Introduction

Marine-derived fungi belonging to the species Aspergillus versicolor are an attractive source of secondary metabolites with great structural diversities and interesting bioactivities, such as nitrobenzyl esters,1 chromones,2 sterigmatocystins3 and cyclopeptides.4 In our continuous search for new bioactive natural products from the marine-derived fungi, the fungus A. versicolor ZLN-60 was selected for the cyctoxicity in vitro against P388 cell line. In our previous report, two new cyclic pentapeptides, versicotides A and B, which showed no antitumor activity, were isolated from one of the fractions (Fr.3) of the ethyl acetate extract.5 Further investigation of the active fraction (Fr.2) led to the discovery of four new prenylated diphenyl ethers, diorcinols B-E (1–4) (Figure 1), together with a known compound, diorcinol (5).6 In this paper, we describe the isolation, structural elucidation and cytotoxic activities of these new compounds.

Figure 1
figure 1

Compounds 1-5 isolated from Aspergillus versicolor ZLN60.

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Results and discussion

The fungal strain was fermented and extracted as previously described.5 The ethyl acetate extract was concentrated in vacuo and then chromatographed by repeated silica column. The subfraction (Fr.2) with cytotoxic activity was further fractionated by Sephadex LH-20 and semipreparative HPLC to yield compounds 1–5 (Figure 1).

Structure determination

Diorcinol B (1) was obtained as yellow powder with a molecular formula of C19H24O5 deduced by the [M+H]+ ion peak at m/z 333.1714 in HR-ESI-MS spectrum, which indicated eight degrees of unsaturation. The 1H- and 13C-NMR spectra of 1 (Tables 1 and 2) displayed resonances that were assigned to four methyls, one methylene, one oxygen-bearing methane and five aromatic methines, five oxygen-bearing quaternary carbons (among them four aromatic carbons). These data suggested that 1 contains a diphenyl ether unit similar to diorcinol (5), which was also supported by the HMBC correlations.6 The major diffrences were the absence of one aromatic proton and the presence of an additional isopentyl chain unit in 1. The isopentyl group was determined by the 1H–1H COSY (H-1′′/H-2′′) and the HMBC correlations from H-4′′ (δH 1.62) and H-5′′ (δH 1.65) to C-2′′ (δC 78.7) and C-3′′ (δC 72.5). The HMBC correlations from H-1′′ (δH 2.63, 2.40) to C-1′ (δC 155.7), C-2′ (δC 121.7) and C-3′ (δC 140.4) located the isopentyl group on C-2′ (Figure 2). Considering the chemical shifts of C-2′′ and C-3′′, together with the molecular formula, the two hydroxyl groups were attached to C-2′′ and C-3′′.

Table 1 1H (600 MHz) NMR data for compounds 1–4 ( recorded in DMSO-d6)a
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Table 2 13C (150 MHz) NMR data for compounds 1–4 ( recorded in DMSO-d6)a
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Figure 2
figure 2

Key HMBC correlations of compounds 1–4.

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The absolute configuration of C-2′′ was assigned as S by employing the modified Mosher’s method (Figure 3).7, 8

Figure 3
figure 3

Δδ Values (δS−δR) of the MTPA esters 1a ((S)-MTPA) and 1b ((R)-MTPA).

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Diorcinol C (2), was separated as yellow powder, and an [M+H]+ molecular ion at m/z 347.1864 (calcd 347.1858) in the HR-ESI-MS spectrum was in agreement with the molecular formula C20H26O5. Their similar UV absorptions suggested that 2 was an analog of 1. On comparison of the 1D-NMR data for the two compounds (Tables 1 and 2), compound 2 showed an additional methoxy and the position was determined by the HMBC correlation between Me (3′′) (δH 3.08) and C (3′′) (δC 77.6) (Figure 2). Thus, compound 2 was elucidated as a methylated analog of morkeyol A (1). As the co-metabolite, 2 was proposed to have the same 2′′S-configuration, which was also supported by the similar specific rotation data (−29.3 in 2 versus −45.1 in 1).

Diorcinol D (3), was a yellow powder, and HR-ESI-MS indicated the molecular formula C19H22O3. Compound 3 displayed similar NMR resonances to morkeyol A (1) and their comparison suggested that they shared the same substructures of phenyl ethers (Tables 1 and 2). However, the absence of three signals for two hydroxy and one methenyl protons in 1 (δH 4.07, OH-2′′; δH 4.08, OH-3′′; and δH 3.35, H-2′′) and the presence of an additional olefinic singlet in 3 (δH 4.97, H-2′′) suggested that compound 3 contained an isopentenyl group. Therefore, the structure of 3 was determined by further analysis the HMBC correlations (Figure 2) and named as diorcinol D.

The molecular formula of 4 was determined as C19H22O4 on the basis of an [M+H]+ ion at m/z 315.1528 (calcd 315.1596) in the HR-ESI-MS. Similarities in the NMR data (Tables 1 and 2) of 4 and 3 showed that their structures were closely related. With the absence of the signals for the double bound and the appearance of an additional ketone (δc 211.8, C-2′′) and a methine (δc 40.4, C-3′′), together with the HMBC correlations from H-4′′ (δH 0.96) and H-5′′ (δH 0.97) to C-2′′ and C-3′′, and from H-1′′ (δH 3.68) to C-2′′ (Figure 2), the structure of 4 was deduced and named as diorcinol E.

Cytotoxic activities

The cytotoxicities of compounds 1–5 were evaluated in vitro on the A-549, BEL-7402, Hela and K562 cells by the SRB or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) methods.9, 10 Compound 3 exhibited moderate cytotoxicities against the Hela and K562 cell lines with the IC50 values of 31.5 and 48.9 μM, respectively, and compound 4 exhibited moderate cytotoxicity only against the Hela cell line with the IC50 value of 36.5 μM, whereas compounds 1, 2 and 5 showed no activities.

Discussion

A number of diphenyl ethers derivatives have been previously isolated and characterized from different organisms, whereas prenylated phenyl ethers are relatively rare in nature. To the best of our knowledge, only nine prenylated diphenyl ethers (tenellic acids) were isolated from several fungi such as Penicillium spp.11, 12 and Dendrospora tenella,13 and six natural transformation prenylated phenyl ethers products (chromenes) were found in the injured fruiting bodies of the mushrooms lactarius fuliginosus and lactarius picinus.14 These structurally similar diphenyl ethers showed diverse biological activities such as anti-microbial,11, 15, 16 cytotoxic17 and radical-scavenging activities.18 To our knowledge, this is the first report for the isolation of prenylated diphenyl ethers from a member of the genus Aspergillus, and compounds 3 and 4 exhibited cytotoxicities.

Materials and Methods

General

Specific rotations were obtained on a JASCO P-1020 digital polarimeter (JASCO, Tokyo, Japan). IR spectra were taken on a Nicolet Nexus 470 spectrophotometer (Thermo Scientific, Beijing, China) in KBr discs. NMR spectra were recorded on a JEOL JNM-ECP 600 spectrometer (JEOL Ltd, Beijing, China) using tetramethylsilane as internal standard, and chemical shifts were recorded as δ values. ESIMS utilized on a Q-TOF Ultima Global GAA076 LC mass spectrometer (Waters Asia, Ltd, Singapore). Semiprepartive HPLC was performed using an ODS column (HPLC:YMC-Pack ODS-A (5 μm, 10 × 250 mm, 4 ml min−1)). TLC and column chromatography were performed on plates precoated with silica gel GF254 (10–40 μm) and over silica gel (200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, Shandong, China). Vacuum–liquid chromatography was carried out over silica gel H (Qingdao Marine Chemical Factory).

Fermentation and extraction of the fungal strain

The fungus strain was fermented and extracted as previously described.5

Purification

The crude extract (13.0 g) was applied to a silica gel (300–400 mesh) column and was separated into six fractions (Fr.1 to Fr.6) using a step-gradient elution of petroleum ether/CHCl3 and CHCl3/CH3OH. Fr.2 (1.28 g) was separated on a silica gel column using CHCl3 and second subfraction (Fr.2-2) further separated into four subfractions (Fr.2-2-1 to Fr.2-2-4) by Sephadex LH-20 (Greenherbs Science and Technology Development Co., Ltd., Beijing, China) using CH3OH as the eluting solvent. Subfraction Fr.2-2-1 was purified by semipreparative HPLC (50:50 MeOH/H2O, 4 ml min−1) to give compound 1 (15 mg, tR=9.5 min), 2 (13 mg, tR=13.0 min) and 4 (8 mg, tR=11.0 min). Subfraction Fr.2-2-3 was purified by semipreparative HPLC (60:50 MeOH/H2O, 4 ml min−1) to obtain compound 3 (19 mg, tR=15.0 min) and 5 (38.0 mg, tR=12.0 min).

The cytotoxic activities of subfractions were tested on 96-well plates and observed under a microscope (Supplementary Figure S1).

Physico-chemical properties

The MS, 1D and 2D NMR spectra of compounds 1–4 can be found in the Supplementary Figures S2–S25. Diorcinol B (1): yellow powder, [α]25D −45.1 (c 0.1, MeOH), UV (HPLC, mobile phase) λmax 210, 262 nm, IR (KBr) νmax 3206, 1588, 1467, 1320, 1144, 1064, 986 cm−1, 1H-NMR and 13C-NMR data (Tables 1 and 2), HR-ESI-MS m/z: 333.1714 [M+H]+ C19H25O5 (calcd 333.1702).

Diorcinol C (2): yellow powder, [α]25D −29.3 (c 0.1, MeOH), UV (HPLC, mobile phase) λmax 210, 262 nm, IR (KBr) νmax 3216, 1710, 1700, 1587, 1492, 1145, 1024, 1001 cm−1, 1H-NMR and 13C-NMR data (Tables 1 and 2), HR-ESI-MS m/z: 347.1864 [M+H]+ C20H27O5 (calcd 347.1858).

Diorcinol D (3): yellow powder, UV (HPLC, mobile phase) λmax 210, 262 nm, IR (KBr) νmax 3316, 1610, 1600, 1587, 1492, 1045, 1024, 985 cm−1, 1H-NMR and 13C-NMR data (Tables 1 and 2), HR-ESI-MS m/z: 299.1642 [M+H]+ C19H23O3 (calcd 299.1647).

Diorcinol E (4): yellow powder, UV (HPLC, mobile phase) λmax 210, 262 nm, IR (KBr) νmax 2964, 2917, 1710, 1700, 1587, 1467, 1322, 1105, 1024 cm−1, 1H-NMR and 13C-NMR data (see Tables 1 and 2), HR-ESI-MS m/z: 315.1582 [M+H]+ C19H23O4 (calcd 315.1596).

In vitro cytotoxicity assays

In the MTT assay, cell lines were grown in RPMI-1640 supplemented with 10% FBS in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Cell suspensions, 200 μl, at a density of 5 × 104 cells per ml were plated in 96-well microtiter plates and incubated for 24 h. Then, 2 μl of the test solutions (in MeOH) were added to each well and further incubated for 72 h. Then, 20 μl of the MTT solution (5 mg ml−1 in IPMI-1640 medium) was added to each well and incubated for 4 h. Old medium containing MTT (150 μl) was gently replaced by dimethylsulfoxide and pipetted to dissolve any formazan crystals formed. Absorbance was then determined on a Spectra Max Plus plate reader at 540 nm.

In the sulforhodanine B (SRB) assay, 200 μl of the cell suspensions were plated in 96-well plates at a density of 2 × 105 cells per ml. Then, 2 μl of the test solutions (in MeOH) was added to each well, and the culture was further incubated for 24 h. The cells were fixed with 12% trichloroacetic acid, and the cell layer was strained with 0.4% SRB. The absorbance of SRB solution was measured at 515 nm. Dose–response curves were generated, and the IC50 values, the concentration of compound required to inhibit cell proliferation by 50%, were calculated from the linear portion of log dose–response curves.