Three Bianthraquinone Derivatives from the Mangrove Endophytic Fungus Alternaria sp. ZJ9-6B from the South China Sea

Three new bianthraquinone derivatives, alterporriol K (1), L (2) and M (3), along with six known compounds were obtained from extracts of the endophytic fungus Alternaria sp. ZJ9-6B, isolated from the mangrove Aegiceras corniculatum collected in the South China Sea. Their structures were elucidated by one- and two-dimensional NMR spectroscopy, MS data analysis and circular dichroism measurements. Compounds 1, 2 and 3 were first isolated alterporriols with a C-2–C-2′ linkage. The crystallographic data of tetrahydroaltersolanol B (7) was reported for the first time. In the primary bioassays, alterporriol K and L exhibited moderate cytotoxic activity towards MDA-MB-435 and MCF-7 cells with IC50 values ranging from 13.1 to 29.1 μM.

The alterporriol family of bianthraquinone derivatives were first reported from Alternaria porri by Suemitsu et al. in 1984 [10]. Over the last 27 years, nine additional alterporriols have been reported from fungi. All the alterporriols except alterporriols G-J were described from Alternaria sp. [5,[11][12][13]. In terms of the underlying monomers, alterporriols can occur as either homodimers or heterodimers. With regard to the coupling positions of the monomers, alterporriols A, B, D, E, I and J feature a C-5-C-5′ linkage, alterporriol C shows a C-1-C-7′ connection, and G and H possess a C-7-C-5′ linkage [5,[11][12][13].
As part of our ongoing program to search for new bioactive natural products from the South China Sea [14][15][16], an endophytic fungus Alternaria sp. ZJ9-6B has been isolated from the fruit of the marine mangrove Aegiceras corniculatum in Zhanjiang, Guangdong, China. Chemical investigation of this fungus led to the isolation of nine metabolites, including three new anthraquinone derivatives 1-3 and six known compounds 4-9 ( Figure 1). It is interesting that compounds 1-3 all possess dimeric structures with a C-2-C-2′ linkage. In this report, we describe the isolation, structural elucidation and biological activity of these new metabolites.
An interesting feature of the isolated bianthquinones is their optical activity. Ab initio calculations of CD spectra for several phenylanthraquinones with chiral axes have recently been reported [17,18]. Specific optical rotation of compounds 2 and 3 showed 20 ] [ D α +60 and −30, respectively. CD spectra of 2 and 3 recorded in methanol showed a near quasi-mirror image pattern (Figure 4). In the case of 2, the CD spectrum consists of a small positive band at 480 nm, followed by two moderate negative bands in the 300-400 nm regions, and a stronger negative band centered at 220 nm, then a stronger positive band at 264 nm. For 3, the sequence of bands is similar but their signs are inverted. The CD spectra of 2 and 3 reflect contributions from a chiral axis and three chiral centers. The chiral axis occurring within the chromophore is expected to dominate the observed CD spectrum [18,19] (Figure 4).The chiral center C-7 is expected to have a much smaller contribution to the observed CD spectrum. With these results, compounds 2 and 3 were deduced as two diastereomers and atropisomers [20]. The known compounds were identified as physcion (4), marcrospin (5), dactylariol (6), tetrahydroaltersolanol B (7), alternariol (AOH) (8) and alternariol methyl ether (AME) (9) by spectral analyses and comparison with reported literature data, respectively [21][22][23][24]. The structure of 7 was confirmed by X-ray diffraction analysis ( Figure 5) and its crystallographic data was reported for the first time.

General
Column chromatography (CC) was performed using silica gel (200-300 mesh, Qingdao marine Chemical). The HPLC system consisted of a Waters 2010 series. A mini ODS column (250 × 10 mm, 10 μm particle size) was used. Melting points were determined on an X-4 micro-melting point apparatus and were uncorrected. Circular dichroism was measured on a Schmidt Haensch Polartronic HH W5 polarimeter and was uncorrected. UV spectra were measured on a Shimadzu UV-2501 PC spectrophotometer. IR spectra were measured on a Bruker EQUINOX55 spectrophotometer. 1 H and 13 C NMR data were recorded on a Varian Inova 500 NB and a Burker AVANCE 400 spectrometer, respectively (TMS as internal standard). EIMS were on a Thermo DSQ EI-mass spectrometer. LC/MS data were acquired using an Applied Biosystems/MDS Sciex and ESI source. HR-EIMS were measured on a Thermo MAT95XP High Resolution mass spectrometer. HR-ESIMS were measured on a Schimadzu LCMS-IT-TOF.

Strain Isolation, Taxonomic Classification and Endophyte Fermentation
The fungus Alternaria sp. ZJ9-6B was isolated from the fruit of a mangrove tree Aegiceras Corniculatum collected in Zhanjiang Mangrove, Guangdong province, P.R. China, in 2008. A voucher specimen (registration number: ZJ9-6B) has been deposited in the Natural Products Laboratory and the Department of Applied Chemistry, Sun Yat-sen University, China. It was identified according to a molecular biological protocol by DNA amplification and sequencing of the ITS region as described previously with an ITS sequence GenBank ID: HM 754629. The fungal strain was cultivated in potato dextrose broth (PDB) medium (20 g of dextrose and 3 g of crude sea salt in 1 L of potato infusion). Starter cultures were maintained on cornmeal seawater agar. Plugs of agar supporting mycelia growth were cut and transferred aseptically into a 500 mL Erlenmeyer flask containing 250 mL of liquid medium, and incubated at 28 °C on a rotary shaker for 5-7 days. The mycelium was aseptically transferred into 1000 mL Erlenmeyer flasks containing 500 mL PDB medium and incubated at 28 ± 1 °C for 30 days under stationary conditions.

Cytotoxic Assays
The cytotoxicity of compounds 1 and 2 were evaluated using human breast cancer cell lines MDA-MB-435 and MCF-7 by MTT assay. Cell viability was measured using the CellTiter 96 aqueous nonradioactive cell proliferation assay. Results were expressed as the mean value of triplicate data points.

Conclusions
Alternaria sp. ZJ9-6B is a prolific producer of bioactive metabolites. Nine compounds have been isolated from this fungal strain, including three new alterporriols. These three alterporriols all possess dimeric structures with a C-2-C-2′ linkage. In the primary bioassay, compounds 1 and 2 showed moderate cytotoxic activity against human breast cancer cell lines.