1 INTRODUCTION

Phenylquinolinones are a family of alkaloids that can be produced by Penicillium (Wei et al., 2011), Aspergillus (Fremlin et al., 2009) and the roots of several different plants (Abbadi et al., 1989). Compounds belonging to this family exhibit a wide range of interesting biological activities (Kobayashi and Harayama, 2009), as exemplified by the farnesyl transferase inhibitor developed by Pfizer as an anticancer agent, which is currently undergoing clinical trials (Andresen et al., 2004). Based on their interesting biological properties, phenylquinolinones have attracted considerable attention in recent years as potential drug candidates. As part of our research towards the isolation of bioactive compounds with novel structures from marine-derived fungi, we investigated the EtOAc extract of the fungal strain Aspergillus versicolor, which was isolated from seawater samples taken from the Indian Ocean. This study resulted in the isolation of one new phenylquinolinone derivative, 4-(3-hydroxyphenyl)-3-methoxyquinolin-2(1H)-one (1), as well as the analogue 3-O-methylviridicatin (2). Both of these compounds represent rare examples of naturally produced phenylquinolinones, which were characterized from the culture extract of the fungus. It is noteworthy that 3-O-methylviridicatin (2) has been reported to inhibit the replication of human immunodeficiency virus (HIV) with an IC₅₀ of 2.5 μmol/L (Heguy et al., 1998). The structures of compounds 1 and 2 were established by spectroscopic analyses. The cytotoxic activities of these compounds were evaluated against five tumor cell lines, including MCF-7 (human breast carcinoma cell line), SMMC-7721 (human liver cancer cell line), HEK293 (human embryonic kidney cancer cell line), A549 (human pulmonary carcinoma) and T-47D (human ductal breast epithelial tumor cell line) cells. Compound 1 exhibited moderate cytotoxicity against MCF-7 and SMMC-7721 with IC₅₀ values of 16.6 and 18.2 μmol/L, respectively. This paper describes the isolation, structural determination and cytotoxicity of the phenylquinolinone compounds isolated from this fungus.

2 EXPERIMENTAL 2.1 General experimental procedures

Ultraviolet (UV) spectra were recorded on an Eppendorf BioSpectrometer system (Eppendorf, Hamburg, Germany). ¹H nuclear magnetic resonance (NMR), ¹³C NMR, distortionless enhancement by polarization transfer, heteronuclear multiple-quantum coherence, heteronuclear multiple-bond correlation (HMBC), correlation spectroscopy (COSY) and nuclear overhauser effect spectroscopy spectra were recorded using a JEOL JNM-ECP 600 spectrometer (JEOL, Tokyo, Japan). Mass spectra were determined on an Agilent Technologies G1969A mass spectrometer (Agilent, Santa Clara, USA). An analytical high performance liquid chromatography (HPLC) system (Hitachi, Tokyo, Japan), consisting of an organizer, UV detector (L-2400), pump (L-2130) and Hitachi software (D-2000) was used for the purification of the compounds with a C₁₈ column (YMC-pack ODS-A, 150 mm×4.6 mm, I.D., S-5 μmol/L, 12 nm, 1 mL/min). Semipreparative HPLC purifications were conducted on the same system using a Prep RP-18 column (YMC-pack ODS-A, 250 mm×10 mm, I.D., S-5 μmol/L, 12 nm, 2.5 mL/min) with UV detection. Purifications by column chromatography were performed over Silica gel (200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China), Lobar LiChroprep RP-18 (40–63 mm, Merck, Darmstadt, Germany) and Sephadex LH-20 (Amersham Biosciences, Piscataway, USA).

2.2 Fungal material

The fungus Aspergillus versicolor Y31-2 was obtained from sea water samples collected from the Indian Ocean at a depth of about 30 m (88°0′6ʺE, 4°0′6ʺS), in 2013. The samples were cultured on Malt Extract Agar (MEA: Malt extract, 17 g; Peptone, 3 g; Agar, 20 g; sea water, 1 L) in a plate containing the antibiotic chloramphenicol (100 μg/mL). A single colony was transferred onto another MEA plate and was identified according to its morphological characteristics and 18S rRNA gene sequences (GenBank accession No. KP872504) (Fan et al., 2013). The pure reference culture was deposited in our laboratory at -80℃, whilst the producing strain was prepared on PDA slants and stored at 4℃.

2.3 Fermentation and extraction

The fungus Aspergillus versicolor Y31-2 was cultured under static conditions in 300 mL of fermentation medium in a 1-L conical flask containing 17 g of malt extract and 3 g of peptone per liter of sea water at 28℃ for 30 days. The whole fermentation broth (15 L) of cultivated medium was extracted exhaustively with EtOAc, yielding 8.2 g of extract; the mycelia were extracted with 80% (v/v) aqueous acetone, yielding 4.2 g of extract (Fan et al., 2013). The extracts of the fermentation broth and the mycelia were combined because the analysis of these extracts by HPLC revealed that their chemical compositions were similar.

2.4 Purification

The combined extracts from Aspergillus versicolor Y31-2 were subjected to column chromatography over silica gel using a vacuum-liquid chromatography column, eluting with a stepwise gradient of petroleum ether (PE) and CH₂C_l2 (2:1 and 0:1, v/v), followed by a stepwise gradient of CH₂C_l2 and MeOH (100:1, 50:1, 30:1, 10:1, 1:1 and 0:1, v/v) to yield eight major primary fractions (Fr.1–8) (Fan et al., 2013). Fraction 6 was purified by reversed-phase C₁₈ silica gel column chromatography eluting with a stepwise gradient of 10%–100% MeOH in H₂O to give 10 sub-fractions (Fr.6.1–6.10). Fraction 6.5 was further purified by Sephadex LH-20 eluting with MeOH to obtain compound 1 (4.4 mg). Fraction 6.6 was also resolved in a similar manner over Sephadex LH-20 eluting with MeOH, followed by HPLC purification to yield compound 2 (15 mg).

2.5 Cytotoxicity assay

The cytotoxic activities of compounds 1 and 2 were evaluated against five tumor cell lines: MCF-7 (human breast carcinoma cell line), SMMC-7721 (human liver cancer cell line), HEK293 (human embryonic kidney cancer cell line), A549 (human pulmonary carcinoma) and T-47D (human ductal breast epithelial tumor cell line) cells. The cytotoxic activities were determined according to a previously reported method (Bergeron et al., 1984).

3 SPECTRAL DATA

4-(3-Hydroxyphenyl)-3-methoxyquinolin-2(1H)-one (1)

White amorphous powder; UV (MeOH) λ_max 203, 221, 279 and 322 nm; ¹H NMR (DMSO-d₆, 600 MHz): δ 7.05(1H, d, J=8.0 Hz, H-6), 7.09 (1H, t, J=7.5 Hz, H-7), 7.42(1H, t, J=7.1 Hz, H-8), 7.37 (1H, d, J=8.1 Hz, H-9), 6.69(1H, s, H-12), 6.86 (1H, d, J=7.8 Hz, H-14), 7.31 (1H, t, J=8.0 Hz, H-15), 6.70 (1H, d, J=7.0 Hz, H-16), 3.70 (3H, s, H-17). ¹³C NMR (150 MHz, DMSO-d₆): δ 158.6 (C, C-2), 144.9 (C, C-3), 137.7 (C, C-4), 119.9 (C, C-5), 125.9 (CH, C-6), 121.9 (CH, C-7), 128.6 (CH, C-8), 115.1 (CH, C-9), 135.8 (C, C-10), 134.7 (C, C-11), 116.1 (CH, C-12), 157.7 (C, C-13), 115.1 (CH, C-14), 129.4 (CH, C-15), 119.4 (CH, C-16), 59.5 (CH₃, C-17). Highresolution electrospray ionisation mass spectrometry m/z 268.0980 [M+H]⁺ (calcd for C₁₆H₁₄NO₃, 268.097 4).

3-O-Methylviridicatin (2)

White amorphous powder; UV (MeOH) λ_max 203, 221, 279 and 322 nm; ¹H NMR (CDCl₃, 600 MHz): δ 7.22 (1H, d, J=8.0 Hz, H-6), 7.12 (1H, t, J=7.5 Hz, H-7), 7.44 (1H, t, J=7.1 Hz, H-8), 7.49 (1H, d, J=8.1 Hz, H-9), 7.38 (1H, d, J=7.1 Hz, H-12), 7.53 (1H, t, J=7.2 Hz, H-13), 7.53 (1H, m, H-14), 7.53 (1H, t, J=7.2 Hz, H-15), 7.38 (1H, d, J=7.1 Hz, H-16), 3.84 (3H, s, H-17). ¹³C NMR (150 MHz, CDCl₃): δ 161.3 (C, C-2), 145.4 (C, C-3), 139.6 (C, C-4), 121.1 (C, C-5), 126.6 (CH, C-6), 122.8 (CH, C-7), 128.4 (CH, C-8), 116.2 (CH, C-9), 135.6 (C, C-10), 133.6 (C, C-11), 128.4 (CH, C-12), 129.6 (CH, C-13), 129.0 (CH, C-14), 129.6 (CH, C-15), 128.4 (CH, C-16), 60.5 (CH₃, C-17).

4 RESULT AND DISCUSSION 4.1 Structure identification of compound 1 and 2

Compound 1 was obtained as a white amorphous powder. The molecular formula of this compound was determined to be C₁₆H₁₃NO₃ by high-resolution electrospray ionisation mass spectrometry, which gave a peak with an m/z value of 268.098 0 [M+H]⁺, indicating 11 degrees of unsaturation. The ¹H NMR, ¹³C NMR and distortionless enhancement by polarization transfer spectra of compound 1 (Table 1) revealed the presence of twelve aromatic carbon atoms (eight methines and four quaternary carbons) with eight protons (three triplets, four doublets and one singlet). These signals were indicative of an ortho-disubstituted benzene ring system, as well as a 1, 3-disubstituted benzene system. This deduction was further verified by the key COSY correlations between H-6 (δ_H 7.05, d, 8.0 Hz)/H-7 (δ_H 7.09, t, 7.5 Hz)/H-8 (δ_H 7.42, t, 7.1 Hz)/H-9 (δ_H 7.37, d, 8.1 Hz) and H-14 (δ_H 6.86, d, 7.8 Hz)/H-15 (δ_H 7.31, t, 8.0 Hz)/H-16 (δ_H 6.70, d, 7.0 Hz), together with the HMBC correlations from H-8 and H-6 to the quaternary carbon (C-10, δ_C 135.8); from H-9 and H-7 to the quaternary carbon (C-5, δ_C 119.9); from H-14 and H-16 to the methine C-12 (δ_C 116.1; δ_H 6.69, s); and from H-15 to the two quaternary carbons, one of which was oxygenated (C-11, δ_C 134.7; C-13, δ_C 157.7) (Fig. 1). The ¹H and ¹³C NMR spectra also revealed the presence of two aromatic carbons (one oxygenated) at δ_C 137.7 and 144.9, a methoxy group (δ_C 59.5; δ_H 3.70, s) and an amido bond at δ_C 158.6, which appeared to be conjugated to another double bond. Having accounted for 10 of 11 degrees of unsaturation in this molecule, we turned out attention to the final degree of unsaturated. With this in mind, we identified a planar structure, which was established by forming a hexatomic ring with the lactam, as evidenced by the HMBC correlations from H-6, H-12, H-16 to C-4 (δ_C 137.7), H-17 to C-3 (δ_C 144.9) and significant fourbond correlations from H-6, H-12 to C-3 (Fig. 1). Compound 1 was therefore determined to be 4-(3-hydroxyphenyl)-3-methoxyquinolin-2(1H)-one (Fig. 2).

The fungal metabolite 3-O-methylviridicatin (2) was identified by detailed spectroscopic analysis and, where possible, through a comparison of its spectroscopic data with literature data. Compound 2 was initially reported in 1963 following its isolation from a strain of P. cyclopium (Birkinshaw et al., 1963).

4.2 Anti-tumor activity of compounds 1 and 2

Compounds 1 and 2 were assayed for their cytotoxic activities against five tumor cell lines, including MCF-7, SMMC-7721, HEK293, A549 and T-47D cells. Compound 1 displayed cytotoxic activities against MCF-7 and SMMC-7721 cells with IC₅₀ values of 16.6 and 18.2 μmol/L, respectively. Compared with adriamycin, which gave IC₅₀ values of 1.2 and 1.5 μmol/L against MCF-7 and SMMC-7721 cells, compound 1 showed reasonable levels of cytotoxicity.

It is envisaged that compound 1 will also show anti-HIV activity because the related compound 2 has been reported to exhibit reasonable activity against HIV with an IC₅₀ value of 2.5 μmol/L, although further work is required to determine the activity of this compound against HIV.