Stereochemical Determination of Fistularins Isolated from the Marine Sponge Ecionemia acervus and Their Regulatory Effect on Intestinal Inflammation.

By activity-guided fractionation based on inhibition of nitric oxide (NO) and prostaglandin E2 (PGE2), six fistularin compounds (1-6) were isolated from the marine sponge Ecionemia acervus (order Astrophorida). Based on stereochemical structure determination using Mosher's method, fistularin-3 was assigned as a new stereoisomer. On the basis of the stereochemistry of fistularin-3, the stereochemical homogeneity of all six compounds was established by comparing carbon and proton chemical shifts. For fistularin-1 (1) and -2 (2), quantum calculations were performed to confirm their stereochemistry. In a co-culture system of human epithelial Caco-2 cells and THP-1 macrophages, all six isolated compounds showed potent anti-inflammatory activities. These bioactive fistularins inhibited the production of NO, PGE2, TNF-α, IL-1ß, and IL-6 induced by lipopolysaccharide and interferon gamma. Inducible NO synthase and cyclooxygenase-2 expression and MAPK phosphorylation were downregulated in response to the inhibition of NF-κB nuclear translocation. Among the compounds tested, fistularin-1 (1) and 19-deoxyfistularin-3 (4) showed the highest activity. These findings suggest the potential use of the marine sponge E. acervus and its metabolites as pharmaceuticals for the treatment of inflammation-related diseases including inflammatory bowel disease.

Naturally Drug-Loaded Chitin: Isolation and Applications.

Naturally occurring three-dimensional (3D) biopolymer-based matrices that can be used in different biomedical applications are sustainable alternatives to various artificial 3D materials. For this purpose, chitin-based structures from marine sponges are very promising substitutes. Marine sponges from the order Verongiida (class Demospongiae) are typical examples of demosponges with well-developed chitinous skeletons. In particular, species belonging to the family Ianthellidae possess chitinous, flat, fan-like fibrous skeletons with a unique, microporous 3D architecture that makes them particularly interesting for applications. In this work, we focus our attention on the demosponge Ianthella flabelliformis (Linnaeus, 1759) for simultaneous extraction of both naturally occurring ("ready-to-use") chitin scaffolds, and biologically active bromotyrosines which are recognized as potential antibiotic, antitumor, and marine antifouling substances. We show that selected bromotyrosines are located within pigmental cells which, however, are localized within chitinous skeletal fibers of I. flabelliformis. A two-step reaction provides two products: treatment with methanol extracts the bromotyrosine compounds bastadin 25 and araplysillin-I N20 sulfamate, and a subsequent treatment with acetic acid and sodium hydroxide exposes the 3D chitinous scaffold. This scaffold is a mesh-like structure, which retains its capillary network, and its use as a potential drug delivery biomaterial was examined for the first time. The results demonstrate that sponge-derived chitin scaffolds, impregnated with decamethoxine, effectively inhibit growth of the human pathogen Staphylococcus aureus in an agar diffusion assay.

Synergistic AML Cell Death Induction by Marine Cytotoxin (+)-1(R), 6(S), 1'(R), 6'(S), 11(R), 17(S)-Fistularin-3 and Bcl-2 Inhibitor Venetoclax.

Treatment of acute myeloid leukemia (AML) patients is still hindered by resistance and relapse, resulting in an overall poor survival rate. Recently, combining specific B-cell lymphoma (Bcl)-2 inhibitors with compounds downregulating myeloid cell leukemia (Mcl)-1 has been proposed as a new effective strategy to eradicate resistant AML cells. We show here that 1(R), 6(S), 1'(R), 6'(S), 11(R), 17(S)-fistularin-3, a bromotyrosine compound of the fistularin family, isolated from the marine sponge Suberea clavata, synergizes with Bcl-2 inhibitor ABT-199 to efficiently kill Mcl-1/Bcl-2-positive AML cell lines, associated with Mcl-1 downregulation and endoplasmic reticulum stress induction. The absolute configuration of carbons 11 and 17 of the fistularin-3 stereoisomer was fully resolved in this study for the first time, showing that the fistularin we isolated from the marine sponge Subarea clavata is in fact the (+)-11(R), 17(S)-fistularin-3 stereoisomer keeping the known configuration 1(R), 6(S), 1'(R), and 6'(S) for the verongidoic acid part. Docking studies and in vitro assays confirm the potential of this family of molecules to inhibit DNA methyltransferase 1 activity.

Small scale separation of isoxazole structurally related analogues by chiral supercritical fluid chromatography.

Chromatographic preparative enantioseparation is now the preferred method to obtain milligram amounts of pure enantiomers in the first step of the development of a therapeutic molecule. Supercritical fluid chromatography has many advantages over liquid chromatography and was therefore chosen for the small scale enantioseparation of four original 3-carboxamido-5-aryl isoxazole molecules, ligands of the CB2 cannabinoid receptors. The preparation of about 10mg of each of the eight enantiomers was achieved successfully on a Chiralpak® AD-H (tris-3,5-dimethylphenylcarbamate of amylose) polysaccharide based stationary phase with various percentages of ethanol as a co-solvent, through mixed-stream injections and touching-band approach. For the all compounds, no peak distortion is observed during the volume overloading, in spite of the injection mode. Production rate (mgmin-1), productivity (kilogram of racemate separated per kilogram of CSP per day (kkd)) and solvent usage were found higher and environmental factors (E Factor) were found lower for compounds 1 and 3. The yields of each purified enantiomer were comprised between 60 and 94%. In order to improve the limit of detection calculated with the diode array detector, the hyphenation with an evaporating light scattering detector was explored and a factor of ten was won. Lastely, the enantiomeric excess and achiral purity of each of the eight individual enantiomer generated was determined and found higher than 98%.

A tale of four kingdoms - isoxazolin-5-one- and 3-nitropropanoic acid-derived natural products.

Covering up to September 2016This review reports on natural compounds that derive from the isoxazolinone ring as well as the 3-nitropropanoic acid (3-NPA) moiety. These structural elements occur in compounds that have been identified in plants, insects, bacteria and fungi. In particular, plants belonging to the family of legumes produce such compounds. In the case of insects, isoxazolin-5-one and 3-NPA derivatives were found in leaf beetles of the subtribe Chrysomelina. A number of these natural products have been synthesized so far. In the case of the single compound 3-NPA, several synthetic strategies have been reported and some of the most efficient routes are reviewed. The toxicity of 3-NPA results from its ability to bind covalently to the catalytic center of succinate dehydrogenase causing irreversible inhibition of mitochondrial respiration. As a motif that is produced by many species of plants, leaf beetles and fungi, different detoxification mechanisms for 3-NPA have evolved in different species. These mechanisms are based on amide formation of 3-NPA with amino acids, reduction to ß-alanine, ester formation or oxidation to malonic acid semialdehyde. The biosynthetic pathways of 3-NPA and isoxazolin-5-one moieties have been studied in fungi, plants and leaf beetles. In the case of fungi, 3-NPA derives from aspartate, while leaf beetles use essential amino acids such as valine as ultimate precursors. In the case of plants, it is supposed that malonate serves as a precursor of 3-NPA, as indicated by feeding of 14C-labeled precursors to Indigofera spicata. In other leguminous plants it is suggested that asparagine is incorporated into compounds that derive from isoxazolin-5-one, which was indicated by 14C-labeled compounds as well. In the case of leaf beetles it was demonstrated that detection of radioactivity after 14C-labeling from a few precursors is not sufficient to unravel biosynthetic pathways.

Enantioseparation of isoxazolines with functionalized perphenylcarbamate cyclodextrin clicked chiral stationary phases in HPLC.

The enantioseparations of 12 isoxazoline racemates were explored with four perphenylcarbamate cyclodextrin (CD) clicked chiral stationary phases (CSPs) in high performance liquid chromatography (HPLC). The results demonstrated that the functionalities on phenylcarbamate moiety greatly determined the chiral separation ability of CD clicked CSPs. Among of them, per(3-chloro-4-methylphenylcarbamate) CD clicked CSP (CCC3M4-CSP) exhibited the best enantioseparation ability, affording 4ClPh-OPr with a chiral resolution over 20 in ternary eluent mobile phases. The optimization of CSPs structures provided wide platform for their chiral separations towards multi-mode HPLC.

Development and Validation of a Normal Phase Chiral HPLC Method for Analysis of Afoxolaner Using a Chiralpak® AD-3 Column.

Afoxolaner is a new antiparasitic molecule from the isoxazoline family that acts on the insect acarine gamma-aminobutyric acid and glutamate receptors. Isoxazoline family of compounds has been employed as active pharmaceutical ingredient in drug products prescribed for control of fleas and ticks in dogs. Afoxolaner with a chiral center at isoxazoline ring exists as a racemic mixture. A normal phase chiral high performance liquid chromatography analytical method has been developed to verify that afoxolaner is a racemic mixture as demonstrated by specific rotation, as well as to determine enantiomeric purity of single enantiomer samples. A Chiralpak® AD-3 column (150 × 4.6 mm I.D.) maintained at 35°C was used in the method. Analytes were analyzed with an isocratic elution using n-Hexane/IPA/MeOH (89:10:1, v/v/v) as the mobile phase with a detection wavelength of 312 nm. Desired separation of the two enantiomers was achieved in <10 minutes with resolution and selectivity factors of 5.0 and 1.54, respectively. The analytical method was appropriately validated according to ICH guidelines for its intended use. ® All marks are the property of their respective owners.

Thioether bridged cationic cyclodextrin stationary phases: Effect of spacer length, selector concentration and rim functionalities on the enantioseparation.

The preparation and evaluation of four single thioether bridged cationic cyclodextrin (CD) chiral stationary phases (CSPs) with different spacer length, selector concentration and rim functionalities are reported. Mono-6-(1-vinyl/allyl/butenylimidazolium)-ß-CDs chloride were synthesized and clicked onto thiol silica to form three novel cationic native-CD-CSPs (CSP1, CSP2 and CSP3) and a post-synthetic phenylcarbamoylation of CSP2 was performed affording CSP4. The enantioseparation ability of the as-prepared CSPs were evaluated in high performance liquid chromatography (HPLC) by separating over forty enantiomers including isoxazolines, dansyl amino acids, flavonoids, tröger's base, 4-chromanol, bendroflumethiazide and styrene oxide. Most of the enantiomers were well resolved with the resolution (Rs) of 4NPh-OPr reaching 12.68. The effects of spacer length, selector concentration and rim functionalities on the enantioseparation were investigated. A comparison of the current CSP with a commercial column (Cyclobond I 2000) was also conducted to reveal the superiors enantioselectivity of the as-prepared CSPs.

Two new isoxazolines from the husks of Xanthoceras sorbifolia Bunge.

Two new isoxazoline compounds, 1-oxa-2-azaspiro[4.5]dec-2-ene-8ß-ol (1) and 1-oxa-2-azaspiro[4.5]dec-2-ene-8α-ol (2), were isolated from the husks of fruits of Xanthoceras sorbifolia Bunge and their structures were determined by spectroscopic analyses, including X-ray crystallography, HRESI-MS, UV, IR, and 1D and 2D NMR (HSQC, HMBC, NOESY) methods. Neither compound showed significant inhibitory effects on butyrylcholinesterase (BuchE) and acetylcholinesterase (AChE), nor the selected tumor cells growth. Based on an online activity prediction program (PASS ONLINE), the structures with isoxazoline skeletons were found to show potential anti-asthmatic (AM) and anti-anaphylaxis (AP) activities; moreover, compounds 1 and 2 were predicted to possess high affinities for many enzymes involved in AM and AP according to the RCSB Protein Data Bank. High-affinity binding to phosphodiesterase IV (PDE-4), an important inflammatory modulator in asthma, was demonstrated experimentally, beside that, the predicted structures based on compounds 1 and 2 were analyzed for PDE-4 interactions using the molecular docking methodology of Discovery Studio 3.0 (DS 3.0). The predicted structure 2A-6 exhibited much higher affinity and stability of PDE-4 binding than the clinical PDE-4 inhibitor rolipram.

Volvalerine A, an unprecedented N-containing sesquiterpenoid dimer derivative from Valeriana officinalis var. latifolia.

Volvalerine A (1), a novel N-containing bisesquiterpenoid derivative with a dihydroisoxazole ring, and its possible biosynthetic precursor, 1-hydroxy-1,11,11-trimethyldecahydrocyclopropane azulene-10-one (2), were isolated from the roots of Valeriana officinalis var. latifolia. Their structures and relative configurations were identified using spectroscopic data and X-ray crystallography. A plausible biosynthetic pathway for 1 is also presented.

A chemical genetic strategy identify the PHOSTIN, a synthetic molecule that triggers phosphate starvation responses in Arabidopsis thaliana.

Plants display numerous strategies to cope with phosphate (Pi)-deficiency. Despite multiple genetic studies, the molecular mechanisms of low-Pi-signalling remain unknown. To validate the interest of chemical genetics to investigate this pathway we discovered and analysed the effects of PHOSTIN (PSN), a drug mimicking Pi-starvation in Arabidopsis. We assessed the effects of PSN and structural analogues on the induction of Pi-deficiency responses in mutants and wild-type and followed their accumulation in plants organs by high pressure liquid chromotography (HPLC) or mass-spectrophotometry. We show that PSN is cleaved in the growth medium, releasing its active motif (PSN11), which accumulates in plants roots. Despite the overaccumulation of Pi in the roots of treated plants, PSN11 elicits both local and systemic Pi-starvation effects. Nevertheless, albeit that the transcriptional activation of low-Pi genes by PSN11 is lost in the phr1;phl1 double mutant, neither PHO1 nor PHO2 are required for PSN11 effects. The range of local and systemic responses to Pi-starvation elicited, and their dependence on the PHR1/PHL1 function suggests that PSN11 affects an important and early step of Pi-starvation signalling. Its independence from PHO1 and PHO2 suggest the existence of unknown pathway(s), showing the usefulness of PSN and chemical genetics to bring new elements to this field.

Bromotyrosine-derived metabolites from an Indonesian marine sponge in the family Aplysinellidae (Order Verongiida).

Seven new bromotyrosine-derived metabolites, purpuramine M-N (1-2), araplysillin VII-XI (3-7) and six known compounds (8-13) were isolated from an Indonesian sponge belonging to the family Aplysinellidae (Order Verongiida). The structures of the new compounds were determined by extensive NMR experiments and mass spectrometric measurements. These compounds were screened against BACE1 and five cancer cell lines.

Bioactive secondary metabolites from the Red Sea marine Verongid sponge Suberea species.

In a continuation of our efforts to identify bioactive compounds from Red Sea Verongid sponges, the organic extract of the sponge Suberea species afforded seven compounds including two new dibrominated alkaloids, subereamollines C and D (1 and 2), together with the known compounds aerothionin (3), homoaerothionin (4), aeroplysinin-1 (5), aeroplysinin-2 (6) and a revised subereaphenol C (7) as ethyl 2-(2,4-dibromo-3,6-dihydroxyphenyl)acetate. The structures of the isolated compounds were assigned by different spectral data including optical rotations, 1D (1H and 13C) and 2D (COSY, multiplicity-edited HSQC, and HMBC) NMR and high-resolution mass spectroscopy. Aerothionin (3) and subereaphenol C (7) displayed potent cytotoxic activity against HeLa cell line with IC50 values of 29 and 13.3 µM, respectively. In addition, aeroplysinin-2 (6) showed potent antimigratory activity against the human breast cancer cell line MDA-MB-231 with IC50 of 18 µM. Subereamollines C and D are new congeners of the previously reported compounds subereamollines A and B with methyl ester functionalities on the side chain. These findings provide further insight into the biosynthetic capabilities of members of the genus Suberea and the chemical diversity as well as the biological activity of these compounds.

Isoxazoline containing natural products as anticancer agents: a review.

Isoxazolines are an important class of nitrogen and oxygen containing heterocycles that belong to the azoles family which have gained much importance in the field of medicinal chemistry as the anticancer agents. Moreover, natural products are always expectedly regarded as an important hoard of a large number of potential chemotherapeutic candidates. Therefore, this review mainly focuses on the existence of isoxazoline derivatives in natural sources, their isolation and uses there of as anticancer agents besides highlighting the synthetic pathways to achieve these compounds. Structural-activity relationship and the influence of stereochemical aspects on anticancer activity of such compounds have also been discussed. It covers the literature upto 2014 and would certainly provide a great insight to scientific community to accelerate further research for the development of some novel anticancer drugs.

Discovering small molecules that inhibit adipogenesis and promote osteoblastogenesis: unique screening and Oncostatin M-like activity.

Oncostatin M (OSM), one of the IL-6 family cytokines, inhibits adipogenic differentiation and stimulates osteoblastogenic differentiation from human bone marrow mesenchymal stem cells (hBMSCs). This functional study of OSM enabled us to develop a two-dimensional small-molecule screen that shifts hBMSC differentiation from adipocyte to osteoblast. Several structurally related compounds (isoxazoles) inhibited the accumulation of intracellular lipid droplets, whereas they promoted alkaline phosphatase activity and extracellular matrix calcification. Isoxazoles also reduced the expression of adipogenic transcription factor PPARγ and increased the levels of osteogenic transcription factors Runx2 and Osterix. They also induced the expression of the Wnt/ß-catenin downstream gene and TOPflash reporter; however, the dephosphorylated ß-catenin-active form was not significantly increased. Interestingly, the slight modification of the active compound led to a complete reversion of the dual differentiation activities. In summary, we have identified isoxazoles with anti-adipogenic and pro-osteogenic activities that provide a potential new tool for exploring the lineage commitment of mesenchymal stem cells and a possible lead for therapeutic intervention in osteopenia and osteoporosis.

Application of surfactant assisted dispersive liquid-liquid microextraction as an efficient sample treatment technique for preconcentration and trace detection of zonisamide and carbamazepine in urine and plasma samples.

A simple, rapid, and efficient method, based on surfactant assisted dispersive liquid-liquid microextraction (SA-DLLME), followed by high performance liquid chromatography (HPLC) has been developed for simultaneous preconcentration and trace detection of zonisamide and carbamazepine in biological samples. A conventional cationic surfactant called cethyltrimethyl ammonium bromide (CTAB) was used as a disperser agent in the proposed approach. 1.5 mL of CTAB (0.45 mmol L(-1)) (disperser solvent) containing 50.0 µL of 1-octanol (extraction solvent) was injected rapidly into the 7.0 mL of water or diluted plasma or urine. A cloudy solution (water, 1-octanol, and CTAB) was formed in the test tube. After formation of cloudy solution, the mixture was centrifuged and 20 µL of collected phase was injected into HPLC for subsequent analysis. Some parameters such as the type and volume of the extraction solvent, the type and concentration of surfactant, pH, ionic strength and centrifugation time were evaluated and optimized. Under optimum extraction conditions, the limits of detections (LODs) were 2.1 and 1.5 µg L(-1) (based on 3Sb/m) for urine samples, and 2.3 and 1.6 µg L(-1) for plasma samples. Linear dynamic range of 5-300 and 5-200 µg L(-1) were obtained for zonisamide and carbamazepine in all samples. Finally, the applicability of the proposed method was evaluated by extraction and determination of the drugs in urine and plasma samples.

Ianthelliformisamines A-C, antibacterial bromotyrosine-derived metabolites from the marine sponge Suberea ianthelliformis.

A high-throughput screening campaign using a prefractionated natural product library and an in vitro Pseudomonas aeruginosa (PAO200 strain) assay identified two antibacterial fractions derived from the marine sponge Suberea ianthelliformis. Mass-directed isolation of the CH(2)Cl(2)/CH(3)OH extract from S. ianthelliformis resulted in the purification of three new bromotyrosine-derived metabolites, ianthelliformisamines A-C (1-3), together with the known natural products aplysamine 1 (4) and araplysillin I (5). The structures of 1-3 were determined following analysis of 1D and 2D NMR and MS spectroscopic data. This is the first report of chemistry from the marine sponge S. ianthelliformis. Ianthelliformisamine A (1) showed inhibitory activity against the Gram-negative bacterium P. aeruginosa with an IC(50) value of 6.8 µM (MIC = 35 µM).

Trade-offs in defensive metabolite production but not ecological function in healthy and diseased sponges.

Diseases of marine organisms, and sponges in particular, are increasingly reported worldwide. Prior research indicates that the survival of sponges on reefs is due largely to their production of biologically active secondary metabolites that provide protection from a diversity of stressors. Aplysina Red Band Syndrome (ARBS) is an emerging disease affecting Caribbean rope sponges (Aplysina spp.), but it is not known whether secondary metabolites play a role in disease susceptibility and resistance. To investigate whether differences in secondary metabolites may explain variability in susceptibility to ARBS in Aplysina cauliformis, we used high performance liquid chromatography (HPLC) to generate chemical profiles from healthy tissue in both healthy and diseased sponges, and quantified peak areas for 15 metabolites. Analyses of healthy and diseased sponges revealed qualitative and quantitative differences in their chemical profiles. Aplysamine-1 and fistularin-3 were produced in significantly higher concentrations by healthy sponges, whereas aerothionin and 11-oxoaerothionin were found only in diseased sponges. At natural concentrations, extracts from both healthy and diseased sponges deterred feeding by an omnivorous reef fish. Fistularin-3 deterred feeding at concentrations found in healthy sponges, but not at concentrations found in diseased sponges. Aerothionin deterred feeding at concentrations found in diseased sponges, and may at least partially replace the loss of fistularin-3 as a feeding deterrent compound following pathogenesis, suggesting a trade-off in the production of feeding deterrent compounds. Extracts from healthy and diseased sponges inhibited bacterial growth, and both aplysamine-1 and fistularin-3 displayed selective antibacterial activity. Despite differences in secondary metabolite production between healthy and diseased sponges, the stress associated with ARBS does not appear to compromise the ability of A. cauliformis to maintain defenses against some of its natural enemies.

High-performance liquid chromatographic enantioseparation of unusual isoxazoline-fused 2-aminocyclopentanecarboxylic acids on macrocyclic glycopeptide-based chiral stationary phases.

The enantiomers of four unusual isoxazoline-fused 2-aminocyclopentanecarboxylic acids were directly separated on chiral stationary phases containing macrocyclic glycopeptide antibiotics teicoplanin (Astec Chirobiotic T and T2), teicoplanin aglycone (Chirobiotic TAG), vancomycin (Chirobiotic V) and vancomycin aglycone (Chirobiotic VAG) as chiral selectors. The effects of the mobile phase composition, the structure of the analytes and temperature on the separations were investigated. Experiments were performed at constant mobile phase compositions in the temperature range 5-45 °C to study the effects of temperature, and thermodynamic parameters were calculated from plots of lnk or lnα versus 1/T. Some mechanistic aspects of the chiral recognition process are discussed with respect to the structures of the analytes. It was found that the enantiomeric separations were in most cases enthalpy-driven. The sequence of elution of the enantiomers was determined in all cases.

[Isolation and identification of brominated alkaloids from the sponge Ircinia sp].

OBJECTIVE: To study the alkaloids from sponge Ircinia sp.. METHODS: The alkaloids were isolated and purified by various chromatographic techniques, and their structures were elucidated by physicochemical and spectral data. RESULTS: Five alkaloids were isolated and identified as fistularin-1 (1), fistularin-3 (2), aerothionin (3), 11 -hydroxyaerothionin (4), suberamolline A (5). CONCLUSION: All the alkaloids are isolated from Ircinia sp. for the first time.