Ten new aurovertins from cultures of the basidiomycete Albatrellus confluens

Aurovertins J-S (1–10), together with four known metabolites, aurovertins B, C, E, and I (11–14), were isolated from cultures of the basidiomycete Albatrellus confluens. The structures of compounds 1–10 were elucidated on the basis of extensive spectroscopic analysis. All compounds were evaluated for their cytotoxic activities on five tumor cell lines.


Introduction
The first four aurovertin-type compounds, aurovertins A-D, were isolated as toxic substances from fermentation broth of the fungus Calcarisporium arbuscula. 1 Aurovertins are a class of compounds with the basic structure of 2,6dioxabicyclo[3.2.1]octane ring system and a conjugated αpyrone moiety originated from polyketide pathway. They have attracted considerable attention of chemists because of their actitivities as potent inhibitors of ATP synthesis and antitumor agents. [2][3][4][5][6][7] There have been nine aurovertins (A-I) structurally elucidated to date, 1,[8][9][10] while the relative and absolute configurations of aurovertin B were determined by spectroscopic analysis and total synthesis, respectively. 11,12 The absolute configuration of aurovertin F was determined by analysis of the CD spectrum recently. 9 It is deduced that aurovertin-type of compounds obtained in literature probably share the same absolute configuration on biosynthetic grounds. 10,13 During our continuing investigation of bioactive microbial secondary metabolites from the fungus, 8,14-23 ten new aurovertin derivatives, named as aurovertins J-S (1-10), along with four previously reported aurovertins B, C, E and I (11)(12)(13)(14), were isolated from cultures of the basidiomycete Albatrellus confluens (Alb. et Schw.: Fr.) Kolt. et Pouz. All compounds were evaluated for their cytotoxicities against five human cancer cell lines. This paper deals with the isolation, structural elucidation, and bioactivities of these isolates.

Results and Discussion
A total of 14 aurovertins including 10 previously-unreported compounds were isolated from cultures of the basidiomycete Albatrellus confluens. Comparison with all the reported nine aurovertin-type metabolites to date, it should be noticed that the methyl (C-22) in the moiety of α-pyrone in aurovertins was missing in aurovertin J (1) and aurovertin K (2) 446.1941), corresponding to ten degrees of unsaturation. The IR spectrum showed absorption bands for hydroxy group (3441 cm -1 ) and double bonds (1632 cm -1 ). The 13 C NMR and DEPT spectra showed 24 carbon signals that attributed to five methyls, one methylene, twelve methines, and six quaternary carbons. The 1 H and 13 C NMR spectra (Tables 1 and 3) of 1 were similar to those of aurovertin B (11) 5 except for the loss of the methyl group at C-16. The upfield shift of C-16 (δ C 101.0, d) clearly indicated that 1 was a demethylated analogue of 11, which was supported by the observed HMBC correlations from δ H 5.84 (1H, d, J = 1.9 Hz, H-16) to δ C 122.0 (d, C-14), 158.6 (s, C-15), 171.0 (s, C-17), and 88.7 (d, C-18). In the light of the evidences mentioned above and the key 1 H-1 H COSY and HMBC correlations (Figure 2), the planar structure of 1 was therefore elucidated as shown in Figure 1, named aurovertin J.
The key ROESY correlations of H-2 with H-8, H-3 with Me-20, and H-5 with H-7 were observed in the ROESY spectrum of compound 1 (Figure 2), which established the relative stereochemistry of 1 as that of 11. Because the NMR data and other physico-chemical properties of 1 are very similar to those of 11, the absolute configuration and geometrical isomerism of both compounds are suggested to be the same. Therefore, the structure of aurovertin J (1) was established, as shown.
Aurovertin K (2) was inferred to possess the molecular formula C 22 H 28 O 7 on the basis of its HREIMS at m/z 404.1832 [M] + . Preliminary analysis of the NMR data also indicated that 2 possessed a similar skeleton to that of aurovertin I (14). 10 The same demethylation at C-16 (δ C 101.0) was concluded by the observed HMBC correlations from H-16 to C-14, C-15, C-17, and C-18 as the same to those of 1. In addition, a methyl carbon at δ C 26.5 (q) was assigned to be placed at C-8 according to the HMBC correlations from δ H 1.26 (3H, s, H-8) to δ C 79.6 (d, C-7), 78.2 (s, C-8), and 147.1 (d, C-9). The ROESY correlations of Me-2 with Me-8, H-3 with Me-20, and H-5 with H-7 indicated the same configuration to that of 1. Thus, compound 2 (aurovertin K) was established, as shown.
Aurovertin L (3) exhibited an ion peak at m/z 324.1564 [M] + in the HREIMS, in agreement with the molecular formula C 17 H 24 O 6 . Preliminary analysis of 1D NMR data suggested that compound 3 was still an aurovertin derivative, as indicated by the characteristic carbon signals for C-1 to C-8, as well as the olefinic carbons for C-9 to C-12 (Tables 1 and 3). However, compound 3 might be identified as a degraded product of 11, 5 in which the double bond between C-13 and C-14 was cleaved, then an aldehyde group was formed at C-13   The NMR data of 4 resembled those of 11 5 except that the acetoxy group at C-5 was replaced by a propionyloxy group, which was supported by the 1 H-1 H COSY correlation between δ H 2.43 (2H, m, H-2') and 1.19 (3H, t, J = 7.6 Hz, Me-3'), as well as the HMBC correlation from Me-3' to δ C 27.5 (t, C-2') and 173.3 (s, C-1'). Accordingly, compound 4 was identified as aurovertin M.
Aurovertin N (5) was isolated as yellow syrup. The molecular formula C 23 H 32 O 8 was determined by HREIMS at m/z 436.2097 [M] + . The 1D and 2D NMR data suggested that the structure of 5 was related to that of aurovertin E (13), 8 except that the double bond between C-9 and C-10 in 14 was opened to form a hydroxymethine at C-10 (δ C 69.8), as implied by the HMBC correlations from δ H 4.53 (1H, br. s, H-10) to δ C 75.9 (d, C-8), 39.6 (t, C-9), and 141.5 (d, C-11). Detailed analysis of other spectroscopic data suggested that the other parts of 5 were the same to those of 13. Thus, compound 5 was established as aurovertin N.
The same to 5, all the spectral data suggested that compound 6 also showed similar patterns to those of aurovertin B (11), 5 except that the double bond between C-9 and C-10 was opened to form a hydroxymethine group at C-10 (δ C 69.8), as implied by the HMBC correlations from δ H 4.53 (1H, br. s, H-10) to δ C 75.9 (d, C-8), 39.6 (t, C-9), and 141.5 (d, C-11). Detailed analysis of other spectroscopic data suggested that the other parts of 6 were the same to those of 11. Thus, compound 6 was established as aurovertin O.
Aurovertin P (7) had the molecular formular C 25 H 34 O 10 , as established by the HREIMS at m/z 494.2157 [M] + . All the data suggested that the structure of 7 was closely related to that of 11. 5 A significant change was that C-9 and C-12 in 7 were two oxygenated methines at δ C 72.3 (C-9) and 71.8 (C-12) instead of two olefinic carbons in 11, which was supported by the HMBC correlations from δ H 4.33 (1H, m, H-9) to δ C 79.3 (d, C-8) and 130.0 (d, C-10), and from δ H 4.84 (1H, m, H-12) to δ C 131.7 (d, C-11) and 138.6 (d, C-13), as well as the 1 H-1 H COSY correlations from H-8 to H-13. Further analysis of other 2D NMR data suggested that the other parts were the same to those of 11. Therefore, compound 7 was established as aurovertin P, as shown.
The NMR data of aurovertin Q (8) were closely related to those of 7, except for the existence of a methoxy group at C-12 (δ C 81.0) in 8 rather than the hydroxy group at C-12 in 7, as established by the HMBC correlation of δ H 3.34 (3H, s, OMe) to δ C 81.0 (d, C-12). Thus, compound 8 was elucidated as aurovertin Q, as shown.
Aurovertin R (9) possessed a molecular formula C 25 H 34 O 10 according to the ion peak at m/z 494.2144 [M] + in the HREIMS. The NMR data suggested that the structure of 9 was related to that of 6, except for one more hydroxy group placed at C-9, as indicated by the HMBC correlation from δ H 3.79 (1H, m, H-9) to δ C 77.0 (d, C-8) and 71.3 (d, C-10), as well as the 1 H-1 H COSY correlation from H-8 to H-10. Thus, compound 9 was determined as aurovertin R, as shown.
The NMR data suggested that aurovertin S (10) had the related structure to that of 9 except for the methoxy group at C-10 in 10 rather than the hydroxy group in 9, as indicated by the signal at δ H 3.34 (3H, s, OMe), as well as the HMBC correlation from δ H 3.34 (3H, s, OMe) to δ C 83.1 (d, C-10). Detailed analysis of other 2D NMR data suggested that the other parts of 10 were the same to those of 9. Compound 10 was, therefore, determined as aurovertin S, as shown.
The structures of the known compounds 11-14 isolated were identified as aurovertin B, 5 aurovertin C, 22 aurovertin E, 8 and aurovertin I, 10 respectively, by comparison of their spectroscopic data with literature values. All aurovertins obtained from cultures of the basidiomycete A. confluens were yellow syrup, and could not obtain as crystals in diverse solution systems. In order to elucidate the absolute configuration of the side chain in compounds 5-10, modified Mosher reaction 24 of compound 5 and acylation of compound 7 with 4-bromobenzoyl chloride 25 for crystals were all in failure. Thus, the stereoconfigurations of compounds 5-10 were not determined wholly. All compounds were evaluated for their cytotoxicities against five human cancer cell lines. The results showed that compounds 7 and 11 exhibited moderate cytotoxicities compared with those of cisplatin (Table 4), while the other compounds were inactive (IC 50 > 40 μM).

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 Avance III 600 or Bruker DRX-500 or Bruker AV 400. ESIMS and HREIMS were measured on Bruker HCT/Esquire and VG Autospec-3000 mass spectrometer respectively. Preparative HPLC was performed on an Agilent 1100 series with a Zorbax SB-C18 (5 µm, 9.  Table 3. 13

no.
1 a 2 b 3 a 4 c 5 c 6 a 7 c 8 c 9 b 10 a 1 11.8 q 11.7 q 11.7 q 11.9 q 11.8 q 11.7 q 11.7 q 11.8 q 11.9 q 2 20.  (HFG0307252) was deposited at the Herbarium of the Kunming Institute of Botany, CAS. 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).

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/ 10.1007/s13659-012-0088-y and is accessible for authorized users.