P2X receptor activation protects in heart failure models. MRS2339 3, a 2-chloro-AMP derivative containing a (N)-methanocarba (bicyclo[3.1.0]hexane) system, activates this cardioprotective channel. Michaelis-Arbuzov and Wittig reactions provided phosphonate analogues of 3, expected to be stable in vivo due to the C-P bond. After chronic administration via a mini-osmotic pump (Alzet), some analogues significantly increased intact heart contractile function in calsequestrin-overexpressing mice (genetic model of heart failure) compared to vehicle-infused mice (all inactive at the vasodilatory P2Y(1) receptor). Two phosphonates, (1'S,2'R,3'S,4'R,5'S)-4'-(6-amino-2-chloropurin-9-yl)-2',3'-(dihydroxy)-1'-(phosphonomethylene)-bicyclo[3.1.0]hexane, 4 (MRS2775), and its homologue 9 (MRS2935), both 5'-saturated, containing a 2-Cl substitution, improved echocardiography-derived fractional shortening (20.25% and 19.26%, respectively, versus 13.78% in controls), while unsaturated 5'-extended phosphonates, all 2-H analogues, and a CH(3)-phosphonate were inactive. Thus, chronic administration of nucleotidase-resistant phosphonates conferred a beneficial effect, likely via cardiac P2X receptor activation. Thus, we have greatly expanded the range of carbocyclic nucleotide analogues that represent potential candidates for the treatment of heart failure.

The purpose of the study was to determine whether novel, selective antagonists of human A3 adenosine receptors (ARs) derived from the A3-selective agonist Cl-IB-MECA lower intraocular pressure (IOP) and act across species. IOP was measured invasively with a micropipette by the Servo-Null Micropipette System (SNMS) and by non-invasive pneumotonometry during topical drug application. Antagonist efficacy was also assayed by measuring inhibition of adenosine-triggered shrinkage of native bovine nonpigmented ciliary epithelial (NPE) cells. Five agonist-based A3AR antagonists lowered mouse IOP measured with SNMS tonometry by 3-5 mm Hg within minutes of topical application. Of the five agonist derivatives, LJ 1251 was the only antagonist to lower IOP measured by pneumotonometry. No effect was detected pneumotonometrically over 30 min following application of the other four compounds, consonant with slower, smaller responses previously measured non-invasively following topical application of A3AR agonists and the dihydropyridine A3AR antagonist MRS 1191. Latanoprost similarly lowered SNMS-measured IOP, but not IOP measured non-invasively over 30 min. Like MRS 1191, agonist-based A3AR antagonists applied to native bovine NPE cells inhibited adenosine-triggered shrinkage. In summary, the results indicate that antagonists of human A3ARs derived from the potent, selective A3 agonist Cl-IB-MECA display efficacy in mouse and bovine cells, as well. When intraocular delivery was enhanced by measuring mouse IOP invasively, five derivatives of the A3AR agonist Cl-IB-MECA lowered IOP but only one rapidly reduced IOP measured non-invasively after topical application. We conclude that derivatives of the highly-selective A3AR agonist Cl-IB-MECA can reduce IOP upon reaching their intraocular target, and that nucleoside-based derivatives are promising A3 antagonists for study in multiple animal models.

A known selective agonist of the A3 adenosine receptors (AR), MRS1898 [(1′R,2′R,3′S,4′R,5′S)-4-{2-chloro-6-[(3-iodophenylmethyl)amino]purin-9-yl}-1-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2,3-diol], was synthesized in radioactive form and characterized pharmacologically. This agonist ligand series, based on nucleoside analogues containing a rigid, bicyclic ring system in place of the ribose moiety, was selected for radiolabeling due to its high A3AR affinity across species, with nanomolar binding at both rat and human A3ARs. The radioiodination of MRS1898 on its N 6–3-iodobenzyl substituent was accomplished in 76% radiochemical yield by iododestannylation of a 3-(trimethylstannyl)benzyl precursor. [125I]MRS1898 bound to the rat A3AR with a Kd value of 0.17 ± 0.04 nM and a Bmax value of 0.66 ± 0.15 pmol/mg protein. The competition binding profiles for other agonists and antagonists obtained with this radioligand are similar to those previously obtained with other radioligands. The advantages of [125I]MRS1898 compared with previously used radioligands are primarily its high selectivity and affinity for the rat A3AR and also its facile synthesis and radiochemical stability; however, a relatively high level of nonspecific binding presents a limitation. Thus, we have introduced the first selective radioligand for the rat A3AR.

Ribose-based nucleoside 5'-diphosphates and triphosphates and related nucleotides were compared in their potency at the P2Y receptors with the corresponding nucleoside 5'-phosphonate derivatives. Phosphonate derivatives of UTP and ATP activated the P2Y(2) receptor but were inactive or weakly active at P2Y(4) receptor. Uridine 5'-(diphospho)phosphonate was approximately as potent at the P2Y(2) receptor as at the UDP-activated P2Y(6) receptor. These results suggest that removal of the 5'-oxygen atom from nucleotide agonist derivatives reduces but does not prevent interaction with the P2Y(2) receptor. Uridine 5'-(phospho)phosphonate as well as the 5'-methylenephosphonate equivalent of UMP were inactive at the P2Y(4) receptor and exhibited maximal effects at the P2Y(2) receptor that were 50% of that of UTP suggesting novel action of these analogues.

The P2Y(14) receptor, a nucleotide signaling protein, is activated by uridine-5'-diphosphoglucose 1 and other uracil nucleotides. We have determined that the glucose moiety of 1 is the most structurally permissive region for designing analogues of this P2Y(14) agonist. For example, the carboxylate group of uridine-5'-diphosphoglucuronic acid proved to be suitable for flexible substitution by chain extension through an amide linkage. Functionalized congeners containing terminal 2-acylaminoethylamides prepared by this strategy retained P2Y(14) activity, and molecular modeling predicted close proximity of this chain to the second extracellular loop of the receptor. In addition, replacement of glucose with other sugars did not diminish P2Y(14) potency. For example, the [500] ribose derivative had an EC50 of 0.24 mu M. Selective monofluorination of the glucose moiety indicated a role for the 2 ''- and 6 ''- hydroxyl groups of 1 in receptor recognition. The beta-glucoside was twofold less potent than the native alpha-isomer, but methylene replacement of the 1 ''-oxygen abolished activity. Replacement of the ribose ring system with cyclopentyl or rigid bicyclo[3.1.0] hexane groups abolished activity. Uridine-5'-diphosphoglucose also activates the P2Y(2) receptor, but the 2-thio analogue and several of the potent modified-glucose analogues were P2Y(14)-selective. (C) 2009 Elsevier Ltd. All rights reserved.

Introduction: Bromine-76-radiolabeled analogues of previously reported high-affinity A(3) adenosine receptor (A(3)AR) nucleoside ligands have been prepared as potential radiotracers for positron emission tomography.Methods: The radiosyntheses were accomplished by oxidative radiobromination on the N-6-benzyl moiety of trimethyltin precursors. Biodistribution studies of the kinetics Of uptake were conducted in awake rats.Results: We prepared an agonist ligand {[Br-76](1'S,2'R,3'S,4'R,5'S)-4'-{2-chloro-6-[(3-bromophenylmethyl)amino]purin-9-yl}-1'-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2',3'-diol (MRS3581)} in 59% radiochemical yield with a specific activity of 19.5 GBq/mu mol and all antagonist ligand {[Br-76](1'R,2'R,3'S,4'R,5'S)-4'-(6-(3-bromobenzylamino)-2-clhloro-9H-purin-9-yl)bicyclo[3.1.0]hexane-2',3'-diol (MRS5147) in 65% radiochemical yield with a specific activity of 22 GBq/mu mol. The resultant products exhibited the expected high affinity (K-i similar to 0.6 nM) and specific binding at the human A(3)AR in vitro. Biodistribution studies in the rat showed uptake in the organs of excretion and metabolism. The antagonist MRS5147 exhibited increasing uptake in testes, an organ that contains significant quantities of A(3)AR, over a 2-h time course, which suggests the presence of a specific A(3)AR retention mechanism.Conclusion: We were able to compare uptake of the [Br-76]-labeled antagonist MRS5147 to [Br-76]agonist MRS3581. The antagonist MRS5147 shows increasing uptake in the testes, an A(3)AR-rich tissue, suggesting that this ligand may have promise as a molecular imaging agent. Published by Elsevier Inc.

Analogues of the P2X7 receptor antagonist KN-62, modified at the piperazine and arylsulfonyl groups, were synthesized and assayed at the human P2X7 receptor for inhibition of BzATP-induced effects, that is, uptake of a fluorescent dye (ethidium bromide) in stably transfected HEK293 cells and IL-1β release in differentiated THP-1 cells. Substitution of the arylsulfonyl moiety with a nitro group increased antagonistic potency relative to methyl substitution, such that compound 21 was slightly more potent than KN-62. Substitution with d-tyrosine in 36 and sterically bulky tyrosyl 3,5-dimethyl groups in 9 enhanced antagonistic potency.

Substitution of the ribose moiety of various nucleosides and nucleotides with the (N)-methanocarba ring system increases the potency and selectivity as ligands at certain subtypes of adenosine and P2 receptors. We have prepared a key intermediate in the synthesis of these derivatives, ethyl (1S,2R,3S,4S,5S)-2,3-O-(isopropylidene)-4-hydroxybicyclo[3.1.0]hexane-carboxylate (15), starting from L-ribose (8) as a readily available, enantiopure building block. L-ribose was converted to the corresponding 5′-iodo derivative (9), which was cleaved reductively with Zn. Improvements were made in subsequent steps corresponding to a published route to biologically important (N)-methanocarba 5′-uronamido nucleosides, and new steps were added to prepare related 5′-nucleotides.

We have prepared 5′-modified derivatives of adenosine and a corresponding (N)-methanocarba nucleoside series containing a bicyclo[3.1.0]hexane ring system in place of the ribose moiety. The compounds were examined in binding assays at three subtypes of adenosine receptors (ARs) and in functional assays at the A3 AR. The H-bonding ability of a group of 9-riboside derivatives containing a 5′-uronamide moiety was reduced by modification of the NH; however these derivatives did not display the desired activity as selective A3 AR antagonists, as occurs with 5′-N,N-dimethyluronamides. However, truncated (N)-methanocarba analogues lacking a 4′-hydroxymethyl group were highly potent and selective antagonists of the human A3 AR. The compounds were synthesized from d-ribose using a reductive free radical decarboxylation of a 5′-carboxy intermediate. A less efficient synthetic approach began with L-ribose, which was similar to the published synthesis of (N)-methanocarba A3AR agonists. Compounds 33b–39b (N6-3-halobenzyl and related arylalkyl derivatives) were potent A3AR antagonists with binding Ki values of 0.7–1.4nM. In a functional assay of [35S]GTPγS binding, 33b (3-iodobenzyl) completely inhibited stimulation by NECA with a KB of 8.9nM. Thus, a highly potent and selective series of A3AR antagonists has been described.

P2Y1 is an ADP-activated G protein-coupled receptor (GPCR). Its antagonists impede platelet aggregation in vivo and are potential antithrombotic agents. Combining ligand and structure-based modeling we generated a consensus model (LIST-CM) correlating antagonist structures with their potencies. We docked 45 antagonists into our rhodopsin-based human P2Y1 homology model and calculated docking scores and free binding energies with the Linear Interaction Energy (LIE) method in continuum-solvent. The resulting alignment was also used to build QSAR based on CoMFA, CoMSIA, and molecular descriptors. To benefit from the strength of each technique and compensate for their limitations, we generated our LIST-CM with a PLS regression based on the predictions of each methodology. A test set featuring untested substituents was synthesized and assayed in inhibition of 2-MeSADP-stimulated PLC activity and in radioligand binding. LIST-CM outperformed internal and external predictivity of any individual model to predict accurately the potency of 75% of the test set.

Phagocytosis is thought to be initiated by activation of phagocytosis-promoting receptors that recognize 'eat-me' signals such as phosphatidylserine or amyloid-β expressed in the apoptotic cells. But now Koizumi et al. demonstrate a novel type of microglial phagocytosis that requires neither typical 'eat-me' signals nor Fc receptor ligands for initiation. Instead, this phagocytosis is promoted by the diffusible extracellular molecule uridine 5′-diphosphate, released by injured cells. The UDP activates P2Y6 receptors on the microglial surface. The clearance of dead cells is crucial to the maintenance of brain function, so these findings may have implications for a range of central nervous system diseases. Microglia, brain immune cells, engage in the clearance of dead cells or dangerous debris, which is crucial to the maintenance of brain functions. When a neighbouring cell is injured, microglia move rapidly towards it or extend a process to engulf the injured cell. Because cells release or leak ATP when they are stimulated1,2 or injured3,4, extracellular nucleotides are thought to be involved in these events. In fact, ATP triggers a dynamic change in the motility of microglia in vitro5,6 and in vivo3,4, a previously unrecognized mechanism underlying microglial chemotaxis5,6; in contrast, microglial phagocytosis has received only limited attention. Here we show that microglia express the metabotropic P2Y6 receptor whose activation by endogenous agonist UDP triggers microglial phagocytosis. UDP facilitated the uptake of microspheres in a P2Y6-receptor-dependent manner, which was mimicked by the leakage of endogenous UDP when hippocampal neurons were damaged by kainic acid in vivo and in vitro. In addition, systemic administration of kainic acid in rats resulted in neuronal cell death in the hippocampal CA1 and CA3 regions, where increases in messenger RNA encoding P2Y6 receptors that colocalized with activated microglia were observed. Thus, the P2Y6 receptor is upregulated when neurons are damaged, and could function as a sensor for phagocytosis by sensing diffusible UDP signals, which is a previously unknown pathophysiological function of P2 receptors in microglia.