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.
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A3 adenosine receptor antagonists have potential as anti-inflammatory, anti-asthmatic, and anti-ischemic agents. We previously reported the preparation of chemical libraries of 1,4-dihydropyridine (DHP) and pyridine derivatives and identification of members having high affinity at A3 adenosine receptors. These derivatives were synthesized through standard three-component condensation/oxidation reactions, which permitted versatile ring substitution at five positions, i.e., the central ring served as a molecular scaffold for structurally diverse substituents. We extended this template approach from the DHP series to chemically stable pyran derivatives, in which the ring NH is replaced by O and which is similarly derived from a stepwise reaction of three components. Since the orientation of substituent groups may be conformationally similar to the 1,4-DHPs, a direct comparison between the structure activity relationships of key derivatives in binding to adenosine receptors was carried out. Affinity at human A3 receptors expressed in CHO cells was determined vs. binding of [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5′-N-methyl-carbamoyladenosine). There was no potency-enhancing effect, as was observed for DHPs, of 4-styryl, 4-phenylethynyl, or 6-phenyl substitutions. The most potent ligands in this group in binding to human A3 receptors were 6-methyl and 6-phenyl analogs, 3a (MRS 1704) and 4a (MRS 1705), respectively, of 3,5-diethyl 2-methyl-4-phenyl-4H-pyran-3,5-dicarboxylate, which had Ki values of 381 and 583 nM, respectively. These two derivatives were selective for human A3 receptors vs. rat brain A1 receptors by 57-fold and 24-fold, respectively. These derivatives were inactive in binding at rat brain A2A receptors, and at recombinant human A2B receptors displayed Ki values of 17.3 and 23.2 μM, respectively. The selectivity, but not affinity, of the pyran derivatives in binding to the A3 receptor subtype was generally enhanced vs. the corresponding DHP derivatives.
G protein-coupled receptors; structure–activity relationships; template; radioligand binding; purines
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.
Ion channels; Oocytes; Purines; Dihydropyridine derivatives; Potentiator
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.
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).
Adenosine receptors; Purines; Nucleosides; GPCR; Efficacy; Structure–activity relationships
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.
adenosine; adenosine agonists; adenosine receptors; agonists; G-protein-coupled receptors; homology modelling; signal transduction
Adenosine analogues modified at the 5′-position as uronamides and/or as N6-benzyl derivatives were synthesized. These derivatives were examined for affinity in radioligand binding assays at the newly discovered rat brain A3 adenosine receptor and at rat brain A1 and A2a receptors. 5′-Uronamide substituents favored A3 selectivity in the order N-methyl > N-ethyl ∞ unsubstituted carboxamide > N-cyclopropyl. 5′-(N-Methylcarboxamido)-N6-benzyladenosine was 37–56-fold more selective for A3 receptors. Potency at A3 receptors was enhanced upon substitution of the benzyl substituent with nitro and other groups. 5′-N-Methyluronamides and N6-(3-substituted-benzyl)adenosines are optimal for potency and selectivity at A3 receptors. A series of 3-(halobenzyl)-5′-N-ethyluronamide derivatives showed the order of potency at A1 and A2a receptors of I ~ Br > Cl > F. At A3 receptors the 3-F derivative was weaker than the other halo derivatives. 5′-N-Methyl-N6-(3-iodobenzyl)adenosine displayed a Ki value of 1.1 nM at A3 receptors and selectivity versus A1 and A2a receptors of 50-fold. A series of methoxybenzyl derivatives showed that a 4-methoxy group best favored A3 selectivity. A 4-sulfobenzyl derivative was a specific ligand at A3 receptors of moderate potency. An aryl amino derivative was prepared as a probe for radioiodination and receptor cross-linking.
The pharmacological profile of adenosine A1 receptors in human, guinea-pig, rat and mouse brain membranes was characterized in a radioligand binding assay by use of the receptor selective antagonist, [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX).The affinity of [3H]-DPCPX binding sites in rat cortical and hippocampal membranes was similar. Binding site affinity was higher in rat cortical membranes than in membranes prepared from guinea-pig cortex and hippocampus, mouse cortex and human cortex. pKD values (M) were 9.55, 9.44, 8.85, 8.94, 8.67, 9.39 and 8.67, respectively. The binding site density (Bmax) was lower in rat cortical membranes than in guinea-pig or human cortical membranes.The rank order of potency of seven adenosine receptor agonists was identical in each species. With the exception of 5′-N-ethylcarboxamidoadenosine (NECA), agonist affinity was 3.5–26.2 fold higher in rat cortical membranes than in human and guinea-pig brain membranes; affinity in rat and mouse brain membranes was similar. While NECA exhibited 9.3 fold higher affinity in rat compared to human cortical membranes, affinity in other species was comparable. The stable GTP analogue, Gpp(NH)p (100 μM) reduced 2-chloro-N6-cyclopentyladenosine (CCPA) affinity 7–13.9 fold, whereas the affinity of DPCPX was unaffected.The affinity of six xanthine-based adenosine receptor antagonists was 2.2–15.9 fold higher in rat cortical membranes compared with human or guinea-pig membranes. The rank order of potency was species-independent. In contrast, three pyrazolopyridine derivatives, (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-2-piperidine ethanol (FK453), (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-piperidin-2-yl acetic acid (FK352) and 6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-pyridazinebutyric acid (FK838) exhibited similar affinity in human, guinea-pig, rat and mouse brain membranes. pKi values (M) for [3H]-DPCPX binding sites in human cortical membranes were 9.31, 7.52 and 7.92, respectively.Drug affinity for adenosine A2A receptors was determined in a [3H]-2-[4-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine ([3H]-CGS 21680) binding assay in rat striatal membranes. The pyrazolopyridine derivatives, FK453, FK838 and FK352 exhibited pKi values (M) of 5.90, 5.92 and 4.31, respectively, compared with pKi values of 9.31, 8.18 and 7.57 determined in the [3H]-DPCPX binding assay in rat cortical membranes. These novel pyrazolopyridine derivatives therefore represent high affinity, adenosine A1 receptor selective drugs that, in contrast to xanthine based antagonists, exhibit similar affinity for [3H]-DPCPX binding sites in human, rat, mouse and guinea-pig brain membranes.
Adenosine receptors; [3H]-DPCPX; [3H]-CGS 21680; radioligand binding; FK453; brain
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.
Purines; Nucleosides; GPCR; Cyclic AMP; Receptor binding; Structure–activity relationships
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.
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.
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.
adenosine; adenosine antagonists; adenosine receptors; antagonist; purine derivatives; substituted adenines
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.
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.
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.
We synthesized phenyl ring-substituted analogues of N6-(1S,2R)-(2-phenyl-1-cyclopropyl)adenosine, which is highly potent in binding to the human A3AR with a Ki value of 0.63 nM. The effects of these structural changes on affinity at human and rat adenosine receptors and on intrinsic efficacy at the hA3AR were measured. A 3-nitrophenyl analogue was resolved chromatographically into pure diastereomers, which displayed 10-fold stereoselectivity in A3AR binding in favor of the 1S,2R isomer. A molecular model defined a hydrophobic region (Phe168) in the putative A3AR binding site around the phenyl moiety. A heteroaromatic group (3-thienyl) could substitute for the phenyl moiety with retention of high affinity of A3AR binding. Other related N6-substituted adenosine derivatives were included for comparison. Although the N6-(2-phenyl-1-cyclopropyl) derivatives were full A3AR agonists, several other derivatives had greatly reduced efficacy. N6-Cyclopropyladenosine was an A3AR antagonist, and adding either one or two phenyl rings at the 2-position of the cyclopropyl moiety restored efficacy. N6-(2,2-Diphenylethyl)adenosine was an A3AR antagonist, and either adding a bond between the two phenyl rings (N6-9-fluorenylmethyl) or shortening the ethyl moiety (N6-diphenylmethyl) restored efficacy. A QSAR study of the N6 region provided a model that was complementary to the putative A3AR binding site in a rhodopsin-based homology model. Thus, a new series of high-affinity A3AR agonists and related nucleoside antagonists was explored through both empirical and theoretical approaches.
Nucleoside; Agonist; Molecular modeling; GPCR; Purine receptor
Study of P2-purinoceptor subtypes has been difficult due to the lack of potent and selective ligands. With the goal of developing high affinity P2-purinoceptor-selective agonists, we have synthesized a series of analogues of adenine nucleotides modified on the purine ring as chain-extended 2-thioethers or as N6-methyl-substituted compounds. Chemical functionality incorporated in the thioether moiety included cyanoalkyl, nitroaromatic, amino, thiol, cycloalkyl, n-alkyl, and olefinic groups. Apparent affinity of the compounds for P2Y-purinoceptors was established by measurement of P2Y-purinoceptor-promoted phospholipase C activity in turkey erythrocyte membranes and relaxation of carbachol-contracted smooth muscle in three different preparations (guinea pig taenia coil, rabbit aorta, and rabbit mesenteric artery). Activity at P2X-purinoceptors was established by measurement of contraction of rabbit saphenous artery and of the guinea pig vas deferens and urinary bladder. All 11 of the 2-thioethers of ATP stimulated the production of inositol phosphates with K0.5 values of 1.5–770 nM, with an (aminophenyl)ethyl derivative being most potent. Two adenosine diphosphate analogues were equipotent to the corresponding ATP analogues. Adenosine monophosphate analogues were full agonists, although generally 4 orders of magnitude less potent. ATP 2-thioethers displayed pD2 values in the range of 6–8 in smooth muscle assay systems for activity at P2Y-receptors. There was a significant correlation for the 2-thioether compounds between the pK0.5 values for inositol phosphate production and the pD2 values for relaxation mediated via the P2Y-purinoceptors in the guinea pig taenia coli, but not for the vascular P2Y-receptors or for the P2X-receptors. At P2X-receptors, no activity was observed in the rabbit saphenous artery, but variable degrees of activity were observed in the guinea pig vas deferens and bladder depending on distal substituents of the thioether moiety. N6-Methyl-ATP was inactive at P2X-receptors, and approximately equipotent to ATP at taenia coli P2Y-receptors. This suggested that hybrid N6-methyl and 2-thioether ATP derivatives might be potent and selective for certain P2Y-receptors, as was shown for one such derivative, N6-methyl-2-(5-hexenylthio)-ATP.
In three quinolone compounds, the 1,4-dihydropyridine (1,4-DHP) rings adopt flat-boat conformations which are bisected by the plane of the pseudo-axial brominated aryl ring. The cyclohexanone rings adopt an envelope conformation. In all three compounds, intermolecular N—H⋯O hydrogen bonds link the molecules into extended chains. Intermolecular halogen bonding between Br and the ester carbonyl O atom is observed in two of the compounds.
Three quinolone compounds were synthesized and crystallized in an effort to study the structure–activity relationship of these calcium-channel antagonists. In all three quinolones, viz. ethyl 4-(4-bromophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate, (I), ethyl 4-(3-bromophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate, (II), and ethyl 4-(2-bromophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate, (III), all C21H24BrNO3, common structural features such as a flat boat conformation of the 1,4-dihydropyridine (1,4-DHP) ring, an envelope conformation of the fused cyclohexanone ring and a bromophenyl ring at the pseudo-axial position and orthogonal to the 1,4-DHP ring are retained. However, due to the different packing interactions in each compound, halogen bonds are observed in (I) and (III). Compound (III) crystallizes with two molecules in the asymmetric unit. All of the prepared derivatives satisfy the basic structural requirements to possess moderate activity as calcium-channel antagonists.
crystal structure; structure–activity relationships; calcium-channel antagonists; quinolone compounds; halogen bonding; 1,4-dihydropyridine rings; hydrogen bonding; bromine scanning
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.
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.
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.
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.
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.
purines; G protein-coupled receptors; cerebral ischemia; hippocampal slices; field EPSP; desensitization
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.
report the synthesis, characterization, and antiproliferative
activity of 15 iridium(III) half-sandwich complexes of the type [(η5-Cp*)Ir(2-(R′-phenyl)-R-pyridine)Cl] bearing either
an electron-donating (−OH, −CH2OH, −CH3) or electron-withdrawing (−F, −CHO, −NO2) group at various positions on the 2-phenylpyridine (2-PhPy)
chelating ligand giving rise to six sets of structural isomers. The
X-ray crystal structures of [(η5-Cp*)Ir(2-(2′-fluorophenyl)pyridine)Cl]
(1) and [(η5-Cp*)Ir(2-(4′-fluorophenyl)pyridine)Cl]
(2) exhibit the expected “piano-stool”
configuration. DFT calculations showed that substituents caused only
localized effects on the electrostatic potential surface of the chelating
2-PhPy ligand of the complexes. Hydrolysis of all complexes is rapid,
but readily reversed by addition of NaCl. The complexes show preferential
binding to 9-ethylguanine over 9-methyladenine and are active catalysts
for the oxidation of NADH to NAD+. Antiproliferative activity
experiments in A2780 ovarian, MCF-7 breast, A549 lung, and HCT116
colon cancer cell lines showed IC50 values ranging from
1 to 89 μM, with the most potent complex, [(η5-Cp*)Ir(2-(2′-methylphenyl)pyridine)Cl] (13)
(A2780 IC50 = 1.18 μM), being 10× more active
than the parent, [(η5-Cp*)Ir(2-phenylpyridine)Cl],
and 2× more active than [(η5-CpxPh)Ir(2-phenylpyridine)Cl]. Intriguingly, contrasting biological
activities are observed between structural isomers despite exhibiting
similar chemical reactivity. For pairs of structural isomers both
the nature and position of the functional group can affect the hydrophobicity
of the complex. An increase in hydrophobicity resulted in enhanced
cellular-iridium accumulation in A2780 ovarian cells, which generally
gave rise to an increase in potency. The structural isomers [(η5-Cp*)Ir(2-(4′-fluorophenyl)pyridine)Cl] (2) and [(η5-Cp*)Ir(2-phenyl-5-fluoropyridine)Cl]
(4) preferentially localized in the cytosol > membrane
and particulate > nucleus > cytoskeleton. This work highlights
strong dependence of biological behavior on the nature and position
of the substituent on the chelating ligand and shows how this class
of organometallic anticancer complexes can be fine-tuned to increase
their potency without using extended cyclopentadienyl systems.