Cytotoxic Polyketides from the Marine Sponge-Derived Fungus Pestalotiopsis heterocornis XWS03F09

Four new compounds, including two new polyketides, heterocornols M and N (1, 2), and a pair of epimers, heterocornols O and P (3, 4), were isolated from the fermentation broth of the marine sponge-derived fungus Pestalotiopsis heterocornis XWS03F09, together with three known compounds (5–7). The new chemical structures were established on the basis of a spectroscopic analysis, optical rotation, experimental and calculated electronic circular dichroism (ECD). All of the compounds (1–7) were evaluated for their cytotoxic activities, and heterocornols M-P (1–4) exhibited cytotoxicities against four human cancer cell lines with IC50 values of 20.4–94.2 μM.

Recently, we reported sixteen new compounds including heterocornols A-L [11], pestaloisocoumarins A and B, isopolisin B, and pestalotiol A [12], from the fungus Pestalotiopsis heterocornis (XWS03F09), which have been shown to exhibit cytotoxic and antibacterial activities in vitro.
Compound 2 was isolated as a white amorphous solid. Its molecular formula was determined as C 14 H 18 O 4 on the basis of HRESIMS at m/z 273.1120 [M + Na] + (calcd 273.1103). A comparison of the 1 H and 13 C-NMR data with those of heterocornol C [11] revealed a high degree of similarity. The major difference was due to the presence of signals of -CH 2 OHCH 3 moiety in 2. Compared to heterocornol C, compound 2 showed an additional oxygenated quaternary carbon (δ C 103.8) atom signal instead of the methine signal. The HMBC correlations from H-11(δ H 3.94) to C-12 (δ C 14.3) and C-10 (δ C 103.8) and from H-13 (δ H 3.90) to C-14 (δ C 14.8), C-10 (δ C 103.8), and C-9 (δ C 130.1) revealed that the -CH 2 OHCH 3 moiety was located at C-10 (δ C 103.8) in 2.
According to the literature [15,16], the absolute configuration of typical pestalospirane B was determined using NOE, a total synthesis, and an X-ray crystallographic analysis. The similar CD spectral data between compound 2 and pestalospirane B indicated that 2 (C-11 and C-13) possessed the same relative configuration as pestalospirane B (C-12, C-12 ). In addition, considering the possible biosynthesis of 2, we suggest that C-11 and C-13 are also in the S-configuration. Moreover, the NOESY spectrum revealed the relative configuration of 2, as shown in Figure 3. Based on the above discussion, the absolute configuration of 2 was concluded to be 11S,13S, in that the calculated ECD curve of (11S,13S)-2 had a good agreement with the experimental data ( Figure 4). Thus, the structure of 2 was assigned, and named heterocornol N.

Biological Activities
All of the compounds were evaluated for their cytotoxic activities against four human cancer cell lines (Table 3)

Fungal Material
The culture of Pestalotiopsis sp. XWS03F09 was isolated from the sponge Phakellia fusca, which was collected from the Xisha Islands of China. The strain was identified as Pestalotiopsis heterocornis by Xiuping Lin based on DNA amplification and an ITS region sequence analysis. The result showed that the sequence was most similar (100%) to the sequence of P. heterocornis (GenBank database). The strain (No. XWS03F09) was deposited in the School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, China.

Fermentation, Extraction, and Isolation
The strain Pestalotiopsis heterocornis XWS03F09 was inoculated into 1000 mL conical flasks containing a seed medium composed of rice at 200 g/L of, artificial sea salt at 5 g/L, and distilled water at 200 mL/L. The mass fermentation of this fungus was cultivated statically for 60 days. The solid cultures were extracted with EtOAc four times at room temperature, after which the EtOAc solutions were evaporated under reduced pressure to afford 52.2 g of crude extract.

Calculation of ECD Spectra
A conformational analysis was performed by Spartan's 14 using the Merk Molecular Force Field (MMFF) (Tripos, San Francisco, CA, USA) level. The low energy conformations (Boltzmann distribution ≥ 5.0%) of the compounds were submitted to the density functional theory (DFT) optimization at the level of b3lyp/6-31g(d,p), using the cpcm solvation model with the dielectric constant representing methanol in the Gaussian 09 software. The optimized structures were further submitted to the Time-dependent density functional theory (TDDFT) calculations at b3lyp/6-31g(d,p). The ORD values were read and Boltzman averaged using the SpecDis 1.53 (Gaussian, Berlin, Germany).

Cytotoxicity Assay
The cytotoxic activities of compounds 1-7 were tested in human carcinoma cell lines including a human carcinoma cell line (Ichikawa), a human gastric carcinoma cell line (BGC-823), a human liver cancer cell line (HepG2), and a human kidney cancer cell line (7860) [11]. The procedure of the cytotoxic activities evaluation described was as previously reported [11]. Adriamycin was assayed as a positive control.

Conclusions
In conclusion, seven compounds, including four new compounds, namely heterocornols M-P (1-4), together with three known compounds (5-7), were isolated from the marine-derived fungus P. heterocornis. The structures of the isolated compounds were elucidated through the detailed analysis of spectroscopic data as well as CD Cotton effects. All of the isolated compounds (1-7) were evaluated for their cytotoxic activities. Compounds 1, 3/4, 5, and 6 showed cytotoxicities against four human cancer cell lines with IC 50 values of 20.4-94.2 µM.