New Dimeric Members of the Phomoxanthone Family: Phomolactonexanthones A, B and Deacetylphomoxanthone C Isolated from the Fungus Phomopsis sp.

Three new phomoxanthone compounds, phomolactonexanthones A (1), B (2) and deacetylphomoxanthone C (3), along with five known phomoxanthones, including dicerandrol A (4), dicerandrol B (5), dicerandrol (6), deacetylphomoxanthone B (7) and penexanthone A (8), were isolated in the metabolites of the fungus Phomopsis sp. HNY29-2B, which was isolated from the mangrove plants. The structures of compounds 1–3 were established on the basis of spectroscopic analysis. All compounds were evaluated against four human cancer cell lines including human breast MDA-MB-435, human colon HCT-116, human lung Calu-3 and human liver Huh7 by MTT assay. The compounds 4, 5, 7 and 8 showed cyctotoxic activities against tested cancer cell lines (IC50 < 10 μM).


Structural Elucidation of New Compounds
Phomolactonexanthone A (1) was an amorphous yellow powder with the molecular formula C 34 H 33 O 14 (Table 1) suggested that the compound 1 may be structurally related to the blennolide G [16]. It was also an asymmetric dimer of the usual tetrahydroxanthone I and an analogue of rearranged tetrahydroxanthone II ( Figure 2). The monomer I of 1 in conjunction with the 1 H-1 H COSY, HSQC and HMBC data unambiguously verified three moieties: the first moiety was C-5/C-6(C-11)/C-7, the second moiety was C-12/C-10a/C-5 and the third moiety was C-3/C-4.The ring a of monomer I was indicated by the H-C long range correlations of H-7/C-8, C-8a; H-5/C-8a, C-10a, C-13; H-6/C-8; H-14/C-13, together with the first moiety and second moiety ( Figure 2). Therefore, the carbons at δ C 101.1 (C-8a) and δ C 82.7 (C-10a) should be the position of attachment for the other ring. The H-C long range correlations of H-3/C-8′, C-4a, C-1; H-4/C-9a, C-2, C-4a, C-9 and 1-OH/C-9a, C-2, C-1, together with the third moiety, indicated the presence of ring b of monomer I ( Figure 2). It was suggested that the carbons at δ C 106.2 (C-9a) and δ C 157.4 (C-4a) were the position of attachment for the other ring. Both of the H-4 and H-5 displayed the weak four-bond correlation to the carbonyl carbon at δ C 187.7 (C-9), together with both of C-4a and C-10a shifting to the higher field, which verified the presence of ring c of monomer I (Figure 2). In the monomer II, the 1 H-1 H COSY and HMBC data unambiguously identified four moieties: the first moiety was C-9′/C-10′(C-13′)/C-11′/C-12′, the second moiety was C-6′/C-7′, the third moiety was C-4′/C-3′/C-2′/C-14′ and the fourth moiety was C-16′/C-15′. The H-C long range correlations of H-9′/C-12′, together with the first moiety, indicated the presence of γ-lactone ring a′ of monomer II. The carbon at δ C 87.3 (C-9′) was the position of attachment for the other ring ( Figure 2).

Cytotoxic Results
These phomoxanthone compounds (1-8) were evaluated for their cyctotoxic activity in vitro against four human cancer cell lines including human breast MDA-MB-435, human colon HCT-116, human lung Calu-3 and human liver Huh7, compared with their effects on the immortalized human breast epithelial cell line MCF-10A by MTT assay using epirubicin (an anticancer drug used widely in the clinic) as positive control [17]. The results are summarized in Table 3. These phomoxanthone analogues were tested on inhibitory activities against the growth of four tumor cell lines along with the immortalized human breast epithelial cell line. From the Table 3, the phomolactonexanthone A (1), phomolatonexanthone B (2) and deacetylphomoxanthone C (3) showed weak/no cytotoxic activities against tested cancer cell lines, but they displayed no cytotoxic effect on the immortalized human breast epithelial cell line. Dicerandrol A (4) showed broad-spectrum antitumor activity against MDA-MB-435, HCT-116, Calu-3 and Huh7 cell lines with IC50 values of 3.03, 2.64, 1.76 and 4.19 μM, respectively. However, Dicerandrol A also displayed cytotoxic effect on immortalized human breast epithelial cell line. The dicerandrol B (5) and penexanthone A (8) exhibited marked cytotoxic activities against MDA-MB-435, HCT-116 and Calu-3 (IC 50 < 10 μM), and displayed less marked cytotoxic effect on the immortalized human breast epithelial cell line. Moreover Deacetylphomoxanthone B (7) displayed excellent selective activities against HCT-116 and Calu-3 cell lines over other cancer cell lines and showed no cytotoxic effect on the immortalized human breast epithelial cell line. In addition, Dicerandrol A (4) possessed the most potent activity against human live cell line Huh7, but it was reported that dicerandrol A exhibited less cytotoxic activities than dicerandrol B across the two cancer cell lines, HCT-116 and A549 (colon and lung tumor, respectively) [9]. Dicerandrols A and B displayed marked anticancer activities against MDA-MB-435, HCT-116 and Calu-3, while Dicerandrol C exhibited weak anticancer activities. The activity profiles suggested that free hydroxyl groups at C-12 and/or C-12′ of phomoxanthones was the key pharmacophore of Dicerandrols.

General
Melting points were determined on a Fisher Johns hot-stage apparatus and are uncorrected. Optical rotations were measured on a MCP 300 (Anton Paar, Graz, Austria) polarimeter at 25 °C . IR spectra were measured on a Nicolet 5DX-FTIR spectrophotometer. UV spectra were measured on the HPLC (Waters 1525, Westbrook, CT, USA) with PDA detection (Waters 2898, Greenville, SC, USA). NMR data were recorded on Bruker Avance 400, 600 and 500 spectrometers using TMS as an internal standard. HRMS data were measured on LTQ Orbitrap LC-MS (Thermo, Rockford, IL, USA). Column chromatography was performed using silica gel (200-300 mesh, Qingdao Marine Chemcials, Qingdao, China). Semipreparative HPLC was operated on a Waters 1525 LC using an Ultimate XB-C18 column (250 × 10 mm, 5 μm; Welch, Shanghai, China).

Fungal Material
The fungus strain HNY29-2B was isolated from a branch of mangrove plant Acanthus llicifolius collected from the South China Sea in Hainan province, China. It was identified as Phomopsis sp. by Dr. Yayue Liu at School of Chemistry and Chemical Engineering Sun Yat-sen University, and the sequence data have submitted to GenBank (accession no. KF387574). A voucher specimen (registration number HNY29-2B) was deposited at Sun Yat-sen University, Guangdong, China.

Extraction and Isolation
The fungus was fermented onto auto autoclaved rice solid-substrate medium (thirty 1000 mL Erlenmeyer flasks, each containing 80 g of rice and 80 mL of distilled water) for 5 weeks at 25 °C . The fermentation mixture was broken up with a spatula and extracted three times with methanol. The methanol layer was filtered and evaporated to yield crude extract (50.9 g).

Assessment of Antitumor Activity by MTT Assay
Cells were harvested during logarithmic growth phase and seeded in 96-well plates at a density of 1 × 104 cells/mL, and cultured at 37 °C in a humidified incubator (5% CO 2 ) for 24 h, followed by exposure to various concentrations of compounds tested for 48 h. Subsequently 20 μL of MTT (Genview, Houston, TX, USA) solution (5 mg/mL) was added to each well and mixed, the cells were then incubated for an additional 4 h. Culture supernatant was removed using the micropipette, 150 μL of DMSO (Sangon Biotech, Shanghai, China) was added to each well to fully dissolve the MTT-formazan crystals. Cell growth inhibition was determined by measuring the absorbance (Abs) at λ = 570 nm using a microplate reader and calculated according to the following equation:

Growth inhibition = (1 − OD of treated cells/OD of control cells) × 100%
The half maximal inhibitory concentrations (IC 50 ) were obtained from linear regression analysis of the concentration-response curves plotted for each tested compound.

Conclusions
The present work was isolation, structure determination and biological activity of three new phomoxanthone analogues and five known compounds. The phomolactonexanthones A and B are two new phomoxanthone analogues, thereby enlarging the phomoxanthone family. Dicerandrol A (4) showed significant cytotoxic activities against all the tested cancer cell lines. Dicerandrol B (5), Deacetylphomoxanthone B (7) and penexanthone A (8) have selectivity in the antitumor biological assay. The cytotoxic results revealed that some of the phomoxanthone analogues are a potential antitumor drug and/or lead to compounds for constructing an antitumor compound library.