Chemical investigation on the cultures of the fungus Xylaria carpophila

A chemical investigation on the cultures of Xylaria carpophila led to the isolation of one known cyclopeptide cyclo(Nmethyl-l-Phe-l-Pro-l-Leu-d-Ile-l-Val) (1), five new sesquiterpenes, named as xylcarpins A–E (2–6), and another known compound (7). The structures were determined by extensive NMR and MS spectroscopic analysis. The absolute configuration of 1 was established by use of Marfey’s method and ROESY spectroscopic data. All compounds were tested for their cytotoxicities against five human cancer cell lines. Compound 7 showed week inhibitory activity. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s13659-011-0011-y and is accessible for authorized users.


Results and Discussion
Compound 1 was obtained as a white powder. The molecular formula was established as C 32 H 49 N 5  The IR absorption bands at 3292, 1642 cm −1 indicated the presence of NH and CO groups. The 1D NMR spectroscopic data (Table 1) revealed the presence of three NH protons (δ H 9.76, 9.29, and 7.70, respectively), six methyl groups as well *To whom correspondence should be addressed. E-mail: jkliu@mail.kib.ac.cn as one amide N-CH 3 signal (δ H 3.47; δ c 31.6), six methylene signals, eight sp 3 methine signals, a monosubstituted benzene ring signals, and five carbonyl carbons. Preliminary analysis of these NMR and MS data suggested that compound 1 might be a pentapeptide. In the 1 H-1 H COSY spectrum (Figure 1), six fragments were established indicating that compound 1 comprised one leucine, one isoleucine, one valine, one proline and one phenylalanine. The HMBC correlation of δ H 3.47 (3H, s, Me-N) with δ C 58.2 (CH, α-carbon of the phenylalanine) suggested the methyl substitution at N-atom of phenylalane. In addition, the HMBC correlations also established the amino acids sequence to be N-methyl-Phe-Pro-Leu-Ile-Val ( Figure 1). These data indicated that compound 1 was structurally the same as cyclo(N-methyl-L -Phe-L -Val-D -Ile-L -Leu-L -Pro). 16 Advanced Marfey's method was applied to identify the configuration of the amino acid, and showed the presence of L-Leu as well as L-Val. The configurations of N-MePhe 1 , Pro 2 , Leu 3 , Ile 4 and Val 5 were determined to be L, L, L, D, L respectively by the application of the Marfey's method, the analysis of ROESY correlations (Table 1 and Figure 1) and the comparison with the data of cyclo(N-methyl-L -Phe-L -Pro-L -Leu-D -Ile-L -Val). 16 Compound 2 was purified as a white powder. The molecular formula was determined as C 15 Table 2) showed 15 carbon resonances ascribable for four methyls, four methylenes, three methines (including one oxygen-bearing methine), and four quaternary carbons (one of which is a carbonyl carbon). Apart from one degree of unsaturation occupied by one carbonyl group, the remaining three degrees of unsaturation indicated that compound 2 should possess a three-ring system including one three-membered carbon ring according to the typical methylene signal at δ C 6.2. In the 1 H-1 H COSY spectrum, two fragments was established as shown with bold line in Figure 2. These data suggested that compound 2 possessed a backbone related to the known compound thujopsene. 17 The major differences including a hydroxy group at C-3 (δ C 78.4) and a carbonyl group at C-2 (δ C 214.5) were established by the HMBC and 1 H-1 H COSY spectra analysis ( Figure 2). The relative configuration of 2 was elucidated by the ROESY experiment ( Figure 2 (Table 2) were similar to those of 2 with the major differences being that the signal of the carbonyl carbon at C-2 was absent, showing instead a pair of double bond functionality between C-1 (δ C 135.5) and C-2 (δ C 114.6), which was supported by the HMBC correlations from δ H 1.80 (3H, s, H-11) to δ C 135.5 (s, C-1) and from δ H 1.76 (1H, m, H-3) to δ C 114.6 (d, C-2). The same ROESY correlations of 3 and 2 indicated their identical relative configuration. Therefore, compound 3 was established and named as xylcarpin B.
Compound 4 was obtained as a white powder. The IR spectrum showed the existence of hydroxy groups (3417 cm −1 ). The NMR data were quite similar to those of 3, except for the methyl at C-11 was oxygenated into a methylene (δ C 67.1), which was supported by the HMBC correlation of δ H 4.13 (2H, d, J = 4.8 Hz, H-11) with δ C 139.3 (s, C-1). Furthermore, the same ROESY data of 3 and 4 suggested the same relative configuration of them. Therefore, compound 4 was established as depicted, and named as xylcarpin C.
Compound 5 was isolated as a colorless oil with the molecular formula of C 15 (Table 3)   were quite similar to those of (1R)-1-hydroxybisabololoxide B. 18 The hydroxy group was attached to C-4 as supported by the HMBC correlation from δ H 1.77 (3H, s, H-15) to δ C 68.8 (d, C-4). By comparison of the NMR spectroscopic data and the optical rotation data of (1R)-1-hydroxybisabololoxide B, 18 the relative configurations of C-6, C-7 and C-10 in 5 were deduced as same as those in (1R)-1-hydroxybisabololoxide B.
In the ROESY spectrum, the correlation between H-4 and H-6 was not observed, indicating that the hydroxy group at C-4 should be α-oriented, which was also supported by the broad singlet of H-4. Compound 5 was established and named as xylcarpin D. Compound 6 was obtained as colorless oil. The molecular formula of C 15  NMR data were very similar to those of compound 5 except for an oxygenated quaternary carbon at δ C 70.0 (s, C-6) in 6 instead of a methine in 5. Analysis of the HMBC spectrum suggested that one hydroxy group was placed at C-6 by the correlation of δ C 2.30 (1H, m, H-1) and 1.96 (1H, m, H-5) with δ C 70.0 (s, C-6). Detailed analysis of other 2D NMR data suggested that other parts of 6 were identical to those of 5. Therefore, compound 6 was established and named as xylcarpin E.
Compounds 17 were evaluated for their cytotoxicities against five hunman cancer cell lines using the MTT method as reported previously 19 . Compound 7 showed week cytotoxicity against four of the tested cell lines (Table 4), while other compounds were inactive to all tested cell lines (IC 50 values of more than 40 μM).

Experimental Section
General Experimental Procedures. Optical rotations (OR) were recorded on a Jasco P-1020 digital polarimeter. Infrared spectroscopy (IR) spectra were obtained on a Bruker Tensor 27 FT-IR spectrometer with KBr pellets. Nuclear Magnetic Resonance (NMR) spectra were obtained on a Bruker Avance III 600 MHz spectrometer with tetramethylsilane (TMS) as an internal standard at room temperature. Electrospray ionizationmass spectra (ESI-MS) and high-resolution (HR) ESI-MS were recorded on a VG Autospec-3000 mass spectrometer and an API QSTAR Pulsar I spectrometer. Silica gel (200-300 mesh, Qingdao Marine Chemical Ltd., China) and Sephadex LH-20 (Amersham Biosciences, Sweden) were used for open column chromatography (CC). MPLC was performed on a Büchi Sepacore system (Büchi Labortechnik AG, Switzerland), and columns packed with RP-18 silica gel (40-75 μm, Fuji Silysia Chemical Ltd., Japan). Preparative HPLC was performed on an Agilent 1100 liquid chromatography system equipped with a Zorbax SB-C18 column (9.4 mm × 150 mm). Fractions were monitored by TLC. Spots were visualized by heating silica gel plates immersed in Vanillin-H 2 SO 4 in ethanol.

Fungal Material and Cultivation Conditions. The fungus
Xylaria carpophila (Pers.) Fr. was collected from Gaoligong Mountains in Yunnan Province, China, in 2008. The fungus was identified by Prof. Zhu-Liang Yang at the Kunming Institute of Botany. A voucher specimen was deposited at the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences. The culture medium consisted of glucose (5%), peptone from porcine meat (0.15%), yeast powder (0.5%), KH 2 PO 4 (0.5%) and MgSO4. Fermentation was carried out on a shaker at 160RPM for 25 days.

Cyclo(N-methyl-L -Phe-L -Pro-L -Leu-D -Ile-L -Val
Cytotoxicity Assay. Human myeloid leukemia HL-60, hepatocellular carcinoma SMMC-7721, lung cancer A-549 cells, breast cancer MCF-7 and colon cancer SW480 cell lines were used in the cytoxic assay. All the cells were cultured in RPMI-1640 or DMEM medium (Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone, USA) in 5% CO 2 at 37 C. The cytotoxicity assay was performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method in 96-well microplates. 19 Briefly, 100 µL 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 initial density of 1 × 10 5 cells/mL. Each tumor cell line was exposed to the test compound dissolved in DMSO at concentrations of 0.0625, 0.32, 1.6, 8, and 40 μM in triplicates for 48 h, with cisplatin (Sigma, USA) as a positive control. After compound treatment, cell viability was detected and a cell growth curve was graphed. IC 50 values were calculated by Reed and Muench's method. 21

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-011-0011-y and is accessible for authorized users.