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1.  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.
PMCID: PMC3446815  PMID: 10411465
2.  In search of selective P2 receptor ligands: interaction of dihydropyridine derivatives at recombinant rat P2X2 receptors 
1,4-Dihydropyridines are regarded as privileged structures for drug design, i.e. they tend to bind to a wide variety of receptor sites. We have shown that upon appropriate manipulation of the substituent groups on a 1,4-dihydropyridine template, high affinity and selectivity for the A3 subtype of adenosine receptors (‘P1 receptors’) may be attained. In the present study we have begun to extend this approach to P2 receptors which are activated by ATP and other nucleotides. Nicardipine, a representative dihydropyridine, used otherwise as an L-type calcium channel blocker, was shown to be an antagonist at recombinant rat P2X2 (IC50 = 25 μM) and P2X4 (IC50 ~ 220 μM) receptors expressed in Xenopus oocytes. Thus, this class of compounds represents a suitable lead for enhancement of affinity through chemical synthesis. In an attempt to modify the 1,4-dihydropyridine structure with a predicted P2 receptor recognition moiety, we have replaced one of the ester groups with a negatively charged phosphonate group. Several 4-phenyl-5-phosphonato-1,4-dihydropyridine derivatives, MRS 2154 (2,6-dimethyl), MRS 2155 (6-methyl-2-phenyl), and MRS 2156 (2-methyl-6-phenyl), were synthesized through three component condensation reactions. These derivatives were not pure antagonists of the effects of ATP at P2X2 receptors, rather were either inactive (MRS 2156) or potentiated the effects of ATP in a concentration-dependent manner (MRS 2154 in the 0.3–10 μM range and MRS 2155 at >1 μM). Antagonism of the effects of ATP at P2X2 receptor superimposed on the potentiation was also observed at >10 μM (MRS 2154) or 0.3–1 μM (MRS 2155). Thus, while a conventional dihydropyridine, nicardipine, was found to antagonize rat P2X2 receptors ninefold more potently than P2X4 receptors, the effects of novel, anionic 5-phosphonate analogues at the receptor were more complex.
PMCID: PMC3425633  PMID: 10869714
Ion channels; Oocytes; Purines; Dihydropyridine derivatives; Potentiator
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.  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).
PMCID: PMC3408601  PMID: 15476669
Adenosine receptors; Purines; Nucleosides; GPCR; Efficacy; Structure–activity relationships
5.  Molecular modelling study of 2-phenylethynyladenosine (PEAdo) derivatives as highly selective A3 adenosine receptor ligands 
Purinergic Signalling  2006;2(4):589-594.
A series of 2-phenylethynyladenosine (PEAdo) derivatives substituted in the N6- and 4′position was synthesised and the new derivatives were tested at the four human adenosine receptors stably transfected into Chinese hamster ovary (CHO) cells, using radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assay (A2B). Binding studies showed that the presence of a phenyl ethynyl group in the 2 position of adenosine favoured the interaction with A3 receptors, resulting in compounds endowed with high affinity and selectivity for the A3 subtype. Additional substitution of the N6- and 4′position increases both A3 affinity and selectivity. The results showed that the new compounds have a good affinity for the A3 receptor and in particular, the N6-methoxy-2-phenylethynyl-5′N-methylcarboxamidoadenosine, with a Ki at A3 of 1.9 nM and a selectivity A1/A3 and A2A/A3 of 4,800- and 8,600-fold, respectively. Therefore, it is one of the most potent and selective agonists at the human A3 adenosine receptor subtype reported so far. Furthermore, functional assays of inhibition of 10 μM forskolin-stimulated cAMP production via the adenosine A3 receptor revealed that the new trisubstituted adenosine derivatives behave as full agonist of this receptor subtype. Docking analysis of these compounds was performed at a homology model of the human A3 receptor based on the bovine rhodopsin crystal structure as template, and the results are in accordance with the biological data.
PMCID: PMC2096654  PMID: 18404461
adenosine; adenosine agonists; adenosine receptors; agonists; G-protein-coupled receptors; homology modelling; signal transduction
6.  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
7.  Sulfur-Containing 1,3-Dialkylxanthine Derivatives as Selective Antagonists at A1-Adenosine Receptors 
Journal of medicinal chemistry  1989;32(8):1873-1879.
Sulfur-containing analogues of 8-substituted xanthines were prepared in an effort to increase selectivity or potency as antagonists at adenosine receptors. Either cyclopentyl or various aryl substituents were utilized at the 8-position, because of the association of these groups with high potency at A1-adenosine receptors. Sulfur was incorporated on the purine ring at positions 2 and/or 6, in the 8-position substituent in the form of 2- or 3-thienyl groups, or via thienyl groups separated from an 8-aryl substituent through an amide-containing chain. The feasibility of using the thienyl group as a prosthetic group for selective iodination via its Hg2+ derivative was explored. Receptor selectivity was determined in binding assays using membrane homogenates from rat cortex [[3H]-N6-(phenylisopropyl) adenosine as radioligand] or striatum [[3H]-5′-(N-ethylcarbamoyl)adenosine as radioligand] for A1- and A2-adenosine receptors, respectively. Generally, 2-thio-8-cycloalkylxanthines were at least as A1 selective as the corresponding oxygen analogue. 2-Thio-8-aryl derivatives tended to be more potent at A2 receptors than the oxygen analogue. 8-[4-[(Carboxymethyl)oxy]phenyl]-1,3-dipropyl-2-thioxanthine ethyl ester was >740-fold A1 selective.
PMCID: PMC3479653  PMID: 2754711
8.  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
9.  2- and 8-alkynyl-9-ethyladenines: Synthesis and biological activity at human and rat adenosine receptors 
Purinergic Signalling  2005;1(2):173-181.
The synthesis of a series of 9-ethyladenine derivatives bearing alkynyl chains in 2- or 8-position was undertaken, based on the observation that replacement of the sugar moiety in adenosine derivatives with alkyl groups led to adenosine receptor antagonists. All the synthesized compounds were tested for their affinity at human and rat A1, A2A, and A3 adenosine receptors in binding assays; the activity at the human A2B receptor was determined in adenylyl cyclase experiments. Biological data showed that the 2-alkynyl derivatives possess good affinity and are slightly selective for the human A2A receptor. The same compounds tested on the rat A1 and A2A subtypes showed in general lower affinity for both receptors. On the other hand, the affinity of the 8-alkynyl derivatives at the human A1, A2A, and A2B receptors proved to be lower than that of the corresponding 2-alkynyl derivatives. On the contrary, the affinity of the same compounds for the human A3 receptor was improved, resulting in A3 selectivity. As in the case of the 2-alkynyl-substituted compounds, the 8-alkynyl derivatives showed decreased affinity for rat receptors. However, it is worthwhile to note that the 8-phenylethynyl-9-ethyladenine was the most active compound of the two series (Ki in the nanomolar range) at both the human and rat A3 subtype. Docking experiments of the 2- and 8-phenylethynyl-9-ethyladenines, at a rhodopsin-based homology model, gave a rational explanation of the preference of the human A3 receptor for the 8-substituted compound.
PMCID: PMC2096531  PMID: 18404502
adenosine; adenosine antagonists; adenosine receptors; antagonist; purine derivatives; substituted adenines
10.  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
11.  Glutamate Residues In the Second Extracellular Loop of the Human A2a Adenosine Receptor Are Required for Ligand Recognition 
Molecular pharmacology  1996;49(4):683-691.
The A2a adenosine receptor, a member of the G protein-coupled receptor family, is important in the regulation of dopaminergic pathways of the brain and in platelet and cardiovascular functions. In this study, the role of extracellular loops in ligand binding to the human A2a receptor was explored through site-directed mutagenesis. Four glutamate/aspartate residues (Glu151, Glu161, Glu169, and Asp170) in the second extracellular loop (E2) and a cysteine residue (Cys262) in the third extracellular loop (E3) were individually replaced with alanine and other amino acids. A proline residue (Pro173) in E2 was mutated to arginine, the homologous amino acid in A3 receptors. The binding properties of the resultant mutant receptors were determined in transfected COS-7 cells. The mutant receptors were tagged at their amino terminus with a hemagglutinin epitope, thus allowing their detection in the plasma membrane with immunological techniques. High affinity specific binding of [3H]2-[4-[(2-carboxyethyl)phenyl]ethyl-amino]-5′-N-ethylcarboxamidoadenosine (15 nm) and [3H]8-[4-[[[[(2-aminoethyl)-amino]carbonyl]methyl]oxy]phenyl]-1,3- dipropylxanthine (4 nm), an A2a agonist and antagonist, respectively, was not observed with four of the mutant receptors, E151A, E151Q, E151D, and E169A, although they were well expressed at the cell surface. The E151A and E169A mutant receptors showed nearly full stimulation of adenylyl cyclase at ~103-fold higher concentrations of 2-[4-[(2-carboxyethyl)phenyl]ethyl-amino]-5′-N-ethylcarboxamidoadenosine. The E161A mutant receptor showed an increase in affinity for the nonxanthine adenosine antagonist 9-chloro-2-(furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (6-fold) but not for other ligands. An E169Q mutant gained affinity (5–22-fold) for adenosine derivatives (agonists) substituted at N6 but not at C2 or C5′ positions. Mutant receptors D170K and P173R were similar to wild-type receptors in binding of both agonist and antagonist radioligands. A C262G mutant also resembled the wild-type receptor in radioligand binding, indicating that a potential disulfide bridge with another cysteine residue in proximity is not required for the structural integrity of the receptor. Our data suggest that certain amino acids in the second extracellular loop may be directly or indirectly involved in ligand binding.
PMCID: PMC3425639  PMID: 8609897
12.  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
13.  Adenosine A3 Receptor Agonists Protect HL-60 and U-937 Cells from Apoptosis Induced by A3 Antagonists 
The effects of novel, selective adenosine (ADO) A3 receptor antagonists of diverse structure on cells of the human HL-60 leukemia and U-937 lymphoma cell lines were examined. Both 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine3,5-dicarboxylate (MRS 1191, 0.5µM) and 6-carboxymethyl-5,9-dihydro-9-methyl-2-phenyl-[1,2,4]-triazolo[5,1-a][2,7]naphthyridine (L-249313, 0.5 µM) induced apoptotic cell death and expression of bak protein. Low concentrations of the A3 receptor agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methyluxonamide (Cl-IB-MECA, 10 nM or 1 µM) protected against antagonist-induced cell death. At concentrations ≥ 10 µM, the agonist alone produced apoptosis and bak expression in various cell lines. It is suggested that there exists a tonic low level of A3 receptor activation, possibly induced by release of endogenous adenosine, that results in cell protection.
PMCID: PMC4309999  PMID: 9125172
14.  Agonist Derived Molecular Probes for A2A Adenosine Receptors 
The adenosine agonist 2-(4-(2-carboxyethyl)phenylethylamino)-5′-N-ethylcarboxamidoadenosine (CGS21680) was recently reported to be selective for the A2A adenosine receptor subtype, which mediates its hypotensive action. To investigate structurelactivity relationships at a distal site, CGS21680 was derivatized using a functionalized congener approach. The carboxylic group of CGS21680 has been esterified to form a methyl ester, which was then treated with ethylenediamine to produce an amine congener. The amine congener was an intermediate for acylation reactions, in which the reactive acyl species contained a reported group, or the precursor for such. For radioiodination, derivatives of p-hydroxyphenylpropionic, 2-thiophenylacetic, and p-aminophenylacetic acids were prepared. The latter derivative (PAPA-APEC) was iodinated electrophilically using [125I]iodide resulting in a radioligand which was used for studies of competition of binding to striatal A, adenosine receptors in bovine brain. A biotin conjugate and an aryl sulfonate were at least 350-fold selective for A, receptors. For spectroscopic detection, a derivative of the stable free radical tetramethyl-1-piperidinyloxy (TEMPO) was prepared. For irreversible inhibition of receptors, meta- and para-phenylenediisothiocyanate groups were incorporated in the analogs. We have demonstrated that binding at A2A receptors is relatively insensitive to distal structural changes at the 2-position, and we report high affinity molecular probes for receptor characterization by radioactive, spectroscopic and affinity labelling methodology.
PMCID: PMC3157953  PMID: 2561548
15.  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
16.  Adenosine Receptor Prodrugs: Synthesis and Biological Activity of Derivatives of Potent, A1-Selective Agonists 
5′-Ester derivatives of the potent adenosine agonists N6-[4-[[[[4-[[[(2-acetylaminoethyl)amino] carbonyl] methyl] anilino] carbonyl] methyl] phenyl] adenosine (N-AcADAC; 1) and N6-cyclopentyladenosine (CPA; 2) were prepared as prodrugs. Both alkyl esters or carbonates (designed to enter the brain by virtue of increased lipophilicity) and 1,4-dihydro-1-methyl-3- [(pyridinylcarbonyl)oxy] esters designed to concentrate in the brain by virtue of a redox delivery system were synthesized. In the 5′-blocked form, the adenosine agonists displayed highly diminished affinity for rat brain A1-adenosine receptors in binding assays. The dihydropyridine prodrug 29 was active in an assay of locomotor depression in mice, in which adenosine agonists are highly depressant. The behavior depression was not reversible by peripheral administration of a non-central nervous system active adenosine antagonist. In an assay of the peripheral action of adenosine (i.e., the inhibition of lipolysis in rats), the parent compounds were highly potent and the dihydropyridine prodrug was much less potent.
PMCID: PMC3459066  PMID: 8138909
17.  ‘Reversine’ and its 2-Substituted Adenine Derivatives as Potent and Selective A3 Adenosine Receptor Antagonists 
Journal of medicinal chemistry  2005;48(15):4910-4918.
The dedifferentiation agent ‘reversine’ (2-(4-morpholinoanilino)-N6-cyclohexyladenine 2) was found to be a moderately potent antagonist for the human A3 adenosine receptor (AR) with a Ki value 0.66 μM. This result prompted an exploration of the structure-activity relationship of related derivatives, synthesized via sequential substitution of 6-chloro-2-fluoropurine with selected nucleophiles. Optimization of substituents at these two positions identified 2-phenylamino-N6-(cyclohexyl)adenine 12, 2-phenylamino-N6-(cycloheptyl)adenine 19, and 2-phenylamino-N6-(endo-norbornyl)adenine 21 as potent A3 AR ligands with Ki values of 51, 42 and 37 nM, respectively, with 30 – 200-fold selectivity in comparison to A1 and A2A ARs. The most selective A3 AR antagonist (>200-fold) was 2-phenyloxy-N6-(cyclohexyl)adenine 22. 9-Methylation of 12, but not 19, was well tolerated in A3 AR binding. Extension of the 2-phenylamino group to 2-benzyl- and 2-(2-phenylethylamino) reduced affinity. In the series of 2-phenylamino, 2-phenyloxy, and 2-phenylthio substitutions, the order of affinity at the A3 AR was oxy ≥ amino > thio. Selected derivatives, including reversine (KB value of 466 nM in Schild analysis), competitively antagonized the functional effects of a selective A3 AR agonist, i.e. inhibition of forskolin-stimulated cAMP production in stably transfected Chinese hamster ovary (CHO) cells. These results are in agreement with other studies suggesting the presence of a lipophilic pocket in the AR binding site that is filled by moderately sized cycloalkyl rings at the N6 position of both adenine and adenosine derivatives. Thus, the compound series reported herein comprise an important new series of selective A3 AR antagonists. We were unable to reproduce the dedifferentiation effect of reversine, previously reported, or to demonstrate any connection between A3 AR antagonist effects and dedifferentiation.
PMCID: PMC3474371  PMID: 16033270
18.  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
19.  2-[2-[4-[2-[2-[ 1,3-Dihydro- 1,1-bis (4-hydroxyphenyl)-3-oxo-5-isobenzofuranthioureidyl]ethylaminocarbonyl]ethyl]phenyl] ethylamino]-5′-N-ethylcarboxamidoadenosine (FITC-APEC): A Fluorescent Ligand For A2a-Adenosine Receptors 
Journal of fluorescence  1992;2(4):217-223.
The fluorescein conjugate, FITC-APEC (2-[2-[4-[2-[2-[1,3-dihydro-l,l-bis(4-hydroxyphenyl)-3-oxo-5-isobenzofuranthioureidyl]ethylaminocarbonyl]ethyl]phenyl]ethylamino]-5′-N-ethylcarboxamidoadenosine), is a novel ligand derived from a series of functionalized congeners that act as selective A2a-adenosine receptor agonists. The binding of FITC-APEC to bovine striatal A2a,-adenosine receptors measured by fluorescence techniques was saturable and of a high affinity, with a Bmax, of 2.3 ± 0.3 pmol/mg protein and KD of 57 ± 2 nM. The KD value estimated by fluorescence was consistent with the Ki (11 ± 0.3 nM) obtained by competition studies with [3H]CGS 21680. Additionally, the Bmax, value found by FITC-APEC measurement was in agreement with Bmax, values obtained using radioligand binding. FITC-APEC exhibited rapid and reversible binding to bovine striatum. The potencies of chemically diverse A2a-adenosine receptor ligands estimated by inhibition of FITC-APEC binding were in good agreement with their potencies determined using radioligand binding techniques (r = 0.97, P = 0.0003). FITC-APEC binding was not altered by purine derivatives that do not recognize A2a-adenosine receptors. These findings demonstrate that the novel fluorescent ligand FITC-APEC can be used in the quantitative characterization of ligand binding to A2a-adenosine receptors.
PMCID: PMC3682427  PMID: 23772170
Fluorescence; A2a-adenosine receptors; receptor binding; bovine striatum
20.  Molecular Probes for the A2A Adenosine Receptor Based on a Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine Scaffold 
Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivatives such as SCH 442416 display high affinity and selectivity as antagonists for the human A2A adenosine receptor (AR). We extended ether-linked chain substituents at the p-position of the phenyl group using optimized O-alkylation. The conjugates included an ester, carboxylic acid and amines (for amide condensation), an alkyne (for click chemistry), a fluoropropyl group (for 18F incorporation), and fluorophore reporter groups (e.g. BODIPY conjugate 14, Ki 15 nM). The potent and A2AAR-selective N-aminoethylacetamide 7 and N-[2-(2-aminoethyl)-aminoethyl]acetamide 8 congeners were coupled to polyamidoamine (PAMAM) G3.5 dendrimers, and the multivalent conjugates displayed high A2AAR affinity. Theoretical docking of an AlexaFluor conjugate to the receptor X-ray structure highlighted the key interactions between the heterocyclic core and the binding pocket of the A2AAR as well as the distal anchoring of the fluorophore. In conclusion, we have synthesized a family of high affinity functionalized congeners as pharmacological probes for studying the A2AAR.
PMCID: PMC3081901  PMID: 21185184
SCH 442416; G protein-coupled receptor; fluorescence; dendrimer; radioligand binding
21.  Selective Allosteric Enhancement of Agonist Binding and Function at Human A3 Adenosine Receptors by a Series of Imidazoquinoline Derivatives 
Molecular pharmacology  2002;62(1):81-89.
We have identified a series of 1H-imidazo-[4,5-c]quinolines as selective allosteric enhancers of human A3 adenosine receptors. Several of these compounds potentiated both the potency and maximal efficacy of agonist-induced responses and selectively decreased the dissociation of the agonist N6-(4-amino-3-[125I]iodobenzyl)-5′-N-methylcarboxamidoadenosine from human A3 adenosine receptors. There was no effect on the dissociation of the antagonist [3H]8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2.1-i]purin-5-one (PSB-11) from the A3 receptors, as well as [3H]N6-[(R)-phenylisopropy-l]adenosine from rat brain A1 receptors and [3H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamidoad-enosine from rat striatal A2A receptors, suggesting the selective enhancement of agonist binding at A3 receptors. The analogs were tested as antagonists of competitive binding at human A3 receptors, and Ki values ranging from 120 nM to 101 μM were observed; as for many allosteric modulators of G protein-coupled receptors, an orthosteric effect was also present. The most promising leads from the present set of analogs seem to be the 2-cyclopentyl-1H-imidazo[4,5-c]quinoline derivatives, of which the 4-phenylamino analog DU124183 had the most favorable degree of allosteric modulation versus receptor antagonism. The inhibition of forskolin-stimulated cyclic AMP accumulation in intact cells that express human A3 receptors was employed as a functional index of A3 receptor activation. The enhancer DU124183 caused a marked leftward shift of the concentration-response curve of the A3 receptor agonists in the presence of antagonist and, surprisingly, a potentiation of the maximum agonist efficacy by approximately 30%. Thus, we have identified a novel structural lead for developing allosteric enhancers of A3 adenosine receptors; such enhancers may be useful for treating brain ischemia and other hypoxic conditions.
PMCID: PMC3953617  PMID: 12065758
22.  Role of Adenosine A3 Receptors on CA1 Hippocampal Neurotransmission During Oxygen-Glucose Deprivation Episodes of Different Duration 
Biochemical pharmacology  2007;74(5):768-779.
The role of adenosine A3 receptors in synaptic transmission under severe (7 min) and shorter (2-5 min) ischemic conditions, obtained by oxygen and glucose deprivation (OGD), was investigated in rat hippocampal slices. The effects of selective A3 agonists or antagonists were examined on field excitatory postsynaptic potentials (fEPSPs) extracellularly recorded at the dendritic level of the CA1 pyramidal region. The novel, selective A3 antagonist LJ1251 ((2R,3R,4S)-2-(2-chloro-6-(3-iodobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol, 0.1-10 nM) protected hippocampal slices from irreversible fEPSP depression induced by severe OGD and prevented or delayed the appearance of anoxic depolarization. Similar results were obtained when severe OGD was carried out with a long, receptor-desensitizing exposure to various selective A3 agonists: 5′-N-methylcarboxamidoadenosine derivatives Cl-IB-MECA (N6-(3-iodobenzyl)-2-chloro), VT72 (N6-methoxy-2-phenylethynyl), VT158 (N6-methoxy-2-phenylethynyl), VT160 (N6-methoxy-2-(2-pyridinyl)-ethynyl), and VT163 (N6-methoxy-2-p-acetylphenylethynyl) and AR132 (N6-methyl-2-phenylethynyladenosine).
The selective A3 antagonist MRS1523 (3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate, 100 nM) reduced fEPSP depression evoked by 2-min OGD and induced a faster recovery of fEPSP amplitude after 5-min OGD. Similar results were obtained for 2- or 5-min OGD applied in the presence of each of the A3 agonists tested. Shorter exposure to A3 agonists significantly delayed the recovery of fEPSP amplitude after 5-min OGD.
This indicates that A3 receptors, stimulated by selective A3 agonists, undergo desensitization during OGD. It is inferred that CA1 hippocampal A3 receptors stimulated by adenosine released during brief ischemia (2 and 5 min) might exert A1-like protective effects on neurotransmission. Severe ischemia would transform the A3 receptor-mediated effects from protective to injurious.
PMCID: PMC2000832  PMID: 17626785
purines; G protein-coupled receptors; cerebral ischemia; hippocampal slices; field EPSP; desensitization
23.  Rational Design of Sulfonated A3 Adenosine Receptor-Selective Nucleosides as Pharmacological Tools to Study Chronic Neuropathic Pain 
Journal of medicinal chemistry  2013;56(14):10.1021/jm4007966.
(N)-Methanocarba (bicyclo[3.1.0]hexane)-adenosine derivatives were probed for sites of charged sulfonate substitution, which precludes diffusion across biological membranes, e.g. blood brain barrier. Molecular modeling predicted that sulfonate groups on C2-phenylethynyl substituents would provide high affinity at both mouse (m) and human (h) A3 adenosine receptors (ARs), while a N6-p-sulfo-phenylethyl substituent would determine higher hA3AR vs. mA3AR affinity. These modeling predictions, based on steric fitting of the binding cavity and crucial interactions with key residues, were confirmed by binding/efficacy studies of synthesized sulfonates. N6-3-Chlorobenzyl-2-(3-sulfophenylethynyl) derivative 7 (MRS5841) bound selectively to h/m A3ARs (Ki hA3AR 1.9 nM) as agonist, while corresponding p-sulfo isomer 6 (MRS5701) displayed mixed A1/A3AR agonism. Both nucleosides administered i.p. reduced mouse chronic neuropathic pain that was ascribed to either A3 or A1/A3ARs using A3AR genetic deletion. Thus, rational design methods based on A3AR homology models successfully predicted sites for sulfonate incorporation, for delineating adenosine’s CNS vs. peripheral actions.
PMCID: PMC3858399  PMID: 23789857
Molecular modeling; G protein-coupled receptor; neuropathic pain; purines; radioligand binding; adenosine receptor
24.  Chemistry and Pharmacological Characterization of Novel Nitrogen Analogs of AMOP-H-OH (Sazetidine-A; 6-[5-(Azetidin-2-ylmethoxy)pyridin-3-yl]hex-5-yn-1-ol) as α4β2-Nicotinic Acetylcholine Receptor-Selective Partial Agonists 
Journal of medicinal chemistry  2010;53(19):6973-6985.
In order to advance therapeutic applications of nicotinic ligands, continuing research efforts are being directed toward the identification and characterization of novel nicotinic acetylcholine receptor (nAChR) ligands that are both potent and subtype selective. Herein we report the synthesis and pharmacological evaluation of members of a new series of 3-alkoxy-5-aminopyridine derivatives that display good selectivity for the α4β2-nAChR subtype based on ligand binding and functional evaluations. The most potent ligand in this series, compound 64, showed high radioligand binding affinity and selectivity for rat α4β2-nAChR with a Ki value of 1.2 nM and 4700-fold selectivity for α4β2-over α3β4-nAChR, and ~100-fold selectivity for functional, high-sensitivity, human α4β2-nAChR over α3β4*-nAChR. In the mouse forced swim test, compound 64 exhibited antidepressant-like effects. Structure-activity relationship (SAR) analyses suggest that the introduction of additional substituents to the amino group present on the pyridine ring of the N-demethylated analogue of compound 17 can provide potent α4β2-nAChR-selective ligands for possible use in treatment of neurological and psychiatric disorders including depression.
PMCID: PMC2957884  PMID: 20822184
25.  2-Substitution of N6-Benzyladenosine-5′-uronamides Enhances Selectivity for A3 Adenosine Receptors 
Journal of medicinal chemistry  1994;37(21):3614-3621.
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 β-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.
PMCID: PMC3468333  PMID: 7932588

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