Fluostatins I–K from the South China Sea-Derived Micromonospora

Nov 8, 2012 - University, 601 West Huangpu Road, Guangzhou 510632, People's Republic of ... China Sea.3−7 From our repertoire of marine-derived acti...
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Fluostatins I−K from the South China Sea-Derived Micromonospora rosaria SCSIO N160 Wenjun Zhang,†,§ Zhong Liu,‡,§ Sumei Li,† Yongzhi Lu,⊥ Yuchan Chen,∥ Haibo Zhang,† Guangtao Zhang,† Yiguang Zhu,† Gaiyun Zhang,† Weimin Zhang,∥ Jinsong Liu,⊥ and Changsheng Zhang*,† †

CAS Key Laboratory of Marine Bio-resources Sustainable Utilization, RNAM Center for Marine Microbiology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, People's Republic of China ‡ Guangzhoujinan Biomedicine Research and Development Center, Guangdong Key Laboratory of Bioengineering Medicine, Jinan University, 601 West Huangpu Road, Guangzhou 510632, People's Republic of China ⊥ State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Guangzhou Science Park, Guangzhou 510530, People's Republic of China ∥ Guangdong Institute of Microbiology, 100 Central Xianlie Road, Guangzhou 510070, People's Republic of China S Supporting Information *

ABSTRACT: The strain SCSIO N160 was isolated from a South China Sea sediment sample and was characterized as a Micromonospora rosaria species on the basis of its 16S rRNA gene sequence. Three new fluostatins, I−K (1−3), were isolated from the culture of M. rosaria SCSIO N160, together with six known compounds, fluostatins C−F (4−7), rabelomycin (8), and phenanthroviridone (9). The structure of fluostatin D (5) was confirmed by an X-ray crystallographic study. The absolute configuration of 1 and 3 was assigned by electronic circular dichroism calculations. Compounds 8 and 9 exhibited good antimicrobial activities against Staphylococcus aureus ATCC 29213 with MIC values of 1.0 and 0.25 μg/mL, respectively. Compound 9 also exhibited significant in vitro cytotoxic activities toward SF-268 (IC50 0.09 μM) and MCF-7 (IC50 0.17 μM).

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A

RESULTS AND DISCUSSION The strain SCSIO N160 was isolated from a sediment sample (E 116°17.754′, N 22°41.083′) at a depth of 30 m from the South China Sea and was identified as an M. rosaria species (GenBank accession number JF508525 for its 16S rRNA gene sequence) on the basis of the phylogenetic tree constructed by a neighbor-joining method (Figure S1). The butanone extracts of the fermentation broths of M. rosaria SCSIO N160 led to the isolation of nine compounds, 1−9 (Figure 1). Compounds 4−9 were identified as fluostatins C−E (4−6)16,17 and F (7),18 rabelomycin (8),19,20 and phenanthroviridone (9),21 respectively, on the basis of the comparison of their 1H and 13C NMR spectroscopic data with those previously reported (Tables S1, S2). In a previous report,17 the absolute configuration of fluostatin D (5) was suggested to be 1R, 2S, 3S from its structural similarity to fluostatin C (4). The relative configuration of 4 was established by its X-ray structure, and the absolute

ctinobacteria are an important member of the marine bacterial communities and are well known for their diversity and ability to produce novel natural products of high pharmaceutical significance.1 For example, several compounds isolated from marine actinobacteria are currently in clinical trials against various diseases,2 highlighting the value of marinederived actinobacteria. In recent years, many novel genera and species of actinobacteria have been uncovered from the South China Sea.3−7 From our repertoire of marine-derived actinobacteria, we have recently discovered several new compounds with antibacterial, antitumor, and antimalarial bioactivities.8−15 Herein we report the isolation, structural elucidation, and biological activities of nine secondary metabolites from the strain SCSIO N160, which was identified as a Micromonospora rosaria species by the 16S rRNA gene sequence analysis. These nine metabolites included three new members of the fluostatin family, fluostatins I−K (1−3), the known fluostatins C−F (4− 7), and two structurally related known compounds, rabelomycin (8) and phenanthroviridone (9). © 2012 American Chemical Society and American Society of Pharmacognosy

Received: July 20, 2012 Published: November 8, 2012 1937

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Figure 1. Chemical structures of isolated compounds from Micromonospora rosaria SCSIO N160.

configuration at C-1 of 4 was determined by the application of the Helmchen method.17,22 Herein, the structure of compound 5 was confirmed for the first time by X-ray crystallographic analysis (Figure 2, accession number CCDC 905071), and the

Figure 2. X-ray crystal structure of fluostatin D (5).

Figure 3. Comparison of experimental ECD spectrum of 5 with the calculated ECD spectra of (1R, 2S, 3S)-5 (5a) and (1S, 2R, 3R)-5 (5b).

X-ray diffraction data with Cu Kα radiation supported the assignment of the absolute configuration of 5 as 1R, 2S, 3S on the basis of the Flack parameter value x = −0.2(3)23 and Hooft parameter of −0.11(7).24,25 The comparisons of the experimental electronic circular dichroism (ECD) spectrum of 5 with that of 4 (Figure S2) and with the calculated ECD spectra for (1R, 2S, 3S)-5 (5a) and (1S, 2R, 3R)-5 (5b) (Figure 3) further supported this assignment. A random conformational search was performed for 5a and 5b using the MMFF94 molecular mechanics force field after the energy minimization by SYBYL 8.0 software package, which yielded seven conformers for 5a and six for 5b, respectively, within a 10 kcal/mol energy window. The following geometry optimizations at the B3LYP/ 6-31G(d) level afforded five conformers for 5a (Figure S3) and six for 5b (Figure S4), respectively, within a 2 kcal/mol energy window. Subsequently, the conformers were collected and submitted to ECD calculations by time-dependent density functional theory (TDDFT) calculations at the B3LYP/6-31G (d) level, considering the lowest 80 excited states with the CPCM solvent model for methanol. The resulting ECD spectra of 5a and 5b were energetically weighted according to the respective conformational distribution (Figures S3 and S4) by

the Boltzmann statistics. The comparison of the experimental ECD spectrum of 5 with the calculated ones (Figure 3) revealed that the Cotton effects (CEs) of 5a were in good accordance with the experimental CEs of 5 in the region 220− 400 nm. Therefore, the independent TDDFT calculation method supported the 1R, 2S, 3S absolute configuration for 5, consistent with the previous assignment.17 Fluostatin I (1) was established to have the molecular formula C22H20O7 (m/z 395.1162 [M − H]−, calcd for 395.1131) on the basis of HRESIMS, indicating 13 indices of hydrogen deficiency. The 1H and 13C NMR spectroscopic data of 1 (Table 1, Figures S5) are similar to those of fluostatin D (5).17 The difference was that compound 1 had one less index of hydrogen deficiency than 5. Also, the signal of an oxygenated quaternary carbon in 5 was substituted by an sp3 methine at δC 44.1 in 1, and an 11.7 ppm downfield shift of the C-2 (δC 72.8) in 1 was observed. These data indicated that the oxirane ring in 5 was reductively cleaved and transformed to the secondary alcohol in 1. Consistent with this structural feature of 1, 1H−1H COSY correlations were observed between H-2/H-3 and H-3/ 1938

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Table 1. 1H NMR (500 MHz) and 13C NMR (125 MHz) Assignments of Compounds 1−3 (J in Hz) 1a no.

δH multi (J in Hz)

1 2 3 4 4a 5 6 6a 6b 7 8 9 10 10a 11 11a 11b 12 13 14 15

6.50, d (3.0) 4.30, dd (3.0, 2.5) 3.03, m

16 17 a

7.60, s

7.05, d (8.0) 7.28, dd (8.0, 7.0) 7.21, d (7.0)

1.29, d (6.5) 2.58, m 1.15, d (7.0) 1.17, d (7.0)

2b δC multi 68.4, 72.8, 44.1, 198.5, 135.8, 121.1, 152.1, 134.4, 127.2, 152.5, 125.2, 132.9, 117.7, 136.3, 193.8, 136.9, 129.6, 11.7, 177.4, 35.1, 19.5,

CH CH CH C C CH C Cd C C CH CH CH C C Cd C CH3 C CH CH3

19.6, CH3

δH multi (J in Hz) 6.97, d (2.5) 3.82, d (2.5)

7.46, s

6.91, d (7.5) 7.12, me 7.13, me

1.55, s 2.29, 1.56, 1.35, 0.80, 1.05,

m m m t (7.5) d (7.0)

3c δC multi 62.8, 60.0, 58.4, 193.3, 132.2, 121.1, 150.9, 135.4, 125.8, 150.8, 124.0, 131.5, 116.9, 135.4, 192.2, 132.4, 126.3, 14.7, 176.0, 40.9, 26.4,

CH CH C C C CH C C C C CH CH CH C C C C CH3 C CH CH2

δH multi (J in Hz) 7.57, d (10.0) 6.29, d (10.0)

7.54, s

7.11, d (8.0) 7.31, dd (8.0, 7.0) 7.18, d (7.0)

1.27, s

δC multi 117.4, 141.2, 72.6, 200.1, 127.8, 121.1, 149.0, 133.7, 125.3, 150.9, 123.9, 131.9, 116.3, 135.3, 192.6, 130.7, 128.6, 26.6,

CH CH C C C CH C C C C CH CH CH C C C C CH3

11.2, CH3 16.2, CH3

Recorded in methanol-d4. bCDCl3/methanol-d4. cDMSO-d6. dExchangeable. Multi: multiplicity. eOverlapping.

OH and 12-Me were trans-oriented (Figure 4), suggested by the small coupling constants of H-1/H-2 (3JH1−H2 3.0 Hz) and H-2/H-3 (3JH2−H3 2.5 Hz).26 The absolute configuration of 1 was deduced by the comparison of the experimental and calculated ECD spectra for (1R,2R,3R)-1 (1a) and (1S,2S,3S)-1 (1b) (Figure 5). The computational procedure was performed by the same approach as described for 5. The random

H-12 (Figure S5). The planar structure of compound 1 (Figure 1) was further supported by the HMBC correlations from H-3 to C-1/C-2/C-4a and H-12 to C-2/C-3/C-4 (Figure 4). The NOE correlation between H-2 and H-12 indicated that the 2-

Figure 4. 1H−1H COSY and selected HMBC correlations of fluostatins I (1), J (2), and K (3) and NOESY correlation of fluostatins I (1).

Figure 5. Comparison of experimental ECD spectrum of 1 with the calculated ECD spectra of (1R,2R,3R)-1 (1a) and (1S,2S,3S)-1 (1b). 1939

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conformational search within a 10 kcal/mol energy window yielded fourteen and thirteen conformers for 1a and 1b, respectively. The following geometry optimizations within a 1 kcal/mol energy window afforded five conformers for 1a (Figure S6) and four for 1b (Figure S7), respectively. The experimental ECD spectrum of 1 agreed well with the calculated ECD spectrum of (1R,2R,3R)-1 (1a), both showing negative and positive CEs in the 200−250 and 280−350 nm regions, respectively (Figure 5). Therefore, 1 was deduced to have the 1R, 2R, 3R absolute configuration. The molecular formula of fluostatin J (2) was determined to be C23H20O7 (m/z 407.1136 [M − H]−, calcd for 407.1131) by HRESIMS. The 1H and 13C NMR spectra of 2 (Table 1, Figure S8) were also similar to those of fluostatin D (5);17 the difference was that one of the methyl doublets in 5 [δH 1.03 (d, J = 7.0 Hz, Me-16), δC 18.5 (Me-16)] was substituted by the resonances of an ethyl group [δH 0.80 (t, J = 7.5 Hz, Me-16), 1.35 (m, H-15a), 1.56 (m, H-15b), δC 11.2 (Me-16), 26.4 (C15)] in 2. In the 1H−1H COSY spectrum of 2, the spin system from H-14 to H-17 suggested that the isobutyryl group in 5 was substituted by an isovaleryl group in 2, which was supported by HMBC correlations from H-17 to C-14/C-15 and from H-16 to C-13/C-15 (Figure 4). On the basis of these data, the planar structure of fluostatin J (2) was determined as shown in Figure 1. The relative configuration of 2 was deduced from J values of H-1/H-2 (3JH1−H2 2.5 Hz).26 On the basis of the similarity of the experimental ECD spectra of 2, 4, and 5 (Figure S2), the 1R, 2S, 3S absolute configuration could be deduced for compound 2. However, the C-14 configuration could not be determined by quantum ECD spectra calculations. Although the conformational search and geometry optimizations yielded six and three possible conformers within a 1 kcal/mol energy window (Figures S9 and S10), the overall weighted ECD spectra calculated for diastereomers (1R,2S,3S,14R)-2 (2a) and (1R,2S,3S,14S)-2 (2b) were almost identical (Figure S11). The molecular formula of fluostatin K (3) was established as C18H12O5 (m/z 307.0641 [M − H]−, calcd for 307.0606) by HRESIMS, exhibiting 13 indices of hydrogen deficiency. The 1 H and 13C NMR spectroscopic data of 3 (Table 1, Figure S12) were similar to those of fluostatin C (4).17,27 The two oxygenated methine carbons in 4 (δC 61.3, C-1; 64.0, C-2) were replaced in 3 by a pair of olefinic carbons [δH 6.29 (d, J = 10.0 Hz, H-2), 7.57 (d, J = 10.0 Hz, H-1); δC 117.4 (CH, C-1), 141.2 (CH, C-2)]. Also, C-3 in 3 shifted downfield by 14.2 ppm compared to 4 (Table 1, Table S2). These observations indicated that the oxirane ring in fluostatin C (4) was absent in 3. The presence of a double bond between C-1 and C-2 in 3 was deduced from the 1H−1H COSY correlation of H-1/H-2 and was further supported by HMBC correlations from H-12 to C-2/C-3 and from H-1 to C-4a/C-11a (Figure 4). Therefore, the planar structure of 3 was determined as shown in Figure 1. The 3S absolute configuration was deduced by comparison of the experimental ECD spectrum with the calculated ECD spectra for enantiomers (3R)-3 (3a) and (3S)-3 (3b) (Figure 6). The random conformational search resulted in only one possible conformer for 3a and 3b (Figure S13), respectively, for the lack of a rotatable bond. The experimental ECD spectrum of 3 was in good agreement with the calculated ECD spectrum of 3b (Figure 6). Therefore, the absolute configuration of C-3 of 3 was deduced as S. The new fluostatins I−K (1−3), as well as the known fluostatins C−F (4−7), showed weak antimicrobial activities against five indicator strains (Table 2). Rabelomycin (8) and

Figure 6. Comparison of the experimental ECD spectrum of 3 with the calculated ECD spectra of (3R)-3 (3a) and (3S)-3 (3b).

phenanthroviridone (9) inhibited the growth of Staphylococcus aureus ATCC 29213, with MIC values of 1.0 and 0.25 μg/mL. Both compounds 8 and 9 also showed antibacterial activities against Escherichia coli ATCC 25922, Bacillus thuringiensis SCSIO BT01, and B. subtilis SCSIO BS01, but no antifungal activities against Candida albicans ATCC 10231 (Table 2). The in vitro cytotoxicities of compounds 1−9 were evaluated against three human cancer cell lines: SF-268, MCF-7, and NCI-H460. Phenanthroviridone (9) exhibited moderate activities toward SF-268 (IC50 0.09 μM) and MCF-7 (IC50 0.17 μM) and weak activity against NCI-H460. Compound 8 showed moderate in vitro cytotoxicities against these three cell lines. However, the fluostatin-type compounds (1−7) showed no cytotoxicities (IC50 > 100 μM, Table 2). Prior to this study, eight fluostatin-type compounds have been described, including fluostatins A and B,28,29 C−E,17 and F−H.18 Fluostains A−E were isolated from readily culturable actinobacterial strains, while fluostatins F−H were produced by the heterologous expression of a type II PKS gene cluster, which was cloned and reassembled from environmental DNA fragments.18 Consistent with previous reports,16−18 the new members of the fluostatin-type compounds 1−3, as well as the known 4−7, showed negligible antimicrobial and cytotoxic activities. Rabelomycin (8) was previously shown to have potent inhibitory activity in the inducible nitric oxide synthase (iNOS) assay.30 Phenanthroviridone (9), isolated from a soil Streptomyces strain,31 was shown to be active against lung carcinoma in mice.32 In this study, we demonstrated for the first time that compounds 8 and 9 had antibacterial activities against Staphylococcus aureus and compound 9 had significant in vitro cytotoxicities toward SF-268 and MCF-7 (Table 2).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a 341 polarimeter (Perkin-Elmer, Inc., Norwalk, CT, USA). UV was recorded on an U-2900 spectrophotometer (Hitachi, Tokyo, Japan), and ECD spectra were recorded on a JASCO J-810 spectropolarimeter (JASCO, Tokyo, Japan). IR spectra were recorded

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Table 2. Antimicrobial and Cytotoxic Activities of Compounds 1−9a MIC (μg mL−1)

4 7 8 9 CPb

IC50 (μM)

Escherichia coli ATCC 25922

Staphylococcus aureus ATCC 29213

Bacillus thuringiensis SCSIO BT01

Bacillus subtilis SCSIO BS01

>64 >64 16 32

16 32 1 0.25

>64 >64 8 4

>64 >64 16 64

SF-268

MCF-7

NCI-H460

>100 >100 5.54 ± 0.05 0.09 ± 0.04 3.99 ± 0.49

>100 >100 4.28 ± 0.08 0.17 ± 0.02 9.23 ± 0.41

>100 >100 9.91 ± 0.08 2.18 ± 0.01 1.53 ± 0.12

a The MIC values of compounds 1−3, 5, and 6 against four bacterial strains were >64 μg/mL. The MIC values of compounds 1−9 against Candida albicans ATCC10231 were >64 μg/mL. The IC50 values of compounds 1−3, 5, and 6 against three cancer cell lines were >100 μM. bCisplatin, positive control.

on a Nicolet*6700 FT-IR spectrometer (Thermo Scientific, USA). 1H, 13 C, and 2D NMR spectra were recorded on a Bruker AV-500 MHz NMR spectrometer with TMS as internal standard. Deuterated NMR solvents were purchased from Cambridge Isotopes (Andover, MA, USA). MS data were obtained on an LCQDECA XP HPLC/MS spectrometer for ESIMS. HRESIMS data were obtained on a quadrupole time-of-flight mass spectrometer (Waters). Materials for CC were silica gel (100−200 mesh; 300−400 mesh; Jiangyou Silica Gel Development, Inc., Yantai, P. R. China), Sephadex LH-20 (40−70 μm; Amersham Pharmacia Biotech AB, Uppsala, Sweden), and YMC*gel ODS-A (12 nm S-50 μm; Japan). TLC (0.1−0.2 or 0.3− 0.4 mm) was conducted with precoated glass plates (silica gel GF254, 10−40 nm, Yantai). MPLC was performed on automatic flash chromatography (CHEETAH MP 200, Bonna-Agela Technologies Co., Ltd. China) with a monitor wavelength of 238 nm and collector wavelength of 254 nm. Bacterial Material. The strain SCSIO N160 was isolated from a sediment sample (E 116°17.754′, N22°41.083′) at the depth of 30 m from the South China Sea by incubation at 28 °C for two weeks on Gauze’s Medium No. 1 (soluble starch 2%, KNO3 0.1%, K2HPO4 0.05%, MgSO4·7H2O 0.05%, FeSO4·7H2O 0.01%, pH 7.4) and was deposited in the type culture collection of the Center for Marine Microbiology of the South China Sea Institute of Oceanology. The strain was identified as a Micromonospora rosaria species by the 16S rRNA gene sequence analysis (GenBank accession number JF508525). Fermentation, Extraction, and Isolation. The strain SCSIO N160 was grown and maintained on the agar slant containing yeast extract 0.4%, glucose 0.4%, malt extract 0.5%, and agar 1.5−1.8% (adjusted to pH 7.2−7.4 before sterilization). A few loops of cells of strain SCSIO N160 were inoculated into 50 mL of seed medium consisting of starch 1%, glucose 2%, yeast extract 1%, corn powder 0.3%, beef extract 0.3%, MgSO4·7H2O 0.05%, K2HPO4 0.05%, CaCO3 0.2%, and sea salt 3% (adjusted to pH 7.2−7.4 before sterilization) in a 250 mL Erlenmeyer flask. The fermentation was carried out on a rotary shaker (200 rpm) at 28 °C for 7 days. A 40 mL portion of the seed cultures was transferred to a 2 L Erlenmeyer flask containing 400 mL of the production medium (the same as the seed medium), and the fermentation was carried out on a rotary shaker (200 rpm) at 28 °C for 4 days. A 20 mL (5 vol %) portion of the sterilized polystyrene resin (Amberlite XAD-16) was added into the production medium (400 mL/2 L), and the fermentation was prolonged for another day. The polystyrene resin was separated by filtration through a metal sieve (40 mesh). The resin was then washed twice with H2O and transferred to a glass column. The glass column was eluted with 2 L of acetone, and the acetone fractions were concentrated under vacuum to afford an aqueous residue, which was further extracted four times with 1.5 L of butanone. The organic extracts were then concentrated under vacuum to afford the crude extracts. The crude extracts were dissolved in 50 mL of CHCl3/MeOH (8:2 v/v) and were subjected to a silica gel column (45 × 2.6 cm, silica gel 300−400 mesh). The silica gel column was eluted with a gradient of CHCl3/MeOH (100:0 → 0:100) to give four fractions (Fr.1−Fr.4), which were concentrated under vacuum to yield 300, 124, 195, and 144 mg of crude materials, respectively. Fr.1 was further purified sequentially by MPLC and semipreparative HPLC

to get compounds 2 (16.2 mg), 4 (10.0 mg), and 8 (3.2 mg). Fr.2 was further purified by semipreparative HPLC to get compounds 3 (3.2 mg) and 6 (5.0 mg). Fr.3 was further purified by silica gel and preparative TLC to obtain compound 5 (22.0 mg). Fr.4 were further purified by SephadexLH-20 and semipreparative HPLC to get compounds 1 (2.0 mg), 7 (3.0 mg), and 9 (2.5 mg). The semipreparative HPLC was performed on an HPLC (Hitachi-L2130, diode array detector, Hitachi L-2455, Tokyo, Japan) using a Phenomenex ODS column (250 mm × 10.0 mm i.d., 5 μm; Phenomenex, USA). X-ray Crystallographic Analysis of Fluostatin D (5). A light brown crystal of 5 was obtained in MeOH. The crystal data were recorded on an Oxford Diffraction Gemini R Ultra system at the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, with Cu Kα radiation (λ = 1.54184 Å) was used. The structures were solved by direct methods (SHELXS-97) and refined using full-matrix least-squares difference Fourier techniques. Crystallographic data have been deposited in the Cambridge Crystallographic Data Center with the deposition number CCDC 905071 (5). A copy of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB21EZ, U.K. (fax: +44(0)-1233-336033; e-mail: [email protected]). Quantum Chemical ECD Calculation. The quantum chemical ECD calculation methods were used to support or establish the absolute configurations of 1−3 and 5.33−35 The compounds were charged using the Gasteiger-Hückel method and minimized by the SYBYL 8.0 software package using the TRIPOS force field, followed by a random conformational search using the MMFF94 molecular mechanics force field. The geometry optimizations were performed by using DFT at the B3LYP/6-31G(d) level as implemented in the Gaussian 09 program package.36 The stable conformers obtained were subsequently submitted to ECD calculations by time-dependent DFT calculations (B3LYP/6-31G(d)) with Gaussian 09. Fluostatin I (1): red powder; [α]20D +34.8 (c 2.5, MeOH); UV (MeOH) λmax (log ε) 263 nm (4.16), 207 nm (4.46); ECD (c 2.5 × 10−5 M, MeOH) λmax (Δε) 217 (−10.62), 322 (5.07), and 396 (−2.15) nm; IR (KBr) νmax 3435, 1701, 1384 cm−1; 1H NMR (500 MHz, methanol-d4) and 13C NMR (125 MHz, methanol-d4) data, see Table 1; ESIMS m/z 395 [M − H]−; HRESIMS m/z 395.1162 [M − H]− (calcd for C22H20O7, 395.1131). Fluostatin J (2): red powder; [α]20D −10.7 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 249 nm (3.60), 206 nm (4.14); ECD (c 4.6 × 10−5 M, MeOH) λmax (Δε) 220 (−5.31), 270 (3.24), and 397 (−1.25) nm; IR (KBr) νmax 3396, 1702, 1687, 1075 cm−1; 1H NMR (500 MHz, CDCl3/methanol-d4) and 13C NMR (125 MHz, CDCl3/methanol-d4) data, see Table 1; ESIMS m/z 407 [M − H]−, 815 [2 M − H]−; HRESIMS m/z 407.1136 [M − H]− (calcd for C23H20O7, 407.1131). Fluostatin K (3): red powder; [α]20D +16.2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 266 nm (4.20), 209 nm (4.69); ECD (c 3.6 × 10−5 M, MeOH) λmax (Δε) 208 (−8.35), 336 (10.47), and 393 (−3.16) nm; IR (KBr) νmax 3345, 1647, 1687, 1021 cm−1; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data, see Table 1; ESIMS m/z 307 [M − H]−; HRESIMS m/z 307.0641 [M − H]− (calcd for C18H12O5, 307.0606). 1941

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Biological Assays. Antimicrobial activities were measured against five indicator strains, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Bacillus thuringensis SCSIO BT01, Bacillus subtilis SCSIO BS01, and Candida albicans ATCC 10231, by the broth microdilution method.37 E. coli, Staph. aureus, B. thuringensis, and B. subtilis were grown for 16 h on a rotary shaker at 37 °C, while C. albicans was grown at 28 °C. Cultures were diluted with sterile medium to achieve an optical absorbance of 0.04−0.06 at 600 nm, then further diluted 10-fold before distributing into 96-well microtiter plates. Two replicates of each compound were tested in dilution series ranging from 64 to 0.125 μg/mL. The optical absorbance at 600 nm was measured after 18 h cultivation. The lowest concentrations that completely inhibited visible growth of the tested strains were recorded from two independent experiments. In vitro cytotoxic activities were evaluated against three tumor cell lines, MCF-7 (human breast adenocarcinoma cell line), NCI-H460 (human non-small-cell lung cancer cell line), and SF-268 (human glioma cell line), by the SRB assays according to a previously described protocol.38

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ASSOCIATED CONTENT

S Supporting Information *

1

H, 13C, and 2D NMR spectra of 1−3, NMR spectroscopic data for compounds 4−9. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86 20 89023038. E-mail: [email protected]. Author Contributions §

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported in part by the Chinese Academy of Sciences for Key Topics in Innovation Engineering (KZCX2YW-JC202, KSCX2-EW-G-12), the Ministry of Science and Technology of China (2012AA092104, 2010CB833805), National Science Foundation of China (41006089, 41106143, 31125001), and Natural Science Funds of South China Sea Institute of Oceanology for Young Scholar (SQ200903). C.Z. is a scholar of the “100 Talents Project” of the Chinese Academy of Sciences (08SL111002). We are grateful to analytical facilities at the South China Sea Institute of Oceanology.

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