Cyathane diterpenoids from fruiting bodies of Phellodon niger

Four new cyathane-type diterpenoids, nigernins C-F (1–4), together with four known compounds, were isolated from the fruiting bodies of the basidiomycete Phellodon niger. The structures of these new compounds were established on the basis of spectroscopic analysis, including 1D and 2D NMR experiments. In addition, nigernin F (4) with an unusual 3,4-seco cyathane diterpenoid skeleton was found to occur in nature for the first time. It was suggested to be as an oxidation product of C-3-C-4 bond cleavage of nigernin E (3). Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s13659-011-0002-z and is accessible for authorized users.


Introduction
Phellodon niger is an edible fungus belonging to the family Hydnaceae. 1 In our continuing search for novel and secondary metabolites from higher fungi of Yunnan province in China, we have previously isolated two new cyathane diterpenoids, nigernins A and B from this fungus. 2 Further research for the cyathane-type diterpenoids in the fruiting bodies of P. niger led to the isolation of four new cyathanes, nigernins C-F (1-4), along with four known compounds, sarcodonin δ, 3 1,2diacetoxy-3-(4′-hydroxyphenyl)-4,7,8-trihydroxy-dibenzofuran (Bl-V), 4 grifolic acid 5 and uridine. 6 Herein, we report the isolation and structure elucidation of the new compounds.

Results and Discussion
Compound 1 was isolated as white amorphous powder. The molecular formula was established to be C 30 166.5 (s)]. The remaining 20 carbons were ascribable for four methyls, six methylenes, four methines, five quaternary carbons, and one carbonyl group. Comparison of NMR data of 1 with those for nigernin A, previously isolated from this fungus, 2 revealed the presence of the characteristic signals of a cyathane-type diterpenoid. The absence of a methylene resonance at δ C 43.4 in the 13 C NMR spectrum of nigernin A, and the appearance of the signals at δ C 77.8 and δ H 5.02 (d, J = 7.1 Hz) in the NMR spectra of 1, suggested the existence of an oxygenated methine attributable to C-14 in 1. This was supported by the coupling constant (J = 7.1 Hz) of H-13 at δ H 7.22, and the HMBC correlations from H-14 to C-5, C-12 and C-13, and from H-16 to C-14 ( Figure   *To whom correspondence should be addressed. E-mail: jkliu@mail.kib.ac.cn 1). In addition, the HMBC correlation from H-14 to C-9′ suggested that the p-methoxycinnamoyloxyl ester unit was linked to C-14. The ROESY correlations between H-5 and H-17, H-17 and H-8, H-16 and H-8, H-16 and H-14 were observed in 1. It indicated H-14 to be α-oriented ( Figure 2). Thus, the structure of 1 was elucidated as 14β-(pmethoxycinnamoyloxyl)-cyatha-3,12-diene-15-oic acid, and named as nigernin C.
Compound 3 was isolated as white amorphous powder. Its molecular formula was determined to be C 28 H 36 O 5 on the basis of molecular ion peak at m/z 452.2554 in the HREIMS (calcd for C 28 H 36 O 5 [M] + , 452.2563), in combination with the 13 C NMR and DEPT spectra. The 1 H and 13 C NMR spectroscopic data of 3 (Table 1) were very similar to those of 1, except for a p-methoxybenzoyloxyl group in 3 instead of the pmethoxycinnamoyloxyl group in 1. The proton signals at δ H 6.94 (2H, d, J = 8.9 Hz, H-3′ and 5′) and 8.04 (2H, d, J = 8.9 Hz, H-2′ and 6′) in the 1 H NMR spectrum, together with the 13 C-NMR signals at δ C 55.5 (q), 113.8 (d × 2), 122.4 (s), 131.7 (d × 2), 163.5 (s), 165.5 (s) were determined readily as a pmethoxybenzoyloxyl unit. The location of the substituent and the stereochemistry of 3 were the same as those in 1 on the basis of analysis of HMBC and ROESY data. Therefore, compound 3 was identified as 14β-(p-methoxybenzoyloxyl)cyatha-3,12-diene-15-oic acid, and named as nigernin E.
Compound 4 + , 484.2461). Comparison of the 1 H and 13 C NMR data of 4 (Table 1) with those of 3 indicated that their structures were similar. The main differences between these two compounds were the appearance of two keto carbonyl signals at δ C 215.0 (s, C-3) and 215.5 (s, C-4) in 4 and the absence of two olefinic quaternary carbons [δ C 140.4 (s, C-3) and 138.1(s, C-4)] in 3. In addition, the EIMS of 4 with molecular ion peak [M] + at m/z 484 suggested more 32 mass units than that of 3. On the basis of above evidence and the literature, 7 compound 4 should be a 3,4-seco cyathane diterpenoid due to an oxidation cleavage of C-3-C-4 double bond of 3. This was also confirmed by HMBC correlations (Figure 1) from H-2, H-18, H-19 and H-20 to C-3, and from H-5 and H-17 to C-4. The ROESY (Figure 2) correlations between H-5 and H-17 of 4 indicated that the methyl at C-9 is . Therefore, the structure of 4 was determined to be 3,4-seco nigernin E, and named as nigernin F. To the best of our knowledge, this is the first report of the 3,4-seco cyathane skeleton from nature.

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-011-0002-z and is accessible for authorized users.