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Short Paper | Regular issue | Vol. 87, No. 4, 2013, pp. 861-867
Received, 15th January, 2013, Accepted, 4th February, 2013, Published online, 20th February, 2013.
DOI: 10.3987/COM-13-12667
Aculeatusquinones A-D, Novel Metabolites from the Marine-Derived Fungus Aspergillus aculeatus

Li Chen,* Wei-Wei Zhang, Qiu-Hong Zheng, Qin-Ying Liu, Ping Zhong, Xiao Hu, Zhe-Xiang Fang, and Qi-Qing Zhang*

Institute of Biomedical and Pharmaceutical Technology & College of Chemistry and Chemical Engineering, Fuzhou University, No. 523, Gongye Road, Fuzhou City, 350002, China

Abstract
Four new aculeatusquinones A–D (14) and five known compounds, (5aS,6S,7S)-3,7-dihydroxy-6-methoxy-1,4,6,9-tetramethyl-6,7-dihydro-5aH-dibenzo[b,e][1,4]dioxepine-8,11-dione (5), 3,8-dihydroxy-1,4,6,9-tetramethyldibenzo[b,e][1,4]dioxepin-11-one (6), 4-O-demethylbarbatic acid (7), atraric acid (8), and 2,5-dimethyl-1,3-benzenediol (9), were isolated from the marine-derived fungus Aspergillus aculeatus. The structures of the new compounds were elucidated by spectroscopic methods, including one- and two-dimensional NMR and high-resolution mass spectrometric analyses. Two new compounds (2 and 4) showed cytotoxic effects on the HL-60, K562, and A-549 cell lines, with IC50 values ranging from 5.4 μM to 76.1 μM.

Numerous natural products with novel structures and distinct biological activities have been discovered as the secondary metabolites of marine-derived microbes,1 and some have been used as drugs, for example, echinocandins (antifungal drugs), ergot alkaloids (for the treatment of migraine), cyclosporine (an immunosuppressive drug), and lovastatin (a cholesterol-lowering drug).2 To search for new anticancer compounds, more than 300 microbial strains isolated from sediment samples collected from the Min River estuary in China were screened for cytotoxicity against HL-60 cell.3 Among these strains, a fungal strain identified as Aspergillus aculeatus showed significant cytotoxic activity. The broth extract of A. aculeatus was separated by chromatography on Si gel and Sephadex LH-20 columns and then purified by reversed-phase HPLC to yield four new benzoquinone derivatives, aculeatusquinones A–D (14) and five known compounds, (5aS,6S,7S)-3,7-dihydroxy-6-methoxy-1,4,6,9-tetramethyl-6,7-dihydro-5aH-dibenzo- [b,e][1,4]dioxepine-8,11-dione (5),4 3,8-dihydroxy-1,4,6,9-tetramethyl-dibenzo[b,e][1,4]dioxepin-11-one (6),5 4-O-demethylbarbatic acid (7),6 atraric acid (8),7 and 2,5-dimethyl-1,3-benzenediol (9).8
Fungi of the genus
Aspergillus are sources of many bioactive compounds.9 Previous chemical investigations on A. aculeatus have afforded various bioactive secondary metabolites, such as asperaculin A,9 aculeacins A–G,10 CJ-15,183,11 aspergillusol A,12 secalonic acids D and F,13 and okaramines H and I,14 which exhibit antifungal,10,11 enzymatic inhibitory,11,12 and antimicrobial activities.13 However, benzoquinone derivatives isolated from A. aculeatus are reported for the first time. Herein, we report the isolation, structural elucidation and bioactivities of compounds 19.

The molecular formula of aculeatusquinone A (1) was determined to be C16H16O5 on the basis of HRESIMS (m/z: 287.0920 [M − H], calcd for C16H15O5, 287.0919). The 13C NMR and DEPT spectra of 1 (Table 2) displayed 16 carbons assignable to one benzene ring, one quinone ring and four methyl groups. The HMBC correlations from 3'-OH to C-2' and C-3', from CH3-7' to C-1', C-2', and C-3', and from CH3-8' to C-4', C-5', and C-6' (Figure 2) suggested the presence of a tetrasubstituted benzene ring. Moreover, the HMBC from CH3-7 to C-2, C-3, and C-4, as well as the HMBC from CH3-8 to C-1, C-5, and C-6 revealed the presence of a symmetric quinone ring. FT-IR microscopy data was useful in determining that 1 was the 1,4-p-quinone rather than the 4,5-o-quinone, since p-quinones typically exhibit strong IR absorptions near 1645 and 1585 cm-1.15 Furthermore, an absorption at 3383 cm-1 indicated the presence of an intramolecularly H-bonded hydroxyl group, which also proved that 1 was the 1,4-p-quinone. The two moieties were further connected via an oxygen atom after full consideration of the molecular formula and the 1D and 2D NMR spectral data. Thus, the structure of aculeatusquinone A was established to be 1, as shown in Figure 1.
Aculeatusquinone
B (2) was obtained as yellow oil. The molecular formula was established by negative HRESIMS (m/z: 319.1192 [M − H], calcd for C17H19O6, 319.1187). The structure of 2 was found to be similar to that of 1 through the 1H and 13C NMR data comparison, except for the appearance of a methoxy group (δH 3.08 and δC 52.7), a methine (δH 5.42 and δC 81.0), and a quaternary carbon (δC 83.3), and for the disappearance of two aromatic carbons (δC 128.6 and 152.1). In addition, the chemical shifts of the two carbonyl carbons (δC 184.1 and 182.4) were mobiled to lower field (δC 194.5 and 193.9). After the 1D and 2D NMR spectral data analyses of 1 and 2, the benzene ring was reserved in 2. The differences in the quinone ring were determined by the HMBC of Me-7 with C-2, C-3, and C-4, as well as the HMBC of OMe with C-3 (Figure 2). The two rings were further connected via an oxygen atom, which was determined by its molecular formula and the HMBC correlations between H-2 and C-1', C-1, C-3, and C-7. In the NOE experiment for 2, enhancement of the signal of the methoxy protons (OMe) occurred on irradiation of the signal of the methine proton (H-2). Therefore, the structure of aculeatusquinone B (2) was elucidated, as shown in Figure 1.

The structure of aculeatusquinone C (3) was found by comparing its NMR and MS data with those of 2. The molecular formula of 3 was determined to be C17H22O6 based on HRESIMS (m/z: 321.1349 [M − H], calcd for C17H21O6, 321.1344). Its degree of unsaturation is seven, whereas that of 2 is eight. After comparing the 1H, 13C NMR, and DEPT data of 3 and 2 (Tables 1 and 2), an oxygenic methine (δH 4.68 and δC 69.3) in 3 was found to replace the carbonyl (δC 193.9) in 2. This finding was confirmed by the HMBC and HMQC data. In the NOE experiments for 3, the signals of H-2 (δH 5.04) and H-4 (δH 4.68) were enhanced upon irradiation of the signal of OMe (δH 3.12), indacating that H-2, H-4, and the methoxy group were on the same side of the plane. Thus, aculeatusquinone C (3) was established to be a reduction product of aculeatusquinone B (2), as shown in Figure 1.
Aculeatusquinone D (
4) exhibited an anion peak at m/z 345.0981 in HRESIMS (calcd for C18H17O7, 345.0974). The 13C NMR and DEPT spectral data showed four methyl groups, a methoxy group, a methine, and 12 quaternary carbons. The HMBC from Me-7' to C-1', C-2', and C-3', from Me-8' to C-4', C-5', and C-6', and from 3'-OH to C-2' and C-3' indicated the presence of a pentasubstituted phenyl ring. The HMBC of Me-7 with C-2, C-3, and C-4, Me-8 with C-1, C-5, and C-6, and OMe with C-3 indicated the presence of a quinone ring similar to that of 2, except for a loss of H-2. In addition, the 1,4-p-quinone structure of 4 was determined by its IR data. Considering the weak HMBC from H-4' and Me-8' to C-1'', as well as the lower field of C-2 and C-1' than those of 2, the two moieties were finally connected via an oxygen atom and a carbonyl (C-1''), which is consistent with its molecular formula. In the NOE experiment for 4, enhancement of the signal of Me-8' occurred on irradiation of the signal of OMe, revealing the relative configurations of C-2 and C-3. Thus, the structure of aculeatusquinone D was established to be 4, as shown in Figure 1.

Structurally, compounds 17 are composed of two moieties, either a benzene ring and a quinone ring or two benzene rings. The benzene ring can be divided into two types, depending on the presence or absence of the carbonyl group, whereas the quinone ring may be regarded as a derivative product of multi-substituted benzoquinone. Ultimately, these two moieties are connected by dehydration reaction. For a detailed explanation of the interrelationship of these metabolites, a plausible biosynthetic pathway is proposed in Figure 3.
The new compounds (
14) were tested for cytotoxic effects on the HL-60 and K562 cell lines using the MTT method and on the A-549 cell line using the SRB method.16 Compounds 2 and 4 showed moderate cytotoxicity against HL-60 cell, with IC50 values of 7.3 and 5.4 μM, and weak activity against A-549 and K562 cells, with IC50 values of 42.3, 76.1, 37.2, and 29.7 μM. By contrast, compounds 1 and 3 were inactive (Table 3).

EXPERIMENTAL
General Experimental Procedures.
Optical rotations were obtained from a Shenguang SGW-1 digital polarimeter. UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer. IR spectra were recorded on a Nicolet Avatar 670 spectrophotometer. 1H NMR, 13C NMR, DEPT spectra and 2D NMR were recorded on a BRUKER BIOSPIN AVANCE III spectrometer using TMS as the internal standard. HRESIMS were obtained by an Agilent Q-TOF 6520 LC mass spectrometer. Semipreparative HPLC was performed using an ODS column (ODS-A, 10×250 mm, 5 µm) at 5 mL/min.
Fungal Material. The fungus A. aculeatus was isolated from marine sediments collected from Langqi Island, Fujian, China. It was identified according to its morphological characteristics and ITS by Beijing Sunbiotech Co. Ltd, and preserved in our laboratory at −80 °C. The producing strain was prepared on Martin medium and stored at 4 °C.
Fermentation and Extraction. The fungus A. aculeatus was cultured under static conditions at 28 °C for 30 days in 1000 mL conical flasks containing the liquid medium (400 mL/flask), composed of glucose (10 g/L), maltose (20 g/L), mannitol (20 g/L), monosodium glutamate (10 g/L), KH2PO4 (0.5 g/L), MgSO4·7H2O (0.3 g/L), yeast extract (3 g/L), and seawater. The fermented whole broth (60 L) was filtered through cheese cloth to separate supernatant from mycelia. The former was extracted two times with EtOAc to yield an EtOAc solution that was concentrated under reduced pressure to give a broth extract (48.9 g).
Purification. The crude extract (48.9 g) of the fungus A. aculeatus was separated into five fractions on a Si gel column using a step gradient elution of petroleum ether, CH2Cl2, and MeOH. Fraction 3 (5.6 g) was further purified on a Sephadex LH-20 column (CHCl3:MeOH, 1:2) to afford four subfractions. Subfraction 3-2 (1.3 g) was subjected on a Si gel column using a step gradient elution of CH2Cl2 and MeOH, followed by semipreparative HPLC (40% MeCN, 0.1% TFA) to yield compounds 1 (8.7 mg, tR 21.2 min), 2 (10.3 mg, tR 15.1 min), 3 (9.4 mg, tR 11.6 min), 4 (6.5 mg, tR 13.4 min), and 5 (6.1 mg, tR 18.5 min). Subfraction 3-3 (850 mg) was purified by semipreparative HPLC (50% MeCN, 0.1% TFA), yielding compounds 6 (5.5 mg, tR 9.3 min), 7 (4.2 mg, tR 11.4 min), 8 (7.4 mg, tR 7.1 min), and 9 (9.4 mg, tR 5.2 min).
Aculeatusquinone A (
1): yellow oil (MeOH); UV (MeOH) λmax 277 nm; IR (KBr) νmax 3383, 2989, 1645, 1585, 1296, 1108 cm1; 1H and 13C NMR data (see Tables 1 and 2); HRESIMS (m/z: 287.0920 [M – H], calcd for C16H15O5, 287.0919).
Aculeatusquinone B (
2): yellow oil (MeOH); [α]25D +213.7 (c 0.11, MeOH); UV (MeOH) λmax 278 nm; 1H and 13C NMR data (see Tables 1 and 2); HRESIMS (m/z: 319.1192 [M − H], calcd for C17H19O6, 319.1187).
Aculeatusquinone C (
3): yellow oil (MeOH); [α]25D +245.4 (c 0.17, MeOH); UV (MeOH) λmax 263 nm; 1H and 13C NMR data (see Tables 1 and 2); HRESIMS (m/z: 321.1349 [M − H], calcd for C17H21O6, 321.1344).
Aculeatusquinone D (
4): colorless oil (MeOH); [α]25D +379.6 (c 0.15, MeOH); UV (MeOH) λmax 292 nm; IR (KBr) νmax 3409, 2937, 1699, 1658, 1590, 1381, 1092 cm1; 1H and 13C NMR data (see Tables 1 and 2); HRESIMS (m/z: 345.0981 [M − H], calcd for C18H17O7, 345.0974).
Biological Assays. The cytotoxic activity for the HL-60 and K-562 cancer cell lines was evaluated by the MTT method, whereas that for the A-549 cell line was evaluated by the SRB method.16 Doxorubicin was used as the reference drug.

ACKNOWLEDGEMENTS
This research was supported by the Chinese National Natural Science Fund (21102015 and 31201034), Natural Science Foundation of Fujian Province (2012J05138), the Scientific Research Foundation in Fuzhou University (2009-XY-16 and 022229), Scientific Key Research Project of Fujian Province (2010N0015), Key Laboratory of Biomedical Material of Tianjin and Scientific Major Research Project of Fujian Province (2010NZ0001-1).

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