Two goitrogenic 1,3-oxazolidine-2-thione derivatives from Brassicales taxa: Challenging identification, occurrence and immunomodulatory effects.

1,3-Oxazolidine-2-thione derivatives are glucosinolate-related food constituents known to impart (thyreo)toxic properties to some cruciferous vegetables. In this work, 5,5-dimethyl-1,3-oxazolidine-2-thione and (-)-(R)-5-phenyl-1,3-oxazolidine-2-thione, known goitrogens, were isolated from Draba lasiocarpa Rochel (Brassicaceae) and Reseda luteola L. (Resedaceae), respectively, and were fully spectrally characterized. Subsequently, the occurrence of the two 1,3-oxazolidine-2-thiones was verified in six additional taxa out of in total 78 screened Serbian Brassicales taxa. The stereochemistry of 5-phenyl-1,3-oxazolidine-2-thione was inferred from nuclear magnetic resonance experiments with a chiral lanthanide-shift reagent, employed in this work for the first time for this type of compounds. Unexpectedly, during gas chromatography, 5-phenyl-1,3-oxazolidine-2-thione underwent an unreported thermal core isomerization (1,3-oxazolidine-2-thione to 1,3-thiazolidine-2-one). These goitrogenic volatile glucosinolate products were tested for their effect on rat macrophage viability (three assays) and nitric oxide production. It was shown that the compounds displayed different levels of cytotoxicity. All tested compounds caused a significant lactate dehydrogenase leakage, but only (R)-5-phenyl-1,3-oxazolidine-2-thione statistically significantly reduced macrophage mitochondrial activity, whereas the racemic 5-phenyl-1,3-oxazolidine-2-thione and 5,5-dimethyl-1,3-oxazolidine-2-thione had little or no effect. Again only (R)-5-phenyl-1,3-oxazolidine-2-thione exerted nitric oxide production-inhibiting properties, suggesting the higher immunomodulatory potential of this enantiomer compared with its antipode and racemic mixture.

Novel Concept of Discrimination of 1,2,4-Triazole-3-thione and 3-Thiol Tautomers.

Till now, three major spectroscopic techniques, fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, have been used for determination of thiol-thione tautomeric forms. Therefore, a novel analytical concept of discrimination of 1,2,4-triazole-3-thione and 3-thiol tautomers using two high-resolution mass spectrometers has been proposed. It comprises the high-performance liquid chromatography (HPLC) hyphenated with electrospray ionization (ESI)-time-of-flight mass spectrometry (ESI-TOF-MS, positive ion mode) followed by in-source collision-induced dissociation (CID) and low-energy CID-tandem mass spectrometry (MS/MS) analysis measured with a Quadruple-TOF-MS instrument. The HPLC column was a Zorbax Stable Bond RP-18 and a rapid isocratic elution. Selected 3-thione and 3-thiol tautomers were rapidly separated, within 6 min and detected both by in-source CID ESI(+)-TOF-MS and CID ESI(+)-QTOF-MS with a high mass accuracy and high sensitivity. The method limits of detection were of 2.8-5.6 pg/µl (at S/N 3:1) for ESI-TOF-MS and 0.25-0.55 pg/µl for ESI-QTOF-MS. The tautomeric form could be easily discriminated by both methods and by the different gas-phase fragmentation patterns. Differences and similarities between in-source CID MS and CID MS/MS spectra have also been presented. These findings were also supported by recorded FT-IR spectra in solid state. The developed methodology using both the high-resolution MS systems is considerably the most sensitive among the others.

Isolation of a novel antibacterial phenyl thioketone from the seagrass, Cymodocea serrulata.

A total of 40 extract types of varying polarities from commonly occurring seagrasses were tested for their antibacterial efficiency against 14 clinically isolated human pathogens using agar well diffusion technique. The extracts from acetone of Cymodocea serrulata expressed moderate broad span of activity against a range of gram-positive and gram-negative isolates that were at least resistant to five of the commercially available antibiotics at a minimal concentration of 10 µg. The active extracts of C. serrulata that showed maximal inhibitions were purified using column chromatography that afforded six compounds (a-f). Compound f elicited pronounced inhibitions against Escherichia coli with minimal inhibitory concentration values of 1-3 µg concentration using micro-dilution method. The active compound was identified as phenyl thioketone using various spectral analyses. This is the first investigation that reveals thioketone functionality from this seagrass species possessing antibacterial actions. This study indicates that there are thiocarbonyl groups from marine floral sources too, which could be possibly used for therapeutic purposes.

Identification of a novel sildenafil analogue in an adulterated herbal supplement.

iErect, a new dietary supplement marketed as "100% natural" and sold over the Internet, was analyzed. It contains thiosildenafil, a sildenafil analogue already reported as an adulterant in herbal formulations, and a new compound whose structure was elucidated after isolation using NMR, MS and IR. It was named depiperazinothiosildenafil as it results from the hydrolytic cleavage of the S-N bond of the sulfonamide group of thiosildenafil. A capsule of iErect contains a very high amount (≈220mg) of thiosildenafil and ≈30mg of depiperazinothiosildenafil, which places consumers at risk for potentially serious side-effects.

Precursors and formation of pyrithione and other pyridyl-containing sulfur compounds in drumstick onion, Allium stipitatum.

Two novel, structurally unusual cysteine derivatives were isolated from the bulbs of Allium stipitatum (Allium subg. Melanocrommyum) and shown to be S-(2-pyridyl)cysteine N-oxide and S-(2-pyridyl)glutathione N-oxide. The former compound is the first example of a naturally occurring alliinase substrate that contains an N-oxide functionality instead of the S-oxide group. In addition, S-methylcysteine S-oxide (methiin) and S-(methylthiomethyl)cysteine 4-oxide (marasmin) were found in the bulbs. Presented data suggest that the previously reported identification of S-(2-pyridyl)cysteine S-oxide was most likely erroneous. The alliinase-mediated formation of pyridyl-containing compounds following disruption of A. stipitatum bulbs was studied by a combination of HPLC-MS, HPLC-PDA, DART-MS, and NMR techniques. It was found that no pyridyl-containing thiosulfinates are present in homogenized bulbs in detectable quantities. Instead, various pyridine N-oxide derivatives are formed, including N-hydroxypyridine-2(1H)-thione (pyrithione), 2-(methyldithio)pyridine N-oxide, 2-[(methylthio)methyldithio]pyridine N-oxide, di(2-pyridyl) disulfide N-oxide, and di(2-pyridyl) disulfide N,N'-dioxide. This represents the first report of pyrithione formation as a natural product.

Thioperoxy derivative generated by UV-induced transformation of N-hydroxypyridine-2(1H)-thione isolated in low-temperature matrixes.

Photochemical transformations of N-hydroxypyridine-2(1H)-thione and its deuterated isotopologue were studied using the matrix-isolation technique. Low-temperature Ar and N2 matrixes containing monomers of this compound were irradiated with continuous-wave near-UV light. Photogeneration of two products was observed in these experiments. The relative population of these photogenerated species was found to be dependent on the wavelength of the UV light used for irradiation. By comparison of the IR spectra of the photoproducts with the spectra simulated theoretically at the DFT(B3LYP)/6-311++G(d, p) level, the final and the intermediate products were identified as rotameric forms of 2-hydroxysulfanyl-pyridine. This is the first report on generation of this thioperoxy derivative of pyridine. The mechanism of photogeneration of 2-hydroxysulfanyl-pyridine involves a photoinduced cleavage of the N-O bond in N-hydroxypyridine-2(1H)-thione, generation of the .OH radical weakly bound with the remaining pyridylthiyl radical, and recombination of these two radicals by formation of the new -S-O- bond. A theoretical model supporting this interpretation was constructed on the basis of approximate coupled cluster (CC2) calculations of the potential energy surfaces of the ground and first excited singlet electronic states of the system. After electronic excitation of the monomeric N-hydroxypyridine-2(1H)-thione, the molecule evolves to the conical intersection with the potential energy surface of the ground state and then to the global minimum corresponding to 2-hydroxysulfanyl-pyridine.

Identification of a novel class of dithiolethiones that prevent hepatic insulin resistance via the adenosine monophosphate-activated protein kinase-p70 ribosomal S6 kinase-1 pathway.

UNLABELLED: Several established liver diseases of various causes are highly associated with hepatic insulin resistance, which is characterized by the desensitization of target cells to insulin. Peripheral insulin resistance is observed in most patients who have cirrhosis. Conversely, insulin-resistant diabetic patients are at increased risk for developing liver disease. Current therapeutic interventions in insulin resistance are limited and therefore likely to be advanced by new tailor-made drugs. Oltipraz, a prototype dithiolthione, inhibits transforming growth factor beta1 and has the ability to regenerate cirrhotic liver. We investigated the effects of oltipraz and synthetic dithiolthiones on hepatic insulin resistance and the molecular basis of action. Oltipraz and other dithiolethione compounds were tested on tumor necrosis factor alpha (TNF-alpha)-induced insulin resistance and glucose homeostasis in vitro and in vivo via immunoblotting, plasmid transfection, kinase analysis, and functional assays. Oltipraz treatment inhibited the ability of TNF-alpha to activate p70 ribosomal S6 kinase-1 (S6K1) downstream of mammalian target of rapamycin, thus preventing insulin receptor substrate-1 serine phosphorylation and protecting insulin signals. Moreover, oltipraz activated AMP-activated protein kinase (AMPK), whose inhibition by a dominant negative mutant abolished S6K1 inhibition and protected insulin signaling, indicating that AMPK activation leads to S6K1 inhibition. In hepatocyte-derived cell lines, oltipraz inhibited glucose production. Oltipraz prevented hepatic insulin resistance in C57BL/6 mice challenged with endotoxin (or TNF-alpha), leptin-deficient mice, and mice fed a high-fat diet. Synthetic dithiolethiones comparably inhibited insulin resistance. CONCLUSION: Our findings led to the identification of dithiolethione compounds that prevent insulin resistance through a mechanism involving AMPK-mediated S6K1 inhibition and thereby sensitize hepatic insulin response.

Rapid preparation of the mitotic kinesin Eg5 inhibitor monastrol using controlled microwave-assisted synthesis.

We present here a protocol for the synthesis of the dihydropyrimidine (DHPM) derivative monastrol, which is known to be a specific mitotic kinesin Eg5 inhibitor. By applying controlled microwave heating under sealed-vessel conditions, the synthesis via the one-pot three-component Biginelli condensation can be performed in a shorter reaction time (30 min) compared with conventional heating methods that normally require several hours of reflux heating. For the purification of the crude target compound, two different methods are presented. The first protocol includes a simple precipitation/filtration step to provide monastrol in 76% isolated yield and high purity so that no recrystallization step is necessary. This can be ascribed to the microwave heating technology in which less side-product formation is typically one of the advantages. In an alternative purification step, column chromatography is performed, which provides the product in a slightly higher yield (86%). Monastrol synthesis can be conducted in approximately 2 h by employing the precipitation/filtration purification method.

Kinetic resolution of protected alpha-amino acid derivatives by a chiral O-nucleophilic acyl transfer catalyst.

The kinetic resolution of alpha-trifluoroacetamido N-acyl oxazolidinethiones using 5-10% of the chiral, nonracemic O-nucleophilic acyl transfer catalyst 2 is described. A variety of substrates participate in this reaction in excellent yields, with s-factors ranging from 20 to 86.

A fluorimetric assay for the spontaneous release of an N7-alkylguanine residue from duplex DNA.

A fluorimetric assay for monitoring depurination of the N7-alkylguanine adduct derived from the anticancer natural product leinamycin is described. This general approach could potentially provide the foundation for a high throughput assay that detects DNA-alkylating agents or a convenient continuous fluorimetric assay for base excision repair enzymes.

The identification of 1,3-oxazolidine-2-thiones and 1,3-thiazolidine-2-thiones from the reaction of glucose with benzyl isothiocyanate.

The structure of interaction products resulting from the reaction of unmodified glucose with benzyl isothiocyanate is reported. Prior to their identification, the main products of this reaction were isolated using solid-phase extraction (SPE) as well as preparative HPLC. They were then identified by NMR and MS as 3-benzyl-4-hydroxy-5-(D-arabino-1,2,3,4-tetrahydroxybutyl)-1,3-oxazolidine-2-thione, 3-benzyl-4-hydroxy-4-hydroxymethyl-5-(D-erythro-1,2,3-trihydroxypropyl)-1,3-oxazolidine-2-thione, N-benzyl-(D-gluco-4,5-dihydroxy-6-hydroxymethyl-tetrahydropyrano)[2,3-b]oxazolidine-2-thione and 3-benzyl-4-(N-benzyl amino)-5-(D-arabino-1,2,3,4-tetrahydroxybutyl)-1,3-thiazolidine-2-thione. The identity of the last compound was secured by X-ray crystal structure data.

Combining synthetic and analytical strategies for preparative HPLC enantioseparation of monastrol racemic mixture.

Large-scale resolution of racemic monastrol has been carried out by normal-phase mode HPLC on an amylose-based chiral stationary phase. Because monastrol solubility, in media of proper compositions for normal-mode HPLC separations (in terms of retention factors and selectivity), was significantly low and impractical for preparative scopes, racemic monastrol was transformed into the corresponding O-tert-butyldimethylsilyl derivative. The tert-butyldimethylsilyl group was chosen as a suitable derivatizing agent because it induced approximatively a six-times higher solubility and allowed for an almost quantitative recovery of pure monastrol from the derivatization-deprotection sequence. The competitive isotherms of the O-tert-butyldimethylsilyl compounds, measured through frontal analysis, were fitted to competitive Langmuir and four-parameter bi-Langmuir models. The equilibrium dispersive model of chromatography was used for modeling the nonlinear separation of the racemate and to optimize the experimental conditions for collection of highly concentrated fractions of pure (R,S)-O-tert-butyldimethylsilyl compounds, from which significant amounts of the corresponding enantiomers of monastrol (about 100 mg of each enantiomer with 30 runs on an analytical-scale column) were obtained by quantitative back-derivatization.

2-[3-(2-Thioxopyrrolidin-3-ylidene)methyll-tryptophan, a novel yellow pigment in salted radish roots.

The structure of the yellow pigment found in salted radish roots was studied. It was found that 1-(2-thioxopyrrolidin-3-yl)-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (TPCC) was unstable under neutral pH, and was easily converted into the yellow pigment. The yellow pigment was isolated and identified as 2-[3-(2-thioxopyrrolidin-3-ylidene)methyl]-tryptophan (TPMT) by IR, MS, 1H-, and 13C-NMR spectroscopy. In addition, we proved that this compound was the main yellow pigment in salted radish roots. This compound induced no mutagenicity in Salmonella typhimurium TA98 and TA100, either with or without prior activation.

Potential anticancer agents vi. Constituents of Ailanthus excelsa (Simaroubaceae).

Ailanthione (1), glaucarubinone (2) and a mixture of glaucarubol 15-isovalerate (3) and 13,18-dehydroglaucarubol 15-isovalerate (4) were found to be the compounds responsible for the antitumor and cytotoxic activities of extracts of the root bark of Ailanthus excelsa (Simaroubaceae). The latter compound, 4, is new.