Feature-based molecular networking in the GNPS analysis environment.

Molecular networking has become a key method to visualize and annotate the chemical space in non-targeted mass spectrometry data. We present feature-based molecular networking (FBMN) as an analysis method in the Global Natural Products Social Molecular Networking (GNPS) infrastructure that builds on chromatographic feature detection and alignment tools. FBMN enables quantitative analysis and resolution of isomers, including from ion mobility spectrometry.

Health risk associated with the oral consumption of "Chiniy-tref", a traditional medicinal preparation used in Martinique (French West Indies): Qualitative and quantitative analyses of aristolochic acids contained therein.

"Chiniy-tref" (CT) is a traditional preparation used in folk medicine in Martinique Island (French West Indies) that is nowadays mainly taken orally to prevent or act against any "manifestation of evil". CT is easily prepared at home by macerating larvae of the endemic swallowtail Battus polydamas (ssp.) cebriones (Dalman, 1823), sometimes accompanied by a leaf of its host-plant Aristolochia trilobata L., in commercial rum. We have previously reported the detection of nephrotoxic and carcinogenic aristolochic acids (AAs) I and II in CT, leading the Regional Health Agency (ARS) of Martinique to issue an alert regarding the potential risks associated with its consumption in 2015. In order to complete the toxicity risk assessment for oral consumption of CT, a full qualitative analysis of AAs and their analogues (AAAs) was performed, as well as a quantitative determination of the major AAs, namely AAs I and II. The phytochemical profiling of AAAs present in CT, that also corresponds to that of B. polydamas cebriones larvae feeding on A. trilobata, has been established for the first time by ultra-high performance liquid chromatography/electrospray ionization quadrupole time of-flight tandem mass spectrometry. AAs I and II were quantified in a small panel of tinctures by using a validated UHPLC/UV method, allowing us to estimate the probable daily intakes of these toxins by CT consumers. The results proved the existence of a real risk of renal toxicity and carcinogenicity associated with the chronic oral consumption of CT in Martinique, and more generally of similar "snake bottles" throughout the Caribbean.

Severity of manchineel fruit (Hippomane mancinella) poisoning: A retrospective case series of 97 patients from French Poison Control Centers.

In this retrospective series of 97 cases of manchineel fruit ingestion reported to French Poison Control Centers between 2009 and 2017, we investigated cases of poisoning due to manchineel fruit (from the Hippomane mancinella tree). This fruit is known to be responsible for oropharyngeal and gastrointestinal tract lesions and possibly hypotension and bradycardia (previously attributed to the presence of physostigmine). The most commonly observed clinical signs were oropharyngeal pain, abdominal pain, diarrhea and oropharyngeal irritation. No major gastrointestinal tract lesions were observed in the five cases in which upper gastrointestinal (GI) endoscopy was performed. One case of laryngeal edema and one case of bradycardia were observed, but analysis of the harvested fruits did not confirm the presence of physostigmine. Ingestion of manchineel fruit can cause mild abdominal pain and digestive irritation, requiring medical attention. Rarely, when several fruits have been ingested, severe oropharyngeal injury or hemodynamic disorders may require otorhinolaryngological consultation or cardiac monitoring for several hours, respectively.

Clerodane furanoditerpenoids as the probable cause of toxic hepatitis induced by Tinospora crispa.

Tinospora crispa is a popular traditional herbal plant commonly used throughout the world for treatment of various diseases, in particular type 2 diabetes mellitus. We report here a new case of toxic hepatitis in a 57-year old male patient in the French West Indies following the consumption of two aqueous extracts of fresh Tinospora crispa stems. It thus differs from two previously reported cases that concerned the chronic intake of powdered dry stems delivered in solid oral dosage forms (i.e. pellets and tablets). Liquid Chromatography-Diode Array Detection-Mass Spectrometry (LC/DAD/MS) analyses were performed on an aqueous extract of the offending sample that mimics the swallowed preparation. They revealed the presence of species-specific molecular marker borapetoside C (1) and thus enabled an unambiguous phytochemical identification. The exploration of tandem MS/MS data obtained by ultra-high performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-HRMS) allowed the identification of 17 additional cis-clerodane-type furanoditerpenoid lactones, analogues of 1. These results support the hypothesis that the mechanisms underlying hepatotoxicity of Tinospora crispa are the same as those encountered with furanoditerpenoids-containing plants such as Teucrium chamaedrys or Dioscorea bulbifera. In the context of type 2 diabetes treatment, we recommend that Tinospora crispa intake should be more closely monitored for signs of hepatotoxicity.

Environmentally Friendly Procedure Based on Supercritical Fluid Chromatography and Tandem Mass Spectrometry Molecular Networking for the Discovery of Potent Antiviral Compounds from Euphorbia semiperfoliata.

A supercritical fluid chromatography-based targeted purification procedure using tandem mass spectrometry and molecular networking was developed to analyze, annotate, and isolate secondary metabolites from complex plant extract mixture. This approach was applied for the targeted isolation of new antiviral diterpene esters from Euphorbia semiperfoliata whole plant extract. The analysis of bioactive fractions revealed that unknown diterpene esters, including jatrophane esters and phorbol esters, were present in the samples. The purification procedure using semipreparative supercritical fluid chromatography led to the isolation and identification of two new jatrophane esters (13 and 14) and one known (15) and three new 4-deoxyphorbol esters (16-18). The structure and absolute configuration of compound 16 were confirmed by X-ray crystallography. This compound was found to display antiviral activity against Chikungunya virus (EC50 = 0.45 µM), while compound 15 proved to be a potent and selective inhibitor of HIV-1 replication in a recombinant virus assay (EC50 = 13 nM). This study showed that a supercritical fluid chromatography-based protocol and molecular networking can facilitate and accelerate the discovery of bioactive small molecules by targeting molecules of interest, while minimizing the use of toxic solvents.

Bioactive Natural Products Prioritization Using Massive Multi-informational Molecular Networks.

Natural products represent an inexhaustible source of novel therapeutic agents. Their complex and constrained three-dimensional structures endow these molecules with exceptional biological properties, thereby giving them a major role in drug discovery programs. However, the search for new bioactive metabolites is hampered by the chemical complexity of the biological matrices in which they are found. The purification of single constituents from such matrices requires such a significant amount of work that it should be ideally performed only on molecules of high potential value (i.e., chemical novelty and biological activity). Recent bioinformatics approaches based on mass spectrometry metabolite profiling methods are beginning to address the complex task of compound identification within complex mixtures. However, in parallel to these developments, methods providing information on the bioactivity potential of natural products prior to their isolation are still lacking and are of key interest to target the isolation of valuable natural products only. In the present investigation, we propose an integrated analysis strategy for bioactive natural products prioritization. Our approach uses massive molecular networks embedding various informational layers (bioactivity and taxonomical data) to highlight potentially bioactive scaffolds within the chemical diversity of crude extracts collections. We exemplify this workflow by targeting the isolation of predicted active and nonactive metabolites from two botanical sources (Bocquillonia nervosa and Neoguillauminia cleopatra) against two biological targets (Wnt signaling pathway and chikungunya virus replication). Eventually, the detection and isolation processes of a daphnane diterpene orthoester and four 12-deoxyphorbols inhibiting the Wnt signaling pathway and exhibiting potent antiviral activities against the CHIKV virus are detailed. Combined with efficient metabolite annotation tools, this bioactive natural products prioritization pipeline proves to be efficient. Implementation of this approach in drug discovery programs based on natural extract screening should speed up and rationalize the isolation of bioactive natural products.

Detection of aristolochic acids I and II in "Chiniy-trèf", a traditional medicinal preparation containing caterpillars feeding on Aristolochia trilobata L. in Martinique, French West Indies.

"Chiniy-trèf" is a traditional medicinal preparation used in Martinique, French West Indies, for the prevention of all kinds of attempted poisoning and hex. It is produced by the maceration in alcohol (mostly rum) of larvae (caterpillars) of the butterfly Battus polydamas ssp. cebriones, feeding on the leaves of Aristolochia trilobata. Aristolochic acids I and II that are well-known nephrotoxic and carcinogenic substances were identified on two samples of "chiniy-trèfl" by chromatographic methods.

Antibacterial Labdane Diterpenoids from Vitex vestita.

A large-scale in vitro screening of tropical plants using an antibacterial assay permitted the selection of several species with significant antibacterial activities. Bioassay-guided purification of the dichloromethane extract of the leaves of the Malaysian species Vitex vestita, led to the isolation of six new labdane-type diterpenoids, namely, 12-epivitexolide A (2), vitexolides B and C (3 and 4), vitexolide E (8), and vitexolins A and B (5 and 6), along with six known compounds, vitexolides A (1) and D (7), acuminolide (9), 3ß-hydroxyanticopalic acid (10), 8α-hydroxyanticopalic acid (11), and 6α-hydroxyanticopalic acid (12). Their structures were elucidated on the basis of 1D and 2D NMR analyses and HRMS experiments. Both variable-temperature NMR spectroscopic studies and chemical modifications were performed to investigate the dynamic epimerization of the γ-hydroxybutenolide moiety of compounds 1-4. Compounds were assayed against a panel of 46 Gram-positive strains. Vitexolide A (1) exhibited the most potent antibacterial activity with minimal inhibitory concentration values ranging from 6 to 96 µM, whereas compounds 2 and 6-9 showed moderate antibacterial activity. The presence of a ß-hydroxyalkyl-γ-hydroxybutenolide subunit contributed significantly to antibacterial activity. Compounds 1-4 and 6-9 showed cytotoxic activities against the HCT-116 cancer cell line (1 < IC50s < 10 µM) and human fetal lung fibroblast MRC5 cell line (1 < IC50s < 10 µM for compounds 1, 2, 7, 8, and 9).

A new 3,4-seco-cycloartane from the leaves of Hopea odorata Roxb.

A new 3,4-seco-cycloartane, identified as (24R,25S)-dihydroxy-26-O-nonadecylcarbonyloxy-3,4-secocycloarta-4(28)-en-3-oic acid (1), has been isolated from the leaves of Hopea odorata Roxb. (Dipterocarpaceae), together with the rare 3,4-seco-cycloart-4(28),24-diene-3,26-dioic acid (2 or abiesatrine J) and six other known compounds (3-8). Their structures were elucidated on the basis of both chemical and spectroscopic methods.

Synthesis of a new bis(indolyl)methane that inhibits growth and induces apoptosis in human prostate cancer cells.

The synthesis and the antiproliferative activity against the human breast MCF-7, SkBr3 and the prostate LNCaP cancer cell lines of a series of bis(indolyl)methane derivatives are reported. The synthesis of new compounds was first accomplished by the reaction of different indoles with trimethoxyacetophenone in the presence of catalytic amounts of hydrochloric acid. A second procedure involving the use of oxalic acid dihydrate [(CO2H)2·2H2O] and N-cetyl-N,N,N-trimethylammonium bromide in water was carried out and led to better yields. Compound 5b significantly reduced LNCaP prostate cancer cell viability in a dose-dependent manner, with an IC50 of 0.64 ± 0.09 µM. To determine whether the growth inhibition was associated with the induction of apoptosis, treated cells were stained using DAPI. LNCaP cells treated with 1 µM of 5b showed the morphological changes characteristic of apoptosis after 24 h of incubation.

Large scale purification of the SERCA inhibitor thapsigargin from Thapsia garganica L. roots using centrifugal partition chromatography.

Thapsigargin (Tg) is a selective and irreversible inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA)-dependent pump at subnanomolecular concentrations. As such, it has become a powerful tool in the study of Ca(2+) signaling pathway. Purification of Tg from Thapsia species requires repeated chromatographic steps with normal-phase alumina or silica and reverse phase chromatography. We thus developed an innovative procedure coupling high pressure automatized extraction with centrifugal partition chromatography allowing a fast and safe large-scale isolation of highly pure Tg, in two steps from Thapsia garganica L. roots. Comparison of influence of extraction procedures, storage conditions and harvesting areas on Tg content in different Algerian specimens of Thapsia garganica L. roots has been precised by mean of HPLC quantification procedure. Highest Tg content were found in the fresh material of the sample from Setif area.

Tetra-hydro-alstonine.

In the title compound, C21H24N2O3 [systematic name: methyl (20α)-16,17-dide-hydro-19α-methyl-18-oxayohimban-16-carb-oxy-l-ate], the mol-ecule adopts an L-type conformation. The crystal packing is governed by one N-H⋯π and one C-H⋯π inter-actions. The crystal cohesion is ensured by inter-molecular van der Waals contacts [shortest O⋯O contact = 3.199 (2) Å].

A one-pot synthesis of 7-phenylindolo[3,2-a]carbazoles from indoles and ß-nitrostyrenes, via an unprecedented reaction sequence.

A six-step one-pot reaction was designed for synthesizing homodimeric 7-phenylindolo[3,2-a]carbazoles from 1H-indoles and ß-nitrostyrenes, in the presence of SnCl(2)·2H(2)O. The reactions proceeded under very mild conditions and the desired heterocycles were obtained in moderate to good yields. An unprecedented mechanism involving sequential indole dimerization, regioselective nucleophilic conjugate addition of the resulting 2,3'-biindole to ß-nitrostyrene and formal intramolecular [4 + 2]-cycloaddition is proposed.

Structure-activity relationships of indole compounds derived from combretastatin A4: synthesis and biological screening of 5-phenylpyrrolo[3,4-a]carbazole-1,3-diones as potential antivascular agents.

A series of 5-(3',4',5'-trimethoxyphenyl)pyrrolo[3,4-a]carbazole-1,3(2H,10H)-diones was designed as cis-restricted analogues of 3-aroylindoles, arylthioindoles and 3-benzylidoneindolin-2-ones derived from combretastatin A4 (CA-4). Starting from various indoles, compounds were synthesized by means of a convenient two-step procedure involving a one-pot multicomponent reaction as key step. Intermediate tetrahydro[3,4-a]carbazoles and their corresponding carbazoles were submitted to biological screening tests involved in antivascular action, including the cytotoxicity against murine B16 melanoma cells, the rounding up of endothelial cells (EA.hy 926) and the inhibition of tubulin polymerization. Of the 31 compounds screened, those bearing a methoxy group at the 8-position endowed significant biological activities. A carbazole compound 30 was identified as a promising candidate for further development of novel vascular targeting agents.

A novel series of cytotoxic iridoid glucosides derived from aucubin: design, synthesis and structure-activity relationships.

Five new unsaturated iridolactones 3-7 related to natural cytotoxic oxylipins, Tei 9826, and iridolactone 2, were prepared by parallel synthesis from natural aucubin. It was found that perpivaloyl iridoid glucosides 2, 3, and 4 were markedly cytotoxic against both L1210 and KB-3-1 cell lines.

Synthesis and biological evaluation of new disubstituted analogues of 6-methoxy-3-(3',4',5'-trimethoxybenzoyl)-1H-indole (BPR0L075), as potential antivascular agents.

6-Methoxy-3-(3',4',5'-trimethoxybenzoyl)-1H-indole (BPR0L075) (1) is a potent inhibitor of tubulin polymerization which exhibits both in vitro and in vivo activities against a broad spectrum of solid tumors. This compound was designed as a heterocyclic analogue of combretastatin A4 (CA-4), a natural stilbene derivative that disrupts the tumor vasculature and causes tumor regression. In the present work, we describe the design and synthesis of several new disubstituted analogues of 1, along with their biological evaluation as potential antivascular agents. Compound 13, bearing a hydroxyl group at the 7-position of the indole nucleus that mimics the hydroxyl group at the 3-position of the B-ring of CA-4, was identified as a potent inhibitor of tubulin polymerization and also as a cytotoxic agent against B16 melanoma cells at sub-micromolar concentration. In addition, compound 13 displayed marked morphological activity (rounding up) at nanomolar concentrations on endothelial cells (EA.hy 926 cells), which is indicative of potential antivascular activity. Interestingly, the corresponding 7-O-mesylate derivative 28 (an intermediate in the synthesis of 13), was also found active in cellular assays, although it was moderately active in the tubulin polymerization inhibition test. Finally, in order to better understand the SAR of disubstituted analogues of 1, two other position isomers (10 and 14), were synthesized and evaluated for their biological activities. It was noted that the 7-hydroxysubstituted analogue 13 was more potent than the 5-hydroxysubstituted analogue 10. In conclusion, this work has allowed the identification of biologically potent CA-4 analogues (13 and 28) and also contributes to a better understanding of the SAR of 1.

Synthesis and biological evaluation of (3,4,5-trimethoxyphenyl)indol-3-ylmethane derivatives as potential antivascular agents.

Combretastatin A-4 (CSA-4), a stilbene derivative, is a potent vascular disrupting agent (VDA) with the structural requirement of a cis-configuration to maintain a molecular geometry and a correct orientation of both phenyl groups. A series of indolic analogues of CSA-4 was synthesized by means of an efficient strategy. Six compounds (20b, 25b-27b, 32b, and 35b) were identified as potent inhibitors of tubulin polymerization and also displayed cytotoxic activities on B16 melanoma cells at a nanomolar level. Both activities were well correlated with the ability to induce morphological changes of EA.hy 926 endothelial cells. In conclusion, the cis-stilbene skeleton of CSA-4 could conveniently be replaced by the 3-aroylindolic moiety, thus avoiding any isomerization leading to inactive trans compounds.

New antitubulin derivatives in the combretastatin A4 series: synthesis and biological evaluation.

Two series of combretastatin A4 derivatives (acrylamide=carboxamide and carbamate) were synthesized in order to improve the water solubility and stabilize the cis-configuration of the double bond. Their cytotoxic effects were evaluated against MCF-7, KB-3-1 and IGROV human cancer cell lines, as well as their inhibitory activity on tubulin polymerization. Results were compared to those of carboxamide 1, chosen as reference. Potent inhibitions were observed on both tests in the carboxamide series, particularly for compound 4d bearing a fluorine group in replacement of the 3-hydroxyl of CA4. In contrast, most of the carbamates were either inactive or displayed only moderate cytotoxicities. Interestingly, a submicromolar IC(50) was measured on MCF-7 cells for 6g, although this compound was totally devoid of antitubulin activity.