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We have investigated the gas-phase fragmentation reactions of 11 synthetic 4-aryl-3,4-dihydrocoumarins by electrospray ionization tandem mass spectrometry (ESI-MS/MS) on a quadrupole-time-of flight (Q-TOF) hybrid mass spectrometer. We have also estimated thermochemical data for the protonated coumarins (precursor ion A) and product ion structures by computational chemistry at a B3LYP level of theory to establish the ion structures and to rationalize the fragmentation pathways. The most abundant ions in the product ion spectra of coumarins 1-11 resulted from C8H8O2, CO2, C4H4O3, C8H10O3, C8H8O2, and CH3OH eliminations through retro-Diels-Alder (RDA) reactions, remote hydrogen rearrangements (ß-eliminations), and ß-lactone ring contraction. Although the investigated coumarins shared most of the fragmentation pathways, formation of a benzylic product ion and its corresponding tropylium ion was diagnostic of the substituents at ring C. The thermochemical data revealed that the nature and position of the substituents at ring C played a key role in the formation of this product ion and determined its relative intensity in the product ion spectrum. The results of this study contribute to knowledge of the gas-phase ion chemistry of this important class of organic compounds.

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RATIONALE: 4,7-Dichloroquinoline (DCQ) represents a group of synthetic molecules inspired by natural products with important roles in biological and biomedical areas. This work aimed to characterize DCQ and its derivatives by high-resolution electrospray ionization (ESI) mass spectrometry and tandem mass spectrometry (ESI-MS/MS), supported by theoretical calculations. Biological assays were carried out with DCQ and its derivatives to determine LC50 values against Aedes aegypti larvae. METHODS: Five DCQ derivatives were synthesized by using previously described protocols. ESI-MS/MS analyses were carried out with a quadrupole/time-of-flight and ion-trap instrument. The proposed gas-phase protonation sites and fragmentation were supported by density functional theory calculations. The larvicidal tests were performed with the Ae. aegypti Rockefeller strain, and the LC50 values were determined by employing five test concentrations. Larval mortality was determined after treatment for 48 h. RESULTS: DCQ bromides or aldehydes (C-3 or C-8 positions), as well as the trimethylsilyl derivative (C-3 position), were prepared. Detailed ESI-MS/MS data revealed heteroatom elimination through an exception to the even-electron rule, to originate open-shell species. Computational studies were used to define the protonation sites and fragmentation pathways. High activity of DCQ and its derivatives against Ae. aegypti larvae was demonstrated. CONCLUSION: Our results provided a well-founded characterization of the fragmentation reactions of DCQ and its derivatives, which can be useful for complementary studies of the development of a larvicidal product against Ae. aegypti.

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Licarin A, a dihydrobenzofuranic neolignan presents in several medicinal plants and seeds of nutmeg, exhibits strong activity against protozoans responsible for Chagas disease and leishmaniasis. From biomimetic reactions by metalloporphyrin and Jacobsen catalysts, seven products were determined: four isomeric products yielded by epoxidation from licarin A, besides a new product yielded by a vicinal diol, a benzylic aldehyde, and an unsaturated aldehyde in the structure of the licarin A. The incubation with rat and human liver microsomes partially reproduced the biomimetic reactions by the production of the same epoxidized product of m/z 343 [M + H]+. In vivo acute toxicity assays of licarin A suggested liver toxicity based on biomarker enzymatic changes. However, microscopic analysis of tissues sections did not show any tissue damage as indicative of toxicity after 14 days of exposure. New metabolic pathways of the licarin A were identified after in vitro biomimetic oxidation reaction and in vitro metabolism by rat or human liver microsomes.

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RATIONALE: Oxazolines are important compounds for drug development, synthesis, and pharmaceutical applications. Interest in analyzing and developing methods to characterize reaction products from these small heterocyclics has led us to study the gas-phase reactivity and fragmentation of seven 2-arene-2-oxazolines compounds using computational chemistry combined with mass spectrometry. METHOD: Protonation sites were investigated using computed proton affinity, gas-phase basicity, and some quantum chemistry descriptors of reactivity; the B3LYP/6-31+G(d,p) computational model was used. Fragmentation mechanisms were suggested by employing data from collision-induced dissociation (CID), energy-resolved plots from MS/MS spectra, multiple-stage experiments, and survival-yield method. RESULTS: Protonation studies based on quantum theory of atoms in molecules (QTAIM) and computational thermochemistry were useful to describe the reactivity of the investigated 2-arene-2-oxazolines, which can be protonated at the nitrogen atom. Three major fragmentation pathways were identified for the protonated molecules: formation of (a) benzoylium or (b) nitrilium ions through elimination of 71 and 72 u from the protonated molecules, respectively, and (c) elimination of 54 u from [M+H]+ . These pathways were exploited by the density functional theory calculations combined with QTAIM studies. CONCLUSIONS: Our results can help in identifying 2-arene-2-oxazoline derivatives using electrospray ionization tandem mass spectrometry (ESI-MS/MS), which can be applied for monitoring reactions through the identified diagnostic ions (product ions). Also, we can suggest that benzoylium and nitrilium ions emerge during fragmentation under CID conditions.

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This article discusses the reactivity of 6-azaindazole (1) and 2,6-naphthyridine (2), proposed to be "heteroaromatic rings of the future," which would be useful for fragment-based drug discovery (FBDD) campaigns, developing growth vectors for fragment elaboration by selectively functionalizing different positions on the rings. The pyridone oxygens and pyrazole nitrogen can be functionalized selectively. Arylation at the α-carbon of the pyridone moiety was achieved by a transition metal-free radical cross-coupling using aryl hydrazines. This method proceeded under mild conditions without the need for protection of the hydroxypyridine. Additionally, we developed a method for the regioselective C-3 functionalization of heterocycle 1via N-sulfonamide rearrangement. This method involved a novel regioselective base-mediated N-C migration of the N-1 sulfonamide to yield the C-3 sulfone. This procedure is also applicable for indazole C-3 functionalization and mechanistic studies of the rearrangement suggest that an intermolecular process is involved. These reactions enable the fragment elaboration of heterocycles 1 and 2 in several growth vectors to facilitate their use in FBDD.

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We have accomplished regioselective deprotometalation of aromatic and heteroaromatic nitriles via (TMP)2Zn·2MgCl2·2LiCl and TMPMgCl·LiCl (TMP = 2,2,6,6-tetramethylpiperidyl) with the exploration of new and scarcely investigated metalation positions. Regioselectivity was rationalized by DFT calculations. The quenching of the generated organozinc and organomagnesium intermediates with various electrophiles gave access to 47 highly functionalized nitriles with yields up to 95%. Additionally, we report a difunctionalization strategy and the use of functionalized nitriles as building blocks to construct relevant heterocycles.

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We have prepared a library of functionalized quinolines through the magnesiation of 7-chloroquinolines under mild conditions, employing both batch and continuous flow conditions. The preparation involved the generation of mixed lithium-magnesium intermediates, which were reacted with different electrophiles. Mixed lithium-zinc reagents allowed the synthesis of halogenated and arylated derivatives. Some of the synthesized 4-carbinol quinolines have shown interesting antiproliferative properties, their hydroxyl group being a suitable amino group bioisostere. We also report a two-step approach for optically active derivatives.

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RATIONALE: Although dihydrobenzofuran neolignans (DBNs) display a wide diversity of biological activities, the identification of their in vivo metabolites using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) remains a challenge to be overcome. Recently, ESI-MS/MS data of protonated DBNs have been reported, but they were shown to be limited due to the scarcity of diagnostic ions. METHODS: The gas-phase fragmentation pathways of a series of biologically active synthetic benzofuran neolignans (BNs) and DBNs were elucidated by means of negative ESI accurate-mass tandem and sequential mass spectrometry, and thermochemical data estimated using computational chemistry and the B3LYP/6-31+G(d,p) model. RESULTS: Deprotonated DBNs produced more diagnostic product ions than the corresponding protonated molecules. Moreover, a series of odd-electron product ions (radical anions) were detected, which has not been reported for protonated DBNs. Direct C2 H3 O2 • elimination from the precursor ion (deprotonated molecule) only occurred for the BNs and can help to distinguish these compounds from the DBNs. The mechanism through which the [M - H - CH3 OH]- ion is formed is strongly dependent on specific structural features. CONCLUSIONS: The negative ion mode provides much more information than the positive ion mode (at least one diagnostic product ion was detected for all the analyzed compounds) and does not require the use of additives to produce the precursor ions (deprotonated molecules).

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We have prepared novel highly functionalized benzene derivatives by regioselective metalation of ester-, amide-, carbamate-, and carbonate-substituted 2-phenyl-2-oxazolines with mixed lithium-magnesium amides followed by reaction with different electrophiles. While a complementary metalation site can be accessed by using different bases, steric and electronic effects promoted by the aromatic ring substituents also play an important role in reaction regioselectivity. Computational calculations of the aromatic hydrogen pKa values have helped us to rationalize the metalation preference by the complex-induced proximity effect concept.

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RATIONALE: Solanum paniculatum L., popularly known as jurubeba, has traditionally been used in Brazilian folk medicine for liver diseases. However, there is a lack of knowledge about the chemical characterization of 3-aminospirostane alkaloids, an important class related to pharmacological activities. This work aimed to characterize the alkaloids using liquid chromatography with tandem mass spectrometry (LC/MS/MS) supported by molecular networking and theoretical calculations as well as to evaluate the contribution to hepatoprotective activity. METHODS: S. paniculatum roots were collected and macerated with MeOH/H2 O (8:2) obtaining the crude extract (SP-CE). From this, partition using EtOAc with pH variation yielded the alkaloidic fraction (SP-AF). Both were evaluated in an acute liver injury model (100 and 200 mg/kg), after intraperitoneal administration of carbon tetrachloride (CCl4 ) in mice. AST (aspartate transaminase) and ALT (alanine transaminase) serum levels were investigated, as well as the histopathological characteristics. The SP-CE and SP-AF were analyzed by LC/MS/MS, using quadrupole/time-of-flight and ion-trap systems. The alkaloids annotated by the GNPS molecular network had their structures defined using gas-phase ionization and fragmentation reaction supported by theoretical calculations. RESULTS: The SP-CE and SP-AF decreased the ALT serum levels compared with the negative control. The group treated with the SP-CE (at the highest dose) demonstrated a significant decrease of ALT. Hepatic cell degeneration decrease was observed mainly at the highest dose of the treatment. Detailed electrospray ionization MS/MS data allowed us to identify alkaloids not previously reported, to propose their gas-phase reactions and to redefine the initial open ring fragmentation mechanism of the steroidal alkaloids with the jurubidine moiety. CONCLUSIONS: The results allowed us to identify seven steroidal alkaloids from jurubeba and redefine the initial mechanism of fragmentation. A significant hepatoprotective effect was also demonstrated, corroborating its traditional use.

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RATIONALE: Although monoketone curcuminoids (MKCs) have been largely investigated due to their biological activities, data on the gas-phase fragmentation reactions of protonated MKCs under collision-induced dissociation (CID) conditions are still scarce. Here, we combined electrospray ionization tandem mass spectrometry (ESI-MS/MS) data, multiple-stage mass spectrometry (MSn ), deuterium exchange experiments, accurate-mass data, and thermochemical data estimated by computational chemistry to elucidate and to rationalize the fragmentation pathways of eleven synthetic MKCs. METHODS: The MKCs were synthesized by Claisen-Schmidt condensation under basic (1-9) or acidic (10-11) conditions. ESI-CID-MS/MS analyses and deuterium-exchange experiments were carried out on a triple quadrupole mass spectrometer. MSn analyses on an ion trap mass spectrometer helped to elucidate the fragmentation pathways. Accurate-mass data and thermochemical data, obtained at the B3LYP/6-31+G(d,p) level of theory, were used to support the ion structures. RESULTS: The most intense product ions were the benzyl ions ([C7 H2 R1 R2 R3 R4 R5 ]+ ) and the acylium ions ([M + H - C8 H3 R1 R2 R3 R4 R5 ]+ ), which originated directly from the precursor ion as a result of two competitive hydrogen rearrangements. Product ions [M + H - H2 O]+ and [M + H - C6 HR1 R2 R3 R4 R5 ]+ , which are formed after Nazarov cyclization, were also common to all the analyzed compounds. In addition, •Br and •Cl eliminations were diagnostic for the presence of these halogen atoms at the aromatic ring, whereas •CH3 eliminations were useful to identify the methyl and methoxy groups attached to this same ring. Nazarov cyclization in the gas phase occurred for all the investigated MKCs and did not depend on the presence of the hydroxyl group at the aromatic ring. However, the presence and the position of a hydroxyl group at the aromatic rings played a key role in the Nazarov cyclization mechanism. CONCLUSIONS: Our results reinforce some aspects of the fragmentation pathways previously published for 1,5-bis-(2-methoxyphenyl)-1,4-pentadien-3-one and 1,5-bis-(2-hydroxyphenyl)-1,4-pentadien-3-one. The alternative fragmentation mechanism proposed herein can explain the fragmentation of a wider diversity of monoketone curcuminoids.

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Natural 2H-chromenes were isolated from the crude extract of Piper aduncum (Piperaceae) and analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) applying collision-induced dissociation. Density functional theory (DFT) calculations were used to explain the preferred protonation sites of the 2H-chromenes based on thermochemical parameters, including atomic charges, proton affinity, and gas-phase basicity. After identifying the nucleophilic sites, the pathways were proposed to justify the formation of the diagnostic ions under ESI-MS/MS conditions. The calculated relative energy for each pathway was in good agreement with the energy-resolved plot obtained from ESI-MS/MS data. Moreover, the 2H-chromene underwent proton attachment on the prenyl moiety via a six-membered transition state. This behavior resulted in the formation of a diagnostic ion due to 2-methylpropene loss. These studies provide novel insights into gas-phase dissociation for natural benzopyran compounds, indicating how reactivity is correlated to the intrinsic acid-base equilibrium and structural aspects, including the substitution pattern on the aromatic moiety. Therefore, these results can be applied in the identification of benzopyran derivatives in a variety of biological samples.

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We have investigated gas-phase fragmentation reactions of protonated benzofuran neolignans (BNs) and dihydrobenzofuran neolignans (DBNs) by accurate-mass electrospray ionization tandem and multiple-stage (MSn ) mass spectrometry combined with thermochemical data estimated by Computational Chemistry. Most of the protonated compounds fragment into product ions B ([M + H-MeOH]+ ), C ([B-MeOH]+ ), D ([C-CO]+ ), and E ([D-CO]+ ) upon collision-induced dissociation (CID). However, we identified a series of diagnostic ions and associated them with specific structural features. In the case of compounds displaying an acetoxy group at C-4, product ion C produces diagnostic ions K ([C-C2 H2 O]+ ), L ([K-CO]+ ), and P ([L-CO]+ ). Formation of product ions H ([D-H2 O]+ ) and M ([H-CO]+ ) is associated with the hydroxyl group at C-3 and C-3', whereas product ions N ([D-MeOH]+ ) and O ([N-MeOH]+ ) indicate a methoxyl group at the same positions. Finally, product ions F ([A-C2 H2 O]+ ), Q ([A-C3 H6 O2 ]+ ), I ([A-C6 H6 O]+ ), and J ([I-MeOH]+ ) for DBNs and product ion G ([B-C2 H2 O]+ ) for BNs diagnose a saturated bond between C-7' and C-8'. We used these structure-fragmentation relationships in combination with deuterium exchange experiments, MSn data, and Computational Chemistry to elucidate the gas-phase fragmentation pathways of these compounds. These results could help to elucidate DBN and BN metabolites in in vivo and in vitro studies on the basis of electrospray ionization ESI-CID-MS/MS data only.

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Copaifera (Leguminoseae) species produce a commercially interesting oleoresin that displays several biological activities, including antimicrobial and anti-inflammatory properties. Labdane-type diterpenes are the main chemical constituents of these oleoresins, and copalic acid is the only compound that has been detected in all Copaifera oleoresins. In this study, we investigate some aspects of the gas-phase fragmentation reactions involved in the formation of the product ions from the deprotonated compounds (-)-ent-copalic acid (1), (-)-ent-3ß-hydroxy-copalic acid (2), (-)-ent-3ß-acetoxy-copalic acid (3), and (-)-ent-agathic acid (4) by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and multiple stage mass spectrometry (MSn ). Our results reveal that the product ion with m/z 99 is common to all the analyzed compounds, whereas the product ion with m/z 217 is diagnostic for compounds 2 and 3. Moreover, only compound 4 undergoes CO2 (44 u) and acetic acid (60 u) elimination from the precursor ion. Thermochemical data obtained by computational chemistry at the B3LYP/6-31G(d) level of theory support the proposed ion structures. These data helped us to identify these compounds in a crude commercial Copaifera langsdorffii oleoresin by selective multiple reaction monitoring (MRM). Finally, a precursor ion scan (PIS) strategy aided screening of labdane-type acid diterpenes other than 1 to 4 in the same Copaifera oleoresin sample and led us to propose the structures of 8,17-dihydro-ent-agathic acid (5) and 3-keto-ent-copalic acid (6), which have not been previously reported in Copaifera oleoresins.

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Pyrrolizidine alkaloids are natural molecules playing important roles in different biochemical processes in nature and in humans. In this work, the electron ionization mass spectrum of retronecine, an alkaloid molecule found in plants, was investigated computationally. Its mass spectrum can be characterized by three main fragment ions having the following m/z ratios: 111, 94, and 80. In order to rationalize the mass spectrum, minima and transition state geometries were computed using density functional theory. It was showed that the dissociation process includes an aromatization of the originally five-membered ring of retronecine converted into a six-membered ring compound. A fragmentation pathway mechanism involving dissociation activation barriers that are easily overcome by the initial ionization energy was found. From the computed quantum chemical geometric, atomic charges, and energetic parameters, the abundance of each ion in the mass spectrum of retronecine was discussed.

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We prepared a number of di- and trifunctionalized quinolines by selective metalation of chloro-substituted quinolines with metal amides followed by reaction with different electrophiles. Metalation of the C-3 position of the quinolinic ring with lithium diisopropylamide at -70 °C is easy to achieve, whereas reaction with lithium-magnesium and lithium-zinc amides affords C-2 or C-8 functionalized derivatives in a regioselective fashion. These complementary methods could be rationalized by DFT calculations and are convenient strategies toward the synthesis of bioactive quinoline derivatives such as chloroquine analogues.

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We investigated the fragmentation of the eudesmanolide-type sesquiterpene lactones 1α-(4-hydroxymethacryloyloxy)-3α,4α-epoxy-8α-hydroxyeudesm-11(13)-6α,12-olide (1) and 1α-(2,3-epoxyangeloyloxy)-4α,15-epoxy-8α-hydroxyeudesm-11(13)-6α,12-olide (2) by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The elimination of the different ester substituent at C(1) directly from protonated 1 and 2 (A) led to the formation of two regioisomer product ions B (A - RCO2H). Further fragmentation of B resulted from consecutive eliminations of H2O and CO molecules. However, we identified four product ions that allowed for the differentiation between 3,4- and 4,15-epoxyeudesmanolides. The formation of these diagnostic ions was associated with the C(3)-O bond of compound 1, which propitiates the participation of the lone pair of the oxygen epoxide in the formation of B through a Grob-Wharton-type fragmentation, then resulting in an alternative fragmentation pathway. These data can be useful for the fast differentiation between epoxyeudesmanolide regioisomers directly from Dimerostemma extracts by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as an alternative to NMR, or even for quantitation studies of these compounds using multiple reaction monitoring (MRM) scan.

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We investigated the gas-phase fragmentation reactions of a series of 2-aroylbenzofuran derivatives by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The most intense fragment ions were the acylium ions m/z 105 and [M+H-C6 H6 ]+ , which originated directly from the precursor ion as a result of 2 competitive hydrogen rearrangements. Eliminations of CO and CO2 from [M+H-C6 H6 ]+ were also common fragmentation processes to all the analyzed compounds. In addition, eliminations of the radicals •Br and •Cl were diagnostic for halogen atoms at aromatic ring A, whereas eliminations of •CH3 and CH2 O were useful to identify the methoxyl group attached to this same ring. We used thermochemical data, obtained at the B3LYP/6-31+G(d) level of theory, to rationalize the fragmentation pathways and to elucidate the formation of E, which involved simultaneous elimination of 2 CO molecules from B.

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BACKGROUND AND OBJECTIVES: ß-Lapachone is a drug candidate in phase II clinical trials for treatment of solid tumors. The therapeutic efficacy of ß-lapachone is closely related to its metabolism, since this o-naphthoquinone produces cytotoxic effect after intracellular bioreduction by reactive oxygen species formation. The aim of this study was to produce ß-lapachone human blood phase I metabolites to evaluate their cytotoxic activities. METHODS: The biotransformation of ß-lapachone was performed using Mucor rouxii NRRL 1894 and Papulaspora immersa SS13. The metabolites were isolated and their chemical structures determined from spectrometric and spectroscopic data. Cell cytotoxicity assays were carried out with ß-lapachone and its metabolites using the neoplastic cell line SKBR-3 derived from human breast cancer and normal human fibroblast cell line GM07492-A. RESULTS: Microbial transformation of ß-lapachone by filamentous fungi resulted in the production of five metabolites identical to those found during human blood metabolism, a novel metabolite and a product stated before only in a synthetic procedure. The analysis of the results showed that ß-lapachone metabolites were not cytotoxic for the neoplastic cell line SKBR-3 derived from human breast cancer and the normal human fibroblast cell line GM07492-A. The cytotoxic activity assay against the neoplastic cell line SKBR-3 revealed that the lowest half-maximal inhibitory concentration (IC50) values of these ß-lapachone metabolites were 33- to 52-fold greater than IC50 values of ß-lapachone. CONCLUSIONS: The cytotoxic activity of ß-lapachone in vivo may be reduced due to its swift conversion in blood.

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