Secondary Metabolites from the Deep-Sea Derived Fungus Acaromyces ingoldii FS121

Activity-guided isolation of the fermentation broth of the deep-sea derived fungus Acaromyces ingoldii FS121, which was obtained from the China South Sea, yielded a new naphtha-[2,3-b]pyrandione analogue, acaromycin A (1) and a new thiazole analogue, acaromyester A (2), as well as the known compound (+)-cryptosporin (3). Their structures, including absolute configurations, were determined by extensive spectroscopic analysis and electronic circular dichroism (ECD) spectra. Compounds 1–3 were evaluated for in vitro growth inhibitory activities against four tumor cell lines (MCF-7, NCI-H460, SF-268 and HepG-2), wherein compounds 1 and 3 exhibited considerable growth inhibitory effects, with IC50 values less than 10 µM.


Introduction
Marine organisms live in a biologically competitive environment with unique environmental conditions, therefore marine-derived fungi have been proven to be a rich source of structurally unique and biologically active secondary metabolites [1][2][3][4][5][6][7][8][9][10]. In spite of the fact that the investigations of fungal metabolites and their derivatives have not yet led to a clinical cancer drug, significant research efforts have revealed a large number of fungi-derived natural products with promising anticancer activity [11][12][13]. Such chemical entities not only provide insight into the biogenetic landscape, revealing new structure classes and new biosynthetic pathways, but also enlighten the approach to biomimetic synthesis and further bioactivity investigations, suggesting potential candidates for the development of new pharmaceutical agents [14].
In the course of our search for new bioactive natural products from marine-derived fungi, we found that a culture broth of Acaromyces ingoldii FS121 showed potent cell growth inhibitory effects against SF-268, MCF-7, NCI-H460 and HepG-2 human tumor cell lines [15]. Further chemical investigation of this strain led to the isolation of two new secondary metabolites-acaromycin A (1) and acaromyester A (2)-along with the known compound (+)-cryptosporin (3) (Figure 1). Herein, details of the isolation, structure elucidation, and cell growth inhibition effects of these compounds are described.
The relative configuration of 1 was established by the comparison of its 1 H-NMR coupling constants [H-2 (J = 6.7, 1. . This indicated that these protons were cofacial and designated as β-oriented, whereas the Me-11 was α-oriented in 1. The absolute configuration of 1 was determined by the CD spectrum. The absolute configuration of (+)-cryptosporin (3) was established by synthesis and its CD spectra data was reported in the literature [17]. The CD spectrum ( Figure S19) of 1 showed a positive Cotton effect at 287, 314, 370 nm and a negative Cotton effect at 239, 425 nm, which was similar to that of 3. Thus, the absolute stereochemistry of 1 was established as 2R,3R,4R and given the trivial name as acaromycin A.
Compound 2 was obtained as a light pink oil. The HRESIMS (m/z 278.0845, [M + H] + , calcd. for C14H16NO3S, 278.0851) allowed the molecular formula C14H15NO3S to be assigned to 2, corresponding to eight degrees of unsaturation. The IR spectrum exhibited absorption bands for OH (3309 cm −1 ) and carboxyl groups (1732 and 1716 cm −1 ). The 1D NMR data of 2 (Table 1)
The . This indicated that these protons were cofacial and designated as β-oriented, whereas the Me-11 was α-oriented in 1. The absolute configuration of 1 was determined by the CD spectrum. The absolute configuration of (+)-cryptosporin (3) was established by synthesis and its CD spectra data was reported in the literature [17]. The CD spectrum ( Figure S19) of 1 showed a positive Cotton effect at 287, 314, 370 nm and a negative Cotton effect at 239, 425 nm, which was similar to that of 3. Thus, the absolute stereochemistry of 1 was established as 2R,3R,4R and given the trivial name as acaromycin A.
Compound 2 was obtained as a light pink oil. This assumption was further confirmed by the HMBC correlations from H-2 to C-1, C-3 and C-8, H-5 to C-3 and C-7, and H-8 to C-6 and C-4. Besides, the 1 H-NMR of 2 also exhibited a methyl signal at δ H 2.24 (3H, s), two methylene protons at δ H 3.02 (2H, t, J = 7.1 Hz) and 3.84 (2H, m), and an olefinic proton due to the thiazole ring at δ H 8.61 (1H, s), suggesting the presence of a 4-methyl-5-thiazoleethanol substructure in 2, which has been reported in the literature [18]. Furthermore, the key COSY correlations of H-7 1 /H-8 1 and HMBC correlations from H-2 1 to C-4 1 and C-5 1 , as well as Me-6 1 to C-4 1 , and C-5 1 also supported this deduce (Figure 2). Finally, the two substructures was connected by the HMBC correlations between H-8 to C-1 and C-5 1 . Thus, compound 2 was determined as depicted and given the trivial name as acaromyester A.
The known compound (+)-cryptosporin (3) was identified by comparison of its NMR data with those in the literature [19]. , and an olefinic proton due to the thiazole ring at δH 8.61 (1H, s), suggesting the presence of a 4-methyl-5-thiazoleethanol substructure in 2, which has been reported in the literature [18]. Furthermore, the key COSY correlations of H-7′/H-8′ and HMBC correlations from H-2′ to C-4′ and C-5′, as well as Me-6′ to C-4′, and C-5′ also supported this deduce ( Figure 2). Finally, the two substructures was connected by the HMBC correlations between H-8 to C-1 and C-5′. Thus, compound 2 was determined as depicted and given the trivial name as acaromyester A. The known compound (+)-cryptosporin (3) was identified by comparison of its NMR data with those in the literature [19].

In Vitro Growth Inhibition Assay
All of the isolates 1-3 were tested for in vitro growth inhibitory activities against the MCF-7, NCI-H460, SF-268 and HepG-2 tumor cell lines at an initial concentration of 100 µM. Then, compounds with inhibition greater than 50% were further analyzed to determine their corresponding IC 50 values. Both 1 and 3 exhibited considerable inhibitory activities against the growth of all four cell lines (Table 2). Especially, the inhibitory effects of 1 and 3 against the MCF-7 cells were found to be comparable to that of cisplatin with IC 50 values of 6.7 and 4.1 µM, respectively. The growth inhibitory curves of 1, 3 and cisplatin against MCF-7 were presented in Figure 3.

Fungal Material
The marine fungal strain FS121 was isolated from a marine sediment sample, which was collected

Fungal Material
The marine fungal strain FS121 was isolated from a marine sediment sample, which was collected in the South China Sea (18˝44.606 1 N, 119˝44.263 1 E; 3415 m depth) in September 2011. The strain was identified by the sequence analysis of the rDNA ITS (internal transcribed spacer) region. The sequence of the ITS region of the strain FS121 has been submitted to GenBank (Accession No. KT989306). By using BLAST (nucleotide sequence comparison program) to search the GenBank database, FS121 has 99.8% similarity with Acaromyces ingoldii CBS 10536 (Accession No. AM991023). The strain is preserved at the Guangdong Provincial Key Laboratory of Microbiology Culture Collection and Application, Guangdong Institute of Microbiology.

Fermentation, Extraction and Compound Isolation
The fungal strain FS121 was maintained on potato dextrose agar (PDA) medium at 28˝C for 7 days, and then three pieces (0.5ˆ0.5 cm 2 ) of mycelial agar plugs were inoculated into 20ˆ500 mL Erlenmeyer flasks, each containing 250 mL potato dextrose broth (potato 20%, glucose 2%, K 2 HPO 4 0.3%, MgSO 4 ‚7H 2 O 0.15%, vitamin B 1 10 mg/L, sea salt 1.5%). After 6 days of incubation at 28˝C on a rotary shaker at 120 r/m, 20 mL seed cultures were aseptically transferred into each of a total of 220 flasks (1000 mL) containing 500 mL of potato dextrose broth. The liquid cultivation that followed was kept for 7 days at 28˝C and 120 r/m on a rotary shaker. The culture (110 L) was centrifuged to give the broth and mycelia. The broth was exhaustively extracted with EtOAc four times, then the EtOAc layers were combined and evaporated under reduced pressure at a temperature not exceeding 40˝C to yield a dark brown gum (35.8 g). The crude extract was subjected to silica gel (200-300 mesh) column chromatography (CC) with a gradient system of increasing polarity (petroleum ether/EtOAc, 50:1Ñ1:2) to afford 33 fractions, Fr.

In Vitro Growth Inhibition Assay
Cell growth inhibitory effects of compounds 1-3 were tested against four tumor cell lines, including MCF-7 (human breast adenocarcinoma cell line), NCI-H460 (human non-small cell lung cancer cell line) and SF-268 (human glioma cell line), HepG-2 (human hepatomacarcinoma cell line) by the SRB method [20]. Cells (180 µL) with a density of 3ˆ10 4 cells/mL of media were seeded onto 96-well plates and incubated for 24 h at 37˝C, 5% CO 2 . Various concentrations of compounds (20 µL) were added to the plate wells, and plates were further incubated for 72 h. After incubation, cell monolayers were fixed with 50% (w/v) trichloroacetic acid (50 µL) and stained for 30 min with 0.4% (w/v) SRB dissolved in 1% acetic acid. Unbound dye was removed by washing repeatedly with 1% acetic acid. The protein-bound dye was dissolved in 10 mM Tris base solution (200 µL) for OD determination at 570 nm using a microplate reader. Cisplatin was used as a positive control. All data were obtained in triplicate and are presented as means˘S.D. IC 50 values were calculated with the SigmaPlot 10.0 software (San Jose, California, CA, USA) using a nonlinear curve-fitting method.