Structure-Activity Relationship of Purine and Pyrimidine Nucleotides as Ecto-5'-Nucleotidase (CD73) Inhibitors.

Cluster of differentiation 73 (CD73) converts adenosine 5'-monophosphate to immunosuppressive adenosine, and its inhibition was proposed as a new strategy for cancer treatment. We synthesized 5'- O-[(phosphonomethyl)phosphonic acid] derivatives of purine and pyrimidine nucleosides, which represent nucleoside diphosphate analogues, and compared their CD73 inhibitory potencies. In the adenine series, most ribose modifications and 1-deaza and 3-deaza were detrimental, but 7-deaza was tolerated. Uracil substitution with N3-methyl, but not larger groups, or 2-thio, was tolerated. 1,2-Diphosphono-ethyl modifications were not tolerated. N4-(Aryl)alkyloxy-cytosine derivatives, especially with bulky benzyloxy substituents, showed increased potency. Among the most potent inhibitors were the 5'- O-[(phosphonomethyl)phosphonic acid] derivatives of 5-fluorouridine (4l), N4-benzoyl-cytidine (7f), N4-[ O-(4-benzyloxy)]-cytidine (9h), and N4-[ O-(4-naphth-2-ylmethyloxy)]-cytidine (9e) ( Ki values 5-10 nM at human CD73). Selected compounds tested at the two uridine diphosphate-activated P2Y receptor subtypes showed high CD73 selectivity, especially those with large nucleobase substituents. These nucleotide analogues are among the most potent CD73 inhibitors reported and may be considered for development as parenteral drugs.

Pyrimidine Nucleotides Containing a (S)-Methanocarba Ring as P2Y6 Receptor Agonists.

Both agonists and antagonists of the UDP-activated P2Y6 receptor (P2Y6R) have been proposed for therapeutic use, in conditions such as cancer, inflammation, neurodegeneration and diabetes. Uracil nucleotides containing a South-bicyclo[3.1.0]hexane ((S)-methanocarba) ring system in place of the ribose ring were synthesized and shown to be potent P2Y6R agonists in a calcium mobilization assay. The (S)-methanocarba modification was compatible with either a 5-iodo or 4-methoxyimino group on the pyrimidine, but not with a α,ß-methylene 5´-diphosphate. (S)-Methanocarba dinucleotide potency was compatible with a N4-methoxy modification on the proximal nucleoside that is assumed to bind at the P2Y6R similarly to UDP; (N)-methanocarba was preferred on the distal nucleoside moiety. This suggests that the distal dinucleotide P2Y6R binding site prefers a ribose-like group that can attain a (N) conformation, rather than (S). Dinucleotide binding was modeled by homology modeling, docking and molecular dynamics simulations, which suggested the same ribose conformational preferences found empirically.

Structure-Based Design of 3-(4-Aryl-1H-1,2,3-triazol-1-yl)-Biphenyl Derivatives as P2Y14 Receptor Antagonists.

UDP and UDP-glucose activate the P2Y14 receptor (P2Y14R) to modulate processes related to inflammation, diabetes, and asthma. A computational pipeline suggested alternatives to naphthalene of a previously reported P2Y14R antagonist (3, PPTN) using docking and molecular dynamics simulations on a hP2Y14R homology model based on P2Y12R structures. By reevaluating the binding of 3 to P2Y14R computationally, two alternatives, i.e., alkynyl and triazolyl derivatives, were identified. Improved synthesis of fluorescent antagonist 4 enabled affinity quantification (IC50s, nM) using flow cytometry of P2Y14R-expressing CHO cells. p-F3C-phenyl-triazole 65 (32) was more potent than a corresponding alkyne 11. Thus, additional triazolyl derivatives were prepared, as guided by docking simulations, with nonpolar aryl substituents favored. Although triazoles were less potent than 3 (6), simpler synthesis facilitated further structural optimization. Additionally, relative P2Y14R affinities agreed with predicted binding of alkynyl and triazole analogues. These triazoles, designed through a structure-based approach, can be assessed in disease models.

Design, synthesis, pharmacological characterization of a fluorescent agonist of the P2Y14 receptor.

The P2Y14R is a G(i/o)-coupled receptor of the P2Y family of purinergic receptors that is activated by extracellular UDP and UDP-glucose (UDPG). In an earlier report we described a P2Y14R fluorescent probe, MRS4174, based on the potent and selective antagonist PPTN, a naphthoic acid derivative. Here, we report the design, preparation, and activity of an agonist-based fluorescent probe MRS4183 (11) and a shorter P2Y14R agonist congener, which contain a UDP-glucuronic acid pharmacophore and BODIPY fluorophores conjugated through diaminoalkyl linkers. The design relied on both docking in a P2Y14R homology model and established structure activity relationship (SAR) of nucleotide analogs. 11 retained P2Y14R potency with EC50 value of 0.96 nM (inhibition of adenylyl cyclase), compared to parent UDPG (EC50 47 nM) and served as a tracer for microscopy and flow cytometry, displaying minimal nonspecific binding. Binding saturation analysis gave an apparent binding constant for 11 in whole cells of 21.4±1.1 nM, with a t1/2 of association at 50 nM 11 of 23.9 min. Known P2Y14R agonists and PPTN inhibited cell binding of 11 with the expected rank order of potency. The success in the identification of a new P2Y14R fluorescent agonist with low nonspecific binding illustrates the advantages of rational design based on recently determined GPCR X-ray structures. Such conjugates will be useful tools in expanding the SAR of this receptor, which still lacks chemical diversity in its collective ligands.

Deprotonation in Mixed-Valent Diiron(II,III) Complexes with Aniline or Benzimidazole Ligands.

We have previously investigated cis/trans isomerization processes in phenoxido-bridged mixed-valent Fe(II)Fe(III) complexes that contain either one aniline or one anilide ligand. In this work, we compare the properties of similar complexes bearing one terminal protic ligand, either aniline or 1H-benzimidazole. Whatever the ligand, (1)H NMR spectroscopy clearly evidences that the complexes are present in CH3CN as a mixture of cis- and trans-isomers in a close to 1:1 ratio. We show here that addition of NEt3 indeed allows the deprotonation of these ligands, the resulting complexes bearing either anilide or benzimidazolide that are coordinated to the ferric site. The latter are singular examples of a high-spin ferric ion coordinated to a benzimidazolide ligand. Whereas the trans-isomer of the anilide complex is the overwhelming species, benzimidazolide species are mixtures of cis- and trans-isomers in equal proportions. Moreover, cyclic voltammametry studies show that Fe(III)Fe(III) complexes with 1H-benzimidazole are more stable than their aniline counterparts, whereas the reverse is observed for the deprotonated species.

John Daly Lecture: Structure-guided Drug Design for Adenosine and P2Y Receptors.

We establish structure activity relationships of extracellular nucleosides and nucleotides at G protein-coupled receptors (GPCRs), e.g. adenosine receptors (ARs) and P2Y receptors (P2YRs), respectively. We synthesize selective agents for use as pharmacological probes and potential therapeutic agents (e.g. A3AR agonists for neuropathic pain). Detailed structural information derived from the X-ray crystallographic structures within these families enables the design of novel ligands, guides modification of known agonists and antagonists, and helps predict polypharmacology. Structures were recently reported for the P2Y12 receptor (P2Y12R), an anti-thrombotic target. Comparison of agonist-bound and antagonist-bound P2Y12R indicates unprecedented structural plasticity in the outer portions of the transmembrane (TM) domains and the extracellular loops. Nonphosphate-containing ligands of the P2YRs, such as the selective P2Y14R antagonist PPTN, are desired for bioavailability and increased stability. Also, A2AAR structures are effectively applied to homology modeling of closely related A1AR and A3AR, which are not yet crystallized. Conformational constraint of normally flexible ribose with bicyclic analogues increased the ligand selectivity. Comparison of rigid A3AR agonist congeners allows the exploration of interaction of specific regions of the nucleoside analogues with the target and off-target GPCRs, such as biogenic amine receptors. Molecular modeling predicts plasticity of the A3AR at TM2 to accommodate highly rigidified ligands. Novel fluorescent derivatives of high affinity GPCR ligands are useful tool compounds for characterization of receptors and their oligomeric assemblies. Fluorescent probes are useful for characterization of GPCRs in living cells by flow cytometry and other methods. Thus, 3D knowledge of receptor binding and activation facilitates drug discovery.

Enhancement of glucose uptake in mouse skeletal muscle cells and adipocytes by P2Y6 receptor agonists.

Glucose uptake by peripheral tissues such as skeletal muscles and adipocytes is important in the maintenance of glucose homeostasis. We previously demonstrated that P2Y6 receptor (P2Y6R) agonists protect pancreatic islet cells from apoptosis and stimulate glucose-dependent insulin release. Here, we investigated the effects of P2Y6R activation on glucose uptake in insulin target tissues. An agonist of the P2Y6R, P1-(5'-uridine)-P3-(5'-N4-methoxycytidine)-triphosphate (MRS2957), significantly increased the uptake of [3H]2-deoxyglucose in mouse C2C12 myotubes and 3T3-L1 adipocytes, and this stimulation was significantly decreased by a selective P2Y6R antagonist N,N″-1,4-butanediyl-bis[N'-(3-isothiocyanatophenyl)thiourea] (MRS2578). Pre-incubation with Compound C (an inhibitor of 5'-AMP-activated protein kinase, AMPK), or AMPK siRNA abolished the stimulatory effect of MRS2957 on glucose uptake. Also, MRS2957 (60 min incubation) increased recruitment of the facilitated glucose transporter-4 (GLUT4) to the cell membrane, which was blocked by MRS2578. Treatment of C2C12 myotubes with MRS2957 induced significant phosphorylation of AMPK, which increase GLUT4 expression through histone deacetylase (HDAC)5 signaling. Glucose uptake in primary mouse adipocytes from wild-type mice was stimulated upon P2Y6R activation by either MRS2957 or native agonist UDP, and the P2Y6R effect was antagonized by MRS2578. However, in adipocytes from P2Y6R-knockout mice P2Y6R agonists had no effect on glucose uptake, and there was no change in the glucose uptake by insulin. Our results indicate that the P2Y6R promotes glucose metabolism in peripheral tissues, which may be mediated through AMPK signaling.

Cis/trans isomerizations in diiron complexes involving aniline or anilide ligands.

We have recently reported a deprotonation-induced valence inversion within a phenoxido-bridged mixed-valent diiron(II,III) complex. The initial aniline coordinated to the Fe(II) site reacts with triethylamine, and the resulting complex contains an anilide ligand coordinated to the Fe(III) ion. The behavior of these complexes in acetonitrile is indeed more intricate. Owing to the very distinctive spectroscopic signatures of the complexes, the conjunction of NMR, Mössbauer, and UV-visible absorption spectroscopies allows one to evidence two isomerization reactions, one involving the aniline linked to Fe(II) and the other the anilide on Fe(III). Theoretical calculations sustain this conclusion. Aniline in the cis position versus the bridging phenoxide is shown to be the most stable isomer while the anilide trans to the phenoxido bridge is favored. The trans isomer of the aniline complex is more acidic than the cis one by 1 pKa unit. Isomerization of the anilide complex is 10 times faster than the analogous isomerization of the aniline complex. Both reactions are proposed to proceed through a unique mechanism. This is the first time that such isomerization reactions are evidenced in dinuclear complexes.

Probing biased/partial agonism at the G protein-coupled A(2B) adenosine receptor.

G protein-coupled A(2B) adenosine receptor (AR) regulates numerous important physiological functions, but its activation by diverse A(2B)AR agonists is poorly profiled. We probed potential partial and/or biased agonism in cell lines expressing variable levels of endogenous or recombinant A(2B)AR. In cAMP accumulation assays, both 5'-substituted NECA and C2-substituted MRS3997 are full agonists. However, only 5'-substituted adenosine analogs are full agonists in calcium mobilization, ERK1/2 phosphorylation and ß-arrestin translocation. A(2B)AR overexpression in HEK293 cells markedly increased the agonist potency and maximum effect in cAMP accumulation, but less in calcium and ERK1/2. A(2B)AR siRNA silencing was more effective in reducing the maximum cAMP effect of non-nucleoside agonist BAY60-6583 than NECA's. A quantitative 'operational model' characterized C2-substituted MRS3997 as either balanced (cAMP accumulation, ERK1/2) or strongly biased agonist (against calcium, ß-arrestin). N6-substitution biased against ERK1/2 (weakly) and calcium and ß-arrestin (strongly) pathways. BAY60-6583 is ERK1/2-biased, suggesting a mechanism distinct from adenosine derivatives. BAY60-6583, as A(2B)AR antagonist in MIN-6 mouse pancreatic ß cells expressing low A(2B)AR levels, induced insulin release. This is the first relatively systematic study of structure-efficacy relationships of this emerging drug target.

4-Alkyloxyimino derivatives of uridine-5'-triphosphate: distal modification of potent agonists as a strategy for molecular probes of P2Y2, P2Y4, and P2Y6 receptors.

Extended N(4)-(3-arylpropyl)oxy derivatives of uridine-5'-triphosphate were synthesized and potently stimulated phospholipase C stimulation in astrocytoma cells expressing G protein-coupled human (h) P2Y receptors (P2YRs) activated by UTP (P2Y2/4R) or UDP (P2Y6R). The potent P2Y4R-selective N(4)-(3-phenylpropyl)oxy agonist was phenyl ring-substituted or replaced with terminal heterocyclic or naphthyl rings with retention of P2YR potency. This broad tolerance for steric bulk in a distal region was not observed for dinucleoside tetraphosphate agonists with both nucleobases substituted. The potent N(4)-(3-(4-methoxyphenyl)-propyl)oxy analogue 19 (EC50: P2Y2R, 47 nM; P2Y4R, 23 nM) was functionalized for chain extension using click tethering of fluorophores as prosthetic groups. The BODIPY 630/650 conjugate 28 (MRS4162) exhibited EC50 values of 70, 66, and 23 nM at the hP2Y2/4/6Rs, respectively, and specifically labeled cells expressing the P2Y6R. Thus, an extended N(4)-(3-arylpropyl)oxy group accessed a structurally permissive region on three Gq-coupled P2YRs, and potency and selectivity were modulated by distal structural changes. This freedom of substitution was utilized to design of a pan-agonist fluorescent probe of a subset of uracil nucleotide-activated hP2YRs.

A dangerous twist of the 'T' wave: A case of Wellens' Syndrome.

Wellens' syndrome is a condition in which electrocardiographic (ECG) changes indicate critical proximal left anterior descending artery narrowing occurring during the chest pain-free period. Due to the severity of the obstruction, if such cases are managed by early invasive revascularisation therapy, a major threat in the form of a massive myocardial infarction or sudden death may be averted. We present the case of a patient with previous chest pain, whose ECG showing subtle ischemic changes was initially overlooked. A repeat ECG taken during the painless period showed a biphasic T wave, suggestive of Wellen's' syndrome. This was confirmed by an immediate coronary angiogram.

AMP-activated protein kinase as regulator of P2Y(6) receptor-induced insulin secretion in mouse pancreatic ß-cells.

5'-AMP-activated protein kinase (AMPK) and its pharmacological modulators have been targeted for treating type 2 diabetes. Extracellular uridine 5'-diphosphate (UDP) activates P2Y6 receptors (P2Y6Rs) in pancreatic ß-cells to release insulin and reduce apoptosis, which would benefit diabetes. Here, we studied the role of P2Y6R in activation of AMPK in MIN6 mouse pancreatic ß-cells and insulin secretion. Treatment with a potent P2Y6R dinucleotide agonist MRS2957 (500nM) activated AMPK, which was blocked by P2Y6R-selective antagonist MRS2578. Also, MRS2957 induced phosphorylation of acetyl-coenzyme A carboxylase (ACC), a marker of AMPK activity. Calcium chelator BAPTA-AM, calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-069 and IP3 receptor antagonist 2-APB attenuated P2Y6R-mediated AMPK phosphorylation revealing involvement of intracellular Ca(2+) pathways. P2Y6R agonist induced insulin secretion at high glucose, which was reduced by AMPK siRNA. Thus, P2Y6R has a crucial role in ß-cell function, suggesting its potential as a therapeutic target in diabetes.

A2B adenosine receptor blockade inhibits growth of prostate cancer cells.

The role of the A2B adenosine receptor (AR) in prostate cell death and growth was studied. The A2B AR gene expression quantified by real-time quantitative RT-PCR and Western blot analysis was the highest among four AR subtypes (A1, A2A, A2B, and A3) in all three commonly used prostate cancer cell lines, PC-3, DU145, and LNCaP. We explored the function of the A2B AR using PC-3 cells as a model. The A2B AR was visualized in PC-3 cells by laser confocal microscopy. The nonselective A2B AR agonist NECA and the selective A2B AR agonist BAY60-6583, but not the A2A AR agonist CGS21680, concentration-dependently induced adenosine 3',5'-cyclic monophosphate (cyclic AMP) accumulation. NECA diminished lactate dehydrogenase (LDH) release, TNF-α-induced increase of caspase-3 activity, and cycloheximide (CHX)-induced morphological changes typical of apoptosis in PC-3 cells, which were blocked by a selective A2B AR antagonist PSB603. NECA-induced proliferation of PC-3 cells was diminished by siRNA specific for the A2B AR. The selective A2B AR antagonist PSB603 was shown to inhibit cell growth in all three cell lines. Thus, A2B AR blockade inhibits growth of prostate cancer cells, suggesting selective A2B AR antagonists as potential novel therapeutics.

4-Alkyloxyimino-cytosine nucleotides: tethering approaches to molecular probes for the P2Y6 receptor.

4-Alkyloxyimino derivatives of pyrimidine nucleotides display high potency as agonists of certain G protein-coupled P2Y receptors (P2YRs). In an effort to functionalize a P2Y6R agonist for fluorescent labeling, we probed two positions (N4 and γ-phosphate of cytidine derivatives) with various functional groups, including alkynes for click chemistry. Functionalization of extended imino substituents at the 4 position of the pyrimidine nucleobase of CDP preserved P2Y6R potency generally better than γ-phosphoester formation in CTP derivatives. Fluorescent Alexa Fluor 488 conjugate 16 activated the human P2Y6R expressed in 1321N1 human astrocytoma cells with an EC50 of 9 nM, and exhibited high selectivity for this receptor over other uridine nucleotide-activated P2Y receptors. Flow cytometry detected specific labeling with 16 to P2Y6R-expressing but not to wild-type 1321N1 cells. Additionally, confocal microscopy indicated both internalized 16 (t1/2 of 18 min) and surface-bound fluorescence. Known P2Y6R ligands inhibited labeling. Theoretical docking of 16 to a homology model of the P2Y6R predicted electrostatic interactions between the fluorophore and extracellular portion of TM3. Thus, we have identified the N4-benzyloxy group as a structurally permissive site for synthesis of functionalized congeners leading to high affinity molecular probes for studying the P2Y6R.

Virtual screening leads to the discovery of novel non-nucleotide P2Y1 receptor antagonists.

The P2Y(1) receptor (P2Y(1)R) is a G protein-coupled receptor naturally activated by extracellular ADP. Its stimulation is an essential requirement of ADP-induced platelet aggregation, thus making antagonists highly sought compounds for the development of antithrombotic agents. Here, through a virtual screening campaign based on a pharmacophoric representation of the common characteristics of known P2Y(1)R ligands and the putative shape and size of the receptor binding pocket, we have identified novel antagonist hits of µM affinity derived from a N,N'-bis-arylurea chemotype. Unlike the vast majority of known P2Y(1)R antagonists, these drug-like compounds do not have a nucleotidic scaffold or highly negatively charged phosphate groups. Hence, our compounds may provide a direction for the development of receptor probes with altered physicochemical properties.

Novel fluorescent antagonist as a molecular probe in A(3) adenosine receptor binding assays using flow cytometry.

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.

G protein-coupled adenosine (P1) and P2Y receptors: ligand design and receptor interactions.

The medicinal chemistry and pharmacology of the four subtypes of adenosine receptors (ARs) and the eight subtypes of P2Y receptors (P2YRs, activated by a range of purine and pyrimidine mono- and dinucleotides) has recently advanced significantly leading to selective ligands. X-ray crystallographic structures of both agonist- and antagonist-bound forms of the A(2A)AR have provided unprecedented three-dimensional detail concerning molecular recognition in the binding site and the conformational changes in receptor activation. It is apparent that this ubiquitous cell signaling system has implications for understanding and treating many diseases. ATP and other nucleotides are readily released from intracellular sources under conditions of injury and organ stress, such as hypoxia, ischemia, or mechanical stress, and through channels and vesicular release. Adenosine may be generated extracellularly or by cellular release. Therefore, depending on pathophysiological factors, in a given tissue, there is often a tonic activation of one or more of the ARs or P2YRs that can be modulated by exogenous agents for a beneficial effect. Thus, this field has provided fertile ground for pharmaceutical development, leading to clinical trials of selective receptor ligands as imaging agents or for conditions including cardiac arrhythmias, ischemia/reperfusion injury, diabetes, pain, thrombosis, Parkinson's disease, rheumatoid arthritis, psoriasis, dry eye disease, pulmonary diseases such as cystic fibrosis, glaucoma, cancer, chronic hepatitis C, and other diseases.

Proton-coupled intervalence charge transfer: concerted processes.

The kinetics of proton-induced intervalence charge transfer (IVCT) may be measured electrochemically by generating one of the members of the IVCT couple in situ and following its conversion by means of the electrochemical signature of the other member of the couple. In the case of the diiron complex taken as an example, the reaction kinetics analysis, including the H/D isotope effect, clearly points to the prevalence of the concerted proton-intervalence charge transfer pathway over the stepwise pathways. A route is thus open toward systematic kinetic studies of proton-induced IVCT aiming at uncovering the main reactivity parameters and the factors that control the occurrence of concerted versus stepwise pathways.

Pyrimidine ribonucleotides with enhanced selectivity as P2Y(6) receptor agonists: novel 4-alkyloxyimino, (S)-methanocarba, and 5'-triphosphate gamma-ester modifications.

The P2Y(6) receptor is a cytoprotective G-protein-coupled receptor (GPCR) activated by UDP (EC(50) = 0.30 microM). We compared and combined modifications to enhance P2Y(6) receptor agonist selectivity, including ribose ring constraint, 5-iodo and 4-alkyloxyimino modifications, and phosphate modifications such as alpha,beta-methylene and extension of the terminal phosphate group into gamma-esters of UTP analogues. The conformationally constrained (S)-methanocarba-UDP is a full agonist (EC(50) = 0.042 microM). 4-Methoxyimino modification of pyrimidine enhanced P2Y(6), preserved P2Y(2) and P2Y(4), and abolished P2Y(14) receptor potency, in the appropriate nucleotide. N(4)-Benzyloxy-CDP (15, MRS2964) and N(4)-methoxy-Cp(3)U (23, MRS2957) were potent, selective P2Y(6) receptor agonists (EC(50) of 0.026 and 0.012 microM, respectively). A hydrophobic binding region near the nucleobase was explored with receptor modeling and docking. UTP-gamma-aryl and cycloalkyl phosphoesters displayed only intermediate P2Y(6) receptor potency but had enhanced stability in acid and cell membranes. UTP-glucose was inactive, but its (S)-methanocarba analogue and N(4)-methoxycytidine 5'-triphospho-gamma-[1]glucose were active (EC(50) of 2.47 and 0.18 microM, respectively). Thus, the potency, selectivity, and stability of pyrimidine nucleotides as P2Y(6) receptor agonists may be enhanced by modest structural changes.

Structure-activity relationship of (N)-Methanocarba phosphonate analogues of 5'-AMP as cardioprotective agents acting through a cardiac P2X receptor.

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.