Synthesis and cellular characterization of novel isoxazolo- and thiazolohydrazinylidene-chroman-2,4-diones on cancer and non-cancer cell growth and death

Coumarins are extensively studied anticoagulants that exert additional effects such as anticancerogenic and even anti-inflammatory. In order to find new drugs with anticancer activities, we report here the synthesis and the structural analysis of new coumarin derivatives which combine the coumarin core and five member heterocycles in hydrazinylidene-chroman-2,4-diones. The derivatives were prepared by derivatization of the appropriate heterocyclic amines which were used as electrophiles to attack the coumarin ring. The structures were characterized by spectroscopic techniques including IR, NMR, 2D-NMR and MS. These derivatives were further characterized especially in terms of a potential cytotoxic and apoptogenic effect in several cancer cell lines including the breast and prostate cancer cell lines MCF-7, MDA-MB-231, PC-3, LNCaP, and the monocytic leukemia cell line U937. Cell viability was determined after 48 h and 72 h of treatment with the novel compounds by MTT assay and the 50% inhibitory concentrations (EC50 values) were determined. Out of the 8 novel compounds screened for reduced cell viability, 4c, 4d and 4e were found to be the most promising and effective ones having EC50 values that were several fold reduced when compared to the reference substance 4-hydroxycoumarin. However, the effects were cancer cell line dependent. The breast cancer MDA-MB-231 cells, the prostate cancer LNCaP cells, and U937 cells were most sensitive, MCF-7 cells were less sensitive, and PC-3 cells were more resistant. Reduced cell viability was accompanied by increased apoptosis as shown by PARP-1 cleavage and reduced activity of the survival protein kinase Akt. In summary, this study has identified three novel coumarin derivatives that in comparison to 4-hydroxycoumarin have a higher efficiency to reduce cancer cell viability and trigger apoptosis and therefore may represent interesting novel drug candidates

Natural Variation in Maize Aphid Resistance Is Associated with 2,4-Dihydroxy-7-Methoxy-1,4-Benzoxazin-3-One Glucoside Methyltransferase Activity

Plants differ greatly in their susceptibility to insect herbivory, suggesting both local adaptation and resistance tradeoffs. We used maize (Zea mays) recombinant inbred lines to map a quantitative trait locus (QTL) for the maize leaf aphid (Rhopalosiphum maidis) susceptibility to maize Chromosome 1. Phytochemical analysis revealed that the same locus was also associated with high levels of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) and low levels of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc). In vitro enzyme assays with candidate genes from the region of the QTL identified three O-methyltransferases (Bx10a-c) that convert DIMBOA-Glc to HDMBOA-Glc. Variation in HDMBOA-Glc production was attributed to a natural CACTA family transposon insertion that inactivates Bx10c in maize lines with low HDMBOA-Glc accumulation. When tested with a population of 26 diverse maize inbred lines, R. maidis produced more progeny on those with high HDMBOA-Glc and low DIMBOA-Glc. Although HDMBOA-Glc was more toxic to R. maidis than DIMBOA-Glc in vitro, BX10c activity and the resulting decline of DIMBOA-Glc upon methylation to HDMBOA-Glc were associated with reduced callose deposition as an aphid defense response in vivo. Thus, a natural transposon insertion appears to mediate an ecologically relevant trade-off between the direct toxicity and defense-inducing properties of maize benzoxazinoids.

Crystal structure of 2,4,6-tris(cyclohexyloxy)-1,3,5-triazine

The title compound, C21H33N3O3, is a tri-substituted cyclo­hex­yloxy triazine. In the crystal, the triazine rings form (C3i-PU) Piedfort units. The inter-centroid distance of the [pi]-[pi] inter­action involving the triazine rings is 3.3914 (10) Å. In the crystal, mol­ecules are linked by C-H...O hydrogen bonds, forming ribbons propagating along [1-10]. There are also weak C-H...N and C-H...O contacts present, linking inversion-related ribbons, forming a three-dimensional structure.

Composition Space Analysis in the Development of Copper Molybdate Hybrids Decorated by a Bifunctional Pyrazolyl/1,2,4-Triazole Ligand

A bitopic ligand, 4-(3,5-dimethylpyrazol-4-yl)-1,2,4-triazole (Hpz-tr) (1), containing two different heterocyclic moieties was employed for the design of copper(II)–molybdate solids under hydrothermal conditions. In the multicomponent CuII/Hpz-tr/MoVI system, a diverse set of coordination hybrids, [Cu(Hpz-tr)2SO4]·3H2O (2), [Cu(Hpz-tr)Mo3O10] (3), [Cu4(OH)4(Hpz-tr)4Mo8O26]·6H2O (4), [Cu(Hpz-tr)2Mo4O13] (5), and [Mo2O6(Hpz-tr)]·H2O (6), was prepared and characterized. A systematic investigation of these systems in the form of a ternary crystallization diagram approach was utilized to show the influence of the molar ratios of starting reagents, the metal (CuII and MoVI) sources, the temperature, etc., on the reaction products outcome. Complexes 2–4 dominate throughout a wide crystallization range of the composition triangle, while the other two compounds 5 and 6 crystallize as minor phases in a narrow concentration range. In the crystal structures of 2–6, the organic ligand behaves as a short [N–N]-triazole linker between metal centers Cu···Cu in 2–4, Cu···Mo in 5, and Mo···Mo in 6, while the pyrazolyl function remains uncoordinated. This is the reason for the exceptional formation of low-dimensional coordination motifs: 1D for 2, 4, and 6 and 2D for 3 and 5. In all cases, the pyrazolyl group is involved in H bonding (H-donor/H-acceptor) and is responsible for π–π stacking, thus connecting the chain and layer structures in more complicated H-bonding architectures. These compounds possess moderate thermal stability up to 250–300 °C. The magnetic measurements were performed for 2–4, revealing in all three cases antiferromagnetic exchange interactions between neighboring CuII centers and long-range order with a net moment below Tc of 13 K for compound 4.

Determination of ethyl glucuronide in human serum by capillary zone electrophoresis and an immunoassay

Ethyl glucuronide (EtG) is a marker of recent alcohol consumption. For the optimization of the analysis of EtG by CZE with indirect absorbance detection, the use of capillaries with permanent and dynamic wall coatings, the composition of the BGE, and various sample preparation procedures, including dilution with water, ultrafiltration, protein precipitation, and SPE, were investigated. Two validated screening assays for the determination of EtG in human serum, a CZE-based approach and an enzyme immunoassay (EIA), are described. The CZE assay uses a coated capillary, 2,4-dimethylglutaric acid as an internal standard, and a pH 4.65 BGE comprising 9 mM nicotinic acid, epsilon-aminocaproic acid and 10% v/v ACN. Proteins are removed via precipitation with ACN prior to analysis and the LOQ is 0.50 mg/L. The EIA is based upon commercial reagents which are promoted for the determination of urinary EtG. Krebs-Ringer solution containing 5% BSA is used as a calibration matrix. All samples are ultrafiltered prior to analysis of the ultrafiltrate on a Mira Plus analyzer. Assay calibration ranged between 0 and 2 mg/L and the upper reference limit was determined to be 0.05 mg/L. Both assays proved to be suitable for the analysis of samples from different individuals. For EtG levels above 0.50 mg/L, good agreement was observed for the comparison of the results of the two methods.

Evaluation of a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugated bombesin-based radioantagonist for the labeling with single-photon emission computed tomography, positron emission tomography, and therapeutic radionuclides

PURPOSE: G protein-coupled receptor agonists are being used as radiolabeled vectors for in vivo localization and therapy of tumors. Recently, somatostatin-based antagonists were shown to be superior to agonists. Here, we compare the new [111In/68Ga]-labeled bombesin-based antagonist RM1 with the agonist [111In]-AMBA for targeting the gastrin-releasing peptide receptor (GRPR). EXPERIMENTAL DESIGN: IC50, Kd values, and antagonist potency were determined using PC-3 and HEK-GRPR cells. Biodistribution and imaging studies were done in nude mice transplanted with the PC-3 tumor. The antagonist potency was assessed by evaluating the effects on calcium release and on receptor internalization monitored by immunofluorescence microscopy. RESULTS: The IC50 value of [(nat)In]-RM1 was 14 +/- 3.4 nmol/L. [(nat/111)In]-RM1 was found to bind to the GRPR with a Kd of 8.5 +/- 2.7 nmol/L compared with a Kd of 0.6 +/- 0.3 nmol/L of [111In]-AMBA. A higher maximum number of binding site value was observed for [111In]-RM1 (2.4 +/- 0.2 nmol/L) compared with [111In]-AMBA (0.7 +/- 0.1 nmol/L). [(nat)Lu]-AMBA is a potent agonist in the immunofluorescence-based internalization assay, whereas [(nat)In]-RM1 is inactive alone but efficiently antagonizes the bombesin effect. These data are confirmed by the calcium release assay. The pharmacokinetics showed a superiority of the radioantagonist with regard to the high tumor uptake (13.4 +/- 0.8% IA/g versus 3.69 +/- 0.75% IA/g at 4 hours after injection. as well as to all tumor-to-normal tissue ratios. CONCLUSION: Despite their relatively low GRPR affinity, the antagonists [111In/68Ga]-RM1 showed superior targeting properties compared with [111In]-AMBA. As found for somatostatin receptor-targeting radiopeptides, GRP-based radioantagonists seem to be superior to radioagonists for in vivo imaging and potentially also for targeted radiotherapy of GRPR-positive tumors.

Biomechanical evaluation of the interfacial strength of a chemically modified sandblasted and acid-etched titanium surface

The functional capacity of osseointegrated dental implants to bear load is largely dependent on the quality of the interface between the bone and implant. Sandblasted and acid-etched (SLA) surfaces have been previously shown to enhance bone apposition. In this study, the SLA has been compared with a chemically modified SLA (modSLA) surface. The increased wettability of the modSLA surface in a protein solution was verified by dynamic contact angle analysis. Using a well-established animal model with a split-mouth experimental design, implant removal torque testing was performed to determine the biomechanical properties of the bone-implant interface. All implants had an identical cylindrical shape with a standard thread configuration. Removal torque testing was performed after 2, 4, and 8 weeks of bone healing (n = 9 animals per healing period, three implants per surface type per animal) to evaluate the interfacial shear strength of each surface type. Results showed that the modSLA surface was more effective in enhancing the interfacial shear strength of implants in comparison with the conventional SLA surface during early stages of bone healing. Removal torque values of the modSLA-surfaced implants were 8-21% higher than those of the SLA implants (p = 0.003). The mean removal torque values for the modSLA implants were 1.485 N m at 2 weeks, 1.709 N m at 4 weeks, and 1.345 N m at 8 weeks; and correspondingly, 1.231 N m, 1.585 N m, and 1.143 N m for the SLA implants. The bone-implant interfacial stiffness calculated from the torque-rotation curve was on average 9-14% higher for the modSLA implants when compared with the SLA implants (p = 0.038). It can be concluded that the modSLA surface achieves a better bone anchorage during early stages of bone healing than the SLA surface; chemical modification of the standard SLA surface likely enhances bone apposition and this has a beneficial effect on the interfacial shear strength.

Ascorbic acid for amelioration of reperfusion injury in a lung autotransplantation model in sheep

BACKGROUND: Reperfusion injury is the leading cause of early graft dysfunction after lung transplantation. Activation of neutrophilic granulocytes with generation of free oxygen radicals appears to play a key role in this process. The efficacy of ascorbic acid as an antioxidant in the amelioration of reperfusion injury after lung transplantation has not been studied yet. METHODS: An in situ autotransplantation model in sheep is presented. The left lung was flushed (Euro-Collins solution) and reperfused; after 2 hours of cold storage, the right hilus was then clamped (group R [reference], n = 6). Group AA animals (n = 6) were treated with 1 g/kg ascorbic acid before reperfusion. Controls (group C, n = 6) underwent hilar preparation and instrumentation only. RESULTS: In group R, arterio-alveolar oxygen difference (AaDO2) and pulmonary vascular resistance (PVR) were significantly elevated after reperfusion. Five of 6 animals developed frank alveolar edema. All biochemical parameters showed significant PMN activation. In group AA, AaDO2, PVR, work of breathing, and the level of PMN activation were significantly lower. CONCLUSIONS: The experimental model reproduces all aspects of lung reperfusion injury reliably. Ascorbic acid was able to weaken reperfusion injury in this experimental setup.

Presence of UV filters in surface water and the effects of phenylbenzimidazole sulfonic acid on rainbow trout (Oncorhynchus mykiss) following a chronic toxicity test

UV filters belong to a group of compounds that are used by humans and are present in municipal waste-waters, effluents from sewage treatment plants and surface waters. Current information regarding UV filters and their effects on fish is limited. In this study, the occurrence of three commonly used UV filters - 2-phenylbenzimidazole-5-sulfonic acid (PBSA), 2-hydroxy-4-methoxybenzophenone (benzophenone-3, BP-3) and 5-benzoyl-4-hydroxy-2-methoxy-benzenesulfonic acid (benzophenone-4, BP-4) - in South Bohemia (Czech Republic) surface waters is presented. PBSA concentrations (up to 13μgL(-1)) were significantly greater than BP-3 or BP-4 concentrations (up to 620 and 390ngL(-1), respectively). On the basis of these results, PBSA was selected for use in a toxicity test utilizing the common model organism rainbow trout (Oncorhynchus mykiss). Fish were exposed to three concentrations of PBSA (1, 10 and 1000µgL(-1)) for 21 and 42 days. The PBSA concentrations in the fish plasma, liver and kidneys were elevated after 21 and 42 days of exposure. PBSA increased activity of certain P450 cytochromes. Exposure to PBSA also changed various biochemical parameters and enzyme activities in the fish plasma. However, no pathological changes were obvious in the liver or gonads.

Effects of buffering properties and undissociated acid concentration on dissolution of dental enamel in relation to pH and acid type

To quantify the relationships between buffering properties and acid erosion and hence improve models of erosive potential of acidic drinks, a pH-stat was used to measure the rate of enamel dissolution in solutions of citric, malic and lactic acids, with pH 2.4-3.6 and with acid concentrations adjusted to give buffer capacities (β) of 2-40 (mmol·l(-1))·pH(-1) for each pH. The corresponding undissociated acid concentrations, [HA], and titratable acidity to pH 5.5 (TA5.5) were calculated. In relation to β, the dissolution rate and the strength of response to β varied with acid type (lactic > malic ≥ citric) and decreased as pH increased. The patterns of variation of the dissolution rate with TA5.5 were qualitatively similar to those for β, except that increasing pH above 2.8 had less effect on dissolution in citric and malic acids and none on dissolution in lactic acid. Variations of the dissolution rate with [HA] showed no systematic dependence on acid type but some dependence on pH. The results suggest that [HA], rather than buffering per se, is a major rate-controlling factor, probably owing to the importance of undissociated acid as a readily diffusible source of H(+) ions in maintaining near-surface dissolution within the softened layer of enamel. TA5.5 was more closely correlated with [HA] than was β, and seems to be the preferred practical measure of buffering. The relationship between [HA] and TA5.5 differs between mono- and polybasic acids, so a separate analysis of products according to predominant acid type could improve multivariate models of erosive potential.

Effect of a chitosan additive to a Sn(2+)-containing toothpaste on its anti-erosive/anti-abrasive efficacy-a controlled randomised in situ trial

OBJECTIVES It is well known that Sn(2+) is a notable anti-erosive agent. There are indications that biopolymers such as chitosan can enhance the effect of Sn(2+), at least in vitro. However, little information exists about their anti-erosive/anti-abrasive in situ effects. In the present in situ study, the efficacy of Sn(2+)-containing toothpastes in the presence or absence of chitosan was tested. METHODS Ten subjects participated in the randomised crossover study, wearing mandibular appliances with human enamel specimens. Specimens were extraorally demineralised (7 days, 0.5 % citric acid, pH 2.6; 6 × 2 min/day) and intraorally exposed to toothpaste suspensions (2 × 2 min/day). Within the suspension immersion time, one half of the specimens were additionally brushed intraorally with a powered toothbrush (5 s, 2.5 N). Tested preparations were a placebo toothpaste (negative control), two experimental toothpastes (F/Sn = 1,400 ppm F(-), 3,500 ppm Sn(2+); F/Sn/chitosan = 1,400 ppm F(-), 3,500 ppm Sn(2+), 0.5 % chitosan) and an SnF2-containing gel (positive control, GelKam = 3,000 ppm Sn(2+), 1,000 ppm F(-)). Substance loss was quantified profilometrically (μm). RESULTS In the placebo group, tissue loss was 11.2 ± 4.6 (immersion in suspension) and 17.7 ± 4.7 (immersion in suspension + brushing). Immersion in each Sn(2+)-containing suspension significantly reduced tissue loss (p ≤ 0.01); after immersion in suspension + brushing, only the treatments with GelKam (5.4 ± 5.5) and with F/Sn/chitosan (9.6 ± 5.6) significantly reduced loss [both p ≤ 0.05 compared to placebo; F/Sn 12.8 ± 6.4 (not significant)] CONCLUSION Chitosan enhanced the efficacy of the Sn(2+)-containing toothpaste as an anti-erosive/anti-abrasive agent. CLINICAL RELEVANCE The use of Sn(2+)- and chitosan-containing toothpaste is a good option for symptomatic therapy in patients with regular acid impacts.