Five New Guanacastane-Type Diterpenes from Cultures of the Fungus Psathyrella candolleana

Five new guanacastane-type diterpenes, named guanacastepenes P–T (1–5), were isolated from cultures of the fungus Psathyrella candolleana. Their structures were elucidated on the basis of extensive spectroscopic methods. All of the compounds were tested for their 11β-hydroxysteroid dehydrogenase (11β-HSD1) inhibitory activity. Compound 3 exhibited inhibitory activity against both human and mouse isozymes of 11β-HSD1 with IC50 values of 6.2 and 13.9 μM, respectively.


Introduction
Guanacastepene A, was the first member of the guanacastane family isolated from an unidentified endophytic fungus (CR 115) [1]. This was followed by isolation of guanacastepenes B-O from the same fungus by the same group [2]. Heptemerones A-G were later isolated from cultures of Coprinus heptemerus [3,4], as well as radianspenes A-M from Coprinus radians [5], which attracted interest of the guanacastanes in the synthetic organic community [6][7][8][9][10][11][12][13][14][15][16][17][18]. The total synthesis of guanacastepenes with a novel 5/7/6 ring system was considered a challenging synthetic target [19], whereas synthesis of analogues offered the best prospects for capitalizing on the promising antibiotic activity, as well as avoiding the side effect of hemolytic activity against human red blood cells [10,20]. However, no other bioactivities, except for antibiotic effects were reported for this type of diterpenes. As a part of a search for naturally occurring secondary metabolites with diverse structures from higher fungi in China, investigations of chemical components from Psathyrella candolleana cultures were carried out, which led to the isolation of a series of new guanacastane-type diterpenes, guanacastepenes P-T (1-5, Fig. 1). Their structures were elucidated by means of spectroscopic methods. These compounds shared a larger conjugated system including an a,b-unsaturated ketone moiety in the highly oxygenated five-member ring system comparing to the known guanacastane-type diterpenes. All of these compounds were evaluated for their cytotoxic and antiherpes simplex viruses (HSV) activities, while compounds 1, 3, and 5 were evaluated for inhibitory activities against one isozyme of 11b-hydroxysteroid dehydrogenases (11b-HSD1).

Results and Discussion
Guanacastepene P (1) was obtained as a light yellow oil. Its molecular formula C 20 (Table 2), classified as five methyl groups, four aliphatic methylenes, three methines (one olefinic and one oxygenated), and eight quaternary carbons (one carbonyl and five olefinic). Thus, four degrees of unsaturation were accounted for by the three double bonds and the one carbonyl group, while the remaining three degrees of unsaturation suggested that compound 1 should possess a three-ring system. Further inspection of the 1 H NMR spectrum and 1 H-1 H COSY correlations (Fig. 2) resulted in the deduction of an isopropyl group (Me-19-H-18-Me-20), two connected methylene groups (H 2 -9-H 2 -10), a chain of CH 2 -CH 2 -CH-OH (H 2 -7-H 2 -6-H-5-OH), an isolated olefinic hydrogen (H-2), and three isolated methyl groups (H-15, H-16 and H-17).
Connectivity among the above mentioned fragments and the contribution of the three-ring system were established by analysing 1 H, 13 C long-range correlations extracted from HMBC experiments (Fig. 2). The correlations of H-18 to C-11, C-12 and C-13 and HO-13 to C-12 and C-14 established the connection of C-11-C-12-C-13-C-14, while the correlations of H-17 to C-11, C-12 and C-1, and H-2 to C-1 and C-11 indicated the presence of a fivemembered ring A. Subsequently, the correlations of H-9 to C-8 and H-10 to C-11 supported the connection of C-8-C-9-C-10-C-11. Besides, correlations of H-16 to C-8 and C-3, as well as H-2 to C-8 were evident for a sevenmembered ring B. The last ring C and the links of remaining blocks were clearly shown by HMBC correlations of H-15 to C-3, C-4 and C-5, H-5 to C-4 and C-6, H-6 to C-4 and C-5, and H-7 to C-8 and C-3. Finally, compound 1 possessed a backbone of a 5/7/6 ring system related to that of guanacastepene A [1]. Biogenetically, the stereoconfigurations of methyls of Me-16 and Me-17 were a and b oriented, respectively [4]. In the ROESY spectrum (  (Table 2) spectroscopic data were similar to those of compound 1. The main difference was that an oxygenated methine at d C 70.3 (C-5) in 1 was replaced by a keto carbonyl group at d C 197.5 in 2, constructing a 3,4-unsaturated-5-keto moiety, which led to the downfield shift of C-3 (Dd 20.8 ppm) and C-6 (Dd 4.4 ppm), and the upfield shift of C-4 (Dd 6.4 ppm) in compound 2, consistent with the HMBC correlations of H-6, H-7 and H-15 to C-5. Detailed analysis of 1D and 2D NMR data (HSQC, HMBC, 1 H-1 H COSY, ROESY) suggested that the other parts of 2 were the same as those of 1. Therefore, compound 2 was established as shown.
Guanacastepene R (3), a light yellow oil, had a molecular formula of C 20 H 26 O 4 on the basis of its HRESIMS at m/z 353.1737 ([M?Na] ? ), 16 mass units higher than that of 2. The 1D NMR spectroscopic data (Tables 1 and 2) were quite similar to those of 2, except that the signals for a methyl group in 2 were replaced by the signals for an oxygenated methylene group, which was confirmed by the HMBC correlations of d Guanacastepene S (4) had the same molecular formula of C 20 H 28 O 3 (HRESIMS ([M?Na] ? at m/z 339.1927) as 1 and was obtained as a colorless oil. Its 13 C NMR spectrum ( Table 2) displayed 20 carbon signals corresponding to five methyls (three singlets and two doublets), five methylenes, two methines and eight quaternary carbons (four olefinic and two carbonyl carbons), consistent with a similar structure of compound 2. Significant differences were one less double bond and absence of an olefinic hydrogen in 4.  (Table 2) together with analysis of its HSQC spectrum indicated 20 carbons, including four methyls, five methylenes (one olefinic), six methines (two oxygenated and one olefinic), and five quaternary carbons (two olefinic and one carbonyl carbons). Comprehensive analysis of the 1 H-1 H COSY and HMBC spectra (Fig. 2), suggested that 5 shared the same  , which was also supported by the HMBC correlations of H-15 to C-3 and C-4, and H-3 to C-2 and C-4. The 1 H-1 H COSY data, as well as HMBC correlations of H-5 to C-3 and C-15 also gave the information that C-5 was a methine substituted by a hydroxy group. Likewise, C-9 was confirmed as a hydroxy substituted methine based on the 1 H-1 H COSY and HMBC correlations as shown in Fig. 2. Based on the proposed biogenetic orientation of Me-16 and Me-17, the configurations of H-3 and HO-9 were determined to be b and a oriented, respectively, owing to the strong correlation signals of H-3 with H-17 and H-9, and H-9 with H-17 in the ROESY spectrum. In addition, the stereoconfiguration of H-5 was determined to be a oriented, as indicated by the observation of the ROESY correlations the same to those of compound 1. Consequently, compound 5 was elucidated as guanacastepene T. Compounds 1-5 were evaluated for their cytotoxicity against five human cancer cell lines. None was found to possess significant activity with IC 50 values less than 40 lM. In addition, they were evaluated for anti-HSV (herpes simplex viruses) activity. However, none exhibited activity. Furthermore, compounds 1, 3 and 5 were tested for inhibitory activities against one isozyme of 11b-HSD1. Of these, only compound 3 exhibited inhibitory activity against both human and mouse isozymes of 11b-HSD1 with IC 50 values of 6.2 and 13.9 lM, while glycyrrhizinic acid (positive control) had IC 50 values of 4.2 and 6.5 nM, respectively.
Five new guanacastane-type diterpenes, guanacastepenes P-T, each containing a 5/7/6 ring system, were isolated from cultures of fungus P. candolleana. Until now, totally 33 guanacastane-type diterpenes have been obtained from kinds of fungi, and some of them showed antibiotic and antitumor activities. Our present research enriched the structure diversity of the guanacastane family and this is  also the first report to show that guanacastane-type diterpenes possessed inhibitory activity against human 11b-HSD1.

General Experimental Procedures
Optical rotations (OR) were recorded on a JASCO P-1020 digital polarimeter, while the UV and IR spectra were obtained on a Shimadzu UV2401PC and a Bruker Tensor 27 FT-IR (KBr pellets) spectrometers. NMR spectra were acquired on Bruker AM-400, DRX-500 and Avance III 600 MHz spectrometers with tetramethylsilane (TMS) used as an internal standard at room temperature. Basidiomata small to medium-sized. Pileus 3-7 cm in diam, campaniform then flattened, with moist umbonate center; surface glabrous, light honey-yellow to brown, yellow-brown at the apex, fading to grayish when dry; white universal veil gradually fall off with age. Context white, relatively thin. Lamellae adnate, narrow and length unequal, crowded, dirty white, grayish to pallid purplebrown; edge dirty white, coarse. Stipe slender and fistulous, cylindrical, 3-8 9 0.2-0.7 cm, white, weak and fragile, surface densely floccose-fibrillose or reticulate. Spores print dark purple-brown; spores smooth, ellipsoid, 6.5-9.0 9 3.5-5.0 lm, germ pore was visible. Pleurocystidia smooth and thin-walled, hyaline in KOH, more or less lageniform and apex rounded, 34-50 9 8-16 lm.

Cytotoxicity Assay
Human myeloid leukemia HL-60, hepatocellular carcinoma SMMC-7721, lung cancer A-549 cells, breast cancer MCF-7 and colon cancer SW480 cell lines were used in the cytotoxic assay. All cell lines were cultured in RPMI-1640 or DMEM medium (Hyclone, USA), supplemented with 10 % fetal bovine serum (Hyclone, USA) in 5 % CO 2 at 37°C . The cytotoxicity assay was performed according to the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide[ method in 96-well microplates [21]. Briefly, adherent cells (100 lL) were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition with initial density of 1 9 10 5 cells/mL. Each tumor cell line was exposed to the test compound dissolved in DMSO at concentrations of 0.0625, 0.32, 1.6, 8, and 40 lM in triplicates for 48 h, with cisplatin (Sigma, USA) as a positive control. After compound treatment, cell viability was detected and a cell growth curve was graphed. IC 50 values were calculated by Reed and Muench's method [22].

Antiviral Assay
Confluent Vero cells in a 96-well tissue plate were inoculated in triplicate with virus suspension (50 lL) [HSV/ Blue, at multiplicity of infection (MOI) 1] and culture medium (50 lL) containing testing compounds at different concentrations. Cells were lysed with 1 % Nonidet P-40 in DMEM at 24 h postinfection. Lysates from each well were mixed with chlorophenol red-b-D-galactopyranoside [CPRG; Boehringer, Ingelheim, Germany), and b-galactosidase (b-Gal)] activity was measured by taking absorbance readings at 570 nm every 2 min for a total of 25 readings. The slope of the line was used to quantify b-Gal activity as milli-optical density units/min (mOD/min). The 50 % inhibitory concentration (IC 50 ) was defined as the concentration of the antiviral drug that reduced the mOD/min values by 50 % relative to the virus control. Inhibitory concentrations were calculated using the probit regression method [23].

Inhibitory Activities Against 11b-HSD1 Assay
Inhibitory activities of the compounds on human or mouse 11b-HSD1 were determined using scintillation proximity assay (SPA). Microsomes containing 11b-HSD1 were used according to our previous studies [24,25]. Full-length cDNAs of human or murine 11b-HSD1 were isolated from cDNA libraries provided by the NIH Mammalian Gene Collection. The cDNAs were cloned into pcDNA3 expression vectors. HEK-293 cells were transfected with the pcDNA3-derived expression plasmid and selected by cultivation in the presence of G418 (700 lg/mL). The microsomal fraction overexpressing 11b-HSD1 was prepared from the HEK-293 cells, which were stable transfected with 11b-HSD1. The fraction was then used as the enzyme source for SPA. Microsomes containing human or mouse 11b-HSD1 were incubated with NADPH and [ 3 H] cortisone. The product, [ 3 H] cortisol, was specifically captured by a monoclonal antibody coupled to protein A-coated SPA beads. All tests were done in twice with glycyrrhizinic acid as a positive control. IC 50 (X ± SD, n = 2) values were calculated by using Prism Version 4 (GraphPad Software, San Diego, CA, USA).