Title: Mass spectrometry for characterization of homologous piperidine alkaloids and their activity as acetylcholinesterase inhibitors Freitas TR, Danuello A, Viegas Junior C, Bolzani VS, Pivatto M Ref: Rapid Commun Mass Spectrom, 32:1303, 2018 : PubMed
RATIONALE: Piperidine alkaloids from Senna spectabilis constitute a rare class of natural products with several biological activities. However, the absence of chromophores makes their structural elucidation by conventional methods a great challenge. In this context, mass spectrometry emerges as a powerful tool for metabolomics studies. METHODS: The piperidine alkaloids (-)-cassine and (-)-spectaline and the semisynthetic derivatives (-)-3-O-acetylcassine and (-)-3-O-acetylspectaline were investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the positive mode and electron ionization mass spectrometry (EI-MS). ESI fragmentation studies were performed with a quadrupole time-of-flight instrument; N2 was used as collision gas. The acetylcholinesterase inhibitory activity of the investigated compounds was evaluated by bioautography and microplate screening assays. RESULTS: ESI-MS/MS and EI-MS provided valuable and complementary information about the structure of the piperidine compounds. Collision-induced dissociation experiments (MS/MS) revealed that neutral elimination of water or acetic acid is the major fragmentation pathway, which agrees with the stereochemistry proposed for (-)-cassine and (-)-spectaline and the semisynthetic derivatives (-)-3-O-acetylcassine and (-)-3-O-acetylspectaline. CONCLUSIONS: The ESI-MS/MS and EI-MS studies allowed us to propose fragmentation mechanisms for piperidine alkaloids and derivatives. Therefore, mass spectrometry is an important tool for characterizing the structure of these compounds and for supporting further metabolomics studies.
Chemical investigation of an acetonitrile fraction from the endophytic fungus Phomopsis sp. led to the isolation of the new natural product 2-hydroxy-alternariol (7) together with the known compounds cytochalasins J (1) and H (2), 5'-epialtenuene (3) and the mycotoxins alternariol monomethyl ether (AME, 4), alternariol (AOH, 5) and cytosporone C (6). The structure of the new compound was elucidated by using 1-D and 2-D NMR (nuclear magnetic resonance) and high resolution mass spectrometry. The cytochalasins J (1) and H (2) and AOH (5) exhibited potent inhibition of the total ROS (reactive oxygen species) produced by stimulated human neutrophils and acted as potent potential anti-inflammatory agents. Moreover, cytochalasin H (2) demonstrated antifungal and acetylcholinesterase enzyme (AChE) inhibition in vitro.
In this study, eight endophytic fungi were isolated from the leaves, stems and roots of Michelia champaca. The isolates were screened and evaluated for their antifungal, anticancer and acetylcholinesterase (AChE) inhibitory activities. All of the extracts exhibited potent activity against two evaluated phytopathogenic fungi. Chemical investigation of EtOAc extracts of the endophytic fungus Colletotrichum gloeosporioides resulted in the isolation of one new compound, 2-phenylethyl 1H-indol-3-yl-acetate (1), and seven known compounds: uracil (2), cyclo-(S*-Pro-S*-Tyr) (3), cyclo-(S*-Pro-S*-Val) (4), 2(2-aminophenyl)acetic acid (5), 2(4-hydroxyphenyl)acetic acid (6), 4-hydroxy- benzamide (7) and 2(2-hydroxyphenyl)acetic acid (8). All of the compound structures were elucidated using 1D and 2D NMR and MS analyses. The antifungal and AChE inhibitory activities of compounds 1-8 were evaluated in vitro. Compound 1 exhibited promising activity against Cladosporium cladosporioides and C. sphaerospermum that was comparable to that of the positive control nystatin.
As part of an ongoing research project on Senna and Cassia species, five new pyridine alkaloids, namely, 12'-hydroxy-7'-multijuguinol (1), 12'-hydroxy-8'-multijuguinol (2), methyl multijuguinate (3), 7'-multijuguinol (4), and 8'-multijuguinol (5), were isolated from the leaves of Senna multijuga (syn. Cassiamultijuga). Their structures were elucidated on the basis of spectroscopic data analysis. Mass spectrometry was used for confirmation of the positions of the hydroxy groups in the side-chains of 1, 2, 4, and 5. All compounds exhibited weak in vitro acetylcholinesterase inhibitory activity as compared with the standard compound physostigmine.
In the search for acetylcholinesterase inhibitors as a potential target for the discovery of anthelmintic drugs, a series of 27 pyridinic and pyrazinic compounds have been designed on the basis of molecular hybridization of two known AChE inhibitors, namely, tacrine and (-)-3-O-acetylspectaline, and on the concept of isosterism. The synthesized compounds generally presented moderate anticholinesterasic activities when compared with the positive control physostigmine, but one compound (ethyl 2-[(6-chloropyrazin-2-yl)sulfanyl] acetate) exhibited an in vitro ability to immobilize the root-knot nematode Meloidogyne incognita that was highly comparable to that of the positive control Temik. Moreover, in anthelmintic assays against the gastrointestinal parasitic nematode Nippostrongylus brasiliensis (L4), some of the compounds, such as (6-chloropyrazin-2-yl)sulfanyl ethanol (32, EC(5)(0) = 33 nM), presented activities that were considerably stronger than that of the positive control albendazole (EC(5)(0) = 340 nM). In the light of the positive results obtained in the anthelmintic evaluations, the acute oral toxicity of the representative compound diethyl 2,2'-[(3-nitropyridine-2,6-diyl) bissulfanediyl] diacetate was determined in rats, and the drug was shown to be non-toxic at a dose of 2000 mg/kg. These results, allied with the relatively simple structures of the active compounds and their facile synthesis, highlight their potential use as anthelmintic or nematicidic agents.
Two unusual pyridine alkaloids, 7'-multijuguinone (1) and 12'-hydroxy-7'-multijuguinone (2), were isolated from the leaves of Senna multijuga, together with the known flavonoid rutin. The structures of the new alkaloids were established on the basis of spectroscopic data interpretation. Compounds 1 and 2 exhibited moderate in vitro acetylcholinesterase (AChE) inhibitory activity, in comparison with the standard compound physostigmine.
LASSBio-767 [(-)-3-O-acetyl-spectaline] and LASSBio-822 [(-)-3-O-tert-Boc-spectaline] were recently described as cholinesterase inhibitors derived from the natural piperidine alkaloid (-)-spectaline, obtained from the flowers of Senna spectabilis (Fabaceae). We investigated their mechanism of inhibition of acetylcholinesterase and their efficacy in reversing scopolamine-induced amnesia. Competition assays with the substrate acetylthiocholine showed a concentration-dependent reduction in rat brain cholinesterase Vmax without changes in apparent Km. The kinetic data for LASSBio-767 and LASSBio-822 were best fit by a model of simple linear noncompetitive inhibition with Ki of 6.1 microM and 7.5 microM, respectively. A dilution assay showed a fast and complete reversal of inhibition, independent of incubation time. Simulated docking of the compounds into the catalytic gorge of Torpedo acetylcholinesterase showed interactions with the peripheral anionic site, but not with the catalytic triad. Anti-amnestic effects in mice were assessed in a step-down passive avoidance test and in the Morris water maze 30 min after injection of scopolamine (1 mg/kg i.p.). Saline, LASSBio-767, or LASSBio-822 was administered 15 min before scopolamine. Both compounds reversed the scopolamine-induced reduction in step-down latency at 0.1 mg/kg i.p. LASSBio-767 reversed scopolamine-induced changes in water maze escape latency at 1 mg/kg i.p. or p.o., while its cholinergic side effects were absent or mild up to 30 mg/kg i.p. (LD50 above 100 mg/kg i.p.). Thus, the (-)-spectaline derivatives are potent cholinergic agents in vivo, with a unique profile combining noncompetitive cholinesterase inhibition and CNS selectivity, with few peripheral side effects.
Five new piperidine alkaloids were designed from natural (-)-3-O-acetyl-spectaline and (-)-spectaline that were obtained from the flowers of Senna spectabilis (sin. Cassia spectabilis, Leguminosae). Two semi-synthetic analogues (7 and 9) inhibited rat brain acetylcholinesterase, showing IC50 of 7.32 and 15.1 microM, and were 21 and 9.5 times less potent against rat brain butyrylcholinesterase, respectively. Compound 9 (1mg/kg, i.p.) was fully efficacious in reverting scopolamine-induced amnesia in mice. The two active compounds (7 and 9) did not show overt toxic effects at the doses tested in vivo.
