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Mono- and Dimeric Naphthalenones from the Marine-Derived Fungus Leptosphaerulina chartarum 3608

Five new naphthalenones, two enantiomers (−)-1 and (+)-1 leptothalenone A, (−)-4,8-dihydroxy-7-(2-hydroxy-ethyl)-6-methoxy-3,4-dihydro-2H-naphthalen-1-one ((−)-2), (4S, 10R, 4’S)-leptotha-lenone B (5), (4R, 10S, 4’S)-leptothalenone B (6), and a new isocoumarine, 6-hydroxy-5,8-dimethoxy-3-methyl-1H-isochromen-1-one (4), along with two known compounds (+)-4,8-dihydroxy-7-(2-hydroxy-ethyl)-6-methoxy-3,4-dihydro-2H-naphthalen-1-one ((+)-2) and (+)-10-norparvulenone (3) were isolated from the marine-derived fungus Leptosphaerulina chartarum 3608. The structures of new compounds were elucidated by HR-ESIMS, NMR, and ECD analysis. All compounds were evaluated for cytotoxicity and anti-inflammatory activity. Compound 6 showed moderate anti-inflammatory activity by inhibiting the production of nitric oxide (NO) in lipopolysaccharide-stimulated RAW264.7 cells, with an IC50 value of 44.5 μM.

Compound 1 was of racemic nature because it lacked any CD (circular dichroism) maximum and optical rotation. Subsequent chiral HPLC purification of (±)-1 led to the separation of the two enantiomers, (−)-1 and (+)-1 (Figure 2a
The known compound, (+)-10-norparvulenone (3) was identified by NMR, MS and optical rotation data analysis and comparison of spectroscopic data with the literature [9]. The optical rotation of 10- which indicated their absolute configurations were opposite. The absolute configuration of 3 was 4S supported by X-ray single-crystal diffraction using anomalous scattering of Cu Kα radiation with Flack parameter = 0.00(6) ( Figure S45). Thus, compound 3 was named as (+)-10-norparvulenone. All the compounds were tested for their inhibition activity against LPS-activated NO production in RAW264.7 cells using the Griess assay. Compound 6 displayed moderate inhibitory effects on the production of NO with an IC 50 value of 44.5 ± 1.1 µM, compared to the positive control indomethacin (IC 50 = 37.5 ± 1.6 µM), while the other compounds showed no significant anti-inflammatory activity (IC 50 > 100 µM). In addition, some of naphthalenones have been found to have cytotoxic activity according to previous studies [2,5,11,12]. Thus, all the isolated compounds were evaluated for their cytotoxicity against A549 (lung cancer), HeLa (cervical cancer), and MCF-7 (breast cancer) human cancer cell lines using MTT assay and displayed no cytotoxicity against all three cell lines at 50 µM.

General Experimental Procedures
Optical rotations were measured on an MCP 200 polarimeter by using an Na lamp (Shimadzu). UV spectra were obtained on a Blue Star A spectrophotometer. A Fourier transformation infra-red spectrometer coupled with infra-red microscope EQUINOX 55 (Bruker, Rheinstetten, Germany) was used to record the IR spectra. NMR spectra were obtained on a Bruker Avance 400 MHz spectrometer with tetramethylsilane as the internal standard. HR-ESIMS data were obtained on a LTQ-Orbitrap LC-MS spectrometer (Thermo Corporation, Waltham, MA, USA). ESIMS spectra were obtained on an ACQUITY QDA (Waters Corporation, Milford, MA, USA). HPLC was carried out on an Essentia LC-16 with an SPD-16 Detector (Shimadzu, Shanghai, China). Column chromatography was carried out on silica gel (100-200 mesh, 200-300 mesh, Qing dao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (GE Healthcare, Littile Chalfont, UK).

Fungal Material
The fungal strain 3608 was isolated from a crinoid collected in Xuwen, Zhanjiang City, Guangdong Province, China, in August 2014. It was identified as L. chartarum by ITS sequence, and the sequence data have been submitted to and deposited in the GenBank database under accession number MF980969. The fungal strain has been preserved at the school of marine science, Sun Yat-Sen University.

Extraction, Isolation, and Characterization
The fungus L. chartarum 3608 was cultured at room temperature (25-30 • C) for one month in 1000 mL Erlenmeyer flasks containing rice medium, composed of 60 mL rice, 80 mL H 2 O, and 3% sea salt. After 30 days of cultivation, the fermented rice substrate of 170 flasks was extracted three times with MeOH to yield the organic extract (70 g). The organic extract was portioned into three phases by successive extraction with n-hexane (43 g), CHCl 3 (8 g), EtOAc (17 g).