A new Secondary metabolites of the crinoid (Comanthina schlegeli) associated fungus Alternaria brassicae 93

Abstract Fungus Alternaria brassicae 93 isolated from crinoid (Comanthina schlegeli), which was collected from the South China Sea. Six compounds were isolated from A. brassicae 93, including one new compound (1), along with five known compounds, ochratoxin A methyl ester (2), cis-4-hydroxym-ellein (3), (R)-7-hydroxymellein (4), trans-2-anhydromevalonic (5) and protocatechuic acid (6). Their structures were determined by spectroscopic methods and comparison with reported data. Cytotoxicity against two human cancer cell lines and antibacterial activity against twelve aquatic bacteria of compound 1 were also tested. The fungus Alternaria brassicae 93 separated from crinoid (Comanthina schlegeli) produced one new compound (1).


Introduction
The Crinoidea is one of the most primitive echinoderm species, including the feather stars, the sessile sea lilies and stemmed sea lilies which live in the deep sea. Most of existing Crinoidea are feather stars with a large number of flexible brachiopods in various colours, which are promising sources of new natural products and provide suitable habitats for marine micro-organisms. Some bioactive constituents have been isolated from marine crinoid (Shao et al. 2007;Bokesch et al. 2010;Wangun et al. 2010). However, there are few reports about crinoid associated fungi and their metabolites. In addition, Alternaria species play a significant role in new compounds discovery Mu et al. 2015;. Accordingly, we took the crinoid-associated fungus Alternaria brassicae 93 as a new resource for novel compounds.
A. brassicae 93 was isolated from a crinoid (Comanthina schlegeli), which was collected from the South China Sea. Six metabolites ( Figure 1) were isolated from the ethyl acetate extract of the fermentation culture of the strain 93. Herein, the structure elucidation and bioactivities of compound 1 were reported. Unfortunately, compound 1 exhibited no cytotoxicity against human tumour cell line (MDA-MB-435) and human lung cancer cell line (A549), nor antibacterial activity against twelve aquatic pathogenic bacteria.

Structure determination
Compound 1 was obtained as a light yellow solid with specific optical rotation of [ ] 30 D + 14.1 (c = 0.59, MeOH). The molecular formula of 1 was determined as C 14 H 18 O 3 by HReSI-MS at m/z 234.1211 [M] − , being indicative of 6° of unsaturation. The UV spectrum showed absorption maximum at 200 and 232 nm, which indicated a α,β-unsaturated carbonyl group. The nMR data were shown in Table 1. The 13 C nMR (100 MHz, Acetone-d 6 ) and HSQC spectra showed 14 carbon signals including five quaternary carbons (two carbonyl carbons δ C 204.0 and 198.2; two olefinic carbons δ C 163.5 and 137.8; one quaternary sp 3 carbon δ C 88.4) and five tertiary carbons (four olefinic carbons δ C 147.1, 133.9, 125.5, and 121.3; one tertiary sp 3 carbon δ C 54.6) as well as four methyls (δ C 19.6, 18.6, 11.7, and 8.9). The 14 carbon signals showed 5° of unsaturation, which indicated a ring in the molecule.
According to the HMBC correlations of C-6 (δ C 198.2) with H-7, H-8 and H-9, C-7 should be coupled with the carbonyl group C-6, which was supported by the chemical shifts of C-7 (δ C 125.5) and C-8 (δ C 147.1). There are strong HMBC correlations between H-10 and C-1 (δ C 204.0) as well as between H-11 and C-2, which indicated that C-2 connected with C-10. The HMBC correlations of H-14 with C-1, C-4 and C-5 showed that C-5 connected with both C-1 and C-4. And the HMBC correlation of H-13 (δ H 1.89) with C-4 confirmed that C-13 connected with C-4. Moreover, the HMBC correlations of H-13 with C-1, C-2 and C-3, and of H-10 with C-1, C-2 and C-3 implied that C-1, C-2, C-3, C-4 and C-5 formed a five-member ring, which was supported by the remaining 1° of unsaturation. Furthermore, according to no nOe correlation between H-13 and H-14, we tentatively supposed they were not in the same plane. Thus, compound 1 was identified as a new 2,3-substituted-cyclopentone (Figure 1), for which we propose the name Alterbrasone.
Compound 2 was obtained as red oil. The molecular formula of 2 was assigned as C 21 H 20 ClnO 6 on the basis of the nMR data and eSI-MS at m/z 417.6 [M-H] − . This formula demanded 12° of unsaturation. It was identified as ochratoxin A methyl ester (Xu et al. 2013) by comparing the spectroscopic data with previously reported data.
Compound 3 was isolated as a white crystal. Its molecular formula of C 10 H 10 O 4 was deter-  indicative of 6° of unsaturation. It was identified as (R)-7-hydroxymellein (Oliveira et al. 2011) by comparing the spectroscopic data with reported data. Compound 5 was isolated as a white solid. The molecular formula of 5 was determined as C 6 H 10 O 3 on the basis of the nMR data and eSI-MS at m/z 129.2 [M-H] − , which suggested 2° of unsaturation. After comparing the data with those reported in the literature (Xu et al. 2011), compound 5 was identified as trans-2-anhydromevalonic acid.
Compound 6 was got as a white solid. Its molecular formula of C 7 H 6 O 4 was determined from the nMR data and eSI-MS at m/z 153.2 [M-H] − , showing 5° of unsaturation. It was identified as protocatechuic acid (Meng et al. 2012) by the comparison with literature values.

Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1-S11.