Additional New Cytotoxic Triquinane-Type Sesquiterpenoids Chondrosterins K–M from the Marine Fungus Chondrostereum sp

By the method of 1H NMR prescreening and tracing the diagnostic proton signals of the methyl groups, three additional new triquinane-type sesquiterpenoids—chondrosterins K–M (1–3) and the known sesquiterpenoid anhydroarthrosporone (4)—were isolated from the marine fungus Chondrostereum sp. Their structures were elucidated on the basis of MS, 1D, and 2D NMR data. Chondrosterin K is a rare hirsutane sesquiterpenoid, in which a methyl group was migrated from C-2 to C-6 and has a double bond between C-2 and C-3. Compounds 1–3 showed significant cytotoxicities against various cancer cell lines in vitro.


Introduction
In recent decades, a large number of novel compounds were isolated from the soft corals collected from the South China Sea, which have significant biological activities, such as antitumor, antivirus, anti-hypertension, anti-inflammatory, and analgesic [1]. However, the limited supply of the soft corals and their pharmaceutical lead compounds makes the drug development a very slow process; so, searching for alternative drug resources has become a crucial task. Marine fungi associated with the soft corals can be expected to metabolize biologically interesting and chemically diverse compounds and draw much attention [2,3]. Naturally occurring sesquiterpenoids with hirsutane frameworks are the typical metabolites of some fungi. Up to now, about fifty hirsutane-type compounds have been reported, and some of them have significant biological activities, such as antibacterial [4][5][6][7], cytotoxic [6][7][8][9], and antimalarial activities [9]. The fungal strain Chondrostereum sp. was isolated from the soft coral of Sarcophyton tortuosum. Previous isolation of metabolites led to the discovery of hirsutane sesquiterpenoid compounds, chondrosterins A-F [10,11], I-J [12], hirsutanols A [13], C [10], E [13], and F [13], incarnal [11], and arthrosporone [11]. Among them, hirsutanol A, incarnal, and chondrosterin A and J ( Figure 1) showed potent cytotoxicities. Hirsutanol A inhibited the growth of cancer cells by increasing the level of reactive oxygen species (ROS) [14][15][16].
In our continued research project, the fungal strain Chondrostereum sp. was cultured in a large-scale glucose-peptone-yeast (GPY) medium. By tracing the characteristic proton NMR signals of the methyl groups around 1.00-1.40 ppm, chemical investigation of the extract led to the discovery of three new triquinane-type sesquiterpenoids, chondrosterins K-M (1)(2)(3), and the known sesquiterpenoid anhydroarthrosporone (4) (Figure 1) from the fungal culture extract. The structures of these metabolites were assigned on the basis of the detailed NMR and MS spectroscopic analysis. The isolation, structure identification, and cytotoxicities of these compounds are reported herein.

Structure Elucidation
Chondrosterin K (1) was isolated as a colorless oil. The HR-EI-MS data at m/z 250.1568 [M] + (Supplementary Figure S1), along with the NMR data (Tables 1 and 2, Supplementary Figures S2-S7) revealed the molecular formula of compound 1 to be C15H22O3, and the degrees of unsaturation are five. The UV absorption at λmax 241 nm indicated a conjugated system formed by the carbonyl group and the double bond. So, this molecule must be tricyclic to count the five degrees of unsaturation. According to the 1 H and 13 C NMR and DEPT data (Tables 1 and 2), compound 1 had three methyls, four methylenes, three methines, and five quaternary carbons. The typical functional groups included one carbonyl carbon (δC 210.1), one tetrasubstituted double bond (δC 186.4 and 135.2), three methyl group singlets (δH 1.01, 1.16, and 1.33), and two hydroxyl groups (δH 2.15, brs, 2H).   Figure 3) revealed that H-1, H-7, H-8, and H-15 have an α-orientation. No NOESY correlation between H-13 and H-1, H-8 was observed, so C-13 was placed at the β position. Compound 1 is an unprecedented hirsutane-type sesquiterpenoid having a C-2/C-3 double bond in the molecule.           Chondrosterin L (2) was isolated as a yellowish oil. The molecular formula of compound 2 was determined as C 15 (Tables 1 and 3, Supplementary Figures S18-S25). Compound 3 has four methyls, three methylenes, two methines, and six quaternary carbons. 1 H NMR data recorded in CDCl 3 revealed three methyl groups with singlets (δ H 1.12, 1.15, and 1.21), and one methyl group with doublet (δ H 1.06) which connected with the methine carbon at C-3 (δ C 52.1, δ H 1.93); these are the diagnostic resonance signals of hirsutane sesquiterpenoids. By comparison, looking at the NMR data with compound 2, quick identification was made that a fragment of the CH 3    Compound 4 was identified as anhydroarthrosporone, which was firstly isolated by Amouzou E and co-workers from a basidiomycete fungus Ceratocystis ulmi [17]. Our NMR data are obviously different from the reference data, although both of them were recorded in the same solvent (CDCl 3 ). For example, our 13 C NMR data of C-1, C-3, C-6, C-7, and C-9 are 63.4, 57.7, 190.8, 43.9, and 55.9, respectively. As a comparison, the corresponding reference values are 57.7, 63.4, 177.0, 55.9, and 44.0, respectively [17].

Biological Evaluation
Seven cancer cell lines were used to examine the cytotoxicities of compounds 1-4 in vitro. This assay revealed that 1-3 had significant cytotoxic effects (Table 4). In contrast, 4 were apparently inactive in this assay (IC 50 values > 100 µM). Hirsutanol A was used as a positive control.

Fungal Material
The marine fungus Chondrostereum sp. was isolated from the inner tissue of a soft coral of the species Sarcophyton tortuosum collected from the Hainan Sanya National Coral Reef Reserve, China. This fungal strain was deposited at School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China, and maintained in sterile aqueous solution of 15% (v/v) glycerol at −80 • C.

Cytotoxic Assay
The in vitro cytotoxicities of 1-4 were determined by means of the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) 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, Hercules, CA, USA), and the data were analyzed with the SPSS 13.0 software package. Hirsutanol A-a potent anticancer agent isolated from marine fungal metabolites-was used as a positive control, and its cytotoxicities against the tested cancer cell lines are shown in Table 4.