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Jun 3, 2015 - the Marine-Derived Fungus Penicillium adametzioides AS-53 on ... Key Laboratory of Experimental Marine Biology, Institute of Oceanology,...
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Bisthiodiketopiperazines and Acorane Sesquiterpenes Produced by the Marine-Derived Fungus Penicillium adametzioides AS-53 on Different Culture Media Yang Liu,†,‡ Xiao-Ming Li,† Ling-Hong Meng,†,‡ Wen-Li Jiang,§ Gang-Ming Xu,† Cai-Guo Huang,*,§ and Bin-Gui Wang*,† †

Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Nanhai Road 7, Qingdao 266071, People’s Republic of China ‡ University of Chinese Academy of Sciences, Yuquan Road 19A, Beijing 100049, People’s Republic of China § Department of Biochemistry and Molecular Biology, Second Military Medical University, Xiangyin Road 800, Shanghai 200433, People’s Republic of China S Supporting Information *

ABSTRACT: Chemical investigation of the marine-spongederived fungus Penicillium adametzioides AS-53 resulted in the identification of two new bisthiodiketopiperazine derivatives, adametizines A (1) and B (2), from cultivation in a liquid potato−dextrose broth (PDB) culture medium, whereas two new acorane sesquiterpenes, adametacorenols A (3) and B (4), were isolated from a rice solid culture medium. The structures of these compounds were elucidated on the basis of spectroscopic analysis. The absolute configuration of compound 1 was determined by X-ray crystallographic analysis, and that of 3 was determined by modified Mosher’s method. Compound 1 exhibited lethality against brine shrimp (Artemia salina) with an LD50 value of 4.8 μM and inhibitory activities against Staphyloccocus aureus, Aeromonas hydrophilia, Vibrio spp. V. harveyi and V. parahaemolyticus, and Gaeumannomyces graminis with minimum inhibitory concentration values of 8, 8, 32, 8, and 16 μg/ mL, respectively. Chlorination at C-7 significantly increased the brine shrimp lethality and antimicrobial activity of the bisthiodiketopiperazines. arine filamentous fungi are recognized as an important source of structurally unique and biologically active natural products.1 Recently, the study of whole genome sequences of microorganisms has demonstrated the presence of cryptic biosynthetic pathways, which are not always expressed under standard fermentation conditions.2,3 As part of our ongoing efforts to discover bioactive metabolites from marine-derived fungi,4−8 a fungal strain, Penicillium adametzioides AS-53, which was isolated from an unidentified sponge collected at Hainan Island in the South China Sea, attracted our attention. Primary investigation of this fungus has resulted in the isolation of a new spiroquinazoline derivative, Nformyllapatin A, from the organic extract of the liquid potato−dextrose broth (PDB) culture medium.8 Further work on this strain indicated that alteration of fermentation media affected its chemical profiles (Figure S31). As a result, two new bisthiodiketopiperazine derivatives, adametizines A (1) and B (2), along with one known bisthiodiketopiperazine derivative and seven known meroterpenes, were identified from the PDB culture medium. In contrast, two new acorane sesquiterpenes, adametacorenols A (3) and B (4), together with dimethyl-N-(phenylacetyl)-glutamate, were characterized from the rice solid culture medium. This paper describes the

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© XXXX American Chemical Society and American Society of Pharmacognosy

isolation, structure elucidation, and biological activity of the new compounds 1−4.

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RESULTS AND DISCUSSION

Compound 1 was initially obtained as colorless needles. The molecular formula C21H23ClN2O8S2, implying 11 degrees of Received: February 6, 2015

A

DOI: 10.1021/acs.jnatprod.5b00102 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data for Compounds 1 and 2 in DMSO-d6 1 position

δC, type

1 2 3 4 5

165.3, C 65.4, CH 163.8, C 69.0, C 33.5, CH2

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 N-CH3 6-OH 7-OH 10-OH 14-OH

70.1, C 67.0, CH 126.4, CH 131.2, CH 64.2, CH 85.4, CH 41.2, CH 116.3, C 147.6, C 135.8, C 152.9, C 103.2, CH 122.8, CH 60.2, CH3 55.7, CH3 32.6, CH3

2 δH (J in Hz)

δC, type

4.62, br s

α 2.07, d (15.5) β 2.27, d (15.5) 4.87, 5.57, 5.62, 4.34, 4.05, 4.62,

br s d (10.4) d (10.4) br d (7.1) d (7.1) br s

6.55, 7.34, 3.68, 3.79, 3.00,

d (8.8) d (8.8) s s s

9.46, s

unsaturation, was established by HRESIMS. The 13C NMR and DEPT spectroscopic data (Table 1) exhibited the presence of 21 carbon signals containing three methyls (with one nitrogenated and two oxygenated), one sp3 methylene, nine methines (with four sp2 and five sp3), and eight nonprotonated (with six sp2 and two sp3) carbon atoms. In examining the 1H NMR and HSQC data, an exchangeable proton at δH 9.46 (14OH), two aromatic methine protons attributed to a 1,2,3,4tetrasubstituted phenyl unit at δH 6.55 (d, J = 8.8 Hz, H-17) and 7.34 (d, J = 8.8 Hz, H-18), and two olefinic protons derived from a cis-coupled double bond at δH 5.57 (d, J = 10.4 Hz, H-8) and 5.62 (d, J = 10.4 Hz, H-9) were observed in the deshielded region of the spectrum, whereas shielded signals for five methines (H-2, H-7, and H-10−H-12) and one methylene (H25) were also present. In addition, one N-methyl (H3-21) and two O-methyl groups (H3-19 and H3-20) were also observed in the 1H NMR spectrum of 1. Detailed analysis of the above NMR data as well as 2D NMR correlations indicated that the structure of compound 1 was similar to that of FA-2097 (Nmethylgliovirin), a bisthiodiketopiperazine derivative obtained from the culture broth of Eupenicillium abidjanum IFO 8939.9,10 However, signals for the epoxy nonprotonated carbon (C-6) and methine (CH-7) resonating at δC 57.7 (C-6) and δH 3.13/ δC 52.4 (CH-7) in N-methylgliovirin10 were deshielded at δC 70.1 (C-6) and δH 4.87/δC 67.0 (CH-7), respectively, in the NMR spectra of compound 1 (Table 1). The above evidence along with the molecular formula suggested that 1 was the 6hydroxy-7-chloro derivative of N-methylgliovirin.9,10 Detailed analysis of the key HMBC and COSY correlations (Figure 1 and Table S2) resulted in the elucidation of the planar structure of 1.

165.6, C 65.5, CH 164.0, C 69.8, C 32.3, CH2 70.8, C 74.0, CH 129.6, CH 128.6, CH 64.6, CH 85.4, CH 41.0, CH 116.4, C 147.6, C 135.8, C 152.9, C 103.2, CH 122.8, CH 60.2, CH3 55.7, CH3 32.5, CH3

δH (J in Hz) 4.59, br s

α 1.98, d (15.9) β 2.19, d (15.9) 4.18, 5.42, 5.48, 4.22, 3.93, 4.59,

br s d (10.4) d (10.4) br d (7.1) d (7.1) br s

6.55, 7.35, 3.68, 3.79, 2.99, 5.16, 5.29, 5.16, 9.44,

d (8.8) d (8.8) s s s br s d (5.0) d (6.4) br s

Figure 1. Key HMBC (arrows) and COSY (bold lines) correlations of compounds 1−4.

The coupling constant between H-10 and H-11 (7.1 Hz) supports the idea that these two protons are in an axial relationship, whereas the observed NOE correlation (Figure S6) between H-7 and H-11 indicated the two protons are on the same side of the molecule. However, the relative configurations at C-2, C-4, C-6, and C-12 for compound 1 B

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could not be unambiguously solved because of a lack of diagnostic J-coupling constants or NOE correlations. Upon slow evaporation of the solvent MeOH, which was achieved by storing the sample in a refrigerator for 3 weeks, single crystals of adequate quality of 1 were obtained, making an X-ray diffraction study possible that could unequivocally confirm the chemical structure of 1. The absolute configuration was determined on the basis of measuring the anomalous dispersion effects by collecting Friedel pair reflections in the X-ray diffraction experiment (Figure 2). The presence of heavy sulfur

Figure 3. ECD spectra of compounds 1 and 2.

correlations from the terminal protons H2-12 to C-1 and C13 as well as from H-1 and H3-13 to C-12 indicated the presence of the isopropenyl group on C-1, whereas the HMBC correlations from H3-15 to C-7, C-8, and C-9, from H2-6 and H2-10 to C-8, and from H-9 to C-5 and C-7 revealed the presence of a trisubstituted double bond at C-8. The COSY data and other HMBC correlations fully supported the structure assignment of compound 3 (Figure 1 and Table S3). The relative configuration was established by analysis of NOESY data. The observed NOE effects from H3-14 to H-1, H-6β, and H-7 established the relative configurations at C-1, C4, C-5, and C-7, whereas NOE correlations from H-2 to H-4 determined the relative configuration at C-2 and C-4 (Figure 4). To unequivocally confirm the structure and absolute configuration by X-ray crystallographic analysis, compound 3 was esterified with p-bromobenzoyl chloride,13 but unfortunately, attempts to get quality crystals of the p-bromobenzoyl ester of 3 failed. The absolute configuration of compound 3 was then determined by the modified Mosher’s method, which proved the S configuration for C-7 (Figure 5). Consequently, the absolute configurations of compound 3 were deduced to be 1R, 2R, 4S, 5S, and 7S. Compound 3 was named adametacorenol A. Compound 4 was isolated as a colorless oil. The HRESIMS data of 4 determined the molecular formula C17H26O4, with one oxygen atom more than 3. The NMR spectroscopic data (Table 2) were consistent with the presence of one more hydroxy group in 4, and the assignments of the 1H and 13C NMR chemical shifts of 4 matched well with those of the corresponding signals for 3. However, the methyl group resonating at δC 19.2/δH 1.64 (CH3-15) in 3 was replaced by a hydroxymethyl unit resonating at δC 66.2/δH 4.19 (CH2−15) in 4, suggesting that the methyl group CH3-15 in 3 was replaced by a hydroxymethyl group in 4. This deduction was supported by the observed HMBC correlations from H2-15 to C-7, C-8, and C-9 (Figure 1 and Table S3). The relative configuration of 4 was determined to be same as that of 3 as shown in Figure 4 on the basis of a NOESY experiment. From a biosynthetic point of view, the absolute configuration of 4 was assumed to be the same as that of 3. The trivial name adametacorenol B was assigned to 4. In addition to the new compounds 1−4, one known bisthiodiketopiperazine derivative, gliovictin,14 and seven known meroterpenes including austinol,15 austinolide,16 dehydroaustinol,15 dehydroaustin,15 acetoxydehydroaustin,15 neoaustin,15 and isoaustinone16 were also isolated from the PDB culture medium, whereas dimethyl-N-(phenylacetyl)glutamate17 was characterized from the rice solid medium.

Figure 2. X-ray crystallographic structure of compound 1, with two inclusive H2O molecules.

atoms and a chlorine atom in 1 had a measurable anomalous dispersion effect. The Flack parameter11 was 0.02(9) in the final refinement, which allowed the assignment of the absolute configuration of 1 as 2S, 4R, 6R, 7R, 10R, 11S, and 12S. The trivial name adametizine A was assigned to compound 1. Compound 2, a colorless solid, was assigned the molecular formula C21H24N2O9S2 on the basis of HRESIMS data, implying the replacement of the Cl atom in 1 by an OH unit in 2. The 1D NMR data of 2 (Table 1) exhibited signals similar to those presented in compound 1. Detailed comparison of the NMR data of 2 with those of 1 indicated that signals for the chloromethine at δC 67.0 (C-7)/δH 4.87 (H-7) in the NMR spectra of 1 were replaced by an oxymethine unit at δC 74.0 (C7)/δH 4.18 (H-7) in 2, indicating that the Cl atom at C-7 of 1 was replaced by an OH group in 2. The 2D NMR correlations supported this deduction (Figure 1 and Table S2). The coupling constant between H-10 and H-11 (7.1 Hz) supports an axial relationship for these protons, whereas the observed NOE correlation (Figure S13) between H-7 and H-11 indicated that the proton pair is on the same side of the molecule. From a biosynthetic viewpoint, compounds 1 and 2 should possess the same relative configurations. As might be expected, compounds 1 and 2 showed similar ECD spectra, with a very weak, positive Cotton effect (CE) at approximately 302 nm and two strong, negative CEs at approximately 260 and 216 nm (Figure 3). The absolute configuration of 2 was thus assigned as 2S, 4R, 6S, 7R, 10R, 11S, and 12S, and 2 was named adametizine B. Compound 3, a colorless oil, was found to have the molecular formula C17H26O3 on the basis of HRESIMS data. Its 13 C NMR data (Table 2) displayed the presence of 17 carbon signals, which were assigned by DEPT and HSQC experiments as four methyls, one terminal and three aliphatic methylenes, five methines (with one olefinic and two oxygenated), and four nonprotonated (with one aliphatic, two olefinic, and one carbonyl) carbon atoms. Detailed analysis of the 2D NMR correlations showed that compound 3 is an acorane-type sesquiterpenoid derivative, which bears a spiro-fused cyclopentane-cyclohexene ring system.12 The observed HMBC C

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Table 2. 1H (500 MHz) and 13C (125 MHz) NMR Data for Compounds 3 and 4 3a

a

4b

Position

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

1 2 3

56.8, CH 76.6, CH 37.8, CH2

57.6, CH 77.6, CH 38.60, CH2

4 5 6

37.2, CH 46.9, C 38.5, CH2

2.69, d (8.3) 5.14, td (8.3, 3.6) α 2.45, dt (15.0, 7.7) β 1.13, d (15.0) 1.88, m

2.76, d (8.4) 5.20, td (8.4, 4.0) α 2.50, dt (15.0, 8.7) β 1.18, dt (15.0, 5.5) 1.92, overlapped

7 8 9 10

66.4, CH 136.6, C 120.9, CH 31.0, CH2

11 12

142.3, C 113.9, CH2

13 14 15 16 17

23.9, CH3 17.6, CH3 19.2, CH3 170.1, C 20.9, CH3

38.1, CH 47.2, C 38.61, CH2

α 1.50, dd (12.6, 9.4) β 1.76, dd (12.6, 5.4) 3.96, br s

67.9, CH 138.1, C 126.3, CH 31.6, CH2

5.25, br s α 1.59, br d (16.8) β 1.83, br d (16.8)

142.4, C 114.7, CH2

Z 4.68, br s E 4.96, br s 1.73, s 0.98, d (7.1) 1.64, s

24.6, CH3 17.8, CH3 66.2, CH2 171.2, C 21.4, CH3

1.95, s

α 1.68, m β 1.92, overlapped 4.50, br s 5.63, br s α 1.66, br d (15.1) β 2.06, br d (15.1) Z 4.71, br s E 4.98, br s 1.76, s 1.02, d (7.1) 4.19, m 1.99, s

Measured in DMSO-d6. bMeasured in CDCl3.

Figure 5. Values of ΔδH(S−R) (measured in DMSO-d6) of the MTPA esters of compound 3.

human-pathogenic bacteria (Escherichia coli and Staphyloccocus aureus) and seven aquacultural bacteria (Aeromonas hydrophilia, Edwardsiella tarda, Micrococcus luteus, Vibrio spp. V. alginolyticus, V. anguillarum, V. harveyi, and V. parahaemolyticus) as well as four plant-pathogenic fungi (Alternaria brassicae, Colletotrichum gloeosprioides, Fusarium graminearum, and Gaeumannomyces graminis), compound 1 was found to be active against S. aureus, A. hydrophilia, V. harveyi, V. parahaemolyticus, and G. graminis with minimum inhibitory concentration (MIC) values of 8, 8, 32, 8, and 16 μg/mL, respectively, whereas compound 2 only showed activity against S. aureus with an MIC value of 64 μg/mL. These data indicated that the Cl substitution at C-7 significantly increased the brine shrimp lethality and antimicrobial activity. Compounds 1−4 were also examined for cytotoxicity against 14 tumor cell lines (A549, DU145, HeLa, HepG2, Huh7, L02, LM3, MA, MCF-7, NCI-H446, SGC-7901, SW1990, SW480, and U251). None of them showed significant activity (IC50 > 10 μM) except compound 4, which showed selective activity against the NCI-H446 cell line (IC50 = 5.0 μM).

Figure 4. Key NOESY (arrows) correlations of compounds 3 and 4.

The structures of these known compounds were identified by comparison of their spectroscopic and physical data (1H and 13 C NMR, MS, and [α]D) with those reported in the literature. The new compounds 1−4 were evaluated for brine shrimp lethality, antimicrobial potency, and cytotoxic activity. Compound 1 exhibited brine shrimp lethality with an LD50 value of 4.8 μM, which was stronger than that of the positive control colchicine (LD50 = 8.1 μM). In the evaluation against two

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined with an SGW X-4 micromelting-point apparatus. Optical rotations were measured on an Optical Activity AA-55 polarimeter. D

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1030, 961, 820 cm−1; 1H and 13C NMR data, Table 1; ESIMS m/z 513 [M + H]+; HRESIMS m/z 513.0987 [M + H]+ (calcd for C21H25N2O9S2, 513.0996). 27 Gliovictin: colorless solid; [α]25 D −27.9 (c 0.86, MeOH); Lit. [α]D −47.0 (c 0.13, MeOH).14 Fermentation, Extraction, and Isolation of New Compounds from Rice Culture Medium. The rice culture fermentation growth of the fungus was accomplished by incubation in a 1 L Erlenmeyer flasks containing rice medium (rice, 70 g; corn steep powder, 0.1 g; peptone, 0.3 g; and natural seawater, 100 mL; 30 flasks) at rt for 30 days. The whole rice culture of the fungal strain was extracted three times with EtOAc, and the extract (26 g) was fractionated by VLC on silica gel using different solvents of increasing polarity from petroleum ether (PE) to MeOH to yield eight fractions (Frs. 1−8) on the basis of TLC analysis. Fr. 3 (1.5 g) was further purified by CC on silica gel eluting with CHCl3/acetone (from 100:1 to 3:1) to afford six subfractions. Fr. 3-3 (120 mg) was purified by CC on Sephadex LH-20 (MeOH) to afford 3 (21.8 mg). Fr. 5 (4.7 g) was further purified by CC on Lobar LiChroprep RP-18 from MeOH/H2O (1:9 to 1:0) to yield 10 subfractions. Fr. 5-5 (MeOH/H2O 1:1, 48 mg) was further fractionated by CC on Sephadex LH-20 (MeOH) and then by CC on silica gel eluting with CHCl3/MeOH (from 80:1 to 50:1) to give compound 4 (13.2 mg). Adametacorenol A (3): colorless oil; [α]25 D −2.17 (c 0.46, MeOH); IR vmax 3413, 2938, 1734, 1447, 1372, 1242, 1028, 892 cm−1; 1H and 13 C NMR data, Table 2; ESIMS m/z 301 [M + Na]+; HRESIMS m/z 279.1959 [M + H]+ (calcd for C17H27O3, 279.1955). Adametacorenol B (4): colorless oil; [α]25 D − 6.67 (c 0.15, MeOH); IR vmax 3399, 2928, 1731, 1449, 1374, 1242, 1026, 892 cm−1; 1H and 13 C NMR data, Table 2; ESIMS m/z 295 [M + H]+; HRESIMS m/z 295.1910 [M + H]+ (calcd for C17H27O4, 295.1904). X-ray Crystallographic Analysis. All crystallographic data were collected on a Bruker Smart-1000 CCD diffractometer equipped with a graphite-monochromatic Mo Kα radiation (λ = 0.71073 Å) at 298(2) K. The data were corrected for absorption by using the program SADABS.19 The structure was solved by direct methods with the SHELXL-97 software package.20 All non-hydrogen atoms were refined anisotropically. The H atoms were located by geometrical calculations, and their positions and thermal parameters were fixed during the structure refinement. The structure was refined by fullmatrix least-squares techniques.21 Crystallographic data of compound 1 have been deposited in the Cambridge Crystallographic Data Centre as CCDC 1040973. The data can be obtained free of charge via http://www.ccdc.cam.ac.uk/data_request/cif (or from the CCDC, 12 Union Road, Cambridge CB21EZ, United Kingdom; fax: +44-1223336-033; e-mail: [email protected]). Crystal Data for Compound 1: C21H27ClN2O10S2; fw = 567.02; orthorhombic space group P2(1)2(1)2(1); unit cell dimensions a = 12.1222(9) Å, b = 12.1222(11) Å, c = 16.3859(16) Å, V = 2407.9(4) Å3, α = β = γ = 90°, Z = 4, dcalcd = 1.564 mg/m3; crystal dimensions = 0.31 × 0.18 × 0.11 mm3; μ = 0.393 mm−1; F(000) = 1184. The total number of reflections collected was 12 153, which yielded 4261 independent reflections after equivalent data were averaged and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.0431 and wR2 = 0.0820 (I > 2σ(I)). Preparation of the (R)- and (S)-MTPA Ester Derivatives of Compounds 3.22 To a solution of 3 (2.0 mg) in pyridine (400 μL) was added 4-(dimethylamino)pyridine (4.0 mg) and (S)-(+)-αmethoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl, 10 μL). The reaction took place at rt for 20 h and was stopped by adding three drops of distilled H2O. The reaction mixture was then purified by preparative TLC (PE/EtOAc 15:1) to give the respective (R)-Mosher ester 3r. Treatment of 3 (2.0 mg) with (R)-MTPA-Cl (10 μL) as described above yielded the corresponding (S)-Mosher ester 3s. Brine Shrimp Assay. Evaluation of the isolated compounds for brine shrimp (A. salina) lethality was determined as described previously.4 Colchicine was used as a positive control. Antimicrobial Assays. The antimicrobial activities of the new compounds 1−4 against two human-pathogenic bacteria (E. coli

UV spectra were measured on a PuXi TU-1810 UV−visible spectrophotometer. ECD spectra were acquired on a Chirascan spectropolarimeter. IR spectra were obtained on a Thermo Scientific Nicolet iS10 spectrophotometer. 1D and 2D NMR spectra were recorded at 500 and 125 MHz for 1H and 13C, respectively, on a Bruker Avance 500 MHz spectrometer with TMS as an internal standard. Mass spectra were determined on a VG Autospec 3000 or an API QSTAR Pulsar 1 mass spectrometer. Analytical and semipreparative HPLC were performed using a Dionex HPLC system equipped with a P680 pump, an ASI-100 automated sample injector, and a UVD340U multiple wavelength detector controlled by Chromeleon software (version 6.80). Commercially available Si gel (200−300 mesh, Qingdao Haiyang Chemical Co.), Lobar LiChroprep RP-18 (40−63 μm, Merck), and Sephadex LH-20 (Pharmacia) were used for open-column chromatography. All solvents used were distilled prior to use. Fungal Material. The fungus P. adametzioides AS-53 was isolated from the fresh tissue of an unidentified marine sponge that was collected from Hainan Island of China in September 2011. Fungal identification was performed on the basis of sequencing of the ITS regions.18 The sequence data of 5.8S rDNA and ITS regions derived from the fungal strain have been submitted to and deposited at GenBank with accession no. KJ906543. The nucleotide BLAST search result showed that the sequence was the most similar (99%) to the sequence of P. adametzioides strain DTO115I8 (compared to accession no. KC773824.1). The strain is preserved at the Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences (IOCAS). Fermentation, Extraction, and Isolation of New Compounds from PDB Culture Medium. Mass growth of the fungus was accomplished by incubation in 1 L Erlenmeyer flasks containing 300 mL of the PDB medium (4% potato powder, 2% mannitol, 2% sucrose, 0.5% peptone, 0.3% yeast extract, and naturally sourced and filtered seawater from the Huiquan gulf of the Yellow Sea near the campus of IOCAS, pH 6.5−7.0, 300 mL/flask, 60 flasks) at room temperature (rt) for 30 days. The whole fermented cultures (18 L) were filtered to separate the broth from the mycelia. The former was extracted three times with EtOAc, whereas the latter was extracted three times with a mixture of 80% acetone and 20% H2O. The acetone solution was evaporated under reduced pressure to afford an aqueous solution, which was then extracted three times with EtOAc. Because the TLC and HPLC profiles of the two EtOAc solutions were almost identical, they were combined and concentrated under reduced pressure to give an extract (37 g), which was fractionated by silica-gel vacuum liquid chromatography (VLC) using different solvents of increasing polarity from petroleum ether (PE) to MeOH to yield eight fractions (Frs. 1−8) on the basis of TLC analysis. Fr. 6 (5.6 g) was further purified by CC on Lobar LiChroprep RP-18 eluting with MeOH/H2O (1:9−1:0) to yield 10 subfractions. Fr. 6-4 (MeOH/H2O 2:3, 850 mg) was further fractionated by CC on silica gel eluting with CHCl3/MeOH (from 100:1 to 10:1) to afford four subfractions. Fr. 64-3 (140 mg) was purified by semipreparative HPLC (Elite ODS-BP column, 10 μm; 20 × 250 mm2; 45% MeOH/H2O, 16 mL/min) to afford compound 2 (16.8 mg, tR = 24.223 min). Fr. 6-5 (MeOH/H2O 1:1, 380 mg) was further fractionated by CC on silica gel eluting with CHCl3/MeOH (from 100:1 to 10:1) to afford five subfractions. Fr. 65-5 (75 mg) was purified by preparative TLC (CHCl3/acetone, 1:1) to give compound 1 (32.3 mg, Rf = 0.5). For the isolation of known compounds, please refer to the Supporting Information. Adametizine A (1): colorless needles; mp 346−348 °C; [α]25 D −171 (c 0.17, MeOH); UV (MeOH) λmax (log ε) 205 (4.80), 336 (2.84) nm; ECD (0.24 mM, MeOH) λmax (Δε) 216 (−31.25), 261 (−6.29), 303 (+0.52) nm; IR vmax 3403, 2942, 2835, 1686, 1613, 1506, 1468, 1360, 1278, 1098, 1034, 951, 827 cm−1; 1H and 13C NMR data, Table 1; ESIMS m/z 533 and 531 [M + H]+; HRESIMS m/z 531.0648 [M + H]+ (calcd for C21H2435ClN2O8S2, 531.0657). Adametizine B (2): colorless solid; [α]25 D −75.0 (c 0.56, MeOH); UV (MeOH) λmax (log ε) 202 (4.52), 332 (3.64) nm; ECD (0.23 mM, MeOH) λmax (Δε) 216 (−17.22), 259 (−4.15), 302 (+0.65) nm; IR vmax 3326, 2917, 2849, 1673, 1607, 1504, 1462, 1336, 1274, 1092, E

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Journal of Natural Products

Article

EMBLC-1 and S. aureus EMBLC-2, from the Key Laboratory of the Experimental Marine Biology (KLEMB) at the IOCAS) and seven aquacultural bacteria (A. hydrophilia QDIO-1, E. tarda QDIO-2, M. luteus QDIO-3, V. alginolyticus QDIO-5, V. anguillarum QDIO-6, V. harveyi QDIO-7, and V. parahemolyticus QDIO-8, from the KLEMB at the IOCAS) as well as four plant-pathogenic fungi (A. brassicae QDAU-1, C. gloeosprioides QDAU-2, G. graminis QDAU-3, and F. graminearum QDAU-4, from Qingdao Agricultural University) were evaluated using the methods described previously.23 Chloramphenicol and amphotericin B were used as positive controls against bacteria and fungi, respectively. Cytotoxicity Assays. The cytotoxic activities of the new compounds 1−4 against 14 tumor cell lines including A549 (human lung adenocarcinoma cell), Du145 (human prostate carcinoma cell line), HeLa (human cervical carcinoma cell line), HepG2 (human liver hepatocellular cells), Huh7 (human hepatocarcinoma cell line), L02 (human hepatic L02 cells), LM3 (murine LM3 breast cells), MA (mouse Leydig tumor cell line), MCF-7 (human breast carcinoma cell line), NCI-H446 (human small-cell lung carcinoma cell line), SGC7901 (human gastric carcinoma cell line), SW1990 (human pancreatic cancer cell line), SW480 (human colon carcinoma cancer), and U251 (human glioma cells) were determined according to previously reported methods.24,25

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(7) Meng, L. H.; Li, X. M.; Liu, Y.; Wang, B. G. Org. Lett. 2014, 16, 6052−6055. (8) Liu, Y.; Li, X. M.; Meng, L. H.; Wang, B. G. Phytochem. Lett. 2014, 10, 145−148. (9) Miyamoto, C.; Yokose, K.; Furumai, T.; Maruyama, H. B. J. Antibiot. 1982, 35, 374−377. (10) Yokose, K.; Nakayama, N.; Miyamoto, C.; Furumai, T.; Maruyama, H. B.; Stipanovic, R. D.; Howell, C. R. J. Antibiot. 1984, 37, 667−669. (11) Flack, H. D. Acta Crystallogr. 1983, A39, 876−881. (12) Li, G. H.; Yang, Z. S.; Zhao, P. J.; Zheng, X.; Luo, S. L.; Sun, R.; Niu, X. M.; Zhang, K. Q. Phytochem. Lett. 2011, 4, 86−88. (13) An, C. Y.; Li, X. M.; Li, C. S.; Gao, S. S.; Shang, Z.; Wang, B. G. Helv. Chim. Acta 2013, 96, 682−687. (14) Michel, K. H.; Chaney, M. O.; Jones, N. D.; Hoehn, M. M.; Nagarajan, R. J. Antibiot. 1974, 27, 57−64. (15) Hayashi, H.; Mukaihara, M.; Murao, S.; Arai, M.; Lee, A. Y.; Clardy, J. Biosci., Biotechnol., Biochem. 1994, 58, 334−338. (16) Fill, T. P.; Pereira, G. K.; Geris dos Santos, R. M.; Rodrigues-Fo, E. Z. Naturforsch. 2007, 62B, 1035−1044. (17) Heuring, V. P.; Scott, K. N. J. Magn. Reson. 1970, 2, 302−304. (18) Wang, S.; Li, X. M.; Teuscher, F.; Li, D. L.; Diesel, A.; Ebel, R.; Proksch, P.; Wang, B. G. J. Nat. Prod. 2006, 69, 1622−1625. (19) Sheldrick, G. M. SADABS, Software for Empirical Absorption Correction; University of Göttingen: Germany, 1996. (20) Sheldrick, G. M. SHELXTL, Structure Determination Software Programs; Bruker Analytical X-ray System, Inc.: Madison, WI, 1997. (21) Sheldrick, G. M. SHELXL-97 and SHELXS-97, Program for X-ray Crystal Structure Solution and Refinement; University of Göttingen: Germany, 1997. (22) Wang, B. G.; Ebel, R.; Wang, C. Y.; Wray, V.; Proksch, P. Tetrahedron Lett. 2002, 43, 5783−5787. (23) Pierce, C. G.; Uppuluri, P.; Tristan, A. R.; Wormley Jr, F. L., Jr.; Mowat, E.; Ramage, G.; Lopez-Ribot, J. L. Nat. Protoc. 2008, 3, 1494− 1500. (24) Bergeron, R. I.; Cavanaugh, P. F., Jr.; Kline, S. J.; Hughes, R. G., Jr.; Elliott, G. T.; Porter, C. W. Biochem. Biophys. Res. Commun. 1984, 121, 848−854. (25) Mosmann, T. J. Immunol. Methods. 1983, 65, 55−63.

ASSOCIATED CONTENT

S Supporting Information *

Detailed procedures for the isolation and purification of all new and known compounds, chemical structures of all isolated compounds, assigned NMR data for the p-bromobenzoyl ester of 3, HMBC data for the new compounds 1−4, specific rotations for the known compounds, 1D and 2D NMR spectra of compounds 1−4, HPLC profiles of compounds 1 and 2 and extracts from different media cultivation, and a packing diagram of compound 1 at 298(2) K. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00102.

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86-21-81870966. Fax: +86-21-65334344. E-mail: [email protected]. *Tel. and Fax: +86-532-82898553. E-mail: [email protected]. ac.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the NSFC-Shandong Joint Fund for Marine Science Research Centers (U1406402) and from the Ministry of Science and Technology of China (2010CB833802 and 2013AA092901) is gratefully acknowledged.

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REFERENCES

(1) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.; Prinsep, M. R. Nat. Prod. Rep. 2013, 30, 237−323. (2) Wang, W. J.; Li, D. Y.; Li, Y. C.; Hua, H. M.; Ma, E. L.; Li, Z. L. J. Nat. Prod. 2014, 77, 1367−1371. (3) Scheffler, R. J.; Colmer, S.; Tynan, H.; Demain, A. L.; Gullo, V. P. Appl. Microbiol. Biotechnol. 2013, 97, 969−978. (4) An, C. Y.; Li, X. M.; Luo, H.; Li, C. S.; Wang, M. H.; Xu, G. M.; Wang, B. G. J. Nat. Prod. 2013, 76, 1896−1901. (5) Meng, L. H.; Li, X. M.; Lv, C. T.; Li, C. S.; Xu, G. M.; Huang, C. G.; Wang, B. G. J. Nat. Prod. 2013, 76, 2145−2149. (6) Zhang, P.; Mándi, A.; Li, X. M.; Du, F. Y.; Wang, J. N.; Li, X.; Kurtán, T.; Wang, B. G. Org. Lett. 2014, 16, 4834−4837. F

DOI: 10.1021/acs.jnatprod.5b00102 J. Nat. Prod. XXXX, XXX, XXX−XXX