Sesquiterpenoids from the Mangrove-Derived Aspergillus ustus 094102

Four new drimane sesquiterpenoids (1–4) and three known ones (5–7) were isolated from the fermentation broth of the mangrove-derived Aspergillus ustus 094102. Compound 5 was further resolved as four purified compounds 5a–5d. By means of extensive spectroscopic and ECD analysis as well as the chemical transformation, their structures were identified as (2R,3R,5S,9R,10S)-2,3,9,11-tetrahydroxydrim-7-en-6-one (ustusol F, 1), (2R,3R,5R,9S,10R)-2,3,11-trihydroxydrim-7-en-6-one (9-deoxyustusol F, 2), (3S,5R,9R,10R)-3,11,12-trihydroxydrim-7-en-6-one (ustusol G, 3), (5S,6R,9S,10S, 11R,2′E,4′E)-(11-dideoxy-11-hydroxystrobilactone A-6-yl)-5-carboxypenta-2,4-dienoate (ustusolate H, 4), ((5S,6R,9S,10S)-strobilactone A-6-yl) (2E,4E)-6,7-dihydroxyocta-2,4-dienoate (ustusolate I, 5), (2′E,4′E;6′,7′-erythro)-ustusolate I (5a) and (2′E,4′E;ent-6′,7′-erythro)-ustusolate I (5b), (2′E,4′E,6′R,7′R)-ustusolate I (5c) and (2′E,4′E,6′S,7′S)-ustusolate I (5d), (5S,6R,9S,10S,2′E,4′E)-(strobilactone A-6-yl)-5-carboxypenta-2,4-dienoate (ustusolate J, 6), and (2S,5S,9R,10S)-2,9,11-trihydroxydrim-7-en-6-one (ustusol B, 7), respectively. Compound 5 showed antiproliferation against the human tumor cells CAL-62 and MG-63 with the IC50 values of 16.3 and 10.1 µM, respectively.

The drimane sesquiterpenoids 1-7 were postulated to be biosynthesized from farnesyl-PP (I) which generated intermediate II, III and IV after cyclization and oxidation. The intermediates II and III were subjected to further oxidation to form compounds 1, 2, 3, and 7. The intermediate II was further oxidized to intermediate IV, and the latter was subjected to oxidation, hemi acetalization, and esterification to form compounds 4, 5, and 6 ( Figure 5).
The antiproliferations of compounds 1-7 were evaluated against 29 human cancer cell lines and a normal cell line (the names of cell lines are listed in the Supplementary Files) by the cell counting  methods [18,19]. Only compound 5, the mixture of 5a/5b/5c/5d, showed antiproliferative activity against the human thyroid cancer cells (CAL-62) and human osteosarcoma cells (MG-63) with the IC 50 values of 16.28 ± 1.01 and 10.08 ± 0.04 µM, respectively, while the pure compounds 5a-5d were inactive (IC 50 ≥ 50 µM). The IC 50 values of doxorubicin (positive control) against CAL-62 and MG-63 were 0.062 ± 0.022 and 0.096 ± 0.012 µM, respectively. The bacteriostatic activities of compounds 1-7 against 6 human pathogenic bacteria and 6 aquatic pathogenic bacteria (the names are listed in the Supplementary Files) were tested by the diffusion method of filter paper, but no inhibition zone was observed at the concentration of 100 µg/mL for compounds 1-7.
viously reported ustusol B [4] (Table S1) and displayed the same retention times in the co-HPLC ( Figure S91). Thus, the structure of ustusol B was revised as structure 7, which was named ustusol B.
The drimane sesquiterpenoids 1-7 were postulated to be biosynthesized from farnesyl-PP (I) which generated intermediate II, III and IV after cyclization and oxidation. The intermediates II and III were subjected to further oxidation to form compounds 1, 2, 3, and 7. The intermediate II was further oxidized to intermediate IV, and the latter was subjected to oxidation, hemi acetalization, and esterification to form compounds 4, 5, and 6 ( Figure 5).

Fungal Material
The mangrove fungal strain A. ustus 094102 was isolated from the rhizosphere soil of the mangrove plant Bruguiera gymnorrhiza grown in Wenchang, Hainan Province of China. It was identified according to the morphological characteristics and the ITS sequences [4,5].

Cultivation and Extraction
The fungus A. ustus 094102 was statically cultured at 25 • C for 28 days in one hundred 1000 mL conical flasks, each containing 300 mL of the liquid medium that was prepared by dissolving maltose (20 g), mannitol (20 g), glucose (10 g), monosodium glutamate (10 g), yeast extract (3 g), corn steep liquor (1 g), CaCO 3 (2 g), KH 2 PO 4 (0.5 g), MgSO 4 ·7H 2 O (0.3 g), and sea salt (33 g) in 1 L of tap water (pH 7.0). The whole fermentation broth (30 L) was filtered by cheesecloth to separate the mycelia from the filtrate. The mycelia were extracted three times with an 80% volume of aqueous acetone. The acetone solution was concentrated under reduced pressure to give an aqueous solution. The aqueous solution was extracted three times with an equivalent volume of ethyl acetate (EtOAc), while the filtrate was extracted three times with an equivalent volume of EtOAc. All EtOAc extracts were combined and concentrated under vacuum to give 240 g of crude gum.

The Preparation of Acetonide (5e) for Relative Configuration
According to our procedure [16], compound 5a (5 mg) in acetone (3 mL) was added to the mixture of 2,2-dimethoxypropane (1 mL), pyridinium p-toluenesulfonate (PPTS, 26 mg) and N,N-dimethylformamide (DMF, 1 mL). The resulting solution was stirred at room temperature (rt) for 12 h, and then 5 mL of H 2 O was added. The reaction solution was extracted with 15mL of CH 2 Cl 2 , and the organic phase was concentrated under reduced pressure. The residue was purified by semipreparative HPLC (95% MeOH-H 2 O) to yield the acetonide 5e (3.4 mg, t R 5.7 min). Its structure was identified by ESIMS ( Figure S65) and NMR data (Tables 3 and 4, Figures 4 and S66-S71).