Systematic Studies on Anti-Cancer Evaluation of Stilbene and Dibenzo[b,f]oxepine Derivatives.

Cancer is one of the most common causes of human death worldwide; thus, numerous therapies, including chemotherapy, have been and are being continuously developed. In cancer cells, an aberrant mitotic spindle-a microtubule-based structure necessary for the equal splitting of genetic material between daughter cells-leads to genetic instability, one of the hallmarks of cancer. Thus, the building block of microtubules, tubulin, which is a heterodimer formed from α- and ß-tubulin proteins, is a useful target in anti-cancer research. The surface of tubulin forms several pockets, i.e., sites that can bind factors that affect microtubules' stability. Colchicine pockets accommodate agents that induce microtubule depolymerization and, in contrast to factors that bind to other tubulin pockets, overcome multi-drug resistance. Therefore, colchicine-pocket-binding agents are of interest as anti-cancer drugs. Among the various colchicine-site-binding compounds, stilbenoids and their derivatives have been extensively studied. Herein, we report systematic studies on the antiproliferative activity of selected stilbenes and oxepine derivatives against two cancer cell lines-HCT116 and MCF-7-and two normal cell lines-HEK293 and HDF-A. The results of molecular modeling, antiproliferative activity, and immunofluorescence analyses revealed that compounds 1a, 1c, 1d, 1i, 2i, 2j, and 3h were the most cytotoxic and acted by interacting with tubulin heterodimers, leading to the disruption of the microtubular cytoskeleton.

Oxepinamide F biosynthesis involves enzymatic D-aminoacyl epimerization, 3H-oxepin formation, and hydroxylation induced double bond migration.

Oxepinamides are derivatives of anthranilyl-containing tripeptides and share an oxepin ring and a fused pyrimidinone moiety. To the best of our knowledge, no studies have been reported on the elucidation of an oxepinamide biosynthetic pathway and conversion of a quinazolinone to a pyrimidinone-fused 1H-oxepin framework by a cytochrome P450 enzyme in fungal natural product biosynthesis. Here we report the isolation of oxepinamide F from Aspergillus ustus and identification of its biosynthetic pathway by gene deletion, heterologous expression, feeding experiments, and enzyme assays. The nonribosomal peptide synthase (NRPS) OpaA assembles the quinazolinone core with D-Phe incorporation. The cytochrome P450 enzyme OpaB catalyzes alone the oxepin ring formation. The flavoenzyme OpaC installs subsequently one hydroxyl group at the oxepin ring, accompanied by double bond migration. The epimerase OpaE changes the D-Phe residue back to L-form, which is essential for the final methylation by OpaF.

Cytochrome P450 Can Epoxidize an Oxepin to a Reactive 2,3-Epoxyoxepin Intermediate: Potential Insights into Metabolic Ring-Opening of Benzene.

Dimethyldioxirane epoxidizes 4,5-benzoxepin to form the reactive 2,3-epoxyoxepin intermediate followed by very rapid ring-opening to an o-xylylene that immediately isomerizes to the stable product 1H-2-benzopyran-1-carboxaldehyde. The present study demonstrates that separate incubations of 4,5-benzoxepin with three cytochrome P450 isoforms (2E1, 1A2, and 3A4) as well as pooled human liver microsomes (pHLM) also produce 1H-2-benzopyran-1-carboxaldehyde as the major product, likely via the 2,3-epoxyoxepin. The reaction of 4,5-benzoxepin with cerium (IV) ammonium nitrate (CAN) yields a dimeric oxidized molecule that is also a lesser product of the P450 oxidation of 4,5-benzoxepin. The observation that P450 enzymes epoxidize 4,5-benzoxepin suggests that the 2,3-epoxidation of oxepin is a major pathway for the ring-opening metabolism of benzene to muconaldehyde.

Chemical Equivalent of Arene Monooxygenases: Dearomative Synthesis of Arene Oxides and Oxepines.

Direct epoxidation of aromatic nuclei by cytochrome P450 monooxygenases is one of the major metabolic pathways of arenes in eukaryotes. The resulting arene oxides serve as versatile precursors to phenols, oxepines, or trans-dihydrodiol-based metabolites. Although such compounds have an important biological and chemical relevance, the lack of methods for their production has hampered access to their utility. Herein, we report a general arenophile-based strategy for the dearomative synthesis of arene oxides. The mildness of this method permits access to sensitive monocyclic arene oxides without any noticeable decomposition to phenols. Moreover, this method enables direct conversion of polycyclic arenes and heteroarenes into the corresponding oxepines. Finally, these studies provided direct connection between simple aromatic precursors and complex small organic molecules via arene oxides and oxepines.

Structural and Mechanistic Basis of an Oxepin-CoA Forming Isomerase in Bacterial Primary and Secondary Metabolism.

Numerous aromatic compounds are aerobically degraded in bacteria via the central intermediate phenylacetic acid (paa). In one of the key steps of this widespread catabolic pathway, 1,2-epoxyphenylacetyl-CoA is converted by PaaG into the heterocyclic oxepin-CoA. PaaG thereby elegantly generates an α,ß-unsaturated CoA ester that is predisposed to undergo ß-oxidation subsequent to hydrolytic ring-cleavage. Moreover, oxepin-CoA serves as a precursor for secondary metabolites (e.g., tropodithietic acid) that act as antibiotics and quorum-sensing signals. Here we verify that PaaG adopts a second role in aromatic catabolism by converting cis-3,4-didehydroadipoyl-CoA into trans-2,3-didehydroadipoyl-CoA and corroborate a Δ3,Δ2-enoyl-CoA isomerase-like proton shuttling mechanism for both distinct substrates. Biochemical and structural investigations of PaaG reveal active site adaptations to the structurally different substrates and provide detailed insight into catalysis and control of stereospecificity. This work elucidates the mechanism of action of unusual isomerase PaaG and sheds new light on the ubiquitous enoyl-CoA isomerases of the crotonase superfamily.

Development of Dihydrodibenzooxepine Peroxisome Proliferator-Activated Receptor (PPAR) Gamma Ligands of a Novel Binding Mode as Anticancer Agents: Effective Mimicry of Chiral Structures by Olefinic E/ Z-Isomers.

A novel class of PPARγ ligand 1 (EC50 = 197 nM) with a dibenzoazepin scaffold was identified through high-throughput screening campaign. To avoid the synthetically troublesome chiral center of 1, its conformational analysis using the MacroModel was conducted, focusing on conformational flip of the tricyclic ring and the conformational restriction by the methyl group at the chiral center. On the basis of this analysis, scaffold hopping of dibenzoazepine into dibenzo[ b, e]oxepine by replacing the chiral structures with the corresponding olefinic E/ Z isomers was performed. Consequently, dibenzo[ b, e]oxepine scaffold 9 was developed showing extremely potent PPARγ reporter activity (EC50 = 2.4 nM, efficacy = 9.5%) as well as differentiation-inducing activity against a gastric cancer cell line MKN-45 that was more potent than any other well-known PPARγ agonists in vitro (94% at 30 nM). The X-ray crystal structure analysis of 9 complexed with PPARγ showed that it had a unique binding mode to PPARγ ligand-binding domain that differed from that of any other PPARγ agonists identified thus far.

Genome-based deletion analysis in Aspergillus terreus reveals the acetylaranotin bis-thiomethyltransferase gene.

Acetylaranotin is an epipolythiodiketopiperazine (ETP) secondary metabolite with a broad range of bioactivities. We demonstrated that ATEG_01465.1 located outside of acetylaranotin gene cluster is responsible for catalyzing the S-methylation of its biosynthetic pathway. Combining the previous characterization of acetylaranotin biosynthetic gene cluster together with the identification of its S-methyltransferase provides a means to obtain second-generation acetylaranotin derivatives previously inaccessible. By permutations of targeted deletions of ATEG_01465.1, acetyltransferase (AtaH), and benzoate hydroxylase (AtaY), three novel acetylaranotin derivatives were produced by Aspergillus terreus.

Evaluation of Anti-cancer Activity of Stilbene and Methoxydibenzo[b,f] oxepin Derivatives.

BACKGROUND: Stilbenes, 1,2-diphenylethen derivatives, including resveratrol and combretastatins, show anticancer features especially against tumor angiogenesis. Fosbretabulin, CA-4, in combination with carboplatin, is in the last stages of clinical tests as an inhibitor of thyroid cancer. The mode of action of these compounds involves suppression of angiogenesis through interfering with tubulin (de)polymerization. OBJECTIVE: We have previously synthesized five E-2-hydroxystilbenes and seven dibenzo [b,f]oxepins in Z configuration, with methyl or nitro groups at varied positions. The aim of the present work was to evaluate the anticancer activity and molecular mechanism(s) of action of these compounds. RESULTS: Two healthy, EUFA30 and HEK293, and two cancerous, HeLa and U87, cell lines were treated with four newly synthetized stilbenes and seven oxepins. Two of these compounds, JJR5 and JJR6, showed the strongest cytotoxic effect against cancerous cells tested and these two were selected for further investigations. They induced apoptosis with sub-G1 or S cell cycle arrest and PARP cleavage, with no visible activation of caspases 3 and 7. Proteomic differential analysis of stilbene-treated cells led to the identification of proteins involved almost exclusively in cell cycle management, apoptosis, DNA repair and stress response, e.g. oxidative stress. CONCLUSION: Among the newly synthesized stilbene derivatives, we selected two as potent anticancer compounds triggering late apoptosis/necrosis in cancerous cells through sub-G1 phase cell cycle arrest. They changed cyclin expression, induced DNA repair mechanisms, enzymes involved in apoptosis and oxidative stress response. Compounds JJR5 and JJR6 can be a base for structure modification(s) to obtain even more active derivatives.

Lichen Secondary Metabolite, Physciosporin, Inhibits Lung Cancer Cell Motility.

Lichens produce various unique chemicals that can be used for pharmaceutical purposes. To screen for novel lichen secondary metabolites showing inhibitory activity against lung cancer cell motility, we tested acetone extracts of 13 lichen samples collected in Chile. Physciosporin, isolated from Pseudocyphellaria coriacea (Hook f. & Taylor) D.J. Galloway & P. James, was identified as an effective compound and showed significant inhibitory activity in migration and invasion assays against human lung cancer cells. Physciosporin treatment reduced both protein and mRNA levels of N-cadherin with concomitant decreases in the levels of epithelial-mesenchymal transition markers such as snail and twist. Physciosporin also suppressed KITENIN (KAI1 C-terminal interacting tetraspanin)-mediated AP-1 activity in both the absence and presence of epidermal growth factor stimulation. Quantitative real-time PCR analysis showed that the expression of the metastasis suppressor gene, KAI1, was increased while that of the metastasis enhancer gene, KITENIN, was dramatically decreased by physciosporin. Particularly, the activity of 3'-untranslated region of KITENIN was decreased by physciosporin. Moreover, Cdc42 and Rac1 activities were decreased by physciosporin. These results demonstrated that the lichen secondary metabolite, physciosporin, inhibits lung cancer cell motility through novel mechanisms of action.

Biosynthetic pathway for the epipolythiodioxopiperazine acetylaranotin in Aspergillus terreus revealed by genome-based deletion analysis.

Epipolythiodioxopiperazines (ETPs) are a class of fungal secondary metabolites derived from diketopiperazines. Acetylaranotin belongs to one structural subgroup of ETPs characterized by the presence of a seven-membered 4,5-dihydrooxepine ring. Defining the genes involved in acetylaranotin biosynthesis should provide a means to increase the production of these compounds and facilitate the engineering of second-generation molecules. The filamentous fungus Aspergillus terreus produces acetylaranotin and related natural products. Using targeted gene deletions, we have identified a cluster of nine genes (including one nonribosomal peptide synthetase gene, ataP) that is required for acetylaranotin biosynthesis. Chemical analysis of the wild-type and mutant strains enabled us to isolate 17 natural products from the acetylaranotin biosynthesis pathway. Nine of the compounds identified in this study are natural products that have not been reported previously. Our data have allowed us to propose a biosynthetic pathway for acetylaranotin and related natural products.

Protuboxepin A, a marine fungal metabolite, inducing metaphase arrest and chromosomal misalignment in tumor cells.

Previously we reported the identification of a new oxepin-containing diketopiperazine-type marine fungal metabolite, named protuboxepin A which showed antiproliferative activity in several cancer cell lines. In this study we elucidated the mechanism by which protuboxepin A induces cancer cell growth inhibition. Here we report that protuboxepin A induced round-up morphology, M phase arrest, and an increase in the subG(1) population in tumor cells in a dose dependent manner. Our investigations revealed that protuboxepin A directly binds to α,ß-tubulin and stabilizes tubulin polymerization thus disrupting microtubule dynamics. This disruption leads to chromosome misalignment and metaphase arrest which induces apoptosis in cancer. Overall, we identified protuboxepin A as a microtubule-stabilizing agent which has a distinctly different chemical structure from previously reported microtubule inhibitors. These results indicate that protuboxepin A has a potential of being a new and effective anti-cancer drug.

New stress metabolite from Bulbophyllum kwangtungense.

A new dibenz[b,f]oxepin (1) was found to be produced as a stress metabolite from the leaves and stems of Bulbophyllum kwangtungense Schlecht, in response to abiotic stress elicitation by CuCl2. The structure of 1 was established by spectroscopic and spectrometric means.

Studies on the mechanism of ring hydrolysis in phenylacetate degradation: a metabolic branching point.

The widespread, long sought-after bacterial aerobic phenylalanine/phenylacetate catabolic pathway has recently been elucidated. It proceeds via coenzyme A (CoA) thioesters and involves the epoxidation of the aromatic ring of phenylacetyl-CoA, subsequent isomerization to an uncommon seven-membered C-O-heterocycle (oxepin-CoA), and non-oxygenolytic ring cleavage. Here we characterize the hydrolytic oxepin-CoA ring cleavage catalyzed by the bifunctional fusion protein PaaZ. The enzyme consists of a C-terminal (R)-specific enoyl-CoA hydratase domain (formerly MaoC) that cleaves the ring and produces a highly reactive aldehyde and an N-terminal NADP(+)-dependent aldehyde dehydrogenase domain that oxidizes the aldehyde to 3-oxo-5,6-dehydrosuberyl-CoA. In many phenylacetate-utilizing bacteria, the genes for the pathway exist in a cluster that contains an NAD(+)-dependent aldehyde dehydrogenase in place of PaaZ, whereas the aldehyde-producing hydratase is encoded outside of the cluster. If not oxidized immediately, the reactive aldehyde condenses intramolecularly to a stable cyclic derivative that is largely prevented by PaaZ fusion in vivo. Interestingly, the derivative likely serves as the starting material for the synthesis of antibiotics (e.g. tropodithietic acid) and other tropone/tropolone related compounds as well as for ω-cycloheptyl fatty acids. Apparently, bacteria made a virtue out of the necessity of disposing the dead-end product with ring hydrolysis as a metabolic branching point.

Apoptosis-inducing effect of diketopiperazine disulfides produced by Aspergillus sp. KMD 901 isolated from marine sediment on HCT116 colon cancer cell lines.

AIMS: Research is to identify the bioactive secondary metabolites produced by Aspergillus sp. KMD 901 isolated from marine sediment and to assess their apoptosis-inducing effects. METHODS AND RESULTS: Aspergillus sp. KMD 901 was isolated from marine sediment obtained from the East Sea of Korea. An ethyl acetate extract of KMD 901 exhibited potent cytotoxic activity towards five cancer cell lines (HCT116, AGS, A549, MCF-7 and HepG2). Sequencing of the internal transcribed spacer (ITS) region in this strain allowed us to identify KMD 901 as a strain of Aspergillus versicolor. The cytotoxic compounds from Aspergillus sp. KMD 901 were purified by reversed-phase high-performance liquid chromatography and identified as diketopiperazine disulfides through spectroscopic analyses including extensive 2D NMR and mass spectrometry. The diketopiperazine disulfides were found to induce apoptosis in HCT116 cells based on cell morphology, DNA fragmentation observed by agarose gel electrophoresis, Annexin-V/PI staining using a flow cytometer and cleavage of poly (ADP-ribose) polymerase (PARP), caspase-3, caspase-8, caspase-9 and Bcl-2 family proteins (Bcl-2, Bcl-xL and Bax) using Western blotting analysis. Further study using an in vivo xenograft model showed inhibitory effects of acetylapoaranotin (2) on tumour proliferation. CONCLUSION: A new diketopiperazine disulfide, deoxyapoaranotin (3), along with previously described acetylaranotin (1) and acetylapoaranotin (2) was separated from Aspergillus sp. KMD 901 and found to have direct cytotoxic and apoptosis-inducing effects towards HCT116 colon cancer cell lines. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that the diketopiperazine disulfides produced from Aspergillus sp., KMD 901, could be candidates for the development of apoptosis-inducing antitumour agents. Also, this study indicates that marine natural products as potential source of pharmaceuticals.

Modeling the formation and reactions of benzene metabolites.

One or more of the muconaldehyde isomers is a putative product of benzene metabolism. As muconaldehydes are highly reactive dienals and potentially mutagenic they might be relevant to the carcinogenicity of benzene. Muconaldehydes may be derived through the action of a cytochrome P450 mono-oxygenase on benzene oxide-oxepin, which are established metabolites of benzene. Oxidation of benzene oxide-oxepin either by the one-electron oxidant cerium(IV) ammonium nitrate (CAN) or by iron(III) tris(1,10-phenanthroline) hexafluorophosphate in acetone at -78 degrees C or acetonitrile at -40 degrees C gave (E,Z)-muconaldehyde, which was a single diastereoisomer according to analysis by (1)H NMR spectroscopy. Reaction of toluene-1,2-oxide/2-methyloxepin with CAN gave (2E,4Z)-6-oxo-hepta-2,4-dienal. Similarly, the action of CAN on 1,6-dimethylbenzene oxide-2,7-dimethyloxepin gave (3Z,5E)-octa-3,5-diene-2,7-dione. In vivo, benzene oxide-oxepin could suffer one-electron oxidation by cytochrome P450 mono-oxygenase giving (E,Z)-muconaldehyde. The observations presented may be relevant to the toxicology of benzene oxide-oxepin and other arene oxide-oxepins as we have previously shown that (E,Z)-muconaldehyde, analogously to (Z,Z)-muconaldehyde, affords pyrrole adducts with the exocyclic amino groups of the DNA bases adenine and guanine. Independent of their possible toxicological significance, the experiments described provide preparatively useful routes to (E,Z)-muconaldehyde and its congeners. Methods are also described for the trapping and analysis of reactive benzene metabolites, e.g. using the Diels-Alder reaction with the dienophile 4-phenyl-1,2,4-triazoline-3,5-dione to trap arene oxides and with the diene 1,3-diphenylisobenzofuran to trap enals.

Microbial Baeyer-Villiger oxidation of terpenones by recombinant whole-cell biocatalysts--formation of enantiocomplementary regioisomeric lactones.

Recombinant whole-cell expression systems for Baeyer-Villiger monooxygenases of various bacterial origin were utilized in the regiodivergent biooxidation of cyclic terpenones enabling access to enantio- and regioisomeric lactones on preparative scale.

1,5-Benzodioxepin derivatives as a novel class of muscarinic M3 receptor antagonists.

The structure-activity relationships of novel 1,5-benzodioxepin derivatives as muscarinic M(1)-M(3) receptor antagonists are reported. Some of these compounds were found to possess high binding affinity for the muscarinic M(3) receptor and potent effect on rhythmic increase in bladder pressure in unanesthetized rats following oral administration. These compounds displayed selectivity for the bladder over the salivary gland.

Simultaneous analytical method for the determination of TCH346 and its four metabolites in human plasma by liquid chromatography/tandem mass spectrometry.

TCH346 (dibenzo[b,f]oxepin-10-ylmethyl-prop-2-ynylamine) is a novel propargylamine compound under investigation as a putative agent in the treatment of chronic neurodegenerative illnesses. To support clinical studies an analytical method was developed for TCH346 plus its three amine metabolites and a carboxylic acid metabolite in human plasma. Using a two-step liquid-liquid extraction, one under acidic and one under basic conditions, by pH-switching both the basic and acidic analytes were extracted from 0.5 mL of plasma. All these basic and acidic compounds could be analyzed simultaneously using gradient high-performance liquid chromatographic (HPLC) separation with positive/negative selected reaction monitoring mass spectrometry. As a result of the validation study, the analytical method was shown to be appropriate for the determination of TCH346 and its metabolites CGP70861, GP42120, CGP71090, and GP54840 in plasma for forthcoming clinical studies. The LLOQs were set to 2, 200, 20, 20, and 200 pg/mL for TCH346, CGP70861, GP42120, CGP71090, and GP54840, respectively, and the ULOQ for all analytes was 20 000 pg/mL. All analytes were stable in 50% MeOH at 4 degrees C for at least one year, in human plasma stored below -70 degrees C for at least 7 months, in human plasma below -18 degrees C for at least 6 months, in human plasma at room temperature for at least 1 day, and in the final extract solution at 4 degrees C for at least 3 days.

Dimethyldioxirane converts benzene oxide/oxepin into (Z,Z)-muconaldehyde and sym-oxepin oxide: modeling the metabolism of benzene and its photooxidative degradation.

Oxidation of 7-oxabicyclo[4.1.0]hepta-2,4-diene (benzene oxide)/oxepin with dimethyldioxirane (DMDO) gave mainly (Z,Z)-muconaldehyde, with complete diastereoselectivity. Similarly, 2-methyl-7-oxabicyclo[4.1.0]hepta-2,4-diene (toluene 1,2-epoxide)/2-methyloxepin gave (Z,Z)-1,6-dioxohepta-2,4-diene, while 2,6-dimethyl-7-oxabicyclo[4.1.0]hepta-2,4-diene (1,2-dimethylbenzene 1,2-epoxide)/2,7-dimethyloxepin gave (Z,Z)-2,7-dioxo-3,5-octadiene. By monitoring the DMDO oxidation of benzene oxide/oxepin by 1H NMR spectroscopy, a significant byproduct was identified as 4,8-dioxabicyclo[5.1.0]octa-2,5-diene (sym-oxepin oxide). This observation supports the hypothesis that the route to (Z,Z)-muconaldehyde proceeds from oxepin via 6,8-dioxabicyclo[5.1.0]octa-2,4-diene (oxepin 2,3-oxide), with a minor pathway leading to sym-oxepin oxide. The DMDO oxidations described provide model systems for the cytochrome P450-dependent metabolism of benzene and for the atmospheric photooxidation of benzenoid hydrocarbons.

Structure-activity relationships in the mutagenicity and cytotoxicity of putative metabolites and related analogs of benzene derived from the valence tautomers benzene oxide and oxepin.

A series of putative metabolites and related analogs of benzene, derived from the valence tautomers benzene oxide and oxepin, was tested for mutagenicity (reversions to histidine prototrophy and forward mutations to resistance to 8-azaguanine) and for cytotoxicity by the Ames Salmonella mutagenicity test. Benzene was not mutagenic in either assay. The benzene oxide-oxepin system and benzene dihydrodiol induced point mutations but not frameshifts. 4,5-sym-Oxepin oxide, which is a putative metabolite of the oxepin valence tautomer; 3,6-diazo-cyclohexane-1,6-3,4-dioxide, a synthetic precursor of sym-oxepin oxide; and transoid-4,11-dioxatricyclo(5.1 0)undeca-1,6-diene, a stable bridge-head diene analog of sym-oxepin oxide, were toxic but not mutagenic in both assays. 4H-Pyran-4-carboxaldehyde, a stable acid catalyzed rearrangement product of sym-oxepin oxide, was not mutagenic and much less cytotoxic than sym-oxepin oxide. Stable analogs of the valence tautomer benzene oxide, namely syn-indan-3a,7a-oxide and syn-2-hydroxyindan-3a,7a-oxide, were mutagenic and induced point mutations. All compounds were cytotoxic to Salmonella. Firstly, the apparent decay times of these chemicals, especially that of sym-oxepin oxide, were surprisingly longer than expected, as judged by quantitative plate diffusion assays. Secondly, it is concluded that if benzene oxide is further metabolized in its oxepin tautomeric form, toxic but not mutagenic products are formed. Thirdly, the relatively weak mutagenicity of benzene oxide may be mainly due to its instability and corresponding low probability to reach intracellular polynucleotide targets, whereas stable analogs of benzene oxide are relatively more potent mutagens.