Добірка наукової літератури з теми "Lc-Ms/ms | 13c rmn"

Two methods for measuring ochratoxin A in corn, oat, and grape juice were developed and compared. Flow injection (FI) and on-line liquid chromatography (LC) performances were evaluated separately, with both methods using a triple quadrupole tandem mass spectrometer (MS/MS) for quantitation. Samples were fortified with 13C uniformly labeled ochratoxin A as the internal standard (13C-IS) and prepared by dilution and filtration, followed by FI- and LC-MS/MS analysis. For the LC-MS/MS method, which had a 10 min run time/sample, recoveries of ochratoxin A fortified at 1, 5, 20, and 100 ppb in corn, oat, red grape juice, and white grape juice ranged from 100% to 117% with RSDs < 9%. The analysis time of the FI-MS/MS method was <60 s/sample, however, the method could not detect ochratoxin A at the lowest fortification concentration, 1 ppb, in all tested matrix sources. At 5, 20, and 100 ppb, recoveries by FI-MS/MS ranged from 79 to 117% with RSDs < 15%. The FI-MS/MS method also had ~5× higher solvent and matrix-dependent instrument detection limits (0.12–0.35 ppb) compared to the LC-MS/MS method (0.02–0.06 ppb). In the analysis of incurred corn and oat samples, both methods generated comparable results within ±20% of reference values, however, the FI-MS/MS method failed to determine ochratoxin A in two incurred wheat flour samples due to co-eluted interferences due to the lack of chromatographic separation.

Abstract Background: Measurement of porphobilinogen (PBG) is useful in the diagnosis of the acute neurologic porphyrias. Currently used colorimetric assays lack analytical and clinical sensitivity and specificity. Methods: We developed a liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the measurement of PBG in 1 mL of urine, using 5-(aminoethyl)-4-(carboxymethyl) 1H-2,4-[13C]pyrolle-3-propanoic acid ([2,4-13C]PBG; 2.75 μg) as internal standard. After solid-phase extraction, LC-MS/MS analysis was performed in the selected-reaction monitoring (SRM) mode. PBG and [2,4-13C]PBG were monitored through their own precursor and product ion settings (m/z 227 to 210 and m/z 229 to 212, respectively). The retention time of PBG and [2,4-13C]PBG was 1.0 min in a 2.3-min analysis. Results: Daily calibrations (n = 6) between 0.1 and 2.0 mg/L were linear and reproducible. Inter- and intraassay CVs were 3.2–3.5% and 2.6–3.1%, respectively, at mean concentrations of 0.24, 1.18, and 2.15 mg/L. The regression equation for the comparison between an anion-exchange column method (y) and the LC-MS/MS method (x) was: y = 0.84x + 0.74 (Sy|x = 5.8 mg/24 h; r = 0.85; n = 100). In 47 volunteers, PBG excretion was 0.02–0.42 mg/24 h, lower than reported reference intervals (up to 2.0 mg/24 h) based on colorimetric methods. In 85 samples with PBG ≤0.5 by LC-MS/MS, 8 (9.4%) had values ≥2.0 mg/24 h by the anion-exchange method (mean ± SD, 4.3 ± 1.8 mg/24 h). In 11 patients with confirmed diagnoses of acute porphyria and increased PBG by LC-MS/MS, 2 had values within the reported reference intervals by a quantitative anion-exchange method. Conclusions: The quantitative LC-MS/MS method for PBG measurement exhibits greater analytical specificity and improved clinical sensitivity and specificity than currently available methods.

A partir de 6-amino-3-metil-2-(metiltio)pirimidin-4(3H)-ona, aldehídos aromáticos y benzoquinona, por medio de una reacción de ciclodeshidratación de Michael asistida por radiación de microondas, se realizó la síntesis y caracterización de la serie de derivados tricomponentes pirimido[4,5-b]quinolínicos, y se evaluó la posible actividad antileishmania. Los nuevos compuestos se caracterizaron por análisis espectroscópico de IR, RMN 1H, RMN 13C y MS.

Subcellular compartmentation has been challenging in plant 13C-metabolic flux analysis. Indeed, plant cells are highly compartmented: they contain vacuoles and plastids in addition to the regular organelles found in other eukaryotes. The distinction of reactions between compartments is possible when metabolites are synthesized in a particular compartment or by a unique pathway. Sucrose is an example of such a metabolite: it is specifically produced in the cytosol from glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). Therefore, determining the 13C-labeling in the fructosyl and glucosyl moieties of sucrose directly informs about the labeling of cytosolic F6P and G6P, respectively. To date, the most commonly used method to monitor sucrose labeling is by nuclear magnetic resonance, which requires substantial amounts of biological sample. This study describes a new methodology that accurately measures the labeling in free sugars using liquid chromatography tandem mass spectrometry (LC-MS/MS). For this purpose, maize embryos were pulsed with [U-13C]-fructose, intracellular sugars were extracted, and their time-course labeling was analyzed by LC-MS/MS. Additionally, extracts were enzymatically treated with hexokinase to remove the soluble hexoses, and then invertase to cleave sucrose into fructose and glucose. Finally, the labeling in the glucosyl and fructosyl moieties of sucrose was determined by LC-MS/MS.

Abstract An interlaboratory study was conducted to evaluate stable isotope dilution and LC tandem MS (MS/MS) for the determination of aflatoxins B1, B2, G1, G2, and M1 (AFB1, AFB2, AFG1, AFG2, and AFM1) in milk, milk-based infant formula (formula), and feed. Samples were first fortified with five 13C uniformly labeled aflatoxins {[13C]–internal standard (IS)} corresponding to the five native aflatoxins, which were subsequently extracted with acetonitrile–water (50 + 50, v/v), followed by centrifugation, filtration, and LC-MS/MS analysis. In addition to certified milk powder and animal feed, the three participating laboratories also analyzed milk, formula, and feed fortified with the five aflatoxins at concentrations ranging from 0.5 to 50 ng/g. The majority of recoveries ranged from 80 to 120%, with RSDs &lt; 20%. Method LOQs were determined by the three laboratories using the three sample matrixes in replicates (n = 8), and the determined LOQs of AFB1, AFB2, AFG1, AFG2, and AFM1 ranged from 0.1 to 0.91, 0.24 to 0.64, 0.28 to 1.52, 0.19 to 3.80, and 0.12 to 0.45 ng/g, respectively. For detected aflatoxins in the certified materials, all measured concentrations were within ±25% of the certified values. Using [13C]–IS eliminated the need for matrix-matched calibration standards for quantitation, simplified sample preparation, and achieved simultaneous identification and quantitation of the aflatoxins in a simple LC-MS/MS procedure.

Background: The acetate ion has important physiological functions and important therapeutic applications. A rapid LC–MS/MS method is described to measure acetate ions in human plasma without chemical derivatization. Materials & methods: A 200 μl sample was spiked with the internal standard 1,2-13C-acetate and proteins precipitated with trichloroacetic acid. The supernatant was recovered and separated under acidic conditions on a C18-column. The eluent was alkalinized by post-column infusion of methanolic ammonium hydroxide. Acetate ions were monitored on a low resolution mass spectrometer in negative ion mode. Results: Method was validated for accuracy and precision with a lower limit of quantitation of 9.7 μM and linear dynamic range up to 339.6 μM. Conclusion: The method is open for analytical improvement and adapts with metabolomic and pharmacometabolomic studies on chemicals of similar nature.

Abstract An analog of aildenafil, which is a potent and highly selective inhibitor of phosphodiesterase 5, was found in a dietary supplement marketed for enhancement of sexual function. The compound was isolated by silica gel column chromatography, and its structure was identified by means of 13C-NMR spectrometry, 1H-NMR spectrometry, high-resolution MS, and X-ray structure determination. The compound was identified to be sulfoaildenafil (other names: thioaildenafil, dimethyl sildenafil thione, and thiomethisosildenafil). Sulfoaildenafil is very similar to the compound thiohomosildenafil. As it is difficult to distinguish between them by LC-photodiode array detector analysis, ultra-performance LC (UPLC)/MS, ion trap LC/MS/MS (LC/IT-MS/MS), and GC/MS were performed. The mass spectra of thiohomosildenafil by UPLC/MS and LC/IT-MS/MS showed mass fragments of m/z 58, 72, and 355, and the mass spectrum by GC/MS showed mass fragments of m/z 56, 72, and 420. Some of these fragments had low intensities, but they were useful for distinguishing between the two compounds. The relationship between aildenafil (other names: dimethylsildenafil and methisosildenafil) and homosildenafil is similar to that between sulfoaildenafil and thiohomosildenafil. Therefore, these were also examined.

The application of internal standards in quantitative and qualitative bioanalysis is a commonly used procedure. They are usually isotopically labeled analogs of the analyte, used in quantitative LC-MS analysis. Usually, 2H, 13C, 15N and 18O isotopes are used. The synthesis of deuterated isotopologues is relatively inexpensive, however, due to the isotopic effect of deuterium and the lack of isotopologue co-elution, usually they are not considered as good internal standards for LC-MS quantification. On the other hand, the preparation of 13C, 15N and 18O containing standards of drugs and their metabolites requires a complicated multistep de novo synthesis, starting from the isotopically labeled substrates, which are usually expensive. Therefore, there is a strong need for the development of low-cost methods for isotope-labeled standard preparations for quantitative analysis by LC-MS. The presented review concentrates on the preparation of deuterium-labeled standards by hydrogen−deuterium exchange reactions at the carbon centers. Recent advances in the development of the methods of isotopologues preparation and their application in quantitative analysis by LC-MS are evaluated.

Background: Mycotoxins are big concerns in food safety. Analytical methods are important for the evaluation of mycotoxins in different food commodities. Objective: In this study, stable isotope dilution assay (SIDA) was compared with a matrix-matched calibration method for the quantification of mycotoxins in four different commercially available commodities and two reference materials. Methods: All samples were extracted with water–acetonitrile (50+50, v/v), followed by filtration and LC–tandem MS analysis. Results: SIDA calibration accuracies ranged from 78.6 to 112% with relative SDs (RSDs) ≤16% across all four matrices. The majority of the recoveries across all matrices ranged from 70 to 120% with RSDs <11%. Of the four mycotoxins in the reference materials analyzed, only three had 13C-internal standard (IS), whereas the fourth was quantified using a closely eluting 13C-IS for a different mycotoxin. Mycotoxins paired with their corresponding 13C-IS had accuracies >90%, whereas accuracies for the mismatched mycotoxin/13C-IS were <14%. Conclusions: When 13C-IS are not available, matrix-matched calibration was also evaluated as an alternative to quantitating target mycotoxins. The use of 13C-IS is the best way to dynamically account for prevalent matrix effects, but matrix matching provides a viable alternative. Highlights: The study compared SIDA and matrix-matched calibration methods in terms of recovery, efficiency, advantages, and limitations for LC-MS based mycotoxin analysis.

Abstract Stable isotope dilution with LC/MS/MS was used to determine the following 11 mycotoxins in food grade gums: aflatoxins B1, B2, G1, and G2; deoxynivalenol; fumonisins B1, B2, and B3; ochratoxin A; T-2 toxin; and zearalenone. Samples were fortified with 11 [13C]-uniformly labeled internal standard ([13C]-IS) mycotoxins that corresponded to the 11 target mycotoxins and extracted by acetonitrile–water (4 + 1, v/v), followed by LC/MS/MS analysis. Mycotoxins were quantitated with the fortified [13C]-IS in each sample. The average recoveries of aflatoxins B1, B2, G1, and G2 (1, 5, and 25 μg/kg); deoxynivalenol and fumonisins B1, B2, and B3 (25, 100, and 500 μg/kg); and ochratoxin A, T-2 toxin, and zearalenone (10, 50, and 250 μg/kg) ranged from 84 to 117% with RSDs less than 20%. Method-dependent LOQs were from 0.1 (aflatoxin B1) to 25 μg/kg (fumonisin B3). Among 20 market samples, aflatoxin B1 (&lt; LOQ) was detected in a Guar gum and a Tragacanth gum, and zearalenone (6 ± 0.6 μg/kg) was detected in a Xanthan gum. The detected mycotoxins were further confirmed by comparing their enhanced product ion spectra to those of reference standards. The single laboratory validated stable isotope dilution and LC/MS/MS method provides sufficient selectivity, sensitivity, accuracy, and reproducibility with a simple sample preparation to screen the 11 mycotoxins in gums.

Parmi les nombreux pathogènes fongiques susceptibles d’infecter les épis de maïs, les espèces appartenant au genre Fusarium sont particulièrement préoccupantes pour la filière maïsicole. La fusariose est susceptible d’induire des pertes de rendement considérables et est fréquemment associée à une contamination des épis par des mycotoxines. Un des leviers prometteur repose sur la sélection génétique de plantes résistantes à Fusarium et à l’accumulation de mycotoxines. Plusieurs Quantitative Trait Loci (QTL) ont été identifiés d’après la caractérisation moléculaire de la résistance à la fusariose chez le maïs. Cependant malgré les progrès des approches génétiques, les mécanismes moléculaires impliqués restent en grande partie inconnus. L’identification de métabolites majeurs associés à la résistance reste donc indispensable pour la création d’un outil d’aide à la sélection variétale. Une approche métabolomique combinant de la spectrométrie de masse et de la 1H-RMN a été mise au point pour identifier un ensemble de métabolites de défense, constitutifs ou induits par l’infection, susceptibles d’intervenir dans la résistance à Fusarium. Cette approche a été appliquée aux grains à deux stades de développement sur 20 variétés présentant des degrés de résistance contrastés inoculés ou non avec une souche de Fusarium graminearum toxinogène par le canal des soies. Les résultats obtenus mettent en évidence un panel de métabolites liés à la résistance ou la sensibilité des variétés de maïs
Fusarium graminearum is the main causal agent of maize ear rot or Gibberella ear rot (GER), an important fungal disease affecting maize. GER leads to significant economic loss and serious health issues due to the ability of F. graminearum to produce mycotoxins such as type B trichothecenes. One promising approach to control Giberella Ear Rot and reduce mycotoxins contamination is to promote host-genetic resistance. Several Quantitative Trait Loci (QTLs) have been identified in maize. However molecular basis to resistance to Fusarium infection remains largely unknown and the success of selection for GER resistance is still challenging. Biochemical approaches can provide valuable insights in the mechanisms crops employ against F. graminearum and its production of mycotoxins. A biochemical profiling could actually be an efficient way to decipher plant-pathogen interactions and progress in screening resistant maize lines. This study aims to elucidate the metabolic profiling of F. graminearum resistance and toxin accumulation in kernels toward the combination of high resolution mass spectrometry and 1H NMR to identify a large set of metabolites, preformed, constitutive as well as inducible defense metabolites that could play a key role in GER resistance. This approach was applied to kernels harvested at two developmental stages. Twenty genotypes with contrasting levels of resistance were inoculated, or not, with a toxigenic Fusarium graminearum strain through the silk channel. The obtained data allowed highlighting a set of biochemical compounds linked to the resistance or susceptibility of maize genotypes

Une importante proportion de formes atypiques de parkinsonismes a été rapportée en Guadeloupe en 1999. Il ressort des études épidémiologiques menées sur place, que tous les patients atteints étaient de grands consommateurs de produits alimentaires et médicinaux de la famille des Annonaceae, et plus particulièrement du genre Annona. De nombreux genres appartenant à cette famille renferment des molécules fortement cytotoxiques : les acétogénines d’Annonaceae. Leur présence dans les fruits d’Annona muricata a déjà été mise en évidence. Cependant, peu de données existent sur leur teneur en acétogénines dans ces fruits ou dans ceux d’espèces proches. Par ailleurs, bien que l’annonacine, acétogénine principale d’A. muricata soit neurotoxique in vivo, aucune donnée quantitative de son accès au cerveau n’est disponible. Nous nous sommes donc attachés au cours de ce travail, à développer des méthodes de dosage de l’annonacine par LC-MS/MS dans le plasma et le cerveau de Rat pour déterminer sa biodisponibilité et la fraction à tropisme cérébral, dans le but de comprendre pourquoi des molécules si fortement cytotoxiques n’entraînaient pas d’intoxication aigue lors de l’exposition alimentaire. Nous avons par ailleurs développé une méthode de quantification des acétogénines totales par 1H RMN dans des extraits bruts de fruits, appliquée à des lots d’A. muricata d’origines variées. Une méthode par LC-MS/MS a également été développée pour une description plus approfondie des acétogénines présentes dans des extraits bruts de fruits, appliquée à différents lots d’A. squamosa. Les fruits d’A. reticulata et d’A. glabra ont également fait l’objet d’investigations. Ces deux approches combinées ont contribué à améliorer l’estimation de l’exposition aux acétogénines dans le cadre de l’alimentation
Abstract : A high proportion of atypical parkinsonisms was reported in French West Indies in 1999. Epidemiological studies pointed out an association with the consumption of fruits and medicinal herbs from Annonaceae of the Annona genera. Numerous Annonaceae members contain Annonaceous acetogenins (AAGs), which are highly cytotoxic molecules. They were found in the pulp fruit of Annona muricata. However, scarce only quantitative exist for this fruit and those of related species. Moreover, although annonacin, the major AAG of A. muricata proved neurotoxic in vivo, no quantitative data is available towards its distribution to the brain. We therefore developed a method for annonacin quantitation in Rat plasma and brain homogenate, in order to determine its bioavailability and the fraction reaching the brain, to understand why those highly cytotoxic molecules are not responsible for acute toxicity when fruits are ingested. We then developed a quantitation method for global estimation of AAGs in crude fruit extracts by 1H NMR, which we applied to the fruit pulp of A. muricata batches from diverse locations. An LC-MS/MS method was also developed for the qualitative study of AAGs. It was applied to different batches from A. squamosa fruits. The species A. reticulata and A. glabra were also examined. Those two approaches contributed in a better estimation of AAGs exposure by fruit consumption

Os fungos são microrganismos amplamente dispersos, podendo ser encontrados em vegetais, animais, solo e ambientes aquáticos, participando do ciclo de elementos na natureza. Embora muitos papéis ecológicos tenham sido estudados e descritos para os fungos terrestres, a ecologia de fungos marinhos ainda é pouco conhecida. Assim, os oceanos, que representam aproximadamente metade da biodiversidade global, são uma fonte enorme e virtualmente inexplorada de microrganismos produtores de novos produtos naturais. O objetivo deste trabalho foi isolar linhagens de fungos derivados de uma espécie de alga marinha do gênero Sargassum, visando à avaliação do seu potencial para a produção de metabólitos secundários bioativos. Ao todo foram isoladas 58 linhagens, das quais 52 foram crescidas em meio de cultura líquido e, após a extração com solventes orgânicos, deram origem a 99 extratos. Tais extratos foram avaliados por ensaios de atividade biológica, cromatografia em camada delgada (CCD), ressonância magnética nuclear (RMN) e cromatografia líquida acoplada a detectores de arranjo de diodos, espalhamento de luz evaporativo e espectrômetro de massas (LC - PDA - ELSD - MS). A avaliação pelo ensaio antibiótico foi o que resultou no maior número de extratos ativos (n = 13), seguido dos ensaios enzimático (n = 8), citotóxico (n = 3) e anti-tuberculose (n = 1). O extrato AS Fub 39, que apresentou atividade antibiótica, foi selecionado para estudos adicionais. Este extrato foi purificado por HPLC, e o seu composto majoritário identificado como sendo o 8-metóxi-3,5-dimetilisocroman-6-ol. Posteriormente, a linhagem AS Fub 39 foi taxonomicamente identificada como pertencendo à espécie Penicillium steckii.
Fungi are widely disperse microorganisms, typically associated with plants, animals, soil and aquatic environments (fresh and sea water), participating in the elements cycling. Although many ecological roles have been described for terrestrial fungi, ecological studies of marine derived fungi are still scarce. Therefore, oceans, which represent approximately half of the global biodiversity, are a huge and virtually unexplored source of microorganisms producers of interesting metabolites. The aim of this study was to isolate fungal strains derived from a marine algae of the Sargassum genus, and the evaluation of their metabolical potential for the production of secondary metabolites. Overall, 58 strains were isolated, of which 52 were grown in liquid culture media and extracted with organic solvents, originating 99 crude extracts. These extracts were analyzed by bioassays, thin layer chromatography (TLC), nuclear magnetic resonance (NMR) and liquid chromatography coupled with a photo diode array, an evaporative light scattering, and a mass spectrometry detectors (LC - PDA - ELSD - MS). The evaluation with the antibiotic assay resulted in the largest number of active extracts (n = 13), followed by the enzymatic (n = 8), the cytotoxic (n = 3) and the antituberculosis (n = 1) assays. The crude extract AS Fub 39, which presented antibiotic activity, was selected for additional studies. This extract was purified by HPLC, and its major compound identified as the 8-methoxy-3,5-dimethylisocroman-6-ol. Later, the AS Fub 39 strain was taxonomically identified as Penicillium steckii.

La saveur sucrée est à l’origine de l’équilibre gustatif des vins secs. On observe uneaugmentation de son intensité au cours de la macération post-fermentaire et de l’élevage enbarrique. Nous montrons que ces phénomènes sont respectivement liés à la libération depeptides de la levure et de composés non-volatils du bois de chêne dans les vins.Le rôle de la protéine Hsp12 de S. cerevisae sur le gain de sucrosité est établi enutilisant des techniques de biologie moléculaire et d’analyse sensorielle.Le développement d’un couplage chromatographie de partage centrifuge –gustatométrie permet de fractionner un extrait de bois de chêne et de purifier plusieurscomposés sapides. L’utilisation de la LC-FT/MS et de la RMN nous a permis d’identifierquatre nouvelles molécules, appelées quercotriterpénosides (QTT), deux d’entre elles (QTTI et III) possédant une saveur douce. Les seuils de perception du QTT I et d’un lignane amer,le lyonirésinol, sont respectivement 590 μg/L et 1.52 mg/L.La mise au point d’une méthode de quantification de ces composés en LC-FT/MS nous apermis de démontrer l’impact organoleptique du lyonirésinol dans les vins.Il est probable que les QTT I et III contribuent, directement ou indirectement, au gain desucrosité conféré par le bois de chêne
Sweetness contributes to the balance in taste of dry wines. An increase in sweet taste isobservable during post-fermentation maceration and oak-barrel aging. We have revealed thatthese phenomena are respectively due to the release in wines of yeast peptides and nonvolatileoak wood compounds.The role of Hsp12 protein from S.cerevisae on the increase in sweetness is establishedwith both molecular biology and sensorial analysis techniques.The development of a method coupling centrifugal partition chromatography andgustatometry has enabled us to fractionate an oak-wood extract and to purify several sapidcompounds. Thanks to both the LC-FTMS and the NMR spectroscopy methods, we havehighlighted four new molecules, called quercotriterpenosides (QTT), out of which QTT Iand III are responsible for a sweet taste. The perception thresholds of QTT I and a bitterlignan, lyoniresinol, are respectively 590 μg/L and 1.52 mg/L.LC-FT/MS method has been used to develop a quantification method for these compoundsand we have demonstrated the organoleptic impact of lyoniresinol in wines.QTT I and III are likely to contribute, directly or indirectly, to the increase in sweetnessconsecutive to barrel aging in dry wines

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This work reports studies of in vitro metabolism involving the compound 3-(2-chloro-6-fluorobenzyl)- imidazolidine-2,4-dione (LPSF-PT-31), a new 2-adrenoceptor agonist and, studies of enzyme phenotyping of montelukast, a drug used for the treatment of asthma. The results of this study revealed that LPSF-PT-31 is metabolized via CYP P450s in rat and human liver microsomes, producing only one major hydroxy-metabolite. LPSFPT- 31 showed a higher rate of in vitro metabolism in rats, which suggests a greater exposure to the drug in humans. The structural identification of LPSF-PT-31 metabolite’s was achieved through LC-MSn and 1H-NMR analysis that provided data to conclude that the hydroxylation occurred in the 5th position of the imidazolidine ring yielding to the production of 3-(2-chloro-6-fluorobenzyl)-5-hydroxyimidazolidine-2,4- dione. Related to the studies of enzyme phenotyping of montelukast, it was observed that the glucuronidation is the main clearance pathway of montelukast accounting for ~85% of the total apparent in vitro Clint (CYPs +UGTs) and that the CYP-mediated oxidation accounts only for ~15% to the overall metabolism of the drug, being montelukast acyl-β-D-glucuronide and montelukast 1,2 diol the major metabolites formed via UGTs and CYPs, respectively. Kinetic studies, correlation analysis, inhibition studies and, experiments in expressed CYPs and UGTs revealed that the CYP2C9 and CYP2C8 are comparably involved in the formation of montelukast 1,2- diol. CYP3A4 was responsible for the formation of 21(R)-OH montelukast and 21(S)- OH montelukast, while multiple CYPs catalyzed the formation of 25-OH montelukast (CYP2C8>2C9>3A4>2C19). The direct glucuronidation of montelukast resulted in the formation of montelukast acyl-β-D-glucuronide and of a new metabolite (Mglucuronide) not reported previously and was exclusively catalyzed by isoform UGT1A3. In conclusion, the in vitro data suggest that the applicability of montelukast as a probe of CYP2C8 activity in vitro and in vivo may be severely compromised due to important role of UGT1A3 and involvement of multiple CYPs in its metabolism. In addition, considering the lack of selective markers for UGT1A3, montelukast may be used as a selective marker of the UGT1A3 in vitro and in vivo.
Este trabalho relata estudos de metabolismo in vitro envolvendo o composto 3-(2-cloro-6- fluorobenzil)-imidazolidina-2,4-diona (LPSF-PT-31), um novo agonista adrenérgico 2A, e estudos de fenotipagem enzimática do montelucaste, fármaco utilizado no tratamento da asma. Os resultados do presente estudo revelaram que LPSF-PT-31 é metabolizado via CYP P450s em microssomas de fígado de ratos e humanos, produzindo apenas um hidroxi-metabólito principal. LPSF-PT-31 apresentou uma maior taxa de metabolismo in vitro em ratos, o que sugere uma maior exposição ao fármaco em seres humanos. A identificação estrutural do metabólito do LPSF-PT-31 foi estabelecida através de análises por LC-MSn e 1H-RMN, o que indicou que a reação de hidroxilação ocorreu na posição 5 do anel da imidazolidina levando a produção do metabólito 3-(2-cloro-6-fluorobenzil)-5-hidroxi-imidazolidina-2,4-diona. Em relação aos estudos de fenotipagem enzimática do montelucaste foi observado que a glucuronidação é o principal mecanismo de eliminação deste fármaco, representando ~85% do Clint in vitro aparente total (CYPs +UGTs) e que a oxidação via CYPs representa somente ~15% do Clint in vitro, sendo os metabólitos majoritários formados via UGTs e CYPs o montelucaste acil-β-D-glucuronídeo e o montelucaste 1,2 diol, respectivamente. Estudos cinéticos, de correlação com a atividade enzimática, de inibição e empregando CYPs e UGTs expressas indicaram que as CYP2C9 e CYP2C8 estão comparavelmente envolvidas na formação do montelucaste 1,2 diol. A CYP3A4 foi responsável pela formação dos metabólitos 21(R)-OH montelucaste e 21(S)-OH montelucaste, enquanto múltiplas CYPs catalisam a formação do 25-OH montelucaste (CYP2C8>2C9>3A4>2C19). A glucuronidação direta do montelucaste resultou na formação do montelucaste acil-β- D-glucuronídeo e de um novo metabólito (M-glucuronídeo) não reportado previamente e foi catalisada exclusivamente pela isoforma UGT1A3. Deste modo, os dados in vitro sugerem que a aplicabilidade do montelucaste como marcador da CYP2C8 in vitro e in vivo pode ser severamente comprometida devido ao importante papel da UGT1A3 e o envolvimento de múltiplas CYPs no seu metabolismo. Ainda, considerando a falta de marcadores seletivos para a UGT1A3, montelucaste pode ser utilizado como um marcador seletivo da UGT1A3 in vivo e in vitro.

Les travaux de cette thèse portent sur la caractérisation chimique des ambres provenant de plusieurs gisements d’âges et d’origines géographiques variés, dont certains sont inédits. Des protocoles identiques à tous les échantillons et combinant les analyses spectroscopiques (IR et RMN 13C) et chromatographiques (THM-CPG-SM) ont été appliqués, permettant d’identifier l’origine botanique des ambres et fournissant des indices pour la reconstitution des paléoenvironnements terrestres. La caractérisation chimique des gisements d’ambre du Jurassique supérieur (Kimméridgien) jusqu’au Crétacé supérieur (Santonien) du Liban, de Jordanie, du Congo, d’Equateur et de France, permet de proposer des biomarqueurs pour les résines de Cheirolepidiaceae, une famille exclusivement mésozoïque de Conifères. Une évolution des sources botaniques des résines produites durant le Mésozoïque et le Cénozoïque est alors discutée. Une production dominée par les familles de Conifères Araucariaceae et Cheirolepidiaceae est remarquée au Jurassique supérieur et Crétacé inférieur. La production au Crétacé supérieur est plutôt dominée par des Cupressaceae. Au Cénozoïque, les origines botaniques des ambres sont plus variées, et des familles d’Angiospermes sont à l’origine de nombreux gisements, dont l’ambre du Pérou produit par une Fabaceae. La production par des Conifères reste toutefois importante au Tertiaire, à l’exemple des ambres de Nouvelle-Zélande qui ont pour origine les Araucariaceae. Les données obtenues ont permis une ré-évaluation de la classification des ambres par Py-GC-MS. Ainsi, une nouvelle molécule dont la structure est inconnue encore, a été identifiée dans les chromatogrammes d’ambres de classe Ib et Ic, ajoutant un caractère discriminant entre ces deux sous-classes. Enfin, la relation âge / maturation des résines fossiles est discutée, qui dépend avant tout des conditions d’enfouissement des résines. Une large base de données moléculaires est ainsi établie pour un grand nombre de gisements d’âges et d’origines botaniques variés, qui permettra une comparaison globale dans les travaux futurs
This work focuses on the chemical characterisation of amber from different outcrops from different localities, and varied ages. Some of these outcrops had never been studied. All the amber samples were analysed with the same analytical techniques. The combination of the data obtained from spectroscopic (IR and 13C NMR) and chromatographic (THM-GC-MS) analysis allows the identification of the botanical origin of the amber and provide some information, for the reconstruction of the palaeoenvironment. Biomarkers for the cheirolepidiaceous resins were proposed based on the chemical characterisation of different amber outcrops dating from the Upper Jurassic (Kimmeridgian) to the Upper Cretaceous (Santonian) from Lebanon, Jordan, Congo, Ecuador and France. The Cheirolepidiaceae familt was exclusively present in the Mesozoic era. Hence, the evolution of the botanical origins of the produced resins during the Mesozoic and Cenozoic eras was discussed. It seems that Araucariaceae and Cheirolepidiaceae were the dominant resin producing trees during the Upper Jurassic and the Lower Cretaceous. While, cupressaceous resiniferous plants were dominant during the Upper Cretaceous. Howerver, resins dating from the Cenozoic era, were produced by a wider variety of plants, as resiniferous families of Angiosperm intensively participated in the resin production, i.e. the Peruvian amber produced by Fabaceae. Conifer resins traces were also detected in the Tertiary, such as the amber from the Araucariaceae found in New Zealand. The obtained data allowed a re-evaluation of the classification of ambers by Py-GC-MS, leading to the discovery of a novel molecule. This molecule of an unknown structure brings a new discrimination factor between the classes Ib and Ic. Finally, the age / maturity relationship is showed to be dependent on the burial and the conservation conditions of the resins. A broad molecular database is established based a large group of amber outcrops from different ages, and having diverse botanical origins. This database could be used as a comparative platform for further work in the future

Depuis deux décennies, l’humanité est confrontée à une augmentation spectaculaire du nombre de bactéries résistantes et multirésistantes aux antibiotiques, mettant en péril l’arsenal thérapeutique au complet. La tuberculose est particulièrement concernée par l’apparition de souches multirésistantes aux antibiotiques, obligeant très souvent les médecins à prolonger de plusieurs mois la prise en charge des patients et à jongler avec différents antibiotiques en cours de traitement, ce qui retarde d’autant plus la guérison. En outre, si les souches pan-résistantes restent, pour le moment, exceptionnelles, elles illustrent clairement les dangers à laisser dégénérer une situation clinique déjà préoccupante. Il est donc aujourd’hui nécessaire de développer des approches thérapeutiques innovantes afin de lutter plus efficacement contre ces phénomènes de résistance. A la différence de la plupart des antibiotiques conventionnels, un grand nombre de médicaments antituberculeux possèdent la particularité d’être des prodrogues. Ils nécessitent, en effet, d’être bioactivés au sein de la mycobactérie par l’action d’enzymes spécifiques afin de pouvoir exercer leur action antibiotique. Au cours de ces dernières années, les équipes des unités de recherche U1177 et U1019 ont identifié plusieurs familles de composés qui permettent de potentialiser ou de reprogrammer les voies de bioactivation de l’éthionamide. Ces composés agissent via l’inhibition des régulateurs transcriptionnels de la bactérie impliqués dans la bioactivation de l’éthionamide. Ces équipes ont ainsi montré qu’en plus d’augmenter la sensibilité de Mycobacterium tuberculosis à l’éthionamide, il est également possible de resensibiliser des souches cliniques résistantes à l’éthionamide en stimulant des voies de bioactivation cryptiques de cet antibiotique.Le premier objectif de cette thèse a été d’évaluer la spécificité de substrats des trois voies de bioactivation actuellement connues de l’éthionamide. Cette étude a été réalisée en produisant des dérivés de l’éthionamide et en étudiant leur activation en stimulant alternativement chacune des trois voies à l’aide de boosters spécifiques. Pour ce faire, une nouvelle voie de synthèse de thioisonicotinamides a été mise au point afin d’obtenir rapidement un grand nombre d’analogues structuraux de l’éthionamide. Nous avons alors pu explorer l’activité antimycobactérienne de ces analogues, seuls ou en combinaison avec les boosters stimulant individuellement les trois voies d’activation. Cette étude nous a permis de mettre en évidence les spécificités propres à chaque voie. Parallèlement, une étude par voltampérométrie cyclique réalisée sur une partie de ces composés nous a permis de corréler le potentiel d’oxydation des analogues avec leurs activités antimycobactériennes.Dans un deuxième axe, nous avons tenté de suivre, par RMN et par LC-MS/MS, la bioactivation de l’éthionamide dans un modèle d’expression hétérologue des enzymes de bioactivation de l’éthionamide chez E. coli.Dans un troisième axe, nous nous sommes intéressés à la recherche et la caractérisation de nouvelles molécules bioactivables par les différentes voies de bioactivation de l’éthionamide.Enfin, un criblage phénotypique a été réalisé sur Mycobacterium tuberculosis afin d’étendre le concept de réversion de résistance à d’autres pro-antibiotiques antituberculeux. Cette approche a débouché sur l’identification d’une molécule capable de reprogrammer la bioactivation de nitro-imidazolés d’importance clinique majeure, tels que le prétomanide et le délamanide. Sur cette base, nous avons entrepris un travail de pharmacomodulation de ce hit, ce qui nous a permis de déterminer les premières relations structure-activité dans cette famille chimique
For the last two decades, humanity has faced a dramatic increase in the number of resistant and multi-resistant bacteria, putting at risk the entire therapeutic arsenal. Tuberculosis is particularly affected by the spread of multi-resistant strains of antibiotics, very often forcing doctors to prolong patients care for several months and juggling with different antibiotics in the course of the treatment, which further delays recovery. In addition, if pan-resistant strains remain exceptional for the moment, they clearly illustrate the dangers of allowing an already worrying clinical situation to degenerate. It is therefore necessary today to develop innovative therapeutic approaches to fight more effectively against these phenomena of resistance. Unlike most conventional antibiotics, a large number of antituberculous drugs have the distinction of being prodrugs. They require bioactivation within the mycobacterium by the action of specific enzymes in order to be able to exercise their antibiotic effect. Over the last few years, research teams U1177 and U1019 have identified several families of compounds that potentiate or reprogram the bioactivation pathways of ethionamide. These compounds act via the inhibition of the transcriptional regulators of the bacterium involved in the bioactivation of ethionamide. These teams have shown that in addition to increasing the sensitivity of Mycobacterium tuberculosis to ethionamide, it is also possible to resensitize ethionamide-resistant clinical strains by stimulating cryptic bioactivation pathways of this antibiotic.The first objective of this thesis was to evaluate the substrate specificity of the three currently known pathways of ethionamide. This study was performed by producing ethionamide derivatives and studying their activation by alternately stimulating each of the three pathways using specific boosters. To do this, a new synthesis route of thioisonicotinamides has been developed in order to quickly obtain a large number of structural analogues of ethionamide. We were then able to explore the antimycobacterial activity of these analogues, alone or in combination with boosters which individually stimulate the three activation pathways. This study allowed us to highlight the specificities of each pathway. In parallel, a cyclic voltammetric study carried out on some of these compounds allowed us to correlate the oxidation potential of the analogues with their antimycobacterial activities.In a second axis, we tried to follow, by NMR and by LC-MS/MS, the bioactivation of ethionamide in a heterologous expression model of ethionamide bioactivation enzymes in E. coli.In a third part, we were interested in the identification and the characterization of new bioactivatable molecules by the different bioactivation pathways of ethionamide.Finally, phenotypic screening was performed on Mycobacterium tuberculosis to extend the concept of resistance reversal to other anti-tuberculosis pro-antibiotics. This approach has led to the identification of a molecule capable of reprogramming the bioactivation of nitroimidazoles of major clinical importance, such as pretomanid and delamanid. On this basis, we undertook a work of pharmacomodulation of this hit, which allowed us to determine the first structure-activity relationships in this chemical family

La connaissance du comportement sous irradiation γ et en présence d'eau des Résines Echangeuses d'Ions est nécessaire pour prévoir leur impact sur l'environnement pendant la phase d'entreposage et dans un éventuel stockage en profondeur géologique. Les REI étudiées sont la résine MB400 en lit mélangé ainsi que ses composants anionique et cationique « purs ». La stratégie expérimentale suivie a été basée sur l'utilisation d'outils chimiométriques qui ont permis d'étudier l'effet du milieu d'irradiation, du débit de dose, de la dose et de la température de lixiviation. Les produits de radiolyse gazeux et hydrosolubles ont été analysés par Spectrométrie de Masse gaz et Chromatographie Ionique. Les REI génèrent principalement du H2g, du CO2g et des amines dont les quantités dépendent de la nature de la résine et des conditions d'irradiation. L'analyse des résines solides irradiées a été effectuée par spectroscopie Infrarouge à Transformée de Fourrier et par Résonance Magnétique Nucléaire. Ces techniques révèlent des modifications structurales différentes suivant les conditions d'irradiation. Le comportement sous eau des REI a été étudié sur une période de 143 jours en caractérisant la matière organique relarguée après lixiviation post-irradiation. Les études cinétiques montrent qu'au premier contact avec l'eau, toutes les espèces hydrosolubles sont relarguées. La quantité de Carbone Organique Total dépend, selon la nature de la résine, soit de la dose, soit du milieu d'irradiation. Le débit de dose n'a pas d'effet sur la dégradation et la lixiviation de la résine MB400 qui, néanmoins se comporte d'une façon différente de ses composants pris séparément
The knowledge of the behavior under irradiation and in presence of water of Ion Exchange Resins (IER) is very necessary to predict their impact on the environment during the storage phase and in a possible deep geological disposal. The IER studied are the MB400 mixed bed resin and its « pure » anionic and cationic components. The experimental strategy used in this work was based on the use of chemometric tools permitting to estimate the effect of the irradiation atmosphere, the dose rate, the absorbed dose and the leaching temperature. The gaseous and water-soluble radiolysis products were analyzed by gas Mass Spectrometry (MS) and Ion Chromatography (IC). The IER generated principally H2g, CO2g and amines for which quantities depended of the resin nature and the irradiation conditions. The analysis of solid irradiated resins was investigated by Fourier Transformed Infrared (FTIR) and Nuclear Magnetic Resonance (13C NMR) techniques. The last ones revealed structural modifications of the IER solid matrix in function of the experimental conditions. Their behavior in presence of water was studied during 143 days by characterization of the organic matter released after their post-irradiation leaching. The kinetics showed that all the water-soluble components were releasing at the first contact with water. The Total Organic Carbon (TOC) quantity released depends, according to the resin nature, either on the dose, either on the irradiation atmosphere. The dose rate has no effect on the degradation and the leaching of the MB400 resin, which behaved differently than its pure components

Les écorces d’arbres, co-produit de la sylviculture, constituent une source abondante et durable de substances naturelles. Toxoplasma gondii est le parasite responsable de la toxoplasmose, présentant une menace chez les fœtus, les nouveau-nés et les personnes immunodéprimées. Les thérapies actuelles, limitées et mal tolérées, font désormais face à des phénomènes de chimiorésistance. Ce travail de thèse a pour but d’explorer l’espace chimique associé aux écorces d’essences champardennaises, et les cibles protéiques essentielles à T. gondii. Une première évaluation in silico par docking inverse (AMIDEv2.0) a été réalisée afin d’identifier la cible biologique de triterpènes dérivés de la bétulone, isolés de l’Aulne glutineux ayant montré une activité anti-toxoplasmose in vitro. La CDPK3 a été identifiée comme étant la cible la plus probable parmi 87 protéines de T. gondii. Puis, une protéothèque de 25 structures protéiques 3D essentielles à la survie du parasite, 19 ayant été modélisées par homologie, a été constituée. Les composés de la Chimiothèque Nationale Essentielle ont été évalués ensuite sur cette protéothèque en utilisant AMIDEv2.0. Deux protéines ont été identifiées comme de potentielles cibles, dont ATG3, une structure reconstruite à partir d'homologues avec un pourcentage d'identité inférieur à 50%. Deuxièmement, les écorces du Mélèze d'Europe, dont l’extrait n-heptane avait démontré une activité significative (58 % d’inhibition de croissance parasitaire à 100 µg/ml), ont été soumises à un profilage chimique impliquant un fractionnement par Chromatographie de Partage Centrifuge et de déréplication combinant les données issues de la résonance magnétique nucléaire et de la spectrométrie de masse. Les outils VersaDB et CATHEDRAL ont été développés pour faciliter la création de bases de données modulables et l’évaluation du niveau de confiance des annotations. 52 molécules ont ainsi pu être annotées et associées à un score de confiance. En parallèle, des tests in vitro ont démontré que 2 des 12 fractions CPC, majoritairement composées de terpènes, inhibaient à plus de 40% la survie du parasite à 25 µg/ml. Les composés annotés chez L. decidua ont été soumis à AMIDEv2.0. Le croisement des résultats in vitro et in silico, reposant sur le calcul d'un score d'activité biologique, a mis en évidence l'acide 7-oxo-déhydroabiétique et l'acide daniellique, fortement corrélés à l'activité inhibitrice in vitro des écorces. La CDPK1 et la protéine SET containing Protein ont été identifiées comme leurs cibles protéiques probables, fournissant ainsi de premières informations sur leurs mécanismes d'action. Ces deux hits font actuellement l’objet d’une évaluation in vitro afin d’attester l’efficacité de la démarche développée au cours de ces travaux de thèse
Tree barks, by-product of forestry industry, constitute an abundant and sustainable source of natural compounds. Toxoplasma gondii is the parasite responsible for toxoplasmosis, posing a threat to fetuses, newborns, and immunocompromised individuals. The current therapeutics, limited and poorly tolerated, are now confronted to chemoresistant phenomena. This doctoral project aims to explore the chemical space associated with tree barks from the Champagne-Ardenne region, as relevant protein targets to fight T. gondii. An initial in silico evaluation using reverse docking (AMIDEv2.0) was carried out to identify biological target for triterpenes derived from betulone, isolated from the European alder, which had exhibited in vitro anti-toxoplasmosis activity. Among 87 proteins of T. gondii, CDPK3 was identified as the most probable target. Subsequently, a bank of 25 essential 3D protein structures for parasite survival, including 19 homology-modeled structures, was compiled. Thereafter, compounds from the Essential National Chemical Library were screened against this protein bank, using AMIDEv2.0. Two proteins were identified as potential targets; one of them was ATG3, a protein structure modeled from homologs with less than 50% identity. Subsequently, the barks of European Larch, whose n-heptane extract had shown significant activity (58% inhibition of parasitic growth at 100 µg/ml), were subjected to a chemical profiling. First, through a fractionation process using Centrifugal Partition Chromatography, and then a dereplication approach combining data from nuclear magnetic resonance and mass spectrometry. Tools like VersaDB and CATHEDRAL were developed to facilitate the creation of custom-databases and assess the confidence level of annotations. 52 molecules were annotated and associated with a confidence score. Simultaneously, in vitro tests demonstrated that 2 out of the 12 CPC fractions, primarily composed of terpenic derivatives, inhibited the parasite's survival by more than 40% at 25 µg/ml. Ultimately, the annotated compounds from L. decidua were subjected to AMIDEv2.0. The overlap between in vitro and in silico results highlighted 7-oxo-dehydroabietic acid and daniellic acid, strongly correlated with the in vitro inhibitory activity of the barks. CDPK1 and the SET-containing Protein are likely protein targets for these two ligands, thereby providing initial insights into their mechanism of action. These two hits are currently undergoing in vitro evaluation to verify the efficiency of developed approach during this doctoral project

De nos jours, il y a une volonté générale de se tourner vers une agriculture plus respectueuse de l’environnement et du consommateur se traduisant notamment par une démarche de réduction des intrants chimiques. Dans un contexte de développement durable, la recherche de produits naturels pour lutter contre les maladies et les ravageurs suscite un regain d’intérêt. Dans cette thèse, des extraits hydro-alcooliques issus de coproduits de la vigne (sarment, cep, racine) et d’essences forestières (écorce d'épicéa, nœud de pin) se sont révélés être une excellente source de polyphénols bioactifs, en particulier en stilbènes complexes. En effet, ces extraits ont démontré un large spectre d’activités contre différentes maladies végétales. En particulier, un potentiel oomycide contre le mildiou de la vigne et une capacité insecticide contre un parasite des Solanacées sont rapportés. En outre, la pertinence de l'utilisation de la « chimie verte » pour extraire les stilbènes comme méthode alternative aux solvants organiques a été mise en évidence. Les présents résultats renforcent une voie de recherche originale pour faire progresser une viticulture et une agriculture plus durables, en utilisant des produits de biocontrôle moins toxiques et biodégradables, constituant ainsi une solution possible et réaliste pour lutter contre les pathogènes des plantes
Nowadays, is a priority to turn towards a more eco- and consumer friendly agriculture resulting in the reduction of the chemical inputs. In a context of a sustainable development, the investigation of natural products to fight against diseases and pests raised a renewed interest. In this thesis, hydroalcoholic extracts derived from grapevine (cane, wood, root) and forest species (spruce bark, pine knot) by-products have demonstrated to be a great source of bioactive polyphenols, and particularly in complex stilbenes. Indeed, these extracts have proved to confer a broad spectrum of activities against different major plant diseases. In particular, an oomycide potential against downy mildew of the vine and an insecticidal capacity against Solanaceae pest were reported. Furthermore, the relevant use of “green chemistry” to extract stilbenes as an alternative method of organic solvents has been highlighted. The present findings strengthen an original line of research to advance in a more sustainable viticulture and agriculture, using less toxic and biodegradable biocontrol products, being this a possible and realistic solution to combat plant pathogens

The essential dietary fatty acid α-linolenic acid (ALA) can be converted into anti-inflammatory 18 carbon oxylipins or into longer chain n€‘3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The partitioning of ALA between these alternative metabolic fates is not understood. To address this, peripheral blood CD3+ T cells from healthy volunteers (18-30 years; n=10) were cultured for 48h, with or without concanavalin A (10µg/ml) in 10% (v/v) pooled donor plasma with low ALA (20 µM) or high ALA (40 µM) concentrations (1:10 [13C]€‘labelled/unlabelled). [13C]ALA metabolites were detected either by GC-isotope ratio mass spectrometry for intracellular PUFA or by LC-MS/MS for oxylipins in cell culture supernatant. The ratio of the labelled metabolites hydroxyoctatrienoic acid ([13C]HOTrE) and dihydroxyoctadecaenoic acid ([13C]DiHODE) to [13C]ALA were 1.8±0.2 / 7.2±1.1 and 0.9±0.2 / 4.3±0.6 for low / high ALA, respectively, compared to the eicosatrienoic acid ([13C]20:3n€‘3) to [13C]ALA ratio of 0.002±0.0001 / 0.02±0.003 in stimulated T cells. Results from unstimulated cells were similar. Furthermore, oxylipins from all PUFA precursors were analysed in the culture supernatant of the T cells. The ratio of oxylipin concentrations in high compared to low ALA cultures was 1.4±0.1 for EPA-derived dihydroxyeicosatetraenoic acid (DiHETE), 5.6±0.9 for DHA-derived dihydroxydocosapentaenoic acid (DiHDPE), 7.9±2.7 for resolvin RvE1 and 2.0±0.3 for resolvin RvD1. The total oxylipin profile was not altered significantly by mitogen stimulation. These findings show that ALA is used primarily by T cells for constitutive production of anti-inflammatory lipid mediators rather than synthesis of longer chain PUFA. Further, ALA addition changes the secreted oxylipins towards a less-inflammatory profile. This has implications for understanding the effects of dietary PUFA on immune function.

Prostate cancer (PCa) is the second most frequently diagnosed malignancy, as well as a leading cause of cancer-related mortality in men globally. Despite the initial response to hormonal targeted therapy, the majority of patients ultimately progress to a lethal form of the disease, termed as castration-resistant prostate cancer (CRPC), which currently lacks curative therapeutic options and is associated with poor prognosis. Therefore, the development of novel treatment modalities for PCa is urgently needed. Chalcones, also known as 1,3-diphenyl-2-propen-1-ones, are among the highly attractive scaffolds being investigated for their antitumor activities. Three series of 18 cyclic (tetralone-based) and two acyclic chalcone analogs, in which ring B was either substituted with nitrogen mustard or replaced by pyrrole or pyridine heterocyclic rings, were designed, synthesized and evaluated as potential therapies for CRPC. Compounds were synthesized by Claisen-Schmidt condensation reaction, purified using columnchromatography or recrystallization and characterized by 1H-NMR, 13C-NMR and LC-MS. The compounds' in-vitro cytotoxicity was evaluated against three prostate cancer cell lines (PC3, DU145, and LNCaP). Among the tested compounds, OH14, OH19 and OH22 showed potent antiproliferative activities at low micromolar levels with IC50 values ranging between 4.4 and 10 µM against PC3 and DU145 cell lines. Detailed biological studies of the lead molecule OH19 revealed that it significantly induces apoptosis through upregulation of Bax and downregulation of BCL-2. In addition, OH19 potently inhibits colony formation and reduces cell migration of androgen-independent PCa cell lines (PC3 and DU145). The molecular pathway analysis show that the anticancer activity of OH19 is associated with attenuation in the phosphorylation of Akt and ERK. Furthermore, OH19 inhibits blood vessel formation in the chick chorioallantoic membrane (CAM) model as compared to control. These results indicate that OH19 could serve as a potential promising lead molecule for the treatment of CRPC and thus, further in-vitro and invivo studies are warranted.