Synthesis and Target Identification of Benzoxepane Derivatives as Potential Anti-Neuroinflammatory Agents for Ischemic Stroke.

Benzoxepane derivatives were designed and synthesized, and one hit compound emerged as being effective in vitro with low toxicity. In vivo, this hit compound ameliorated both sickness behavior through anti-inflammation in LPS-induced neuroinflammatory mice model and cerebral ischemic injury through anti-neuroinflammation in rats subjected to transient middle cerebral artery occlusion. Target fishing for the hit compound using photoaffinity probes led to identification of PKM2 as the target protein responsible for anti-inflammatory effect of the hit compound. Furthermore, the hit exhibited an anti-neuroinflammatory effect in vitro and in vivo by inhibiting PKM2-mediated glycolysis and NLRP3 activation, indicating PKM2 as a novel target for neuroinflammation and its related brain disorders. This hit compound has a better safety profile compared to shikonin, a reported PKM2 inhibitor, identifying it as a lead compound in targeting PKM2 for the treatment of inflammation-related diseases.

Semisynthesis and antibacterial activities of nidulin derivatives.

Derivatives of the fungal depsidone, nidulin, have been synthesized in order to evaluate the potential of the chemical skeleton as antibacterial agents. Alkylation, acylation, and arylation reactions of nornidulin underwent in a regioselective manner to predominantly produce 8-O-substituted derivatives. Many of the semisynthetic derivatives showed more potent antibacterial activities than nidulin, In particular, 8-O-aryl ether derivatives displayed significant activities against Gram-positive bacteria, including Methicillin-resistant Staphylococcus aureus.

Expanding antibiotic chemical space around the nidulin pharmacophore.

Reinvestigating antibiotic scaffolds that were identified during the Golden Age of antibiotic discovery, but have long since been "forgotten", has proven to be an effective strategy for delivering next-generation antibiotics capable of combatting multidrug-resistant superbugs. In this study, we have revisited the trichloro-substituted depsidone, nidulin, as a selective and unexploited antibiotic lead produced by the fungus Aspergillus unguis. Manipulation of halide ion concentration proved to be a powerful tool for modulating secondary metabolite production and triggering quiescent pathways in A. unguis. Supplementation of the culture media with chloride resulted in a shift in co-metabolite profile to dichlorounguinols and nornidulin at the expense of the non-chlorinated parent, unguinol. Surprisingly, only marginal enhancement of nidulin was observed, suggesting O-methylation may be rate-limiting. Similarly, supplementation of the media with bromide led to the production of the corresponding bromo-analogues, but also resulted in a novel family of depsides, the unguidepsides. Unexpectedly, depletion of chloride from the media halted the biosynthesis of the non-chlorinated parent compound, unguinol, and redirected biosynthesis to a novel family of ring-opened analogues, the unguinolic acids. Supplementation of the media with a range of unnatural salicylic acids failed to yield the corresponding nidulin analogues, suggesting the compounds may be biosynthesised by a single polyketide synthase. In total, 12 new and 11 previously reported nidulin analogues were isolated, characterised and assayed for in vitro activity against a panel of bacteria, fungi and mammalian cells, providing a comprehensive structure-activity profile for the nidulin scaffold.

Effects of two lichen acids isolated from Pseudevernia furfuracea (L.) Zopf in cultured human lymphocytes.

The present study aims at assessing the efficacies of olivetoric acid (OA) and physodic acid (PA) isolated from Pseudevernia furfuracea (L.) Zopf (Parmeliaceae) in human lymphocytes (HLs) in vitro. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays were performed to establish cytotoxicity in HLs. Besides, oxidative stress and genotoxicity were monitored by estimating the changes of total oxidative stress (TOS) and 8-hydroxy-2'-deoxyguanosine (8-OH-dG) levels, respectively, in HLs. At the same time, OA- and PA-induced total antioxidant capacity (TAC) levels in HLs were determined. Although especially low concentrations of OA (IC50=109.94 mg/L) and PA (IC50=665.49 mg/L) did not show cytotoxic effect at high levels in HLs, it was revealed that cytotoxicity was significantly (p<0.05) associated with oxidative stress and genotoxicity via correlation analysis. While TOS level in HLs did not statistically (p>0.05) increase in the presence of all treatments (0.5-100 mg/L) of PA, TAC level was increased by PA applications in certain concentrations (0.5-10 mg/L). Overall, the obtained data indicate that OA and especially PA as lichen compounds that do not cause oxidative stress can be a new resource of therapeutics as recognized in the present study with their high antioxidant features.

Lichen-derived caperatic acid and physodic acid inhibit Wnt signaling in colorectal cancer cells.

Lichens are a source of secondary metabolites which possess important biological activities, including antioxidant, antibacterial, anti-inflammatory, and cytotoxic effects. The anticancer activity of lichens was shown in many types of tumors, including colorectal cancers (CRC). Several studies revealed that the application of lichen extracts diminished the proliferation of CRC cells and induced apoptosis. Colon carcinogenesis is associated with aberrations in Wnt signaling. Elevated transcriptional activity of ß-catenin induces cell survival, proliferation, and migration. Thus, the inhibition of Wnt signaling is a promising therapeutic strategy in colorectal cancer. The aim of this study was the evaluation of the effects of lichen-derived depsides (atranorin, lecanoric acid, squamatic acid) and depsidones (physodic acid, salazinic acid) and a poly-carboxylic fatty acid-caperatic acid, on Wnt signaling in HCT116 and DLD-1 colorectal cancer cell lines. HCT116 cells were more sensitive to the modulatory effects of the compounds. PKF118-310, which was used as a reference ß-catenin inhibitor, dose-dependently reduced the expression of the classical ß-catenin target gene-Axin2 in both cell lines. Lecanoric acid slightly reduced Axin2 expression in HCT116 cells while caperatic acid tended to reduce Axin2 expression in both cell lines. Physodic acid much more potently decreased Axin2 expression in HCT116 cells than in DLD-1 cells. Physodic acid and caperatic acid also diminished the expression of survivin and MMP7 in a cell line and time-dependent manner. None of the compounds affected the nuclear translocation of ß-catenin. This is the first report showing the ability of caperatic acid and physodic acid to modulate ß-catenin-dependent transcription.

Tubulin-binding dibenz[c,e]oxepines: Part 2. Structural variation and biological evaluation as tumour vasculature disrupting agents.

5,7-Dihydro-3,9,10,11-tetramethoxybenz[c,e]oxepin-4-ol 1, prepared from a dibenzyl ether precursor via Pd-catalysed intramolecular direct arylation, possesses broad-spectrum in vitro cytotoxicity towards various tumour cell lines, and induces vascular shutdown, necrosis and growth delay in tumour xenografts in mice at sub-toxic doses. The biological properties of 1 and related compounds can be attributed to their ability to inhibit microtubule assembly at the micromolar level, by binding reversibly to the same site of the tubulin αß-heterodimer as colchicine 2 and the allocolchinol, N-acetylcolchinol 4.

Potential anticancer activity of lichen secondary metabolite physodic acid.

Secondary metabolites present in lichens, which comprise aliphatic, cycloaliphatic, aromatic and terpenic compounds, are unique with respect to those of higher plants and show interesting biological and pharmacological activities. However, only a few of these compounds, have been assessed for their effectiveness against various in vitro cancer models. In the present study, we investigated the cytotoxicity of three lichen secondary metabolites (atranorin, gyrophoric acid and physodic acid) on A375 melanoma cancer cell line. The tested compounds arise from different lichen species collected in different areas of Continental and Antarctic Chile. The obtained results confirm the major efficiency of depsidones. In fact, depsides atranorin and gyrophoric acid, showed a lower activity inhibiting the melanoma cancer cells only at more high concentrations. Whereas the depsidone physodic acid, showed a dose-response relationship in the range of 6.25-50 µM concentrations in A375 cells, activating an apoptotic process, that probably involves the reduction of Hsp70 expression. Although the molecular mechanism, by which apoptosis is induced by physodic acid remains unclear, and of course further studies are needed, the results here reported confirm the promising biological properties of depsidone compounds, and may offer a further impulse to the development of analogues with more powerful efficiency against melanoma cells.

Depsidones from Lichens as Natural Product Inhibitors of M-Phase Phosphoprotein 1, a Human Kinesin Required for Cytokinesis.

M-Phase Phosphoprotein 1 (MPP1), a microtubule plus end directed kinesin, is required for the completion of cytokinesis. Previous studies have shown that MPP1 is upregulated in various types of bladder cancer. This article describes inhibitor screening leading to the identification of a new class of natural product inhibitors of MPP1. Two compounds with structural similarity, norlobaridone (1) and physodic acid (2), were found to inhibit MPP1. Physodic acid is not competitive with ATP, indicating the presence of an allosteric inhibitor-binding pocket. Initial drug-like property screening indicates that physodic acid is more soluble than norlobaridone and has more favorable lipophilicity. However, both suffer from high clearance in human microsomal stability assays mediated by the lability of the lactone ring as well as hydroxylation of the alkyl chains as shown by metabolite identification studies. In cell-based assays physodic acid is a weak inhibitor with EC50 values of about 30 µM in a range of tumor cell lines. The two depsidones identified and characterized here could be used for future improvement of their activity against MPP1 and will be useful chemical probes for studying this unique molecular motor in more depth.

Crystal structure-based discovery of a novel synthesized PARP1 inhibitor (OL-1) with apoptosis-inducing mechanisms in triple-negative breast cancer.

Poly (ADP-ribose) polymerase-1 (PARP1) is a highly conserved enzyme focused on the self-repair of cellular DNA damage. Until now, numbers of PARP inhibitors have been reported and used for breast cancer therapy in recent years, especially in TNBC. However, developing a new type PARP inhibitor with distinctive skeleton is alternatively promising strategy for TNBC therapy. In this study, based on co-crystallization studies and pharmacophore-docking-based virtual screening, we discovered a series of dihydrodibenzo[b,e]-oxepin compounds as PARP1 inhibitors. Lead optimization result in the identification of compound OL-1 (2-(11-(3-(dimethylamino)propylidene)-6,11- dihydrodibenzo[b,e]oxepin )-2-yl)acetohydrazide), which has a novel chemical scaffold and unique binding interaction with PARP1 protein. OL-1 demonstrated excellent potency (inhibiting PARP1 enzyme activity with IC50 = 0.079 µM), as well as inhibiting PARP-modulated PARylation and cell proliferation in MDA-MB-436 cells (BRAC1 mutation). In addition, OL-1 also inhibited cell migration that closely related to cancer metastasis and displayed remarkable anti-tumor efficacy in MDA-MB-436 xenograft model without apparent toxicities. These findings highlight a new small-molecule PAPR1 inhibitor (OL-1) that has the potential to impact future TNBC therapy.

Oxidative C-H bond functionalization and ring expansion with TMSCHN2 : a copper(I)-catalyzed approach to dibenzoxepines and dibenzoazepines.

Tricyclic dibenzoxepines and dibenzazepines are important therapeutic agents for the pharmaceutical industry and academic research. However, their syntheses are generally rather tedious, requiring several steps that involve a Wagner-Meerwein-type rearrangement under harsh conditions. Herein, we present the first copper(I)-catalyzed oxidative CH bond functionalization and ring expansion with TMSCHN2 to yield these important derivatives in a facile and straightforward way.

Chromatographic analysis of olopatadine in hydrophilic interaction liquid chromatography.

In this paper, chromatographic analysis of active substance olopatadine hydrochloride, which is used in eye drops as antihistaminic agent, and its impurity E isomer by hydrophilic interaction liquid chromatography (HILIC) and application of design of experiments (DoE) methodology are presented. In addition, benzalkonium chloride is very often used as a preservative in eye drops. Therefore, the evaluation of its chromatographic behavior in HILIC was carried out as well. In order to estimate chromatographic behavior and set optimal chromatographic conditions, DoE methodology was applied. After the selection of important chromatographic factors, Box-Behnken design was utilized, and on the basis of the obtained models factor effects were examined. Then, multi-objective robust optimization is performed aiming to obtain chromatographic conditions that comply with several quality criteria simultaneously: adequate and robust separation of critical peak pair and maximum retention of the first eluting peak. The optimal conditions are identified by using grid point search methodology. The experimental verification confirmed the adequacy of the defined optimal conditions. Finally, under optimal chromatographic conditions, the method was validated and applicability of the proposed method was confirmed.

FeCl3-catalyzed synthesis of functionally diverse dibenzo[b,f]oxepines and benzo[b]oxepines via alkyne-aldehyde metathesis.

An efficient synthesis of dibenzo[b,f]oxepines and benzo[b]oxepines via FeCl3-catalyzed alkyne-aldehyde metathesis reaction is described. Structurally diverse dibenzo[b,f]oxepines and benzo[b]oxepines have been achieved in good yields with high regio- and chemoselectivity under mild conditions. Notably, among the various catalysts such as Fe(III), Au(III), In(III), Zn(II), Ag(I) and triflic acid, the alkyne-aldehyde metathesis reaction of 2-(2'-phenylethynyl-phenyloxy)-benzaldehyde is only catalyzed by environmentally friendly and sustainable iron(III) chloride.

Metabolically stable dibenzo[b,e]oxepin-11(6H)-ones as highly selective p38 MAP kinase inhibitors: optimizing anti-cytokine activity in human whole blood.

Five series of metabolically stable disubstituted dibenzo[b,e]oxepin-11(6H)-ones were synthesized and tested in a p38α enzyme assay for their inhibition of tumor necrosis factor-α (TNF-α) release in human whole blood. Compared to the monosubstituted dibenzo[b,e]oxepin-11(6H)-one derivatives, it has been shown that the additional introduction of hydrophilic residues at position 9 leads to a substantial improvement of the inhibitory potency and metabolic stability. Using protein X-ray crystallography, the binding mode of the disubstituted dibenzoxepinones and the induction of a glyince flip in the hinge region were confirmed. The most potent compound of this series, 32e, shows an outstanding biological activity on isolated p38α, with an IC50 value of 1.6 nM, extraordinary selectivity (by a factor >1000, Kinase WholePanelProfiler), and low ATP competitiveness. The ability to inhibit the release of TNF-α from human whole blood was optimized down to an IC50 value of 125 nM. With the promising dibenzoxepinone inhibitor 3i, a pharmacokinetic study in mice was conducted.

Effect of four lichen acids isolated from Hypogymnia physodes on viability of rat thymocytes.

Four lichen acids, physodalic acid (F1), physodic acid (F2), 3-hydroxyphysodic acid (F3), and isophysodic acid (F4), were isolated from Hypogymnia physodes methanol extract using preparative reversed-phase high performance liquid chromatography and their structures were determined by UV, MS, (1)H NMR and (13)C NMR. This is the first report on the isolation of F4 from H. physodes. Isolated rat thymocytes were cultivated with increasing F1-F4 concentrations (0.1, 1, 10µg/well) and proliferative activity, viability, ROS (reactive oxygen species) production and MMP (mitochondrial membrane potential) disturbances were evaluated. Obtained results show significantly decreased thymocytes proliferation was observed when cells were treated with F1 (1µg, p<0.05; 10µg; p<0.001), F2 (10µg, p<0.05) and F3 compound (10µg, p<0.05). Significantly increased cytotoxicity was detected when cells were incubated with F1 (1µg, p<0.05; 10µg, p<0.01), F2 (10µg, p<0.05) and F3 compound (10µg, p<0.001). Increased H2DCF-DA fluorescence intensity, when cells were treated with F1 (1µg, p<0.001; 1µg, p<0.01; 10µg, p<0.001) and F2 (1µg, p<0.05; 10µg, p<0.01) compound, indicating the increase of intracellular ROS production. Simultaneously, increased ROS levels were followed with significantly decreased MMP when thymocytes were cultivated with F1 (0.1µg, p<0.001; 1µg, p<0.001; 10µg, p<0.001) and F2 compound (10µg, p<0.001). Thymocytes exposure to increased (0.1, 1, 10µg) concentrations of F3 and F4 compounds did not result with significant alterations in MMP and intracellular ROS production. We have shown that higher F1 and F2 concentrations induce thymocytes toxicity mainly through induction of oxidative stress, while cytotoxicity effect of F3 is followed with altered antioxidant/oxidant balance. The rigid 11H-dibenzo[b,e][1,4]dioxepin-11-one ring in the depsidone structure may play a important role for the examined biological activities.

Novel dibenzo[b,e]oxepinones from the freshwater-derived fungus Chaetomium sp. YMF 1.02105.

Six new dibenzo[b,e]oxepinone metabolites, chaetones A-F (1-6), as well as three known compounds, 1-hydroxy-6-methyl-8-hydroxymethylxanthone (7), citreorosein (8), and emodin (9), were obtained from a freshwater-derived fungal strain Chaetomium sp. YMF 1.02105. Their structures were established on the basis of extensive spectroscopic data analysis and comparison with spectroscopic data reported. Compounds 1-6 are further additions to the small group of dibenzo[b,e]oxepinones represented by arugosins A-H. Compounds 1-7 were tested for their cytotoxic activities against A549, Raji, HepG2, MCF-7, and HL-60 cell lines. The results showed that compound 3 had significant cytotoxicity with IC50 values of 1.2, 1.8, 1.9, 2.3, and 1.6 µg/mL, respectively, against the five cancer cell lines. All compounds showed modest antimicrobial activity against Staphylococcus aureus (ATCC 6538) in standard disk assays.

First total synthesis of dioxepine bastadin 3.

The synthesis of dioxepine bastadin 3, a tyrosine-tyramine derivative with a dibenzo-1,3-dioxepine scaffold that is rarely present among natural products, is described. The dibenzo-1,3-dioxepine ring was formed early in the sequence and the (E)-2-(hydroxyimino)-N-alkylamide was generated in the last step by oxidation of the 2-amino-N-alkylamide precursor. The presumably biogenetic late-stage ring formation starting from congener bastadin 3 failed. A new synthesis of this alkaloid was also developed. This new route requires a minimal use of protecting groups and the order of the two key steps was reversed relative to the route to dioxepine bastadin 3.

Stereoselective syntheses of the antihistaminic drug olopatadine and its E-isomer.

Practical stereoselective synthetic routes to the antihistaminic drug olopatadine and its E-isomer have been developed, the key steps being a trans stereoselective Wittig olefination using a nonstabilized phosphorus ylide and a stereoselective Heck cyclization. The stereoselectivity of the Wittig reaction depends on both the phosphonium salt anion and the cation present in the base used to generate the ylide.

Determination of fatty alcohol ethoxylates and alkylether sulfates by anionic exchange separation, derivatization with a cyclic anhydride and liquid chromatography.

A method for the separation, characterization and determination of fatty alcohol ethoxylates (FAE) and alkylether sulfates (AES) in industrial and environmental samples is described. Separation of the two surfactant classes was achieved in a 50:50 methanol-water medium by retaining AES on a strong anionic exchanger (SAX) whereas most FAE were eluted. After washing the SAX cartridges to remove cations, the residual hydrophobic FAE were eluted by increasing methanol to 80%. Finally, AES were eluted using 80:20 and 95:5 methanol-concentrated aqueous HCl mixtures. Methanol and water were removed from the FAE and AES fractions, and the residues were dissolved in 1,4-dioxane. In this medium, esterification of FAE and transesterification of AES with a cyclic anhydride was performed. Phthalic and diphenic anhydrides were used to derivatizate the surfactants in industrial samples and seawater extracts, respectively. Separation of the derivatized oligomers was achieved by gradient elution on a C8 column with acetonitrile/water in the presence of 0.1% acetic acid. Good resolution between both the hydrocarbon series and the successive oligomers within the series was achieved. Cross-contamination of FAE with AES and vice versa was not observed. Using dodecyl alcohol as calibration standard, and correction of the peak areas of the derivatized oligomers by their respective UV-vis response factors, both FAE and AES were evaluated. After solid-phase extraction on C18, the proposed method was successfully applied to the characterization and determination of the two surfactant classes in industrial samples and in seawater.

Tubulin-binding dibenz[c,e]oxepines as colchinol analogues for targeting tumour vasculature.

Various methoxy- and hydroxy-substituted dibenz[c,e]oxepines were prepared via the copper(I)-induced coupling of ether-tethered arylstannanes or the dehydrative cyclisation of 1,1'-biphenyl-2,2'-dimethanols, assembled using the Ullmann cross-coupling of ortho-bromoaryl carbonyl compounds. The dibenzoxepines were screened for their ability to inhibit tubulin polymerisation and the in vitro growth of K562 human chronic myelogenous leukemia cells. The most active was 5,7-dihydro-3,9,10,11-tetramethoxydibenz[c,e]oxepin-4-ol, whose tubulin inhibitory and cytotoxicity (IC(50)) values were 1 µM and 40 nM, respectively.