New Eudesmane-Type Sesquiterpenoids from the Mangrove-Derived Endophytic Fungus Penicillium sp. J-54

Four new eudesmane-type sesquiterpenoids, penicieudesmol A–D (1–4), were isolated from the fermentation broth of the mangrove-derived endophytic fungus Penicillium sp. J-54. Their structures were determined by spectroscopic methods, the in situ dimolybdenum CD method, and modified Mosher’s method. The bioassays results showed that 2 exhibited weak cytotoxicity against K-562 cells.


Introduction
Mangrove forests, the unique forest ecosystems distributed in most tropical and subtropical regions, are an important resource of endophytic fungi that have been proved to be an important source of structurally and biologically diverse substances [1][2][3][4][5][6][7][8][9] such as peniphenones A-D, aniquinazolines A-D, phomazines A-C, and so on [10][11][12]. In order to pursue bioactive products from mangrove fungus, the secondary metabolites of mangrove endophytic fungus Penicillium sp. FJ-1 isolated from the stem of Ceriops tagal were studied, and a new drimane-type sesquiterpene [13] with antibacterial activity has been reported in our previous research. In our continuous research, four eudesmane-type new sesquiterpenoids, penicieudesmol A-D (1-4) (Figure 1), were obtained from the culture broth of the Penicillium sp. J-54 isolated from the healthy leaves of Ceriops tagal collected in Dong Zhai Gang Mangrove Reserve in Hainan. Herein, we described the isolation, structure determination, and biological activities of the new sesquiterpenoids 1-4.

Structural Elucidation
Penicieudesmol A (1), a white powder, had the molecular formula of C 15 Figure S2) implied a total of 15 carbon resonances including three methyl carbons (δ C 21.1, 16.0, 15.6), five methylene carbons (including one sp 2 methylene carbon and four sp 3 methylene carbons), five methine carbons (including two oxygen bearing methine carbons and three sp 3 methine carbons), and two quaternary carbons (δ C 150.3, 39.2). The 1D-NMR data of 1 (Table 1) combined with the sequential 1 H-1 H COSY correlations of H-1/H-2/H-3/H-4/H-5/H-6/ H-7/H-8/H-9, as well as the key HMBC from H 3 -14 to C-3/C-4/C-5, H 3 -15 to C-1/C-9/C-5, and H 3 -13 to C-7/C-11/C-12, suggested an eudesmane-type skeleton for 1. By comparison, above data (Table 1) were very close to that of the known compound nardoeudesmol A [14] with the eudesmane-type skeleton. The major difference between them pointed to the additional of a methine (δ C 33.7, C-4) and a methyl (δ C 15.6, C-14), as well as the absence of two olefinic carbon (δ C 146.4, C-4 and δ C 109.7, C-14) in 1 based on the key HMBC from H 3 -14 to C-3/C-4/C-5. The relative configuration of 1 was identical to the ROESY experiment ( Figure 3), such that the observed cross-correlation peaks from H 3 -15 and H 3 -14 to H-2, as well as from H-1 and H-7 to H-5, proved H 3 -15, H 3 -14, and H-2 were on the same side of the molecular plane and H-1, H-5, and H-7 were on the same side. The large coupling constants (9.2 Hz) between H-1 and H-2 characterised the trans-diaxial relationship. Moreover, the absolute configuration of the 1,2-diol moiety in 1 was determined by the in situ dimolybdenum CD method developed by Snatzke and Frelek [14][15][16]. On the basis of the empirical rule proposed by Snatzke, the positive Cotton effect observed at around 310 and 400 nm, respectively, in the induced CD spectrum (Figure 4a) permitted one to assign the 1S and 2S absolute configuration. Therefore, the absolute configuration of penicieudesmol A was deduced to be 1S, 2S, 4S, 5S, 7R, and 10R.
Penicieudesmol B (2) was isolated as a white powder with a molecular formula C 15 H 26 O 3 determined by its HREIMS at m/z 254.1878 [M] + (calcd. for m/z 254.1882). The similarity of 1D and 2D NMR data between 2 ( Table 1) and 1 indicated their similar planar structure. The only difference between these two compounds was that H-7 in 1 was substituted by a hydroxyl in 2, which was proved by the obvious downfield shift of C-7 (δ C 72.7) and the HMBC correlations from 7-OH to C-7/C-8 and H 3 -13 to C-7, together with the HREIMS. The relative configuration of 2 was identical with that of 1 by the large coupling constants (9.1 Hz) between H-1 and H-2, as well as the ROESY correlations ( Figure 3). In addition, the 2S configuration of compound 2 was clearly defined by the observed chemical shift differences ∆δ S−R by the modified Mosher's method (Figure 4b) [12]. So, the stereogenic centers of penicieudesmol B were determined as 1S, 2S, 4S, 5S, 7S, and 10R. Penicieudesmol C (3) was obtained as yellow oil with the molecular formula C 15 H 26 O 3 determined according to the HREIMS peak at m/z 254.1880 [M] + (calcd. for m/z 254.1882), indicating an isomer of 2. The 1 H and 13 C NMR data of 3 (Table 2) showed high similarity to those of 2, except for the location of hydroxyl in the two compounds. The sequential 1 H-1 H COSY correlations of H-6/H-7/H-8/H-9, together with the key HMBC correlations from 5-OH, H 3 -14, and H 3 -15, as well as H-7 to C-5, from H 3 -15 to C-9, and from H 3 -13 to C-7 displayed that 7-OH in 2 shifted to 5-OH in 3. The relative and absolute configuration of 3 was determined to be consistent with that of 2 through the same method (Figures 3 and 4b). Hence, the stereogenic centers of penicieudesmol C were determined as 1S, 2S, 4S, 5R, 7R, and 10S.
Penicieudesmol D (4) was also obtained as yellow oil. The HREIMS displayed a quasi-molecular ion peak at m/z 270.  Table 2 suggests that the substituent hydrogen atoms were H-7 in 4.
The relative and absolute configuration of 4 was determined to be consistent with that of 2 and 3 via the same method (Figures 3 and 4b). Consequently, the stereogenic centers of penicieudesmol D were determined to be 1S, 2S, 4S, 5R, 7S, and 10S.

The Bioactivities of Compounds 1-4 from Penicillium sp. J-54
All the compounds (1-4) were evaluated for their cytotoxic activity against K-562, SEL-7420, and SGC-7721 cell lines using the MTT method in vitro [17] and antimicrobial activity against Candida albicans and Staphylococcus aureus using the filter paper disc agar diffusion method [18]. The results showed that compound 2 exhibited weak cytotoxicity against K-562 with IC 50 value of 90.1 µM, with paclitaxel as the positive control (IC 50 = 9.5 µM). Unfortunately, none of these compounds showed antimicrobial activity.

Fungal Material
Penicillium sp. J-54 was isolated from the healthy leaves of Ceriops tagal, which were collected in Dong Zhai Gang Mangrove Reserve in Hainan province, in July 2011. The endophytic fungus was identified based on the DNA sequences of 18S rDNA gene. For identification of its 18S rDNA gene sequences, the Penicillium sp. J-54 was cultured in potato dextrose agar for five days. The mycelium was ground to a fine powder in liquid N 2 , then genomic DNA was extracted, and 18S rDNA region was amplified by PCR using primers NS1 (5 -GTAG TCATATGCTTGTCTC-3 ) and NS6 (5 -GCATCACAGACCTGTTATTGCCTC-3 ). PCR products were sequenced (Applid Biosystems 3730 XL Genetic Analyzer, Applied Biosystems Inc., Foster City, CA, USA). The producing strain was prepared on PDA medium and stored in our Lab. at 4 • C.

Fermentation and Extraction
Penicillium sp. J-54 was cultured in PDB (the potato liquid media consisting of 200.0 g/L potato, 20.0 g/L glucose, and 1000 mL deionized water) at 29 • C and 130 rpm for 72 h. 20 mL of the seed culture was inoculated into each 1000 mL Erlenmeyer flask of production medium composed of (per litre) 20.0 g potato, 0.4 g glucose, and 400 mL deionized water; the pH was adjusted 7.0. They were cultivated in static for 4 weeks after being incubated at 29 • C for 7 days on a rotary shaker at 130 rpm. The liquid filtrate from 100 L of fermentation broth was collected and extracted four times with ethyl acetate (1000 mL × 4 times) at room temperature.

Preparation of S-MTPA and R-MTPA Esters
Compound 2 (1 mg) was dissolved in 1 mL CH 2 Cl 2 , and 4-dimethylaminopyridine (3 mg) and (R)-MTPACl (10 µL) were added. The reaction was stirred for 5 h at room temperature. Then, 1 mL of H 2 O was added to stop the reaction and to extract the solution three times with CH 2 Cl 2 (5 mL each). A mixture of diol-Mo 2 (OAc) 4 (1:1.3) for 1 was subjected to CD measurements at a concentration of 0.5 mg/mL in HPLC grade DMSO dried with 4 Å molecular sieves, according the literature report [19]. The first CD spectrum was recorded after mixing immediately, and the CD spectrum was recorded again after mixing for 10 min. The inherent CD was subtracted. The observed signs of the diagnostic bands at about 310 and 400 nm in the induced CD spectrum were correlated to the absolute configuration of the 1, 2-diol moiety.

Bioassays
The cytotoxic activity for compounds 1-4 were tested against three cell lines including human hepatic carcinoma cell lines (SEL-7420), gastric cell lines (SGC-7721), and leukemia cell lines (K-562). These cell lines were purchased from Shang Hai Cell Bank of Chinese Academy of Sciences. The purity of the tested compounds and paclitaxel (PTX) was determined to be over 95% using the chromatography. The cytotoxic effects on these tests cell were assessed by the IC 50 values and determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] colometric method as described in reference [17]. Each set of tests was conducted three times to confirm reproducibility of the results. These compounds were dissolved in DMSO, PTX was used as a positive control, and the medium without test compound was used as a negative control in the bioassay.
The antimicrobial activity of compounds 1-4 against C. albicans and S. aureus were also evaluated using the 2-fold dilution method [18]. The tested strains were cultivated in YPD broth for C. albicans and LB broth for bacteria at 28 • C. The test compounds were dissolved in DMSO at different concentrations from DMSO at different concentrations from 1000 to 7.8 µg/mL (from 6.25 to 0.025 µg/mL for the positive controls) by the continuous 2-fold dilution methods in 96-well plates. Each well contains 100 µL of contents composed of 20 µL of inoculums (5 × 10 5 CFU/mL), test compounds, and YPD or LB media. The microtiter plates were incubated at 28 • C for 24 h and were examined for microbes' growth by turbidity in daylight. Chlorhexidine acetate and kanamycin sulfate were used as positive controls for C. albicans and S. aureus, respectively.

Conclusions
Four new eudesmane-type sequiterpenes (1-4) were isolated from the PDB fermentation broth of the mangrove-derived endophytic fungus Penicillium sp. J-54 originated from the healthy leaves of Ceriops tagal collected in Dong Zhai Gang Mangrove Reserve in Hainan. Their structures were determined by spectroscopic methods, the in situ dimolybdenum CD method, and the modified Mosher's method. Compound 2 exhibited weak cytotoxicity against K-562 with an IC 50 value of 90.1 µM. The results proved that mangrove endophytic fungi are the source of new bioactive substances.