Chondrosterins A–E, Triquinane-Type Sesquiterpenoids from Soft Coral-Associated Fungus Chondrostereum sp.

The marine fungus Chondrostereum sp. was collected from a soft coral Sarcophyton tortuosum from the South China Sea. This fungus was cultured in potato dextrose broth medium and the culture broth was extracted with EtOAc. Five new triquinane-type sesquiterpenoids, chondrosterins A–E (1–5), and the known sesquiterpenoid hirsutanol C (6), were isolated. The structures were elucidated mainly on the basis of NMR, MS, and X-ray single-crystal diffraction data. Chondrosterin A (1) showed significant cytotoxic activities against cancer lines A549, CNE2, and LoVo with IC50 values of 2.45, 4.95, and 5.47 μM, respectively.


Introduction
Sarcophyton tortuosum is the most abundant soft coral in the shallow water regions of Hainan Sanya National Coral Reef Reserve, China.Our previous metabolites isolation work on this soft coral afforded six novel tetraterpenoids, methyl sartortuoate [1][2][3], methyl isosartortuoate [4], and methyl tortuoate A-D [5][6][7].Our current studies concentrate on microorganisms, e.g., bacteria and fungi, OPEN ACCESS associated with Sarcophyton tortuosum, with the main goal to discover novel metabolites with potent pharmacological properties.Forty-nine fungal strains were purified in our primary isolation.These fungi purifications were conducted using small-scale fermentation.The EtOAc extracts of their culture broth were screened for their cytotoxicity.The fungal strain, Chondrostereum sp.nov.(collection No. SF002), was cultured in glucose-peptone-yeast extract (GPY) medium and potato dextrose broth (PDB) medium.Both of the metabolites extracts showed significant cytotoxic activities with a 90% inhibitor ratio against the CNE2 cell line at 20 μg/mL.Investigation of the metabolites of Chondrostereum sp.cultured in GPY medium led to the isolation and structural determination of a new hirsutane sesquiterpenoid hirsutanol E, together with the known compounds hirsutanol A and gloeosteretriol.Hirsutanol A exhibited potent cytotoxic activities against various cancer cell lines, and can induce autophagic cell death by increasing ROS production [8,9].The HPLC-MS data of GPY and PDB culture extracts showed distinct differences.This indicated that some metabolites in PDB medium were not present in GPY medium.Furthermore, the 13 C NMR spectrum of the semi-pure EtOAc extract of Chondrostereum sp. in PDB medium showed many carbonyl carbon resonance signals (δ C > 200).To the best of our knowledge, the carbonyl group is common in the naturally occurring hirsutane sesquiterpenoids.Therefore, the crude extract was assumed to be rich in hirsutane-type compounds, especially containing carbonyl groups.The isolation work on the extract from PDB medium resulted in the characterization of five new triquinane-type sesquiterpenoids, chondrosterins A-E (1-5), and the known sesquiterpenoid hirsutanol C (6).The structures were elucidated mainly based on the NMR, MS, and X-ray single-crystal diffraction experiments data.Chondrosterin A (1) showed significant cytotoxic activities.Herein we describe the structure elucidation and biological evaluation of these compounds (1-6, Figure 1).

Results and Discussion
Chondrosterin A (1) was obtained as yellowish oil.The molecular formula of 1 was established as C 15 H 20 O 2 , based on the HREIMS peak at m/z 232.1456 [M] + and 13 C NMR data (Table 1).The strong IR absorptions at 3433 and 1693 cm −1 indicated the presence of hydroxyl and conjugated carbonyl groups, respectively.The 13 C NMR and DEPT spectra displayed three methyls, four methylenes, two methines and six quaternary carbons.One carbonyl carbon (δ C 197.3), one trisubstituted double bond (δ C 186.9; δ C 125.0, δ H 6.02, d, J = 1.5 Hz), and one terminal double bond (δ C 154.0; δ C 113.4, δ H 5.89, s, and 5.16, s) represented three double bond equivalents.Thus, 1 must be tricyclic to account for the six double bond equivalents required by the molecular formula.The hydroxyl group was attached to quaternary carbon C-8 (δ C 92.5), based on the large chemical shift.Two methyl groups with singlets at δ H 1.12 and 1.19 were connected to quaternary carbon C-10 (δ C 43.4), the other methyl group with singlet at δ H 1.14 was connected to C-2 (δ C 52.6), on the basis of their HMBC correlations (Figure 2).The cross-peaks of H-1/H-11 in 1 H-  Chondrosterin B (2) was isolated as yellowish oil.The HREIMS displays a molecular ion peak at m/z 246.1250 corresponding to the molecular formula C 15 H 18 O 3 .The UV λ max 301 nm indicated the presence of a long conjugated system.Two carbonyl carbons (δ C 210.5 and 207.6) and two trisubstituted double bonds (δ C 126.9, δ H 6.20, s, and δ C 187.5; δ C 126.2, δ H 7.12, s, and δ C 158.0) suggested that 2 also possessed a tricyclic system.Three methyl groups with singlets (δ H 1.04, 1.18 and 1.40) and one methyl group with doublet (δ H 1.16, d, J = 7.0 Hz) which connected with methine carbon C-3 (δ C 48.5, δ H 2.99, q, J = 7.0 Hz) are diagnostic resonance signals of hirsutane sesquiterpenoids.The hydroxyl group was placed at quaternary carbon C-1 (δ C 84.0).The methylene at δ C 41.7 was assigned to 11-position due to the HMBC correlations between H-11 and C-1, C-10, C-14, C-15.The HMBC correlations (Figure 3) of H-5/C-4, H-5/C-6, H-7/C-5, H-7/C-6, H-7/C-8, and H-7/C-9 allowed to establish the large conjugated system.Compound 6 was identified as hirsutanol C, which was firstly isolated by Crews and co-workers from an unidentified fungus [10].Its NMR data recorded in DMSO-d 6 (Tables 1 and 2    Three cancer cell lines: human lung cancer cell line A549, human nasopharyngeal carcinoma cell line CNE2, and human colon cancer cell line LoVo, were used to evaluate the cytotoxic activities of 1-6 in vitro.As a result, 1 showed potent cytotoxicity against these cancer cell lines with the IC 50 values of 2.45, 4.95, and 5.47 μM, respectively.In contrast, 2-6 were apparently inactive in this assay (IC 50 > 200 μM).

General Experimental Procedures
Preparative HPLC was conducted on a Shimadzu LC-20AT HPLC pump equipped with a SPD-20A dual λ absorbance detector and Shim-pack PRC-ODS HPLC column (250 × 20 mm).Melting points were measured on an X-6 micro-melting-point apparatus (Beijing Fukai Science and Technology Development, Beijing, China) and were uncorrected.Optical rotations were acquired using a Schmidt and Haensch polartronic HNQW5 optical rotation spectrometer.IR spectra were recorded on a Nicolet Avatar 330 FT-IR spectrophotometer.UV spectra were recorded on a Shimadzu UV-Vis-NIR spectrophotometer.1D and 2D NMR spectra were recorded on a Varian Inova-500 spectrometer and Bruker Avancell-400 spectrometer.The chemical shifts were referenced to the residual solvent signal (CDCl 3 : δ H 7.26 and δ C 77.0; DMSO-d 6 : δ H 2.50 and δ C 39.43).HPLC-MS analyses were performed with Thermo Finnigan LCQ TM DECA XP liquid chromatography-mass spectrometry.Mass spectra were obtained on Thermo DSQ EI low resolution mass spectrometer and Thermo MAT95XP EI high resolution mass spectrometer.X-ray diffraction data were acquired on a Bruker SMART 1000 CCD X-ray single crystal diffractometer.

Fungal Strain and Culture Method
Chondrostereum sp. was isolated from the inner tissue of soft coral Sarcophyton tortuosum collected from Hainan Sanya National Coral Reef Reserve, China.This fungal strain was maintained on potato dextrose agar (PDA) slants.Fermentation medium was potato dextrose broth (PDB, potatoes 200 g, dextrose 20 g, seawater 1 L).Plugs of agar supporting mycelia growth were cut and transferred aseptically to a 500 mL Erlenmeyer flask containing 200 mL PDB liquid medium.The liquid medium was sterilized at 120 °C for 30 min.The flask was incubated at 28 °C on a rotary shaker (120 rpm) for 5 days.The mycelia were aseptically transferred to 500 mL Erlenmeyer flasks containing 200 mL of the same liquid medium.The flasks were incubated at 28 °C on a rotary shaker (120 rpm) for 20 days.
CCDC 847843 and 847844 contain the supplementary crystallographic data of compounds 4 and 6 respectively [13].

Cytotoxicity Assay
The in vitro cytotoxicity of 1-6 was determined by means of the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay.The tested human cancer cell lines were seeded in 96-well plates at a density of 3 × 10 7 cells/L, and the compounds were added at various concentrations (0.125-50 mg/L).After 48 h, MTT was added to the culture medium at a final concentration of 0.5 mg/mL, and the plates were incubated for 4 h at 37 °C.The supernatant was removed.The formazan crystals were dissolved in DMSO (150 μL) with gentle shaking at room temperature.The absorbance at 570 nm was recorded with a microplate reader (Bio-Rad, USA), and the data were analyzed with the SPSS [14].

Conclusions
The marine fungus Chondrostereum sp. was cultured in PDB medium and afforded five new sesquiterpenoids, chondrosterins A-E (1-5), and the known compound hirsutanol C (6). 1-4 and 6 are hirsutane-type sesquiterpenoids, 5 has a novel rearranged hirsutane skeleton, which could be derived by migration of a methyl group from C-2 to C-6.Chondrosterin A (1), with the typical α-methylene ketone group, showed significant cytotoxic activities.These results indicated the metabolites produced by Chondrostereum sp. in PDB medium were different from those in GPY medium.By altering the fermentation conditions, e.g.carbon and nitrogen source, inorganic salts, Chondrostereum sp. can produce highly functionalized hirsutane derivatives with a surprising chemodiversity.Furthermore, the metabolites isolation work based on 13 C NMR screening seems effectively to obtain the novel hirsutane-type compounds containing carbonyl groups.

Figure 6 .
Figure 6.Molecular structure of 4 in the crystal.Thermal ellipsoids are plotted at 30% probability level.
) were slightly different from the reference data recorded in CD 3 OD and dioxane-d 8 .The relative configuration was established by single-crystal X-ray diffraction.In the crystal structure of 6, the molecules related by a simple translation along the b-axis are connected by pairs of O3-H•••O1 hydrogen bonds to form a ribbon, which is further consolidated by the bridging water molecules with pairs of O1w-H•••O3 and O1-H•••O1w hydrogen bonds.The composite ribbons are further joined together with O1w-H•••O2 hydrogen bonds to form a double layer parallel to the (001) family of planes.Adjacent layers are stacked together with the hydrophobic hydrocarbon skeleton pointing outwards (Figure 8).
a Measured in CDCl 3 , and CDCl 3 was used as an internal standard (δ H 7.26); b Measured in DMSO-d 6 , and DMSO-d 6 was used as an internal standard (δ H 2.50).