004)49:4<253::AID-DDR4>3.0.CO;2-1
PMCID: PMC3393598  PMID: 22791931
ion channel; oocytes; purines; ATP derivatives; bisphosphates; deoxyadenosine derivatives
24.  Binary Drugs: Conjugates of Purines and a Peptide That Bind to Both Adenosine and Substance P Receptors 
Journal of medicinal chemistry  1987;30(8):1529-1532.
A “functionalized congener” approach to adenosine receptor antagonists has provided a means to synthesize highly potent peptide conjugates of 1,3-dialkylxanthines. The antagonist XAC, such a functionalized xanthine amine congener, has been attached to a segment derived from the neurotransmitter peptide substance P (SP) to form a binary drug that binds to both receptors with Ki values of 35 nM (central A1-adenosine) and 300 nM (striatal SP). Coupling of the functionalized adenosine agonist N6-[p-(carboxymethyl)phenyl]adenosine to an SP C-terminal peptide also resulted in a binary drug that binds to both receptors. The demonstration that the biochemical properties of two unrelated drugs, both of which act through binding at extracellular receptors, may be combined in the same molecule suggests a novel strategy for drug design. In principle, a combined effect of the two different substances that produce the same final effect (e.g., hypotension by adenosine agonists and by SP analogues) might occur in vivo. Adenosine analogues have analgesic properties, and the binary drug derived from substance P and adenosine agonists or antagonists might provide useful tools for probing interrelationships of SP pathways and sites for the antinociceptive action of adenosine.
PMCID: PMC3413949  PMID: 2441057
25.  Species differences in structure-activity relationships of adenosine agonists and xanthine antagonists at brain A1 adenosine receptors 
FEBS letters  1986;209(1):122-128.
A series of 28 adenosine analogs and 17 xanthines has been assessed as inhibitors of binding of N6-R-[3H]-phenylisopropyladenosine binding to A1 adenosine receptors in membranes from rat, calf, and guinea pig brain. Potencies of N6-alkyl- and N6-cycloalkyladenosines are similar in the different species. However, the presence of an aryl or heteroaryl moiety in the N6 substituent results in marked species differences with certain such analogs being about 30-fold more potent at receptors in calf than in guinea pig brain. Potencies at receptors in rat brain are intermediate. Conversely, 2-chloroadenosine and 5′-N-ethylcarboxamido-adenosine are about 10-fold less potent at receptors in calf brain than in guinea pig brain. Potencies of xanthines, such as theophylline, caffeine and 1,3-dipropylxanthine are similar in the different species. However, the presence of an 8-phenyl or 8-cycloalkyl substituent results in marked species differences. For example, a xanthine amine conjugate of 1,3-dipropyl-8-phenylxanthine is 9-fold more potent at receptors in calf than in rat brain and 110-fold more potent in calf than in guinea pig brain. Such differences indicate that brain A1 adenosine receptors are not identical in recognition sites for either agonists or antagonists in different mammalian species.
PMCID: PMC3469266  PMID: 3803571
Adenosine agonist; Xanthine antagonist; Adenosine receptor; Structure-activity

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