Synthesis and Antiangiogenic Activity of N-Alkylated Levamisole Derivatives

Inhibition of angiogenesis is a promising addition to current cancer treatment strategies. Neutralization of vascular endothelial growth factor by monoclonal antibodies is clinically effective but may cause side effects due to thrombosis. Low molecular weight angiogenesis inhibitors are currently less effective than antibody treatment and are also associated with serious side effects. The discovery of new chemotypes with efficient antiangiogenic activity is therefore of pertinent interest. (S)-levamisole hydrochloride, an anthelminthic drug approved for human use and with a known clinical profile, was recently shown to be an inhibitor of angiogenesis in vitro and exhibited tumor growth inhibition in mice. Here we describe the synthesis and in vitro evaluation of a series of N-alkylated analogues of levamisole with the aim of characterizing structure-activity relationships with regard to inhibition of angiogenesis. N-methyllevamisole and p-bromolevamisole proved more effective than the parent compound, (S)-levamisole hydrochloride, with respect to inhibition of angiogenesis and induction of undifferentiated cluster morphology in human umbilical vein endothelial cells grown in co-culture with normal human dermal fibroblasts. Interestingly, the cluster morphology caused by N-methyllevamisole was different than the clusters observed for levamisole, and a third "cord-like" morphology resembling that of the known drug suramin was observed for an aniline-containing derivative. New chemotypes exhibiting antiangiogenic effects in vitro are thus described, and further investigation of their underlying mechanism of action is warranted

An inverse association between plasma benzoxazinoid metabolites and PSA after rye intake in men with prostate cancer revealed with a new method

Prostate cancer (PC) is a common cancer among men, and preventive strategies are warranted. Benzoxazinoids (BXs) in rye have shown potential against PC in vitro but human studies are lacking. The aim was to establish a quantitative method for analysis of BXs and investigate their plasma levels after a whole grain/bran rye vs refined wheat intervention, as well as exploring their association with PSA, in men with PC. A quantitative method for analysis of 22 BXs, including novel metabolites identified by mass spectrometry and NMR, was established, and applied to plasma samples from a randomized crossover study where patients with indolent PC (n = 17) consumed 485 g whole grain rye/rye bran or fiber supplemented refined wheat daily for 6 wk. Most BXs were significantly higher in plasma after rye (0.3-19.4 nmol/L in plasma) vs. refined wheat (0.05-2.9 nmol/L) intake. HBOA-glc, 2-HHPAA, HBOA-glcA, 2-HPAA-glcA were inversely correlated to PSA in plasma (p < 0.04). To conclude, BXs in plasma, including metabolites not previously analyzed, were quantified. BX metabolites were significantly higher after rye vs refined wheat consumption. Four BX-related metabolites were inversely associated with PSA, which merits further investigation

Simple and selective spectrophotometric assay of diethylcarbamazine citrate using 2,3-dichloro-5,6-dicyano-p-benzoquinone and 2,4-dinitro phenol

Two simple, rapid and inexpensive spectrophotometric methods are described for the determination of diethylcarbamazine citrate (DEC) in bulk drug and formulations. The methods are based on the charge-transfer (CT) complexation reaction involving DEC as the n- donor and 2,3-dichloro-5,6-dicyano-p-benzoquinone DDQ] (method A) and 2,4-dinitro phenol DNP] (method B) as pi-acceptors in chloroform. The absorbance of CT complexes was measured at 480 nm for method A, and 420 nm for method B. Under optimum conditions, Beer's law was obeyed over the concentration ranges 4-90 and 4-100 mu g mL(-1) for methods A and B, respectively

Detection of Seasonal Variation in Aloe Polysaccharides Using Carbohydrate Detecting Microarrays

Aloe vera gel is a globally popular natural product used for the treatment of skin conditions. Its useful properties are attributed to the presence of bioactive polysaccharides. Nearly 25% of the 600 species in the genus Aloe are used locally in traditional medicine, indicating that the bioactive components in Aloe vera may be common across the genus Aloe. The complexity of the polysaccharides has hindered development of relevant assays for authentication of Aloe products. Carbohydrate detecting microarrays have recently been suggested as a method for profiling Aloe polysaccharide composition. The aim of this study was to use carbohydrate detecting microarrays to investigate the seasonal variation in the polysaccharide composition of two medicinal and two non-medicinal Aloe species over the course of a year. Microscopy was used to explore where in the cells the bioactive polysaccharides are present and predict their functional role in the cell wall structure. The carbohydrate detecting microarrays analyses showed distinctive differences in the polysaccharide composition between the different species and carbohydrate detecting microarrays therefore has potential as a complementary screening method directly targeting the presence and composition of relevant polysaccharides. The results also show changes in the polysaccharide composition over the year within the investigated species, which may be of importance for commercial growing in optimizing harvest times to obtain higher yield of relevant polysaccharides

1H NMR-based metabolomics combined with HPLC-PDA-MS-SPE-NMR for investigation of standardized Ginkgo biloba preparations

Commercial preparations of Ginkgo biloba are very complex mixtures prepared from raw leaf extracts by a series of extraction and prepurification steps. The pharmacological activity is attributed to a number of flavonoid glycosides and unique terpene trilactones (TTLs), with largely uncharacterized pharmacological profiles on targets involved in neurological disorders. It is therefore important to complement existing targeted analytical methods for analysis of Ginkgo biloba preparations with alternative technology platforms for their comprehensive and global characterization. In this work, 1H NMR-based metabolomics and hyphenation of high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) were used for investigation of 16 commercially available preparations of Ginkgo biloba. The standardized extracts originated from Denmark, Italy, Sweden, and United Kingdom, and the results show that 1H NMR spectra allow simultaneous assessment of the content as well as identity of flavonoid glycosides and TTLs based on a very simple sample-preparation procedure consisting of extraction, evaporation and reconstitution in acetone-d6. Unexpected or unwanted extract constituents were also easily identified in the 1H NMR spectra, which contrasts traditional methods that depend on UV absorption or MS ionizability and usually require availability of reference standards. Automated integration of 1H NMR spectral segments (buckets or bins of 0.02 ppm width) provides relative distribution plots of TTLs based on their H-12 resonances. The present study shows that 1H NMR-based metabolomics is an attractive method for non-selective and comprehensive analysis of Ginkgo extracts

Assessing specialized metabolite diversity in the cosmopolitan plant genus Euphorbia l.

Coevolutionary theory suggests that an arms race between plants and herbivores yields increased plant specialized metabolite diversity and the geographic mosaic theory of coevolution predicts that coevolutionary interactions vary across geographic scales. Consequently, plant specialized metabolite diversity is expected to be highest in coevolutionary hotspots, geographic regions, which exhibit strong reciprocal selection on the interacting species. Despite being well-established theoretical frameworks, technical limitations have precluded rigorous hypothesis testing. Here we aim at understanding how geographic separation over evolutionary time may have impacted chemical differentiation in the cosmopolitan plant genus Euphorbia. We use a combination of state-of-the-art computational mass spectral metabolomics tools together with cell-based high-throughput immunomodulatory testing. Our results show significant differences in specialized metabolite diversity across geographically separated phylogenetic clades. Chemical structural diversity of the highly toxic Euphorbia diterpenoids is significantly reduced in species native to the Americas, compared to Afro-Eurasia. The localization of these compounds to young stems and roots suggest a possible ecological relevance in herbivory defense. This is further supported by reduced immunomodulatory activity in the American subclade as well as herbivore distribution patterns. We conclude that computational mass spectrometric metabolomics coupled with relevant ecological data provide a strong tool for exploring plant specialized metabolite diversity in a chemo-evolutionary framework

Characterization of Antileishmanial Compounds from Lawsonia inermis L. Leaves Using Semi-High Resolution Antileishmanial Profiling Combined with HPLC-HRMS-SPE-NMR

This work describes an analytical platform based on semi-high-resolution antileishmanial profiling combined with hyphenation of high-performance liquid chromatography – high-resolution mass spectrometry – solid-phase extraction – nuclear magnetic resonance spectroscopy, i.e., semiHR-antileishmanial assay/HPLC-HRMS-SPE-NMR. The platform enables fast pinpointing of HPLC peaks representing Leishmania tropica inhibitors in complex matrices, with subsequent structural identification of targeted inhibitors. Active analytes were cumulatively trapped on SPE cartridges and the structures elucidated by analysis of NMR spectra obtained in the HPLC-HRMS-SPE-NMR mode. This led to the identification of six known compounds 2,4,6-trihydroxyacetophenone-2-O-β-D-glucopyranoside (1), lalioside (2), luteolin-4′-O-β-D-glucopyranoside (3), apigenin-4′-O-β-D-glucopyranoside (4), luteolin (5), and apigenin (6). IC50 of the active compounds were determined with luteolin being the most potent inhibitor with an IC50 value of 4.15 μg/ml. The platform proved to be an efficient method for the identification of L. tropica inhibitors

High-Resolution Inhibition Profiling Combined with HPLC-HRMS-SPE-NMR for Identification of PTP1B Inhibitors from Vietnamese Plants

Protein tyrosine phosphatase 1B (PTP1B) plays a key role as a negative regulator in insulin signal transduction by deactivating the insulin receptor. Thus, PTP1B inhibition has emerged as a potential therapeutic strategy for curing insulin resistance. In this study, 40 extracts from 18 different plant species were investigated for PTP1B inhibitory activity in vitro. The most promising one, the EtOAc extract of Ficus racemosa, was investigated by high-resolution PTP1B inhibition profiling combined with HPLC-HRMS-SPE-NMR analysis. This led to the identification of isoderrone (1), derrone (2), alpinumisoflavone (3) and mucusisoflavone B (4) as PTP1B inhibitors. IC50 of these compounds were 22.7 ± 1.7, 12.6 ± 1.6, 21.2 ± 3.8 and 2.5 ± 0.2 µM, respectively. Kinetics analysis revealed that these compounds inhibited PTP1B non-competitively with Ki values of 21.3 ± 2.8, 7.9 ± 1.9, 14.3 ± 2.0, and 3.0 ± 0.5 µM, respectively. These findings support the important role of F. racemosa as a novel source of new drugs and/or as a herbal remedy for treatment of type 2 diabetes