Synthesis, biological activity, and molecular modeling investigation of new pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives as human A(3) adenosine receptor antagonists

A new series of pyrazolotriazolopyrimidines bearing different substitutions on the phenylcarbamoyl moieties at the N5 position, being highly potent and selective human A(3) adenosine receptor antagonists, is described. The compounds represent an extension and an improvement of our previous work on this class of compounds (J. Med. Chem. 1999, 42, 4473-4478; J. Med. Chem. 2000, 43, 4768-4780). All the synthesized compounds showed A(3) adenosine receptor affinity in the subnanomolar range and high levels of selectivity in radioligand binding assays at the human A(1), A(2A), A(2B), and A(3) adenosine receptors. In particular, the effect of the substitution and its position on the phenyl ring have been studied. From binding data, it is evident that the unsubstituted derivatives on the phenyl ring (e.g., compound 59, hA(3) = 0.16 nM, hA(1)/hA(3) = 3713, hA(2A)/hA(3) = 2381, hA(2B)/hA(3) = 1388) showed the best profile in terms of affinity and selectivity at the human A(3) adenosine receptors. The introduction of a sulfonic acid moiety at the para position on the phenyl ring was attempted in order to design water soluble derivatives. However, this substitution led to a dramatic decrease of affinity at all four adenosine receptor subtypes. A computer-generated model of the human A(3) receptor was built and analyzed to better interpret these results, demonstrating that steric control, in particular at the para position on the phenyl ring, plays a fundamental role in the receptor interaction. Some of the synthesized compounds proved to be full antagonists in a specific functional model, where the inhibition of cAMP-generation by IB-MECA was measured in membranes of CHO cells stably transfected with the human A(3) receptor with IC(50) values in the nanomolar range, with a statistically significative linear relationship with the binding data.     

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives as highly potent and selective human A(3) adenosine receptor antagonists: influence of the chain at the N(8) pyrazole nitrogen

An enlarged series of pyrazolotriazolopyrimidines previously reported, in preliminary form (Baraldi et al. J. Med. Chem. 1999, 42, 4473-4478), as highly potent and selective human A(3) adenosine receptor antagonists is described. The synthesized compounds showed A(3) adenosine receptor affinity in the sub-nanomolar range and high levels of selectivity evaluated in radioligand binding assays at human A(1), A(2A), A(2B), and A(3) adenosine receptors. In particular, the effect of the chain at the N(8) pyrazole nitrogen was analyzed. This study allowed us to identify the derivative with the methyl group at the N(8) pyrazole combined with the 4-methoxyphenylcarbamoyl moiety at the N(5) position as the compound with the best binding profile in terms of both affinity and selectivity (hA(3) = 0.2 nM, hA(1)/hA(3) = 5485, hA(2A)/hA(3) = 6950, hA(2B)/hA(3) = 1305). All the compounds proved to be full antagonists in a specific functional model where the inhibition of cAMP generation by IB-MECA was measured in membranes of CHO cells stably transfected with the human A(3) receptor. The new compounds are among the most potent and selective A(3) antagonists so far described. The derivatives with higher affinity at human A(3) adenosine receptors proved to be antagonists, in the cAMP assay, capable of inhibiting the effect of IB-MECA with IC(50) values in the nanomolar range, with a trend strictly similar to that observed in the binding assay. Also a molecular modeling study was carried out, with the aim to identify possible pharmacophore maps. In fact, a sterically controlled structure-activity relationship was found for the N(8) pyrazole substituted derivatives, showing a correlation between the calculated molecular volume of pyrazolo[4,3-e]1,2, 4-triazolo[1,5-c]pyrimidine derivatives and their experimental K(i) values.     

Synthesis and 3D QSAR of new pyrazolo[3,4-b]pyridines: potent and selective inhibitors of A1 adenosine receptors

A number of 4-aminopyrazolo[3,4-b]pyridines 5-carboxylic acid esters (2-8) were synthesized and evaluated for their binding affinity at the A1, A2A, and A3 adenosine receptors (AR), in bovine cortical membranes, as well as for their affinity toward human A1AR (hA1AR). Some of the new compounds were characterized by a high affinity and selectivity toward the A1 receptor subtype, showing a significant improvement in comparison with other pyrazolo-pyridines previously reported in the literature. In particular the methyl ester 2h as well as the isopropyl ester 5h, both of them bearing a p-methoxyphenylethylamino side chain at the position 4, presented Ki values of 6 and 7 nM, respectively. To rationalize the relationships between structure and affinity of the novel compounds, a 3D QSAR model was also generated starting from compounds belonging to different classes of known A1AR antagonists.     

N6-methoxy-2-alkynyladenosine derivatives as highly potent and selective ligands at the human A3 adenosine receptor

A new series of N6-methoxy-2-(ar)alkynyladenosine derivatives has been synthesized and tested at the human recombinant adenosine receptors. Binding studies demonstrated that the new compounds possess high affinity and selectivity for the A3 subtype. Among them, compounds bearing an N-methylcarboxamido substituent in the 4'-position showed the highest A3 affinity and selectivity. In particular, the N6-methoxy-2-p-acetylphenylethynylMECA (40; Ki A3 = 2.5 nM, A3 selectivity versus A1 = 21 500 and A2A = 4200) results in one of the most potent and selective agonists at the human A3 adenosine receptor reported so far. Furthermore, functional assay, performed with selected new compounds, revealed that the presence of an alkylcarboxamido group in the 4'-position seems to be essential to obtain full agonists at the A3 subtype. Finally, results of molecular docking analysis were in agreement with binding and functional data and could explain the high affinity and potency of the new compounds.     

Design,synthesis, and biological evaluation of C9- and C2-substituted pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines as new A2A and A3 adenosine receptors antagonists

In the past few years, our group has been involved in the development of A(2A) and A(3) adenosine receptor antagonists which led to the synthesis of SCH58261 (5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine, 61), potent and very selective at the A(2A) receptor subtype, and N(8)-substituted-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines-N(5)-urea or amide (MRE series, b), very selective at the human A(3) adenosine receptor subtype. We now describe a large series of C(9)- and C(2)-substituted pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines to represent an extension of structure-activity relationship work on this class of tricyclic compounds. The introduction of a substituent at 9 position of the tricyclic antagonistic structure led to retention of receptor affinity but a loss of selectivity in respect to the lead compounds b, N(8)-substituted-pirazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines-N(5)-urea or -amide. The substitution of the furanyl moiety of compound 61, necessary for receptor binding, with a phenyl or a substituted aromatic ring (compounds 5a-d, 6-8), caused a complete loss of the affinity at all the adenosine receptor subtypes, demonstrating that the furanyl ring is a necessary structural element to guarantee interaction with the adenosine receptor surface. The introduction of an ethoxy group at the ortho position of the aromatic ring to mimic the oxygen of the furan (compound 5c, 5-amino-7-(2-phenylethyl)-2-(2-ethoxyphenyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) did not enhance affinity. The introduction of the cycloaminomethyl function by Mannich reaction at the 5' position of the furanyl ring of 61 and the C(9)-substituted compound 41 (5-amino-8-methyl-9-methylsulfanyl-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) resulted in complete water solubility but a loss of receptor affinity. We can conclude that modifications or substitutions at the furanyl ring are not allowed and the introduction of a substituent at the 9-position of the core pyrazolo-triazolo-pyrimidine structure caused a severe loss of selectivity, probably due to an increased steric hindrance of the radical introduced.     

Synthesis, CoMFA analysis, and receptor docking of 3,5-diacyl-2, 4-dialkylpyridine derivatives as selective A3 adenosine receptor antagonists

3,5-Diacyl-2,4-dialkyl-6-phenylpyridine derivatives have been found to be selective antagonists at both human and rat A3 adenosine receptors (Li et al. J. Med. Chem. 1998, 41, 3186-3201). In the present study, ring-constrained, fluoro, hydroxy, and other derivatives in this series have been synthesized and tested for affinity at adenosine receptors in radioligand binding assays. Ki values at recombinant human and rat A3 adenosine receptors were determined using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyladenosine). Selectivity for A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors, and structure-activity relationships at various positions of the pyridine ring (the 3- and 5-acyl substituents and the 2- and 4-alkyl substituents) were probed. At the 5-position inclusion of a beta-fluoroethyl (7) or a gamma-fluoropropyl ester (26) was favorable for human A3 receptor affinity, resulting in Ki values of 4.2 and 9.7 nM, respectively, while the pentafluoropropyl analogue was clearly less potent at human A3 receptors. At the 2-, 3-, and 4-positions, fluoro or hydroxy substitution failed to enhance potency and selectivity at human A3 receptors. Several analogues were nearly equipotent at rat and human A3 receptors. To further define the pharmacophore conformationally, a lactam, a lactone, and thiolactones were tested in adenosine receptor binding. The most potent analogue in this group was compound 34, in which a thiolactone was formed between 3- and 4-positions and which had a Ki value of 248 nM at human A3 receptors. Using affinity data and a general pharmacophore model for A3 adenosine receptor antagonists recently proposed, we applied comparative molecular field analysis (CoMFA) to obtain a three-dimensional quantitative structure-activity relationship for pyridine derivatives, having good predictability (r2pred = 0.873) for compounds in the test set. A rhodopsin-based model of the human A3 receptor was built, and the pyridine reference ligand 2,3,4, 5-tetraethyl-6-phenyl-pyridine-3-thiocarboxylate-5-carboxylate (MRS 1476) was docked in the putative ligand binding site. Interactions between receptor transmembrane domains and the steric and the electrostatic contour plots obtained from the CoMFA analysis were analyzed.     

Characterization of the A2 adenosine receptor labeled by [3H]NECA in rat striatal membranes

[3H]NECA (1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuronamide) is known to bind to both the A1 and A2 subtypes of adenosine receptor in rat striatal membranes. In order to study the putative A2 component of [3H]NECA binding, we examined several compounds for the ability to selectively eliminate the A1 component of binding; N6-cyclopentyladenosine was found to give the most satisfactory results. Binding of [3H]NECA in the presence of 50 nM N6-cyclopentyladenosine was characterized. The rank order of potency for inhibition of [3H]NECA binding was NECA much greater than 2-chloroadenosine greater than N6-[(R)-1-methyl-2-phenyl-ethyl]adenosine (R-PIA) greater than N6-cyclohexyladenosine greater than S-PIA, indicating that binding was to an A2 adenosine receptor. When affinities of compounds in [3H]NECA binding to A2 receptors were compared to their affinities in [3H]N6-cyclohexyladenosine binding to A1 receptors, N6-cyclopentyladenosine was the most A1-sensitive agonist (A1 Ki, 0.59 nM; A2 Ki, 460 nM; Ki ratio, 780), whereas the selective coronary vasodilator 2-(phenylamino)adenosine was the most A2-selective agonist (A1, 560 nM; A2, 120 nM; ratio, 0.21). The antagonist 8-cyclopentyltheophylline had considerable A1 selectivity (A1, 11 nM; A2, 1400 nM; ratio, 130), whereas alloxazine had slight A2 selectivity (A1, 5200 nM; A2, 2700; ratio, 0.52). [3H]NECA binding to A2 receptors was highest in striatum but was detectable at much lower levels in each of seven other brain areas. The regional distribution of [3H]NECA binding and the affinities of adenosine agonists and antagonists for inhibition of binding indicate that the site labeled by [3H]NECA belongs to the high affinity, or A2a, subclass of A2 receptor.     

Synthesis, biological and modeling studies of 1,3-di-n-propyl-2,4-dioxo-6-methyl-8-(substituted) 1,2,3,4-tetrahydro [1,2,4]-triazolo [3,4-f]-purines as adenosine receptor antagonists

A new series of potential adenosine receptor antagonists with a [1,2,4]-triazolo-[3,4-f]-purine structure bearing at the 1 and 3 position n-propyl groups have been synthesized, and their affinities at the four human adenosine receptor subtypes (A(1), A(2A), A(2B) and A(3)) have been evaluated. In this case the presence of n-propyl groups seems to induce potency at the A(2A) and A(3) adenosine receptor subtypes as opposed to our previously reported series bearing methyl substituents at the 1 and 3 positions. In particular the non-acylated derivative 17 showed affinity at these two receptor subtypes in the micromolar range. Indeed, preliminary molecular modeling investigations according to the experimental binding data indicate a modest steric and electrostatic antagonist-receptor complementarity.     

(3)H]MRE 3008F20: a novel antagonist radioligand for the pharmacological and biochemical characterization of human A(3) adenosine receptors

The lack of a radiolabeled selective A(3) adenosine receptor antagonist is a major drawback for an adequate characterization of this receptor subtype. This paper describes the pharmacological and biochemical characterization of the tritiated form of a new potent A(3) adenosine receptor antagonist, the pyrazolo triazolo pyrimidine derivative [(3)H]5N-(4-methoxyphenylcarbamoyl)amino-8-propyl-2-(2-furyl )pyrazolo [4,3-e] -1,2,4- triazolo[1,5-c]pyrimidine ([(3)H]MRE 3008F20). [(3)H]MRE 3008F20 bound specifically to the human adenosine A(3) receptor expressed in CHO cells (hA(3)CHO), and saturation analysis revealed a single high affinity binding site, K(D) = 0.80 +/- 0.06 nM, with a B(max) = 300 +/- 33 fmol/mg protein. This new ligand displayed high selectivity (1294-, 165-, and 2471-fold) in binding assay to human A(3) versus A(1), A(2A), and A(2B) receptors, respectively, and binds to the rat A(3) receptors with a K(i) > 10 microM. The pharmacological profile of [(3)H]MRE 3008F20 binding to hA(3)CHO cells was evaluated using known adenosine receptor agonists and antagonists with a rank order of potency consistent with that typically found for interactions with the A(3) adenosine receptors. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency identical with that observed in binding experiments. Thermodynamic data indicated that [(3)H]MRE 3008F20 binding to hA(3)CHO is entropy- and enthalpy-driven in agreement with the typical behavior of other adenosine antagonists to A(1) and A(2A) receptors. These results show that [(3)H]MRE 3008F20 is the first antagonist radioligand with high affinity and selectivity for the human A(3) adenosine receptor and may be used to investigate the physiopathological role of A(3) adenosine receptors.     

Pharmacological and biochemical characterization of purified A2a adenosine receptors in human platelet membranes by [3H]-CGS 21680 binding.

1. The binding properties of human platelet A2a adenosine receptors, assayed with the A2a-selective agonist, [3H]-2-[p-(2-carboxyethyl)-phenethylamino]-5''-N-ethylcarboxamidoad enosine ([3H]-CGS 21680), are masked by a non-receptorial component, the adenotin site. In order to separate A2a receptors from adenotin sites, human platelet membranes were solubilized with 1% 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulphonate (CHAPS). The soluble platelet extract was precipitated with polyethylene glycol (PEG) and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. 2. The present paper describes the binding characteristics of the selective A2a agonist, [3H]-CGS 21680, to this purified platelet membrane preparation. In addition, receptor affinity and potency of several adenosine agonists and antagonists were determined in binding and adenylyl cyclase studies. 3. Saturation experiments revealed a single class of binding site with Kd and Bmax values of 285 nM and 2.07 pmol mg-1 of protein respectively. Adenosine receptor ligands competed for the binding of 50 nM [3H]-CGS 21680 to purified protein, showing a rank order of potency consistent with that typically found for interactions with the A2a adenosine receptors. In the adenylyl cyclase assay the compounds examined exhibited a rank order of potency very close to that observed in binding experiments. 4. Thermodynamic data indicated that [3H]-CGS 21680 binding to the purified receptor is totally entropy-driven in agreement with results obtained in rat striatal A2a adenosine receptors. 5. It is concluded that in the purified platelet membranes there is a CGS 21680 binding site showing the characteristic properties of the A2a receptor. This makes it possible to use this compound for reliable radioligand binding studies on the A2a adenosine receptor of human platelets.     

Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives as adenosine receptor antagonists. Influence of the N5 substituent on the affinity at the human A 3 and A 2B adenosine receptor subtypes: a molecular modeling investigation

A new series of pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines bearing various substituents at both the N5-pyrimidinyl and N8-pyrazolyl positions have been synthesized, and their binding affinities at the four human adenosine receptor subtypes (hA(1), hA(2A), hA(2B), and hA(3)) have been evaluated. All the described compounds contain arylacetyl moieties at the N5 position and arylalkyl substituents at the N8 position. Surprisingly, all the compounds present their most potent affinities at the hA(2B) adenosine receptor with a range of selectivities against the other subtypes. When bulky groups are present simultaneously at the N5 and N8 positions (e.g., compound 9), the best selectivity for the hA(2B) receptor was observed (K(i)(hA(1)) = 1100 nM; K(i)(hA(2A)) = 800 nM; K(i)(hA(2B)) = 20 nM; K(i)(hA(3)) = 300 nM, K(i)(hA(1)/A(2B)) = 55, K(i)(hA(2A)/A(2B)) = 40, K(i)(hA(3)/hA(2B)) = 15). To understand the molecular significance of these results, we compared the putative TM (transmembrane) binding motif of compound 9 on both hA(2B) and hA(3) receptors. From our docking studies, compound 9 fits neatly inside the TM region of the hA(2B) receptor but not in the corresponding hA(3) region, illustrating significant differences between the two subtypes. The study herein presented permits an understanding of why the bioisosteric replacement of an -NH, present in previously reported hA(3) receptor antagonists, with a -CH(2) group at the N5 position induces such large differences in hA(2B)/hA(3) affinity. In the molecular structure of the hA(3) receptor, two residues, Ser243 (TM6) and Ser271 (TM7), create a hydrophilic region, which seems to permit a better accommodation of the phenylurea series into this putative hA(3) binding site than the phenylacetyl series.     

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

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

Synthesis and adenosine receptor affinity of a series of pyrazolo[3,4-d]pyrimidine analogues of 1-methylisoguanosine

Pyrazolo[3,4-d]pyrimidines are pyrazolo analogues of purines. They have been shown to be a general class of compounds which exhibit A1 adenosine receptor affinity. Two series of pyrazolo[3,4-d]pyrimidine analogues of 1-methylisoguanosine have been synthesized. The first involved substitution of the N1-position while the second involved substitution of the N5-position. Both alkyl and aryl substituents were examined. All compounds were tested for A1 adenosine receptor affinity by using a (R)-[3H]-N6-(phenylisopropyl)adenosine binding assay. The 3-chlorophenyl group showed the greatest activity in the N1-position and the butyl group produced the greatest activity in the N5-position. Combination of the best substituent in each of these positions enhanced the overall activity. The most potent compound was 4-amino-5-N-butyl-1-(3-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-6(5H)- one with an IC50 of 6.4 x 10(-6) M. Selectivity at the receptor subclasses was examined by performing an A2 adenosine receptor affinity assay with [3H]CGS 21680. This series of compounds were slightly less potent at A2 receptors. 4-Amino-5-N-butyl-1-(3-chlorophenyl-1H-pyrazolo[3,4-d]pyrimidin-6(5H)-one was the most potent compound with an IC50 of 19.2 x 10(-6) M.     

N6,5'-Disubstituted adenosine derivatives as partial agonists for the human adenosine A3 receptor

5'-(Alkylthio)-substituted analogues of N6-benzyl- and N6-(3-iodobenzyl)adenosine were synthesized in 37-61% overall yields. The affinities of these compounds for the adenosine A1, A2A, and A3 receptors were determined using rat brain cortex, rat brain striata, and stably transfected human A3 receptors in HEK 293 cells, respectively. The compounds proved to be selective for the adenosine A3 receptor and displayed affinities in the nanomolar range. Compounds 8, 10, and 11 had the highest affinities for the A3 receptor with Ki values ranging from 8.8 to 27.7 nM. In the N6-benzyl series, compound 4 (LUF 5403), with a 5'-methylthio group, maintained a reasonable affinity and had the highest selectivity for the A3 receptor. Compound 12 (LUF 5411), with an N6-(3-iodobenzyl) group and a 5'-(n-propylthio) substituent, had the highest A3 selectivity of all of the compounds and also displayed high affinity for this receptor (Ki = 44.3 nM). The compounds were also evaluated for their ability to stimulate [35S]GTPgamma[S] binding in cell membranes expressing the human adenosine A3 receptor. It appeared that the N6,5'-disubstituted adenosine derivatives behaved as partial agonists. Compounds 2, 4, 8, and 10 had the highest intrinsic activities. Additionally, when tested in a cAMP assay, these compounds also behaved as partial agonists.     

2-Substituted N-ethylcarboxamidoadenosine derivatives as high-affinity agonists at human A3 adenosine receptors

A number of 2-substituted 5'-N-ethylcarboxamidoadenosine (NECA) derivatives was investigated for their affinity and selectivity at human A3 adenosine receptors. The compounds were tested in radioligand competition studies and modulation of adenylyl cyclase activity on membranes from CHO cell lines stably transfected with the four human adenosine receptor subtypes. In binding studies the most potent compound, 2-(3-hydroxy-3-phenyl)propyn-1-yl-NECA (PHPNECA), exhibited a subnanomolar affinity for A3 adenosine receptors with a Ki value of 0.4 nM. As opposed to the limited A3 selectivity of PHPNECA, a 100-fold selectivity compared to both A1 and A2A receptors was found for 2-(2-phenyl)ethynyl-NECA (PENECA; Ki 6 nM). The EC50 values for activation of adenylyl cyclase via A2A adenosine receptors were in good agreement with the respective Ki values from binding experiments. In contrast, IC50 values for A1 and A3 receptor-mediated inhibition of adenylyl cyclase were shifted to higher values compared to the respective affinities determined in radioligand competition studies. Similar discrepancies between binding and functional data have been observed for the inhibitory A1 adenosine receptor in previous studies. Therefore, the same A3 selectivity of PENECA compared to A1 receptors was found in binding and adenylyl cyclase inhibition whereas the selectivity compared to A2A receptors that was detected in ligand binding was obscured in the functional assay. The series of compounds presented in this study identifies 2-substitution of the purine system as a promising target for the development of A3-selective high-affinity ligands.     

7-Substituted 5-amino-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines as A2A adenosine receptor antagonists: a study on the importance of modifications at the side chain on the activity and solubility.

It was demonstrated in the early 1990s that adenosine exerts many physiological functions through the interaction with four different receptors, named A1, A2A, A2B, and A3. In the past few years, our group has been involved in the development of A2A antagonists, which led to the synthesis of SCH 58261 (1), the first potent and selective adenosine A2A antagonist, which has been widely used as a reference compound. In this paper, we present an extended series of pyrazolotriazolopyrimidines synthesized with the aim to investigate the influence of the substitutions on the pyrazole ring. The choice of the substituents was based on their capability to improve water solubility while retaining high affinity and selectivity at the human A2A adenosine receptor subtype. In this series, some structural characteristics that are important for activity, i.e., tricyclic structure, free amino group at 5-position, furan ring, and substituent at 7-position on the pyrazole moiety, have been maintained. We focused our attention on the nature of the phenyl ring substituent to improve water solubility. Following this strategy, we developed new compounds with good affinity and selectivity for A2A adenosine receptors, such as 8d (K(i) 0.12 nM; hA1/hA2A ratio = 1025; R(m) = 2.8), 8h (K(i) 0.22; hA1/hA2A ratio = 9818; R(m) = 3.4), 8i (K(i) 0.18 nM; hA1/hA2A ratio = 994; R(m) = 2.8), 8k (K(i) 0.13 nM; hA1/hA2A ratio = 4430; R(m) = 3.6), and 14b (K(i) 0.19 nM; hA1/hA2A ratio = 2273; R(m) = 2.7). All the new synthesized compounds have no significant interaction with either A2B or A3 receptor subtypes. This new series of compounds deeply enlightens some structural requirements to display high affinity and selectivity for the A2A adenosine receptor subtype, although our goal of identifying new compounds with increased water solubility was not completely achieved. On this basis, other strategies will be devised to improve this class of compounds with a profile that appears to be promising for treatment of neurodegenerative disorders, such as Parkinson's disease.     

4-Amino[1,2,4]triazolo[4,3-a]quinoxalines. A novel class of potent adenosine receptor antagonists and potential rapid-onset antidepressants

A series of 4-amino[1,2,4]triazolo[4,3-a]quinoxalines has been prepared. Many compounds from this class reduce immobility in Porsolt's behavioral despair model in rats upon acute administration and may therefore have therapeutic potential as novel and rapid acting antidepressant agents. Optimal activity in this test is associated with hydrogen, CF3, or small alkyl groups in the 1-position, with NH2, NH-acetyl, or amines substituted with small alkyl groups in the 4-position, and with hydrogen or 8-halogen substituents in the aromatic ring. Furthermore, many of these 4-amino[1,2,4]triazolo[4,3-a]quinoxalines bind avidly, and in some cases very selectively, to adenosine A1 and A2 receptors. A1 affinity of these compounds was measured by their inhibition of tritiated CHA (N6-cyclohexyladenosine) binding in rat cerebral cortex membranes and A2 affinity by their inhibition of tritiated NECA (5'-(N-ethylcarbamoyl)adenosine) binding to rat striatal homogenate in the presence of cold N6-cyclopentyladenosine. Structure-activity relationship (SAR) studies show that best A1 affinity is associated with ethyl, CF3, or C2F5 in the 1-position, NH-iPr or NH-cycloalkyl in the 4-position, and with an 8-chloro substituent. Affinity at the A2 receptor is mostly dependent on the presence of an NH2 group in the 4-position and is enhanced by phenyl, CF3, or ethyl in the 1-position. The most selective A1 ligand by a factor of greater than 3000 is 121 (CP-68,247; 8-chloro-4-(cyclohexyl-amino)-1- (trifluoromethyl)[1,2,4]triazolo[4,3-a]quinoxaline) with an IC50 of 28 nM at the A1 receptor. The most potent A2 ligand is 128 (CP-66,713; 4-amino-8-chloro-1- phenyl[1,2,4]triazolo[4,3-a]quinoxaline) with an IC50 of 21 nM at the A2 receptor and a 13-fold selectivity for this receptor. Representatives from this series appear to act as antagonists at both A1 and A2 receptors since they antagonize the inhibiting action of CHA on norepinephrine-stimulated cAMP formation in fat cells and they decrease cAMP accumulation induced by adenosine in limbic forebrain slices. Thus certain members of this 4-amino[1,2,4]triazolo[4,3-a]quinoxaline series are among the most potent and A1 or A2 selective non-xanthine adenosine antagonists known.     

1,2,4-triazolo[4,3-a]quinoxalin-1-one moiety as an attractive scaffold to develop new potent and selective human A3 adenosine receptor antagonists: synthesis, pharmacological, and ligand-receptor modeling studies

In the past few years much effort in our laboratory has been directed toward the study of adenosine receptor antagonists, and recently we focused our attention on 2-aryl-1,2,4-triazolo[4,3-a]quinoxaline-1,4-diones and 2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-4-amino-1-ones, some of which were potent and/or selective A(3) receptor antagonists. In the present paper, a new series of triazoloquinoxaline derivatives is described. Most of the new compounds, biologically evaluated in radioligand binding assays at bovine (b) A(1) and A(2A) and at human (h) A(1) and A(3) adenosine receptors, showed high hA(3) adenosine receptor affinity and selectivity. In particular, 2-(4-nitrophenyl)-1,2,4,5-tetrahydro-1,2,4-triazolo[4,3-a]quinoxaline-1,4-dione (1), also tested at the hA(2A) ARs, shows the best binding profile with a high hA(3) affinity (K(i) = 0.60 nM) and strong selectivity vs hA(1) and vs hA(2A) receptors (both selectivity ratios greater than 16 600). To interpret our experimental results, we decided to theoretically depict the putative transmembrane binding motif of our triazoloquinoxaline analogues on hA(3) receptor. Structure-activity relationships have been explained analyzing the three-dimensional structure of the antagonist-receptor models obtained by molecular docking simulation.     

5-amino-2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1-one: a versatile scaffold to obtain potent and selective A3 adenosine receptor antagonists

Binding assays on human A1, A2A, and A3 adenosine receptors (ARs) and functional studies on A2B ARs revealed that various 2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1,5(6H)-diones VIII, previously reported as ligands at the central benzodiazepine receptor (BzR), possess nanomolar affinity at the A3 AR. Replacement of the amide of VIII with an amidine moiety gave the 5-amino-2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1-ones IX, which maintain a nanomolar potency at the A3 AR with selectivity over the BzR. Insertion of a p-methoxybenzoyl at the 5-amino moiety enhanced A3 AR affinity and selectivity over the A1, A2A, and A2B ARs. The best result of our lead optimization efforts is 9-chloro-5-(4-methoxybenzoyl)amino-2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1-one (23), which displayed a Ki of 1.6 nM at the A3 AR and no significant affinity at the other ARs or the BzR. Docking simulations on selected ligands into a model of the A3 AR allowed us to rationalize the structure-activity relationships of phenyltriazolobenzotriazindiones VIII and aminophenyltriazolobenzotriazinones IX at the molecular level.     

5'-O-alkyl ethers of N,2-substituted adenosine derivatives: partial agonists for the adenosine A1 and A3 receptors

New N,5'-di- and N,2,5'-trisubstituted adenosine derivatives were synthesized in good overall yields. Appropriate 5-O-alkyl-substituted ribose moieties were coupled to 6-chloropurine or 2,6-dichloropurine via Vorbrüggen's glycosylation method. Subsequent amination and deprotection of the intermediates yielded compounds 18-35. Binding affinities were determined for rat adenosine A1 and A2A receptors and the human A3 receptor. The ability of compounds 18-35 to inhibit forskolin-induced (10 microM) cyclic AMP (cAMP) production and their ability to stimulate guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding, via either the adenosine A1 receptor or the adenosine A3 receptor, were assessed. N-Cyclopentyl-substituted adenosine derivatives displayed affinities in the low nanomolar range for the adenosine A1 receptor, whereas N-(3-iodobenzyl)-substituted derivatives had high affinity for the adenosine A3 receptor. Compound 22 had the highest affinity for the adenosine A1 receptor (K(i) value of 16 nM), and compounds 20 and 26 had the highest affinities for the adenosine A3 receptor (K(i) values of 4 and 3 nM, respectively). A chlorine substituent at the 2-position either did not affect or slightly increased the adenosine A1 receptor affinity, whereas the A3 receptor affinity was affected differently, depending on the N-substituent. Furthermore, the introduction of chlorine slightly increased the A3/A1 selectivity ratio. At the 5'-position, an O-methyl substituent induced the highest adenosine A1 receptor affinity, whereas an O-ethyl substituent did so for the A3 receptor. All compounds showed partial agonistic effects in both the cAMP and [35S]GTPgammaS assays, although more marked in the latter assay. In general, the 2-chloro derivatives seemed to have lower intrinsic activities compared to the 2-H-substituted compounds on both the adenosine A1 and the adenosine A3 receptors. The compounds with an N-(3-iodobenzyl) substituent displayed the lowest intrinsic activities. Finally, all compounds also showed partially antagonistic behavior in the [35S]GTPgammaS assay.