Five New Secondary Metabolites Produced by a Marine-Associated Fungus, Daldinia eschscholzii

Five new compounds, including a benzopyran ribonic glycoside, daldiniside A (1), two isocoumarin ribonic glycosides, daldinisides B (2) and C (3), and two alkaloids, 1-(3-indolyl)-2R,3-dihydroxypropan-1-one (4) and 3-ethyl-2,5-pyrazinedipropanoic acid (5), along with five known compounds (6–10), were isolated from the EtOAc extract of the marine-associated fungus, Daldinia eschscholzii. Their structures were elucidated by extensive physicochemical and spectroscopic properties, besides comparison with literature data. The absolute configurations of compounds 1–3 were corroborated by chemical transformation, GC analysis and X-ray crystallographic analysis. Meanwhile, the absolute configuration of compound 4 and the planar structure of compound 6 were also determined based on the X-ray diffraction analysis. The cytotoxicity of compounds 1–10, antifungal and anti-HIV activities of compounds 1–5 and the in vitro assay for glucose consumption of compounds 1–3 were done in the anti-diabetic model, whereas none showed obvious activity.


Introduction
Marine fungi are known as a rich source of structurally diverse and biologically active secondary metabolites, including polyketides, steroids, terpenes and alkaloids. Nevertheless, the potential chemical investigations on marine fungi are limited. In recent decades, bioactive natural products obtained from the marine-derived fungi have attracted the rising attention of organic chemists for discovering new drugs [1].
It was amazing that slight variations of traditional cultivation conditions, such as media compositions, temperature, aeration or the shape of the culturing flask, might lead to the discovery of various types of new natural products by microorganism [2]. As was reported, Daldinia eschscholzii was well-known to produce abundant polyketides as a mantis-associated fungus [3,4], which motivated us to investigate the secondary metabolites produced by the marine-associated fungus, D. eschscholzii. As part of our ongoing research for structurally unique and bioactive natural products from the D. eschscholzii, we obtained a new benzopyran ribonic glycoside (1), two new isocoumarin ribonic glycosides (2 and 3) and two new alkaloids (4 and 5), together with five known derivatives (6-10) from the scaled-up fermentation of the D. eschscholzii. Herein, we describe the isolation, structural elucidation and biological evaluations of these compounds.
Analysis of the key 1 H-1 H COSY and HMBC correlations ( Figure 2) was used to establish the planar structure of 1. In the HMBC spectrum, a diagnostic long-range correlation from the anomeric proton H-1′ to C-5 (δC 158.6) suggested that the sugar moiety was linked to the C-5 of aglycone. The remaining one degree of unsaturation, together with the 1 H-1 H COSY correlations of H-9/H-2, H-2/H-3 and H-3/H-4 and the HMBC correlations from H-9 to C-2, C-3 and from H-4 to C-2, C-4a, C-5 and C-8a, indicating that a pyranoid ring was linked to C-4a and C-8a, and the methyl and hydroxyl groups were located at C-2 and C-4, respectively. Thus, the planar structure of 1 was established. Acid hydrolysis of 1 gave the sugar motif, and then, it was unambiguously established as D-ribose by chemical transformation and GC analysis. The coupling constant of the anomeric proton at δH 5.70 (H-1′, d, J = 4.5 Hz) in the 1 H NMR spectrum of 1 indicated the D-ribose unit to be in the α-configuration [10]. In the NOESY experiment, the correlations of H-2/H-4 or H-9/H-4 were not observed; Thus, it was difficult to determine the configurations at C-2 and C-4. Fortunately, we obtained the crystal of 1, and a single crystal X-ray diffraction experiment was carried out with Cu Kα radiation (Figure 3), allowing an explicit assignment of the absolute structure as 2R and 4R. Hence, the absolute configuration of 1 was elucidated and named daldiniside A. Compound 2 was isolated as a yellowish solid with the molecular formula C15H16O8, as deduced by the HRESIMS result ([M + Na] + at m/z 347.0731, calcd. for C15H16O8Na, 347.0743). The presence of hydroxyl, carbonyl and double bond groups were shown by IR absorption bands at 3429, 1689 and 1573 cm −1 , respectively. The α-D-ribose group of 2 was confirmed by NMR experiment (Table 1) and acid hydrolysis. The attachment of the α-D-ribose at C-6 was determined on the basis of the HMBC correlation from H-1′ (δH 5.74) to C-6 (δC 166.1). Apart from the signals of the sugar moiety, the 1 H NMR spectrum showed proton signals at δH 6.58 (1H, d, J = 1.8 Hz) and 6.62 (1H, d, J = 1.8 Hz), indicating the presence of a 1,2,3,5-tetrasubstituted aromatic ring. This structural assignment was further established by the HMBC correlations from H-5 to C-6, C-7 and C-8a, and from H-7 to C-5, C-6, C-8 and C-8a. The 13 C NMR spectrum showed one ester carbon signal at C-1 (δC 167.8), one olefinic carbon signal at C-4 (δC 105.8) and one methyl carbon signal at C-9 (δC 19.4). The HMBC correlations ( Figure 2) from H-9 to C-3, C-4 and from H-4 to C-3, C-4a, C-5, C-8a and C-9 suggested that there existed an isocoumarin unit, in which the hydroxyl and methyl groups were located at C-8 and C-3, respectively. Thus, the structure of 2 was established, namely, daldiniside B.
Compound 3 was determined to be C17H20O9 by the HRESIMS data, which showed a molecular ion at m/z 391.0994 [M + Na] + (calcd. for C17H20O9Na, 391.1005). The NMR data of 3 were very similar to those of 2 ( Table 1), suggesting that they shared the same basic skeleton. Moreover, the signals for a methylene at C-9 (δC 44.3), an oxygenated methine at C-10 (δC 65.4) and a methyl at C-11 (δC 24.4) were observed in the 13 C NMR of 3, from which we deduced that a -CH2(9)-CH(10)OH-CH3(11)-group in 3 replaced a -CH3 group in 2. Hence, the planar structure of 3 was determined ( Figure 2). To ascertain the absolute configuration at C-10, an acid hydrolysis experiment was carried out. By the chemical transformation and GC analysis, we established the sugar moiety to be α-D-ribose. In addition, the CHCl3 layer was evaporated to dryness, and the NMR data of the residual compound was identical to de-O-methyldiaporthin ([α] 20 D : +20.0, c 0.09, MeOH). Therefore, the absolute configuration of 3 was established, namely daldiniside C.
Compound 4 was obtained as a colorless crystal. The molecular formula C11H11NO3 was determined upon analysis of the HRESIMS peak at m/z 228.0628 [M + Na] + (calcd. for C11H11NO3Na, 228.0637). UV absorption bands at 210, 243, 257 and 300 nm and IR absorption bands at 3394, 3325 and 1607 cm −1 implied the presence of amine, hydroxy and conjugated carbonyl functionalities. In the 1 H NMR spectrum (Table 2) Figure 4). The -CO(8)-CH(9)OH-CH2(10)OH-subunit was established by analysis of the 1 H-1 H COSY correlation of H-9/H-10 and HMBC correlation from H-10 to C-8 and C-9 and linked to the indole moiety by C-3, determined by the HMBC correlations from H-2 to C-3 and C-8. The configuration at C-9 was unequivocally established to be R by the single-crystal X-ray diffraction using Cu Kα radiation ( Figure 5). Consequently, the absolute configuration of 4 was established and named 1-(3-indolyl)-2R,3-dihydroxypropan-1-one.   Compound 5 was isolated as a yellow oil with the molecular formula C10H12N2O4 as determined by the HRESIMS peak at m/z 275.0998 [M + Na] + (calcd. for C12H16N2O4Na, 275.1008). In the 2D NMR spectra of 5 (Figure 4), the 1 H-1 H COSY correlations of H-7/H-8 and H-10/H-11 and the HMBC correlations from H-8 to C-2 and C-9, from H-11 to C-5 and C-12 and from H-6 to C-2 and C-5 indicated the existence of the 2,5-pyrazinedipropanoic acid group. An additional ethyl moiety was located at C-3 by the 1 H-1 H COSY correlation of H-13/H-14 and a long-rang HMBC correlation from H-14 to C-3. Thus, the structure of 5 was established and named 3-ethyl-2,5-pyrazinedipropanoic acid, whose signals were similar to 6 ( Table 2), confirmed by a single-crystal X-ray diffraction using Mo Kα radiation ( Figure 6).

General Experimental Procedures
UV spectra were measured on a Varian Cary 50 spectrophotometer or a Shimadzu UV-2401A spectrophotometer. Optical rotations were recorded on a Perkin-Elmer PE-341LC polarimeter. IR spectra were determined on a Bruker Vertex 70 FT-IR spectrophotometer. The 1 H, 13 C, and 2D NMR spectroscopic data were recorded on Bruker AM-400 and DRX-600 spectrometers using TMS as the internal standard. HRESIMS data were acquired using an APIQSTAR Pulsar spectrometer. X-ray data were collected using a Bruker APEX DUO diffractometer. Column chromatography was performed on silica gel (100-200 mesh and 200-300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), RP-C18 silica gel (50 μm, YMC, Kyoto, Japan) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). Semi-preparative HPLC was conducted on an Agilent 1100 liquid chromatography with a YMC-Pack ODS-A (10 × 250 mm, 5μm, YMC Co., Ltd., Kyoto, Japan) column. GC analysis was performed with an Agilent Technologies 6890N gas chromatography system. Solvents were distilled prior to use, and spectroscopic grade solvents were used. TLC was performed with silica gel 60 F254 (Yantai Chemical Industry Research Institute, Yantai, China) and RP-C18 F254 plates (Merck, Darmstadt, Germany). Peptone (produced by protamine with enzymatic hydrolysis and drying into a pale yellow powder) was purchased from Beijing Shuangxuan Microbial Medium Plant (Product ID: 02-31A, Specification: BR).

Fungal Material and Fermentation
The strain of fungus D. eschscholzii was isolated from the branches of Scaevola sericea Vahl, collected from the mangrove forest nature reserve in Haikou, Hainan province, China. The fungus was identified by sequence analysis of the ITS region of its rDNA, as described previously [11], and the sequence data have been deposited in NCBI with Accession Number FJ624265. A voucher specimen (MCCC 3J00088) was deposited in a public collection, the Marine Culture Collection of China, MCCC. All of the information and strains collected can be shared at the website http://www.mccc.org.cn/ and the collection center.

Extraction and Isolation
The fermented rice substrate was extracted four times with EtOAc (4 × 25 L) at room temperature. After concentration in vacuo, the total extract (145.0 g) was suspended in water and then extracted exhaustively with petroleum ether and EtOAc, respectively. The EtOAc organic phase was evaporated under reduced pressure to afford a crude extract (77.0 g), which was subjected to silica gel column chromatography (CC) with a CH2Cl2/CH3OH gradient system (1:0, 50:1, 25:1, 10:1, 6:1, 3:1 and 1:1, v/v, each 8 L) to obtain six main fractions (A−F).

Acid hydrolysis and GC Analysis of 1−3 and Determination of the Absolute Configuration of the Sugar Moiety
Compound 1 (1.2 mg) was hydrolyzed with 2 M aqueous CF3COOH (2.0 mL) at 90 °C for 6 h. The reaction mixture was evaporated to dryness; Then, the residue and L-cysteine methyl ester hydrochloride (2.5 mg) were dissolved in dry pyridine (1.0 mL) and kept at 65 °C for 2 h. The reaction mixture was dried, and then, trimethylsilylimidazole (0.2 mL) was added to the residue, followed by stirring at 65 °C for 1 h [12]. In the end, the resultant solution was extracted with water and n-hexane, and then, the organic phase was submitted to GC analysis by using an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent, Shanghai, China); column temperature, 230 °C; injection temperature, 250 °C; detector FID, detector temperature, 250 °C. A peak at the retention time of 12.61 min for compound 1 was observed. When the corresponding ribose was prepared by the same reaction, the retention times of presilylated D-ribose and L-ribose were 12.66 and 14.09 min, respectively. Hence, the sugar in compound 1 was determined to be D-ribose.
Compounds 2 (1.5 mg) and 3 (9.8 mg) were subjected to a similar treatment as compound 1, and the retention times of ribose were 12.63 and 12.66 min, respectively. Therefore, the sugar in compounds 2 and 3 were determined to be D-ribose. In addition, the reaction mixture of compound 3 was diluted with H2O (1.5 mL) and extracted with CHCl3. The CHCl3 layer was dried to yield the aglycone, whose NMR and optical rotation data were identical to de-O-methyldiaporthin ([α] 20 D : +20.0, c 0.09, MeOH) [7].

Biological Activities
The cytotoxicity of 1-10 against HL-60, SMMC-7721, A-549, MCF-7 and SW-480 was studied using the MTT method [13], and the results showed no obvious inhibitory activity toward the above cancer cells with IC50 > 40 μg/mL. In addition, compounds 1-5 were tested for antifungal activities against Candida albicans (ATCC32354 and ATCC10231) at a concentration of 128 μg/mL and anti-HIV activity according to the described method [14,15]; Unfortunately, none of the compounds exhibited significant activities. Otherwise, the in vitro assay for glucose consumption of compounds 1-3 was done in the anti-diabetic model with DMEM-induced 3T3 fibroblasts [16], whereas none showed obvious activity at the concentration of 20 μg/mL.