New Metabolites and Bioactive Chlorinated Benzophenone Derivatives Produced by a Marine-Derived Fungus Pestalotiopsis heterocornis

Four new compounds, including two isocoumarins, pestaloisocoumarins A and B (1, 2), one sesquiterpenoid degradation, isopolisin B (4), and one furan derivative, pestalotiol A (5), together with one known isocoumarin, gamahorin (3), and three chlorinated benzophenone derivatives, pestalachloride B (6), pestalachloride E (7) and a mixture of pestalalactone atropisomers (8a/8b), were isolated from a culture of the fungus Pestalotiopsis heterocornis associated with sponge Phakellia fusca. These new chemical structures were established using NMR and MS spectroscopic data, as well as single-crystal X-ray crystallographic analysis and CD Cotton effects. All of the isolated compounds were evaluated for their antimicrobial and cytotoxic activities. Isocoumarins 1–3, showed antibacterial activities against Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis with MIC values ranging from 25 to 100 μg/mL and weak antifungal activities. Chlorinated benzophenone derivatives 6–8 exhibited antibacterial activities against S. aureus and B. subtilis with MIC values ranging from 3.0 to 50 μg/mL and cytotoxicities against four human cancer cell lines with IC50 values of 6.8–87.8 μM.

In recent decades, the genus Pestalotiopsis has been isolated as an endophyte from the tropical and subtropical rainforest plants [13]. However, only a few fungi of the genus Pestalotiopsis have been Following the above investigations, with the aim of discovering bioactive substances from marine-derived fungi, the secondary metabolites of a culture fermentation of Pestalotiopsis heterocornis, which was isolated from the sponge Phakellia fusca, were investigated. Four new compounds, including two isocoumarins, pestaloisocoumarin A (1) and pestaloisocoumarin B (2), one sesquiterpenoid degradation, isopolisin B (4), and one furan derivative, pestalotiol A (5), together with four known compounds, gamahorin (3) [15], pestalachloride B (6) [16], pestalachloride E (7) [17] and a mixture of pestalalactone atropisomers (8a/8b) [18,19], were discovered ( Figure 1). The structures of the new compounds were elucidated on the basis of spectroscopic data, circular dichroism (CD) Cotton effects and single-crystal X-ray crystallographic analysis. The cytotoxicities against four human cancer cell lines, antibacterial and antifungal activities against a panel of bacteria and fungi of these isolated compounds were evaluated in the present paper.
The relative configuration of 1 was defined on the basis of single-crystal X-ray diffraction analysis ( Figure 2). The absolute configuration of 1 was determined by the electronic circular dichroism (ECD) spectra with quantum chemical calculations using the time dependent density functional theory (TDDFT) method at the B3 LYP/6-31 + G(d) level. The calculated ECD spectrum showed the same pattern as the experimental ECD spectrum of 1 ( Figure 3). Thus, the absolute configuration of 1 was identified as 3R and 4S and named pestaloisocoumarin A.
The relative configuration of 1 was defined on the basis of single-crystal X-ray diffraction analysis ( Figure 2). The absolute configuration of 1 was determined by the electronic circular dichroism (ECD) spectra with quantum chemical calculations using the time dependent density functional theory (TDDFT) method at the B3 LYP/6-31 + G(d) level. The calculated ECD spectrum showed the same pattern as the experimental ECD spectrum of 1 ( Figure 3). Thus, the absolute configuration of 1 was identified as 3R and 4S and named pestaloisocoumarin A.         The HRESI-MS analysis of 2 showed a deprotonated ion at m/z 279.0865 [M − H] − . Analysis of the 1 H and 13 C NMR data (Table 1) indicated that 2 was very similar to gamahorin (3) [15], except that a doublet methyl in 3 was replaced by a singlet methyl in 2, and an oxygenated quaternary carbon was presented in 2 instead of a methine in 3. Furthermore, one more acetyl appeared in 2. HMBC experiments helped to determine the planar structure. HMBC correlations from H-9 (δ H 1.43) to C-3 (δ C 82.5) and C-4 (δ C 67.9), from H-10 (δ H 1.57) to C-3 (δ C 82.5), C-4 (δ C 67.9) and C-4a (δ C 145.3), from H-11 (δ H 5.17) to C-6 (δ C 136.2), C-7 (δ C 123.6), C-8 (δ C 159.4) and C-13 (δ C 171.1) confirmed that the acetyl was linked with C-11 and that C-4 was oxygenated. Nuclear overhauser effect (NOE) correlations between H-3 (δ H 4.60) and H-10 (δ H 1.57) suggested H-3 and the methyl at C-4 were α-orientated and the methyl at C-3 and the hydroxyl at C-4 were β-orientated. The calculated ECD spectrum of 2 showed excellent agreement with experimental results, and the 3R and 4R configurations of 2 was confirmed ( Figure 3). Thus, Compound 2 was determined and named pestaloisocoumarin B.
All of the isolated compounds were evaluated for their cytotoxic activities against four human cancer cell lines via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) assay (Table 3) and antimicrobial activities against three bacteria and three fungi using a micro broth dilution method (Table 4). Chlorinated benzophenone derivatives 6, 7 and a mixture of 8a/8b exhibited moderate cytotoxicities against four human cancer cell lines with half maximal inhibitory concentration (IC 50 ) values 6.8-87.8 µM; while Compounds 1-5 did not show an obvious inhibition effect against any test cancer cell lines at 100 µM. Isocoumarins 1-3 and chlorinated benzophenone derivatives 6-8 showed antibacterial activities against Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis with minimum inhibitory concentration (MIC) values ranging from 3 to 100 µg/mL. Among them, isocoumarins 1-3 also exhibited weak antifungal activities against three test fungi or part of them with MIC values 100 µg/mL. Compounds 6-8 were inactive against three test fungi at 100 µg/mL, and Compounds 4, 5 did not show antimicrobial activity against any test microorganism at 100 µg/mL.

Fungal Material
The fungal strain, P. heterocornis, was isolated from the sponge P. fusca, which was collected from the Xisha Islands of China in 2012. The strain was identified by Xiuping Lin, and a voucher specimen (No. XWS03F09) was deposited in the CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China.

Fermentation, Extraction and Isolation
The fungal strain P. heterocornis was cultivated in 1000 mL conical flasks containing solid rice medium (each flask contained 200 g of rice, 5 g of artificial sea salt; 200 mL of distilled water, boiled in an autoclave for 20 min at 121 • C, at 28 • C without shaking for 36 days. The total of rice culture was extracted with EtOAc three times. The combined EtOAc extract was evaporated to dryness under reduced pressure to afford 182.5 g of crude extract.

Computational Work
The stable conformational analysis was carried out with SYBYL software (Tripos, San Francisco, CA, USA) using the MMFF94S (Merck Molecular Force Field 94S) force field with an energy cutoff of 10 kcal/mol. The stable conformers were used for geometry optimization at the B3 LYP/6-31G(d) level with a CPCM (conductor-like conductor polarizable continuum model) solvent model for methanol in the Gaussian 09 software. The ECD spectra of stable conformers were then calculated based on the TDDFT method at the B3 LYP/6-31 + G(d) level in methanol [22]. The final ECD curves were generated based on the rotatory strengths with a half-band of 0.3 eV by SpecDis [23] and calculated from the spectra of individual conformers according to their contribution to the Boltzmann weighting. The theoretical spectra have been corrected based on the UV correction.