Antifouling phenyl ethers and other compounds from the invertebrates and their symbiotic fungi collected from the South China Sea

Marine organism-derived secondary metabolites are promising potential sources for discovering environmentally safe antifouling agents. In present study, 55 marine secondary metabolites and their synthesized derivatives were tested and evaluated for their antifouling activities and security. These compounds include 44 natural products isolated from marine invertebrates and their symbiotic microorganisms collected from the South China Sea and 11 structural modified products derived from the isolated compounds. The natural secondary metabolites, covering phenyl ether derivatives, terpenoids, 9, 11-secosteroids, anthraquinones, alkaloids, nucleoside derivatives and peptides, were isolated from two corals, one sponge and five symbiotic fungi. All of the isolated and synthesized compounds were tested for their antifouling activities against the cyprids of barnacle Balanus (Amphibalanus) amphitrite Darwin. Noticeably, five phenyl ether derivatives (9, 11, 13–15) exhibited potent anti-larval settlement activity with the EC50 values lower than 3.05 μM and the LC50/EC50 ratios higher than 15. The study of structure–activity relationship (SAR) revealed that the introduction of acetoxy groups and bromine atoms to phenyl ether derivatives could significantly improve their antifouling activities. This is the first report on the SAR of phenyl ether derivatives on antifouling activity against barnacle B. amphitrite. The polybrominated diphenyl ether derivative, 2, 4, 6, 2′, 4′, 6′-hexabromo-diorcinol (13), which displayed excellent antifouling activity, was considered as a promising candidate of environmentally friendly antifouling agents. Electronic supplementary material The online version of this article (doi:10.1186/s13568-016-0272-2) contains supplementary material, which is available to authorized users.


Isolation of secondary metabolites from marine invertebrates and their symbiotic fungi.
The fresh gorgonian and sponge samples (about 1.0 kg each, wet weight) were immediately chilled to -20°C and kept frozen until they were exhaustively extracted with 95% ethanol/H 2 O (3 × 2000 mL) and then with CH 2 Cl 2 /MeOH (v/v 1:1; 3 × 2000 mL) at room temperature. After removal of the solvent under reduced pressure, the residue was dissolved in H 2 O and extracted with EtOAc three times (3 × 1000 mL).
The EtOAc extracts were evaporated to give the EtOAc residues and were then subjected to silica gel (100-200 mesh) vacuum column chromatography (VLC) and eluted with petroleum ether-EtOAc mixtures of increasing polarity, yielding the fractions. Then the fractions were further isolated and purified by column chromatography on silica gel, Sephadex LH-20 and semi-preparative HPLC until the pure substance was obtained.
The fungi were cultured statically in rice medium (100 mL seawater, 100 g rice) or in normal potato glucose liguid medium (20 g of glucose and 12 g of natural sea salt (from Yangkou saltern, China) in 1 L of potato infusion; 1 L Erlenmeyer flasks each containing 300 mL of culture broth) at 25°C for 4 to 5 weeks. The fermented rice substrate was extracted with EtOAc (3×300 mL for each flask), and the solvent was combined and concentrated in vacuo to afford a residue; While the potato culture was filtered to separate the broth from the mycelia, then the broth was extracted three times with an equal volume of EtOAc, and the mycelia were extracted three times with MeOH to afford the residue. The crude extract was subjected to vacuum liquid chromatography on a silica gel column using step gradient elution with petroleum ether-EtOAc to produce fractions, then isolated and purified through a bioassay-guided chromatograph system as mentioned above.
Brominated derivatives 13 and 14: To a stirred solution of 1 or 3 (10 mg, respectively) in acetone (1.0 mL) was added slowly bromine (2.0 mL) at rt, and the reaction mixture was stirred for 30 min. After the starting material was consumed, the mixture was concentrated in vacuo to give a residue, which was purified by semi-preparative HPLC (70% MeOH/H 2 O) to give compound 13 (11 mg) or 14 (9 mg) as a white solid.

Structure determination.
The structures of all of the compounds were elucidated on the spectroscopic (NMR and MS) analysis comparing with the data in literature. NMR spectra were acquired using a JEOL JEM-ECP NMR spectrometer (JEOL Ltd., Tokyo, Japan; 600 MHz for 1 H and 150 MHz for 13 C) or an Agilent DD2 500MHz NMR spectrometer (Agilent Technologies, Inc., CA, USA; 500 MHz for 1 H and 125 MHz for 13 C). Chemical shifts (δ) were reported in ppm using TMS as internal standard and coupling constant (J) were in Hz. ESIMS spectra were obtained from a Micromass Q-TOF spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).