Six New Vibralactone Derivatives from Cultures of the Fungus Boreostereum vibrans

Graphical Abstract Phytochemical reinvestigation on the cultural broth of Boreostereum vibrans led to the isolation of six new vibralactone derivatives, vibralactone N (1), vibralactone O (2), vibralactone P (3), 10-lactyl vibralactone G (4), (3S*, 4R*)-6-acetoxymethyl-2,2-dimethyl-3,4-dihydro-2H-chromene-3,4-diol (5), vibralactone Q (6). Their structures were elucidated by extensive spectroscopic methods. . Electronic supplementary material The online version of this article (doi:10.1007/s13659-014-0029-z) contains supplementary material, which is available to authorized users.


Introduction
Boreostereum vibrans (synonym Stereum vibrans) is a fungus belonged to the family Boreostereaceae which is characterized by possessing diverse bioactive compounds [1][2][3][4][5][6][7]. Vibralactone, first reported in 2006, is a rare fused blactone isolated from B. vibrans with significant lipase inhibitory activity (IC 50 = 0.4 lg/mL) [8]. This distinguished compound has aroused many follow-up studies. In 2008, first total synthesis of vibralactone was reported [9,10]. In 2011, vibralactone was used as a tool to study the activity and structure of the ClpP1P2 complex from Listeria monocytogenes was published [11]. Our continuous investigations on the chemical constituents of the culture of B. vibrans have led to a series of reports on bioactive vibralactone derivatives [12][13][14][15]. Recently the biosynthetic origin of vibralactone and its biosynthetic pathway which includes several very interesting reactions were established [16]. In order to explore and understand the potential for the production of secondary metabolites by B. vibrans, a scale-up fermentation of this fungus was carried out. Very careful investigation of the culture has resulted in the isolation of six new vibralactone derivatives. This paper deals with the isolation and structure elucidation of these compounds.

Results and Discussion
Compound 1 was isolated as a colorless oil and determined to have a molecular formula of C 13   3.61 (3H, s). The 13 C NMR and DEPT spectra of 1 showed thirteen carbons resonances, including three methyls, three methylenes, four olefinic carbons, an oxymethine, and two quaternary carbons (d C 65.8, C-1; 174.5, C-7; Table 1). Detailed analyses of the 2D NMR spectra of 1 revealed that it was similar to those of vibralactone E [12]. In the HMBC spectrum of 1, an obvious correlation was observed from a methoxy (d H 3.61) to the carboxyl group (d C 174.5, C-7). To draw a conclusion, compound 1 was esterified by a methyl at the carboxyl group of vibralactone E. The relative configuration of 1 was determined by a ROESY experiment. In the ROESY spectrum, H-5 correlated to the two protons of C-8, indicating that both H-5 and the isopentenyl group were b oriented. Compound 1 was named vibralactone N ( Fig. 1).
Compound 2, a colorless oil, was determined to have a molecular formula of C 12 H 20 O 3 according to the HREIMS data, m/z 212.1404 [M] ? (calcd for 212.1412). The IR spectrum revealed the presence of hydroxyl (3388 cm -1 ) and carboxyl (1735 cm -1 ) groups. The 1D NMR spectra demonstrated twelve carbons, which were ascribed to two methyls, four methylenes, four methines and two quaternary carbons (Table 1). These spectroscopic data showed that 2 was very similar to those of vibralactone I [14]. The analyses of 1 H-1 H COSY and HMBC spectra of 2 suggested that 2 possessed a same planar structure with that of vibralactone I. Its 13 C NMR and DEPT spectra showed that the chemical shifts of C-4 (d C 31.0) and C-5 (d C 50.8) of 2 were downfield shifted, while C-1 (d C 218.7) was upfield shifted obviously comparing to the corresponding signals of vibralactone I (C-1, d C 220.2, C-4, d C 28.3; C-5, d C 47.3). These data suggested that 2 was a stereoisomer of vibralactone I. In the ROESY spectrum, a key correlation of H-4 with H-8 and strong correlations from H-8 to H-6 and H-7 suggested H-2, H-3 and H-5 were in the same side (Fig. 2). Therefore, the structure of 2 was elucidated and named vibralactone O (Fig. 1).
The molecular formula of compound 3 was established as C 12 H 18 O 3 by HREIMS (m/z 210.1250, calcd for 210.1256). Compared its 13 C NMR and DEPT spectroscopic data with those of compound 2, the chemical shifts of the carbons in 3 were similar to the corresponding carbons in 2, with the exception of chemical shifts of C-2 (d C 137.8) and C-3 (d C 173.2). Subsequent analysis of the 2D  NMR spectroscopic data of 3 suggested the existence of an a,b-unsaturated ketone, owing to C-2 and C-3 were oxidized to form a double bond which conjugated with the carbonyl group (d C 210.7). The stereo-configuration of C-5 was not determined currently. Compound 3 was named vibralactone P (Fig. 1). Compound 4 was obtained as a colorless oil. Its molecular formula was determined as C 13 H 20 O 5 by HRE-SIMS (m/z 279.1203 [M ? Na] ? , calcd for 279.1208), with four degrees of unsaturation. The IR spectrum revealed the existence of hydroxy (3437 cm -1 ) and carboxyl (1766 cm -1 ) groups. The 13 C NMR and DEPT spectra showed thirteen carbons, including three methyls, three methylenes, four methines (three were oxygenated) and three quaternary carbons (two olefinic carbons and two lactone carbons; Table 2). In the HMBC spectrum, the proton at 1.34 ppm (d, J = 6.6 Hz, H-3 0 ) was correlated to a methine (d C 67.5, C-2 0 ) and a carbonyl group (d C 175.4, C-1 0 ), as well as cross peaks from 2 0 -OH (d H 4.24) and H-3 0 to H-2 0 (d H 4.25) in the COSY spectrum, revealed that the presence of a lactic acid group. Further analyses of the 2D NMR spectroscopic data of 4 suggested that the other parts of 4 were similar to those of vibralactone G both in planar structure and stereo-configuration [14]. From the HMBC spectrum, significant correlations were observed from H-10 . These evidences suggested that the double bond (C8-C9) was a E configuration (Fig. 2). Therefore, compound 4 was identified as 10-lactyl vibralactone G, as shown in Fig. 1.
Compound 6, a colorless oil, was determined to have a molecular formula of C 12 H 18 O 3 based on the HREIMS data, m/z 210.1259 [M] ? (calcd for 210.1256). The strong adsorption bands at 3427 and 1717 cm -1 suggested the presence of hydroxyl and carboxyl groups. The 1D NMR spectroscopic data demonstrated twelve carbons signals, including four olefinic carbons and a carbonyl. According to the HMBC spectrum, correlations can be found from both two methyl singlets (d H 1.62, H-10; 1.70, H-11) to two olefinic carbons (C-8, C-9). Meanwhile, cross peaks from H-7 to H-8 were also displayed in the 1 H-1 H COSY spectrum. These data confirmed the presence of an isopentenyl unit. Furthermore, the 1 H-1 H COSY correlations established connections from C-2/C-3/C-12/C-13. The HMBC spectrum showed that H-7 (d H 2.92) correlated to C-1, C-2 and C-6, H-2 (d H 6.67, d, J = 3.9 Hz) correlated to C-4 and C-6, as well as H-4 (d H 4.11, 1H, dd, J = 10.8, 7.2 Hz; 4.37, 1H, dd, J = 10.8, 4.8 Hz) correlated to C-6. The stereo-configuration of C-3 was not determined currently. Compound 6 was named vibralactone Q (Fig. 1).

General Experimental Procedures
UV spectra were obtained using a Shamashim UV 2401 spectrometer. Optical rotations were recorded on a JASCO P-1020 polarimeter. IR spectra were measured on a Bruker Tensor-27 infrared spectrophotometer with KBr pellets. HREIMS were obtained on a Waters Autospec Premier P776 mass spectrometer. HRESIMS were taken on an Agilent G6230 TOF MS spectrometer. 1D and 2D NMR spectra were recorded on Bruker Avance-600 and Ultrashield-800 spectrometers using TMS as an internal standard. Silica gel 200-300 mesh (Qingdao Marine Chemical Inc., China) and Sephadex LH-20 (Amersham Biosciences, Sweden) were used for column chromatography. Medium pressure liquid chromatography (MPLC) was performed on a Büchi Sepacore System equipping pump manager C-615, pump modules C-605 and fraction collector C-660 (Büchi Labortechnik AG, Switzerland), and columns packed with Chromatorex C-18 (40-75 lm, Fuji Silysia Chemical Ltd., Japan). Preparative HPLC was performed on an Agilent 1260 liquid chromatography system equipped with a Zorbax SB-C18 column (5 lm, 9.4 mm 9 150 mm).

Fungus Material and Cultivation Conditions
The fungus B. vibrans was provided and fermented by Zheng-Hui Li, Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (No. 20120920B) was deposited at the Herbarium of Kunming Institute of Botany. The culture medium to ferment this fungus consist of glucose (5 %), peptone from porcine meat (0.15 %), yeast powder (0.5 %), KH 2 PO 4 (0.05 %) and MgSO 4 (0.05 %). Five hundred 500-mL Erlenmeyer flasks each containing 350 mL of above-mentioned culture medium were inoculated with B. vibrans strains, respectively. Then they were incubated on rotary shakers at 24°C and 150 rpm for 25 days in dark environment.