Perylenequinones from an endophytic Alternaria sp. of Pinus ponderosa.

The differential solvent extraction and further purification of fractions of endophytic Alternaria sp. isolated from Pinus ponderosa led to the isolation of further two perylenequinone compounds as 3,6,6a,9,10-pentahydroxy-7,8-epoxy-4-oxo-4,5,6,6a,6b,7,8,9-octahydroperylene (1) and 3,6,6a,7,10-pentahydroxy-4,9-dioxo-4,5,6,6a,6b,7,8,9-octahydroperylene (2). Structure of compounds 1-2 was determined on the basis of detailed spectroscopic analysis, as well as by comparison with literature reports. The antimicrobial, antileismanial, antimalarial and cytotoxic activities of compound 1 and 2 were evaluated.

A novel dihydroxylated derivative of artemisinin from microbial transformation.

Microbial transformation of artemisinin (1) by Cunninghamella elegans was investigated. Four isolated products were identified as 6ß-hydroxyartemisinin (2), 7α-hydroxyartemisinin (3), 7ß-hydroxyartemisinin (4), and 6ß,7α-dihydroxyartemisinin (5). The structures were elucidated by spectroscopic and X-ray crystallographic analysis. Product 5 is a novel compound and being reported here for the first time. It features two hydroxyl groups in its structure, and this is the first report on dihydroxylation of the artemisinin skeleton. Quantitative structure-activity relationship and molecular modeling studies indicate the modification of artemisinin skeleton will increase antimalarial activity and water solubility. The chemical syntheses of artemisinin derivatives at C6 or C7 position are impossible due to the lack of functional groups. 6ß,7α-Dihydroxyartemisinin is hydroxylated at both 6ß- and 7α-positions of artemisinin skeleton at the same time. Therefore, this new compound would be a good scaffold for further structural modification in the search for more potent antimalarial drugs.

The Challenge of Reproducibility and Accuracy in Nutrition Research: Resources and Pitfalls.

Inconsistent and contradictory results from nutrition studies conducted by different investigators continue to emerge, in part because of the inherent variability of natural products, as well as the unknown and therefore uncontrolled variables in study populations and experimental designs. Given these challenges inherent in nutrition research, it is critical for the progress of the field that researchers strive to minimize variability within studies and enhance comparability between studies by optimizing the characterization, control, and reporting of products, reagents, and model systems used, as well as the rigor and reporting of experimental designs, protocols, and data analysis. Here we describe some recent developments relevant to research on plant-derived products used in nutrition research, highlight some resources for optimizing the characterization and reporting of research using these products, and describe some of the pitfalls that may be avoided by adherence to these recommendations.

Biotransformation of artemisinin by Aspergillus niger.

Biotransformation of artemisinin (1) by Aspergillus niger was investigated. During 12 days at 28 °C and pH 6.0, A. niger transformed artemisinin into four products. They were identified as 3ß-hydroxy-4,12-epoxy-1-deoxyartemisinin (2), artemisinin G (3), 3,13-epoxyartemisinin (4), and 4α-hydroxy-1-deoxyartemisinin (5). Products 2 and 4 are new compounds and are being reported here for the first time. The product 4 contains a 3,13-epoxy structure. This is the first report of epoxidation of artemisinin using microbial strains. The product 4 still has an intact peroxide bridge and therefore can be used as a scaffold for further structural modification using chemical and biological methods in the search for new antimalarial drugs.

Discovery of thienoquinolone derivatives as selective and ATP non-competitive CDK5/p25 inhibitors by structure-based virtual screening.

Calpain mediated cleavage of CDK5 natural precursor p35 causes a stable complex formation of CDK5/p25, which leads to hyperphosphorylation of tau. Thus inhibition of this complex is a viable target for numerous acute and chronic neurodegenerative diseases involving tau protein, including Alzheimer's disease. Since CDK5 has the highest sequence homology with its mitotic counterpart CDK2, our primary goal was to design selective CDK5/p25 inhibitors targeting neurodegeneration. A novel structure-based virtual screening protocol comprised of e-pharmacophore models and virtual screening workflow was used to identify nine compounds from a commercial database containing 2.84 million compounds. An ATP non-competitive and selective thieno[3,2-c]quinolin-4(5H)-one inhibitor (10) with ligand efficiency (LE) of 0.3 was identified as the lead molecule. Further SAR optimization led to the discovery of several low micromolar inhibitors with good selectivity. The research represents a new class of potent ATP non-competitive CDK5/p25 inhibitors with good CDK2/E selectivity.

Efficient synthesis of 4-oxo-4,5-dihydrothieno[3,2-c]quinoline-2-carboxylic acid derivatives from aniline.

The first reported synthesis of potential kinase inhibitors, 4-oxo-4,5-dihydrothieno[3,2-c]quinoline-2-carboxylic acid derivatives starting from aniline is described. This efficient high yielding sequence was carried out in six steps without any chromatographic purification. A tandem nucleophilic aromatic substitution/cyclization reaction was used as a key step in the sequence. The versatile intermediate 2-carboxylic acid was used as a suitable precursor to access the functionalization of the C-ring, by convergent analog synthesis of several novel derivatives.

Determination of antimalarial compound, ARB-89 (7ß-hydroxy-artemisinin carbamate) in rat serum by UPLC/MS/MS and its application in pharmacokinetics.

Among all the antimalarial agents, artemisinin and its semi synthetic family of analogs are the most potent antimalarials available for the treatment of Plasmodium falciparum infections. But these analogs have a few issues such as shorter half-lives and low oral bioavailability values. In order to overcome these inherent problems, novel artemisinin analogs were synthesized from 7ß-hydroxy artemisinin by the Department of Medicinal Chemistry, University of Mississippi using a new synthesis mechanism. Out of all the 7ß-hydroxy artemisinin analogs synthesized, 7ß-hydroxy artemisinin carbamate (ARB-89) was chosen as a lead compound because of its high in vitro and in vivo activity. In this manuscript, a sensitive and rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) method was developed and validated for the quantification of ARB-89 in rat serum. The analysis was carried out on an Acquity™ UPLC BEH C(18) column (1.7 µm, 2.1 mm × 50 mm) with a flow rate of 0.3 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in positive electrospray ionization (ESI) mode. The selected mass-to-charge (m/z) ratio transitions used in the multiple reaction monitoring (MRM) for ARB-89 and artemisinin (internal standard) were m/z 778.4>253.4 and m/z 283.4>151.1 respectively. The calibration curve was linear from 1.00 ng/mL to 10.0 µg/mL (r(2)=0.999). A simple protein precipitation method was used for extraction. Moreover, the inter-day and intra-day precision values were found to be less than 15%. The recoveries of the method ranged from 94.0% to 96.7% at three concentrations. ARB-89 in rat serum was found to be stable at room temperature for 12h. This method was successfully used to quantitate the novel antimalarial compound ARB-89 after intravenous and oral administration to rats.

Xenobiotic biotransformation of 4-methoxy-N-methyl-2-quinolone, isolated from Zanthoxylum monophyllum.

Phytochemical evaluation of Zanthoxylum monophyllum has led to the isolation of the alkaloid 4-methoxy-N-methyl-2-quinolone (1) with a significant activity against methicillin-resistant Staphylococcus aureus (MRSA), with an IC50 value of 1.5 microg/mL. Xenobiotic biotransformation of 1 has been conducted with the general goal of increasing the bioactivity of the compound and contributing new leads for further pharmacological research. Twenty-nine microorganisms were used for screening and two (Aspergillus flavus and Cunninghamella echinulata var. echinulata) were able to transform compound 1 to 4-methoxy-2-quinolone (2). Structural identification of the compounds was based on NMR, IR, and MS data.

A new instrument for thermal electron attachment at high temperature: NF3 and CH3Cl attachment rate constants up to 1100 K.

A new high temperature flowing afterglow Langmuir probe (HT-FALP) apparatus is described. A movable Langmuir probe and a four-needle reactant gas inlet were fitted to an existing high temperature flowing afterglow apparatus. The instrument is suitable for study of electron attachment from 300-1200 K, the upper limit set to avoid softening of the quartz flow tube. We present results for two reactions over extended ranges: NF(3) (300-900 K) and CH(3)Cl (600-1100 K). Electron attachment rate constants for NF(3) had been measured earlier using our conventional FALP apparatus. Those measurements were repeated with the FALP and then extended to 900 K with the HT-FALP. CH(3)Cl attaches electrons too weakly to study with the low temperature FALP but reaches a value of approximately 10(-9) cm(3) s(-1) at 1100 K. F(-) is produced in NF(3) attachment at all temperatures and Cl(-) in CH(3)Cl attachment, as determined by a quadrupole mass spectrometer at the end of the flow tube. Future modifications to increase the plasma density should allow study of electron-ion recombination at high temperatures.

Synthesis, antitubercular activity and docking study of novel cyclic azole substituted diphenyl ether derivatives.

The re-emergence of tuberculosis (TB) as a global health problem over the past few decades, accompanied by the rise of drug-resistant strains of Mycobacterium tuberculosis, emphasizes the need for discovery of new therapeutic drugs against this disease. The emerging serious problem both in terms of TB control and clinical management prompted us to synthesize a novel series of heterocyclic o/m/p substituted diphenyl ether derivatives and determine their activity against H37Rv strain of Mycobacterium. All 10 compounds inhibited the growth of the H37Rv strain of mycobacterium at concentrations as low as 1 microg/mL. This level of activity was found comparable to the reference drugs rifampicin and isoniazid at the same concentration. Molecular modeling of the binding of the diphenyl ether derivatives on enoyl-ACP reductase, the molecular target site of triclosan, indicated that these compounds fit within the binding domain occupied by triclosan. Hence the diphenyl ether derivatives tested in this study were docked to ENR and the binding of the diphenyl ether derivatives was also estimated using a variety of scoring functions that have been compiled into the single consensus score. As the scores ranged from 47.27% to 65.81%, these bioactive compounds appear to have a novel mechanism of action against M. tuberculosis, and their structural features should be studied further for their potential use as new antitubercular drugs.

Ultra-performance liquid chromatography-tandem mass spectrometric method for the determination of Artemisinin in rat serum and its application in pharmacokinetics.

A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to quantify artemisinin in rat serum. The lower limit of quantification (LLOQ) was 4 ng/mL. The calibration curve was linear from 4 ng/mL to 10,000 ng/mL (R=0.998). The assay was based on the selected reaction monitoring (SRM) transitions at m/z 305.4-151.10 for artemisinin and m/z 335.2-163.10 for arteether (internal standard). The artemisinin and internal standard can be separated from endogenous interferences in rat serum. Inter- and intra-day assay variation was less than 15%. The extraction recoveries ranged from 80.0 to 107.3% at the three concentrations (5000, 2000, and 200 ng/mL). This method was successfully applied to pharmacokinetic studies of artemisinin after intravenous and oral administration to rats.

7ß-Hydroxy-artemisinin.

CRYSTALS OF THE TITLE COMPOUND [SYSTEMATIC NAME: (3R,6R,7S,8aR,9R,12aR)-7-hydr-oxy-3,6,9-trimethyl-octa-hydro-3,12-ep-oxy[1,2]dioxepino[4,3-i]isochromen-10(3H)-one], C(15)H(22)O(6), were obtained from microbial transformation of artemisinin by a culture of Cunninghamella elegans. The stereochemistry of the compound is consistent with the spectroscopic findings in previously published works. A weak O-H⋯O hydrogen bond occurs in the crystal structure, together with intermolecular C-H⋯O hydrogen bonds.

Rate constants for the reactions of CO3- and O3- with SO2 from 300 to 1440 K.

Rate constants for the reactions of CO(3) (-) and O(3) (-) with SO(2) have been measured between 300 and 1440 K in a high temperature flowing afterglow apparatus. The CO(3) (-) rate constants near to the collision rate at low temperatures and fall by about a factor of 50 with temperature until a broad minimum is reached at 900-1300 K. The highest temperature point shows the increasing rate constant. Comparison to drift tube data taken in a helium buffer shows that total energy controls the reactivity, presumably because the reaction goes through a long lived complex even at 1440 K. The reaction of O(3) (-) with SO(2) was studied up to 1400 K. The rate constant is collisional until 700 K and then decreases with increasing temperature. Rate constants measured at 1300 and 1400 K appear to show an increase, but that observation is questionable since O(3) (-) could not be made cleanly. The O(3) (-) data at 1200 K and below show that total energy controls reactivity in that range.

Microbial transformation of artemisinin to 5-hydroxyartemisinin by Eurotium amstelodami and Aspergillus niger.

Transformation of the anti-malarial drug artemisinin by the fungi Eurotium amstelodami and Aspergillus niger were investigated. Cultures were grown in sucrose/malt broth with artemisinin for 14 days and extracted with ethyl acetate. Extracts were characterized by liquid chromatography. Two metabolites from each fungal extract were isolated and identified using mass spectrometry and nuclear magnetic resonance. 5Beta-hydroxyartemisinin and 7beta-hydroxyartemisinin were isolated in 63 and 32% yields, respectively, from the extract of E. amstelodami, and 80 and 19%, respectively, from the extract of A. niger.

Hydroxylation of 10-deoxoartemisinin by Cunninghamella elegans.

The microbial metabolism of 10-deoxoartemisinin (1), a derivative of the antimalarial drug artemisinin, was investigated. Various strains of fungi were investigated for their ability to transform 1. Of these microorganisms, only Cunninghamella elegans was capable of transforming 1 to 5beta-hydroxy-10-deoxoartemisinin (2), 4alpha-hydroxy-1,10-deoxoartemisinin (3), and 7beta-hydroxy-10-deoxoartemisinin (4). The metabolites 2 and 4 retained an intact peroxide group and are therefore useful scaffolds for synthetic modification in the search for new antimalarial agents.

Hydroxylation of 10-deoxoartemisinin to 15-hydroxy-10-deoxoartemisinin by Aspergillus niger.

The microbial hydroxylation of 10-deoxoartemisinin was investigated with the aim of obtaining preparative yields of hydroxy derivatives. During 14 d at 28 degrees C and pH 6.5 Aspergillus niger transformed 10-deoxoartemisinin (500 mg l(-1)) to 15-hydroxy-10-deoxoartemisinin (26%) and 7beta-hydroxy-10-deoxoartemisinin (69%).

Three-dimensional quantitative structure-activity relationship and comparative molecular field analysis of dipeptide hydroxamic acid Helicobacter pylori urease inhibitors.

A homology model of Helicobacter pylori urease was developed by using the crystal structure of urease from Klebsiella aerogenes (EC 3.5.1.5) as a template. The acetohydroxamic acid moiety was docked into the active pocket of the enzyme model, followed by relaxation of the complex by use of molecular dynamics. The resulting conformation was used as a template to construct 24 potential dipeptide hydroxamic acid inhibitors with which comparative molecular field analysis (CoMFA) was performed. The resulting model provided a cross-validation correlation coefficient (q(2)(L00)) of 0.610, a conventional r(2) value of 0.988, and an F (Fisher indication of statistical significance) value of 294.88. We were able to validate the CoMFA model by using the 50% inhibitory concentrations of six compounds that were not included in the construction of the model. A very good structural correlation was observed between the amino acids in the model urease's active pocket and the contour maps derived from the CoMFA model. This correlation, accompanied by the validation supplied by use of the CoMFA data, illustrates that the model can aid in the prediction and design of novel H. pylori urease inhibitors.