Synthesis of 4,4-Disubstituted 3-Methylidenechroman-2-ones as Potent Anticancer Agents.

The synthesis of a new library of 4,4-disubstituted 3-methylidene-3,4-dihydro-2H-chroman-2-ones applying Horner-Wadsworth-Emmons methodology for the construction of an exo-methylidene moiety is reported. Corresponding 3-diethoxyphosphorylchroman-2-ones were synthesized in a three-step reaction sequence consisting of O-methylation of ethyl 2-diethoxyphosphoryl-3-oxoalkanoates, followed by reaction of the obtained 2-diethoxyphosphoryl-3-methoxy-2-alkenoates with phenols or 1-naphthol. The resulting 3-diethoxyphosphorylochromen-2-ones proved to be effective Michael acceptors in reactions with various Grignard reagents. Preliminary biological evaluations showed that many of the synthesized 3-methylidenechroman-2-ones possess very high cytotoxic activity against NALM-6 and HL-60 cancer cell lines (IC50 <1.0 µm) as well as high activity against the MCF-7 cancer cell line (IC50 <10 µm). Furthermore, two of the highly active 3-methylidenechroman-2-ones with geminal methyl and ethyl substituents at position 4 showed promising therapeutic indexes of 10 and 13 in tests against human umbilical vein endothelial cells (HUVECs).

The immunomodulatory potential of Leonurus cardiaca extract in relation to endothelial cells and platelets.

The immunomodulatory activity of Leonurus cardiaca L. polyphenol-rich extract (LCE) was tested in vitro on HUVECs to explore its potential therapeutic usefulness in the treatment of inflammatory lesions. The phytochemical composition of LCE, its antioxidant and cytotoxic activity, and the influence of LCE on NO and platelet-activating factor (PAF) secretion by HUVECs and platelet aggregation were all assessed. Total polyphenol contents in LCE reached 137.0 ± 0.8 mg/g, with hydroxycinnamic acid derivatives as the predominant phenolic compounds. LCE expressed antioxidant capacity, which was, however, 13- to 16-fold lower than the antioxidant activity of ascorbic acid. The plant extract was not cytotoxic up to a concentration 4500 µg/ml and did not exhibit proapoptotic activity. LCE significantly increased NO production in HUVECs in a concentration-dependent manner and led to the inhibition of PAF secretion induced by staphylococcal peptidoglycan. The extract used at the concentration of 100 µg/ml significantly reduced platelet aggregation in the presence of arachidonic acid. We provide in vitro data demonstrating the immunomodulatory potential of LCE, which may be beneficial in preventing the development of difficult-to-treat inflammatory lesions within chronically infected tissues.

Quantitative relationships between structure and cytotoxic activity of flavonoid derivatives. An application of Hirshfeld surface derived descriptors.

Quantitative relationships between the structure and cytotoxic activity of series flavonoid derivatives were examined. The first regression-based model, developed for 18 flavanone-2-pyrazoline hybrids, involved two interpretable descriptors: a Mor04v and partial atomic charge. The second model, developed for structurally diverse set of compounds, was based on descriptors derived from Hirshfeld surface analysis. This model suggests that cytotoxic activity of compounds can be successfully predicted based on a fraction of H⋯H contacts and a fraction of interactions involving a halogen atom. For non-halogen derivatives, the data reveal that cytotoxic activity is inversely proportional to the percentage of O⋯H and N⋯H close contacts to Hirshfeld surface, while directly proportional to the percentage of H⋯H interactions. Chlorine (1k) and bromine (1l) derivatives of compounds, containing flavanone fused with N-methyl-2-pyrazoline, exhibited high cytotoxic potential against HL-60 cancer cell line (IC50<10µM). The cytotoxicity of 1k and 1l towards normal cells (HUVEC) was 10 and 25-fold lower, respectively.

Antioxidant and DNA Repair Stimulating Effect of Extracts from Transformed and Normal Roots of Rhaponticum carthamoides against Induced Oxidative Stress and DNA Damage in CHO Cells.

Rhaponticum carthamoides has a long tradition of use in Siberian folk medicine. The roots and rhizomes of this species are used in various dietary supplements or nutraceutical preparations to increase energy level or eliminate physical weakness. This is the first report to reveal the protective and DNA repair stimulating abilities of R. carthamoides root extracts in Chinese hamster ovary (CHO) cells exposed to an oxidative agent. Both transformed root extract (TR extract) and extract of soil-grown plant roots (NR extract) may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, but CHO cells stimulated with extract from the transformed roots demonstrated significantly stronger properties than cells treated with the soil-grown plant root extract. These differences in biological activity may be attributed to the differences in the content of phenolic compounds in these root extracts. Preincubation of the CHO cells with TR and NR extracts showed an increase in gene expression and protein levels of catalase (CAT) and superoxide dismutase (SOD2). R. carthamoides may possess antioxidant properties that protect CHO cells against oxidative stress.

Design, synthesis and cytotoxic evaluation of 4-methylidenepyrazolidin-3-ones.

Three series of new 4-methylidenepyrazolidin-3-ones with various substitution patterns were synthesized and tested for the cytotoxic activity against two human leukemia cell lines NALM-6 and HL-60 as well as MCF-7 breast cancer cell line. Several obtained methylidenepyrazolidinones exhibited high cytotoxic activity with IC50 values below 10 µM, mainly against HL-60 leukemia cell line and two of them, 18d,e, displayed IC50 ≤ 5 µM, against all tested cell lines. Structure-activity relationship studies revealed that the presence of phenyl substituents on both ring nitrogen atoms and vinyl or phenyl substituents in position 5 are crucial for high activity. Selected methylidenepyrazolidinones were also tested on normal human umbilical vein endothelial cells (HUVEC) and pyrazolidinone 18a was found to be 5-fold more toxic against HL-60 than normal cells.

Extract from Ribes nigrum leaves in vitro activates nitric oxide synthase (eNOS) and increases CD39 expression in human endothelial cells

The aim of the present study was to evaluate whether blackcurrant leaf extract (BLE) modulates endothelium antithrombotic function, namely increases the expression/activity of ADPase (CD39) and augments the production of nitric oxide in human umbilical vein endothelial cells (HUVEC). It was found that BLE with proanthocyanidins (60 % of the total polyphenol content) increased the CD39-positive endothelial cell fraction (up to 10 % for 2.5 μg/ml, and up to 33 % for 15 μg/ml, p < 0.05 or less) in a concentration-dependent manner, and enhanced endothelial nitric oxide synthase (eNOS) activation (T495 phosphorylation decreased by 31 ± 6 % for 2.5 μg/ml and 48 ± 6 % for 15 μg/ml; S1177 phosphorylation increased by 13 ± 3 % for 2.5 μg/ml and 18 ± 7 % for 15 μg/ml, compared to untreated cells, p < 0.05 or less). Additionally, incubation for 24 or 48 h with BLE at a lower range of polyphenol concentrations, significantly increased cell viability with a maximal effect at 2.5 μg/ml (viability increased by 24.8 ± 1.0 % for 24 h and by 32.5 ± 2.7 % for 48-h time incubation, p < 0.0001). The increased CD39 expression and the increased eNOS activation in HUVEC can be regarded as the beneficial markers of the improvement of antiplatelet action of endothelial cells. Unexpectedly, these assumptions were not confirmed in the experimental model of platelet-endothelial cell interactions. These observations lead to the conclusion that BLE may improve endothelial cell viability at low physiological concentrations without affecting the antiplatelet action of endothelium

Extract from Ribes nigrum leaves in vitro activates nitric oxide synthase (eNOS) and increases CD39 expression in human endothelial cells.

The aim of the present study was to evaluate whether blackcurrant leaf extract (BLE) modulates endothelium antithrombotic function, namely increases the expression/activity of ADPase (CD39) and augments the production of nitric oxide in human umbilical vein endothelial cells (HUVEC). It was found that BLE with proanthocyanidins (60 % of the total polyphenol content) increased the CD39-positive endothelial cell fraction (up to 10 % for 2.5 µg/ml, and up to 33 % for 15 µg/ml, p < 0.05 or less) in a concentration-dependent manner, and enhanced endothelial nitric oxide synthase (eNOS) activation (T495 phosphorylation decreased by 31 ± 6 % for 2.5 µg/ml and 48 ± 6 % for 15 µg/ml; S1177 phosphorylation increased by 13 ± 3 % for 2.5 µg/ml and 18 ± 7 % for 15 µg/ml, compared to untreated cells, p < 0.05 or less). Additionally, incubation for 24 or 48 h with BLE at a lower range of polyphenol concentrations, significantly increased cell viability with a maximal effect at 2.5 µg/ml (viability increased by 24.8 ± 1.0 % for 24 h and by 32.5 ± 2.7 % for 48-h time incubation, p < 0.0001). The increased CD39 expression and the increased eNOS activation in HUVEC can be regarded as the beneficial markers of the improvement of antiplatelet action of endothelial cells. Unexpectedly, these assumptions were not confirmed in the experimental model of platelet-endothelial cell interactions. These observations lead to the conclusion that BLE may improve endothelial cell viability at low physiological concentrations without affecting the antiplatelet action of endothelium.

Does grape seed extract potentiate the inhibition of platelet reactivity in the presence of endothelial cells?

PURPOSE: Numerous studies have suggested that grape seed extract (GSE) confers vascular protection due to the direct effect of its polyphenol content on endothelial cells. The aim of the study was to determine whether GSE confers vascular protection through the direct effect of its polyphenol content on endothelial cells. MATERIAL/METHODS: After incubation with GSE-treated human umbilical vein endothelial cells (HUVECs), blood platelet reactivity was evaluated with regard to the expression of CD62P and the activated form of GPIIbIIIa in ADP-stimulated platelets. RESULTS: Lower concentrations of GSE were found to enhance the antiplatelet action of HUVECs: 1 µg/ml GSE reduced platelet reactivity by about 10%. While platelet reactivity was not altered by HUVECs incubated with higher concentrations of GSE, HUVEC proliferation was significantly reduced by GSE of up to 10 µg gallic acid equivalent/ml. CONCLUSIONS: The results of the study show that low doses of GSE potentiate the inhibitory action of HUVECs on platelet reactivity, which may account, at least partially, for the protective effects of grape products against cardiovascular diseases. In contrast, high concentrations of GSE significantly impair endothelial cell proliferation in vitro.

Cytotoxic activity of substituted chalcones in terms of molecular electronic properties.

Global chemical reactivity descriptors and lipophilicity (logP) were evaluated via density functional theory in order to clarify the structure-cytotoxic activity relationships of substituted chalcones. Stepwise multiple regression was employed to establish correlation between descriptors and cytotoxic activity against three cancer cell lines (HL-60, NALM-6 and WM-115) for 11 compounds. Regression analysis revealed that electrophilicity index and chemical potential significantly contributed in explaining of chalcones cytotoxic potential. Moreover, the established structure-activity relationships based on electronic structure properties allow indicating the substructures responsible for their cytotoxic activity. The study has also been supported by crystallographic data of 2-chloro-2'-hydroxychalcone.

Comparison of PrestoBlue and MTT assays of cellular viability in the assessment of anti-proliferative effects of plant extracts on human endothelial cells.

INTRODUCTION: PrestoBlue (PB) is a new, simple and extremely fast live assay to monitor cell viability and cytotoxicity. Herein, we compared two in vitro cytotoxicity assays, new (PB) and classic (MTT), in the assessment of viability of human umbilical vein endothelial cells (HUVECs) in the presence of selected plant extracts. METHODS: The anti-proliferative effects of two extracts from medicinal plants, i.e., walnut husk extract and spent hop extract, used at the concentration range of 1-200 µg/ml of gallic acid equivalent, were compared with the effects recorded for resveratrol--a natural polyphenolic compound. Reduction of dyes by endothelial cells was determined colorimetrically (MTT and PB) and fluorometrically (PB). RESULTS: At higher concentrations, all tested compounds caused significant loss of cell viability. Regardless of plant compound, the PB assay, when measured colorimetrically, produced higher EC50 values compared to other modes of measurement, however, the statistically significant differences in EC50 values among the assays were revealed only for spent hop extract. Conversely, the EC50 values for each plant compound obtained in MTT (colorimetric assay) and PB (fluorometric assay) were similar. According to EC50 values, the cytotoxicity of plant compounds ranked as follows: spent hop extract>resveratrol>walnut husk extract. Furthermore, the MTT assay showed overall lower inter-assay variability and higher signal-to-noise ratio compared to PB assay. DISCUSSION: In conclusion, we recommend fluorometric PrestoBlue assay for cytotoxicity assessment in human endothelial cells. Due to substantial differences in EC50 values and S/N ratios between spectrophotometric PB and MTT or fluorometric PB assays, colorimetric quantification of HUVECs' viability with the use of PB reagent should be avoided.

Structure-cytotoxic activity relationship of 3-arylideneflavanone and chromanone (E,Z isomers) and 3-arylflavones.

The E,Z-isomers of 3-arylidene substituted flavanone, chromanone and 3-aryl substituted flavone derivatives were tested in vitro for their cytotoxic activity against three cancer cell lines (HL-60, NALM-6, WM-115) and normal cell line (HUVEC). It was observed that substitution at C3 position led to significant enhance in cytotoxicity. Isomeric configuration of 3-arylideneflavanones had an influence on the cytotoxic potential. Multiple regression analysis combined with variable selection by genetic algorithm was used to model relationships between molecular descriptors and the cytotoxic activity. The most accurate QSAR models were based on a combination between energy of LUMO, experimental value of logP and partial charge on carbonyl oxygen (δO2).

Synthesis and biological evaluation of α-methylidene-δ-lactones with 3,4-dihydrocoumarin skeleton.

A series of new 3-methylidenechroman-2-ones bearing various aromatic moieties and various substituents at position 4 were synthesized in a three step reaction sequence. Friedel-Crafts alkylation of phenols or naphthols using ethyl 3-methoxy-2-diethoxyphosphorylacrylate in the presence of trifluoromethanesulphonic acid gave 3-diethoxyphosphorylchromen-2-ones. These compounds were employed as Michael acceptors in the reaction with Grignard reagents to give adducts which were finally used as Horner-Wadsworth-Emmons reagents for the olefination of formaldehyde. All obtained 3-methylidenechroman-2-ones were tested against two human leukemia cell lines NALM-6 and HL-60 as well as MCF-7 breast cancer and HT-29 colon cancer adenocarcinomas. Several obtained methylidenechromanones displayed high cytotoxic activity with IC(50) values below 1 µM, mainly against leukemia and MCF-7 cell lines. Investigation of structure-activity relationships revealed that the presence of additional, ortho-fused benzene ring and n-butyl or i-propyl group in position 4 enhances the activity. Selected methylidenechromanones were also tested on normal human umbilical vein endothelial cells (HUVEC) and chromanone 14o was found to be eightfold more toxic against MCF-7 than normal cells. Furthermore, antimicrobial assays revealed that chromanone 14n is highly active and bactericidal at concentration equal to MIC or 2MIC against nosocomial and community-associated staphylococci (MRSA) which are resistant to most or all available therapeutic classes of antimicrobial drugs.

An unusual taxodione derivative from hairy roots of Salvia austriaca.

From a root culture of Salvia austriaca, transformed with Agrobacterium rhizogenes, a new diterpenoid was isolated and its chemical structure was determined as 7-(2-oxohexyl)-11-hydroxy-6, 12-dioxo-7,9(11),13- abietatriene [= 7-(2-oxohexyl)-taxodione] on the basis of spectroscopic methods, especially 1D and 2D NMR, and by comparison with structurally related compounds. This compound represents a hitherto unknown 2-oxohexyl diterpenoid derivative. Cytotoxic studies revealed that the new compound exhibited high cytotoxic activity against three cancer cell lines with IC(50) values ranging from 0.63 to 0.72µM. Its cytotoxic effectiveness against the cancer cells was ten fold higher than that of taxodione.

Synthesis, X-ray structure and cytotoxic effect of nickel(II) complexes with pyrazole ligands.

Here we present the synthesis of the new Ni(II) complexes with chelating ligands 1-benzothiazol-2-yl-3,5-dimethyl-1H-pyrazole (a), 5-(2-hydroxyphenyl)-3-methyl-1-(2-pyridylo)-1H-pyrazole-4-carboxylic acid methyl ester (b) and 1-benzothiazol-2-yl-5-(2-hydroxyphenyl)-3-methyl-1H-pyrazole-4-carboxylic acid methyl ester (c). These ligands a-c create solid complexes with Ni(II). The crystal and molecular structures of two complexes were determined by X-ray diffraction method. Thermal stability of two complexes with ligand c by TG/DTG and DSC methods were also shown. Cytotoxic activity of all the complexes against three tumour cell lines and to normal endothelial cells (HUVEC) was also estimated. Complexes with ligand c exhibited relatively high cytotoxic activity towards HL-60 and NALM-6 leukaemia cells and WM-115 melanoma cells. Cytotoxic effectiveness of one of these complexes against melanoma WM-115 cells was two times higher than that of cisplatin. The protonation constant log K=9.63 of ligand b corresponding to the phenol 2-hydroxy group has been determined in 10% (v/v) DMSO/water solution (25°C). The coordination modes (formation of two monomeric species: NiL and NiL(2)) in the complexes with Ni(II) are discussed for b on the basis of the potentiometric and UV/Vis data.

Synthetic 3-arylideneflavanones as inhibitors of the initial stages of biofilm formation by Staphylococcus aureus and Enterococcus faecalis.

The antimicrobial activity of twenty two synthetic flavonoids is reported. Among them three 3-arylideneflavanones, 2b, 2c, and 2i, were shown to be highly active against Staphylococcus aureus, S. epidermidis, and Enterococcus faecalis reference strains, with MIC (minimal inhibitory concentration) values ranging from 4.68 microg/ml (14.3 microM) to 37.5 microg/ml (119.7 microM). The synergy of oxacillin and vancomycin with 2c, evaluated as fractional inhibitory concentration index (FICI) was shown (against planktonic culture of S. aureus A3 and E. faecium 138/09 clinical strains). The presence of 2c in the culture medium diminished the initial adhesion of bacteria to an abiotic surface. Such an effect resulted in a decrease in biofilm formation during prolonged culture. Unfortunately, 2e failed to eradicate the S. aureus mature biofilm which was already preformed, however, decreased the number of live biofilm cells. The biofilm of E. faecalis was more susceptible to the action of 3-arylideneflavanone 2c than the S. aureus biofilm. The finding that 3-arylideneflavanones are lipophilic, cause bacterial aggregation, and influence the integrity of membranes making them permeable to SYTO 9/propidium iodide dyes may implicate the cytoplasmic membrane as a target site for these compounds activity.

Synthesis, X-ray structures and cytotoxic activity of platinum(II), palladium(II) and copper(II) complexes with chelating ligands.

Here we present the synthesis of the new chelating ligands 1-benzothiazol-2-yl-5-(2-hydroxyphenyl)-3-methyl-1H-pyrazole-4-carboxylic acid methyl ester (2a) and 1-(6-chloropyridazin-3-yl)-5-(2-hydroxyphenyl)-3-methyl-1H-pyrazole-4-carboxylic acid methyl ester (2b), obtained in the reaction of 2-methyl-4-oxo-4H-chromene-3-carboxylic acid methyl ester (1) with hydrazine derivatives. These ligands 2a and 2b create solid complexes with Pt(II) (4a, 4b), Pd(II) (5a, 5b) and Cu(II) (6a, 7a, 8a, 8b and 9b) metal ions or can be cyclized to 1-benzothiazol-2-yl-3-methyl-1H-chromeno[4,3-c]pyrazol-4-one (3a) or 1-(6-chloropyridazin-3-yl)-3-methyl-1H-chromeno[4,3-c]pyrazol-4-one (3b). The crystal and molecular structures of ligand 2a, its Cu(II) complexes 6a and 7a were determined by X-ray diffraction method. Cytotoxic activity of the ligands 2a and 2b and their complexes 4a, 4b, 5a, 5a, 6a, 8a, 8b and 9b, and modulation of expression of BAX and P53 genes are also shown.

Extract from Aronia melanocarpa fruits potentiates the inhibition of platelet aggregation in the presence of endothelial cells.

INTRODUCTION: Some polyphenolic compounds extracted from Aronia melanocarpa fruits (AM) have been reported to be cardioprotective agents. In this study we evaluated the ability of AM extract to increase the efficacy of human umbilical vein endothelial cells (HUVECs) to inhibit platelet functions in vitro. MATERIAL AND METHODS: THIS STUDY ENCOMPASSES TWO MODELS OF MONITORING PLATELET REACTIVITY: optical aggregation and platelet degranulation (monitored as the surface CD62P expression) in PRP upon the stimulation with ADP. RESULTS: We observed that only at low concentrations (5 µg/ml) did AM extract significantly improve antiplatelet action of HUVECs towards ADP-activated platelets in the aggregation test. CONCLUSIONS: It is concluded that the potentiating effect of AM extract on the endothelial cell-mediated inhibition of platelet aggregation clearly depends on the used concentrations of Aronia-derived active compounds. Therefore, despite these encouraging preliminary outcomes on the beneficial effects of AM extract polyphenols, more profound dose-effect studies should certainly be considered before the implementation of Aronia-originating compounds in antiplatelet therapy and the prevention of cardiovascular diseases.

Synthesis and cytotoxic evaluation of beta-alkyl or beta-aryl-delta-methyl-alpha-methylene-delta-lactones. Comparison with the corresponding gamma-lactones.

We present a simple and general strategy for the synthesis of beta,delta-disubstituted-alpha-methylene-delta-lactones starting from easily available tert-butyl 2-(diethoxyphosphoryl)alk-2-enoates. The elaborated synthetic protocol includes pyrrolidine-catalyzed Michael addition of acetone, diastereoselective reduction of the carbonyl group, lactonization and finally the Horner-Wadsworth-Emmons reaction with formaldehyde. All alpha-methylene-delta-lactones were evaluated in vitro against mouse leukemia cell line L-1210 and two human leukemia cell lines HL-60 and NALM-6. Comparison of cytotoxic activity with corresponding alpha-methylene-gamma-lactones is also discussed.

Tautomeric forms study of 1H-(2'-pyridyl)-3-methyl-5-hydroxypyrazole and 1H-(2'-pyridyl)-3-phenyl-5-hydroxypyrazole. Synthesis, structure, and cytotoxic activity of their complexes with palladium(II) ions.

In this article the synthesis of new 1H-(2'-pyridyl)-3-methyl-5-hydroxypyrazole and 1H-(2'-pyridyl)-3-phenyl-5-hydroxypyrazole complexes with palladium(II) ions is reported. The structures of obtained compounds have been characterized by X-ray crystallography and DFT (density functional theory) calculations. The cytotoxicity of complexes and ligands has been examined for two human leukemia cell lines (HL-60 and NALM-6) and one human melanoma cell line (WM-115). The palladium(II) complex with 1H-(2'-pyridyl)-3-phenyl-5-hydroxypyrazole has been shown to possess greater activity than carboplatin against the WM-115 melanoma cell line. Additionally, the ligands' tautomeric forms existence in different solvents (chloroform, methanol, DMSO) has been characterized by (1)H nuclear magnetic resonance (NMR) analysis and DFT calculations. The obtained results have been compared with those from other studies of similar compounds.

Cancer procoagulant (CP) analysis in human WM 115 malignant melanoma cells in vitro.

Neoplastic cells produce procoagulants responsible for hypercoagulation states frequently observed in cancer patients. It is accepted that two major procoagulants from malignant tissue are tissue factor (TF) and a direct activator of coagulation factor X called cancer procoagulant (CP). Direct factor X-activating activity of cultured human malignant melanoma WM 115 cells has been analyzed in the cell extracts, whole cells and in the medium after the cell culture. The factor X-activating activity was detected in the malignant cell lysates but not in the cultured medium or intact malignant cells. The lysates contained no TF as determined by Western blotting and enzyme-linked immunosorbent assay (ELISA) using anti-TF monoclonal antibody. The enzymatic characteristics of the activity was typical for CP. The results suggest that cancer procoagulant is an intracellular protein.