Diphenyl Ethers from a Marine-Derived Aspergillus sydowii

Six new diphenyl ethers (1–6) along with eleven known analogs were isolated from the ethyl acetate extract of a marine-derived Aspergillus sydowii guided by LC-UV-MS. Their structures were unambiguously characterized by HRESIMS, NMR, as well as chemical derivatization. Compounds 1 and 2 are rare diphenyl ether glycosides containing d-ribose. The absolute configuration of the sugar moieties in compounds 1–3 was determined by a LC-MS method. All the compounds were evaluated for their cytotoxicities against eight cancer cell lines, including 4T1, U937, PC3, HL-60, HT-29, A549, NCI-H460, and K562, and compounds 1, 5, 6, and 8–11 were found to exhibit selective cytotoxicity against different cancer cell lines.

Compound 4 was obtained as colorless oil. Its molecular formula was determined to be C 18 H 18 O 7 by HRESIMS, having a fragment of C 3 H 4 O 2 more than that of 4-carboxydiorcinal (16). The 1 H and 13 C NMR spectra for 4 (Tables 1 and 2) and 16 showed very similar signals, with the exception that H-6 at δ H 6.17 for 16 was missing for 4, which indicated that the CH at C-6 in 16 was replaced by another substitution in 4. Analysis of the 1 H NMR and 13 C NMR (Tables 1 and 2 (Tables 1 and 2) showed very similar signals to those of 8, with the exception that the tertiary carbon signal at δ C 111.8 for 8 was replaced by a quaternary carbon signal at δ C 140.8 for 6. These data indicated that the hydrogen at C-6 (δ C 111.8) in 8 was substituted by a hydroxy group in 6. The two hydroxy groups in the B ring did not display any correlation in the HMBC spectrum, and therefore the position of the ether linkage between the two benzene rings could not be determined by 2D NMR data at this stage. Therefore, full methylation of 6 was completed with CH 3 I/K 2 CO 3 , which afforded its derivative 6a. HMBC correlations of 6a from H 3 -8 (δ H 3.73) to C-3 (δ C 162.3), from H 3 -8 (δ H 3.78) to C-3 (δ C 154.8) and from H 3 -9 (δ H 3.75) to C-4 (δ C 144.5) (Figure 2) demonstrated that the three free hydroxy groups of 6 were attached to C-3, C-3 and C-4 , respectively. Therefore, 6 was unambiguously determined as 3-hydroxydiorcinol.
The eleven known compounds (7-17) were identified as 4-methoxycarbonyl diorcinol (7) [10], diorcinol (8) [11], glyceryl diorcinolic acid (9) [12], cordyol C (10) [14], aspergilol E (11) [13] Table 3). None of the compounds showed cytotoxicity against the five solid cancer cell lines (4T1, PC3, HT-29 and NCI-H460). Compounds 1, 5, 8 and 9 showed moderate cytotoxicity against A549. These results suggested that glycosylation of the 3-hydroxy group seems to negatively contribute to its cytotoxicity against A549 cell line (2 vs. 8), while substitution at the same 3-OH position by a glycerol group positively compensate for cytotoxicity (16 vs. 9). In addition, compounds 1, 6, 9-11 showed selective cytotoxicity against different nonsolid cancer cell lines (U937, HL-60, and K562). Interestingly, only compounds 6, 10 and 11, which possess two adjacent hydroxy groups in one of the benzene rings and no substitution at C-2 position in the other ring exhibited varied inhibitory cytotoxicity on K562 cells. Moreover, by comparison of the structures of 9 and 3-4, 7, 11-12 and 16 having a carboxyl group, it was found that when the carboxyl group in the benzene ring is adjacent to a free hydroxy group, the cytotoxicity against HL-60 cells is lost. In summary, we found that when the ortho position of the carboxyl group in the diphenyl ethers is a free phenolic hydroxy group, it will lose all cytotoxicity against cancer cells, and the adjacent phenolic hydroxy groups confer selective cytotoxicity against several cell lines.

Fungal Material
The fungal strain FNA026 was isolated from marine water collected in the sea of China, Xiamen. The voucher specimen is deposited in our laboratory at −80 • C. The partial 18S rRNA sequence was compared to sequences in available databases using the Basic Local Alignment Search Tool and strain FNA026 determined to be an Aspergillus sydowii (Supplementary Information Figure S57).

Fermentation
The fungal strain FNA026 was grown on potato dextrose agar at 28 • C for 5 days. Five pieces (0.5 × 0.5 cm 2 ) of mycelial agar plugs were inoculated into 500 mL Erlenmeyer flasks containing 300 mL of potato dextrose broth, which were then incubated on a rotary shaker at 250 rpm and 28 • C for 3 days. Then the seed liquid was spread in 500 mL Roux flasks (30 flasks) containing rice (100 g per flask) and artificial seawater (120 mL per flask). The flasks were incubated at 28 • C for 4 weeks.

Extraction and Isolation
The extraction and isolation procedures were guided by LC-MS screening with UV absorption characteristics (207 nm and 270 nm) and molecular weight (m/z 230-280 and m/z 380-480) as search criteria. The fermented rice inoculated with FNA026 (3 kg) was extracted three times with ethyl acetate (500 mL) at room temperature under sonication to give a crude extract (28.86 g), which was then dissolved in MeOH, and extracted three times using petroleum ether to afford MeOH-soluble (22.

Determination of the Absolute Configuration of Sugar Moieties in 1-3
To determine the absolute configurations of sugar moieties in 1-3, a modified method based on LC-ESI-MS analysis was performed, where the retention time of sugar samples obtained after hydrolysis of the parent compounds were compared with those from standard sugars (D/L) [34]. In detail, compounds 1, 2 and 3 (approximately 0.05 mg, each) were hydrolyzed with 2 mol/L HCl (400 µL) in a 2 mL glass vial at 80 • C for 4 h. The reaction mixture was then diluted with H 2 O (400 µL) and extracted with CHCl 3 (400 µL) three times. The aqueous layer containing monosaccharides was concentrated in vacuo to yield a dried sugar mixture. The resulting sugar mixture (not weighed out) and standard sugar samples (D/L-ribose and D/L-glucose, 0.1 mg for each) respectively, were heated with L-cysteine methyl ester (0.1 mg) in pyridine (400 µL) in a 2 mL glass vial at 60 • C for 60 min, then o-tolyl isothiocyanate (200 µL) was added to the reaction mixture and kept at 60 • C for additional 60 min. Then, the reaction mixture was directly analyzed by LC-ESI-MS (COSMOSIL 5 µm, 4.6 × 250 mm, C 18 column). Analysis was performed at 30 • C with a flow rate of 1.0 mL/min, and the elution was carried out using a gradient of MeCN (0-30 min, 10-50%, linear gradient) in H 2 O. Source parameters in the positive ion mode were set as follows: Capillary entrance voltage = −4500 V, end plate offset = −500 V, nebulizer pressure (N 2 ) = 11.6 psi, dry gas (N 2 ) = 6.0 L/min, dry gas temperature = 220 • C. High-purity nitrogen (N 2 ) were used as the nebulizing gas. Ion Peaks were extracted at m/z 447 for ribose and at m/z 471 for glucose identified by comparison of retention time with those of standards. The retention time of D-ribose and D-glucose derivatives were 22.3 and 21.0 min, respectively.

Cytotoxicity Assay
Cancer cell lines, including 4T1, U937, PC3, HL-60, HT-29, A549, NCI-H460, and K562, were purchased from ATCC. All the cells were maintained in RPMI1640 supplemented with 10% FBS, 100 units/mL Penicillin G and 100 µg/mL streptomycin. All the cancer cells were incubated at 37 • C in humidified air containing 5% CO 2 . MTT assay was used to determine the cell viability. Cells were seeded in 96-well plates at 1.5-3.0 × 10 4 /mL (100 µL/well). After 24 h incubation, 5 different concentrations (final concentrations were 1.6, 3.1, 6.3, 12.5 and 25.0 µM) of tested compounds were added into the wells in triplicate. Five concentrations of doxorubicin were tested, including 2.0, 1.0, 0.5, 0.25, and 0.125 µM. Cells were incubated for 96 h before MTT was added into the cells at a final concentration of 500 µg/mL, and the plates were incubated for an additional 4 h. The resultant formazan crystals were dissolved in 200 µL of DMSO, then a microplate reader (Synergy HT, Bio-Tek) was used to measure the absorbance of the plates at 570 nm for testing the cell viability of serial concentrations of compounds and the IC 50 were estimated.

Conclusions
A total of 17 diphenyl ethers, including 6 new compounds, were isolated from a marine-derived Aspergillus sydowii. Compounds 1 and 2 are rare diphenyl ether glycosides containing a D-ribofuranose moiety. Although natural diphenyl ethers have been extensively investigated, their structures still exhibit variability due to the presence of hydroxy groups, and the diversity and location of sugar moieties. Furthermore, a modified method based on LC-MS analysis was used to determine the absolute configuration of sugar moieties. Comparing to conventional method based on LC-UV, which normally requires 0.5-3 mg of sample [34][35][36][37], our method has higher sensitivity due to the use of ESIMS detection, and as low as 0.05 mg of sample was enough to determine the absolute configuration of the sugar units using this procedure. Moreover, all the compounds were evaluated for their cytotoxicity against eight cancer cell lines, and compounds 1, 5, 6, and 8-11 were found to exhibit highly selective cytotoxicities against different cancer cell lines.