Pyrazolo-triazolo-pyrimidines as adenosine receptor antagonists: Effect of the N-5 bond type on the affinity and selectivity at the four adenosine receptor subtypes.
ABSTRACT: In the last few years, many efforts have been made to search for potent and selective human A(3) adenosine antagonists. In particular, one of the most promising human A(3) adenosine receptor antagonists is represented by the pyrazolo-triazolo-pyrimidine family. This class of compounds has been strongly investigated from the point of view of structure-activity relationships. In particular, it has been observed that fundamental requisites for having both potency and selectivity at the human A(3) adenosine receptors are the presence of a small substituent at the N(8) position and an unsubstitued phenyl carbamoyl moiety at the N(5) position. In this study, we report the role of the N(5)-bond type on the affinity and selectivity at the four adenosine receptor subtypes. The observed structure-activity relationships of this class of antagonists are also exhaustively rationalized using the recently published ligand-based homology modeling approach.
Project description:In the last 5 years, many efforts have been conducted searching potent and selective human A(3) adenosine antagonists. In this field several different classes of compounds, possessing very good affinity (nM range) and with a broad range of selectivity, have been proposed. Recently, our group synthesized a new series of pyrazolo-triazolo-pyrimidines bearing different substitutions at the N(5) and N(8) positions, which have been described as highly potent and selective human A(3) adenosine receptor antagonists. The present review summarizes available data and provides an overview of the structure-activity relationships found for this class of human A(3) adenosine receptor antagonists.
Project description:A series of adenosine receptor antagonists bearing a reactive linker was developed. Functionalization of these derivatives is useful to easily obtain multi-target ligands, receptor probes, drug delivery systems, and diagnostic or theranostic systems. The pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine scaffold was chosen as a pharmacophore for the adenosine receptors. It was substituted at the 5 position with reactive linkers of different lengths. Then, these compounds were used to synthesise probes for the adenosine receptors by functionalization with a fluorescent moiety. Both series of compounds were evaluated for their binding at the four adenosine receptor subtypes. Different affinity and selectivity profiles were observed towards hA1, hA2A and hA3 adenosine receptors. In particular, fluorescent compounds behave as dual hA2A/hA3 ligands. Computational studies suggested different binding modes for developed compounds at the three receptors. Both molecular docking and supervised molecular dynamics (SuMD) simulations confirmed that the preferred binding mode at the single receptor was driven by the substitution present at the 5 position. Obtained results rationalized the compounds' binding profile at the adenosine receptors and pave the way for the development of more potent conjugable and conjugated ligands targeting these membrane receptors.
Project description: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 A(2A) 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 (18)F incorporation), and fluorophore reporter groups (e.g., BODIPY conjugate 14, K(i) 15 nM). The potent and A(2A)AR-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 A(2A)AR 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 A(2A)AR 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 A(2A)AR.
Project description:A new series of pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (PTP) derivatives has been developed in order to explore their affinity and selectivity profile at the four adenosine receptor subtypes. In particular, the PTP scaffold was conjugated at the C2 position with the 1-(3-trifluoromethyl-benzyl)-1H-pyrazole, a group believed to confer potency and selectivity toward the human (h) A2B adenosine receptor (AR) to the xanthine ligand 8-(1-(3-(trifluoromethyl)benzyl)-1H-pyrazol-4-yl)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (CVT 6975). Interestingly, the synthesized compounds turned out to be inactive at the hA2B AR but they displayed affinity at the hA3 AR in the nanomolar range. The best compound of the series (6) shows both high affinity (hA3 AR Ki = 11 nM) and selectivity (A1/A3 and A2A/A3 > 9090; A2B/A3 > 909) at the hA3 AR. To better rationalize these results, a molecular docking study on the four AR subtypes was performed for all the synthesized compounds. In addition, CTV 6975 and two close analogues have been subjected to the same molecular docking protocol to investigate the role of the 1-(3-trifluoromethyl-benzyl)-1H-pyrazole on the binding at the four ARs.
Project description:In the past few decades, medicinal chemistry research towards potent and selective antagonists of human adenosine receptors (namely, A1, A2A, A2B, and A3) has been evolving rapidly. These antagonists are deemed therapeutically beneficial in several pathological conditions including neurological and renal disorders, cancer, inflammation, and glaucoma. Up to this point, many classes of compounds have been successfully synthesized and identified as potent human adenosine receptor antagonists. In this paper, an overview of the structure-activity relationship (SAR) profiles of promising nonxanthine pyrazolo derivatives is reported and discussed. We have emphasized the SAR for some representative structures such as pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidines; pyrazolo-[3,4-c] or -[4,3-c]quinolines; pyrazolo-[4,3-d]pyrimidinones; pyrazolo-[3,4-d]pyrimidines and pyrazolo-[1,5-a]pyridines. This overview not only clarifies the structural requirements deemed essential for affinity towards individual adenosine receptor subtypes, but it also sheds light on the rational design and optimization of existing structural templates to allow us to conceive new, more potent adenosine receptor antagonists.
Project description:The structure-activity relationship (SAR) of 1,2,4-triazolo[1,5-a]-1,3,5-triazine derivatives related to ZM241385 as antagonists of the A(2A) adenosine receptor (AR) was explored through the synthesis of analogues substituted at the 5 position. The A(2A) AR X-ray structure was used to propose a structural basis for the activity and selectivity of the analogues and to direct the synthetic design strategy to provide access to solvent-exposed regions. Thus, we have identified a point of substitution for the attachment of solubilizing groups to enhance both aqueous solubility and physicochemical properties, maintaining potent interactions with the A(2A) AR and, in some cases, receptor subtype selectivity. Among the most potent and selective novel compounds were a long-chain ether-containing amine congener 20 (K(i) 11.5 nM) and its urethane-protected derivative 14 (K(i) 17.8 nM). Compounds 20 and 31 (K(i) 11.5 and 16.9 nM, respectively) were readily water-soluble up to 10 mM. The analogues were docked in the crystallographic structure of the hA(2A) AR and in a homology model of the hA(3) AR, and the per residue electrostatic and hydrophobic contributions to the binding were assessed and stabilizing factors were proposed.
Project description:A pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine antagonist of the A2A adenosine receptor (AR) was functionalized as amine congeners, fluorescent conjugates and a sulfonate, and the A2AAR binding modes were predicted computationally. The optimal n-butyl spacer was incorporated into the following A2AAR-selective (Ki, nM) conjugates: BODIPY630/650 derivative 11 (MRS7396, 24.6) and AlexaFluor488 derivative 12 (MRS7416, 30.3). Flow cytometry of 12 in hA2AAR-expressing HEK-293 cells displayed saturable binding (low nonspecific) and inhibition by known A2AAR antagonists. Water-soluble sulfonate 13 was a highly potent (Ki = 6.2 nM) and selective A2AAR antagonist based on binding and functional assays. Docking and molecular dynamics simulations predicted the regions of interaction of the distal portions of these chain-extended ligands with the A2AAR. The BODIPY630/650 fluorophore of 11 was buried in a hydrophobic interhelical (TM1/TM7) region, while AlexaFluor488 of 12 associated with the hydrophilic extracellular loops. In conclusion, we have identified novel high affinity antagonist probes for A2AAR drug discovery and characterization.
Project description:1. Recent studies have shown beneficial effects of an adenosine A(2A) receptor agonist in dt(sz) mutant hamsters, an animal model of paroxysmal dystonia, in which stress and consumption of coffee can precipitate dystonic attacks. This prompted us to examine the effects of adenosine receptor agonists and antagonists on severity of dystonia in dt(sz) hamsters in more detail. 2. The non-selective adenosine A(1)/A(2A) receptor antagonists, caffeine (10 - 20 mg kg(-1) i.p.) and theophylline (10 - 30 mg kg(-1) s.c.), worsened the dystonia in dt(sz) hamsters. 3. Aggravation of dystonia was also caused by the selective adenosine A(1)/A(2A) antagonist CGS 15943 (9-chloro2-2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine) at a dose of 30 mg kg(-1) i.p. and by the adenosine A(1) antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine; 20 - 30 mg kg(-1) i.p.), while the A(2) antagonist DMPX (3,7-dimethyl-1-propargylxanthine; 2 - 4 mg kg(-1) i.p.) and the highly selective A(2A) antagonist ZM 241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol; 2 - 5 mg kg(-1) i.p.) failed to exert any effects on dystonia. 4. In contrast to the antagonists, both the adenosine A(1) receptor agonist CPA (N(6)-cyclopentyladenosine; 0.1 - 1.0 mg kg(-1) i.p.) and the A(2A) agonist CGS 21680 (2p-(2carboxyethylphen-ethylamino-5'-N-ethylcarboxamindoadenosine; 0.1 - 2.0 mg kg(-1) i.p.) exerted a striking improvement of dystonia. 5. These data suggest that the precipitating effects of methylxanthines are, at least in part, related to their adenosine receptor antagonistic action. 6. Although adenosine receptor agonists can be regarded as interesting candidates for the therapy of paroxysmal dystonia, adverse effects may limit the therapeutic potential of adenosine A(1) agonists, while beneficial effects of the adenosine A(2A) agonist CGS 21680 were already found at well tolerated doses.
Project description:The physiological role of the A(3) adenosine receptor (AR) was explored in cardiac ischaemia, inflammatory diseases and cancer. We report a new fluorophore-conjugated human (h) A(3)AR antagonist for application to cell-based assays in ligand discovery and for receptor imaging. Fluorescent pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-ylamine (pyrazolo-triazolo-pyrimidine, PTP) and triazolo[1,5-c]quinazolin-5-yl)amine (triazolo-quinazoline, TQ) AR antagonists were compared. A chain-extended and click-conjugated Alexa Fluor-488 TQ derivative (MRS5449) displayed a radioligand binding K(i) value of 6.4±2.5nM in hA(3)AR-expressing CHO cell membranes. MRS5449 antagonized hA(3)AR agonist-induced inhibition of cyclic AMP accumulation in a concentration-dependent manner (K(B)=4.8nM). Using flow cytometry (FCM), MRS5449 saturated hA(3)ARs with very high specific-to-nonspecific binding ratio with an equilibrium binding constant 5.15nM, comparable to the K(d) value of 6.65nM calculated from kinetic experiments. K(i) values of known AR antagonists in inhibition of MRS5449 binding in whole cell FCM were consistent with radioligand binding in membranes, but agonist binding was 5-20 fold weaker than obtained with agonist radioligand [(125)I]I-AB-MECA. Further binding analysis of MRS5549 suggested multiple agonist binding states of the A(3)AR. Molecular docking predicted binding modes of these fluorescent antagonists. Thus, MRS5449 is a useful tool for hA(3)AR characterization.
Project description: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 [N(6)-(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 microM.