Bioactive Diphenyl Ethers and Isocoumarin Derivatives from a Gorgonian-Derived Fungus Phoma sp. (TA07-1)

Three new diphenyl ether derivatives—phomaethers A–C (1–3) and five known compounds—including a diphenyl ether analog, 2,3′-dihydroxy-4-methoxy-5′,6-dimethyl diphenyl ether (4); and four isocoumarin derivatives, diaportinol (5), desmethyldiaportinol (6), citreoisocoumarinol (7), and citreoisocoumarin (8)—were isolated from a gorgonian-derived fungus Phoma sp. (TA07-1). Their structures were elucidated by extensive spectroscopic investigation. The absolute configurations of 1 and 2 were determined by acid hydrolysis reactions. It was the first report to discover the diphenyl glycoside derivatives from coral-derived fungi. Compounds 1, 3, and 4 showed selective strong antibacterial activity against five pathogenic bacteria with the minimum inhibiting concentration (MIC) values and minimum bactericidal concentration (MBC) values between 0.156 and 10.0 μM.


Results and Discussion
Phomaether B (2) was isolated as a light brown, amorphous powder. The molecular formula was assigned as C 20 H 24 O 8 (nine degrees of unsaturation) by its HRESIMS data. Detailed inspection of the NMR data (Table 1) of 2 with those of 1 revealed that these two compounds were very similar, except for the sugar moiety. The sugar residue in 2 was defined as a ribose by comparing the NMR data ( Table 1) with those of naphthyl ribofuranoside, isotorachrysone-6-O-α-D-ribofuranoside [20], chromene glycoside, and sterin A [21]. The key HMBC correlation from H-1 to C-3 ( Figure 2) established the connection between the ribose and diphenyl ether moiety. The coupling constant of anomeric proton H-1 (J = 4.5 Hz) in 2 was found to close to that in methyl-α-D-ribofuranoside (J = 4.3 Hz) [22], indicating an α-ribose in 2. The α-ribose was determined as D-configuration by comparing the optical rotation data of the acid hydrolysate of 2 with that of the standard  Phomaether B (2) was isolated as a light brown, amorphous powder. The molecular formula was assigned as C20H24O8 (nine degrees of unsaturation) by its HRESIMS data. Detailed inspection of the NMR data (Table 1) of 2 with those of 1 revealed that these two compounds were very similar, except for the sugar moiety. The sugar residue in 2 was defined as a ribose by comparing the NMR data ( Table 1) with those of naphthyl ribofuranoside, isotorachrysone-6-O-α-D-ribofuranoside [20], chromene glycoside, and sterin A [21]. The key HMBC correlation from H-1″ to C-3′ ( Figure 2) established the connection between the ribose and diphenyl ether moiety. The coupling constant of anomeric proton H-1″ (J = 4.5 Hz) in 2 was found to close to that in methyl-α-D-ribofuranoside (J = 4.3 Hz) [22], indicating an α-ribose in 2. The α-ribose was determined as D-configuration by comparing the optical rotation data of the acid hydrolysate of 2 with that of the standard D-ribose ( α −23.0 (c 0.03, H2O) vs. α −38.7 (c 0.10, H2O)). From above, 2 was determined as 2-hydroxy-3′-O-α-Dribofuranoside-4-methoxy-5′,6-dimethyl diphenyl ether, and named phomaether B.
A literature survey revealed that the diphenyl glycoside derivatives were rare in marine natural products. To the best of our knowledge, a diphenyl glycoside was found from a sponge-derived fungus Metarhizium anisopliae [23]. In present study, the diphenyl glycoside derivatives were reported for the first time isolated from coral-derived fungi.   Phomaether B (2) was isolated as a light brown, amorphous powder. The molecular formula was assigned as C20H24O8 (nine degrees of unsaturation) by its HRESIMS data. Detailed inspection of the NMR data (Table 1) of 2 with those of 1 revealed that these two compounds were very similar, except for the sugar moiety. The sugar residue in 2 was defined as a ribose by comparing the NMR data ( Table 1) with those of naphthyl ribofuranoside, isotorachrysone-6-O-α-D-ribofuranoside [20], chromene glycoside, and sterin A [21]. The key HMBC correlation from H-1″ to C-3′ (Figure 2) established the connection between the ribose and diphenyl ether moiety. The coupling constant of anomeric proton H-1″ (J = 4.5 Hz) in 2 was found to close to that in methyl-α-D-ribofuranoside (J = 4.3 Hz) [22], indicating an α-ribose in 2. The α-ribose was determined as D-configuration by comparing the optical rotation data of the acid hydrolysate of 2 with that of the standard D-ribose ( α −23.0 (c 0.03, H2O) vs. α −38.7 (c 0.10, H2O)). From above, 2 was determined as 2-hydroxy-3′-O-α-Dribofuranoside-4-methoxy-5′,6-dimethyl diphenyl ether, and named phomaether B.
A literature survey revealed that the diphenyl glycoside derivatives were rare in marine natural products. To the best of our knowledge, a diphenyl glycoside was found from a sponge-derived fungus Metarhizium anisopliae [23]. In present study, the diphenyl glycoside derivatives were reported for the first time isolated from coral-derived fungi.
) and HMBC (  Phomaether B (2) was isolated as a light brown, amorphous powder. The molecular formula was assigned as C20H24O8 (nine degrees of unsaturation) by its HRESIMS data. Detailed inspection of the NMR data (Table 1) of 2 with those of 1 revealed that these two compounds were very similar, except for the sugar moiety. The sugar residue in 2 was defined as a ribose by comparing the NMR data ( Table 1) with those of naphthyl ribofuranoside, isotorachrysone-6-O-α-D-ribofuranoside [20], chromene glycoside, and sterin A [21]. The key HMBC correlation from H-1″ to C-3′ (Figure 2) established the connection between the ribose and diphenyl ether moiety. The coupling constant of anomeric proton H-1″ (J = 4.5 Hz) in 2 was found to close to that in methyl-α-D-ribofuranoside (J = 4.3 Hz) [22], indicating an α-ribose in 2. The α-ribose was determined as D-configuration by comparing the optical rotation data of the acid hydrolysate of 2 with that of the standard D-ribose ( α −23.0 (c 0.03, H2O) vs. α −38.7 (c 0.10, H2O)). From above, 2 was determined as 2-hydroxy-3′-O-α-Dribofuranoside-4-methoxy-5′,6-dimethyl diphenyl ether, and named phomaether B.
A literature survey revealed that the diphenyl glycoside derivatives were rare in marine natural products. To the best of our knowledge, a diphenyl glycoside was found from a sponge-derived fungus Metarhizium anisopliae [23]. In present study, the diphenyl glycoside derivatives were reported for the first time isolated from coral-derived fungi.
A literature survey revealed that the diphenyl glycoside derivatives were rare in marine natural products. To the best of our knowledge, a diphenyl glycoside was found from a sponge-derived fungus Metarhizium anisopliae [23]. In present study, the diphenyl glycoside derivatives were reported for the first time isolated from coral-derived fungi.
All the isolated compounds (1)(2)(3)(4)(5)(6)(7)(8) were evaluated for their antibacterial activity against a panel of pathogenic bacteria, including two Gram-positive bacteria, S. albus and S. aureus, and three Gram-negative bacteria E. coli, V. parahaemolyticus, and V. anguillarum (Table 2). Compound 1 exhibited remarkable antibacterial activity against S. albus, S. aureus, E. coli, and V. parahaemolyticus with MIC values ranging from 0.312 to 0.625 µM and MBC values from 0.625 to 2.50 µM. Compound 3 showed strong antibacterial activity to S. albus, S. aureus, and E. coli with MIC values ranging from 0.312 to 1.25 µM and MBC values from 0.625 to 5.00 µM. It was notable that compound 4 showed strong antibacterial activity to all of the tested pathogenic bacteria, with MIC and MBC values ranging from 0.156 to 5.00 µM. "-" means no activity; each experiment of the activity bioassays has been repeated three times and the results were same.
Compounds 1-8 were also tested for their lethality to the brine shrimp, Artemia salina. Compounds 1, 3, and 4 showed moderate lethality to the brine shrimp A. salina with the LC 50 values ranging from 14.01 ± 0.36 to 37.33 ± 0.26 µg/mL.

Fungal Materials
The

Methanolysis of Compound 1
Compound 1 (2.0 mg) was dissolved in 5% HCl-MeOH (5 mL) and refluxed at boiled temperature for 2 h. The reaction mixture was neutralized and evaporated to give the residue. Then, the residue was extracted by 50% EtOAc-H 2 O to obtain aglycone. The configuration of D-glucoside was determined by comparing its rotation with that of the authentic sample.

Methanolysis of Compound 2
The aglycone of compound 2 was obtained by the same method for that of compound 1. The configuration of D-ribose was determined by comparing its rotation with that of a standard sample.

Biological Assays
The antibacterial activity of compounds was evaluated by the conventional broth dilution assay [24,25]. The MICs were tested in 96-well microtiter plates, and the concentrations of the compounds were serial double dilution which were certain in each well. The MICs were determined as the lowest concentrations at which no growth was observed. The MBCs were determined by transferring approximately 0.0015 mL from each well of the microtiter plate with the MIC 2000 inoculator to a petri dish (15 by 150 mm) containing solid LB culture. The plates were incubated at 35 • C for 48 h. The MBCs were read as the lowest concentrations of compounds that prevented growth of more than one colony on subculture. The test range of compounds 1-8 was 0.039-20.0 µM and the test range of positive control was 0.010-10.0 µM. Five bacterial strains S. albus (ATCC 23361), S. aureus (ATCC 27154), E. coli (ATCC 25922), V. parahaemolyticus (ATCC 17802), and V. anguillarum (ATCC 19109) were used, and ciprofloxacin was used as a positive control.
The lethality to the brine shrimp A. salina was tested according to the method in literature [26]. The brine shrimp A. salina eggs (Tianjin Red Sun Aquaculture Co., LTD., Tianjin, China) were incubated in nature seawater from Yellow Sea in Qingdao, China and oxygenated with an aquarium pump at 25 • C for 48 h. The nauplii of brine shrimp were separated from the eggs in small beakers containing sea water. The test compounds were dissolved in DMSO and the serial diluted concentration ranges of the compounds 1-8 were 0.625-100 µg/mL. In 24-well microplates, 15-20 brine shrimp in each well were incubated with the test compounds for 24 h. The lethality rates were observed and the LC 50 values were calculated by Probit analysis.