Artemisinin-Daumone Hybrid Inhibits Cancer Cell-Mediated Osteolysis by Targeting Cancer Cells and Osteoclasts.

BACKGROUND/AIMS: Bone metastasis of cancer cells decreases patient survival and quality of life. Hybridization via the covalent coupling of two bioactive natural products is a useful strategy for developing more potent anticancer agents by enhancing their bioavailability and avoiding drug resistance. METHODS: The in vivo activities of artemisinin-daumone hybrid 15 (ARTD) were estimated in cancer cell-inoculated mice and ovariectomized mice. The viability, migration, and invasion of cancer cells were measured via MTT, wound-healing, and transwell invasion assays. ARTD-regulated transcription factors were detected with an RT2 profiler PCR array kit and Western blotting. Osteoclastogenesis and osteoclast activity were detected with tartrate-resistant acid phosphatase staining, a pit formation assay, gelatin zymography, and a cathepsin K ELISA assay. RESULTS: ARTD blocked cancer-associated osteolysis more potently than artemisinin in mice with intratibially inoculated breast cancer or lung cancer cells. ARTD inhibited the viability, migration, and invasion of breast and lung cancer cells in the absence or presence of transforming growth factor-ß1. ARTD treatment induced the expression of tumor suppressive activating transcription factor 3 and inhibited oncogenic E2F transcription factor 1 expression at the mRNA and protein levels. ARTD inhibited receptor activator of nuclear factor kappa-B ligand-induced osteoclast formation and bone resorbing activity by reducing the secreted levels of matrix metalloproteinase-9 and cathepsin K. Furthermore, ARTD prevented estrogen deficiency-induced bone loss in ovariectomized mice. CONCLUSION: ARTD may be a promising candidate for inhibiting cancer-induced bone destruction. The application of ARTD may be extended to patients with chemotherapy-induced ovarian failure or postmenopausal osteoporosis.

Practical Synthesis of Chalcone Derivatives and Their Biological Activities.

Practical synthesis and biological activities of 4-hydroxy-3-methoxy-2-propene derivatives are described. The novel chalcone derivatives were prepared by acid catalysed one-step condensation of 1,3- or 1,4-diacetylbenzene and 1,3,5-triacetylbenzene with 4-hydroxy-3-methoxybenzaldehyde. They were then evaluated for free radical scavenging activity, suppression of lipopolysaccharides (LPS)-induced NO generation, and anti-excitotoxicity in vitro. It was found that all compounds showed good effects for 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, LPS-induced NO generation, and anti-neurotoxicity. Compounds 6 and 7 were potent suppressor of NO generation with the concentration range 10 µM and especially compound 8 showed very potent anti-inflammatory activity with 1 µM. In addition, the di- and tri-acetylbenzyl derivatives 6, 7, and 8 showed enhanced anti-neurotoxicity activity in cultured cortical neurons. Molecular modelling studies to investigate the chemical structural characteristics required for the enhanced biological activities interestingly revealed that compound 8 has the smallest highest occupied molecular orbital-lowest energy unoccupied molecular orbital (HOMO-LUMO) gap, which signifies easy electron and radical transfer between HOMO and LUMO in model studies.

Total Synthesis and Anticancer Activity of Novel Pulsatilla Saponin D Analogues.

Novel saponins that retain a free carboxyl group at the C-17 position and various sugars linked at the C-3 position of hederagenin aglycone were synthesized via stereospecific glycosylation. Since these natural products represented by Pulsatilla saponin D (PSD) were obtained in very small amounts, the total synthesis developed in this paper will resolve this problem of scarcity. The two types of synthesized arabinose- and rhamnose-cored saponins showed potent anticancer activity against a human lung cancer cell line (A549), and most disaccharide moiety saponins possessed more potent anti-lung cancer activity. Among the novel PSD analogues containing disaccharide saponins, compound 10i showed anti-lung cancer activity (6.6 µM) that was four-fold more potent than the clinical agent Iressa (26.08 µM).

First Synthesis of Novel Aminophenyl Pyridinium-5-(hydroxybenzoyl)-hydrazonomethyl-2-oxothiazol-3-ide Derivatives and Evaluation of Their Anticancer Activities.

The first total synthesis for large-scale production and anticancer activity of novel aminophenylpyridinium-5-(hydroxybenzoyl)hydrazonomethyl-2-oxothiazol-3-ide (PBHT) (1) and its derivatives are reported. The chemical structure of PBHT was unambiguously determined by utilization of the two-dimensional nuclear Overhauser effect (NOE) technique. The anticancer activity against human colon adenocarcinoma (HCT15) cells of all synthesized compounds was approximately four-fold greater than that of 5-fluorouracil, with IC50 values ranging from 10.1 to 14.2 µM. The three structural determinants of hydroxybenzoyl, hydrazinylidene, and pyridinium oxothiazole in the synthesized compounds could be indispensable for exhibiting anticancer activity.

Synthesis of daumone derivatives and their antiangiogenic activities on chorioallantoic membrane.

Daumone, a dauer-inducing pheromone and a series of lipid derivatives were synthesized from daumone to investigate structure-activity trends. Lipid derivatives demonstrated potent in vivo antiangiogenic activity on the chorioallantoic membrane, which exceeded that of fumagillin and thalidomide as reference agents. Among the 11 synthetic compounds tested, new derivatives 3, 11 and 13 showed the most potent antiangiogenic activity, which was twice that of fumagillin and thalidomide, replacing these as the most potent known antiangiogenic agents.

Daumone fed late in life improves survival and reduces hepatic inflammation and fibrosis in mice.

The liver is one of the most susceptible organs to aging, and hepatic inflammation and fibrosis increase with age. Chronic inflammation has been proposed as the major molecular mechanism underlying aging and age-related diseases, whereas calorie restriction has been shown to be the most effective in extending mammalian lifespan and to have anti-aging effects through its anti-inflammatory action. Thus, it is necessary to develop effective calorie restriction mimetics. Daumone [(2)-(6R)-(3,5-dihydroxy-6-methyltetrahydropyran-2-yloxy)heptanoic acid], a pheromone secreted by Caenorhabditis elegans, forces them to enter the dauer stage when facing inadequate conditions. Because Caenorhabditis elegans live longer during the dauer stage under energy deprivation, it was hypothesized that daumone may improve survival in mammals by mimicking calorie restriction. Daumone (2 mg kg(-1) day(-1) ) was administered orally for 5 months to 24-month-old male C57BL/6J mice. Daumone was found to reduce the risk of death by 48% compared with age-matched control mice, and the increased plasma insulin normally presented in old mice was significantly reduced by daumone. The increased hepatic hypertrophy, senescence-associated ß-galactosidase activity, insulin resistance, lipid accumulation, inflammation, oxidative stress, and fibrosis in old mice were significantly attenuated by daumone. From a mechanistic view, daumone reduced the phosphorylation of the IκBα and upregulation of Rela and Nfkbia mRNA in the livers of old mice. The anti-inflammatory effect of daumone was confirmed in lipopolysaccharide-induced liver injury model. Oral administration of daumone improves survival in mice and delivers anti-aging effects to the aged liver by modulating chronic inflammation, indicating that daumone could be developed as an anti-aging compound.

Synthesis and anticancer activity of novel deoxoartemisinin-glycolipid hybrids.

The practical synthesis and anticancer activity of novel deoxoartemisinin-glycolipid hybrids, which incorporate two drugs into a single molecule and can impact multiple targets simultaneously are presented. These hybrids exhibited potent in vitro anticancer activity against several human cancer cell lines. The deoxoartemisinin-glycolipid hybrids generally demonstrated better anticancer activity than either artemisinin or daumone alone and cisplatin.

Therapeutic effects of water soluble danshen extracts on atherosclerosis.

Danshen is a traditional Chinese medicine with many beneficial effects on cardiovascular diseases. The aim of this study was to evaluate the mechanisms responsible for the antiatherogenic effect of water soluble Danshen extracts (DEs). Rat vascular smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs) were treated with DE. To evaluate the effects of DE in vivo, carotid balloon injury and tail vein thrombosis were induced in Sprague-Dawley (SD) rats and iliac artery stent was induced in New Zealand white rabbits. The inhibitory action of DE on platelet aggregation was confirmed with an impedance aggregometer. DE inhibited the production of reactive oxygen species, and the migration and proliferation of platelet-derived growth factor-BB stimulated VSMCs. Furthermore, DE prevented inflammation and apoptosis in HUVECs. Both effects of DE were reconfirmed in both rat models. DE treatment attenuated platelet aggregation in both in vivo and ex vivo conditions. Pretreatment with DE prevented tail vein thrombosis, which is normally induced by κ-carrageenan injection. Lastly, DE-treated rabbits showed decreased in-stent restenosis of stented iliac arteries. These results suggest that water soluble DE modulates key atherogenic events in VSMCs, endothelial cells, and platelets in both in vitro and in vivo conditions.

Quantitative structure-activity relationship study of aromatic inhibitors against rat lens aldose reductase activity using variable selections.

A quantitative structure-activity relationship (QSAR) study of aromatic inhibitors against aldose reductase (AR) activity was performed using variable selection from stepwise multiple linear regression (MLR) and genetic algorithm (GA)-MLR. As a result of variable selection, stepwise MLR and GA-MLR gave the same results with one, two, three and five descriptors and different results with four and six descriptors. GA-MLR produced higher values and was better in explanatory and predictive power than stepwise MLR in four variables. AR activity (pIC50) of aromatic derivatives was expressed with acceptable explanatory (74.6-81.2%) and predictive power (68.8-74.4%) in models 3 and 4. The resulting models with the given descriptors illustrate that hydrophobic and electrostatic interactions play a significant role in inhibition of AR activity. This study suggests that the QSAR models can be used as guidelines to predict improved aldose reductase inhibitory activity and to obtain reliable predictions in structurally diverse compounds.

Synthesis and evaluation of a series of 3,4,5-trimethoxycinnamic acid derivatives as potential antinarcotic agents.

A series of 3,4,5-trimethoxycinnamic acid derivatives was prepared and evaluated for antinarcotic effects on morphine dependence in mice and binding affinities on serotonergic receptors. The key synthetic strategies involve generation of ketones 6-7, esters 9-12 through condensation reaction, and amides 13-19 via coupling reaction using 1-hydroxybenzotriazole/ethyl(dimethylaminopropryl)carbodiimide system in high yield. We found that the naloxone-induced morphine withdrawal syndrome was significantly suppressed by new synthetic 3,4,5-trimethoxycinnamic acid derivatives (20 mg/kg/day). Most of 3,4,5-trimethoxycinnamic acid derivatives were found to have high affinity to 5-HT(1A) receptor. The naloxone-induced morphine withdrawal syndrome was attenuated by (+)8-OH-DPAT (0.1 mg/kg/day, i.p.), a 5-HT(1A) receptor agonist. In cortical neuronal cells, (+)8-OH-DPAT (1 µM) produced an elevation of the pERK 1/2 expression, and the elevated pERK levels were inhibited by WAY 100635, a 5-HT(1A) receptor-specific antagonist. Interestingly, the pERK levels were increased by the 3,4,5-trimethoxycinnamic acid derivatives and the derivatives-mediated changes in pERK levels were blocked by the WAY 100635. These results suggested that new synthetic 3,4,5-trimethoxycinnamic acid derivatives have a potential antinarcotic effect through acting as a 5-HT(1A) receptor agonist in mice.

Simple synthesis of modafinil derivatives and their anti-inflammatory activity.

Simple synthesis of modafinil derivatives and their biological activity are described. The key synthetic strategies involve substitution and coupling reactions. We determined the anti-inflammatory effects of modafinil derivatives in cultured BV2 cells by measuring the inhibition of nitrite production and expression of iNOS and COX-2 after LPS stimulation. It was found that for sulfide analogues introduction of aliphatic groups on the amide part (compounds 11a­d) resulted in lower anti-inflammatory activity compared with cyclic or aromatic moieties (compounds 11e­k). However, for the sulfoxide analogues, introduction of aliphatic moieties (compounds 12a­d) showed higher anti-inflammatory activity than cyclic or aromatic fragments (compounds 12e­k) in BV-2 microglia cells.

Total synthesis of flocoumafen via knoevenagel condensation and intramolecular ring cyclization: general access to natural products.

The total synthesis and structure determination of cis- and trans-flocoumafen was described. The key synthetic steps involve Knoevenagel condensation with p-methoxybenzaldehyde, in situ decarboxylation and intramolecular ring cyclization to construct the tetralone skeleton. Stereospecific reduction of the O-alkylated ketone 13 afforded good yield of precusor alcohol 5. Final coupling of alcohol 5 with 4-hydroxy-coumarin yielded flocoumafen (1). Separation and structure determination of cis- and trans-flocoumafen through 2D NMR analyses-assisted computer simulation techniques for the evaluation of anticoagulant activities are reported for the first time. This method is useful for generating the core tetralone skeleton of 4-hydroxycoumarin derivatives and provides a generalized access to various warfarin type anticoagulants.

Determination of daumone in mouse plasma by HPLC/MS-MS.

Daumone, a pheromone secreted by Caenorhabditis elegans, is an essential regulator of chemosensory processes in development and aging. A quantification method using HPLC/MS-MS was developed for the determination of daumone in mouse plasma. After simple protein precipitation with acetonitrile including methaqualone (an internal standard), the analytes were chromatographed on a reversed-phase column and detected by liquid chromatography/tandem mass spectrometry with electrospray ionization. The accuracy and precision of the assay were in accordance with FDA regulations for validation of bioanalytical methods. This method was applied to measure the plasma daumone concentrations following a 5-week repeated oral administration of daumone in mice.

Convenient synthesis and biological evaluation of modafinil derivatives: benzhydrylsulfanyl and benzhydrylsulfinyl [1,2,3]triazol-4-yl-methyl esters.

Simple synthesis and biological activities of modafinil derivatives are described. The key reactions include condensation of acid and propargyl alcohol, subsequent 1,3-dipolar cycloaddition reaction of alkynes and (3-azido-propyl)cyclohexane or (4-azido-butyl)benzene in the presence of sodium ascorbate and CuSO4·5H2O in excellent yield. They were then evaluated for the suppression of LPS-induced NO generation in vitro. It was found that all compounds showed moderate effects for suppression of LPS-induced NO generation.

Discovery of artemisinin-glycolipid hybrids as anti-oral cancer agents.

Novel artemisinin-glycolipid hybrids were directly synthesized from 12ß (C-C)-type deoxoartemisinin and glycolipid and exhibited exceptional in vitro anticancer activity, particularly against the oral carcinoma cancer cell lines, respectively. The artemisinin-glycolipid hybrids, with effective concentrations under 20 µM, demonstrated better anticancer activity than either artemisinin or glycolipid alone and showed five times more anti-oral cancer activity than either cisplatin or paclitaxel.

Synthesis of a dual-labeled probe of dimethyl lithospermate B with photochemical and fluorescent properties.

Dimethyl lithosermate B (DLB) is a highly potent natural antioxidant and antidiabetic polyphenol with unknown mode of action. To determine its cellular targets, a photochemical and fluorescent dimethyl lithopermate B probe was designed and efficiently synthesized. The dual-labeled chemical probe for biological application was evaluated by UV and fluorescence to determine its electrochemical absorption and emission properties. This probe could be valuable for investigating ligand-protein interactions and subcellular localization.

Practical synthesis and biological evaluation of bergenin analogs.

Here, we describe the practical synthesis and biological properties of bergenin and its structural analogs. Synthetic bergenin compounds were prepared by acylation of bergenin. These compounds were then evaluated for suppression of lipopolysaccharide-induced nitric oxide (NO) generation in cultured cells and anti-narcotic effects on morphine-dependent mice. We found that bergenin derivatives showed potent anti-inflammatory activity (suppression of NO generation) at concentrations ranging from 20 to 30 µmin vitro, and bergenin derivatives (10-20 mg/kg) exhibited significant anti-narcotic effects on morphine dependence in mice. These results suggest the potential utility of bergenin and its analogs as anti-narcotic agents and the design of more potent anti-inflammatory compounds.

Quantitative determination of daumone in rat plasma by liquid chromatography-mass spectrometry.

Daumone, 6-(3,5-dihydroxy-6-methyl-tetrahydro-pyran-2-yloxy)-heptanoic acid is a pheromone secreted by Caenorhabditis elegans, and has been known as a pivotal regulator of chemosensory processes in development and ageing. A quantification method using mass spectrometry was developed for the determination of daumone in rat plasma. After simple protein precipitation with acetonitrile including an internal standard, the analytes were chromatographed on a reversed-phase column and detected by liquid chromatography/tandem mass spectrometry with electrospray ionization. The accuracy and precision of the assay were in accordance with FDA regulations for validation of bioanalytical methods. This method was applied to measure the plasma daumone concentrations after a single intravenous administration of daumone in rats.

Concise synthesis and antiangiogenic activity of artemisinin-glycolipid hybrids on chorioallantoic membranes.

Novel hybrids of non acetal and acetal-type derivatives at C-12 of artemisinin and glycolipids were synthesized via efficient coupling reactions. Some of these hybrids showed potent in vivo antiangiogenic activity on the chorioallantoic membrane (CAM) that was higher than or comparable to those of fumagillin and thalidomide at a concentration of 2.5 nmol.

Natural therapeutic magnesium lithospermate B potently protects the endothelium from hyperglycaemia-induced dysfunction.

AIMS: We have investigated the effects of magnesium lithospermate B (MLB), the active compound of the Oriental herbal remedy, Salvia miltiorrhizae, on endothelial dysfunction associated with diabetes mellitus using cultured endothelial cells and an animal model of type 2 diabetes mellitus. METHODS AND RESULTS: The effect of MLB on vasodilatory function in Otsuka Long-Evans Tokushima Fatty (OLETF) rats was assessed. MLB treatment for 20 weeks starting at 12 weeks attenuated the decrease in endothelium-dependent vasodilation in OLETF rats. MLB treatment also increased serum nitrite level and reduced serum advanced glycation end products concentration. The effect of MLB was greater than an equivalent dose of alpha-lipoic acid (alphaLA), a popular antioxidant treatment. MLB rescued the inhibition of endothelial nitric oxide synthase (eNOS) activity and eNOS phosphorylation in endothelial cells cultured in hyperglycaemia. This effect was dependent on Akt phosphorylation and associated with decreased O-linked N-acetylglucosamine protein modification of eNOS. MLB also increased nuclear factor erythroid 2-related factor-2 (Nrf-2) activation in a phosphoinositide 3-kinase/Akt pathway dependent manner. MLB treatment induced the expression of the Nrf-2-regulated antioxidant enzyme, heme oxygenase-1. The antioxidant alphaLA could not produce this effect. Moreover, MLB decreased oxidative stress and endothelial cell apoptosis caused by hyperglycaemia. CONCLUSION: MLB is a naturally occurring, new generation antioxidant that activates eNOS and ameliorates endothelial dysfunction in diabetes by enhancing vasodilation in addition to reducing oxidative stress. The relative strong performance of MLB makes it an ideal candidate for further, expanded trials as a new generation of antioxidant to treat diabetes-related complications.