Synthesis, antitumor activity and in silico analyses of amino acid derivatives of artepillin C, drupanin and baccharin from green propolis.

Breast cancer has the highest incidence and mortality in females, while prostate cancer has the second-highest incidence in males. Studies have shown that compounds from Brazilian green propolis have antitumor activities and can selectively inhibit the AKR1C3 enzyme, overexpressed in hormone-dependent prostate and breast tumors. Thus, in an attempt to develop new cytotoxic inhibitors against these cancers, three prenylated compounds, artepillin C, drupanin and baccharin, were isolated from green propolis to synthesize new derivatives via coupling reactions with different amino acids. All obtained derivatives were submitted to antiproliferative assays against four cancer cells (MCF-7, MDA MB-231, PC-3, and DU145) and two normal cell lines (MCF-10A and PNT-2) to evaluate their cytotoxicity. In general, the best activity was observed for compound6e, derived from drupanin, which exhibited half-maximal inhibitory concentration (IC50) of 9.6 ± 3 µM and selectivity index (SI) of 5.5 against MCF-7 cells.In silicostudies demonstrated that these derivatives present coherent docking interactions and binding modes against AKR1C3, which might represent a possible mechanism of inhibition in MCF-7 cells.

Chemical Synthesis of Deoxynivalenol-3-ß-d-[(13)C6]-glucoside and Application in Stable Isotope Dilution Assays.

Modified mycotoxins have been gaining importance in recent years and present a certain challenge in LC-MS/MS analysis. Due to the previous lack of a labeled isotopologue of the modified mycotoxin deoxynivalenol-3-glucoside, in our study we synthesized the first (13)C-labeled internal standard. Therefore, we used the Königs-Knorr method to synthesize deoxynivalenol-3-ß-d-[(13)C6]-glucoside originated from unlabeled deoxynivalenol and [(13)C6]-labeled glucose. Using the synthesized isotopically-labeled standard deoxynivalenol-3-ß-d-[(13)C6]-glucoside and the purchased labeled standard [(13)C15]-deoxynivalenol, a stable isotope dilution LC-MS/MS method was firstly developed for deoxynivalenol-3-glucoside and deoxynivalenol in beer. The preparation and purification of beer samples was based on a solid phase extraction. The validation data of the newly developed method gave satisfying results. Intra- and interday precision studies revealed relative standard deviations below 0.5% and 7%, respectively. The recoveries ranged for both analytes between 97% and 112%. The stable isotope dilution assay was applied to various beer samples from four different countries. In summary, deoxynivalenol-3-glucoside and deoxynivalenol mostly appeared together in varying molar ratios but were quantified in rather low contents in the investigated beers.

Metabolism of deoxynivalenol and deepoxy-deoxynivalenol in broiler chickens, pullets, roosters and turkeys.

Recently, deoxynivalenol-3-sulfate (DON-3-sulfate) was proposed as a major DON metabolite in poultry. In the present work, the first LC-MS/MS based method for determination of DON-3-sulfate, deepoxy-DON-3-sulfate (DOM-3-sulfate), DON, DOM, DON sulfonates 1, 2, 3, and DOM sulfonate 2 in excreta samples of chickens and turkeys was developed and validated. To this end, DOM-3-sulfate was chemically synthesized and characterized by NMR and LC-HR-MS/MS measurements. Application of the method to excreta and chyme samples of four feeding trials with turkeys, chickens, pullets, and roosters confirmed DON-3-sulfate as the major DON metabolite in all poultry species studied. Analogously to DON-3-sulfate, DOM-3-sulfate was formed after oral administration of DOM both in turkeys and in chickens. In addition, pullets and roosters metabolized DON into DOM-3-sulfate. In vitro transcription/translation assays revealed DOM-3-sulfate to be 2000 times less toxic on the ribosome than DON. Biological recoveries of DON and DOM orally administered to broiler chickens, turkeys, and pullets were 74%-106% (chickens), 51%-72% (roosters), and 131%-151% (pullets). In pullets, DON-3-sulfate concentrations increased from jejunum chyme samples to excreta samples by a factor of 60. This result, put into context with earlier studies, indicates fast and efficient absorption of DON between crop and jejunum, conversion to DON-3-sulfate in intestinal mucosa, liver, and possibly kidney, and rapid elimination into excreta via bile and urine.

Nucleophilic Addition of Thiols to Deoxynivalenol.

Conjugation of deoxynivalenol (DON) with sulfur compounds is recognized as a significant reaction pathway, and putative DON-glutathione (DON-GSH) conjugates have been reported in planta. To understand and control the reaction of trichothecenes with biologically important thiols, we studied the reaction of DON, T-2 tetraol, and de-epoxy-DON with a range of model thiols. Reaction conditions were optimized for DON with 2-mercaptoethanol. Major reaction products were identified using HRMS and NMR spectroscopy. The results indicate that thiols react reversibly with the double bond (Michael addition) and irreversibly with the epoxide group in trichothecenes. These reactions occurred at different rates, and multiple isomers were produced including diconjugated forms. LC-MS analyses indicated that glutathione and cysteine reacted with DON in a similar manner to the model thiols. In contrast to DON, none of the tested mercaptoethanol adducts displayed toxicity in human monocytes or induced pro-inflammatory cytokines in human macrophages.

Synthesis of non-prenyl analogues of baccharin as selective and potent inhibitors for aldo-keto reductase 1C3.

Inhibitors of a human member (AKR1C3) of the aldo-keto reductase superfamily are regarded as promising therapeutics for the treatment of prostatic and breast cancers. Baccharin [3-prenyl-4-(dihydrocinnamoyloxy)cinnamic acid], a component of propolis, was shown to be both potent (Ki 56 nM) and highly isoform-selective inhibitor of AKR1C3. In this study, a series of derivatives of baccharin were synthesized by replacing the 3-prenyl moiety with aryl and alkyl ether moieties, and their inhibitory activities for the enzyme were evaluated. Among them, two benzyl ether derivatives, 6m and 6n, showed an equivalent inhibitory potency to baccharin. The molecular docking of 6m in AKR1C3 has allowed the design and synthesis of (E)-3-{3-[(3-hydroxybenzyl)oxy]-4-[(3-phenylpropanoyl)oxy]phenyl}acrylic acid (14) with improved potency (Ki 6.4 nM) and selectivity comparable to baccharin. Additionally, 14 significantly decreased the cellular metabolism of androsterone and cytotoxic 4-oxo-2-nonenal by AKR1C3 at much lower concentrations than baccharin.

Methylthiodeoxynivalenol (MTD): insight into the chemistry, structure and toxicity of thia-Michael adducts of trichothecenes.

Methylthiodeoxynivalenol (MTD), a novel derivative of the trichothecene mycotoxin deoxynivalenol (DON), was prepared by applying a reliable procedure for the formal Michael addition of methanethiol to the conjugated double bond of DON. Structure elucidation revealed the preferred formation of the hemiketal form of MTD by intramolecular cyclisation between C8 and C15. Computational investigations showed a negative total reaction energy for the hemiketalisation step and its decrease in comparison with theoretical model compounds. Therefore, this structural behaviour seems to be a general characteristic of thia-Michael adducts of type B trichothecenes. MTD was shown to be less inhibitory for a reticulocyte lysate based in vitro translation system than the parent compound DON, which supports the hypothesis that trichothecenes are detoxified through thia-adduct formation during xenobiotic metabolism.

Effects of oral exposure to naturally-occurring and synthetic deoxynivalenol congeners on proinflammatory cytokine and chemokine mRNA expression in the mouse.

The foodborne mycotoxin deoxynivalenol (DON) induces a ribotoxic stress response in mononuclear phagocytes that mediate aberrant multi-organ upregulation of TNF-α, interleukins and chemokines in experimental animals. While other DON congeners also exist as food contaminants or pharmacologically-active derivatives, it is not known how these compounds affect expression of these cytokine genes in vivo. To address this gap, we compared in mice the acute effects of oral DON exposure to that of seven relevant congeners on splenic expression of representative cytokine mRNAs after 2 and 6h. Congeners included the 8-ketotrichothecenes 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), fusarenon X (FX), nivalenol (NIV), the plant metabolite DON-3-glucoside (D3G) and two synthetic DON derivatives with novel satiety-inducing properties (EN139528 and EN139544). DON markedly induced transient upregulation of TNF-α IL-1ß, IL-6, CXCL-2, CCL-2 and CCL-7 mRNA expressions. The two ADONs also evoked mRNA expression of these genes but to a relatively lesser extent. FX induced more persistent responses than the other DON congeners and, compared to DON, was: 1) more potent in inducing IL-1ß mRNA, 2) approximately equipotent in the induction of TNF-α and CCL-2 mRNAs, and 3) less potent at upregulating IL-6, CXCL-2, and CCL-2 mRNAs. EN139528's effects were similar to NIV, the least potent 8-ketotrichothecene, while D3G and EN139544 were largely incapable of eliciting cytokine or chemokine mRNA responses. Taken together, the results presented herein provide important new insights into the potential of naturally-occurring and synthetic DON congeners to elicit aberrant mRNA upregulation of cytokines associated with acute and chronic trichothecene toxicity.

Stereoselective Luche reduction of deoxynivalenol and three of its acetylated derivatives at C8.

The trichothecene mycotoxin deoxynivalenol (DON) is a well known and common contaminant in food and feed. Acetylated derivatives and other biosynthetic precursors can occur together with the main toxin. A key biosynthetic step towards DON involves an oxidation of the 8-OH group of 7,8-dihydroxycalonectrin. Since analytical standards for the intermediates are not available and these intermediates are therefore rarely studied, we aimed for a synthetic method to invert this reaction, making a series of calonectrin-derived precursors accessible. We did this by developing an efficient protocol for stereoselective Luche reduction at C8. This method was used to access 3,7,8,15-tetrahydroxyscirpene, 3-deacetyl-7,8-dihydroxycalonectrin, 15-deacetyl-7,8-dihydroxycalonectrin and 7,8-dihydroxycalonectrin, which were characterized using several NMR techniques. Beside the development of a method which could basically be used for all type B trichothecenes, we opened a synthetic route towards different acetylated calonectrins.

A synthetic approach for constructing the 3/6/6/5-fused tetracyclic skeleton of tenuipesine A.

An efficient approach toward the 3/6/6/5-fused tetracyclic skeleton of tenuipesine A has been accomplished. The strategy featured 1) a tandem Mitsunobu and 3,3-rearrangement reaction yielding the key intermediate 7 with two adjacent all-carbon quaternary centers with high d.r.; and 2) a tandem DBDMH-mediated semipinacol rearrangement via a 1,2-oxygen migration of an allylic hemiketal to construct the highly substituted tetrahydropyran ring.

Enzyme-assisted synthesis and structural characterization of the 3-, 8-, and 15-glucuronides of deoxynivalenol.

4-Deoxynivalenol is one of the most prevalent mycotoxins in grain-based food and feed products worldwide. Conjugation of deoxynivalenol to glucuronic acid and elimination via the urine appears to be the major metabolism pathway, although with differing efficiency in different species. In order to make pure deoxynivalenol glucuronides for analytical methodologies available we intended to enzymatically synthesize glucuronides of deoxynivalenol using rat and human liver microsomes supplemented with uridine 5'-diphosphoglucuronic acid and alamethicin as detergent. Three glucuronides were isolated and purified using solid-phase extraction of microsomal incubations and subsequent semipreparative hydrophilic interaction chromatography. NMR spectra were obtained for all three compounds from solutions in methanol, showing that deoxynivalenol 3-O-ß-D-glucuronide and deoxynivalenol 15-O-ß-D-glucuronide were the major products from incubations of deoxynivalenol with rat and human liver microsomes, respectively. The NMR spectra of a third glucuronide showed replacement of the C-8 carbonyl by a ketal carbon. This glucuronide was finally identified as deoxynivalenol 8-O-ß-D-glucuronide. The present study provides unequivocal structural evidence for three glucuronides of deoxynivalenol formed by liver enzymes.

Development and validation of a method based on a QuEChERS procedure and heart-cutting GC-MS for determination of five mycotoxins in cereal products.

A new analytical method for the rapid and simultaneous determination of five mycotoxins (zearelenone, deoxynivalenol, Fusarenon X, 15-acetyldeoxynivalenol and nivalenol) in breakfast cereals and flours by heart-cutting GC-MS has been developed and validated. Extraction was performed with MeCN, applying a modified QuEChERS (QUick, Easy, CHeap, Effective, Rugged and Safe) procedure, and the extracts were analyzed after a silylation of the analytes under study. Careful optimization of the parameters of Deans Switch device and GC-MS was achieved in order to attain a fast separation in SIM mode, allowing a total run time of only 8 min. Acceptable recoveries for all mycotoxins at two different spiking levels (20 and 100 microg/kg) were achieved with good repeatability (from 9 to 21%). LOD ranged from 2 to 15 microg/kg and LOQ ranged from 5 to 50 microg/kg, which were lower than the maximum limit legal established by the European Union (EU). The method developed was applied to commercial breakfast cereals and flours; among the mycotoxins studied, deoxynivalenol and zearalenone were the most predominant.

Synthesis of four carbon-13-labeled type a trichothecene mycotoxins and their application as internal standards in stable isotope dilution assays.

The first stable isotope dilution assay (SIDA) for the simultaneous quantitation of the most abundant type A trichothecenes in foods and feeds was developed. Synthesis of carbon-13-labeled T2-toxin, HT2-toxin, diacetoxyscirpenol, and monoacetoxyscirpenol was accomplished by [13C2]-acetylation of T2-triol and scirpentriol, respectively. Scirpentriol was prepared from diacetoxyscirpenol by complete alkaline hydrolysis and subsequently was converted to [13C6]-triacetoxyscirpentriol by peracetylation with [13C4]-acetic anhydride. The latter compound was selectively hydrolyzed using ammonium hydroxide to give [13C4]-diacetoxyscirpenol and [13C2]-monoacetoxyscirpenol in reasonable yields. Analogously, [13C6]-T2-triacetate was prepared from T2-triol and subjected to controlled hydrolysis to yield [13C4]-T2-toxin and [13C2]-HT2-toxin. All synthesized products were characterized by NMR and MS experiments. Using the prepared isotopically labeled standards, SIDAs were developed for the quantitation of type A trichothecenes in food and feeds. The mycotoxins were quantified by LC-single and tandem MS after cleanup on multifunctional columns. The method revealed good sensitivity with low detection and quantification limits along with excellent recovery and good precision in interassay studies. Food samples were analyzed using the developed SIDA and showed substantial contamination of oat products with T2-toxin and HT2-toxin. Diacetoxyscirpenol was detected on potatoes, whereas monoacetoxyscirpenol was not present in the analyzed samples.

Synthesis of stable isotope labeled 3-acetyldeoxynivalenol.

Stable isotope labeled 3-acetyldeoxynivalenol (3-AcDON) was synthesized in excellent yield from deoxynivalenol as starting material. This is the first synthesis of a stable isotope labeled type-B trichothecene suitable as internal standard for HPLC-MS/MS or GC-MS analysis of trichothecene mycotoxins. The isotopic purity of the 3-d(3)-AcDON was determined to be 94.9%.

Biosynthesis of Fusarium culmorum trichothecenes. The roles of isotrichodermin and 12,13-epoxytrichothec-9-ene.

Two different approaches enabled us to unambiguously establish the intermediacy of isotrichodermin and 12,13-epoxytrichothec-9-ene in the biosynthesis of the trichothecenes 3-acetyldeoxynivalenol and sambucinol, respectively. The kinetic pulse-labeling method enabled us to detect a plausible precursor to 3-acetyldeoxynivalenol biosynthesis. Feeding experiments using the pure 14C-labeled intermediate established that it was incorporated 27% into 3-acetyldeoxynivalenol but not to sambucinol. The 14C-precursor was subsequently used as a marker to purify the unlabeled intermediate which was shown to be isotrichodermin by spectroscopic techniques. In order to trace the enriched carbons incorporated into 3-acetylde-oxynivalenol, specifically deuteriated isotrichodermin was synthesized and fed to Fusarium culmorum. The 2H NMR spectrum of the derived 3-acetyldeoxynivalenol proved conclusively the position of the deuteriums and that isotrichodermin is a major biosynthetic precursor. The proof that isotrichodermin is converted in vivo to 3-acetyldeoxynivalenol but not to sambucinol led us to postulate that 12,13-epoxytrichothec-9-ene might have an important role in the biosynthesis of trichothecenes. We synthesized 12,13-epoxytrichothec-9-ene with tritium at C-15 or with two deuteriums at C-4 and two deuteriums at C-15. These labeled compounds enabled us to prove that 12,13-epoxytrichothec-9-ene is a major precursor to sambucinol biosynthesis but is neither converted to isotrichodermin nor to 3-acetyldeoxynivalenol. We also succeeded in isolating a biosynthetic intermediate between 12,13-epoxytrichothec-9-ene and sambucinol and characterized its structure as 3-deoxysambucinol by spectroscopic techniques (1H NMR, 2H NMR, 13C NMR, correlation spectroscopy, two-dimensional heteronuclear correlation experiments, and mass spectroscopy).

[A simple method of synthesis of 3-acetyl-5-(D-arabino-tetrahydroxybutyl)-2-methylpyrrole]. / Prosta metoda syntezy 3-acetylo-5-(D-arabino-tetrahydroksybutylo)-2-metylopirolu.

The reaction of D-fructose with ammonium carbonate and 2,4-pentanedione leads in one step to 3-acetyl-5-(D-arabino-tetrahydroxybutyl)-2-methylpyrrole. This compound has been found to display anticancer activity.

Structure-activity studies of trichothecenes: cytotoxicity of analogues and reaction products derived from T-2 toxin and neosolaniol.

Forty-two analogues and reaction products derived from T-2 toxin or neosolaniol were assayed for their cytotoxicity to cultured mouse lymphoma cells. Structure-activity relationships confirmed the stereospecific nature of the cytotoxic action of T-2. Cytotoxicity was particularly susceptible to changes at C3, C4, C9, and C10 but was relatively unaffected by changes at C8, which appears to represent a region of steric tolerance in the interaction of T-2 with a cellular constituent. The most potent compounds were T-2, diacetoxyscirpenol, and a series of C8 ester analogues 11 and 31-35.

Protein synthesis inhibition by 8-oxo-12,13-epoxytrichothecenes.

The Fusarium mycotoxin, 4-deoxynivalenol, is an abundant, natural contaminant of corn and wheat. 8-Oxo-12,13-epoxytrichothecenes related to 4-deoxynivalenol were synthesized; they either lacked the 7-hydroxyl but contained a hydroxyl at C-4 (7-deoxynivalenol) or lacked substituents at C-3 and C-7 (3,7-dideoxynivalenol). The ability of these synthetic analogs and their acetylated derivatives to inhibit protein synthesis by cultured mammalian cells was compared to that of 4-deoxynivalenol. Whereas the 50% inhibitory dose (ID50) for murine erythroleukemia cells was about 1 microgram/ml for 4-deoxynivalenol and 3,7-dideoxynivalenol, all of the other analogs were at least 10-fold less potent. When tested at their ID50 dose, all of the 8-oxotrichothecenes, except 4-deoxynivalenol and 3,7-dideoxynivalenol, caused polysome 'run-off', indicating that, at this dose, they are inhibitors of polypeptide chain initiation. With 4-deoxynivalenol and 3,7-dideoxynivalenol, polysomes remained at control levels indicating that these toxins prevent polypeptide chain elongation. From these results and comparisons to previous studies of 8-oxo-12,13-epoxytrichothecenes (trichothecolone, trichothecin, nivalenol and fusarenone X), trichothecenes with substituents at both C-3 and C-4 predominantly inhibit polypeptide chain initiation, whereas those lacking one substituent at either site are inhibitors of chain elongation.

Mass spectral investigations on trichothecene mycotoxins. III. Synthesis, characterization and applications of pentafluoropropionyl and trifluoroacetyl esters of simple trichothecenes.

The pentafluoropropionyl, (PFP) and trifluoroacetyl (TFA) esters of several naturally occurring and synthetically modified simple trichothecenes were synthesized in nanogram amounts and characterized. Optimum conditions for the gas chromatographic (GC) separation of these derivatives and their analysis by negative ion chemical ionization (NICI) mass spectrometric technique were determined. These perfluoroacyl derivatives under the NICI conditions undergo limited but characteristic fragmentations similar to the fragmentations of heptafluorobutyryl esters of trichothecenes under the same conditions. Characteristic ions for the specific detection and accurate quantification of these PFP and TFA derivatives were chosen. Preliminary results indicated that the PFP derivatives are better suited for the analysis of simple trichothecenes by GC-NICI-MS technique. Ultra trace (0.5-2.0 pg) amounts of these PFP derivatives were detected by the developed procedure.