Antioxidant Hydroanthraquinones from the Marine Algal-Derived

Article pubs.acs.org/jnp

Antioxidant Hydroanthraquinones from the Marine Algal-Derived Endophytic Fungus Talaromyces islandicus EN-501 Hong-Lei Li,†,‡ Xiao-Ming Li,† Xin Li,†,‡ Chen-Yin Wang,§ Hui Liu,†,‡ Matthias U. Kassack,*,§ Ling-Hong Meng,*,† and Bin-Gui Wang*,† †

Laboratory of Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, 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 § Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany S Supporting Information *

ABSTRACT: Five new polyhydroxylated hydroanthraquinone derivatives, namely, 8-hydroxyconiothyrinone B (1), 8,11-dihydroxyconiothyrinone B (2), 4R,8-dihydroxyconiothyrinone B (3), 4S,8-dihydroxyconiothyrinone B (4), and 4S,8-dihydroxy-10-Omethyldendryol E (5), were isolated and identified from the culture extract of Talaromyces islandicus EN-501, an endophytic fungus obtained from the inner tissue of the marine red alga Laurencia okamurai. The structures of these compounds were established on the basis of detailed interpretation of their NMR and mass spectroscopic data, and the structures and absolute configurations of compounds 1 and 2 were confirmed by X-ray crystallographic analysis, while the absolute configurations of compounds 3−5 were determined by TDDFT calculations of the ECD spectra. The antimicrobial, antioxidant, and cytotoxic activities of compounds 1−5 were evaluated.

H

Compound 1 was obtained as colorless crystals, and its molecular formula was determined as C15H18O5 on the basis of HRESIMS data, implying seven degrees of unsaturation. The 1 H and 13C NMR data of 1 indicated the presence of one methyl, three methylenes, five methines (including one aromatic and two oxygenated), and six nonprotonated (including one ketone carbonyl and five aromatic) carbons, as well as four exchangeable protons (δH 4.71, 6.84, 9.70, and 12.04) (Tables 1 and 2). Detailed analysis of the 1H and 13C NMR spectroscopic data revealed that 1 is a hydroanthraquinone derivative similar to coniothyrinone B.9 However, the resonance for one of the two aromatic protons in the 1H NMR spectrum of coniothyrinone B was not present in that of 1. Instead, a signal for an additional hydroxy group was observed at δH 9.70 (s, OH-8) in the 1H NMR spectrum of 1. Additionally, the resonance of C-8, compared to that of coniothyrinone B, was significantly deshielded (+28.3 ppm), while C-7 and C-8a were shielded in the 13C NMR spectrum of 1. These spectroscopic features suggested that compound 1 was the 8-hydroxylated derivative of coniothyrinone B. The COSY and HMBC data (Figure 1) supported the above deduction. The planar structure of 1 was thus determined.

ydroanthraquione derivatives, which are mainly produced by endophytic fungi,1−9 have been reported to possess various biological effects such as antimalarial,3 antimicrobial,1,5,9 antiviral,4 cytotoxic,6,8 kinase inhibitory,2 and phytotoxic activities.7 In continuation of our efforts to identify new bioactive secondary metabolites from marine-derived fungi,10−14 we performed chemical investigations on the culture extract of Talaromyces islandicus EN-501, an endophytic fungus isolated from the fresh tissue of the marine red alga Laurencia okamurai. As a result, five new hydroanthraquinone derivatives (1−5) were characterized from the fungus. The structures and absolute configurations of compounds 1 and 2 were confirmed by single-crystal X-ray diffraction analysis, while the absolute configurations of compounds 3−5 were determined on the basis of TDDFT calculations of their ECD spectra. This paper describes the isolation, structure determination, stereochemical assignment, and bioactivities of the isolated compounds.

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RESULTS AND DISCUSSION The fermentation culture of T. islandicus EN-501 was exhaustively extracted with EtOAc to afford an organic extract, which was further purified by a combination of column chromatographies including Si gel, Sephadex LH-20, and Lobar LiChroprep RP-18, as well as by semipreparative HPLC, to yield compounds 1−5. © 2016 American Chemical Society and American Society of Pharmacognosy

Received: September 1, 2016 Published: December 19, 2016 162

DOI: 10.1021/acs.jnatprod.6b00797 J. Nat. Prod. 2017, 80, 162−168

Journal of Natural Products

Article

showed a close relationship to that of 1. However, the methyl group in 1 was replaced by a hydroxymethyl in 2, as evidenced by the fact that signals for the methyl (δH 2.15 and δC 16.6) in the NMR spectra of 1 were replaced by the resonances of an oxygenated methylene (δH 4.48 and δC 58.6) in 2. The location of the hydroxymethyl at C-7 was confirmed by the HMBC correlations from H2-11 to C-6, C-7, and C-8. The relative configuration of 2 was deduced to be the same as that of 1, based on the J-coupling constants and NOESY data (Figure 2). Upon slow evaporation of the solvent (MeOH) by storing the sample in a refrigerator, quality single crystals of 2 were obtained, making feasible an X-ray diffraction analysis that unequivocally confirmed its relative and absolute configurations (Figure 3). The final refinement of the Cu Kα data resulted in a 0.0(5) Flack parameter, allowing for the unambiguous assignment of the absolute configuration of 2 as 2S, 4aS and 9R, 9aS, the same as that of 1. 4R,8-Dihydroxyconiothyrione B (3), obtained as a yellowish, amorphous powder, was determined to possess the molecular formula C15H18O6, with one oxygen atom more than 1, on the basis of HRESIMS data. Comprehensive analysis of the 1H and 13 C NMR data of 3 (Tables 1 and 2) suggested that 3 might be a hydroxylated analogue of 1. Signals for one of the three methylene groups at δH 1.25 (Hα-4) and 2.19 (Hβ-4) and δC 23.6 (C-4) in the NMR spectra of 1 were absent in those of 3. Instead, signals for an oxygenated methine resonating at δH 3.89 (H-4) and δC 66.6 (C-4) were observed in the NMR spectra of 3. These differences suggested that the methylene CH2-4 in 1 was hydroxylated in 3, and this observation was supported by the COSY and HMBC correlations (Figure 1). The relative configuration of 3 was deduced to be the same as that of 1 and 2, according to the NOESY experiment (Figure 2) as well as the coupling patterns of the relevant protons (Table 2). The additional OH group at C-4 was deduced to be β-orientated based on the NOE correlations from H-4 to H-9a and H-2 (Figure 2). The ECD spectrum of 3 showed negative Cotton effects (CEs) near 216 and 369 nm and positive CEs around 245 and 275 nm, which were similar to those of 1 and 2, suggesting that the key stereogenic center C-9 in compounds 1−3 has the same R absolute configuration. This was confirmed by electronic circular dichroism (ECD) quantum chemical calculations in Gaussian 09.15 After geometry optimization of

The relative configuration of 1 was determined by analysis of J-values and NOESY data (Figure 2). The large coupling constant between H-9 and H-9a (J = 8.8 Hz) revealed that they were trans oriented, and the key NOE correlation from H-9 to H-4a suggested the cofacial orientation of the two protons, while the NOE correlation from H-2 to H-9a located the proton pair on the other side of the molecule. Consequently, the relative configuration of 1 was determined. The structure and absolute configuration of 1 were unambiguously established by a single-crystal X-ray diffraction experiment using Cu Kα radiation (Figure 3). The Flack parameter 0.0(3) allowed for the establishment of the absolute configuration of 1 as 2S, 4aS and 9R, 9aS. On the basis of the above data, the structure of 1 was determined, and the trivial name 8hydroxyconiothyrinone B was assigned to this compound. 8,11-Dihydroxyconiothyrinone B (2) was originally obtained as an amorphous powder. Its molecular formula, C15H18O6 (seven degrees of unsaturation), was determined by HRESIMS. The 1H and 13C NMR spectroscopic data of 2 (Tables 1 and 2)

Table 1. 13C NMR Data for Compounds 1−5 in DMSO-d6 (125 MHz) no. 1 2 3 4 4a 5 6 7 8 8a 9 9a 10 10a 11 10-OMe

1 38.2, 67.6, 33.9, 23.6, 46.7, 154.7, 117.7, 136.3, 146.5, 126.5, 72.1, 44.4, 204.3, 112.8, 16.6,

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

38.7, 68.1, 34.5, 24.2, 47.3, 155.6, 114.2, 141.3, 145.1, 126.9, 72.6, 45.0, 204.9, 113.4, 58.6,

3 CH2 CH CH2 CH2 CH C CH C C C CH CH C C CH2

37.7, 65.0, 43.0, 66.6, 53.3, 154.3, 117.7, 136.8, 146.4, 126.7, 72.0, 42.0, 205.6, 113.3, 16.5,

163

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

38.9, 63.8, 41.8, 64.5, 52.4, 155.5, 118.1, 137.0, 147.0, 126.7, 72.6, 38.6, 203.1, 114.0, 17.1,

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

35.4, 66.5, 44.9, 66.1, 50.7, 146.8, 126.5, 127.0, 154.2, 114.6, 206.7, 40.7, 66.7, 124.6, 15.6, 57.3,

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



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Table 2. 1H NMR Data for Compounds 1−5 in DMSO-d6 (500 MHz) no. 1α 1β 2 3α 3β 4α 4β 4a 6 9 9a 10 11 2-OH 4-OH 5-OH 8-OH 9-OH 10-OCH3

1 1.09, 2.37, 3.35, 1.12, 1.87, 1.25, 2.19, 2.30, 6.69, 4.77, 1.90,

m br d (11.9) br s m m (overlap) m m (overlap) td (3.5, 12.1) s d (8.8) m (overlap)

2.15, s 4.71, d (4.3) 12.04, s 9.70, s 6.84, br s

2 1.09, 2.35, 3.34, 1.23, 1.88, 1.14, 2.20, 2.37, 6.87, 4.82, 1.91,

m m (overlap) br s m m (overlap) m br d (13.5) m (overlap) s d (10.1) m (overlap)

4.48, s 4.69, d (3.7) 12.08, s

3 1.11, 2.32, 3.41, 1.20, 2.08, 3.89,

m m (overlap) br s m br d (11.8) br s

2.34, 6.72, 4.86, 1.93,

m (overlap) s d (10.0) m

2.16, 4.77, 4.52, 11.61,

s br s s br s

4 1.08, 2.38, 3.81, 1.25, 1.99,

m m (overlap) br s t (11.1) br d (12.6)

4.60, br s 2.42, m (overlap) 6.69, s 4.82,d (10.1) 2.35, m (overlap) 2.15, 4.55, 4.63, 12.33,

s d (4.4) d (3.9) s

5 0.99, 2.42, 3.49, 1.13, 2.10,

m br d (12.2) br s m m (overlap)

3.65, m 1.54, t (11.1) 7.05, s 2.68, 5.03, 2.12, 4.70, 4.81, 9.29, 12.34,

td (3.5, 12.2) s s d (3.8) d (5.8) s s

3.28, s

Figure 1. Key HMBC (arrows) and COSY (bold lines) correlations for compounds 1−5. Figure 2. Key NOESY correlations for compounds 1−5.

each possible isomer of 3, the minimum energy conformers could be obtained, and the time-dependent density functional method was used at the B3LYP/6-31G level to generate calculated ECD spectra of 3. The experimental and calculated ECD spectra for 3 showed agreement for the 2R, 4R, 4aR, 9R, and 9aS-absolute configuration in 3, although the peaks were shifted slightly (Figure 4). The molecular formula of 4S,8-dihydroxyconiothyrinone B (4) was determined to be C15H18O6 by HRESIMS, the same as that of 3. The 1H and 13C NMR spectroscopic data of 4 (Tables 1 and 2) were very similar to those of 3, with small differences in the chemical shifts for C-1 through C-4a and C10. Inspection of the 1H and 13C NMR data suggested that 4 is a diastereomer of 3, epimerized at C-4, and this deduction was further proved by the NOE correlation from 4-OH to H-2 (Figure 2). Similarly, the absolute configuration of 4 was established by ECD quantum chemical calculations in Gaussian

09.15 The TDDFT ECD spectra calculated for the 2R, 4S, 4aR, 9R, and 9aS absolute configuration in 4 gave good agreement with the experimental curve (Figure 4). 4S,8-Dihydroxy-10-O-methyldendryol E (5) was obtained as a yellowish, amorphous powder, and its molecular formula was determined as C16H20O6 by HRESIMS. Its 1H and 13C NMR data revealed the presence of two methyls (one oxygenated), two methylenes, six methines (one aromatic and three oxygenated), and six nonprotonated (one ketone and five aromatic) carbons (Tables 1 and 2). These data suggested that 5 had the same carbon skeleton as that of compounds 1−4 and dendryol E as well.8 Actually, compound 5 was a product from the reduction of the C-10 carbonyl group in the corresponding anthraquinone precursor, not the C-9 carbonyl group as that for compounds 1−4. This was verified by the HMBC 164



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Figure 4. Measured ECD spectra of epimers 3 and 4 in MeOH compared with the Boltzmann-weighted B3LYP/6-31G spectra calculated for the solution conformers of (2R,4R,4aR,9R,9aS)-3 and (2R,4S,4aR,9R,9aS)-4 in MeOH.

Figure 3. X-ray crystallographic structures of compounds 1 and 2.

Figure 5. Measured ECD spectrum of 5 in MeOH compared with the Boltzmann-weighted B3LYP/6-31G, CAM-B3LYP/TZVP, and BH&HLYP/TZVP spectra calculated for the solution conformers of (2S,4S,4aS,10S,9aR)-5 in MeOH.

correlations from H-10 to C-4 and C-5 and from H-9a to C-9, as well as by the COSY correlation between H-10 and H-4. A C-10 methoxy group was identified by the mutual HMBC correlations from H-10 to the carbon of 10-OCH3 as well as from the protons of 10-OCH3 to C-10 (Figure 1). The relative configuration of 5 was assigned by analysis of its NOESY data. NOE correlations from H-9a to H-2 and H-4 and from H-4 to H-10 suggested the cofacial orientation of these protons. The α-orientation of H-4a was determined by the NOE correlations from H-4a to 2-OH (Figure 2). The absolute configuration of 5 was also determined by ECD quantum chemical calculations. The experimental ECD spectrum of 5, which is different from those of compounds 1−4, matched well to the calculated ECD spectra with three functional methods (B3LYP/6-31G, CAM-B3LYP/TZVP, and BH&HLYP/TZVP) for the 2S, 4S, 4aS, 10S, and 9aR absolute configuration in 5 (Figure 5). On the basis of the above discussion, the structure of compound 5 was assigned, and it was named 4S,8-dihydroxy10-O-methyldendryol E.

Compounds 1−5 possess a tetralone chromophore with P or M helicity, similar to those of coniothyriones A−D.9 The M helicity of the tetralone chromophores of compounds 1 and 2 was determined by their single-crystal X-ray diffraction experiments. The B3LYP/6-31G(d) reoptimization of MMFF conformers of (2R,4R,4aR,9R,9aS)-3, (2R,4S,4aR,9R,9aS)-4, and (2S,4S,4aS,10S,9aR)-5 resulted in different conformers above the 2% population (Figures S38−S40), and for all of these conformers, the fused carbocyclic rings had M helicity for compounds 3 and 4 and P helicity for compound 5. The M helicity of the fused ring B of coniothyriones A−D has proved that the universal n−π* helicity rule, correlating the helicity of the fused hetero-ring to the sign of the carbonyl n−π* ECD transition,9,16 was not applicable to them. Accordingly, the helicity of the fused ring B of 1−5 cannot be assigned by the long-wavelength ECD transition. Scrupulous comparisons of the available ECD data and absolute configurations of hydroanthraquinones that possess one reduced carbonyl and a fully saturated C ring, including 165



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well-known antioxidant (IC50 = 61 μM). In addition, compounds 1−5 showed moderate ABTS radical scavenging activity with IC50 values ranging from 8.3 to 34 μM, which are comparable to that of ascorbic acid (IC50 = 16 μM). These results indicated that increasing molecular polarity (1 vs 2) and hydroxylation of nonaromatic carbons (1 vs 2−5) in structures strengthened their antibacterial effects but weakened the antioxidative activities. Compounds 1−5 were further assayed for cytotoxicity against sensitive and cisplatin-resistant human ovarian cancer cell lines A2780 and A2780 CisR, respectively. None of them were active (cytotoxicity = IC50 < 10 μM). Compound 2 showed effects on the cells only at 100 μM concentration (Figure S41).

compounds 1−5 (Figure 6) and coniothyrinones B−D,9 suggested an intimate relationship between the stereoconfigu-

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Figure 6. Measured ECD spectra of compounds 1−5 (hydroanthraquinones that possess one reduced carbonyl and a fully saturated C ring) in MeOH.

ration of C-9 (or C-10) and the signs of the CEs near 217, 246, and 276 nm. Generally, a negative CE near 217 nm and positive CEs around 246 and 276 nm in the ECD spectrum were consistent with the 9R (or 10R) configuration, and opposite signs indicated the 9S (or 10S) configuration. This ECD spectroscopic feature might be the key to assigning the stereoconfiguration of hydroanthraquinones that possess one reduced carbonyl and one fully saturated C ring. Compounds 1−5 were assayed for their antimicrobial activities against two human pathogens (Escherichia coli and Staphylococcus aureus), seven aquatic bacteria (Aeromonas hydrophila, Edwardsiella tarda, Micrococcus luteus, Pseudomonas aeruginosa, Vibrio alginolyticus, V. harveyi, and V. parahemolyticus), and five plant-pathogenic fungi (Alternaria brassicae, Colletotrichum gloeosporioides, Fusarium oxysporum, Gaeumannomyces graminis, and Physalospora piricolav). Each of them showed inhibitory activity against S. aureus, with MIC values ranging from 2 to 8 μg/mL, whereas compounds 2−4 exhibited weak activity against E. coli and E. tarda (Table 3). In the Table 3. Antibacterial Activity of Compounds 1−5 (MIC, μg/mL)a EC ET SA a

1

2

3

4

5

chloramphenicol

64 n.a. 4

16 32 2

32 64 4

32 64 4

n.a. n.a. 8

4 2 2

EC: E. coli. ET: E. tarda. SA: S. aureus. n.a.: no activity.

antioxidant evaluation, compounds 1−5 displayed comparable DPPH radical scavenging activity with IC50 values ranging from 12 to 52 μM (Table 4), which are stronger than that of BHT, a Table 4. Antioxidant Activities of Compounds 1−5 against DPPH and ABTS Radicals (IC50, μM)

DPPH ABTS

1

2

3

4

5

BHT

12 8.3

31 19

42 34

52 31

30 24

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined with an SGW X-4 micro-melting-point apparatus. Optical rotations were measured on an Optical Activity AA-55 polarimeter. UV spectra were measured on a PuXi TU-1810 UV−visible spectrophotometer. ECD spectra were acquired on a Chirascan spectropolarimeter. 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 tetramethylsilane as 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 P680 pump, ASI-100 automated sample injector, and 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 Talaromyces islandicus EN-501 was isolated from the fresh tissue of the marine red alga Laurencia okamurai collected in Qingdao, China, in July 2014, using a protocol described in our previous report.17 Fungal identification was performed by analysis of its ITS region of the rDNA as described previously.17 The resulting sequence data, which were most similar (99%) to the sequence of Talaromyces islandicus CBS 117284 (compared with KF984882.1), have been deposited in GenBank (accession no. KU885935). The strain is preserved at Key Laboratory of Experimental Marine Biology, Institute of Oceanology of the Chinese Academy of Sciences (IOCAS). Fermentation, Extraction, and Isolation. For chemical investigations, the fresh mycelia of T. islandicus EN-501 were grown on PDA medium at 28 °C for 4 days and were then inoculated for 30 days at room temperature in 90 × 1 L conical flasks with solid rice medium (each flask contained 70 g rice, 0.1 g corn flour, 0.3 g peptone, 0.1 g sodium glutamate, and 100 mL naturally sourced and filtered seawater, which was obtained from the Huiquan Gulf of the Yellow Sea near the campus of IOCAS, pH 6.5−7.0). The whole fermented cultures were extracted three times with EtOAc, which was evaporated under reduced pressure to afford an extract (80 g). The extract was fractionated by Si gel vacuum liquid chromatography using different solvents of increasing polarity from petroleum ether (PE) to MeOH to yield nine fractions (Frs. 1−9) based on TLC and HPLC analysis. Purification of Fr. 5 (9.0 g) by reversed-phase column chromatography (CC) over Lobar LiChroprep RP-18 with a MeOH− H2O gradient (from 20:80 to 100:0) yielded six subfractions (Frs. 5.1−5.6). Fr. 5.2 (1.3 g) was further purified by CC on Si gel eluting with a PE−acetone gradient (from 6:1 to 3:1) and then on Sephadex LH-20 (MeOH) to obtain compound 1 (57.4 mg). Fr. 5.3 (1.8 g) was purified by CC on Sephadex LH-20 (MeOH) and then by semipreparative HPLC (Elite ODS-BP column, 10 μm; 20 × 250 mm; 65% MeOH−H2O, 16 mL/min) to afford compound 2 (10.0 mg, tR 31.0 min). Fr. 5.4 (0.8 g) was further purified by CC on Sephadex LH-20 (MeOH) and then by preparative TLC (plate: 20 × 20 cm,

ascorbic acid 16 166



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developing solvents: PE−acetone, 1:1) to yield compound 5 (8.5 mg). Fr. 6 (4.0 g) was fractionated by CC over Lobar LiChroprep RP-18 with a MeOH−H2O gradient (from 20:80 to 100:0) and then purified by CC on Sephadex LH-20 (MeOH) and preparative TLC (plate: 20 × 20 cm, developing solvents: CHCl3−MeOH, 10:1) to yield compounds 3 (10.6 mg) and 4 (6.7 mg). 8-Hydroxyconiothyrinone B (1): colorless crystals; mp 235−237 °C; [α]25 D −16.7 (c 0.36, MeOH); UV (MeOH) λmax (log ε) 206 (2.64), 240 (3.21), 271 (3.08), 366 (2.82) nm; ECD (3.36 mM, MeOH) λmax (Δε) 216 (−15.3), 242 (+9.53), 273 (+8.33), 367 (−1.31) nm; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 279.1229 [M + H]+ (calcd for C15H19O5, 279.1227). 8,11-Dihydroxyconiothyrinone (2): colorless crystals; mp 250−252 °C; [α]25 D −36.4 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 204 (3.07), 238 (3.53), 270 (3.40), 367 (3.17) nm; ECD (3.59 mM, MeOH) λmax (Δε) 218 (−2.82), 243 (+1.78), 270 (+1.68), 364 (−0.23) nm; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 295.1170 [M + H]+ (calcd for C15H19O6, 295.1176). 4R,8-Dihydroxyconiothyrione B (3): yellowish, amorphous powder; [α]25 D −30.8 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 205 (3.62), 241 (3.79), 274 (3.71), 372 (3.42) nm; ECD (3.81 mM, MeOH) λmax (Δε) 216 (−6.02), 245 (+3.49), 275 (+3.52), 369 (−0.73) nm; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 295.1183 [M + H]+ (calcd for C15H19O6, 295.1176). 4S,8-Dihydroxyconiothyrinone B (4): yellowish, amorphous powder; [α]25 D −14 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 204 (3.50), 241 (3.65), 274 (3.53), 370 (3.27) nm; ECD (3.32 mM, MeOH) λmax (Δε) 217 (−2.67), 244 (+2.26), 274 (+2.44), 322 (−0.89), 385 (+0.27) nm; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 295.1186 [M + H]+ (calcd for C15H19O6, 295.1176). 4S,8-Dihydroxy-10-O-methyldendryol E (5): yellowish, amorphous powder; [α]25 D −35.0 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 206 (3.23), 234 (3.49), 270 (3.41), 378 (3.22) nm; ECD (3.47 mM, MeOH) λmax (Δε) 217 (+3.46), 267 (−3.33), 329 (−0.34), 385 (+0.34) nm; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 309.1327 [M + H]+ (calcd for C16H21O6, 309.1333). X-ray Crystallographic Analysis of Compound 1.18 All crystallographic data were collected on an Agilent Xcalibur Eos Gemini CCD plate diffractometer equipped with graphite-monochromatic Cu Kα radiation (λ = 1.541 78 Å) at 293(2) K. The data were corrected for absorption by using the program SADABS.19 The structures were solved by direct methods with the SHELXTL 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 structures were refined by full-matrix least-squares techniques.21 Crystal data for compound 1: C15H18O5, fw = 278.29, monoclinic space group P2(1), unit cell dimensions a = 10.2447(6) Å, b = 8.1822(4) Å, c = 16.0590(8) Å, V = 1323.01(12) Å3, α = γ = 90°, β = 100.634(2)°, Z = 4, dcalcd = 1.397 mg/m3, crystal dimensions 0.45 × 0.34 × 0.30 mm, μ = 0.871 mm−1, F(000) = 592. The 3337 measurements yielded 2825 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.0454 and wR2 = 0.1104 [I > 2σ(I)]. The Flack parameter was 0.0(3) in the final refinement for all 3337 reflections with 2825 Friedel pairs. Crystal data for compound 2: C15H18O6, fw = 294.29, monoclinic space group P1, unit cell dimensions a = 7.4072(8) Å, b = 9.2619(11) Å, c = 10.4835(12) Å, V = 670.13(13) Å3, α = 74.7400(10)°, β = 84.183(2)°, γ = 75.1120(10)°, Z = 2, dcalcd = 1.458 mg/m3, crystal dimensions 0.26 × 0.20 × 0.14 mm, μ = 0.950 mm−1, F(000) = 312. The 2689 measurements yielded 1752 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.0564 and wR2 = 0.1071 [I > 2σ(I)]. The Flack parameter was 0.0(5) in the final refinement for all 2689 reflections with 1752 Friedel pairs. ECD Calculations. Conformational searches for compounds 3−5 were performed via molecular mechanics using the MM+ method in HyperChem 8.0 software, and the geometries were further optimized at the B3LYP/6-31G(d) level via Gaussian 09 software to give the

energy-minimized conformers. Then, the optimized conformers were subjected to the calculations of ECD spectra using the TD-DFT at the B3LYP/6-31G (compounds 3−5), CAM-B3LYP/TZVP (compound 5), and BH&HLYP/TZVP (compound 5) level; solvent effects of the MeOH solution were evaluated at the same DFT level using the SCRF/PCM method.15 Antimicrobial Activity Assay. Antimicrobial evaluation against two human pathogens (E. coli EMBLC-1 and S. aureus EMBLC-2), seven aquatic bacteria (A. hydrophila QDIO-1, E. tarda QDIO-2, M. luteus QDIO-3, P. aeruginosa QDIO-4, V. alginolyticus QDIO-5, V. harveyi QDIO-7, and V. parahemolyticus QDIO-8), and five plantpathogenic fungi (A. brassicae QDAU-1, C. gloeosprioides QDAU-2, F. oxysporum QDAU-5, G. graminis QDAU-3, and P. piricolav QDAU6) was carried out by the microplate assay.22 The human and aquatic pathogens were obtained from the Institute of Oceanology, Chinese Academy of Sciences, while the plant pathogens were provided by the Qingdao Agricultural University. Chloramphenicol and amphotericin B were used as positive controls against bacteria and fungi, respectively. Antioxidant Activity Assay. Evaluations of the pure compounds for antioxidant activity against DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS [2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonate] radicals were performed by the methods described previously.23 BHT (butylated hydroxytoluene) and ascorbic acid were used as positive controls against DPPH and ABTS radicals, respectively. Cytotoxicity Assay. Evaluations for the cytotoxic activity of compounds 1−5 were performed as previously reported.24

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00797. X-ray crystallographic files of 1 (CIF) and 2 (CIF) Selected 1D and 2D NMR and ECD spectra of compounds 1−5 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Phone (M. Kassack): +49-211-8114587. E-mail: Matthias. [email protected]. *Phone (L.-H. Meng): +86-532-82898890. E-mail: menglh@ ms.qdio.ac.cn. *Phone (B.-G. Wang): +86-532-82898553. E-mail: wangbg@ ms.qdio.ac.cn. ORCID

Bin-Gui Wang: 0000-0003-0116-6195 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Natural Science Foundation of China (31330009) and from the Scientific and Technological Innovation Project of Qingdao National Laboratory for Marine Science and Technology (No. 2015ASKJ02) is gratefully acknowledged. B.-G.W. appreciates the support of Taishan Scholar Project from Shandong Province of China. M.U.K. would like to thank the Bundesminsterium fur Forschung (BMBF, Germany) for financial support (BMBF-16GW0108).

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