A New Dibenz[b, e]oxepine Derivative, 1-Hydroxy-10-methoxy-dibenz[b, e]oxepin-6,11-dione, from a Marine-Derived Fungus, Beauveria bassiana TPU942

1-Hydroxy-10-methoxy-dibenz[b, e]oxepin-6,11-dione (1) was obtained from the culture broth of a marine-derived fungus, Beauveria bassiana TPU942, isolated from a marine sponge collected at Iriomote Island in Okinawa, together with two known compounds, chrysazin (2) and globosuxanthone A (3). The structure of 1 was elucidated on the basis of its spectroscopic data (HREIMS, 1D and 2D NMR experiments including 1H–1H COSY, HMQC and HMBC spectra). Dibenz[b, e]oxepines are rare in nature, and only six natural products have been reported. Therefore, compound 1 is the seventh natural product in this class. Compounds 2 and 3 showed an antifungal activity against Candida albicans, and 3 inhibited the cell growth against two human cancer cell lines, HCT-15 (colon) and Jurkat (T-cell lymphoma). Compound 1 did not show an apparent activity in the same bioassays.

In the course of our studies on bioactive components produced by marine-derived fungi isolated from tropical and sub-tropical coral reefs, we found that the EtOAc extract of the culture broth from a marine-derived fungus, Beauveria bassiana TPU942, isolated from a marine sponge collected at Iriomote Island, Okinawa Prefecture, Japan, showed cytotoxicity against a human T-cell lymphoma Jurkat cells. Bioassay-guided separation from the EtOAc extract led to the isolation of a new dibenz[b,e]oxepine derivative (1) and two known anthraquinone and xanthone derivatives, chrysazin (2) and globosuxanthone A (3). The structure of compound 1 was elucidated as 1-hydroxy-10-methoxy-dibenz[b,e]oxepin-6,11-dione ( Figure 1) by the analysis of its spectroscopic data. Natural products possessing a dibenz[b,e]oxepine structure are quite rare, and only six compounds have been reported as natural products in scientific journals [6][7][8][9]. Antifungal and cytotoxic activities were exhibited by two known compounds (2 and 3).
We describe herein the isolation and structure elucidation of compound 1 and biological activities of isolated compounds.

Results and Discussion
The producing fungus, B. bassiana TPU942, was isolated from a piece of an unidentified marine sponge collected at Iriomote Island and cultured as described in Experimental Section. The culture broth and the EtOAc extract of strain TPU942 showed cytotoxicity in the screening bioassay against Jurkat cells and was separated into seven fractions (Fr. 1-Fr. 7) using a silica gel column. Compounds 1 and 2 were isolated from Fr. 1 (eluted with 100% CHCl 3 ) and 3 from Fr. 2 (CHCl 3 -MeOH = 100:1) by HPLC (ODS).
Compound 2 was identified as chrysazin [10], a ubiquitous anthraquinone derivative isolated from several organisms, from the spectroscopic data. The structure of compound 3 was assigned on the basis of its spectral data and comparison of these data with those of the reported values for globosuxanthone A [11]. Globosuxanthone A (3) was originally isolated from Chaetominum globosum, and a cytotoxicity against seven human solid tumor cell lines, accumulation of cells at either G2/M or S phase and induction of apoptosis have been described [11].

General
EI mass spectra were obtained by a JEOL JMS-MS 700 mass spectrometer (Tokyo, Japan). 1 H and 13 C NMR spectra were recorded on a JEOL JNM-AL-400 NMR spectrometer (400 MHz for 1 H and 100 MHz for 13 C) in DMSO-d 6 (δ H 2.49, δ C 39.5) or CDCl 3 (δ H 7.26, δ C 77.0). UV spectra were measured on a Hitach U-3310 UV-Visible spectrophotometer (Tokyo, Japan) and IR spectra on a PerkinElmer Spectrum One Fourier transform infrared spectrometer (Waltham, MA, USA). Preparative HPLC was carried out with a Hitachi L-6200 system.

Fermentation and Isolation
Three pieces of an unidentified marine sponge collected in the coral reef of Iriomote Island in Okinawa, Japan were incubated on a 1/2 PDA plate (Difco Laboratories, Detroit, MI, USA). The strain TPU942 was grown from the sponge body and inoculated into a slant (1/10 YSA). The fungus was identified as Beauveria bassiana by the comparison of 217 bp ITS1 rDNA sequence (100% match).
A slant culture of strain TPU942 grown on 1/10 YSA (0.020% yeast extract, 0.10% soluble starch, and 1.5% agar; dissolved in 90% sea water and adjusted to pH 6.0 before sterilization) was inoculated into a 500-mL Erlenmeyer flask containing 100 mL of the seed medium (2.0% glucose, 0.50% polypeptone, 0.050% MgSO 4 · 7H 2 O, 0.20% yeast extract, 0.10% KH 2 PO 4 and 0.10% agar; adjusted to pH 6.0 before sterilization). The flask was shaken reciprocally for three days at 27 °C to obtain the seed culture, which was then transferred to the production medium (3.0% sucrose, 3.0% soluble starch, 1.0% malt extract, 0.30% Ebios (Asahi Food & Healthcare Co. Ltd., Tokyo, Japan), 0.50% KH 2 PO 4 and 0.050% MgSO 4 · 7H 2 O; adjusted to pH 6.0 before sterilization). The production culture was carried out at 27 °C for seven days under the agitation condition. The seven-day-old whole broth (2.0 L) was extracted with 2.0 L of acetone. The extract was filtered and concentrated to remove acetone, and the aqueous solution was extracted with ethyl acetate. The EtOAc extract was dried over Na 2 SO 4 and concentrated in vacuo to dryness to yield a red brown material (1088.3 mg), and the residue was suspended in CHCl 3 and adsorbed on a silica gel column (100 g). The silica gel column was eluted stepwise with each 500 mL of CHCl 3 , a mixture (v/v) of CHCl 3 -CH 3 OH (10:1, 5:1 and 1:1) and CH 3 OH into seven fractions (Fr. 1-Fr. 7). An active Fr. 1 (CHCl 3 eluate) was concentrated in vacuo to dryness to give a brown oil (112.9 mg). A portion (40 mg) of Fr. 1 was purified by a preparative HPLC [column; PEGASIL ODS (Senshu Scientific. Co. Ltd. Tokyo, Japan), 10 × 250 mm; solvent, 90% CH 3 OH; detection, UV at 254 nm; flow rate, 2.0 mL/min] to give compounds 1 (eluted at 11.0 min) and 2 (eluted at 17.8 min) as a pale yellow solid (3.0 mg) and an orange solid (24.1 mg), respectively.
The second active Fr. 2 (CHCl 3 -CH 3 OH = 100:1) was concentrated to yield a brown oil (368.9 mg), and 50 mg of residue was purified by a preparative HPLC (same conditions as Fr. 1) to yield compound 3 (eluted at 7.6 min) as a white solid (4.7 mg).

Cytotoxicity Assay
HCT-15 and Jurkat cells were obtained from the Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Miyagi, Japan). The cell lines were cultured in RPMI-1640 medium. The medium was supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. Exponentially growing cells, cultured in a humidified chamber at 37 °C containing 5.0% CO 2 , were used for experiments.
Cytotoxic activity was evaluated using the colorimetric MTT assay [12]. HCT-15 (1.0 × 10 4 cells in 100 μL) or Jurkat cells (2.0 × 10 4 cells in 100 μL) were added to each well of a 96-well plastic plate (Corning Inc., Corning, NY, USA). A sample (1.0 μL in CH 3 OH) was added to each well to make the final concentration from 0 to 30 μM, and the cells were incubated for 48 hr at 37 °C. MTT (10 μL of 5.5 mg/mL stock solution), and a cell lysate solution (90 μL, 40% N,N-dimethylformamide, 20% sodium dodecyl sulfate, 2.0% CH 3 COOH and 0.030% HCl) were added to each well and the plate was shaken thoroughly by agitation at room temperature overnight. The optical density of each well was measured at 570 nm using an MTP-500 microplate reader (Corona Electric Co., LTD., Ibaraki, Japan).

Conclusions
A new 1-hydroxy-10-methoxy-dibenz[b,e]oxepine-6,11-dione (1) and two known compounds, chrysazin (2) and globosuxanthone A (3), were obtained from a marine-derived fungus, B. bassiana, strain TPU942, isolated from a marine sponge collected in Iriomote Island, Okinawa. Bioactivities observed by the extract of culture broth were reproduced by two known compounds, but compound 1 was not active in the same bioassays. Compound 1 had a rare dibenz[b,e]oxepine structure, and only six natural products have thus far been reported.