Four New Sesquiterpenoids from Cultures of the Fungus Phellinidium sulphurascens

Four new sesquiterpenoids, namely 12-hydroxy-3-oxodrimenol (1), 11-hydroxyacetoxydrim-7-en-3β-ol (2), 2,6-dimethyl-7,10-epoxy-10-hydroxymethyldodeca-2,11-dien-6-ol (3), and 7,10-epoxy-2,6,10-trimethyldodeca-2,11-diene-4,6-diol (4), along with fourteen known compounds, were isolated from the cultures of Phellinidium sulphurascens. The structures of compounds 1–4 were established on the basis of extensive spectroscopic analysis. All of them were evaluated for their cytotoxic activities. Electronic supplementary material The online version of this article (doi:10.1007/s13659-014-0047-x) contains supplementary material, which is available to authorized users.


Results and Discussion
Compound 1, obtained as amorphous powder, possessed a molecular formula C 15 ). The 13 C NMR spectrum of 1 showed fifteen carbon resonances, which were ascribed to a trisubstituted double bond, three methyl, five methylenes (two oxygenated), two sp 3 methines, two sp 3 quaternary carbons and one carboxyl (d C 215.1) ( Table 1). The chemical shift values of 1D NMR of 1 were quite similar to those of the known compound 3b,12-dihydroxydrimenol (1a) [6], which suggested compound 1 possessed a drimane sesquiterpenoid skeleton. The notable difference between 1a and 1 was that the hydroxy group at C-3 (d C 79.7) in 1a was oxidized into a carbonyl group (d C 215.1) in 1, which caused the downfield shifts of C-4 from d C 39.8 in 1a to 47.9 in 1, and C-2 from d 28.1 in 1a to 38.7 in 1. The above assignment was further supported by the HMBC correlations from d H 1.08 (Me-14, s) and 1.02 (Me-15, s) to d C 215.1 (s, C-3) (Fig. 2), as well as IR absorption band at 1701 cm -1 and mass data analysis. In the ROESY spectrum (Fig. 2), cross peaks between Me-15/H-5, H-5/H-9, Me-13/H-11 were observed, which suggested that both Me-13 and H-11 should be b oriented. Therefore, compound 1 was established as 12-hydroxy-3-oxodrimenol (1).
Compound 2 was isolated as white powder. The molecular formula was established to be C 17 13 C NMR spectrum, along with the DEPT and HSQC spectra, classified the functionalities as a trisubstituted double bond, four methyl, five sp 3 methylenes (two oxygenated), three sp 3 methines (one oxygenated), two sp 3 quaternary carbons, and one carbonyl (d C 173.5) ( Table 1). These NMR spectroscopic data of 2 resembled those of a coexisting known compound acetoxydrim-7-en-3b-ol (2a) [7], except for the chemical shift of C-17 was 60.9 (CH 2 ) in 2 instead of 21.3 ppm (CH 3 ) in 2a. Therefore, compound 2 was established as a hydroxy derivative of 2a, which was also supported by the HMBC correlations from d H 4.13 (1H, d, J = 5.1 Hz) and 4.14 (1H, d, J = 5.1 Hz) to d C 173.5 (C-16, s) ( Fig. 2), as well as mass data analysis. In the ROESY spectrum ( Fig. 2), the cross peak of H-5/Me-15 suggested that C-14 was b oriented, while the cross peak of Me-15/H-3, as well as the constant coupling of H-3 (d H 3.26, dd, J = 11.3, 3.2 Hz), indicated 3-OH to be b oriented. Besides, the cross peaks of Me-14/ H-2, H-2/Me-13, Me-13/H-11 proved that H-9 was a oriented and Me-13 was b oriented. Therefore, compound 2 was elucidated as 11-hydroxyacetoxydrim-7-en-3b-ol.

General Experimental and Procedures
Optical rotations were obtained on a JASCO P-1020 digital polarimeter. IR and UV spectra were recorded on a Bruker Tensor 27 FT-IR spectrometer with KBr pellets and a Shimadzu UV-2401PC instrument, respectively. 1D and 2D NMR spectra were obtained on a Bruker Avance III 600 MHz spectrometer. ESIMS and HREIMS were measured on a Waters Xevo TQ-S spectrometer and a Waters Autospec Premier P776 spectrometer, respectively. HRE-SIMS was measured on an Agilent G6230 TOF MS spectrometer. Silica gel 200-300 mesh (Qingdao Marine Chemical Inc., China) and Sephadex LH-20 (Amersham Biosciences, Sweden) were used for column chromatography. Medium pressure liquid chromatography (MPLC) was performed on a Büchi Sepacore System equipping pump manager C-615, pump modules C-605 and fraction collector C-660 (Büchi Labortechnik AG, Switzerland), and columns packed with Chromatorex C-18 (40-75 lm, Fuji Silysia Chemical Ltd., Japan). Preparative high performance liquid chromatography (Prep-HPLC) was performed on an Agilent 1260 liquid chromatography system equipped with a Zorbax SB-C18 column (5 lm, 9.4 9 150 mm).

Extraction and Isolation
The culture broth (20 L) of P. sulphurascens was filtered, the filtrate was extracted four times with ethyl acetate (EtOAc). Meanwhile, the mycelium was extracted by CHCl 3 /MeOH

Cytotoxicity
The cytotoxicity assay was performed according to the MTT method in 96-well microplates. Five human cancer cell lines: human myeloid leukemia HL-60, hepatocellular carcinoma SMMC-7721, lung cancer A-549, breast cancer MCF-7 and human colon cancer SW480 cells were used in the cytotoxicity assay. All the cells were cultured in RPMI-1640 or DMEM (Hyclone, Logan, UT, USA), supplemented with 10 % fetal bovine serum (Hyclone) in 5 % CO 2 at 37°C. Briefly, 100 lL of adherent cells were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition with an initial density of 1 9 10 5 cells/mL. Each tumor cell line was exposed to the test compound at concentrations of 0.064, 0.32, 1.