Five new 5,6-seco-tremulane sesquiterpenoids from the basidiomycete Conocybe siliginea

Five new 5,6-seco-tremulane sesquiterpenoids (1–5), as well as three known analogues (6–8), were isolated from the basidiomycete Conocybe siliginea. The structures of new compounds were elucidated by extensive spectroscopic methods. The known compounds were identified by comparing their spectroscopic data with those reported in the literature. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s13659-013-0003-1 and is accessible for authorized users.


Results and Discussion
Compound 1 was obtained as colorless oil. H and 13 C NMR data (Table 1) revealed the existence of two methyls, four methylenes (one oxygenated and one olefinic), five methines (two oxygenated and one olefinic), and four quaternary carbons (two olefinic and one carbonyl). Comparison of 1 H and 13 C NMR data of 1 ( Table 1) with those of 10β,11-dihydroxy-5,6-seco-1,6(13)-tremuladien-5,12-olide (8) 1 showed that they are similar in structure, except for the absence of an oxymethylene (δ C 70.8, C-12) instead of the appearance of an acetal carbon (δ H 6.10, δ C 107.9 ) in 1, as *To whom correspondence should be addressed. E-mail: jkliu@mail.kib.ac.cn deduced by the HMBC correlations from H-3 and C-12 and from H-12 to C-5 ( Figure 1). In addition, the HMBC correlation from H-11 to C-12 established an epoxy moiety between C-11 and C-12 ( Figure 1), which was in agreement with the degrees of unsaturation ( Figure 1). According to the biogenetic origin, compound 1 may be biosynthesized via Baeyer-Villiger oxidation, elimination, oxidation, and esterification of conoceol B. 2 Therefore, the relative configuration of 1 should be consistent with that of conoceol B.
Compound 3 was a colorless oil and gave a molecular formula C 15 H 20 O 3 , as assigned by HRESIMS at m/z 271.1316 [M + Na] + (calcd. for 271.1310). Detailed comparison of 1 H and 13 C NMR data ( Table 2) of 3 with those of 6 2 showed that they are similar in structure. The key difference was a CHO group (δ H 9.94, δ C 191.7) in 3 rather than an oxymethylene in 6. The aldehyde was assigned to C-11 on the basis of HMBC correlations from  H 9.94 (1H, s, H-11) to  C 132.9 (s, C-2) and 34.5 (d, C-3). Analysis of 2D NMR data established the structure of 3 to be 11-aldehyde-5,6-seco-1,6(13)-tremuladien-5,12-olide, as shown.
Compound 5 was obtained as a colorless oil with a molecular formula of C 17 H 24 O 4 established by the HRESIMS at m/z 315.1577 [M + Na] + (calcd. for 315.1572). The IR spectrum showed absorption for a carbonyl group (1742 cm -1 ) and C=C double bond (1637 cm -1 ). The 1 H and 13 C NMR data (Table 2) revealed the existence of three methyls, six methylenes (two oxygenated and one olefinic), three methines (one olefinic), and five quaternary carbons (two olefinic and two carbonyl). Detailed comparison of 1D and 2D NMR (HSQC, HMBC, ROESY) data of 5 with those of conocenolide B (7) 2 showed that they were similar in structure, except for an acetoxy substituent at C-12 in 5, as indicated by the HMBC correlations from H-12 to δ C 170.7 (s, CH 3 CO-). Therefore, compound 5 was determined to be 12-acetoxy-5,6-seco-

Experimental Section
General Experimental Procedures. Optical rotations were measured on a Horiba SEPA-300 polarimeter. UV spectra were obtained using a Shimadzu UV-2401A spectrometer. IR spectra were obtained on a Bruker Tensor 27 spectrometer with KBr pellets. 1D and 2D NMR experiments were performed on a Bruker AM-400, DRX-500 or AVANCE III-600 spectrometer with TMS as the internal standard. Chemical shifts (δ) were expressed in ppm with reference to the solvent signals. Mass spectra (MS) were recorded on a VG Auto Spec-3000 or an APIQSTAR time-of-flight spectrometer. Column chromatography (CC) was performed on silica gel Extraction and Isolation. The culture broth of C. siliginea (80 L) was filtered, and the filtrate was extracted four times with EtOAc. The organic layer was concentrated under reduced pressure to give an oily residue (40 g) that was subjected to column chromatography over silica gel (200-300 mesh) eluting with CHCl 3 /MeOH (from 100:0 to 0:100) to afford fractions A-E. Fraction A was separated further by CC over RP-18, eluting with H 2 O/MeOH (from 1:0 to 0:1) to give fractions B 1 -B 4 . Fraction B 4 was purified by repeated CC over silica gel (petroleum ether/EtOAc, 10:1) and then applied to Sephadex LH-20 (Me 2 CO) to yield 2 (40.0 mg), 6 (25.2 mg), 7 (12.1 mg), Fraction B 3 was eluting with petroleum ether/EtOAc to give fractions B 3a -B 3g . Fraction B 3b was purified by CC over silica gel (petroleum ether/EtOAc, 5:1) to yield 1 (48.0 mg), Fraction B 3e by repeated silica gel CC (petroleum ether/EtOAc) and Sephadex LH-20 (Me 2 CO) to yield 3 (4.2 mg), 4 (6.1 mg), 5 (3.5 mg) and 8 (9.5 mg).

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/ 10.1007/s13659-013-0003-1 and is accessible for authorized users.