Synthesis, characterization, and in vitro evaluation of the selective P2Y2 receptor antagonist AR-C118925.

The Gq protein-coupled, ATP- and UTP-activated P2Y2 receptor is a potential drug target for a range of different disorders, including tumor metastasis, inflammation, atherosclerosis, kidney disorders, and osteoporosis, but pharmacological studies are impeded by the limited availability of suitable antagonists. One of the most potent and selective antagonists is the thiouracil derivative AR-C118925. However, this compound was until recently not commercially available and little is known about its properties. We therefore developed an improved procedure for the synthesis of AR-C118925 and two derivatives to allow up-scaling and assessed their potency in calcium mobilization assays on the human and rat P2Y2 receptors recombinantly expressed in 1321N1 astrocytoma cells. The compound was further evaluated for inhibition of P2Y2 receptor-induced ß-arrestin translocation. AR-C118925 behaved as a competitive antagonist with pA 2 values of 37.2 nM (calcium assay) and 51.3 nM (ß-arrestin assay). Selectivity was assessed vs. related receptors including P2X, P2Y, and adenosine receptor subtypes, as well as ectonucleotidases. AR-C118925 showed at least 50-fold selectivity against the other investigated targets, except for the P2X1 and P2X3 receptors which were blocked by AR-C118925 at concentrations of about 1 µM. AR-C118925 is soluble in buffer at pH 7.4 (124 µM) and was found to be metabolically highly stable in human and mouse liver microsomes. In Caco2 cell experiments, the compound displayed moderate permeability indicating that it may show limited peroral bioavailability. AR-C118925 appears to be a useful pharmacological tool for in vitro and in vivo studies.

Focused screening to identify new adenosine kinase inhibitors.

Adenosine kinase (AdK) is a key player in controlling intra- and extracellular concentrations of the signaling molecule adenosine. Extensive evidence points to an important role of AdK in several diseases, and suggests that AdK inhibition might be a promising therapeutic strategy. The development of a new AdK assay and subsequent screening of part of our focused compound library led to the identification of 12 hit compounds (hit rate of 6%) representing six new classes of non-nucleoside human AdK inhibitors. The most potent inhibitor 1 displayed a Ki value of 184nM. Compound screening with a newly developed assay was useful and efficient for discovering novel AdK inhibitors which may serve as lead structures for developing drugs for adenosine augmentation therapy.

A novel receptor cross-talk between the ATP receptor P2Y2 and formyl peptide receptors reactivates desensitized neutrophils to produce superoxide.

Neutrophils express several G-protein coupled receptors (GPCRs) and they cross regulate each other. We described a novel cross-talk mechanism in neutrophils, by which signals generated by the receptor for ATP (P2Y2) reactivate desensitized formyl peptide receptors (FPRs) so that these ligand-bound inactive FPRs resume signaling. At the signaling level, the cross-talk was unidirectional, i.e., P2Y2 ligation reactivated FPR, but not vice versa and was sensitive to the phosphatase inhibitor calyculinA. Further, we show that the cross talk between P2Y2 and FPR bypassed cytosolic Ca(2+) transients and did not rely on the actin cytoskeleton. In summary, our data demonstrate a novel cross-talk mechanism that results in reactivation of desensitized FPRs and, an amplification of the neutrophil response to ATP.

Cardiomyocyte death induced by ischaemic/hypoxic stress is differentially affected by distinct purinergic P2 receptors.

Blood levels of extracellular nucleotides (e.g. ATP) are greatly increased during heart ischaemia, but, despite the presence of their specific receptors on cardiomyocytes (both P2X and P2Y subtypes), their effects on the subsequent myocardial damage are still unknown. In this study, we aimed at investigating the role of ATP and specific P2 receptors in the appearance of cell injury in a cardiac model of ischaemic/hypoxic stress. Cells were maintained in a modular incubator chamber in a controlled humidified atmosphere of 95% N(2) for 16 hrs in a glucose-free medium. In this condition, we detected an early increase in the release of ATP in the culture medium, which was followed by a massive increase in the release of cytoplasmic histone-associated-DNA-fragments, a marker of apoptosis. Addition of either apyrase, which degrades extracellular ATP, or various inhibitors of ATP release via connexin hemichannels fully abolished ischaemic/hypoxic stress-associated apoptosis. To dissect the role of specific P2 receptor subtypes, we used a combined approach: (i) non-selective and, when available, subtype-selective P2 antagonists, were added to cardiomyocytes before ischaemic/hypoxic stress; (ii) selected P2 receptors genes were silenced via specific small interfering RNAs. Both approaches indicated that the P2Y(2) and P2χ(7) receptor subtypes are directly involved in the induction of cell death during ischaemic/hypoxic stress, whereas the P2Y(4) receptor has a protective effect. Overall, these findings indicate a role for ATP and its receptors in modulating cardiomyocyte damage during ischaemic/hypoxic stress.

Preparation of enantiopure (R)-hydroxy metabolite of denbufylline using immobilized Lactobacillus kefiri DSM 20587 as a catalyst.

Lactobacillus kefiri DSM 20587 cells were immobilized in calcium alginate and carrageenan. The immobilized cells were used as biocatalysts for the enantioselective reduction of the methyl ketone group of denbufylline to synthesize the enantiopure (R)-hydroxy metabolite: (-)-1,3-dibutyl-7-((2'R)-hydroxypropyl)-1H-purine-2,6(3H,7H)-dione (1). The experimental conditions for the biotransformation were optimized. As denbufylline is insoluble in aqueous media, the influence of cosolvents (dimethylsulfoxide (DMSO), acetonitrile) and different concentrations of each solvent in the reaction mixture on the yield and enantiomeric excess of the final biotransformation product was studied. The maximum biotransformation yield (96-98%) and highest enantioselectivity (96% ee) for the obtained metabolite were reached using DMSO as a cosolvent at a concentration of 7.5% (v/v) in the presence of L. kefiri immobilized either in calcium alginate or in carrageenan. The absolute configuration of the stereogenic center of 1 was determined by applying Mosher's method.

Recent progress in the development of adenosine receptor ligands as antiinflammatory drugs.

Adenosine receptors belong to the family of G protein-coupled receptors. Four distinct subtypes are known, termed A(1), A(2A), A(2B) and A(3). Adenosine is an important signaling molecule which is released under inflammatory conditions. It can show antiinflammatory as well as proinflammatory activities, and the contribution of the specific adenosine receptor subtypes in various cells, tissues and organs is complex. Agonists selective for adenosine A(1) receptors show antinociceptive activity and are active in animal models of neuropathic and inflammatory pain. Adenosine A(2A) receptor agonists are potent antiinflammatory drugs. A(2A)-selective antagonists have shown antihyperalgesic activity in animal models of inflammatory pain. For A(2B)agonists as well as A(2B) antagonists antiinflammatory activity has been postulated. Selective A(2B) antagonists were shown to decrease (inflammatory) pain, and are promising candidates for the treatment of asthma. Adenosine A(3) receptor agonists appear to be proinflammatory, while there is evidence for an antiinflammatory effect of A(3) antagonists. There are some contradictory findings, and A(3) agonists are being developed for the treatment of inflammatory diseases such as arthritis. Indirect mechanisms increasing the extracellular concentration of adenosine using adenosine kinase inhibitors, adenosine deaminase inhibitors or adenosine uptake inhibitors, or increasing the potency of adenosine at the A(1) receptor subtype by allosteric modulators lead to potent antinociceptive and antiinflammatory activity. The advantage of indirectly acting drugs may be their site- and event-specific action since they are only active where adenosine has been released. In the past decade considerable progress has been made towards the identification of novel lead structures and the development of potent and selective ligands for all four adenosine receptor subtypes. A large number of patents has recently been filed and the field is finally in the process of translating many years of basic science into therapeutic application. This review article will focus on compounds published or patented within the past three years.

Microwave-assisted ring closure reactions: synthesis of 8-substituted xanthine derivatives and related pyrimido- and diazepinopurinediones.

BACKGROUND: Poly-substituted xanthine derivatives are an important class of compounds in medicinal chemistry. Substitution at the 8-position of the purine ring is generally accessible by ring closure of a carboxamido-substituted uracil precursor. Although several procedures to accomplish this synthetic step have been suggested, it still remains difficult in many cases. RESULTS: Ring closure reaction with hexamethyldisilazane was studied under microwave conditions. Reaction times were dramatically reduced by the application of microwaves in the syntheses of the 8-styrylxanthine derivative istradefylline, and in the preparation of 2-substituted pyrimido [1,2,3-cd]purines. Furthermore, the new procedure allowed the preparation of a previously unaccessible diazepino [1,2,3-cd]purine. Yields were generally improved by the new method. The addition of THF as a co-solvent proved to be crucial. CONCLUSION: A new, fast, and efficient ring closure method for the imidazole ring of xanthine derivatives and related tricyclic compounds has been developed. Apart from improving the syntheses of known compounds, some of which are important pharmacological tools or in development as novel drugs, it allows the preparation of 2-substituted diazepino [1,2,3-cd]purines--a new class of tricyclic purine derivatives.

2-Amino[1,2,4]triazolo[1,5-c]quinazolines and Derived Novel Heterocycles: Syntheses and Structure-Activity Relationships of Potent Adenosine Receptor Antagonists.

2-Amino[1,2,4]triazolo[1,5-c]quinazolines were identified as potent adenosine receptor (AR) antagonists. Synthetic strategies were devised to gain access to a broad range of derivatives including novel polyheterocyclic compounds. Potent and selective A3 AR antagonists were discovered, including 3,5-diphenyl[1,2,4]triazolo[4,3-c]quinazoline (17, Ki human A3 AR 1.16 nm) and 5'-phenyl-1,2-dihydro-3'H-spiro[indole-3,2'-[1,2,4]triazolo[1,5-c]quinazolin]-2-one (20, Ki human A3 AR 6.94 nm). In addition, multitarget antagonists were obtained, such as the dual A1 /A3 antagonist 2,5-diphenyl[1,2,4]triazolo[1,5-c]quinazoline (13 b, Ki human A1 AR 51.6 nm, human A3 AR 11.1 nm), and the balanced pan-AR antagonists 5-(2-thienyl)[1,2,4]triazolo[1,5-c]quinazolin-2-amine (11 c, Ki human A1 AR 131 nm, A2A AR 32.7 nm, A2B AR 150 nm, A3 AR 47.5 nm) and 9-bromo-5-phenyl[1,2,4]triazolo[1,5-c]quinazolin-2-amine (11 q, Ki human A1 AR 67.7 nm, A2A AR 13.6 nm, A2B AR 75.0 nm, A3 AR 703 nm). In many cases, significantly different affinities for human and rat receptors were observed, which emphasizes the need for caution in extrapolating conclusions between different species.

Primary prevention of posttraumatic stress disorder: drugs and implications.

Because posttraumatic stress disorder (PTSD) is a highly debilitating condition, prevention is an important research topic. This article reviews possible prevention approaches that involve the administration of drugs before the traumatic event takes place. The considered approaches include drugs that address the sympathetic nervous system, drugs that interfere with the hypothalamic-pituitary-adrenal (HPA) axis, narcotics and other psychoactive drugs, as well as modulators of protein synthesis. Furthermore, some thoughts on potential ethical implications of the use of drugs for the primary prevention of PTDS are presented. While there are many barriers to overcome in this field of study, this paper concludes with a call for additional research, as there are currently no approaches that are well-suited for regular use.

P2Y2 and Gq/G11 control blood pressure by mediating endothelial mechanotransduction.

Elevated blood pressure is a key risk factor for developing cardiovascular diseases. Blood pressure is largely determined by vasodilatory mediators, such as nitric oxide (NO), that are released from the endothelium in response to fluid shear stress exerted by the flowing blood. Previous work has identified several mechanotransduction signaling processes that are involved in fluid shear stress-induced endothelial effects, but how fluid shear stress initiates the response is poorly understood. Here, we evaluated human and bovine endothelial cells and found that the purinergic receptor P2Y2 and the G proteins Gq/G11 mediate fluid shear stress-induced endothelial responses, including [Ca2+]i transients, activation of the endothelial NO synthase (eNOS), phosphorylation of PECAM-1 and VEGFR-2, as well as activation of SRC and AKT. In response to fluid shear stress, endothelial cells released ATP, which activates the purinergic P2Y2 receptor. Mice with induced endothelium-specific P2Y2 or Gq/G11 deficiency lacked flow-induced vasodilation and developed hypertension that was accompanied by reduced eNOS activation. Together, our data identify P2Y2 and Gq/G11 as a critical endothelial mechanosignaling pathway that is upstream of previously described mechanotransduction processes and demonstrate that P2Y2 and Gq/G11 are required for basal endothelial NO formation, vascular tone, and blood pressure.

Development of off-line and on-line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates.

Fast and convenient CE assays were developed for the screening of adenosine kinase (AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260 nm. An MEKC method using borate buffer (pH 9.5) containing 100 mM SDS (method A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH 7.5 or 8.5) was used and a constant current (95 microA) was applied (method B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10 min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method C). After hydrodynamic injection of a plug of reaction buffer (20 mM Tris-HCl, 0.2 mM MgCl2, pH 7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1 mM ATP, 100 microM adenosine, and 20 microM UMP as an internal standard (I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5 kV separation voltage (negative polarity) for 0.20 min to let the plugs interpenetrate. The voltage was turned off for 5 min (zero-potential amplification) and again turned on at a constant current of -60 microA to elute the products within 7 min. The method employing a polyacrylamide-coated capillary of 20 cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose-response curves and calculated K(i) values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay.

Multigram-scale syntheses, stability, and photoreactions of A2A adenosine receptor antagonists with 8-styrylxanthine structure: potential drugs for Parkinson's disease.

The improved multigram-scale syntheses of the important 8-styrylxanthine A(2A) adenosine receptor antagonist MSX-2 (8), its water-soluble prodrug MXS-3 (9), and KW-6002 (16) are described. N-Alkylation reactions at different positions of uracil derivatives were optimized. Two different methods for xanthine formation from 6-amino-5-cinnamoylaminouracil precursors were investigated, (a) the elimination of water by alkaline catalysis and (b) hexamethyldisilazane as a condensing agent; the latter was found to be superior. The photosensitivity of 8-styrylxanthines was studied. The (E)-configurated stryrylxanthine MSX-2 (8) isomerized in diluted solution, and the resulting (Z)-isomer (10a) was isolated and characterized. Furthermore, we describe for the first time that solid 8-styrylxanthines can dimerize upon exposition to daylight or irradiation with UV light. The resulting cyclobutane derivatives with head-to-tail (syn) configuration exhibited a considerably lower A(2A) adenosine receptor affinity than their parent compounds. The dimerization product of MSX-2 was a moderately potent nonselective A(1) and A(2A) antagonist (K(i)(A(1)) = 273 nM, K(i) (A(2A)) = 175 nM) while the dimer of the related compound KW-6002 was inactive at A(1) and only weakly active at A(2A) adenosine receptors (K(i) = 1.57 microM). The light sensitivity of 8-styrylxanthine derivatives, not only in solution, but also in the solid state, has to be considered when using those compounds as pharmacological tools or drugs.

Adenosine receptor agonists: from basic medicinal chemistry to clinical development.

Adenosine is a physiological nucleoside which acts as an autocoid and activates G protein-coupled membrane receptors, designated A(1), A(2A), A(2B) and A(3). Adenosine plays an important role in many (patho)physiological conditions in the CNS as well as in peripheral organs and tissues. Adenosine receptors are present on virtually every cell. However, receptor subtype distribution and densities vary greatly. Adenosine itself is used as a therapeutic agent for the treatment of supraventricular paroxysmal tachycardia and arrhythmias and as a vasodilatatory agent in cardiac imaging. During the past 20 years, a number of selective agonists for A(1), A(2A) and A(3) adenosine receptors have been developed, all of them structurally derived from adenosine. Several such compounds are currently undergoing clinical trials for the treatment of cardiovascular diseases (A(1)and A(2A)), pain (A(1)), wound healing (A(2A)), diabetic foot ulcers (A(2A)), colorectal cancer (A(3)) and rheumatoid arthritis (A(3)). Clinical evaluation of some A(1) and A(2A) adenosine receptor agonists has been discontinued. Major problems include side effects due to the wide distribution of adenosine receptors; low brain penetration, which is important for the targeting of CNS diseases; short half-lifes of compounds; or a lack of effects, in some cases perhaps due to receptor desensitisation or to low receptor density in the targeted tissue. Partial agonists, inhibitors of adenosine metabolism (adenosine kinase and deaminase inhibitors) or allosteric activators of adenosine receptors may be advantageous for certain indications, as they may exhibit fewer side effects.