Residue analysis of tebufenozide and indoxacarb in chicken muscle, milk, egg and aquatic animal products using liquid chromatography-tandem mass spectrometry.

We developed an analytical method using liquid-liquid extraction (LLE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and quantify tebufenozide (TEB) and indoxacarb (IND) residues in animal and aquatic products (chicken muscle, milk, egg, eel, flatfish, and shrimp). The target compounds were extracted using 1% acetic acid (0.1% acetic acid for egg only) in acetonitrile and purified using n-hexane. The analytes were separated on a Gemini-NX C18 column using (a) distilled water with 0.1% formic acid and 5 mm ammonium acetate and (b) methanol with 0.1% formic acid as the mobile phase. All six-point matrix-matched calibration curves showed good linearity with coefficients of determination (R2 ) ≥0.9864 over a concentration range of 5-50 µg/kg. Intra- and inter-day accuracy was expressed as the recovery rate at three spiking levels and ranged between 73.22 and 114.93% in all matrices, with a relative standard deviation (RSD, corresponding to precision) ≤13.87%. The limits of quantification (LOQ) of all target analytes ranged from 2 to 20 µg/kg, which were substantially lower than the maximum residue limits (MRLs) specified by the regulatory agencies of different countries. All samples were collected from different markets in Seoul, Republic of Korea, and tested negative for tebufenozide and indoxacarb residues. These results show that the method developed is robust and may be a promising tool to detect trace levels of the target analytes in animal products.

Identification of Ecdysone Hormone Receptor Agonists as a Therapeutic Approach for Treating Filarial Infections.

BACKGROUND: A homologue of the ecdysone receptor has previously been identified in human filarial parasites. As the ecdysone receptor is not found in vertebrates, it and the regulatory pathways it controls represent attractive potential chemotherapeutic targets. METHODOLOGY/ PRINCIPAL FINDINGS: Administration of 20-hydroxyecdysone to gerbils infected with B. malayi infective larvae disrupted their development to adult stage parasites. A stable mammalian cell line was created incorporating the B. malayi ecdysone receptor ligand-binding domain, its heterodimer partner and a secreted luciferase reporter in HEK293 cells. This was employed to screen a series of ecdysone agonist, identifying seven agonists active at sub-micromolar concentrations. A B. malayi ecdysone receptor ligand-binding domain was developed and used to study the ligand-receptor interactions of these agonists. An excellent correlation between the virtual screening results and the screening assay was observed. Based on both of these approaches, steroidal ecdysone agonists and the diacylhydrazine family of compounds were identified as a fruitful source of potential receptor agonists. In further confirmation of the modeling and screening results, Ponasterone A and Muristerone A, two compounds predicted to be strong ecdysone agonists stimulated expulsion of microfilaria and immature stages from adult parasites. CONCLUSIONS: The studies validate the potential of the B. malayi ecdysone receptor as a drug target and provide a means to rapidly evaluate compounds for development of a new class of drugs against the human filarial parasites.

Suprafenacine, an indazole-hydrazide agent, targets cancer cells through microtubule destabilization.

Microtubules are a highly validated target in cancer therapy. However, the clinical development of tubulin binding agents (TBA) has been hampered by toxicity and chemoresistance issues and has necessitated the search for new TBAs. Here, we report the identification of a novel cell permeable, tubulin-destabilizing molecule--4,5,6,7-tetrahydro-1H-indazole-3-carboxylic acid [1p-tolyl-meth-(E)-ylidene]-hydrazide (termed as Suprafenacine, SRF). SRF, identified by in silico screening of annotated chemical libraries, was shown to bind microtubules at the colchicine-binding site and inhibit polymerization. This led to G2/M cell cycle arrest and cell death via a mitochondria-mediated apoptotic pathway. Cell death was preceded by loss of mitochondrial membrane potential, JNK-mediated phosphorylation of Bcl-2 and Bad, and activation of caspase-3. Intriguingly, SRF was found to selectively inhibit cancer cell proliferation and was effective against drug-resistant cancer cells by virtue of its ability to bypass the multidrug resistance transporter P-glycoprotein. Taken together, our results suggest that SRF has potential as a chemotherapeutic agent for cancer treatment and provides an alternate scaffold for the development of improved anti-cancer agents.

Gold nanoparticle-modified graphite pencil electrode for the high-sensitivity detection of hydrazine.

A novel gold nanoparticle-modified graphite pencil electrode (AuNP-GPE) is prepared just by immersing a bare GPE in AuNP solution, followed by heating for 15 min. The bare and modified GPEs are characterized by FE-SEM imaging and cyclic voltammetry. The AuNP-GPEs showed excellent electrocatalytic activities with respect to hydrazine oxidation, with good reproducibility. To reduce the quantification and detection limits, and increase the hydrazine sensitivity, the pH and square wave voltammetry parameters are optimized. A square wave voltammetry study as a function of the hydrazine concentration showed that the AuNP-GPE detector's quantification limit was 100 nmol L(-1) hydrazine, much lower than the value obtained using amperometry (10 µmol L(-1)). The limits of detection (at 3σ) for hydrazine sensing at AuNP-GPEs using square wave voltammetry and amperometry were 42 nmol L(-1) and 3.07 µmol L(-1). Finally, the modified electrode was used to determine the hydrazine concentration in drinking water, and satisfactory results are obtained. This simple, rapid, low-cost method for fabricating a modified electrode is an attractive approach to the development of new sensors.

Isolation and characterization of unusual hydrazides from Streptomyces sp. impact of the cultivation support and extraction procedure.

Three novel hydrazides, geralcins C-E (1-3), were isolated from Streptomyces sp. LMA-545, together with MH-031 and geralcins A and B. This unusual family of compounds was isolated from liquid-state and agar-supported fermentation using Amberlite XAD-16 solid-phase extraction during the cultivation step. The use of such neutral resin during the cultivation step allowed the specific adsorption of microbial secondary metabolites, avoiding any contamination of the crude extracts by the constituents of the culture medium. The trapped compounds were eluted from the resin with methanol, and their structures elucidated using (1)H, (13)C, and (15)N NMR spectroscopic analysis and high-resolution mass spectrometry. Molecular modeling calculations were applied in order to support structural attributions. No antimicrobial, cytotoxic, or DnaG-inhibition activities were detected for geralcins D and E. Geralcin C has no antimicrobial activity but exhibited an IC(50) of 0.8 µM against KB and HCT116 cancer cell lines. Furthermore, geralcin C inhibited the E. coli DnaG primase, a Gram-negative antimicrobial target, with an IC(50) of 0.7 mM.

Isolation and characterization of α,ß-unsaturated γ-lactono-hydrazides from Streptomyces sp.

Two novel α,ß-unsaturated γ-lactono-hydrazides, geralcin A (2) and geralcin B (3), were isolated from Streptomyces sp. LMA-545. This unusual scaffold consists of the condensation of alkyl-hydrazide with an α,ß-unsaturated γ-lactone, 3-(5-oxo-2H-furan-4-yl)propanoic acid (1), which was isolated from the same broth culture. Amberlite XAD-16 solid-phase extraction was used during the cultivation step, and the trapped compounds (1-3) were eluted from the resin with methanol. The structures were elucidated using (1)H, (13)C, and (15)N NMR spectroscopic analysis and high-resolution mass spectrometry. Geralcin B (3) was cytotoxic against MDA231 breast cancer cells with an IC(50) of 5 µM.

Prediction of the lipophilicity of nine new synthesized selenazoly and three aroyl-hydrazinoselenazoles derivatives by reversed-phase high performance thin-layer chromatography.

Using reversed-phase high-performance thin-layer chromatography and a methanol-water mixture as the mobile phase, the lipophilicity of 12 new synthesized derivatives is studied. The first eight compounds have as a basic chemical structure aryliden-hydrazino-selenazoles, and the second group of the three compounds belongs to aroyl-hydrazinoselenazoles. The linear correlation between R(Mw) and the methanol-water ratios showed high values for the correlation coefficient. The chromatographic hydrophobic index is determined by using the ratio -R(Mw)/S, and the obtained values ranged between 99 and 73. A good linear correlation is obtained between R(Mw) and the slope. The log P values are calculated using ACD/Labs Software. The matrices are formed with R(Mw) and log P and are subjected to a principal component analysis (PCA). The best way to extract information from PCA is graphically, by plotting the obtained matrices. By analyzing the scores, the compounds can be grouped as follows: a group containing nine compounds, and a second one containing three compounds. Each group of compounds has the same basic chemical structure.

Hydrazidomycins, cytotoxic alkylhydrazides from Streptomycesatratus.

Three unusual alkyhydrazide natural products, named hydrazidomycin A (1), B (2) and C (3), were isolated from the chloroform extract of a Streptomycesatratus culture, and their structures were elucidated by MS and NMR techniques. Hydrazidomycins A-C exhibited moderate to strong cytotoxic activities in a panel of 42 cell lines, with hydrazidomycin A being the most potent compound (mean IC(50)=0.37 µM).

Elaiomycins B and C: alkylhydrazide antibiotics from Streptomyces sp. BK 190.

Two novel alkyhydrazides, elaiomycins B and C, together with the azoxy antibiotic elaiomycin were isolated from Streptomyces sp. BK 190. The structures were established by 1D- and 2D-NMR spectroscopy including (15)N NMR studies and high-resolution orbitrap-ESI-mass spectrometry.

Synthesis, stereochemical separation, and biological evaluation of selective inhibitors of human MAO-B: 1-(4-arylthiazol-2-yl)-2-(3-methylcyclohexylidene)hydrazines.

Novel 1-(4-arylthiazol-2-yl)-2-(3-methylcyclohexylidene)hydrazine derivatives have been investigated for their ability to inhibit selectively the activity of the human B isoform of monoamine oxidase. These compounds were obtained as racemates and (R)-enantiomers by a stereoconservative synthetic pattern in high yield and enantiomeric excess. The (S)-enantiomers of the most active derivatives have been separated by enantioselective HPLC. All compounds showed selective activity against hMAO-B with IC(50) ranging between 21.90 and 0.018 microM.

A novel aryl-hydrazide from the marine lichen Lichina pygmaea: isolation, synthesis of derivatives, and cytotoxicity assays.

A new aryl-hydrazide l-glutamic acid derivative, pygmeine (3), was isolated from a methanolic extract of Lichina pygmaea, a marine lichen. Synthetic derivatives obtained via a two-step coupling of l-glutamic acid with phenylhydrazine moieties were useful to elucidate the structure of 3 and to carry out biological assays. Thus, the cytotoxicity of the ortho-, meta-, and para-hydroxyl isomers along with their respective benzyl intermediates, and a natural methoxylated analog, were evaluated on murine and human melanoma cells (B16, A375). The para-hydroxyl isomer 6 was found to be the most active (IC(50)=1.6 microM) on B16 cells.

Anti-genotoxic hydrazide from Crinum defixum.

Crinum defixum Ker-Gawl popularly known as Bon-naharu (meaning wild garlic) in Assam. It is found abundantly growing wild on riverbanks of Dhansiri River in Golaghat District of Assam. It is used as ethnomedicine in this part of India for a number of ailments. Bioassay guided chemical investigation of the bulbs of Crinum defixum Ker-Gawl afforded to isolate a new hydrazide derivative and its structure was determined as (E)-N'-[(E)-2-butenoyl]-2-butenoylhydrazide by spectroscopic methods. The compound was assayed for anti-genotoxic activity by onion root tip assay (by observing different types of chromosomal aberrations such as chromosomal bridges, stickiness, delayed anaphase, polyploidy and vagrant chromosome). The phyto-compound was found to have anti-genotoxic activity and imparted a clear dose dependent protective effect against the genotoxic effect of H(2)O(2). Further, the compound seems to be more effective against clastogenic aberrations than physiological aberration at the highest concentration used (250 ppm).

Isolation and identification of a novel impurity of erythromycin A 9-oxime desosaminehydrazinium salt.

In the manufacturing process for Biaxin (clarithromycin), erythromycin-A oxime, an intermediate, is obtained in high yield, when erythromycin-A is treated with hydroxylamine/isopropyl alcohol in the presence of acetic acid. An unusual impurity, the desosamine hydrazinium salt, is generated in this step of the synthetic pathway, and has been isolated and characterized by using one and two-dimensional NMR spectroscopy in conjunction with MS and EDS.

Separation-friendly Mitsunobu reactions: a microcosm of recent developments in separation strategies.

The Mitsunobu reaction is famous for its scope and power, but infamous for its separation headaches. Typically, the target product is enticed away from the reagent-derived byproducts by careful chromatography. The use of polymer-bound Mitsunobu reagents solves only half of the problem, because polymer-bound diethyl azodicarboxylate (DEAD) and phosphine reagents cannot be employed simultaneously. This article classifies, compares, and contrasts various emerging strategies for product isolation in Mitsunobu reactions. Because so many different strategies have been used, the Mitsunobu reaction is a microcosm for the new field of strategy level separations.

DPPH radical scavenging activity of ten natural p-terphenyl derivatives obtained from three edible mushrooms indigenous to China.

The antioxidant activities of ten natural p-terphenyl derivatives, 1-10, obtained from the fruiting bodies of three edible mushrooms (Thelephora ganbajun, Thelephora aurantiotincta, Boletopsis grisea) indigenous to China were evaluated in comparison with BHA ('butylated hydroxyanisole'=(1,1-dimethylethyl)-4-methoxyphenol) and alpha-tocopherol by the DPPH ('1,1-diphenyl-2-picrylhydrazyl'=2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl) radical-scavenging method. The compounds 1-3 showed significant antioxidant activity. The antioxidant activities of compounds 1-10 and reference compounds followed the order: 2>BHA>1>3>alpha-tocopherol>10>9>6 > 5>8>7>4. The compound 2 exhibited the strongest radical-scavenging activity with an EC(50) value of 0.07 (EC(50)(BHA) 0.09; EC(50)(alpha-tocopherol) 0.25).

DPPH free radical scavenger components from the fruits of Alpinia rafflesiana Wall. ex. Bak. (Zingiberaceae).

The methanol extract of the dried ripe fruits of Alpinia rafflesiana was investigated for its DPPH free radical scavenger constituents. 2',3',4',6'-Tetrahydroxychalcone (7), which has never been isolated from natural sources was found to be most active as a DPPH free radical scavenger with the IC50 value of 55 microM. Other known compounds isolated from this species include 5,6-dehydrokawain (1), flavokawin B (2). 1,7-diphenyl-5-hydroxy-6-hepten-3-one (3), (-)-pinocembrin (4), cardamonin (5) and (-)-pinostrobin (6). The DPPH free radical scavenger compounds were detected using TLC autographic analysis. The percentage inhibition of DPPH free radical scavenging activity was measured on isolates (5-7) using colorimetric analysis.

A re-evaluation of the antioxidant activity of purified carnosine.

The antioxidant activity of carnosine has been re-evaluated due to the presence of contaminating hydrazine in commercial carnosine preparations. Purified carnosine is capable of scavenging peroxyl radicals. Inhibition of the oxidation of phosphatidylcholine liposomes by purified carnosine is greater in the presence of copper than iron, a phenomenon likely to be due to the copper chelating properties of carnosine. Purified carnosine is capable of forming adducts with aldehydic lipid oxidation products. Adduct formation is greatest for alpha,beta-monounsaturated followed by polyunsaturated and saturated aldehydes. While the ability of carnosine to form adducts with aldehydic lipid oxidation products is lower than other compounds such as glutathione, the higher concentrations of carnosine in skeletal muscle are likely to make it the most important molecule that forms aldehyde adducts. Monitoring changes in carnosine concentrations in oxidizing skeletal muscle shows that carnosine oxidation does not occur until the later stages of oxidation suggesting that carnosine may not be as effective free radical scavenger in vivo as other antioxidants like alpha-tocopherol.

A novel fungal metabolite NG-061 enhances and mimics neurotrophic effect of nerve growth factor (NGF) on neurite outgrowth in PC12 cells.

During the course of our screening program for low molecular natural products with their ability to potentiate and/or mimic neurotrophic effect of NGF, a novel fungal metabolite, phenylacetic acid hydrazide derivative NG-061 was isolated from the fermentation broth of Penicillium minioluteum F-4627. NG-061 enhanced and mimicked neurotrophic effect of NGF on neurite outgrowth in a rat pheochromocytoma cell line PC12.

Preparation, conformation, and mild hydrolysis of 1-glycosyl-2-acetylhydrazines of the hexoses, pentoses, 2-acetamido-2-deoxyhexoses, and fucose.

The title compounds were prepared and their conformations studied by 1H-NMR. Their acid hydrolysis under mild conditions was monitored by 1H-NMR.