Investigation of Symphytum cordatum alkaloids by liquid-liquid partitioning, thin-layer chromatography and liquid chromatography-ion-trap mass spectrometry

From the alkalised crude extract of Symphytum cordatum (L.) W.K. roots, pyrrolizidine alkaloids (PAs) were extracted as free tertiary bases and polar N-oxides in a merely one-step liquid-liquid partitioning (LLP) in separation funnel and subsequently pre-fractionated by preparative multiple-development (MD) thin-layer chromatography (TLC) on silica gel plates. In this way three alkaloid fractions of different polarities and retention on silica gel plates were obtained as: the most polar N-oxides of the highest retention, the tertiary bases of medium retention, and diesterified N-oxides of the lowest retention. The former fraction was reduced into free bases by sodium hydrosulfite and purified by LLP on Extrelut-NT3 cartridge. It was further analysed together with the two other fractions by high-performance liquid chromatography (HPLC)-ion-trap mass spectrometry with atmospheric pressure chemical ionization (APCI) interface on XTerra C₁₈ column using a gradient elution. Based on MSⁿ spectra, 18 various alkaloids have been tentatively determined for the first time in this plant as the following types of structure: echimidine-N-oxide (three diasteroisomers), 7-sarracinyl-9-viridiflorylretronecine (two diasteroisomers), echimidine (two diasteroisomers), lycopsamine (two diasteroisomers), dihydroechinatine-N-oxide, dihydroheliospathuline-N-oxide, lycopsamine-N-oxide (three diasteroisomers), 7-acetyllycopsamine-N-oxide, symphytine-N-oxide (two diasteroisomers) and 2″,3″-epoxyechiumine-N-oxide.     

Reactions of 5-nitrospiro[benzimidazole-2,1′-cyclohexane] 1,3-dioxide with nucleophiles

Reactions of 5-nitrospiro[benzimidazole-2,1′-cyclohexane] 1,3-dioxide with aliphatic amines and sodium hydroxide resulted in removal of one N-oxide oxygen atom and formation of 4-alkylamino- or 4-hydroxy-substituted 5-nitrospiro[benzimidazole-2,1′-cyclohexane] 1-oxides, respectively. The title compound reacted with ammonia and methylamine in the presence of MnO₂ with conservation of both N-oxide moieties, and the products were 4-amino- and 4-methylamino-5-nitrospiro[benzimidazole-2,1′-cyclohexane] 1,3-dioxides. The reactions with aromatic amines were accompanied by removal of both N-oxide oxygen atoms with formation of N-aryl-5-nitrospiro[benzimidazole-2,1′-cyclohexane]-4-amines. In the reactions of 5-nitrospiro-[benzimidazole-2,1′-cyclohexane] 1,3-dioxide with sodium azide and aromatic amine hydrochlorides nucleophilic replacement of the 5-nitro group by azido or arylamino occurred, in the first case both N-oxide fragments being conserved. The reactions with aromatic amine hydrochlorides afforded N-aryl-5-nitrospiro[benzimidazole-2,1′-cyclohexan]-4-amine 1-oxides. Treatment of 5-nitrospiro[benzimidazole-2,1′-cyclohexane] 1,3-dioxide with sodium cyanide led to the formation of 5-oxo-3,5-dihydrospiro[benzimidazole-2,1′-cyclohexane]-4-carbonitrile 1-oxide.     

Development and validation of a rapid multiplex ELISA for pyrrolizidine alkaloids and their N-oxides in honey and feed

Pyrrolizidine alkaloids (PAs) are a group of plant secondary metabolites with carcinogenic and hepatotoxic properties. When PA-producing plants contaminate crops, toxins can be transferred through the food chain and cause illness in humans and animals, most notably hepatic veno-occlusive disease. Honey has been identified as a direct risk of human exposure. The European Food Safety Authority has recently identified four groups of PAs that are of particular importance for food and feed: senecionine-type, lycopsamine-type, heliotrine-type and monocrotaline-type. Liquid or gas chromatography methods are currently used to detect PAs but there are no rapid screening assays available commercially. Therefore, the aim of this study was to develop a rapid multiplex ELISA test for the representatives of three groups of alkaloids (senecionine, lycopsamine and heliotrine types) that would be used as a risk-management tool for the screening of these toxic compounds in food and feed. The method was validated for honey and feed matrices and was demonstrated to have a detection capability less than 25 μg/kg for jacobine, lycopsamine, heliotrine and senecionine. The zinc reduction step introduced to the extraction procedure allows for the additional detection of the presence of N-oxides of PAs. This first multiplex immunoassay for PA detection with N-oxide reduction can be used for the simultaneous screening of 21 samples for >12 PA analytes. Honey samples (n?=?146) from various origins were analysed for PA determination. Six samples were determined to contain measurable PAs >25 μg/kg by ELISA which correlated to >10 μg/kg by LC-MS/MS.     

Natural abundance 17O NMR spectroscopy of heterocyclic N-oxides and Di N-oxides. Structural effects

The ¹⁷O chemical shift data for a series of azine N-oxides, diazine N-oxides and di-N-oxides at natural abundance are reported. Isomeric methyl substituted quinoline N-oxides exhibited chemical shifts which are interpreted in terms of electronic and compressional effects. The ¹⁷O chemical shift for 8-methylquinoline N-oxide (370 ppm) is deshielded by 25 ppm more than predicted, based upon electronic considerations. The ¹⁷O chemical shift for the N-oxide of 8-hydroxyquinoline (289 ppm) is substantially shielded as a result of intramolecular hydrogen bonding. The relative ¹⁷O chemical shifts for diazine N-oxides of pyrazine, pyridazine and pyrimidine follow predictions based on back donation considerations. Because of solubility limitations, spectra of only two N,N′-dioxides were obtained. The chemical shift of benzopyrazine di N-oxide in acetonitrile was shielded by 18 ppm compared to that of its mono N-oxide.     

1H NMR studies of solvent and substituent effects on strong intramolecular hydrogen bonds

Chemical shifts of H-bonded protons in tetrabutylammonium hydrogen maleate and 14-substituted picolinic acid N-oxides have been measured in a number of dry solvents, of different activity, in order to distinguish between symmetrical single minimum and asymmetrical hydrogen bonds. In tetrabutylammonium hydrogen maleate the resonance was observed at 20.70 ppm and its was independent of the nature of the solvent used. The chemical shift value of picolinic acid N-oxide varies with the solvent. These observations suggest that the hydrogen bond is symmetrical in tetrabutylammonium hydrogen maleate but that it is asymmetrical in picolinic acid N-oxide. The chemical shifts of substituted picolinic acid N-oxides were correlated with σ_p, σ_m and Δ_pK_a. The substituent and solvent effects are compared and the position of the intramolecular H-bonded protons in picolinic acid N-oxides are estimated and discussed.     

Chemistry of thienopyridines. XXIII. Mass spectra of some N-oxides,sulfoxides, and sulfones

The mass spectral fragmentation patterns of a series of thienopyridine N-oxides, S-oxides, and S,S-dioxides were elaborated as a means of structural determination. Observation of a significant (M-16) peak is diagnostic for the presence of either an N-oxide or an S-oxide function (indistinguishable from one another by this method) but does not occur for an S,S-dioxide function. For a substituted thieno[2,3-b]pyridine 7-oxide, structural rearrangement to a pyridone (followed by emission of carbon monoxide or formyl radical) or side-chain fission may be competitive with de-N-oxygenation. For two tricyclic parent S-oxides, rearrangement and de-S-oxygenation are competing initial processes. For parent S,S-dioxides structural rearrangement precedes fragmentation, wherein the oxygen is ejected in such forms as sulfur monoxide, carbon monoxide, formyl or cyanate radicals, and ketene.     

Polycyclic N-hetero compounds. XXII Reaction of pyridine N-oxides and Pyrazine di-N-oxides with formamide

Reactions of pyridine N-oxides, pyrazine di-N-oxides, and their benzologues with formamide are described. Carbamoylation mainly occurred at aromatic ring with loss of the N-oxide oxygen atom, however, 2,4,6-trimethylpyridine 1-oxide gave 2- and 4-pyrimidinyl derivatives.     

A tandem mass spectrometric study of the N-oxides, quinoline N-oxide, carbadox, and olaquindox, carried out at high mass accuracy using electrospray ionization

A mass spectrometric study of three N-oxides, quinoline N-oxide, and the synthetic antibiotics carbadox and olaquindox, was carried out with a hybrid quadrupole/time-of-flight (TOF) mass spectrometer coupled with electrospray (ES) and atmospheric pressure chemical ionization (APCI) sources. The full scan mass spectra of the N-oxides obtained with ES are similar to those obtained with APCI, and the characteristic fragment ions corresponding to [M+H−O]⁺√ were observed in the full scan mass spectrum of each N-oxide examined. The protonated molecule of each N-oxide was subjected to collision-induced dissociation (CID) and accurate mass measurements were made of each fragment ion so as to determine its elemental composition. Fragment ions generated at enhanced cone voltages upstream of the first mass-resolving element were subjected to CID so as to identify the direct product ion–precursor ion relationship. Plausible structures have been proposed for most of the fragment ions observed. Elimination of OH√ radicals generated from the N→O functional group is a characteristic fragmentation pathway of the N-oxides. The expulsion of radicals and small stable molecules is accompanied by formation and subsequent contraction of heterocyclic rings.     

Synthesis of 2-amino-6-chloro- and 2,6-diammopunne 3-oxides

N-Oxidation of 2-amino-6-chloropurine to the 3-oxide provided a convenient intermediate for the synthesis of 2-amino-6-substituted purine 3-oxides, including the previously unavailable 2,6-diaminopurine 3-oxide. Thiation of the 6-halogen was accompanied by reduction of the N-oxide. The properties of the 1- and 3-oxides of 2,6-diaminopurine are compared.     

Structure-Property Relationships in the Basicity and Lipophilicity of Arylalkylamine Oxides

Homologous N,N-dimethyl-phenylalkylamine oxides and N,N-dimethyl-diphenylalkylamine oxides were prepared. Their basicity and lipophilicity (octan-1-ol/H₂O) were compared to those of the parent amines. In contrast to the amines, the basicity of all N,N-dimethyl-arylalkylamine oxides showed very limited pK_a variations (range 4.65 – 5.01) with increasing chain length and number of Ph groups. The N-oxides in their neutral form had a log P^N value lower by 2.77±0.34 (n=9) units than that of the parent amine. The log P^C of the cationic N,N-dimethyl-diphenylalkylamines was lower than that of their neutral form, with a decrement diff(log P^N−C) that increased from 3.25 to 4.21 in the homologous series. Unexpectedly, the decrement diff(log P^N−C) for the N-oxides was much smaller than for the tertiary amines, being 0.23 for the aliphatic N,N-dimethyl-pentylamine oxide, 0.47±0.13 for the phenylalkylamine oxides, and 0.80±0.07 for the diphenylalkylamine oxides. In fact, the protonated N-oxides had log P^C values that were quite comparable to those of the protonated parent amines. Because of the differences in basicity, the difference in distribution coefficients at physiological pH (log D^7.4) between a tertiary arylalkylamine and its N-oxide was 0.82±0.66 (n=9). The pharmacokinetic implication is that N-oxygenation may have a smaller effect on the urinary excretion of tertiary amines than usually assumed.     

Reactions of functionalized alkyl halides withN-(β-hydroxyalkyl)-N′-hydroxydiazeneN-oxide salts

Some characteristic features of reactions ofN-(β-hydroxyalkyl)-N′-hydroxydiazeneN-oxide salts with various α- and β-functionalized alkyl halides were established. Some α-and β-functionalizedN-(β-hydroxyalkyl)-N′-alkoxydiazeneN-oxides and e ethylenebis[N-(β-hydroxyalkyl)-N′-oxydiazeneN-oxides] were synthesized for the first time. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 123–129, January, 1999.     

1-Aryl- und 1-Alkyl-2-phthalimido-diazen-1-oxide,diacylierte Vertreter von trisubstituierten Triazen-1-oxiden: Bildung,Eigenschaften, Stereoisomerisierung und Fragmentierung

1-Aryl- and 1-Alkyl-2-phathalimido-diazene-1-oxides, Diacylated Examples of Trisubstituted Triazene-1-oxides: Formation, Properties, Stereoisomerization and Fragmentation¹) Oxidatively generated phthalimido-nitrene ( 1 ) reacts with nitrosobenzene ( 8a ), p-nitrosotoluene ( 8b ), o-nitrosotoluene ( 8c ), p-dimethylamino-nitrosobenzene ( 8d ), p-methoxycarbonyl-nitrosobenzene ( 8e ), 1, 1-dimethyl-1-nitrosoethane ( 8f ) and nitrosocyclohexane ( 8g ) to give the respective 1-substituted (Z)-2-phthalimidodiazene-1-oxides 14a-g . The constitution of 2-imidodiazene-1-oxides 14 is deduced from their spectroscopic properties. The UV. spectra of 14a-e are similar with those of the corresponding nitrobenzenes 18 , thus supporting the concept of comparibility of the phthalimido-N group with an O-atom and indicating that the phthalimido group is not in conjugation with the diazene-oxide function. This is in contrast to the situation in the non-acylated trisubstituted triazene-1-oxides 12 , where conjugation is extended over the chain of all three N-atoms as exhibited by the UV. spectra of (Z)-1, 3, 3-triphenyl-, (Z)-3, 3-dimethyl-1-phenyl-, (Z-3-methyl-1, 3-diphenyl-, (Z)-1-(1, 1-dimethylethyl)-3, 3-diphenyl-, and (Z)-1-(1, 1-dimethylethyl)-3, 3-dimethyltriazene-1-oxide ( 12a-e ). These triazene-1-oxides are formed by the reaction of 1, 1-disubstituted hydrazines 11 with three mol-equiv. of nitroso compounds 8 or from equimolar amounts of 11 and 8 in the presence of mercury oxide. Over 90% of 1, 2-diphenyldiazeneoxide ( 19 , R = C₆H₅) are isolated by the reaction 11 + 3 8a . Possible mechanisms of the reaction of 11 with 8 , and of the formation of 12 via triazanols D and of 19 (R = C₆H₅) via N-Phenylhydroxylamine ( 20 , R = C₆H₅) are given in Scheme 6 (in the latter case with experimental evidence). An independent synthesis of 12c confirms the constitutional assignment for 12 .     

Nitrogen-15 nuclear magnetic resonance spectroscopy. Pyridine N-oxides and quinoline N-oxides

The noise-decoupled nitrogen-15 NMR spectra of ten pyridine N-oxides and two quinoline N-oxides have been obtained at the natural-abundance level by high-resolution NMR spectroscopy. Substituents at the 4-ring position of pyridine N-oxide, capable of resonance interaction with the N-O moiety, give fairly large shifts in the expected directions. Spectra taken in dimethyl sulfoxide solution give 5–20 ppm and 33–55 ppm downfield shifts with respect to the solutions of the same substances in 2,2,2-trifluoroethanol and trifluoroacetic acid. Solvent influences are discussed in terms of hydrogen bonding and protonation of the N-oxide oxygen. Carbon-13 chemical shifts and one-bond carbon-hydrogen coupling constants of some substituted pyridine N-oxides are reported and discussed.     

Carbon-13 NMR studies of quinolines and isoquinolines. II. Chlorine–nitrogen interactions and N-oxide effects

Carbon-13 chemical shift assignments are reported for four chloroquinolines, six chloroisoquinolines, one dichloroquinoline, four dichloroisoquinolines, four methylchloroquinolines, two methylchloroisoquinolines, quinoline N-oxide, isoquinoline N-oxide, five methylquinoline N-oxides, two methylisoquinoline N-oxides and three chloroisoquinoline N-oxides. Chlorine substituent chemical shift (SCS) effects are reported for the alpha, ortho, meta, para and peri positions. Consistent patterns are observed for the para and peri positions, a vinylogous ortho pattern is reported and the additivity of these SCS effects is demonstrated. Alpha SCS effects vary widely from 1.1 ppm upfield in 1-chloroisoquinoline to 6.7 ppm downfield in 4-chloroquinoline. These results, together with those in the literature, permit the definition of steric and nitrogen lone-pair contributions which modify the ‘normal’ chlorine SCS effect, and these modifying contributions are shown to be roughly additive. Large (6–16 ppm) upfield shifts are observed for the carbons ortho and para to the N-oxide group. The individual magnitudes of these shifts and their sum are constant and the effects are additive in substituted systems. A 9.5 ppm upfield shift is also observed for C-8 in quinoline N-oxides which is attributed to a space–charge interaction. Substituent chemical shift (SCS) effects for the chloro and methyl groups and the chemical shifts of the methyl carbons are essentially the same in the N-oxides as in the parent heterocycles and are additive, except for those molecules where the substituent is adjacent to the N-oxide moiety, in which cases substantial interactions are observed.     

SNH reactions of 1,2,4-triazine N-oxides,pyrazine N-oxides,and pterin N-oxides with arenethiols*

1,2,4-Triazine 4-oxides were found to enter into the reactions of nucleophilic substitution of hydrogen with S-nucleophiles (arenethiols) in the presence of acylating agents and trifluoroacetic acid. The reactions proceeded with loss of the N-oxide function to form 5-arylthio-1,2,4-triazines. 2-Amino-3-ethoxycarbonylpyrazine 1-oxides and 2-amino-4-oxopterin 8-oxides react with arenethiol analogously.     

A novel hydrogen-bonding N-oxide–sulfonamide–nitro N—H…O synthon determining the architecture of benzenesulfonamide cocrystals

The structures of novel cocrystals of 4-nitropyridine N-oxide with benzenesulfonamide derivatives, namely, 4-nitrobenzenesulfonamide–4-nitropyridine N-oxide (1/1), C₅H₄N₂O₃·C₆H₆N₂O₄S, and 4-chlorobenzenesulfonamide–4-nitropyridine N-oxide (1/1), C₆H₆ClNO₂S·C₅H₄N₂O₃, are stabilized by N—H…O hydrogen bonds, with the sulfonamide group acting as a proton donor. The O atoms of the N-oxide and nitro groups are acceptors in these interactions. The latter is a double acceptor of bifurcated hydrogen bonds. Previous studies on similar crystal structures indicated competition between these functional groups in the formation of hydrogen bonds, with the priority being for the N-oxide group. In contrast, the present X-ray studies indicate the existence of a hydrogen-bonding synthon including N—H…O(N-oxide) and N—H…O(nitro) bridges. We present here a more detailed analysis of the N-oxide–sulfonamide–nitro N—H…O ternary complex with quantum theory computations and the Quantum Theory of Atoms in Molecules (QTAIM) approach. Both interactions are present in the crystals, but the O atom of the N-oxide group is found to be a more effective proton acceptor in hydrogen bonds, with an interaction energy about twice that of the nitro-group O atoms.     

Equilibrium of Acetyl Transfer between Pyridine N-Oxides and Their Acetylonium Salts

The equilibrium constants of acetyl transfer between O-acetylonium salts of a series of pyridine N-oxides and 4-(4-dimethylaminostyryl)pyridine N-oxide in acetonitrile and methylene chloride were determined. The equilibrium constants correlate with the acid-base characteristics of the N-oxides and with the heats of reactions calculated quantum-chemically.     

Syntheses based on anabasine. Preparation and transformations of N-oxides

The oxidation of N-acetyl-and N-benzoylanabasine with the tert-butyl hydroperoxide (TBHP)— MoCl₅ system or MCPBA proceeds selectively at the nitrogen atom of the pyridine ring. The oxidation of N-methylanabasine under similar conditions gives a mixture of stereo-isomeric N-oxides at the piperidine nitrogen atom, their ratio depending on the reagent used. The oxidation of anabasine by TBHP— MoCl₅ or MCPBA is accompanied by dehydrogenation and results in anabaseine N-oxide. The reactions of anabasine and anabaseine pyridine N-oxides with acetic anhydride were investigated. The substituted 1H-3-pyridin-2-ones were prepared. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 2, pp. 322—328, February, 2006.     

On the synthesis of thieno[3,2-c]quinoline N-oxide and thieno-[3,2-C]isoquinoline N-oxide. The nmr spectra of the six isomeric thieno-fused quinoline and isoquinoline N-oxides

The synthesis of the two remaining isomeric monothieno-analogues of phenanthridine N-oxide, thieno-[3,2-c]quinoline N-oxide and thieno[3,2-c]isoquinoline N-oxide, is described. The ¹H and ¹³C nmr spectra of all six isomeric thieno-fused quinoline and isoquinoline N-oxides are discussed.     

Cobalt(II) complexes of the 2-aminopicolineN-oxides and 2-amino-4, 6-lutidineN-oxide

Summary Cobalt(II) complexes of the four 2-aminopicolineN-oxides and 2-amino-4, 6-lutidineN-oxide were prepared from Co(BF₄)₂ and CoCl₂, and characterized by partial elemental analyses, magnetic moments, molar conductivities, thermal analyses, and by plasma desorption mass, i.r., electronic, and e.s.r. spectroscopy. The compounds derived from CoCl₂ are 4-coordinate, tetrahedral, molecular solids with CoO₂Cl₂ chromophores. Dq values range from 332 to 382 cm^–1 and those of B from 758 to 813 cm^–1 for the five solids. Three of the compounds prepared from Co(BF₄)₂ are octahedral with the following stoichiometry: [CoL₆](BF₄)₂ where L=2-amino-4-picolineN-oxide and [CoL₅(H₂O)] (BF₄)₂ where L is either 2-amino-3-or 2-amino-5-picolineN-oxide. Both 2-amino-6-picolineN-oxide and 2-amino-4, 6-lutidineN-oxide gave square planar [CoL ₄ ²⁺ ] complex ions. While numerous square planar cobalt(II) centers are known, those described here are probably the first examples with monodentate ligands and a CoO₄ center. They have weak e.s.r. spectra, magnetic moments between 2 and 3 BM and characteristic d-d spectra.