Trichocadinins B–G: Antimicrobial Cadinane Sesquiterpenes from

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Trichocadinins B−G: Antimicrobial Cadinane Sesquiterpenes from Trichoderma virens QA-8, an Endophytic Fungus Obtained from the Medicinal Plant Artemisia argyi Xiao-Shan Shi,†,‡,§ Ling-Hong Meng,†,⊥ Xiao-Ming Li,†,⊥ Xin Li,†,⊥ Dun-Jia Wang,‡ Hong-Lei Li,†,⊥ Xing-Wang Zhou,*,‡ and Bin-Gui Wang*,†,⊥

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†

Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, and Laboratory of Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Nanhai Road 7, Qingdao 266071, People’s Republic of China ‡ College of Chemistry and Chemical Engineering, Hubei Normal University, Cihu Road 11, Huangshi 435002, People’s Republic of China § University of Chinese Academy of Sciences, Yuquan Road 19A, Beijing 100049, People’s Republic of China ⊥ Center for Ocean Mega-Science, Chinese Academy of Sciences, Nanhai Road 7, Qingdao 266071, People’s Republic of China S Supporting Information *

ABSTRACT: Trichocadinins B−G (1−6), six new cadinanetype sesquiterpene derivatives, each with C-14 carboxyl functionality, were isolated from the culture extract of Trichoderma virens QA-8, an endophytic fungus obtained from the fresh inner tissue of the medicinal plant Artemisia argyi. Their structures were elucidated by interpretation of the NMR spectroscopic and mass spectrometric data. The structures and absolute configurations of compounds 1 and 3 were confirmed by X-ray crystallographic analysis. Compounds 1−3 showed antibacterial and antifungal activity.

T

he fungal genus Trichoderma is a widespread saprophyte that is present in almost all environments, and some species in the genus have been used as biocontrol agents for plant diseases.1 The genus has been found to be a rich source of structurally unique sesquiterpenes including cadinanes,2,3 daucanes,4−6 cyclonerodiol derivatives,7,8 acoranes,9 and other rearranged skeleton types.10 Several investigations on secondary metabolites of the genus have indicated that cadinane sesquiterpenes have a wide range of biological activities including gastroprotective, antibacterial, cytotoxic, anticancer, and fat-accumulation-inhibitory effects.11−14 As part of our continuous search for new bioactive natural products from endophytic fungi,15,16 the fungal strain Trichoderma virens QA8 obtained from the inner root tissue of the medicinal plant Artemisia argyi attracted our attention. The organic extract of the fungal culture exhibited antimicrobial activity against several marine-derived pathogens in our primary screening, and a chemical investigation of the secondary metabolites led to the isolation of six new sesquiterpene derivatives, trichocadinins B−G (1−6). The structures of these compounds were determined by analysis of the spectroscopic data, ECD calculations, and specific optical rotations, and the structures and absolute configurations of compounds 1 and 3 were confirmed by single-crystal X-ray diffraction analysis. All these compounds possess a C-14 carboxyl functionality. The antimicrobial activities against human pathogens, aquatic © XXXX American Chemical Society and American Society of Pharmacognosy

bacteria, and plant pathogenic fungi were determined. This paper describes the isolation, structure determination, stereochemical assignment, and antimicrobial activities of compounds 1−6.

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RESULTS AND DISCUSSION An EtOAc extract from the culture of T. virens QA-8 was fractionated by a combination of column chromatography Received: February 12, 2019

A

DOI: 10.1021/acs.jnatprod.9b00139 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products Table 1.

13

Article

C NMR Data for Compounds 1−6 in DMSO-d6 (125 MHz)

no.

1

2

3

4

5

6

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

151.9, C 109.8, CH 127.4, C 120.6, CH 135.3, C 131.3, C 117.0, C 17.5, CH2 24.8, CH2 42.3, CH 28.9, CH 21.1, CH3 18.8, CH3 168.0, C 141.2, CH

152.2, C 108.9, CH 136.9, C 120.0, CH 133.5, C 128.4, C 116.8, C 18.3, CH2 24.1, CH2 36.4, CH 36.9, CH 12.7, CH3 64.4, CH2 170.2, C 138.8, CH

24.3, CH2 24.6, CH2 131.0, C 138.3, CH 44.7, CH 42.6, CH 151.5, C 35.3, CH2 26.0, CH2 45.4, CH 26.1, CH 21.1, CH3 15.0, CH3 168.2, C 104.2, CH2

65.9, CH 34.8, CH2 148.3, C 135.2, CH 45.3, CH 50.5, CH 148.3, C 36.4, CH2 26.4, CH2 46.0, CH 26.6, CH 21.1, CH3 14.9, CH3 168.5, C 106.1, CH2

21.3, CH2 21.0, CH2 129.0, C 140.1, CH 47.3, CH 70.5, C 42.5, CH2 30.7, CH2 24.0, CH2 48.6, CH 26.9, CH 21.3, CH3 15.3, CH3 168.3, C 15.0, CH3

115.1, CH 161.8, C 116.0, C 119.9, CH 147.1, C 148.4, C 205.2, C 34.9, CH2 23.5, CH2 44.2, CH 29.3, CH 21.3, CH3 19.6, CH3 168.1, C

Table 2. 1H NMR Data for Compounds 1−6 in DMSO-d6 (500 MHz) no. 1α 1β 2α 2β 4 5 6 7 8α 8β 9α 9β 10 11 12 13 15a 15b 14-OH 2-OH 6-OH

1

2

7.88, s

7.81, s

7.67, s

7.73, s

2.78, m (overlap)

2.83, 2.70, 1.77, 1.90, 3.07, 2.23, 0.82, 3.49, 3.42, 7.62,

1.90, m 2.78, 2.09, 0.99, 0.92,

m (overlap) m d (6.6) d (6.7)

7.82, s 12.92, br s

m m m m m dtq (12.8,6.6,6.6) d (6.9) dd (10.5,6.6) dd (10.5,6.6) s

4

5

1.32, 1.09, 2.14, 1.77, 6.97, 2.36, 2.39,

m m m (overlap) m (overlap) s m m

3

3.78, dd (15.4, 9.3)

1.50, m (overlap)

2.67, 1.95, 6.77, 1.79, 1.79,

dd (17.4,5.4) m (overlap) s m (overlap) m (overlap)

2.19, m

1.77, 2.03, 1.96, 1.77, 1.40, 2.14, 0.93, 0.75,

m (overlap) m m m (overlap) m m (overlap) d (6.9) d (6.9)

1.95, 2.30, 1.79, 1.10, 1.35, 2.12, 0.91, 0.73,

m (overlap) m m (overlap) m dd (14.4, 7.6) m d (6.8) d (6.8)

4.70, s 4.57, s 12.21, br s

4.95, s 4.79, s

6

6.86, d (5.4) 1.83, m (overlap) 1.50, 1.03, 1.50, 1.21, 1.03, 1.21, 1.83, 0.85, 0.83,

m (overlap) m (overlap) m (overlap) m (overlap) m (overlap) m (overlap) m (overlap) d (7.0) d (7.0)

7.15, s

7.27, s

2.57, m (overlap) 2.77, m 2.02, m (overlap) 2.57, 2.02, 0.89, 0.93,

m (overlap) m (overlap) d (6.7) d (6.6)

0.88, d (6.7) 12.07, br s 12.40, s 3.95, s

(CC) on Si gel, Lobar LiChroprep RP-18, and Sephadex LH20, as well as by preparative TLC, to obtain compounds 1−6. Compound 1 was isolated as colorless crystals, and its molecular formula was determined as C15H16O3 on the basis of HRESIMS data, implying eight degrees of unsaturation. The 1 H and 13C NMR spectroscopic data of 1 indicated the presence of two methyl groups, two methylenes, five methines (with three aromatic/olefinic), and six nonprotonated carbons (Tables 1 and 2). Detailed inspection of the NMR data revealed a cadinane skeleton with a benzofuran moiety.14,17,18 Two aromatic protons resonating at δH 7.88 (1H, s) and 7.67 (1H, s) were assigned to H-2 and H-4, respectively, while an olefinic proton at δH 7.82 (1H, s) was assigned to the furan at H-15. The isopropyl group was placed at C-10 based on COSY correlations from H-12 and H-13 to H-11 and from H-11 to H-10 and was confirmed by the HMBC correlations from H12 and H-13 to C-10 (Figure 1). The deshielded resonance at δC 168.0 in the 13C NMR spectrum of 1 is typical for a

Figure 1. Key HMBC (red arrows) and COSY (bold blue lines) correlations for compounds 1−6.

carboxyl functionality (C-14), and the HMBC correlations from H-2 and H-4 to C-14 connected the carboxyl moiety with C-3. The structure and absolute configuration of 1 were unambiguously established by a single-crystal X-ray diffraction B

DOI: 10.1021/acs.jnatprod.9b00139 J. Nat. Prod. XXXX, XXX, XXX−XXX

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which showed positive CEs (Cotton effects) around 206 and 224 nm and a negative CE near 266 nm (Figure 3), the absolute configuration at C-10 in 2 was assigned R, as with 1. Further NMR studies were performed to determine the relative configuration of C-11 by using the J-based method developed by Murata and co-workers.19 For C-10 and C-11, the large three-bond homonuclear coupling of 3J(H-10, H-11) = 12.8 Hz indicated an H/H-anti orientation between H-10 and H11, while H-9 and H-13 had NOE correlations in the NOESY spectrum indicating the H/H-anti and C/C-gauche conformation. The absolute configuration at C-11 in 2 was assigned as S. Compound 3 was initially obtained as an amorphous powder. The HRESIMS exhibited a pseudomolecular ion peak [M − H]− at m/z 233.1547, corresponding to the molecular formula of C15H22O2, indicating five degrees of unsaturation. The 1H and 13C NMR data of 3 revealed the presence of two methyls, five methylenes (with one exomethylene), five methines (with one olefinic), and three nonprotonated carbons as well as one exchangeable proton at δH 12.21 (Tables 1 and 2). Detailed analysis of the 1H and 13C NMR spectroscopic data as well as COSY and HMBC correlations (Figure 1) indicated that the planar structure of 3 was the same as that of angelhardic acid,20 whose relative configuration was determined to be 5R*, 6S*, and 10R* by NOESY spectrum. As for compound 3, NOE correlations from H-6 to H-8α, H-2α, and H-10 and from H-10 to H-9α indicated the cofacial orientation of these groups (Figure 4), while correlations from H-5 to H-1β and H-8β suggested that these protons were on the other side of the molecule. The above NOEs allowed the relative configuration of compound 3 to be determined as 5R*, 6R*, and 10S*. To unambiguously clarify the stereochemistry, quality crystals of 3 were cultivated upon slow evaporation of the solvent (MeOH−H2O, 6:1) by storing in a refrigerator. The refinement of the Cu Kα radiation data yielded a Flack parameter of 0.2(4), which not only confirmed the planar structure but also defined the absolute configurations of compound 3 as 5R, 6R, and 10S (Figure 2). Thus, the structure of 3 was fully characterized, and it was named trichocadinin D. Compound 4 was initially isolated as an amorphous powder, and its molecular formula was determined as C15H22O3 (five degrees of unsaturation), with one oxygen atom more than 3. The 1H and 13C NMR spectroscopic data of 4 (Tables 1 and 2) were very similar to those of 3, suggesting that 4 was an analogue of 3. However, resonances for a methylene at δH 1.09/1.32 (2H, H2-1) and δC 24.3 (C-1) in the NMR spectra of 3 disappeared in those of 4. Instead, resonances for an oxygenated methine at δH 3.78 (H-1) and δC 65.9 (C-1) were observed in the NMR spectra of 4 (Tables 1 and 2). This revealed that the methylene group (CH2-1) in 3 was hydroxylated in 4, which was confirmed by the COSY and HMBC correlations (Figure 1). In the NOESY experiments, NOEs from H-1 to H-2α, H-6, and H-8α and from H-10 to H-8α and H-9α indicated the cofacial orientation of these groups (Figure 4), while correlations from H-8β to H-9β suggested that these protons were on the other side of this molecule. However, no other diagnostic NOEs were observed, and thus the relative configuration at C-5 could not be characterized by NOESY experiments. The absolute configuration of 4 was studied by the time-dependent density functional (TDDFT)-ECD calculation in Gaussian 09.21 Geometry optimization of each possible isomer was performed to obtain minimum energy

experiment using Cu Kα radiation (Figure 2). The Flack parameter of 0.1(2) allowed for the establishment of the

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

absolute configuration of 1 as 10R. On the basis of the above data, the structure of 1 was determined. Compound 1 was named trichocadinin B, based on the fact that it shares the same carbon skeleton as that of trichocadinin A, a cadinane sesquiterpene isolated from the marine-alga-epiphytic fungus T. virens Y13-3 by Shi and co-workers in 2018.3 Trichocadinin C (2) was obtained as a colorless oil, and its molecular formula was determined as C15H16O4 (eight degrees of unsaturation), with one oxygen atom more than 1, on the basis of HRESIMS data. Compared to the NMR signals of 1, a hydroxy-substituted methylene at δH 3.49/3.42 (H2-13) appeared in the 1H NMR spectrum of 2 (Table 2), and the doublet signal for the methyl group of H-13 in 1 disappeared in that of 2. Accordingly, the 13C NMR signal at δC 64.4 for an oxygenated methylene carbon in 2 replaced the methyl group (C-13) at δC 18.8 in 1 (Table 1). Thus, compound 2 was deduced to be a C-13 hydroxylated derivative of 1, and this deduction was confirmed by the COSY and HMBC correlations (Figure 1). In view of the virtual identical electronic circular dichroism (ECD) spectra of 1 and 2, C

DOI: 10.1021/acs.jnatprod.9b00139 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 3. Experimental ECD spectra of compounds 1 and 2.

NMR spectrum of 5. HMBC correlation from H-4 to C-14 confirmed the carboxyl moiety at C-3. The relative configuration of compound 5 was determined by NOESY data. NOE correlations from the proton of OH-6 to H-5 and H3-15 indicated the cofacial orientation of these groups (Figure 4), while correlation from H-7 to H-10 suggested that these protons were on the other side of the molecule. The absolute configuration of 5 was established by ECD calculations.21 Geometry optimization of each possible isomer of 5 was performed to generate minimum energy conformers, and then the TDDFT method was employed at the PBE0/TZVP level to obtain calculated ECD spectra. Assessment of the ECD spectra of 5 revealed that the calculated curve for (5S,6R,7R,10R)-5 showed agreement with that of the experimental one (Figure 5). Thus, the structure of compound 5 was fully characterized and was named trichocadinin F. Compound 6 was purified as an amorphous powder. The HRESIMS exhibited a pseudomolecular ion peak [M − H]− at m/z 247.0974, corresponding to the molecular formula C14H16O4, indicating seven degrees of unsaturation. The 1H and 13C NMR data (Tables 1 and 2) closely resembled those of 7-desmethyl-2-hydroxy calamenen-7-on, a desmethyl cadinane sesquiterpene isolated from the aerial parts of Heterotheca grandif lora.17 However, the methyl group resonating at δH 2.31 that is connected at the aromatic ring of 7desmethyl-2-hydroxy calamenen-7-on was replaced by a carboxyl moiety which was detected at δC 168.1 in the 13C NMR spectrum of 6 (Table 1). HMBC correlations from H-2 and H-4 to C-14 evidenced the carboxyl moiety with C-3, and the full structure was further supported by additional HMBC and COSY correlations (Figure 1). Compound 6 has only one chiral center, and the absolute configuration was established by ECD calculation.21 Assessment of the experimental and calculated ECD spectra of 6 revealed agreement with that calculated for (10R)-6 (Figure 5). Thus, the structure of 6 was determined and was named trichocadinin G. Compounds 1−3 showed activity against a broad spectrum of bacteria, while compound 6 had activity against aquatic

Figure 4. Key NOESY correlations for compounds 2−5.

conformers, and then the TDDFT method was employed at the PBE0/TZVP level to generate calculated ECD spectra of 4. The experimental ECD spectrum of 4 showed excellent agreement with that calculated for (1R,5S,6R,10S)-4 (Figure 5). On the basis of the above data, the structure of 4 was determined and was named trichocadinin E. HRESIMS analysis of 5 revealed a pseudomolecular ion peak [M − H]− at m/z 251.1654, consistent with the molecular formula C15H24O3 and indicating the presence of four degrees of unsaturation. The carbon skeleton of compound 5 was determined to be the same as that of cubenol, a cadinane sesquiterpene isolated from the tropical brown alga Dictyopteris delicatula,22 by detailed analysis of the 1H and 13C NMR spectroscopic data (Tables 1 and 2). However, signals at δC/δH 23.6/1.71 (CH3-14) for the methyl group attached to the double bond in cubenol were not detected in the NMR spectra of 5. Instead, a deshielded resonance at δC 168.3 (C-14), which is typical for a carboxyl functionality, was found in the 13C D

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compounds 1−6 showed activity against Fusarium oxysporum f. sp. cucumebrium with MIC values ranging from 1 to 64 μg/mL (Table 4). An analysis of the structure−activity relationship revealed that compound 1, lacking the alcohol function at C13, was more potent than compound 2, while compounds 1 and 2, containing a benzofuran moiety, were generally more antimicrobial when compared to compounds 3−6 (Tables 3 and 4).

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on an SGW X-4 micro-melting-point apparatus. Optical rotations were measured with an Optical Activity AA-55 polarimeter. UV spectra were recorded on a PuXi TU-1810 UV−visible spectrophotometer. The ECD spectra were recorded on a Chirascan spectropolarimeter or a JASCO J-715 spectropolarimeter, using CH3OH as a solvent. 1D and 2D NMR spectra were recorded at 500 for 1H and 125 MHz and 13C in DMSO-d6, respectively, on a Bruker Avance 500 spectrometer with tetramethylsilane as internal standard. Mass spectra were recorded on an API QSTAR Pulsar 1 Orbitrap mass spectrometer or a Thermo Fisher Scientific LTQ Orbitrap XL spectrometer. Analytical HPLC were performed using a Dionex HPLC system equipped with a P680 pump, ASI-100 automated sample injector, and UVD340U multiple wavelength detector controlled by Chromeleon software (version 6.80). Opencolumn chromatography was performed with commercially available Si gel (200−300 mesh, Qingdao Haiyang Chemical Co.), Lobar LiChroprep RP-18 (40−63 μm, Merck), and Sephadex LH-20 (Pharmacia). TLC was carried out using Si gel GF254 (Qingdao Haiyang Chemical Factory) plates. All solvents used were distilled prior to use. Fungal Material. The endophytic fungus T. virens QA-8 was obtained from the inner root tissue of the medicinal plant Artemisia argyi collected at Qichun of Hubei Province in central China, in July 2014, using a protocol as described in our previous report.23 Fungal identification was performed by analysis of its ITS region of the rDNA as described previously.23 The resulting sequence was most similar (99%) to that of T. virens LW23 (compared with KT803076.1) and has been deposited in GenBank (with accession no. MK224593). The strain is preserved at the 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. virens QA-8 were grown on PDA medium at 28 °C for 7 days and used to inoculate solid rice medium (each flask contained 70 g of rice, 0.1 g of corn flour, 0.3 g of peptone, and 100 mL of distilled water) in 1 L conical flasks and incubated 30 days at room temperature. All fermented cultures (180 flasks) were extracted three times with EtOAc, which was evaporated under reduced pressure to afford an extract (97.4 g). The extract was fractionated by Si gel vacuum liquid chromatography (VLC) using different solvents of increasing polarity from petroleum ether (PE) to MeOH to yield 10 fractions (Frs. 1−10) based on TLC and HPLC analysis. Purification of Fr. 3 (3.0 g) by reversed-phase CC over Lobar LiChroprep RP-18 with a MeOH−H2O gradient (from 30:70 to 100:0) yielded six subfractions (Frs. 3.1−3.6). Fr. 3.4 (102 mg) was purified by CC on Sephadex LH-20 (MeOH) and then by preparative TLC (plate: 20 × 20 cm, developing solvents: PE−acetone, 2:1) to obtain compound 6 (6.9 mg). Fr. 3.5 (94 mg) was purified by CC on Sephadex LH-20 (MeOH) to yield compound 1 (10.6 mg), while Fr. 3.6 (143 mg) was purified on Si gel eluting with a PE−acetone gradient (from 20:1 to 2:1) and then by CC on Sephadex LH-20 (MeOH) to afford compound 3 (11.8 mg). Fr. 5 (11.0 g) was fractionated by CC over Lobar LiChroprep RP-18 with a MeOH− H2O gradient (from 10:90 to 100:0) and yielded 10 subfractions (Frs. 5.1−5.10). Fr. 5.8 (436 mg) was purified by CC on Si gel eluting with a CH2Cl2−acetone gradient (from 100:1 to 10:1) and then by CC on Sephadex LH-20 (MeOH) to give compound 4 (4.5 mg). Fr. 5.10 (547 mg) was purified on Si gel eluting with a PE−acetone gradient

Figure 5. Experimental and calculated ECD spectra of compounds 4− 6.

pathogens Edwardsiella tarda and Vibrio anguillarum with MIC values of 1 and 2 μg/mL, respectively, comparable to that of the positive control chloramphenicol (Table 3). Compound 1 exhibited inhibitory activity against the 13 test fungi, while E

DOI: 10.1021/acs.jnatprod.9b00139 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 3. Antibacterial Activity of Compounds 1−6 (MIC, μg/mL)a 1 2 3 4 5 6 chloramphenicol

EC

AH

ET

ML

PA

VAn

VH

VP

VV

8 16 8 − − − 2

8 64 8 − − − 4

−b − − − − 1 0.5

32 − 32 − − − 4

8 8 4 − − − 2

− − − − − 2 1

8 16 2 − − − 1

4 32 8 − − − 4

64 − 64 − − − 1

a

EC: E. coli. AH: A. hydrophilia. ET: E. tarda. ML: M. luteus. PA: P. aeruginosa. VAn: V. anguillarum. VH: V. harveyi. VP: V. parahemolyticus. VV: V. vulnificus. b−: no activity.

Table 4. Antifungal Activity of Compounds 1−6 (MIC, μg/mL)a 1 2 3 4 5 6 amphotericin B

AS

BS

CC

CG

FG

FOC

FOM

FOR

FS

GC

HM

PD

PP

VM

4 32 − − − − 2

1 8 − − − − 0.5

16 − − − − − 8

8 − − − − − 0.5

8 − − − − − 2

1 2 16 32 32 64 0.5

2 − − − − − 1

64 − 64 64 − − 2

4 32 − − − − 4

1 4 1 − − − 0.5

−b − − − 16 − 2

8 8 − 16 − − 2

4 4 8 1 − 4 2

16 − 32 − − − 8

a

AS: A. solani. BS: B. sorokiniana. CC: C. cornigerum. CG: C. gloeosporioides Penz. FG: F. graminearum. FOC: F. oxysporum. f. sp. cucumebrium. FOM: F. oxysporum f. sp. momordicae. FOR: F. oxysporum f. sp. radicis lycopersici. FS: F. solani. GC: G. cingulate. HM: H. maydis. PD: P. digitatum. PP: P. piricola Nose. VM: V. mali. b−: no activity. X-ray Crystallographic Analysis of Compounds 1 and 3.24 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.25 The structures were solved by direct methods with the SHELXTL software package.26 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.27 Crystal data for compound 1: C15H16O3, fw = 244.28, tetragonal space group P4(1), unit cell dimensions a = 17.1550(3) Å, b = 17.1550(3) Å, c = 35.3313(16) Å, V = 10397.8(5) Å3, α = β = γ = 90°, Z = 32, dcalcd = 1.248 mg/m3, crystal dimensions 0.10 × 0.08 × 0.06 mm3, μ = 0.699 mm−1, F(000) = 4160. The 14 076 measurements yielded 6110 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.0562 and wR2 = 0.0896 [I > 2σ(I)]. The Flack parameter was 0.1(2) in the final refinement for all 14 076 reflections with 6110 Friedel pairs. Crystal data for compound 3: C15H22O2, fw = 234.33, monoclinic space group P2(1), unit cell dimensions a = 5.3671(4) Å, b = 16.4292(16) Å, c = 15.9007(13) Å, V = 1395.1(2) Å3, α = γ = 90°, β = 95.710(3)°, Z = 4, dcalcd = 1.116 mg/m3, crystal dimensions 0.40 × 0.13 × 0.10 mm3, μ = 0.565 mm−1, F(000) = 512. The 3678 measurements yielded 2130 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.0559 and wR2 = 0.0990 [I > 2σ(I)]. The Flack parameter was 0.2(4) in the final refinement for all 3678 reflections with 2130 Friedel pairs. Computational Section. Conformational searches 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 in vacuo via Gaussian 09 software to give the energy-minimized conformers. Then, the optimized conformers were subjected to the calculations of ECD spectra using TDDFT at the PBE0/TZVP level; solvent effects of the MeOH solution were evaluated at the same DFT level using the SCRF/PCM method.21

(from 20:1 to 1:1) and then by CC on Sephadex LH-20 (MeOH) to obtain compound 5 (7.5 mg). Purification of Fr. 6 (22.7 g) by reversed-phase CC over Lobar LiChroprep RP-18 with a MeOH− H2O gradient (from 10:90 to 100:0) yielded 15 subfractions (Frs. 6.1−6.15). Fr. 6.9 (2.1 g) was further purified by CC on Si gel eluting with a CH2Cl2−MeOH gradient (from 200:1 to 10:1) and then by preparative TLC (plate: 20 × 20 cm, developing solvents: CH2Cl2− PE, 5:1) and Sephadex LH-20 (MeOH) to afford compound 2 (13.7 mg). Trichocadinin B (1): colorless crystals; mp 109−112 °C; [α]25 D +5.48 (c 0.73, MeOH); UV (MeOH) λmax (log ε) 209 (2.39), 225 (2.57), 276 (2.35) nm; ECD (4.09 mM, MeOH) λmax (Δε) 206 (+15.03), 228 (+11.67), 276 (−6.34) nm; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z [M − H]− 243.1022 (calcd for C15H15O3−, 243.1027). Trichocadinin C (2): colorless oil; [α]25 D +44.29 (c 0.70, MeOH); UV (MeOH) λmax (log ε) 212 (2.63), 222 (2.60), 271 (2.31) nm; ECD (5.00 mM, MeOH) λmax (Δε) 206 (+17.14), 224 (+28.12), 266 (−8.45) nm; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z [M + Na]+ 283.0920 (calcd for C15H16O4Na+, 283.0946). Trichocadinin D (3): colorless, amorphous powder, mp 154−157 °C; [α]25 D −111.11 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 220 (1.74) nm; ECD (3.48 mM, MeOH) λmax (Δε) 217 (−8.70) nm; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z [M − H]− 233.1547 (calcd for C15H21O2−, 233.1547). Trichocadinin E (4): colorless, amorphous powder; [α]25 D −135.71 (c 0.28, MeOH); UV (MeOH) λmax (log ε) 217 (2.24) nm; ECD (3.6 mM, MeOH) λmax (Δε) 210 (−20.66) nm; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z [M − H]− 249.1493 (calcd for C15H21O3−, 249.1496). Trichocadinin F (5): colorless oil; [α]25 D +8.00 (c 0.50, MeOH); UV (MeOH) λmax (log ε) 209 (2.29) nm; ECD (3.17 mM, MeOH) λmax (Δε) 200 (+5.56), 232 (−1.76) nm; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z [M − H]− 251.1654 (calcd for C15H23O3−, 251.1653). Trichocadinin G (6): colorless, amorphous powder; [α]25 D +55.56 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 271 (2.12), 351 (1.51) nm; ECD (3.22 mM, MeOH) λmax (Δε) 218 (+6.84), 277 (+1.80), 336 (−1.85) nm; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z [M − H]− 247.0974 (calcd for C14H15O4−, 247.0976). F

DOI: 10.1021/acs.jnatprod.9b00139 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Antimicrobial Activity Assay. Antimicrobial evaluation against one human pathogen (Escherichia coli EMBLC-1), 10 marine-derived aquatic bacteria (Aeromonas hydrophilia QDIO-1, Edwardsiella tarda QDIO-2, E. ictarda QDIO-10, Micrococcus luteus QDIO-3, Pseudomonas aeruginosa QDIO-4, Vibrio alginolyticus QDIO-5, V. anguillarum QDIO-6, V. harveyi QDIO-7, V. parahemolyticus QDIO-8, and V. vulnificus QDIO-9), and 15 plant-pathogenic fungi (Alternaria solani QDAU-14, Bipolaris sorokiniana QDAU-7, Ceratobasidium cornigerum QDAU-8, Coniothyrium diplodiella QDAU-19, C. gloeosporioides Penz QDAU-9, Fusarium graminearum QDAU-10, F. oxysporum f. sp. cucumebrium QDAU-16, F. oxysporum f. sp. momordicae QDAU-17, F. oxysporum f. sp. radicis lycopersici QDAU-5, F. solani QDAU-15, Glomerella cingulate QDAU-2, Helminthosporium maydis QDAU-18, Penicillium digitatum QDAU-11, P. piricola Nose QDAU-12, and Valsa mali QDAU-13) was carried out by the microplate assay.28 The 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 the positive control against bacteria and fungi, respectively.

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(6) Macias, F. A.; Varela, R. M.; Simonet, A. M.; Cutler, H. G.; Cutler, S. J.; Eden, M. A.; Hill, R. A. J. Nat. Prod. 2000, 63, 1197− 1200. (7) Huang, Q.; Tezuka, Y.; Hatanaka, Y.; Kikuchi, T.; Nishi, A.; Tubaki, K. Chem. Pharm. Bull. 1995, 43, 1035−1038. (8) Fujita, T.; Takaishi, Y.; Takeda, Y.; Fujiyama, T.; Nishi, T. Chem. Pharm. Bull. 1984, 32, 4419−4425. (9) Tezuka, Y.; Tasaki, M.; Huang, Q.; Hatanaka, Y.; Kikuchi, T. Liebigs. Ann. Recl. 1997, 12, 2579−2580. (10) Kawashima, J.; Ito, F.; Kato, T.; Niwano, M.; Koshino, H.; Uramoto, M. J. Antibiot. 1994, 47, 1562−1563. (11) Reyes, M.; Schmeda-Hirschmann, G.; Razmilic, I.; Theoduloz, C.; Yáñez, T.; Rodríguez, J. A. Phytother. Res. 2005, 19, 1038−1042. (12) Almeida, C.; Eguereva, E.; Kehraus, S.; Siering, C.; Konig, G. M. J. Nat. Prod. 2010, 73, 476−478. (13) Rahier, N. J.; Molinier, N.; Long, C.; Deshmukh, S. K.; Kate, A. S.; Ranadive, P.; Verekar, S. A.; Jiotode, M.; Lavhale, R. R.; Tokdar, P.; Balakrishnan, A.; Meignan, S.; Robichon, C.; Gomes, B.; Aussagues, Y.; Samson, A.; Sautel, F.; Bailly, C. Bioorgan. Med. Chem. 2015, 23, 3712−3721. (14) Boonsri, S.; Karalai, C.; Ponglimanont, C.; Chantrapromma, S.; Kanjana-opas, A. J. Nat. Prod. 2008, 71, 1173−1177. (15) Shi, X. S.; Wang, D. J.; Li, X. M.; Li, H. L.; Meng, L. H.; Li, X.; Pi, Y.; Zhou, X. W.; Wang, B. G. RSC Adv. 2017, 7, 51335−51342. (16) Li, H. L.; Li, X. M.; Li, X.; Wang, C. Y.; Liu, H.; Kassack, M. U.; Meng, L. H.; Wang, B. G. J. Nat. Prod. 2017, 80, 162−168. (17) Bohlmann, F.; Zdero, C.; Robinson, H.; King, R. M. Phytochemistry 1979, 18, 1675−1680. (18) Lv, X. Q.; Luo, J. G.; Wang, X. B.; Wang, J. S.; Luo, J.; Kong, L. Y. Chem. Pharm. Bull. 2011, 59, 402−406. (19) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.; Tachibana, K. J. Org. Chem. 1999, 64, 866−876. (20) Wu, C. C.; Peng, C. F.; Tsai, I. L.; Abd El-Razek, M. H.; Huang, H. S.; Chen, I. S. Phytochemistry 2007, 68, 1338−1343. (21) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; et al. Gaussian 09, revision D.01; Gaussian, Inc.: Wallingford, CT, 2013. (22) König, G. M.; Wright, A. D. Magn. Reson. Chem. 1995, 33, 178−183. (23) 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. (24) Crystallographic data of compounds 1 and 3 have been deposited in the Cambridge Crystallographic Data Centre as CCDC 1860046 (for 1) and CCDC 1860045 (for 3). 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, U.K.; fax: +44-1223-336-033; e-mail: [email protected]). (25) Sheldrick, G. M. SADABS, Software for Empirical Absorption Correction; University of Gottingen: Germany, 1996. (26) Sheldrick, G. M. SHELXTL, Structure Determination Software Programs; Bruker Analytical X-ray System Inc.: Madison, WI, 1997. (27) Sheldrick, G. M. SHELXL-97 and SHELXS-97, Program for Xray Crystal Structure Solution and Refinement; University of Gottingen: Germany, 1997. (28) Pierce, C. G.; Uppuluri, P.; Tristan, A. R.; Wormley, F. L., Jr; Mowat, E.; Ramage, G.; Lopez-Ribot, J. L. Nat. Protoc. 2008, 3, 1494−1500.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00139.

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Selected 1D and 2D NMR and HRESIMS spectra of 1− 6; experimental ECD spectra of 3; crystal packing of 1 and 3 (PDF) X-ray crystallographic file of 1 (CIF) X-ray crystallographic file of 3 (CIF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail (X.-W. Zhou): [email protected]. *E-mail (B.-G. Wang): [email protected]. 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 National Natural Science Foundation of China (NSFC grant no. 31870328) is gratefully acknowledged. B.-G.W. appreciates the support of Taishan Scholar Program from Shandong Province of China (ts201511060).

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REFERENCES

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DOI: 10.1021/acs.jnatprod.9b00139 J. Nat. Prod. XXXX, XXX, XXX−XXX