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Negative Ion Electrospray Ionization Mass Spectrometry and Computational Studies on Substituted 7-Hydroxycoumarins
Abstract
Twenty-two substituted 7-hydroxycoumarins were studied by negative ion electrospray ionization collision- induced dissociation (CID) mass spectrometry. Fragmentation pathways were also investigated by computation method using the B3LYP density functional theory. In general, the most important fragmentations of the 7- hydroxycoumarin [M - H](-) ions were the elimination of CO(2) and CO which agreed with the calculated energies of the proposed fragmentation reactions. In most cases, methyl group elimination was also favorable. Methyl group elimination occurred in three different ways, the most interesting being hydrogen rearrangement from a neighboring alkyl group to a ring carbon, which led to a benzyl radical formation. In some cases, CH(2)CO elimination was observed as well. Isomeric compounds gave rise to different CID spectra.
... [14] More recently, electrospray ionization (ESI) has been employed in the MS and MS n studies of coumarins. [15][16][17][18][19][20] Concannon and co-workers analyzed selected coumarin derivatives, in both positive-and negative-ion modes, by using an ion trap instrument. The observed dissociation characteristics in MS n experiments were surprisingly similar to those observed with EI-MS, although the techniques provide different types of precursor ions; that is, odd-electron (radical) ions ([M] +• ) in EI-MS, while even-electron ions (e.g., [M + H] + or [M -H] -) in ESI-MS n . ...
... Many of these compounds have shown promising anti-inflammatory activity in recent lipopolysaccharide-induced inflammation assays (Timonen et al., unpublished results). Following our earlier mass spectrometric study on 7-hydroxycoumarins, [18] we conducted a similar study with 7-acetonyloxycoumarins with the main emphasis on their dissociation characteristics in low-energy CID-MS n . The experimentally observed dissociation pathways were further investigated with density functional theory (DFT) calculations to confirm putative fragment ion structures and their energetics. ...
... The compounds bearing a 8-methoxy substituent had five instead of four oxygen atoms which increased possible dissociation channels. In comparison with 7-hydroxycoumarins studied earlier by negative-ion ESI, [18] the cleavages of CO and CO 2 were not as important as with 7-acetonyloxycoumarins. This is partly due to the differences between positive and negative ionization applied. ...
Coumarins are naturally occurring, oxygen-containing heterocycles with considerable pharmaceutical potential. For structural elucidation of natural or synthetic coumarins, tandem mass spectrometry (MS(n) ) represents an essential tool. In this study, fragmentation characteristics of twenty-two 7-acetonyloxycoumarins, having promising anti-inflammatory properties, were investigated with low-energy collision-induced dissociation (CID).
Accurate mass measurements were performed on a 12-T Fourier transform ion cyclotron resonance (FT-ICR) instrument. Most CID-MS(n) measurements were performed on a quadrupole ion trap (QIT) instrument, except some additional CID-MS(2) measurements performed on the FT-ICR instrument for further confirmation of some fragment ions. Positive-ion electrospray ionization (ESI) was employed throughout. Density functional theory (DFT) calculations (B3LYP) were carried out to analyze putative ion structures/fragmentation channels.
The most favourable dissociation channel for [M + H](+) ions of 7-acetonyloxycoumarins was the elimination of a C3 H5 O(●) radical (57 Da) from the 7-acetonyloxy group via homolytic bond cleavage. The resulting phenolic radical ion was the primary fragment ion for the most compounds studied. Losses of even-electron neutrals, C3 H4 O and C3 H6 O (56 and 58 Da), were also observed. These primary eliminations were accompanied with other characteristic neutral losses from the coumarin skeleton, including H2 O, CO, CO2 , and C2 H2 O (ketene). In addition, propene (C3 H6 ) loss was also observed for 4-propyl or 3-ethyl-4-methyl-substituted compounds.
The studied coumarins showed interesting characteristics in low-energy CID due to the presence of a 7-acetonyloxy group, leading to both even- and odd-electron product ions. The main dissociation channels observed for each compound were highly dependent on the substituents in the benzopyranone ring. The present results will advance our knowledge on the dissociation characteristics of both synthetic and natural coumarins. Copyright © 2013 John Wiley & Sons, Ltd.
... However, a mechanistic explanation of the fragmentation pathways for the formation of these product ions has not yet been provided. Deprotonated molecules of many important species were generated in the negative ion mass spectrometry, and they underwent charge induced and charge-remote fragmentations in the tandem mass spectrometry, which were much different from the fragmentation behaviors of the corresponding protonated species1415161718192021. Many negative ions were favorable to undergo the intramolecular nucleophilic displacement, which led to unusual fragment ions22232425262728. ...
... Many negative ions were favorable to undergo the intramolecular nucleophilic displacement, which led to unusual fragment ions22232425262728. The characteristic fragmentations provided useful information for structural elucidation2829303132 and differentiation of isomers [15,21]. Obviously, the title compounds can be easily ionized in the negative mode, due to the presence of the amide hydrogen function [18]. ...
The gas-phase fragmentation pathways of deprotonated diacylhydrazine derivatives (R1(C = O)-N(t-Bu)NH(C = O)R2, Compounds 1-6) were investigated by the combination of electrospray ionization tandem mass spectrometry (ESI-MS/MS) and theoretical calculations. Upon collisional activation, the deprotonated molecular ions [M - H](-) dissociate in two reaction channels, both of which involve intramolecular rearrangement. The main product ion is confirmed to be an anionic acid species, [R1-CO2](-), generated through intramolecular rearrangement of [M - H](-) initiated by the nucleophilic attack of the amide O6 on the carbonyl C2 (Path-1). The minor fragment channel (Path-2) involves methylpropene elimination of the precursor ion, followed by a similar nucleophilic displacement reaction to produce another acid anion [R2-CO2](-). Density functional theory calculations at the B3LYP/6-31+G(d,p) level indicate that Path-1 is more favorable than Path-2 for dissociation of the deprotonated halofenozide.
In this study; Numerous coumarin compounds were synthesized by Pechmann and Knoevenagel methods, and the substitution of the formyl group was provided by the Duff reaction. The IR spectra and melting points of the synthesized compounds were compared with the literature values. Also confirmed by GC/MS analysis. And the synthesized coumarin derivatives were compared in terms of antioxidant activity according to DPPH and Cuprac methods. The main aim of the study is to determine the effects of substituents on antioxidant activity.
This review summarizes the basic rules for the interpretation of atmospheric pressure ionization (API) mass spectra of small molecules written with the style primarily intended for beginners and low-experienced researchers with the mass spectra interpretation. The first and basic step in any interpretation of mass spectra is always the determination of molecular weight, which is relatively easy in case of soft ionization techniques due to the limited extend of fragmentation and the prevailing presence of (de)protonated molecules in the full scan mass spectra. These [M+H](+) and [M-H](-) ions are often accompanied by low abundant molecular adducts, which can be used as the supplementary information for the unambiguous determination of molecular weights. In certain cases, adduct ions may dominate the spectra. The subsequent interpretation of full scan and tandem mass spectra is more complicated due to a high number of possible functional groups, structural subunits and their combinations resulting in numerous competitive fragmentation pathways. Typical neutral losses and the effect of individual functional groups on the fragmentation are discussed in detail and illustrated with selected examples. Modern mass analyzers have powerful features for the structural elucidation, for example high resolving power, high mass accuracy, multistage tandem mass spectrometry, dedicated softwares for the interpretation of mass spectra and prediction of their fragmentation. Background information on differences among individual ionization techniques suitable for the HPLC-MS coupling and basic types of mass analyzers with consequences for the data interpretation is briefly discussed as well. Selected examples illustrate that the right optimization of chromatographic separation and the use of other than mass spectrometric detectors can bring valuable complementary information.
An improved synthesis of some 7-hydroxycoumarin derivatives is reported. Hydroxybenzenes reacted with β-ketoesters or d,l-malic acid in HClO4 to give good yields of 7-hydroxycoumarins. IR and 1H NMR data for the products are also reported.
To explore the structural requirements of (+)-cis-khellactone derivatives as novel anti-HIV agents, 24 monosubstituted 3‘,4‘-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK) derivatives were synthesized asymmetrically. These compounds included 4 isomeric monomethoxy analogues (3−6), 4 isomeric monomethyl analogues (7−10), 4 4-alkyl/aryl-substituted analogues (11−14), and 12 4-methyl-(+)-cis-khellactone derivatives (15−26) with varying 3‘,4‘-substituents. These (+)-cis-khellactone derivatives were screened against HIV-1 replication in acutely infected H9 lymphocytes. The results demonstrated that the (3‘R,4‘R)-(+)-cis-khellactone skeleton, two (S)-(−)-camphanoyl groups at the 3‘- and 4‘-positions, and a methyl group on the coumarin ring, except at the 6-position, were optimal structural moieties for anti-HIV activity. 3-Methyl- (7), 4-methyl- (8), and 5-methyl- (9) 3‘,4‘-di-O-(S)-camphanoyl-(3‘R,4‘R)-(+)-cis-khellactone showed EC50 and therapeutic index values of <5.25 × 10-5 μM and >2.15 × 106, respectively, in H9 lymphocytes, which are much better than those of DCK and AZT in the same assay. Furthermore, 8 and 9 also showed potent inhibitory activity against HIV-1 replication in the CEM-SS cell line, and most monosubstituted DCK analogues were less toxic than DCK in both assays.
An investigation of the negative-ion chemistry of 13 coumarin and three psoralen compounds is reported. Negative-ion chemical ionization was effected using O−˙ and OH− as the reagent ions; it was found that O−˙ leads preferably to [M – H + O]− whereas OH− mainly furnishes [M – H]−. The results of metastable anion fragmentation and collision-induced dissociation of mass-selected anions are presented.
The aim of this paper is to provide analytical chemical information on a range of naturally occurring and synthetic coumarins. This analytical chemical information on liquid chromatography–electrospray ionisation-mass spectrometry (HPLC–ESI-MS), ion trap mass spectrometry (ESI-MSn), gas chromatography-flame ionisation detection (GLC-FID) and polarographic behaviour is then incorporated into a database which is of use in identifying unknown coumarins isolated from natural sources. This paper is also concerned with understanding the effect of functional groups in coumarins on their analytical chemical behaviour using the above techniques.
Despite the remarkable thermochemical accuracy of Kohn–Sham density-functional theories with gradient corrections for exchange-correlation [see, for example, A. D. Becke, J. Chem. Phys. 96, 2155 (1992)], we believe that further improvements are unlikely unless exact-exchange information is considered. Arguments to support this view are presented, and a semiempirical exchange-correlation functional containing local-spin-density, gradient, and exact-exchange terms is tested on 56 atomization energies, 42 ionization potentials, 8 proton affinities, and 10 total atomic energies of first- and second-row systems. This functional performs significantly better than previous functionals with gradient corrections only, and fits experimental atomization energies with an impressively small average absolute deviation of 2.4 kcal/mol.
Phenylpropanoids are perhaps the most widespread types of natural products occurring in nature. In this account of the chemistry and utilization of phenylpropanoids we emphasize a few of the useful physiological, pharmacological, and chemical properties of flavonoids, lignans, and coumarins which are three of the major classes of compounds containing the C6-C3 phenylpropanoid moiety.
Four DCK-thiolactone analogs (3-6) were synthesized asymmetrically and evaluated for anti-HIV activity against HIV-1 replication in H9 lymphocyte cells. Based on the functionality on the thiolactonecoumarin nucleus, activity was in the order: methyl > H > propyl > benzyl. 4-Methyl-3',4'-di-O-(-)-camphanoyl-(+)-cis-khelthiolactone (4) exhibited extremely potent anti-HIV activity with EC50 and therapeutic index values of 0.00718 microM and > 21,300, respectively.
A correlation-energy formula due to Colle and Salvetti [Theor. Chim. Acta 37, 329 (1975)], in which the correlation energy density is expressed in terms of the electron density and a Laplacian of the second-order Hartree-Fock density matrix, is restated as a formula involving the density and local kinetic-energy density. On insertion of gradient expansions for the local kinetic-energy density, density-functional formulas for the correlation energy and correlation potential are then obtained. Through numerical calculations on a number of atoms, positive ions, and molecules, of both open- and closed-shell type, it is demonstrated that these formulas, like the original Colle-Salvetti formulas, give correlation energies within a few percent.
The metabolism, toxicity and results of tests for carcinogenicity have been reviewed with respect to the safety for humans of coumarin present in foodstuffs and from fragrance use in cosmetic products. Coumarin is a natural product which exhibits marked species differences in both metabolism and toxicity. The majority of tests for mutagenic and genotoxic potential suggest that coumarin is not a genotoxic agent. The target organs for toxicity and carcinogenicity in the rat and mouse are primarily the liver and lung. Moreover, the dose-response relationships for coumarin-induced toxicity and carcinogenicity are non-linear, with tumour formation only being observed at high doses which are associated with hepatic and pulmonary toxicity. Other species, including the Syrian hamster, are seemingly resistant to coumarin-induced toxicity. There are marked differences in coumarin metabolism between susceptible rodent species and other species including humans. It appears that the 7-hydroxylation pathway of coumarin metabolism, the major pathway in most human subjects but only a minor pathway in the rat and mouse, is a detoxification pathway. In contrast, the major route of coumarin metabolism in the rat and mouse is by a 3,4-epoxidation pathway resulting in the formation of toxic metabolites. The maximum daily human exposure to coumarin from dietary sources for a 60-kg consumer has been estimated to be 0.02 mg/kg/day. From fragrance use in cosmetic products, coumarin exposure has been estimated to be 0.04 mg/kg/day. The total daily human exposure from dietary sources together with fragrance use in cosmetic products is thus 0.06 mg/kg/day. No adverse effects of coumarin have been reported in susceptible species in response to doses which are more than 100 times the maximum human daily intake. The mechanism of coumarin-induced tumour formation in rodents is associated with metabolism-mediated, toxicity and it is concluded that exposure to coumarin from food and/or cosmetic products poses no health risk to humans.
The electrospray ionisation (ESI) of selected coumarin derivatives and their subsequent fragmentation using an ion trap mass spectrometer have been investigated. Sequential product ion fragmentation experiments (MS(n)) were performed in order to elucidate the degradation pathways for these compounds. A comparison was also made between these ESI spectra and those obtained under electron impact (EI) conditions. The data presented in this paper provides useful information on the effect of different substituents on the ionisation/fragmentation processes and can be used in the characterisation of these compounds.
Aryl-substituted 4-hydroxycoumarins (1-57) were investigated by electrospray ionisation (ESI) mass spectrometry. Their fragmentation in the ion source or in the collision cell of a triple quadrupole mass spectrometer was investigated. The effect of the substituents in the aromatic ring on the fragmentation of the 4-hydroxycoumarin derivatives is shown. The influence of the tautomerism on the formation of quasimolecular ions and mass spectral fragmentation was explained. Mass spectral studies on some deuterated compounds proved some of the proposed fragmentation pathways. Results obtained are very useful in the process of detection and characterisation of 4-hydroxycoumarins, as well as for structural elucidation of their more complex derivatives.
Coumarin and its derivative 7-hydroxycoumarin (7-OHC) have antitumor and antimetastatic properties. The purpose of this study was to investigate the possible effects of these compounds on expression of the bcl-2 and Bax oncoproteins in two human lung cancer cell lines, A427 and Calu-1. The cells were cultured in vitro for 24 h in RPMI 1640 with 1.5% (v/v) ethanol, 1.0 mM ethanolic coumarin or 1.0 mM ethanolic 7-OHC. Viability was determined in each cell line by an MTT assay. Total protein was extracted from cell lysates and the bcl-2 and Bax oncoproteins were identified. Western blotting showed a decrease in bcl-2 and an increase in Bax in A427 cells cultured with coumarin or 7-OHC. Neither drug changed bcl-2 expression in Calu-1 cells compared to solvent controls, and Bax expression was only slightly increased by coumarin. We conclude that 7-OHC is a more potent inhibitor of cell proliferation than coumarin and has more marked effects on oncoprotein expression. Also, the A427 cell line was more sensitive to the drugs than Calu-1.
Twenty 4-hydroxycoumarin derivatives were synthesized. Five of them are described for the first time. The X-ray crystal structure analysis of 3,3'-(2,3,4-trimethoxyphenylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one) (7) and 3,3'-(3,5-dimethoxy-4-hydroxyphenylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one) (9) confirmed the structure of these compounds. A comparative pharmacological study of the anticoagulant effect with respect to Warfarin showed that the synthesized compounds have different anticoagulant activities. The most prospective compound is 3,3'-(4-chlorophenylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one) (12) with low toxicity, very good index of absorption and dose dependent anticoagulant activity.