Click modification in the N6 region of A3 adenosine receptor-selective carbocyclic nucleosides for dendrimeric tethering that preserves pharmacophore recognition.

Adenosine derivatives were modified with alkynyl groups on N(6) substituents for linkage to carriers using Cu(I)-catalyzed click chemistry. Two parallel series, both containing a rigid North-methanocarba (bicyclo[3.1.0]hexane) ring system in place of ribose, behaved as A(3) adenosine receptor (AR) agonists: (5'-methyluronamides) or partial agonists (4'-truncated). Terminal alkynyl groups on a chain at the 3 position of a N(6)-benzyl group or simply through a N(6)-propargyl group were coupled to azido derivatives, which included both small molecules and G4 (fourth-generation) multivalent poly(amidoamine) (PAMAM) dendrimers, to form 1,2,3-triazolyl linkers. The small molecular triazoles probed the tolerance in A(3)AR binding of distal, sterically bulky groups such as 1-adamantyl. Terminal 4-fluoro-3-nitrophenyl groups anticipated nucleophilic substitution for chain extension and (18)F radiolabeling. N(6)-(4-Fluoro-3-nitrophenyl)-triazolylmethyl derivative 32 displayed a K(i) of 9.1 nM at A(3)AR with ∼1000-fold subtype selectivity. Multivalent conjugates additionally containing click-linked water-solubilizing polyethylene glycol groups potently activated A(3)AR in the 5'-methyluronamide, but not 4' truncated series. N(6)-Benzyl nucleoside conjugate 43 (apparent K(i) 24 nM) maintained binding affinity of the monomer better than a N(6)-triazolylmethyl derivative. Thus, the N(6) region of 5'-methyluronamide derivatives, as modeled in receptor docking, is suitable for functionalization and tethering by click chemistry to achieve high A(3)AR agonist affinity and enhanced selectivity.

Molecular probes for the A2A adenosine receptor based on a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine scaffold.

Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivatives such as SCH 442416 display high affinity and selectivity as antagonists for the human 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.

Design, synthesis, and binding of homologated truncated 4'-thioadenosine derivatives at the human A3 adenosine receptors.

We synthesized homologated truncated 4'-thioadenosine analogues 3 in which a methylene (CH(2)) group was inserted in place of the glycosidic bond of a potent and selective A(3) adenosine receptor antagonist 2. The analogues were designed to induce maximum binding interaction in the binding site of the A(3) adenosine receptor. However, all homologated nucleosides were devoid of binding affinity at all subtypes of adenosine receptors, indicating that free rotation through the single bond allowed the compound to adopt an indefinite number of conformations, disrupting the favorable binding interaction essential for receptor recognition.

Nucleoside conjugates of quantum dots for characterization of G protein-coupled receptors: strategies for immobilizing A2A adenosine receptor agonists.

BACKGROUND: Quantum dots (QDs) are crystalline nanoparticles that are compatible with biological systems to provide a chemically and photochemically stable fluorescent label. New ligand probes with fluorescent reporter groups are needed for detection and characterization of G protein-coupled receptors (GPCRs). RESULTS: Synthetic strategies for coupling the A2A adenosine receptor (AR) agonist CGS21680 (2-[4-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine) to functionalized QDs were explored. Conjugates tethered through amide-linked chains and poly(ethyleneglycol) (PEG) displayed low solubility and lacked receptor affinity. The anchor to the dendron was either through two thiol groups of (R)-thioctic acid or through amide formation to a commercial carboxy-derivatized QD. The most effective approach was to use polyamidoamine (PAMAM) D5 dendrons as multivalent spacer groups, grafted on the QD surface through a thioctic acid moiety. In radioligand binding assays, dendron nucleoside conjugate 11 displayed a moderate affinity at the human A2AAR (Kiapp 1.02 +/- 0.15 muM). The QD conjugate of increased water solubility 13, resulting from the anchoring of this dendron derivative, interacted with the receptor with Kiapp of 118 +/- 54 nM. The fluorescence emission of 13 occurred at 565 nm, and the presence of the pendant nucleoside did not appreciably quench the fluorescence. CONCLUSIONS: This is a feasibility study to demonstrate a means of conjugating to a QD a small molecular pharmacophore of a GPCR that is relatively hydrophobic. Further enhancement of affinity by altering the pharmacophore or the linking structures will be needed to make useful affinity probes.

Novel Alexa Fluor-488 labeled antagonist of the A(2A) adenosine receptor: Application to a fluorescence polarization-based receptor binding assay.

Fluorescence polarization (FP) assay has many advantages over the traditional radioreceptor binding studies. We developed an A(2A) adenosine receptor (AR) FP assay using a newly synthesized fluorescent antagonist of the A(2A)AR (MRS5346), a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivative conjugated to the fluorescent dye Alexa Fluor-488. MRS5346 displayed a K(i) value of 111+/-16nM in radioligand binding using [(3)H]CGS21680 and membranes prepared from HEK293 cells stably expressing the human A(2A)AR. In a cyclic AMP functional assay, MRS5346 was shown to be an A(2A)AR antagonist. MRS5346 did not show any effect on A(1) and A(3) ARs in binding or the A(2B)AR in a cyclic AMP assay at 10microM. Its suitability as a fluorescent tracer was indicated in an initial observation of an FP signal following A(2A)AR binding. The FP signal was optimal with 20nM MRS5346 and 150microg protein/mL HEK293 membranes. The association and dissociation kinetic parameters were readily determined using this FP assay. The K(d) value of MRS5346 calculated from kinetic parameters was 16.5+/-4.7nM. In FP competition binding experiments using MRS5346 as a tracer, K(i) values of known AR agonists and antagonists consistently agreed with K(i) values from radioligand binding. Thus, this FP assay, which eliminates using radioisotopes, appears to be appropriate for both routine receptor binding and high-throughput screening with respect to speed of analysis, displaceable signal and precision. The approach used in the present study could be generally applicable to other GPCRs.

Structure-based discovery of A2A adenosine receptor ligands.

The recent determination of X-ray structures of pharmacologically relevant GPCRs has made these targets accessible to structure-based ligand discovery. Here we explore whether novel chemotypes may be discovered for the A(2A) adenosine receptor, based on complementarity to its recently determined structure. The A(2A) adenosine receptor signals in the periphery and the CNS, with agonists explored as anti-inflammatory drugs and antagonists explored for neurodegenerative diseases. We used molecular docking to screen a 1.4 million compound database against the X-ray structure computationally and tested 20 high-ranking, previously unknown molecules experimentally. Of these 35% showed substantial activity with affinities between 200 nM and 9 microM. For the most potent of these new inhibitors, over 50-fold specificity was observed for the A(2A) versus the related A(1) and A(3) subtypes. These high hit rates and affinities at least partly reflect the bias of commercial libraries toward GPCR-like chemotypes, an issue that we attempt to investigate quantitatively. Despite this bias, many of the most potent new ligands were novel, dissimilar from known ligands, providing new lead structures for modulation of this medically important target.

Polyamidoamine (PAMAM) dendrimer conjugates of "clickable" agonists of the A3 adenosine receptor and coactivation of the P2Y14 receptor by a tethered nucleotide.

We previously synthesized a series of potent and selective A(3) adenosine receptor (AR) agonists (North-methanocarba nucleoside 5'-uronamides) containing dialkyne groups on extended adenine C2 substituents. We coupled the distal alkyne of a 2-octadiynyl nucleoside by Cu(I)-catalyzed "click" chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. A(3)AR activation was preserved in these multivalent conjugates, which bound with apparent K(i) of 0.1-0.3 nM. They were substituted with nucleoside moieties, solely or in combination with water-solubilizing carboxylic acid groups derived from hexynoic acid. A comparison with various amide-linked dendrimers showed that triazole-linked conjugates displayed selectivity and enhanced A(3)AR affinity. We prepared a PAMAM dendrimer containing equiproportioned peripheral azido and amino groups for conjugation of multiple ligands. A bifunctional conjugate activated both A(3) and P2Y(14) receptors (via amide-linked uridine-5'-diphosphoglucuronic acid), with selectivity in comparison to other ARs and P2Y receptors. This is the first example of targeting two different GPCRs with the same dendrimer conjugate, which is intended for activation of heteromeric GPCR aggregates. Synergistic effects of activating multiple GPCRs with a single dendrimer conjugate might be useful in disease treatment.

2-Dialkynyl derivatives of (N)-methanocarba nucleosides: 'Clickable' A(3) adenosine receptor-selective agonists.

We modified a series of (N)-methanocarba nucleoside 5'-uronamides to contain dialkyne groups on an extended adenine C2 substituent, as synthetic intermediates leading to potent and selective A(3) adenosine receptor (AR) agonists. The proximal alkyne was intended to promote receptor recognition, and the distal alkyne reacted with azides to form triazole derivatives (click cycloaddition). Click chemistry was utilized to couple an octadiynyl A(3)AR agonist to azido-containing fluorescent, chemically reactive, biotinylated, and other moieties with retention of selective binding to the A(3)AR. A bifunctional thiol-reactive crosslinking reagent was introduced. The most potent and selective novel compound was a 1-adamantyl derivative (K(i) 6.5nM), although some of the click products had K(i) values in the range of 200-400nM. Other potent, selective derivatives (K(i) at A(3)AR innM) were intended as possible receptor affinity labels: 3-nitro-4-fluorophenyl (10.6), alpha-bromophenacyl (9.6), thiol-reactive isothiazolone (102), and arylisothiocyanate (37.5) derivatives. The maximal functional effects in inhibition of forskolin-stimulated cAMP were measured, indicating that this class of click adducts varied from partial to full A(3)AR agonist compared to other widely used agonists. Thus, this strategy provides a general chemical approach to linking potent and selective A(3)AR agonists to reporter groups of diverse structure and to carrier moieties.

Discovery of New Human A(2A) Adenosine Receptor Agonists: Design, Synthesis, and Binding Mode of Truncated 2-Hexynyl-4'-thioadenosine.

The truncated C2- and C8-substituted-4'-thioadenosine derivatives 4a-d were synthesized from D-mannose, using palladium-catalyzed cross coupling reactions as key steps. In this study, an A(3) adenosine receptor (AR) antagonist, truncated 4'-thioadenosine derivative 3 was successfully converted into a potent A(2A)AR agonist 4a (K(i) = 7.19 ± 0.6 nM) by appending a 2-hexynyl group at the C2-position of a derivative of 3 that was N(6)-substituted. However, C8-substitution greatly reduced binding affinity at the human A(2A)AR. All synthesized compounds 4a-d maintained their affinity at the human A(3)AR, but 4a was found to be a competitive A(3)AR antagonist/A(2A)AR agonist in cyclic AMP assays. This study indicates that the truncated C2-substituted-4'-thioadenosine derivatives 4a and 4b can serve as a novel template for the development of new A(2A)AR ligands.