Microorganisms from marine environments, especially marine-derived fungus, have proven to be an attractive source of new bioactive secondary metabolites [1,2,3,4,5]. Many of the metabolites possess unique chemical structures and interesting biological activities, such as cytotoxicity against cancer cell lines, antimicrobial activity, inhibition of several enzyme activities and so on [1,2,3,4,5].

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.

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% CHCl₃) and 3 from Fr. 2 (CHCl₃–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].

Compound 1 showed a molecular ion peak at m/z 270 [M]⁺ in the EIMS, and the molecular formula C₁₅H₁₀O₅, 11 degrees of unsaturation, was determined from HREIMS [m/z 270.0531 [M]⁺, Δ +0.3 mmu] and NMR data. IR absorptions of 1 at 1735 and 3435 cm⁻¹ suggested the presence of carbonyl and hydroxyl groups in the molecule. The ¹³C NMR spectrum showed 15 resolved signals, which were classified into one oxygenated methyl, six sp² methine, three sp² quaternary, three oxygenated sp² quaternary and two carbonyl carbons by the analysis of 1D and 2D NMR spectra (Table 1). The ¹H NMR spectrum displayed 10 proton signals, and two signals at δ 4.03 and 12.2 were assigned as a hydroxy proton (1-OH) and methoxy protons (10-OMe), respectively (Table 1). The connectivity of carbons and protons was established by the HMQC correlations.

The presence of two 1,2,3-trisubstituted aromatic rings was assigned by the ¹H–¹H COSY and HMBC correlations of the ¹H NMR signals at δ 6.82 (H-2), 7.61 (H-3), 6.94 (H-4), 7.56 (H-7), 7.77 (H-8) and 7.33 (H-9) (Figure 2). HMBC correlations were observed from 1-OH (δ 12.2) to C-1 (δ 161.8), C-2 (δ 111.0) and C-11a (δ 109.0) and from 10-OMe (δ 4.03) to C-10 (δ 169.5). Therefore, an OH group was attached at the C-1 position and an OMe group at C-10. An HMBC correlation from H-7 (δ 7.56) to C-6 (δ 156.1) and from H-8 to C-6 (⁴J), and the ¹³C chemical shift of C-4a (δ 155.8), an oxygenated sp² carbon, revealed that C-4a and C-6a were connected via C-6 by an ester bond. Therefore, one remaining carbonyl group (δ 181.0, C-11) was inevitably attached between C-10a and C-11a [6,7,8,9]. This assignment was supported by an HMBC correlation from H-7 to C-11 (⁴J). Thus, the structure of compound 1 was assigned as 1-hydroxy-10-methoxy-dibenz[b, e]oxepin-6,11-dione.

Dibenz[b, e]oxepines are rare metabolites, and, thus far, only six natural products (4–9, Figure 3) have been reported [6,7,8,9]. Compound 1 is, therefore, the seventh example of this class of natural products. ¹³C and ¹H NMR data for 1 were very similar to those of the reported values for 5 [7].

Compounds 1–3 were tested for their antimicrobial activity against four test microorganisms (fungus Mucor hiemalis, yeast Candida albicans, Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Escherichia coli) using the paper disk method and for their cytotoxicity against two human cancer cell lines (colon adenocarcinoma HCT-15 and T-cell leukemia Jurkat cells) using the MTT method [12] (Table 2). Compounds 2 and 3 showed inhibition zone of 7 mm against C. albicans at the concentration of 10 μg/disk. Compound 3 inhibited the cell proliferation against HCT-15 and Jurkat cells with IC₅₀ values of 10.7 and 2.3 μM, respectively. On the other hand, compound 1 did not show apparent activity in these bioassays. Further study on biological activity of compound 1 is now in progress.

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.