The anticancer/cytotoxic effect of a novel gallic acid derivative in non-small cell lung carcinoma A549 cells and peripheral blood mononuclear cells from healthy individuals and lung cancer patients.

Gallic acid (GA) is a naturally occurring polyphenol with a strong antioxidant capacity. GA stimulates the apoptosis of cancer cells, thereby suppressing cancer cell invasion. However, the low oral permeability of GA limits its therapeutic use. In order to enhance the antioxidant capacity and oral permeability of GA, a series of compounds analogous to GA were synthesized: 4-methoxybenzenesulfonamide (MBS), 3,4-dimethoxybenzenesulfonamide (DMBS) and 3,4,5-trimethoxybenzenesulfonamide (TMBS). In the new compounds, hydroxyl groups were replaced with various numbers of methoxy groups (stronger electron-donating groups), to increase hydrophobicity and oral permeability compared to GA. In addition, the carboxylic group was replaced with a sulfonyl group (a stronger electron-withdrawing group), to increase the molecular polarity and antioxidative activities of the compounds. The cell counting kit-8 (CCK-8) assay was used to detect the effect of GA, MBS, DMBS, and TMBS on cell proliferation and apoptosis in peripheral blood mononuclear cells (PBMCs) from healthy individuals and non-small cell lung carcinoma A549 cells. Additionally, the comet assay was used to assess the genotoxicity of these compounds in PBMCs from healthy individuals, lung cancer patients, and A549 cells. Compared to untreated cells, TMBS reduced DNA damage more effectively than GA in PBMCs from lung cancer patients and healthy donors. Furthermore, in comparison to GA, TMBS was more cytotoxic in A549 cells. Moreover, TMBS was not cytotoxic in healthy PBMCs, suggesting that TMBS demonstrates therapeutic potential in cancer.

Hammett correlation in competition experiments in dissociation of ionised substituted benzophenones and dibenzylideneacetones.

A convenient method of applying competition experiments to devise a Hammett correlation in the dissociation by α-cleavage of 17 ionised 3- and 4-substituted benzophenones, YC6H4COC6H5 [Y=F, Cl, Br, CH3, CH3O, NH2, CF3, OH, NO2, CN and N(CH3)2] is reported and discussed. The results given by this approach, which rely on the relative abundance of [M-C6H5]+ and [M-C6H4Y]+ ions in the electron ionisation spectra of the substituted benzophenones, are compared with those obtained by previous methods. Various refinements of the method are considered, including reducing the ionising electron energy, making allowance for the relative abundance of ions such as C6H5+ and C6H4Y+, which may be formed to some extent by secondary fragmentation, and using substituent constants other than the standard σ constants. The reaction constant, ρ, of 1.08, which is in good agreement with that deduced previously, is consistent with a considerable reduction in electron density (corresponding to an increase in positive charge) at the carbon of the carbonyl group during fragmentation. This method has been successfully extended to the corresponding cleavage of 12 ionised substituted dibenzylideneacetones, YC6H4CH=CHCOCH=CHC6H5 (Y=F, Cl, CH3, OCH3, CF3, and NO2), which may fragment to form either a substituted cinnamoyl cation, [YC6H4CH=CHCO]+, or the cinnamoyl cation, [C6H5CH=CHCO]+. The derived ρ value of 0.76 indicates that the substituent, Y, influences the stability of the cinnamoyl cation somewhat less strongly than it does the analogous benzoyl cation.

Proximity effects in the electron ionisation mass spectra of substituted cinnamamides.

The electron ionisation mass spectra of an extensive set of 53 ionised monosubstituted and disubstituted cinnamamides [XC6H4CH=CHCONH2, X = H, F, Cl, Br, I, CH3, CH3O, CF3, NO2, CH3CH2, (CH3)2CH and (CH3)3C; and XYC6H3CH=CHCONH2, X = Y = Cl; and X, Y = F, Cl or Br] are reported and discussed. Particular attention is paid to the significance of loss of the substituent, X, from the 2-position, via a rearrangement that is sometimes known as a proximity effect, which has been reported for a range of radical-cations, but is shown in this work to be especially important for ionised cinnamamides. When X is in the 2-position of the aromatic ring, [M - X]+ is formed to a far greater extent than [M - H]+; in contrast, when X is in the 3-position or 4-position, [M - H]+ is generally much more important than [M - X]+. Parallel trends are found in the spectra of XYC6H3CH=CHCONH2: the signal for [M - X]+ dominates that for [M - Y]+ when X is in the 2-position and Y in the 4-position or 5-position, irrespective of the nature of X and Y. Further insight is obtained by studying the competition between expulsion of X· and alternative fragmentations that may be described as simple cleavages. Loss of ·NH2 results in the formation of a substituted cinnamoyl cation, [XC6H4CH=CHCO]+ or [XYC6H3CH=CHCO]+; this process competes far less effectively with the proximity effect when X is in the 2-position than when it is in the 3-position or 4-position. Additional information has been obtained by investigating the competition between formation of [M - H]+ by the proximity effect and loss of CH3· by cleavage of a 4-alkyl group to give a benzylic cation, [R1R2CC6H4CH=CHCONH2]+ (R1, R2 = H, CH3).

Proximity Effects in Mass Spectra of Benzanilides.

The analytical value of peaks arising by a proximity effect in the electron ionization mass spectra of benzanilides has been established by examining the spectra of numerous examples of general structure XC6H4NHCOC6H4Y. Significant [M-X]+ signals are observed only when X = Cl, Br, I or CH3O in the 2-position. The presence of strong [M-X]+ signals, but negligibly weak [M-Y]+ peaks, even when the C-Y bond would be expected to break more readily than the C-X bond, indicates that these diagnostically useful signals do not arise by simple cleavage. Similarly, the presence of an appreciable [M-Cl]+ signal, but no [M-Br]+ signal, in the spectra of representative examples of 4-Br-2ClC6H3NHCOC6H4Y, reveals that loss of a substituent from the 2-position occurs much more rapidly than fission of a weaker bond to a substituent in the 4-position. These trends are interpreted in terms of cyclization of the ionized 2-substituted benzanilide, followed by elimination of the substituent originally in the 2-position, to form a protonated 2-arylbenzoxazole.

Hammett Correlation in the Accelerated Formation of 2,3-Diphenylquinoxalines in Nebulizer Microdroplets.

The accelerated formation of 2,3-diphenylquinoxalines in microdroplets generated in a nebulizer has been investigated by competition experiments in which equimolar quantities of 1,2-phenylenediamine, C6H4(NH2)2, and a 4-substituted homologue, XC6H3(NH2)2 [X = F, Cl, Br, CH3, CH3O, CO2CH3, CF3, CN or NO2], or a 4,5-disubstituted homologue, X2C6H2(NH2)2 [X = F, Cl, Br, or CH3], compete to condense with benzil, (C6H5CO)2. Electron-donating substituents (X = CH3 and CH3O) accelerate the reaction; in contrast, electron-attracting substituents (X = F, Cl, Br and particularly CO2CH3, CN, CF3 and NO2) retard it. A structure-reactivity relationship in the form of a Hammett correlation has been found by analyzing the ratio of 2,3-diphenylquinoxaline and the corresponding substituted-2,3-diphenylquinoxaline, giving a ρ value of -0.96, thus confirming that the electron density in the aromatic ring of the phenylenediamine component is reduced in the rate-limiting step in this accelerated condensation. This correlation shows that the phenylenediamine acts as a nucleophile in the reaction.

Experimental and computational evidence for C=O π-bonding in [CH2OH]+ and related oxonium ions.

The question of whether [CH2OH]+ should be described as the hydroxymethyl cation, +CH2OH, or protonated formaldehyde, CH2=OH+, is reconsidered in the light of experimental information and new computational evidence. Previous arguments that the charge distribution in [CH2OH]+ may be probed by considering the incremental stabilisation of [CH2OH]+ induced by homologation on carbon (to give [CH3CHOH]+) or oxygen (to produce [CH2OCH3]+) are critically examined. Cation stabilisation energies are shown to be better indicators of the nature of these oxonium ions. Further insight into the structure of larger CnH2n+1O+ oxonium ions is obtained by considering the site of protonation of enol ethers and related species. Computational information, including AIM (Atoms and Molecules) and NBA (Natural Bond Analysis) charges on the carbon and oxygen atoms in [CH2OH]+ and related species, is considered critically. Particular attention is focused on the calculated bond lengths and barriers to rotation about the C-O bond(s) in [CH2OH]+, [CH3CHOH]+, [(CH3)2COH]+, CH3OH and [CH2OCH3]+ and the C-N bond in [CH2NH2]+. Trends in these data are consistent with appreciable π-bonding only in the C-O connections which correspond to the C=O bond in the parent aldehyde or ketone from which the oxonium ion may be considered to be derived by protonation or alkyl cationation.

Structure reactivity relationship in the accelerated formation of 2,3-diarylquinoxalines in the microdroplets of a nebuliser.

Competition experiments in which 1,2-phenylenediamine, C6H4(NH2)2, condenses with equimolar quantities of benzil, (C6H5CO)2, and a 3,3'- or 4,4'-disubstituted benzil (XC6H4CO)2 (X = F, Cl, Br, CH3 or CH3O) to form a mixture of 2,3-diphenylquinoxaline and the corresponding 2,3-diarylquinoxaline (Ar = XC6H4) in the microdroplets produced in a nebuliser allow a Hammett relationship with a ρ value of 1.85 to be developed for this accelerated condensation in the nebuliser. This structure reactivity relationship reveals that an appreciable amount of negative charge builds up on the carbon of the carbonyl group of the benzil during the rate-limiting step of the reaction, thus confirming that this process involves nucleophilic addition of the 1,2-phenylenediamine to the benzil. In general, the presence of an electron donating substituent, particularly in the 4 and 4' positions, in the benzil retards the reaction, whereas an electron attracting substituent, especially in the 3 and 3' position, accelerates it.

Accelerated generation of (protonated) imines and quinoxalines by formation of C=N bonds in the microdroplets of a nebuliser.

Ions corresponding to protonated imines appear in the positive ion electrospray mass spectra of mixtures of the parent aromatic aldehyde and arylamine. The formation of these imine products occurs readily in the electrospray source nebuliser, even without the application of a spray potential. This accelerated formation of C=N bonds in the nebuliser has been extended to encompass the preparation of quinoxalines from a range of substituted phenylenediamines and benzils. The condensation may be induced either under conventional positive ion electrospray conditions (to give the protonated quinoxalines) or when the nebuliser is disconnected from the mass spectrometer (to give the neutral quinoxaline). Ions corresponding to intermediate adducts formed by condensation of the phenylenediamine component with the protonated benzil are observed in many cases when the condensation occurs in the mass spectrometer. This finding supports an interpretation based on nucleophilic addition in droplets generated by the nebuliser.

Peter J Derrick and the Grand Scale 'Magnificent Mass Machine' mass spectrometer at Warwick.

The value of the Grand Scale 'Magnificent Mass Machine' mass spectrometer in investigating the reactivity of ions in the gas phase is illustrated by a brief analysis of previously unpublished work on metastable ionised n-pentyl methyl ether, which loses predominantly methanol and an ethyl radical, with very minor contributions for elimination of ethane and water. Expulsion of an ethyl radical is interpreted in terms of isomerisation to ionised 3-pentyl methyl ether, via distonic ions and, possibly, an ion-neutral complex comprising ionised ethylcyclopropane and methanol. This explanation is consistent with the closely similar behaviour of the labelled analogues, C3H7CH2CD2OCH3+. and C3H7CD2CH2OCH3+., and is supported by the greater kinetic energy release associated with loss of ethane from ionised n-propyl methyl ether compared to that starting from directly generated ionised 3-pentyl methyl ether.

The mechanism of alkene elimination from protonated toluenesulphonamides generated by electrospray ionisation.

The positive ion electrospray mass spectra of a range of sulphonamides of general structure CH3C6H4SO2NHR1 [R1 = CnH2n+1 (n = 1-7), CnH2n-1 (n = 3, 4), C6H5, C6H5CH2 and C6H5CH(CH3)] and CH3C6H4SO2NR1R2 [R1, R2 = CnH2n+1 (n = 1-8)] are reported and discussed. The protonated sulphonamides derived from saturated primary and secondary aliphatic amines generally fragment to only a limited extent unless energised by collision. Two general fragmentations are observed: firstly, elimination of an alkene, CnH2n, obtained by hydrogen abstraction from one of the CnH2n+1 alkyl groups on nitrogen; secondly, cleavage to form CH3C6H4SO2+. The mechanism by which an alkene is lost has been probed by studying the variation of the intensity of the [M + H - CnH2n]+ signal with the structure of the alkyl substituent(s) on nitrogen and by monitoring the competition between the loss of different alkenes from protonated unsymmetrical sulphonamides in which two different alkyl groups are attached to nitrogen. This fragmentation is favoured by branching of the alkyl group at the carbon atom directly attached to nitrogen, thus suggesting that it involves a mechanism in which the stability of the cation obtained by stretching the bond connecting the nitrogen atom to the alkyl group is critical. This interpretation also explains the competition between alkene elimination and cleavage to form CH3C6H4SO2+ (and, in some cases, cleavage to form C6H5CH2+ or [C6H5CHCH3]+).

Application of positive mode atmospheric chemical ionisation to distinguish epimeric oleanolic and ursolic acids.

A new and more reliable method is reported for distinguishing the equatorial and axial epimers of oleanolic and ursolic acids and related triterpenoids based primarily on the relative abundance of the [M+H](+) and [M+-H(2)O](+) signals in their positive mode atmospheric pressure chemical ionisation mass spectra. The rate of elimination of water, which is the principal primary fragmentation of protonated oleanolic and ursolic acids, depends systematically on the stereochemistry of the hydroxyl group in the 3 position. For the b-epimer, in which the 3-hydroxyl substituent is in an equatorial position,[M+-H(2)O](+) is the base peak. In contrast, for the α-epimer, where the 3-hydroxyl group is axial, [M + H](+) is the base peak. This trend, which is general for a range of derivatives of oleanolic and ursolic acids, including the corresponding methyl esters, allows epimeric triterpenoids in these series to be securely differentiated. Confirmatory information is available from the collision-induced dissociation of the [M+-H(2)O](+) primary fragment ions, which follow different pathways for the species derived from axial and equatorial epimers of oleanolic and ursolic acids. These two pieces of independent spectral information permit the stereochemistry of epimeric oleanolic and ursolic acids (and selected derivatives) to be assigned with confidence without relying either on chromatographic retention times or referring to the spectra or other properties of authentic samples of these triterpenoids.

Novel formation of [2M-H](+) species in positive electrospray mass spectra of indoles.

RATIONALE: When subjected to positive ion electrospray ionisation (ESI+) mass spectrometry (MS), indoles with a 3-alkyl substituent show a propensity to form novel [2M-H](+) 'covalently bound dimers'. This process, which appears to be initiated in the nebuliser of the instrument, is mechanistically interesting, analytically useful and potentially significant in organic synthesis. METHODS: A selection of 2- and 3-substituted indoles have been synthesised and analysed by ESI-MS. The formation of the 'homo' and 'hetero' dimers of these compounds has been investigated using ESI+ mode. The mechanism of formation of the observed 'dimeric' species has been probed by synthesising authentic samples of the dimeric compounds. RESULTS: 'Dimeric' species corresponding to [2M-H](+) have been observed for all 3-substituted indoles studied, but not for indoles substituted in just the 2-position. By infusing equimolar mixtures of labelled and unlabelled indoles through the instrument, the expected approximately statistical mixture of homo- and heterodimeric species has been observed. Further experiments have established that this novel dimerisation occurs in the droplets formed in the nebuliser of the instrument. CONCLUSIONS: It has been shown that 3-substituted indoles form [2M-H](+) dimers in high abundance in the spray obtained from the nebiliser of an ESI+ instrument. The mechanism for the dimerisation does not involve the known 2M dimeric species that is readily formed in the solution-phase chemistry of indoles.

Influence of a methyl substituent on the Raman spectrum of but-3-enyl methyl ether.

The Raman spectrum of but-3-enyl methyl ether, CH(2)CHCH(2)CH(2)OCH(3) is reported and compared with those of its homologues in which a methyl group is substituted for a hydrogen atom on one of the carbon atoms of the alkenyl chain. Attention is focused on the influence of this methyl group on the bands in the spectrum associated with specific CH, skeletal stretching and bending vibrations. The use of ab initio DFT quantum mechanical calculations to assist in making these assignments reveals a high degree of mode-mixing in the skeletal vibrations. The value of model studies of this kind in refining the correlations between the presence and absence of specific bands in a Raman spectrum with molecular structure is emphasised.

Evidence of structure-selective fragmentations in the tandem mass spectra of protonated hydroxyalkylamino-1,4-naphthoquinones formed by electrospray ionisation.

The collision-induced dissociation of protonated hydroxyalkylamino-1,4-naphthoquinones depends strongly on the structure of the substituent [NHCH(2)(CH(2))(n)OH, n = 1-5; or NHCH(2)CH(CH(3))OH] on the quinone ring. Protonated naphthoquinones with an unbranched hydroxypropylamino side chain (n = 3) undergo facile and characteristic CH(2)O loss, whereas isomeric [M + H](+) ions with a branched hydroxypropylamino side chain do not. When n = 1, CH(2)O elimination occurs less readily, accompanied by CH(3)N loss, thus allowing this shorter side chain to be identified. Higher homologous species (n = 3-5) do not expel CH(2)O, but instead eliminate C(n + 1)H(2n)O, C(n + 1)H(2n + 2)O and (for n = 5) C(2n + 1)H(2n + 1)O.

Elimination of H2 from CH3CH=N+HCH3: a synchronous, concerted 1,4-H2 elimination.

Most H2 eliminations from cations in the gas phase are formally 1,1- or 1,2- processes. Larger ring size H2 eliminations are rare and little studied. Thus, whether the 6-center, 1,4- elimination CH3CH=N+HCH3-->CH2=CHN+H=CH2+H2 is concerted and synchronous, as indicated by isotope effects and predicted by conservation of orbital symmetry, is a significant question. This reaction is characterized here by application of QCI and B3LYP theories. CH bond-breaking and H-H bond-making in this reaction are found by theory to be highly synchronized, consistent with previously established isotope effects and in contrast to "forbidden" 1,2-eliminations from organic cations in the gas phase. This reaction is made feasible by its conservation of orbital symmetry, the energy supplied by formation of the H-H bond, and a favorable geometry of the ion for eliminating H2.

Differentiation of isomeric allylic alkenyl methyl ethers by Raman spectroscopy.

The Raman spectra of several pairs of alkenyl methyl ethers of general structure R(1)R(2)C=CR(5)C(R(3)R(4))OCH3 and R(1)R(2)C(OCH3)C(R(5))=CR(3)R(4) (R(1), R(2), R(3), R(4), R(5) = H or C(n)H(2n+1), n = 1-3) are reported and discussed, with a view to establishing whether Raman spectroscopy offers a viable means of distinguishing between these isomeric unsaturated species. Key bands associated with the nu(sp2C-H) and nu(C=C) stretching modes are found to be particularly useful in this connection: R(1)R(2)C=CHCH2OCH3 and R(1)R(2)C(OCH3)CH=CH2 ethers (R(1), R(2) = CH3, C2H5) are easily distinguished on this basis. Differentiation of their lower homologues, R(1)CH=CHCH2OCH3 and R(1)CH(OCH3)CH=CH2 (R(1) = CH3, C2H5, C3H7), by similar means is also quite straightforward, even in cases where cis and trans isomers are possible. Pairs of isomeric ethers, such as CH3CH=C(CH3)CH2OCH3 and CH3CH(OCH3)C(CH3)=CH2, in which the structural differences are more subtle, may also be distinguished with care. Deductions based on bands ascribed to the stretching vibrations are usually confirmed by consideration of the signals associated with the corresponding delta(sp2C-H) deformation vibrations. Even C2H5CH=CHCH(C3H7)OCH3 and C3H7CH=CHCH(C2H5)OCH3 are found to have distinctive Raman spectra, but differentiation of these closely related isomers requires additional consideration of the low wavenumber region.

Raman spectroscopic and structural investigation of 1,4-diphenylbuta-1,3-diene and selected monomethyl and dimethyl substituted homologues.

The Raman and mass spectra of 1,4-diphenylbuta-1,3-diene and several of its monomethyl and dimethyl homologues are reported and discussed, with a view to developing a spectroscopic protocol for detecting the presence and position of a methyl group in these compounds. Raman spectroscopy and mass spectrometry are shown to provide complementary information, by which the four available monomethyl homologues may be readily distinguished from each other and 1,4-diphenylbuta-1,3-diene itself. The utility of these 1,4-diarylbutadienes as model compounds for carotenoids and related materials, which may serve as indicators of extinct or extant extraterrestrial life, is considered.

Differentiation of ionised benzimidazole from its isomeric alpha-distonic ion by collision-induced dissociation and neutralisation-reionisation mass spectrometry.

Ionised benzimidazole and its isomeric alpha-distonic ion (or ionised ylid) have been examined by recording their metastable ion, collision-induced dissociation and neutralisation-reionisation mass spectra. These tautomers may be distinguished by careful consideration of key features of the collision-induced dissociation spectra, with or without prior neutralisation and reionisation. Formation of doubly-charged ions by charge stripping occurs preferentially when the alpha-distonic ion is subjected to collision. This alpha-distonic ion survives neutralisation and reionisation, thus establishing that the corresponding ylid is stable on the microsecond time frame. The effects of benzannulation on the ease of differentiation of classical and distonic radical cations derived from biologically important heterocycles are considered.

Raman spectroscopic and structural studies of indigo and its four 6,6'-dihalogeno analogues.

The Raman and electron impact mass spectra of synthetic indigo and its four 6,6'-dihalogeno analogues are reported and discussed. The influence of varying the halogen on these Raman spectra is considered. Particular emphasis is laid on distinguishing indigo from 6,6'-dibromoindigo and differentiating between the dihalogenocompounds, so as to develop protocols for determining whether artefacts are coloured with dyes of marine or terrestrial origin and whether such artefacts are dyed with genuine "Tyrian Purple" or with dihalogenoindigo substitutes that do not contain bromine. The value of even low resolution electron impact mass spectrometry in a forensic context as a means of identifying authentic 6,6'-dibromoindigo and distinguishing it from its dihalogenoanalogues is emphasised

Reactions of the ionized enol tautomer of acetanilide: elimination of HNCO via a novel rearrangement.

The reactions of ionised acetanilide, C(6)H(5)NH(=O)CH(3)(.+), and its enol, C(6)H(5)NH(OH)=CH(2)(.+), have been studied by a combination of tandem mass spectrometric and computational methods. These two isomeric radical cations have distinct chemistries at low internal energies. The keto tautomer eliminates exclusively CH(2)=C=O to give ionised aniline. In contrast, the enol tautomer loses H-N=C=O, via an unusual skeletal rearrangement, to form predominantly ionised methylene cyclohexadiene. Hydrogen atom loss also occurs from the enol tautomer, with the formation of protonated oxindole. The mechanisms for H-N=C=O and hydrogen atom loss both involve cyclisation; the former proceeds via a spiro transition state formed by attachment of the methylene group to the ipso position, whereas the latter entails the formation of a five-membered ring by attachment to the ortho position. The behaviour of labelled analogues reveals that these two processes have different site selectivities. Hydrogen atom loss involves a reverse critical energy and is subject to an isotope effect. Surprisingly, attempts to promote the enolisation of ionised acetanilide by proton-transport catalysis were unsuccessful. In a reversal of the usual situation for ionised carbonyl compounds, ionised acetanilide is actually more stable than its enol tautomer. The enol tautomer was resistant to proton-transport catalysed ketonisation to ionised acetanilide, possibly because the favoured geometry of the encounter complex with the base molecule is inappropriate for facilitating tautomerisation.