Three New Oblongolides from Phomopsis sp. XZ-01, an Endophytic Fungus from Camptotheca acuminate

Four new metabolites, including three new oblongolides named C1, P1, and X1 (1-3) and 6-hydroxyphomodiol (10), along with eight known compounds – oblongolides B (4), C (5), D (6), O (7), P (8) and U (9), (3R,4aR,5S,6R)-6-hydroxy-5-methylramulosin (11), and (3R)-5-methylmellein (12) – were isolated from the endophytic fungal strain Phomopsis sp. XZ-01 of Camptotheca acuminate. Their structures were elucidated by spectroscopic analyses, including 1H- and 13C-NMR, 2D NMR (HSQC, HMBC, 1H-1H COSY and NOESY) and HR-FT-MS. Cytotoxic activities of these compounds were evaluated. Some of them showed weak selective activities.


Results and Discussion
We obtained oblongolide C1 (1) as white needles and determined it to have the molecular formula C 14 H 20 O 4 by HR-FT-MS. The 13 C-NMR, DEPT and HSQC spectra of compound 1 showed 14 carbon signals: two methyl groups, three methylene groups, three methine groups, one hemiacetal methine (δ C 100.6), a disubstituted olefin (δ C 137.8 and 124.2), an oxygenated quaternary carbon (δ C 78.7), a lactone carbonyl (δ C 176.6), and a quaternary carbon. The 1 H-1 H COSY correlations between H-4 and H-5, H-5 and H-5a, H-5a and H-6, H-5a and H-9a, H-6 and H-7, H-7 and H-1′, H-8 and H-7, H-8 and H-9, H-9a and H-9 established the structure of a 9-carbon moiety ( Figure 2, in green). Key HMBC correlations from H-1″ to C-1, C-3a, C-9a and C-9b, from H-5 to C-3a, from H-4 to C-9b, and from H-3 to C-3a established the planar structure of 1. The relative configuration of 1 was deduced on the basis of NOESY spectroscopic data. The NOE correlations between H-7 and H-5a and between H-5a and H-1″ established the α-orientations of H-5a, H-7 and H-1″. NOESY cross-peaks from H-3 to H-9a and from H-9a to H-1′ indicated the β-orientations of H-3, H-9a and H-1′. A comparison of the 1 H and 13 C-NMR spectra of 1 with that of oblongolide C indicated that 1 was the 3α-hydroxy derivative of oblongolide C [4]. Therefore, we determined the structure of 1 to be 3α-hydroxyoblongolide C and it was named as oblongolide C1 for consistency with the literature [4].  , and there was an acetyl group in 2. Key HMBC correlations from H-8 to C-8a, C-1′ and C-9a, from H-1′ to C-6, C-7 and C-8, from H-1″ to C-1, C-3a, C-9a and C-9b indicated the planar structure of 2. We determined the relative configuration of 2 by analysis of the NOESY spectrum. The NOE correlations between H-8 and H-1′, between H-8 and H-9a, between H-8 and H-9β, and between H-1′ and H-6β established the β-orientations of H-1′, H-8 and H-9a. The NOE correlations between H-3a and H-1″, between H-3α and H-3a and between H-1″ and H-5a indicated the α-orientations of H-1″, H-3a and H-5a. A comparison of the 1 H-and 13 C-NMR data of 2 with those of oblongolide P [3] revealed that these two compounds had similar structures, except that an acetyl group was attached to the C-8 hydroxyl group in 2. Therefore, we determined 2 to be 8-acetylobolngolide P and named it oblongolide P1. Oblongolide X1 (3) was obtained as white oil. Its molecular formula, C 16 H 24 O 5 , was deduced on the basis of HR-FT-MS and 13 C-NMR data. A comparison of the NMR data of 3 with those of known compound oblongolide X [7] indicated that 3 was a hydroxy-derivative of the latter. The HMBC correlations from H-1″′ to C-1 and C-2 located the hydroxyl substitution at C-2. The NOE correlations between H-10a and H-1′, between H-6a and H-8 and between H-6a and H-1″ determined the relative configuration of 3. Therefore, we determined 3 to be 1″′-hydroxyoblongolide X and named it oblongolide X1.  Compound 10 had the molecular formula C 16 H 26 O 4 , as established by HR-FT-MS and 13 C-NMR spectra. 1 H-and 13 C-NMR data of 10 were similar to those of phomodiol [8], except that the methine signal [δ H 1.46 (1H, m), CH-6] was replaced by a quaternary carbon (δ C 70.0, C-6). Key HMBC correlations from H-15 to C-5, C-6 and C-7, from H-11 to C-1, C-2, C-9 and C-12, from H-16 to C-1, C-2 and C-3, from H-4 to C-2, C-5 and C-9 and from H-10 to C-3, C-6 and C-8 indicated the planar structure of 10. The relative configuration of 10 was deduced on the basis of NOESY spectroscopic data. The NOE correlations between H-10 and H-11, between H-2 and H-11, between H-13 and H-11, between H-15 and H-10 and between H-9 and H-16 indicated β-orientation of the hydroxyl group (6-OH) and the α-orientation of the side chain attached to C-1. Therefore, the structure of 10 was determined. We named it 6-hydroxyphomodiol [8]. Besides the nine oblongolides, including three new ones, we isolated two more polyketides. We determined 11 to be (3R,4aR,5S,6R)-6-hydroxy-5-methylramulosin (11) [5] by a comparison of NMR data. This compound was previously isolated from a marine-derived fungus which was derived from the green alga Codium fragile [5]. The spectroscopic data of 12 were identical to those of the known compound (3R)-5-methylmellein, first isolated as the main phytotoxic metabolite of Fusicoccum amygdale [6].

Cytotoxicity
The results of cytotoxic tests of compounds 1-12 are shown in Table 4. They exhibited no significant activity against the three tested cancer cell lines.

General
Optical rotations were measured with a Perkin-Elmer 341 automatic polarimeter in methanol. IR spectra were recorded on a Nicolet AVATAR 330FT spectrometer. NMR spectra were taken on a Bruker Avance III-600 NMR spectrometer with TMS as an internal standard. HR-FT-MS data were acquired by using En Apex ultra 7.0 FT-MS. TLC was carried out using glass-precoated silica gel GF254 (Qingdao) and visualized under UV light or by spraying with vanillin (contains H 2 SO 4 ) ethanol reagent. Sephadex LH-20 (40-70 µm, Amersham Pharmacia Biotech AB, Uppsala, Sweden), silica gel (200-300mesh, Qingdao Marine Chemical, Inc., Qingdao, China), and lichroprep reversed-phase RP-18 silica gel (40-63 µm, Merck, Darmstadt, Germany) were used for column chromatography (CC).

Fungal Material
The fungus (XZ-01) was isolated from current-year twigs (8-12 × 1-2 cm, length × diameter) of Camptotheca acuminate collected from the Jiangshi Natural Reserve, Shaowu, Fujian, China. It was identified as a non-sporulating fungus by traditional morphology. A BLAST search result showed that the internal transcribed spaces (ITS) sequence of XZ-01 was highly homologous (98% percent similarity) to that of a Phomopsis species (BCC 9789 [GU086404]), indicating that XZ-01 belongs to this genus.

Fermentation and Extraction
XZ-01 was cultivated on potato dextrose agar at 28 °C. The agar blocks were chopped and transferred into Erlenmeyer flasks (10 × 3 L), each containing 1 L of potato dextrose broth (PDB), and then fermented at 28 °C on a rotary shaker (150 rpm) for 7d. The culture was filtered to separate broth and mycelia. The culture broth was extracted with EtOAc (6 × 10 L) for six times. The combined organic layer was concentrated under vacuum to afford 3.2 g of residue.

Biological Assay
Cancer cell lines were derived from the cell bank of The Chinese Academy of Sciences. Cells were seeded at a density of 5 × 10 3 /100 µL medium in 96-well microtiter plate and treated with the compounds at the concentration of 20 µg/mL. Viable cells were incubated with MTT (5 mg/mL) for 4 h and formazan precipitate was dissolved in 100 µL DMSO and the absorbance at 490 nm was measured by Multimode Detector DTX880 (Beckman Coulter).