Five new polyketides from the basidiomycete Craterellus odoratus

Five new polyketides, craterellones A–E (1–5), were isolated from cultures of basidiomycete Craterellus odoratus, together with five known compounds (6–10). Structures of 1–5 were elucidated on the basis of extensive spectroscopic analysis. All compounds were evaluated for their inhibitory activities against one isozyme of 11β-hydroxysteroid dehydrogenase (11β-HSD1) and cytotoxic activities on five tumor cell lines. Compound 10 exhibited significant cytotoxicity against HL-60, SMMC-7721, A-549, MCF-7, and SW-480, with IC50 values of 0.50, 0.69, 0.64, 1.10, 0.54 µM, respectively. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s13659-012-0057-5 and is accessible for authorized users.


Introduction
Craterellus odoratus (Schwein.) Fr. (Cantharellaceae) is an edible fungus, which is widespread in mainland China and characterized by possessing a bright orange or yellow cap. Our previous study on the secondary metabolites of C. odoratus has reported a series of merosesquiterpenoids, while one of them demonstrated significant inhibitory activities against human 11β-HSD2. 1 As a part of our continuous search for further new bioactive constituents, an enlarged culture on the same resource was investigated, which resulted in the isolation of five new compounds, craterellones A-E (1)(2)(3)(4)(5), together with five known compounds. The structures of the new compounds were established on the basis of extensive spectroscopic methods, while the known compounds were identified as decumbenones A and B (6 and 7), 2 versiol (8), 3 calbistrin A (9), 4 and calbistrin C (10), 4 respectively, by comparison with data as reported in the literature. All compounds were evaluated for their cytotoxicities against five human cancer lines and their inhibitory activities against one isozyme of 11β-hydroxysteroid dehydrogenase (11β-HSD1). This paper deals with the isolation, structural elucidation, and bioactivities of these isolates.

Results and Discussion
Craterellone A (1) was obtained as white powder. Its molecular formula was determined to be C 16 H 20 O 3 by HRESIMS, corresponding to seven degrees of unsaturation.
The IR spectrum showed absorption bands for a hydroxy group (3431 cm -1 ) and double bonds (1635 cm -1 ). The 13 C NMR and DEPT spectra showed 16 carbon signals that attributed to three methyls, one methylene, eight methines, and four quaternary carbons. Comparison of the NMR data of 1 with those of decumbenone A (6) 2 revealed that compound 1 was a polyketide. The 13 C NMR data indicated that the carbon signals corresponding to two sp 3 methylenes and a carbonyl group (δ C 215.0) in 6 were not present in 1. Instead, one sp 2 quaternary carbon at δ C 181.3 (s, C-3), one sp 2 methine at δ C 102.8 (d, C-2), and an aldehyde carbon at δ C 188.9 (d, C-1) were observed. The 1 H-1 H COSY correlation of H-1/H-2 and the HMBC correlations ( Figure 1) of H-1/C-2 (δ C 102.8), and H-2/C-3 (δ C 181.3) and C-4 (δ C 53.6) concluded C-1, C-2 and C-3 as an α,β-unsaturated aldehyde group. In addition to six *To whom correspondence should be addressed. E-mail: jkliu@mail.kib.ac.cn degrees of unsaturation occupied by two rings, three double bonds, and one aldehyde, the remaining one degree of unsaturation required that compound 1 had an ether ring in the form of a 3,6-ether moiety, which was in agreement with the significant downfield signals for C-3 (δ C 181.3) and C-6 (δ C 79.3) and the HMBC correlation from H-6 to C-3. The relative configuration of 1 was assigned on basis of the ROESY experiment ( Figure 1). ROESY correlations of Me-14 with H-2 and H-5, of Me-16 with H-6 and H-7β, and of Me-15 with H-7α indicated that 1 had the same configuration as that of 6. Therefore, the structure of 1 was determined as shown, and named as craterellone A.
Craterellone B (2) exhibited the molecular formula C 15 H 24 O 2 , as determined by its HRESIMS at m/z 259.1678 ([M + Na] + ), corresponding to four degrees of unsaturation. Comparison of NMR data suggested that 2 still possessed a polyketide skeleton related to that of 1. Analysis of the 1 H-1 H COSY spectrum revealed a partial structure, giving rise to the same bicyclic system to that of 1. Continuous analysis of HMBC spectrum revealed the main difference to be the length of the side chain of carbons at C-1, C-2, and C-3. Carbon resonances at δ C 27.5 (q, C-2) and δ C 211.4 (s, C-3) are typical for an acetyl group, which was suggested to be connected to C-4 by the HMBC correlations of H-2 to C-4. Analysis of other 2D NMR data established compound 2 to be a 1-norpolyketide of 1. According to the ROESY experiment, compound 2 was found to possess the same relative configuration with that of 1. In addition, the ROESY correlation of H-9 with H-5 and Me-16 suggested the α-orientation of OH-9, while the ROESY correlation of H-10 with H-15 suggested the α-orientation of H-10. Thus, the structure of 2 (craterellone B) was established as shown.
Craterellone C (3) was isolated as a yellow oil and found to possess a molecular formula of C 16 H 26 O 5 , as deduced from its HRESIMS at m/z 321.1668 [M + Na] + . The 1 H and 13 C NMR spectra of 3 were similar to those of 7. 2 Careful comparison of their NMR data indicated that 3 was a hydroxy derivative of 7, as explained by the oxygenated quaternary carbon at δ C 71.5. This quatermary carbon at δ C 71.5 (s) was assigned to C-10 according to the HMBC correlations from H-5, H-9, and H-11 to C-10. The ROESY correlation of OH-10 with Me-15 implied the OH-10 to be α-oriented. Consequently, the structure of craterellone C was proposed as 3.
Craterellone D (4) was obtained as yellow, amorphous powder. Its molecular formula was assigned to be C 16 H 26 O 4 , the same to that of 7. Comparison of the spectroscopic data of 4 with those of 7 also indicated the similar patterns except for signals of an oxygenated methylene [δ H 3.70 (2H, d, J = 5.9 Hz, H-16); δ C 67.8 (t, C-16)] in 4 instead of those of the oxygenated methine in 7. Analysis of 1 H-1 H COSY and HMBC spectra revealed the hydroxy substitution at C-16. Detailed analysis of other spectroscopic data (HSQC, HMBC, 1 H-1 H COSY, ROESY) established the structure of compound 4 (craterellone D) as shown.
The molecular formula of craterellone E (5) was inferred to be C 16 H 20 O 3 on the basis of its positive HRESIMS. The IR spectrum indicated the presence of hydroxy (3431 cm -1 ), carbonyl group (1701 cm -1 ), and conjugated terminal double bonds (1630 cm -1 ). Preliminary analysis of the NMR data indicated that 5 possessed the same skeleton to that of 7. HMBC correlations of δ H 3.07 (1H, m, H-9) with C-8 and C-10 indicated the OH substitution at C-9. In addition, 1 H NMR signals of the terminal double bonds at δ H 4.99 and 4.88 (each 1H, s, H-14) showed the HMBC correlations to C-13, C-4, and C-12, suggesting that the terminal double bond was constructed at C-13 and C-14. The ROESY correlation of H-9 with H-5 indicated H-9 to be β-oriented, the same to that of 2. Thus, compound 5 (craterellone E) was established as shown.

Experimental Section
General Experimental Procedures. Optical rotations were measured on a Jasco-P-1020 polarimeter. UV spectra were measured on a Shimadzu UV-2401 PC spectrophotometer. IR spectra were obtained by using a Bruker Tensor 27 FT-IR spectrometer with KBr pellets. NMR spectra were acquired with instruments of Bruker DRX-500 or Bruker AV 400. Institute of Botany. Culture medium: glucose (5%), pork peptone (0.15%), yeast (0.5%), KH 2 PO 4 (0.05%), MgSO 4 (0.05%), The initial pH was adjusted to 6.0, the fermentation was first carried out on an erlenmeyer flask for six days till the mycelium biomass reached to the maximum. Later it was transferred to a fermentation tank (100 L) at 24 o C and 250 rpm for twenty days, ventilation was settled to 1.0 vvm (vvm: air volume/culture volumn/min).