A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes.

Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.

UV-induced hydrogen transfer in DNA base pairs promoted by dark nπ* states.

Dark nπ* states were shown to have substantial contribution to the destructive photochemistry of pyrimidine nucleobases. Based on quantum-chemical calculations, we demonstrate that the characteristic hydrogen bonding pattern of the GC base pair could facilitate the formation of a wobble excited-state charge-transfer complex. This entails a barrierless electron-driven proton transfer (EDPT) process which enables damageless photodeactivation of the base pair. These photostabilizing properties are retained even when guanine is exchanged to hypoxanthine. The inaccessibility of this process in the AT base pair sheds further light on the reasons why cytosine is less susceptible to the formation of photodimers in double-stranded DNA.

Tracking the origin of photostability in purine nucleobases: the photophysics of 2-oxopurine.

This work scrutinizes the relaxation mechanism of 2-oxopurine. Contrary to its ancestor, purine, which is a UVC chromophore, 2-oxopurine shows a red-shifted absorption spectrum centered in the UVA region. In 2-oxopurine, relaxation along the ππ* spectroscopic state directs the population from the Franck-Condon (FC) region towards a minimum, which acts as a crossroad for the further decay of the system either to triplet states or, alternatively, to the ground state through a C6-puckered S1/S0 funnel. A comparison of the optical properties and excited state potential energy surfaces of purine, 2-oxopurine, 2-aminopurine, 6-oxopurine and adenine, allows establishing how the position and nature of substituent tune the photophysics of purine. For this series, we conclude that both C2 and C6 substitution redshift the absorption spectrum of purine, with 2-oxo substitution exhibiting the largest shift. An important exception is the canonical nucleobase adenine, which presents a blue shifted absorption spectrum. The topography of purine's ππ* potential energy surface experiences major changes when functionalized at the C6 position. In particular, the disappearance of the minimum along the ππ* potential energy surface efficiently funnels the excited state population from the FC region to the ground state and increases the photostability of 6-aminopurine (adenine) and 6-oxopurine (hypoxanthine) nucleobases.

Design, synthesis of novel purin-6-one derivatives as phosphodiesterase 2 (PDE2) inhibitors: The neuroprotective and anxiolytic-like effects.

Phosphodiesterase 2 (PDE2) has received much attention for the potential treatment of the central nervous system (CNS) disorders. Herein, based on the existing PDE2 inhibitors and their binding modes, a series of purin-6-one derivatives were designed, synthesized and evaluated for PDE2 inhibitory activities, which led to the discovery of the best compounds 6p and 6s with significant inhibitory potency (IC50: 72 and 81 nM, respectively). Docking simulation was performed to insert compound 6s into the crystal structure of PDE2 at the active site to determine the binding mode. Furthermore, compound 6s significantly protected HT-22 cells against corticosterone-induced cytotoxicity and rescued corticosterone-induced decreases in cAMP and cGMP levels. It also produced anxiolytic-like effect in the elevated plus-maze test and exhibited favorable pharmacokinetic properties in vivo. These results might bring significant instruction for further development of potent PDE2 inhibitors.

Purin-6-one and pyrrolo[2,3-d]pyrimidin-4-one derivatives as potentiating agents of doxorubicin cytotoxicity.

AIM: DNA damage response plays an eminent role in patients' response to conventional chemotherapy and radiotherapy. Its inhibition is of great interest as it can overcome cancer cell resistance and reduce the effective doses of DNA damaging agents. Results & methodology: We have focused our research on phosphatidylinositol 3-kinase-related kinases and prepared 35 novel compounds through a scaffold hopping approach. The newly synthesized inhibitors were tested on a panel of nine cancer and one healthy cell lines alone and in combination with appropriate doses of doxorubicin. CONCLUSION: Five novel compounds 4f, 10b, 15g, 7e and 15f in combination with doxorubicin showed significant antiproliferative effect on seven cancer cell lines while not affecting the cell growth alone.

Novel butanehydrazide derivatives of purine-2,6-dione as dual PDE4/7 inhibitors with potential anti-inflammatory activity: Design, synthesis and biological evaluation.

A novel butanehydrazide derivatives of purine-2,6-dione designed using a ligand-based approach were synthesized and their in vitro activity against both PDE4B and PDE7A isoenzymes was assessed. The 7,8-disubstituted purine-2,6-dione derivatives 31, 34, 37, and 40 appeared to be the most potent PDE4/7 inhibitors with IC50 values in the range of that of the reference rolipram and BRL-50481, respectively. Moreover, docking studies explained the importance of N-(2,3,4-trihydroxybenzylidene)butanehydrazide substituent in position 7 of purine-2,6-dione core for dual PDE4/7 inhibitory properties. The inhibition of both the cAMP-specific PDE isoenzymes resulted in a strong anti-TNF-α effect. Compounds 31, 34, and 37 in the in vivo study in rats with LPS-induced endotoxemia decreased the maximum concentration of this proinflammatory cytokine by 53, 84 and 88%, respectively.

Synthesis and biological evaluation of 2-fluoro and 3-trifluoromethyl-phenyl-piperazinylalkyl derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione as potential antidepressant agents.

A series of 2-fluoro and 3-trifluoromethylphenylpiperazinylalkyl derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (4-21) were synthesized and evaluated for their serotonin (5-HT1A/5-HT7) receptor affinity and phosphodiesterase (PDE4B and PDE10A) inhibitor activity. The study enabled the identification of potent 5-HT1A, 5-HT7 and mixed 5-HT1A/5-HT7 receptor ligands with weak inhibitory potencies for PDE4B and PDE10A. The tests have been completed with the determination of lipophilicity and metabolic stability using micellar electrokinetic chromatography (MEKC) system and human liver microsomes (HLM) model. In preliminary pharmacological in vivo studies, selected compound 8-(5-(4-(2-fluorophenyl)piperazin-1-yl)pentyl)-1,3,7-trimethyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (9) behaved as a potential antidepressant in forced swim test (FST) in mice. Moreover, potency of antianxiety effects evoked by 9 (2.5 mg/kg) is greater than that of the reference anxiolytic drug, diazepam. Molecular modeling revealed that fluorinated arylpiperazinylalkyl derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione have major significance for the provision of lead compounds for antidepressant and/or anxiolytic application.

Discovery, synthesis and biochemical profiling of purine-2,6-dione derivatives as inhibitors of the human poly(A)-selective ribonuclease Caf1.

Eukaryotic mRNA contains a 3' poly(A) tail, which plays important roles in the regulation of mRNA stability and translation. Well-characterized enzymes involved in the shortening of the poly(A) tail include the multi-subunit Ccr4-Not deadenylase, which contains the Caf1 (Pop2) and Ccr4 catalytic components, and poly(A)-specific ribonuclease (PARN). Two Mg(2+) ions present in the active sites of these ribonucleases are required for RNA cleavage. Here, we report the discovery, synthesis and biochemical profiling of purine-2,6-dione derivatives as (sub)micromolar inhibitors of Caf1.

Crucial positively charged residues for ligand activation of the GPR35 receptor.

GPR35 is a G protein-coupled receptor expressed in the immune, gastrointestinal, and nervous systems in gastric carcinomas and is implicated in heart failure and pain perception. We investigated residues in GPR35 responsible for ligand activation and the receptor structure in the active state. GPR35 contains numerous positively charged amino acids that face into the binding pocket that cluster in two distinct receptor regions, TMH3-4-5-6 and TMH1-2-7. Computer modeling implicated TMH3-4-5-6 for activation by the GPR35 agonists zaprinast and pamoic acid. Mutation results for the TMH1-2-7 region of GPR35 showed no change in ligand efficacies at the K1.32A, R2.65A, R7.33A, and K7.40A mutants. However, mutation of arginine residues in the TMH3-4-5-6 region (R4.60, R6.58, R3.36, R(164), and R(167) in the EC2 loop) had effects on signaling for one or both agonists tested. R4.60A resulted in a total ablation of agonist-induced activation in both the ß-arrestin trafficking and ERK1/2 activation assays. R6.58A increased the potency of zaprinast 30-fold in the pERK assay. The R(167)A mutant decreased the potency of pamoic acid in the ß-arrestin trafficking assay. The R(164)A and R(164)L mutants decreased potencies of both agonists. Similar trends for R6.58A and R(167)A were observed in calcium responses. Computer modeling showed that the R6.58A mutant has additional interactions with zaprinast. R3.36A did not express on the cell surface but was trapped in the cytoplasm. The lack of surface expression of R3.36A was rescued by a GPR35 antagonist, CID2745687. These results clearly show that R4.60, R(164), R(167), and R6.58 play crucial roles in the agonist initiated activation of GPR35.

The formamidopyrimidines: purine lesions formed in competition with 8-oxopurines from oxidative stress.

DNA is constantly exposed to agents that induce structural damage, from sources both internal and external to an organism. Endogenous species, such as oxidizing chemicals, and exogenous agents, such as ultraviolet rays in sunlight, together produce more than 70 distinct chemical modifications of native nucleotides. Of these, about 15 of the lesions have been detected in cellular DNA. This kind of structural DNA damage can be cytotoxic, carcinogenic, or both and is being linked to an increasingly lengthy list of diseases. The formamidopyrimidine (Fapy) lesions are a family of DNA lesions that result after purines undergo oxidative stress. The Fapy lesions are produced in yields comparable to the 8-oxopurines, which, owing in part to a perception of mutagenicity in some quarters, have been subjected to intense research scrutiny. But despite the comparable abundance of the formamidopyrimidines and the 8-oxopurines, until recently very little was known about the effects of Fapy lesions on biochemical processes involving DNA or on the structure and stability of the genomic material. In this Account, we discuss the detection of Fapy lesions in DNA and the mechanism proposed for their formation. We also describe methods for the chemical synthesis of oligonucleotides containing Fapy·dA or Fapy·dG and the outcomes of chemical and biochemical studies utilizing these compounds. These experiments reveal that the formamidopyrimidines decrease the fidelity of polymerases and are substrates for DNA repair enzymes. The mutation frequency of Fapy·dG in mammals is even greater than that of 8-oxodGuo (8-oxo-7,8-dihydro-2'-deoxyguanosine, one of the 8-oxopurines), suggesting that this lesion could be a useful biomarker and biologically significant. Despite clear similarities, the formamidopyrimidines have lived in the shadow of the corresponding 8-oxopurine lesions. But the recent development of methods for synthesizing oligonucleotides containing Fapy·dA or Fapy·dG has accelerated research on these lesions, revealing that the formamidopyrimidines are repaired as efficiently and, in some cases, more rapidly than the 8-oxopurines. Fapy·dG appears to be a lesion of biochemical consequence, and further study of its mutagenicity, repair, and interactions with DNA structure will better define the cellular details involving this important product of DNA stress.

Idiopathic Recurrent Calcium Urolithiasis (IRCU): pathophysiology evaluated in light of oxidative metabolism, without and with variation of several biomarkers in fasting urine and plasma--a comparison of stone-free and -bearing male patients, emphasizing mineral, acid-base, blood pressure and protein status.

BACKGROUND: IRCU is traditionally considered as life?style disease (associations with, among others, overweight, obesity, hypertension, type-2 diabetes), arising from excess, in 24 h urine, of calcium (Ca) salts (calcium oxalate (CaOx), calcium phosphate (CaPi)), supersaturation of, and crystallization in, tubular fluid and urine, causing crystal-induced epithelial cell damage, proteinuria, crystal aggregation and uroliths. METHODS: Another picture emerges from the present uncontrolled study of 154 male adult IRCU patients (75 stone-bearing (SB) and 79 age-matched stone-free (SF)), in whom stone-forming and other parameters in fasting urine and plasma were contrasted with five biomarkers (see footnote) of oxidative metabolism (OM), without and with variation of markers. RESULTS: 1) In SB vs. SF unstratified OM biomarkers were statistically unchanged, but the majority of patients was overweight; despite, in SB vs. SF urine pH, total and non-albumin protein concentration were elevated, fractional urinary uric acid excretion and blood bicarbonate decreased, whereas urine volume, sodium, supersaturation with CaOx and CaPi (as hydroxyapatite) were unchanged; 2) upon variation of OM markers (strata below and above median) numerous stone parameters differed significantly, among others urine volume, total protein, Ca/Pi ratio, pH, sodium, potassium, plasma Ca/Pi ratio and parathyroid hormone, blood pressure, renal excretion of non-albumin protein and other substances; 3) a significant shift from SF to SB patients occurred with increase of urine pH, decrease of blood bicarbonate, and increase of diastolic blood pressure, whereas increase of plasma uric acid impacted only marginally; 4) in both SF and SB patients a strong curvilinear relationship links a rise of urine Ca/Pi to urine Ca/Pi divided by plasma Ca/Pi, but in SB urine Ca/Pi failed to correlate significantly with urine hydroxyapatite supersaturation; 5) also in SB, plasma Ca/Pi and urinary nitrate were negatively correlated, whereas in SF plasma Ca/Pi ratio, PTH and body mass index correlated positively; 6) multivariate regression analysis revealed that PTH, body mass index and nitrate together could explain 22 (p = 0.002) and only 7 (p = 0.06) per cent of variation of plasma Ca/Pi in SF and SB, respectively. CONCLUSIONS: In IRCU a) numerous constituents of fasting urine, plasma, blood and blood pressure change in response to variation of OM biomarkers, suggesting involvement of OM imbalance as factor in functional deterioration of tissue; b) in the majority of patients a positive exponential relationship links urine Ca/Pi to urine Ca/Pi divided by plasma Ca/Pi, presumably to accumulate Ca outside tubular lumen, thereby minimizing intratubular and urinary Ca salt crystallization; c) alteration of interactions of low urine nitrate, PTH and Ca/Pi in plasma may be of importance in formation of new Ca stone and co-regulation of dynamics of blood vasculature; d) overweight, combined with OM-modified renal interstitial environment appears to facilitate these processes, carrying the risk that CaPi mineral develops within or/and close to blood vessel tissue, and spreads towards urothelium. - For future research focussing on IRCU pathogenesis studies are recommended on the role of affluent lifestyle mediated renal ischemia, mild hypertensive nephropathy, rise of uric acid precursor oxypurines and uricemia, clarifying also why loss of significance of interrelationships of OM biomarkers with traditional Ca stone risk factors is characteristic for SB patients.

Synthesis and evaluation of novel carbon-11 labeled oxopurine analogues for positron emission tomography imaging of translocator protein (18 kDa) in peripheral organs.

To develop a PET ligand for imaging TSPO in peripheral organs, we designed three novel oxopurine analogues [(11)C]3a-c (LogD: 1.81-2.17) by introducing a pyridine ring in place of a benzene ring in the lead compound [(11)C]2 (LogD: 3.48). The desmethyl precursors 10 for radiosynthesis were synthesized by reacting glycine 7 with picolylamines 4, followed by hydrolysis and by Curtius rearrangement with diphenylphosphoryl azide. Methylation of 10a-c with methyl iodide produced unlabeled compounds 3a-c. The radiosynthesis of [(11)C]3a-c was performed by reacting 10a-c with [(11)C]methyl iodide. Compounds 3a-c displayed high or moderate in vitro binding affinities (K(i): 5-40 nM) for TSPO. PET with [(11)C]3a-c in rats showed high uptake in the lung, heart, and kidney, which are organs with high TSPO expression. Metabolite analysis with [(11)C]3a showed that radioactivity in these organs mainly corresponded with unchanged [(11)C]3a. PET with [(11)C]3a using a rat model of lung inflammation showed a significant signal in the lipopolysaccharide-treated lung.

Structural asymmetry of phosphodiesterase-9, potential protonation of a glutamic acid, and role of the invariant glutamine.

PDE9 inhibitors show potential for treatment of diseases such as diabetes. To help with discovery of PDE9 inhibitors, we performed mutagenesis, kinetic, crystallographic, and molecular dynamics analyses on the active site residues of Gln453 and its stabilizing partner Glu406. The crystal structures of the PDE9 Q453E mutant (PDE9Q453E) in complex with inhibitors IBMX and (S)-BAY73-6691 showed asymmetric binding of the inhibitors in two subunits of the PDE9Q453E dimer and also the significant positional change of the M-loop at the active site. The kinetic analysis of the Q453E and E406A mutants suggested that the invariant glutamine is critical for binding of substrates and inhibitors, but is unlikely to play a key role in the differentiation between substrates of cGMP and cAMP. The molecular dynamics simulations suggest that residue Glu406 may be protonated and may thus explain the hydrogen bond distance between two side chain oxygens of Glu453 and Glu406 in the crystal structure of the PDE9Q453E mutant. The information from these studies may be useful for design of PDE9 inhibitors.

Structure of neutral molecules and monoanions of selected oxopurines in aqueous solutions as studied by NMR spectroscopy and theoretical calculations.

A methodology enabling investigation of a multicomponent tautomeric and acid-base equilibria by (13)C NMR spectroscopy supported by theoretical calculations has been proposed. The effectiveness of this method has been illustrated in a study of 2-oxopurine, 6-oxopurine (hypoxanthine), 8-oxopurine, and 2,6-dioxopurine (xanthine) in neutral and alkaline aqueous solutions. For each compound a series of (13)C NMR spectra were recorded at pH ranges in which neutral molecules, monoanions and/or dianions occurred in dynamic equilibrium. The carbon chemical shifts for these three forms of the investigated compounds were retrieved from the analysis of pH-dependence of the measured, dynamically averaged values of these parameters. The structures of several stable tautomers of the neutral and monoanionic oxopurine forms were predicted from theoretical calculations and nuclear magnetic shielding constants for (13)C nuclei in these tautomers were calculated. At both calculation steps (molecular geometry optimization and calculation of NMR parameters) the PBE1PBE/6-311++G(2d,p) level of theory was used. The populations of the most stable tautomers were determined from the experimental data analysis exploiting the fact that they were population-weighted averages of the chemical shifts of particular tautomers. It has been shown that only the oxo forms of the investigated oxopurines are present in aqueous solutions and that the determined populations in most cases remain in a qualitative agreement with the calculated free energies of the appropriate tautomers. The obtained results are in general agreement with other literature reports on oxopurine tautomerism and confirm importance of the hydration phenomena for the investigated systems. The data analysis has shown that the best compliance between theory and experiment is obtained when the hydration phenomenon is modeled by discrete hydration augmented by PCM (polarizable continuum solvation model).

Structure and mechanism of the 6-oxopurine nucleosidase from Trypanosoma brucei brucei.

Trypanosomes are purine-auxotrophic parasites that depend upon nucleoside hydrolase (NH) activity to salvage nitrogenous bases necessary for nucleic acid and cofactor synthesis. Nonspecific and purine-specific NHs have been widely studied, yet little is known about the 6-oxopurine-specific isozymes, although they are thought to play a primary role in the catabolism of exogenously derived nucleosides. Here, we report the first functional and structural characterization of the inosine-guanosine-specific NH from Trypanosoma brucei brucei. The enzyme shows near diffusion-limited efficiency coupled with a clear specificity for 6-oxopurine nucleosides achieved through a catalytic selection of these substrates. Pre-steady-state kinetic analysis reveals ordered product release, and a rate-limiting structural rearrangement that is associated with the release of the product, ribose. The crystal structure of this trypanosomal NH determined to 2.5 Å resolution reveals distinctive features compared to those of both purine- and pyrimidine-specific isozymes in the framework of the conserved and versatile NH fold. Nanomolar iminoribitol-based inhibitors identified in this study represent important lead compounds for the development of novel therapeutic strategies against trypanosomal diseases.

Imidazopurinones are markers of physiological genomic damage linked to DNA instability and glyoxalase 1-associated tumour multidrug resistance.

Glyoxal and methylglyoxal are reactive dicarbonyl metabolites formed and metabolized in physiological systems. Increased exposure to these dicarbonyls is linked to mutagenesis and cytotoxicity and enhanced dicarbonyl metabolism by overexpression of glyoxalase 1 is linked to tumour multidrug resistance in cancer chemotherapy. We report herein that glycation of DNA by glyoxal and methylglyoxal produces a quantitatively important class of nucleotide adduct in physiological systems-imidazopurinones. The adduct derived from methylglyoxal-3-(2'-deoxyribosyl)-6,7-dihydro-6,7-dihydroxy-6/7-methylimidazo-[2,3-b]purine-9(8)one isomers-was the major quantitative adduct detected in mononuclear leukocytes in vivo and tumour cell lines in vitro. It was linked to frequency of DNA strand breaks and increased markedly during apoptosis induced by a cell permeable glyoxalase 1 inhibitor. Unexpectedly, the DNA content of methylglyoxal-derived imidazopurinone and oxidative marker 7,8-dihydro-8-oxo-2'-deoxyguanosine were increased moderately in glyoxalase 1-linked multidrug resistant tumour cell lines. Together these findings suggest that imidazopurinones are a major type of endogenous DNA damage and glyoxalase 1 overexpression in tumour cells strives to counter increased imidazopurinone formation in tumour cells likely linked to their high glycolytic activity.

Sildenafil protects epithelial cell through the inhibition of xanthine oxidase and the impairment of ROS production.

Xanthine oxidase (XO) plays an important role in various forms of ischemic and vascular injuries, inflammatory diseases and chronic heart failure. The XO inhibitors allopurinol and oxypurinol held considerable promise in the treatment of these conditions both in experimental animals and in human clinical trials. More recently, an endothelium-based protective effect of sildenafil has been reported in preconditioning prior to ischemia/reperfusion in healthy human subjects. Based on the structural similarities between allopurinol and oxypurinol with sildenafil and with zaprinast the authors have investigated the potential effects of these latter compounds on the buttermilk XO and on non-tumourigenic (HMEC) and malignant (MCF7) human mammary epithelial cells. Both sildenafil and zaprinast induced a significant and consistent decrease of XO expression and activity in either cell line. In MCF7 cells only, this effect was associated with the abrogation of xanthine-induced cytotoxicity. Overall, the data suggest that the protective effect of sildenafil on epithelial cells is a consequence of the inhibition of the XO and of the resulting decrease of free oxygen radical production that may influence the expression of NADPH oxidase and PDE-5.

Structures, ionization equilibria, and tautomerism of 6-oxopurines in solution.

6-Oxopurine and its analogues form an important class of biological molecules that include nucleobases and their precursors and are substrates of a wide range of enzymes. Solution structures of purines have been debated in the literature because of the many possible tautomers and protonation states in which they can exist in solution. Substitutions on the pyrimidine and imidazole rings alter tautomerization and protonation equilibria, and as a consequence, the solution compositions and structures of closely related analogues can be significantly different. We have obtained resonance Raman spectra of 6-oxopurines: hypoxanthine, xanthine, their riboside phosphates, guanine monophosphate in the protonated and deprotonated forms with UV excitation at 260 nm. The species present in solution under different pH conditions were identified by isotopic labeling with deuterium as well as by comparison with extensive density functional theoretical calculations. At physiological pH, while N7H and N9H tautomeric forms of hypoxanthine exist in equilibrium, in xanthine, the additional carbonyl group at C2 shifts the equilibrium in favor of the N7H tautomer. The corresponding nucleotide of xanthine, xanthosine monophosphate, on the other hand, is in the anionic form (pK(a) 5.5). We find that Raman spectra show systematic shifts with change in the protonation state and substitution on the ring. In general, deprotonation of the neutral molecule is marked by a downshift in the observed Raman wavenumbers, and protonation is accompanied by an upshift.

A simplified green chemistry approaches to synthesis of 2-substituted 1,2,3-triazoles and 4-amino-5-cyanopyrazole derivatives conventional heating versus microwave and ultrasound as ecofriendly energy sources.

Cyanoacetamides 3 were prepared via reacting ethyl cyanoacetate with benzylamine. Yields and reaction times needed for reaction completion at room temperature, by microwaves (muomega) heating and under ultrasound (US) irradiations are compared. The formed cyanoacetamides were coupled with aromatic diazonium salts and the formed arylhydrazones were used as precursors to title triazoles and pyrazoles via reacting the former with hydroxylamine and chloroacetonitrile. Yields of products formed via conventional heating are compared with those of muomega and US irradiation.

Structure-activity relationship studies of a new series of imidazo[2,1-f]purinones as potent and selective A(3) adenosine receptor antagonists.

We recently described the synthesis of 1-benzyl-3-propyl-1H,8H-imidazo[2,1-f]purine-2,4-diones, new potent and selective A(3) adenosine receptor antagonists containing a xanthine core. The present work can be considered an extension of our SAR studies on related structures in which the effect of different kind of substitutions at the 1-, 3- and 8-positions has been evaluated in order to improve both the potency and the hydrophilicity of the originally synthesised molecules. The A(3) binding disposition of these compounds was also investigated through docking and 3D-QSAR studies.